Poster Abstracts with Authors and Numbers

POSTER ABSTRACTS

APPLIED PLANT BIOLOGY - Zone 100 Applied: Plants and Human/Societal Health 100-001-Y A Nationally Coordinated Effort to Mitigate Food Safety Concerns Related to Acrylamide in Fried Potato Products Though Identification of Superior Germplasm Paul Bethke – USDA ARS and University of Wisconsin-Madison Yi Wang – University of Idaho, Kimberly Research and Extension Center, Jeffrey Endelman – Department of Horticulture, University of Wisconsin-Madison Food safety concerns were raised worldwide when acrylamide, a suspected human carcinogen, was found in many widely consumed foods. Acrylamide is formed from reducing sugars, such as glucose and fructose, and asparagine during high temperature cooking. Several frequently consumed potato products, including French fries and potato chips, make a substantial contribution to total dietary acrylamide. Health safety concerns related to acrylamide in food encouraged the potato industry to take a proactive response toward acrylamide mitigation. The National Fry Processing Trial (NFPT) is a cooperative effort in which researchers and experts in industry are addressing the need for potato cultivars that have lower acrylamide-forming potential than standard varieties through systematic evaluation of elite potato germplasm. This effort began in 2011 with research plots in WA, ID and ND and was expanded in 2012 to include sites in WI and ME as part of the USDA Specialty Crop Research Initiative on acrylamide reduction in potato. Agronomic data were collected for over 140 advanced breeding lines in field years 2011-2013. Acrylamide content of fries produced after one to eight months of tuber storage was determined, as were tuber contents of glucose and asparagine. Promising clones were subjected to in-depth consumer attribute testing. Glucose content in raw tubers was highly predictive of acrylamide content in fries at three post-harvest sampling times (R2 = 0.64 – 0.77). An overwhelming majority of NFPT clones produced fries with less acrylamide than industry standard varieties. Many clones failed to meet processor specifications for size distribution and dry matter content, but the greater challenge was meeting end-user requirements for sensory specifications. Tuber dry matter content variation was identified as a key indicator of a clone’s ability to produce acceptable products, and phenotyping methods are being developed to assess this trait efficiently.

100-002-Z The Role of the Target of Rapamycin (TOR) Kinase and Nutrient Restriction on Lifespan of Arabidopsis Thaliana Sibdas Ghosh – Iona College Debashree Banerjee – Dominican University of California, Pankaj Kapahi – Buck Institute for Research on Aging, Noor Nahar – Systems Biology Research Center, School of Bioscience, University of Skovde The target of rapamycin (TOR) kinase is evolutionarily conserved and is a key regulator, controlling many cellular functions to promote survival and growth in all eukaryotes. Abundant nutrient availability promotes rapid growth and development, whereas depletion of nutrients reduces the activity of the pathways that are involved in growth and nutrient processing. This reduction in activity increases life expectancy. The confirmed presence of the TOR homolog (a major regulator of growth and cell proliferation) in Arabidopsis thaliana makes this plant an ideal candidate to study for the effects of reduced nutrients in the lifespan extension of plants. To study the role of TOR pathway in lifespan extension in Arabidopsis thaliana, mutant plants with reduced and overexpressed TOR activity were grown in hydroponic culture systems, to monitor the effects of varying nutrient conditions and several senescence associated phenotypes (SAP). During this study we found plants with reduced TOR activity displayed delayed flowering, shattering of siliques, and yellowing of leaves as compared to the wild type plants, whereas plants overexpressing TOR activity displayed early SAP. Furthermore, the molecular and physiological analyses shed light on the link between TOR signaling pathway restriction on Arabidopsis lifespan. We hypothesize that modulating TOR may slow the aging process in Arabidopsis, through downstream processes including mRNA translation, protein synthesis, autophagy, and stress

responses. However, the general processes of biological aging in animals differ fundamentally from those are noted in plants. Therefore, identifying the mechanisms through which Arabidopsis thaliana TOR functions in plants may aid in developing stress resistant strains that could provide tools for improving crop yield and creating long lasting plants yielding long lasting fruits and vegetables such as fruits and vegetables, as well as may benefit the floral industry by providing fresh and long lasting flowers.

100-003-Z Characterization of Plant Produced Monoclonal Antibody for Treatment of Flavivirus Infections Jonathan Hurtado – Arizona State University Adrian Esqueda, Huafang Lai, Qiang Chen – Arizona State University Flavivirus infections are emerging as significant threats to human health around the globe. Among them West Nile(WNV) and Dengue Virus (DV) are the most prevalent in causing human disease with WNV outbreaks occurring in all areas around the world and DV epidemics in more than 100 countries. WNV is a neurotropic virus capable of causing meningitis and encephalitis in humans. Currently, there are no therapeutic treatments or vaccines available. DV causes severe hemorrhaging diseases especially in people exposed to secondary DV infection from a heterotypic strain. It is hypothesized that sub-neutralizing cross-reactive antibodies from the first exposure aid the second infection in a process called antibody-dependent enhancement (ADE). ADE depends on the ability of mAb to bind Fc receptors (FcγRs), and has become a major roadblock for developing mAb-based therapeutics against DV. We aim to produce an anti-Dengue mAb (E60) in different glycoengineered plant lines that exhibit reduced/differential binding to FcγRs, therefore, reducing or eliminating ADE. We have successfully cloned the molecular constructs of E60, and expressed it in two plant lines with different glycosylation patterns. We demonstrated that both plant-derived E60 mAb glycoforms retained specific recognition and neutralization activity against DV. Overall, our study demonstrates great strives to develop efficacious therapeutics and potent vaccine candidates against Flaviviruses in plant expression systems.

100-004-Y Do Nucleotides Flanking miRNA Target Sites Affect Repression Efficacy? Seth Polydore – The Pennsylvania State University Michael Axtell – The Pennsylvania State University Manipulation of plant physiology is important to bioengineer crops that are able to adapt to the changing abiotic (higher CO2 levels, soil salinity, changing climates, etc.) and biotic (pathogenic and parasitic) stresses. Artificial micro RNAs (amiRNAs) are one such way to achieve this, especially in species for which targeted gene knockout may prove difficult; this is often the case for many societially relevant plant species. Much research in plants miRNAs has elucidated the complementarity requirements to maximize repression efficacy. Recently, some studies have begun to identify factors beyond complementarity that affects the miRNA repression efficacy – one such factor is the nucleotide context surrounding the miRNA target site. These factors need to be properly studied in order to optimize a-miRNA design and targeting algorithms. Using a dual luciferase assay, we intend to determine how and to what extent nucleotides flanking an a-miRNA target site are able to impair/enchance repression efficacy. Our preliminary results show that the changing flanking sequences do indeed seem to change repression; however, underlying biological effects, such as RNA secondary structure and steric hinderances, in relation to these results have yet to be discovered. Overall, our research may reveal provide the first direct evidence linking miRNA repression and oft-ignored factors.

100-005-Y Detection of an Abundant Plant-based Small RNA in Consumers Kendal Hirschi – Baylor College of Medicine Jian Yang – Baylor College of Medicine, Mark Yarmarkovich – University of Pennsylvania, Lisa Farmer – Baylor College of Medicine, Abia Agyekum – Baylor College of Medicine, Ismail Elbaz-Younes – Baylor College of Medicine Mechanisms of delivery of plant small RNAs to consumers must be addressed in order to harness this technology to positively impact agbiotechnology. Two groups have used honeysuckle (Lonicera japonica) feeding regimes to detect a plant-based small RNA, termed MIR2911, in sera. Meanwhile, numerous groups have failed to detect dietary plantbased small RNAs in consumers. Here we catalog levels of MIR2911 in different herbs, and suggest that in particular herbs MIR2911 levels are elevated. Feeding these different herb-based diets to mice, we found MIR2911 levels in the sera and urine were associated with dietary intake levels. Abundance was not the sole determinate of apparent RNA bioavailability, as gavage-feeding large-doses of synthetic MIR2911 permitted only small transient increases in serum levels. Dietary MIR2911 were not modified in circulation by association with the host’s RNA-induced silencing complex, as the RNA did not co-immunoprecipitate with AGO2. The stability of dietary MIR2911 in circulation differed from synthesized small RNAs, as tail vein administration of various synthetic plant-based small RNAs resulted in rapid clearance. However, synthetic MIR2911 appeared to be more stable than the other plant miRNAs tested. Notably, the uptake of dietary MIR2911 was not related to perturbations in the host’s microbiome or gut permeability. We will present preliminary studies regarding more detailed mechanisms of plant-based dietary RNAs uptake and evidence that MIR2911 can regulate gene expression in mammalian tissues. We suggest dietary uptake of MIR2911 is commonplace in healthy consumers, and reproducible detection of plant-based small RNAs in consumers depends on dietary abundance, RNA stability and digestion from within the food-matrix.

100-006-Z Alpha Glucosidase Inhibition Effects, Antioxidant Activities, and Cytotoxicity of Philippine Herbal Weeds Containing Polyphenols and Carotenoids Richard Licayan – Rutgers University New Use Agriculture and Natural Plant Products Program Heather Healy – Rutgers University New Use Agriculture and Natural Plant Products Program, Daniel Giurleo – Rutgers University New Use Agriculture and Natural Plant Products Program, Thomas Villani – Rutgers University New Use Agriculture and Natural Plant Products Program Diabetes mellitus is a group of metabolic disorders characterized by high blood glucose levels resulting from defects in insulin secretion, insulin action, or both. In the present study, sponsored by the USAID STRIDE, the total phenolics, total carotenoids, α-glucosidase inhibition, antioxidant, and cytotoxic activities of seven Philippine herbal weeds including Coccinia grandis, Mikania micrantha, Ruellia tuberosa, Chromolaena odorata, Momordica chuchinensis, Clitorea ternatea, and Passiflora foetida were investigated. The antioxidant activity was accomplished by ABTS, FRAP, DPPH, Hydroxyl radical, and Superoxide radical inhibition assays. Cytotoxicity of the leaf extract was evaluated in terms of LC50 using brine shrimp lethality test. Results revealed that C. odorata had the highest polyphenolic content (119.26 mg GAE per 100 gram dry weight) while C. grandis had a high total carotenoid content (9160.23 IU/100 g dry weight). The antioxidant activities of the seven herbal weeds showed great variations in five methods used for radical scavenging assays. The C. grandis, M. micrantha, R. tuberosa, C. odorata, M. chuchinensis, C. ternatea, and P. foetida leaf extracts demonstrated strong α-glucosidase inhibition with IC50 values of 61.87, 118.26, 99.28, 44.34, 70.1, 84.77, and 437.57 μg/mL, respectively. Results from the cytotoxicity screens revealed that the C. odorata leaf extract was the most lethal against the brine shrimp with LC50 value of 1.0 μg/mL. These plants appear to be rich sources of natural antioxidants and may be useful in treatment of diabetes mellitus and diseases caused by oxidative stress.

100-007-Z Minimum Inhibitory Concentration of Moringa Oleifera Extracts on Gram Positive and Gram Negative Bacteria Grace Miller – Indiana Wesleyan University Blake Russell – Indiana Wesleyan University, Angela Davidson – Indiana Wesleyan University, Erick Holder – Calvin College, Robert Burchell – Indiana Wesleyan University Native to India, Moringa oleifera is hailed as the “Miracle Tree” for its impressive resume of nutritional, hygienic, and medicinal uses. Nutritionally, Moringa leaf powder contains significant levels of proteins, iron, calcium, potassium, βcarotene, vitamin C, vitamin E, fibers, and sugars. It has been used in many feeding programs where malnutrition is a problem. Moringa seed powder is also used as a coagulant in water purification and has antibiotic and antifungal qualities. It also boasts an incredible growth rate, averaging about 3-4m in one season. Previous data from our lab has shown that seed and leaf extracts of Moringa are effective against several bacteria. The purpose of the research was to further characterize and evaluate Moringa’s ability to kill bacteria. We wanted to see at what concentration does the extract become an effective antibacterial agent. The Minimum Inhibitory Concentration of bacteria was tested on gram positive and gram negative bacteria.

100-008-Y Beyond Corn and Beans – Shedding Light on Iowa’s Fields of Opportunities Emily Heaton – Iowa State University Elke Brandes – Iowa State University, Gabe McNunn – Iowa State University, Ian Bonner – Idaho National Laboratory, David Muth – AgSolver Inc., Lisa Schulte-Moore – Iowa State University, Kara Cafferty – Idaho National Laboratory The USA has a large potential to reduce carbon emissions and increase community resilience by scaling up renewable energy production. Despite of numerous advantages, lignocellulosic biomass production and utilization lags behind mandates. With record commodity prices for maize and soybeans in 2011 and 2012, farmers had little incentive to change management from grain crops to lignocellulosic energy feedstock. However, the recent drop in commodity prices decreased profit margins in Iowa, especially on constantly low yielding soils. In addition to the economic impairment, concerns about the negative impacts of mono-cropped agricultural systems on water quality and soil health are voiced. The question arises how variability in farmland quality across the state influences spatial variability of row crop profitability. We investigated the Iowa agricultural landscape dedicated to maize and soybeans for opportunities to mitigate economic losses. We applied a modeling framework that integrates publically avaialble geospatial and statistic data with agronomic computing tools to evaluate sub-field profitability. We ran a retrospective analysis for 2010 to 2013 and a projection for 2015, including a sensitivity analysis of varying crop production cost, yield, and grain price. In 2010 to 2013, the amount of row-cropped farmland operating at a loss of 500 US$ ha-1 or more was negligible, however, low commodity prices and high production costs in 2015 shifted >6% of row-crop land this category. These sub-field areas show high sensitivity to crop production cost increases, as well as yield and grain price variability. Our findings suggest substantial opportunities to strategicaly place less extensive management options, such as dedicated bioenergy grasses or reconstruction of highly diverse prairie, into unprofitable sub-field areas strictly to improve overall field profitability. Considering the environmental benefits of perennial cropping systems in subfield areas will open up another dimension of valorization to the agricultural landscape.

100-009-Y Genetic Basis of Biosynthesis and Cytotoxic Activity of Medicago Truncatula Triterpene Saponins Brynn Lawrence – Department of Plant Pathology, University of Arkansas Cindi R. Brownmiller – Department of Food Science, University of Arkansas, Sun-ok Lee – Department of Food Science,

University of Arkansas, Ken Korth – Department of Food Science, University of Arkansas Saponins are a large family of specialized metabolites produced in many plants. They have been demonstrated to have negative effects on a number of plant pests and are generally thought to play a role in plant defense. Used often in traditional folk medicine, saponins have also been shown to be hypocholesterolemic, hypoglycemic, immunostimulatory, antioxidative, anti-inflammatory, and cytotoxic. In addition to applications in medicine and agriculture, saponins are used in industry as food and cosmetic additives, acting as foaming and surface-active agents. In spite of their usefulness, our understanding of the genetic basis for saponin biosynthesis is still incomplete. We generated two populations based on parents from genetically distinct accessions of Medicago truncatula with varying levels and profiles of constitutive saponins. Accession-specific markers were developed based on the sequence of genes potentially involved in the biosynthesis of M.truncatula aglycone sapogenins. These genes are predicted to encode cytochrome P450 enzymes, and their transcript levels appear to correlate well with accumulation of some sapogenins. Primers developed from SNPs found in the sequence were used to confirm the presence of male parent DNA in putative crosses. We conducted HPLC-MS analysis of saponin-rich mixtures extracted from four M. truncatula accessions, A17, ESP105, GRC43, and PRT178 to characterize and quantify mixture components. The effects of saponin extracts on the proliferation of human colon adenocarcinoma (Caco-2) cell cultures were investigated. This research will provide information on cytotoxicity of M. truncatula saponins, and the developed populations will assist in determination of genetic components controlling saponin synthesis.

100-010-Z Elicitation for Enhanced Lycopene Biosynthesis in Tomato (Solanum Lycopersicum ) Plants by Exogenous Application of Methyl Jasmonate Anne Osano – Bowie State University Norman Fultang – Bowie State University, Jonathan Harper – Bowie State University, Daniel Ballah – Bowie State University, Ezekiel Alakah – Bowie State University, Eric Bonsu – Bowie State University In this study, we used exogenous treatment of tomato plants with methyl Jasmonate to elicit lycopene biosynthesis and stimulate plant growth. Lycopene is a bright red carotene and carotenoid pigment found in tomatoes, carrots and other red fruits and vegetables. It is a powerful antioxidant that protects cells from damage and blocks cancer cell growth. Many studies suggest eating lycopene-rich foods may be linked to reduced risk of cancer, heart disease and age-related eye disorders and hence the need to enhance the levels in tomatoes. Tomato seeds were soaked in Methyl Jasmonate solutions of varying concentrations and planted. The resulting plants were irrigated with the solutions used to soak the seeds, accordingly. The heights of the plants were recorded on a tri-weekly basis and high performance liquid chromatography was used to observe the effects of the Methyl Jasmonate solutions on lycopene yields in the tomato fruits. Analysis of chromatograms obtained from tomato samples in the MeJA-treated group showed enhanced levels of lycopene. The plants in the MeJA-treated group also grew faster and stood significantly taller than those in the control group.

100-011-Z Elicitation for Enhanced Lycopene Biosynthesis in Tomato (Solanum Lycopersicum ) Plants by Exogenous Application of Methyl Jasmonate Anne Osano – Bowie State University Norman Fultang – Bowie State University, Jonathan Harper – Bowie State University, Daniel Ballah – Bowie State University, Ezekiel Alakah – Bowie State University, Eric Bonsu – Bowie State University In this study, we used exogenous treatment of tomato plants with methyl Jasmonate to elicit lycopene biosynthesis and

stimulate plant growth. Lycopene is a bright red carotene and carotenoid pigment found in tomatoes, carrots and other red fruits and vegetables. It is a powerful antioxidant that protects cells from damage and blocks cancer cell growth. Many studies suggest eating lycopene-rich foods may be linked to reduced risk of cancer, heart disease and age-related eye disorders and hence the need to enhance the levels in tomatoes. Tomato seeds were soaked in Methyl Jasmonate solutions of varying concentrations and planted. The resulting plants were irrigated with the solutions used to soak the seeds, accordingly. The heights of the plants were recorded on a tri-weekly basis and high performance liquid chromatography was used to observe the effects of the Methyl Jasmonate solutions on lycopene yields in the tomato fruits. Analysis of chromatograms obtained from tomato samples in the MeJA-treated group showed enhanced levels of lycopene. The plants in the MeJA-treated group also grew faster and stood significantly taller than those in the control group.

Applied: Biotechnology, Molecular Breeding 100-012-Y Application of Biotechnology for Environmental Cleanup: Engineering Plants for Improved Phytoremediation Vinod Kumar – Kuwait Institute For Scientific Research Hamed Al-Aqeel – Kuwait Institute For Scientific Research, Sindhu Nair – Kuwait Institute For Scientific Research, Anisha Shajan – Kuwait Institute For Scientific Research, Aisha Al-Shatti – Kuwait Institute For Scientific Research, Abdul Mohsen Ali – Kuwait Institute For Scientific Research, Sudhersan Chellan – Kuwait Institute For Scientific Research, Fadila AlSalameen – Kuwait Institute For Scientific Research, Salwa Al-Mouqati – Kuwait Institute For Scientific Research, Sabah Al-Momen – Kuwait Institute For Scientific Research Remediation of oil contaminated sites requires multidisciplinary approach. Phytoremediation is an attractive tool to remediate sites contaminated with moderate amount of crude oil and associated heavy metals. Plants possess unique mechanisms to detoxify or accumulate harmful pollutants including heavy metals. These mechanisms are governed by a set of genes encoding specialized proteins. Understanding the regulation of these genes in model plants would open innovative ways to improve the efficiency of phytoremediation technology. This requires biochemical characterization and better understanding of these genes and their expression patterns. Arabidopsis was used as a model species to elucidate the gene expression patterns in response to exposure to heavy metals such as vanadium and lead. Barley, alfalfa, Indian mustard and Atriplex species were screened to test their efficiency to tolerate and remediate soil contaminated with crude oil and associated heavy metals. The experiments conducted in growth chambers are highly encouraging with potential scope for application at the field level. A transgenic approach has been adopted to increase the efficiency of phytoremediation of alfalfa plants. Adenosine triphosphate (ATP) sulfurylase gene is known to be involved in the regulation of metal uptake and metal tolerance in plants. We have cloned the native Arabidopsis ATP sulfurylase gene and also created the synthetic version of the same. This gene will be engineered to improve the phytoremediation efficiency of selected target plant species that can grow in moderately oil contaminated soil under arid conditions.

100-013-Y Improving Photosynthesis Through Engineering Chloroplast Proteins Maureen Hanson – Cornell University Myat Lin – Cornell University, Alessandro Occhialini – Rothamsted Research, John Andralojc – Rothamsted Research, Kevin Hines – Cornell University, Martin Parry – Rothamsted Research Photosynthetic efficiency of C3 plants suffers from the slow catalytic rate and the reaction of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) with O2 instead of CO2, leading to the costly process of photorespiration. Cyanobacteria and other photosynthetic prokaryotes increase the concentration of CO2 around Rubisco within

microcompartments named carboxysomes for more efficient incorporation of inorganic carbon. A report on modeling indicates that implementation of the cyanobacterial carbon concentrating mechanism (CCM) in chloroplasts could increase yields as much as 60%. We have explored the possibility of producing β-carboxysomes containing the faster cyanobacterial Rubisco enzyme from Synechococcus elongatus PCC7942, a model freshwater cyanobacterium. Using the agroinfiltration technique, we have transiently expressed multiple β-carboxysomal proteins (CcmK2, CcmM, CcmL, CcmO and CcmN) in Nicotiana benthamiana with fusions that target these proteins into chloroplasts and that provide fluorescent labels for visualizing the resultant structures. By confocal and electron microscopic analysis, we have observed that the shell proteins of the β-carboxysome are able to assemble in plant chloroplasts into highly organized structures resembling empty microcompartments. We demonstrate that a foreign protein can be targeted with a Cterminal 17-amino-acid peptide of CcmN to the shell proteins inside chloroplasts. Using chloroplast transformation, we have shown that the tobacco Rubisco enzyme can be replaced with a kinetically faster cyanobacterial enzyme, which assembles and confers autotrophic growth. Our experiments establish the feasibility of introducing carboxysomes into chloroplasts for potential compartmentalization of a faster Rubisco or other proteins.

100-014-Z Homo-dimerization and Ligand Binding by the Leucine-rich Repeat Domain at RHG1/RFS2 Underlying Partial Resistance to Two Soybean Pathogens David Lightfoot – SIUC The protein encoded by GmRLK18-1 (Glyma_18_02680 on chromosome 18) and GmRLK11-1 were nearly identical, syntenic, receptor like kinases (RLK) encoded within and outside the soybean (Glycine max L. Merr.) Rhg1/Rfs2 locus respectively. The locus underlies resistance to the soybean cyst nematode (SCN) Heterodera glycines (I.) and causal agent of sudden death syndrome (SDS) Fusarium virguliforme (Aoki). The aims here were to localize the RLKs; and evaluate 4 mutant alloproteins LRRs ability to; homo-dimerize; bind larger proteins; and bind to small peptides. In planta proteins were localized to the pericycle by immunohistochemistry. The purified LRR domain, from residue 131485, was seen to form a mixture of monomers and homo-dimers in vitro. Cross-linking experiments in vitro showed the H274N,Q and V region was close ( < 11.1 A) to the highly conserved cysteine residue C196 on the second homo-dimer subunit. Binding constants of 20-142 nM for peptides found in plant and nematode secretions were depended on peptide and mutant sequences. A LRR from GmRLK08-1 (Glyma_08_g11350) did not show these strong interactions. The LRR domain bound avidly to 4 different CLE peptides, a cyclophilin and a methionine synthase in a sequence dependent fashion. The CLE peptides GmTGIF and HgCLE were shown to alter stem morphology and resistance to SCN and SDS. Therefore, the LRR domains of GmRLK18-1 and GmRLK11-1 might underlie both root development and disease resistance in soybean and provide an avenue to develop new variants and ligands that might promote reduced losses to SCN and SDS.

100-015-Z Transcriptome-analysis Enabled the Identification of a Molecular Marker Closely Linked to Nic1 Gene in Tobacco Shengming Yang – University of Kentucky Qiuling Qin – University of Kentucky, Dandan Li – University of Kentucky, Xinbin Dai – The Samuel Roberts Noble Foundation, Anne Jack – University of Kentucky, Patrick Zhao – The Samuel Roberts Noble Foundation With the impending FDA regulation, it is expected that tobacco plants with low alkaloid levels will be more and more favored in future tobacco breeding. Nicotine is the predominant alkaloid in most commercial varieties of tobacco. Therefore, an understanding of the mechanisms underlying nicotine biosynthesis is particularly critical for tobacco

breeders and industry to lower nicotine content in plants and cigarettes. Nicotine is synthesized in the tobacco root under the control of two independent genes, Nic1 and Nic2. Genetic studies revealed that nic1 and nic2 mutations are semi‐dominant and act synergistically, with effects of nic1 2.4 times stronger than those of nic2. Even though the nic mutants have long been available, selection is difficult, and without efficient molecular markers the introgression of nic1 and nic2 into commercial varieties has been seriously hampered. Based on the RNA-seq analysis of four near-isogenic lines with various nicotine levels, one co-dominant marker closely linked to Nic1 gene was identified. Our results not only benefit breeders in the harm-reduction breeding, but also provide a robust foundation for molecular cloning of Nic1 gene which contributes significantly to nicotine biosynthesis.

100-016-Y Mutation Breeding Approaches to Domesticate Field Pennycress (Thlaspi Arvense) into a New Winter Oilseed Crop Kevin Dorn – University of Minnesota Evan B. Johnson – University of Minnesota, Erin Daniels – University of Minnesota, M. David Marks – University of Minnesota Pennycress (Thlaspi arvense) is being targeted as a new winter-grown oilseed cover crop and biodiesel feedstock. Pennycress is a member of the Brassicaceae family and is related to the model species Arabidopsis thaliana as well as current oilseed crop species Brassicas napus and rapa. Unlike these latter two species, pennycress can be grown in the interval between the corn/soybean rotation in the Midwestern United States. Pennycress was chosen for domestication as it naturally has a high oilseed yield (1100 to 2250 kg/ha), a high seed oil content (30-40% by seed weight), an oil composition adequate for biodiesel production, and extreme cold tolerance. However, many improvements are needed to make pennycress a successful new crop species, such as a reduction in seed dormancy and optimization of flowering time. Both forward mutant screens and reverse genetic approaches being used will rely on recently developed genomic resources for pennycress, which include both transcriptome and genome assemblies. It is known from work with Arabidopsis that mutations can confer agronomically desirable phenotypes. For example, various mutations in Arabidopsis have been shown to increase seed size, decrease seed dormancy, improve oil quality, speed up flowering, and reduce seed shattering. To begin our mutation breeding program, seeds were treated with ethyl methanesulfonate, fast neutrons, or gamma rays. Currently, M2 plants derived from 10,000 M1 plants are being phenotyped. Over 100 mutants having phenotypes of interest have already been identified, including mutants with traits such as early maturation and larger seeds. Seeds and tissues are being banked for TILLING, with the ultimate goal of directly identifying mutations in genes that are known to produce desirable traits when mutated in Arabidopsis.

100-017-Y HVA1 Regulated by a Stress-inducible Composite Promoter Enhances Root Growth and Abiotic Stress Tolerance in Rice Without Yield Penalty Su-May Yu – Academia Sinica Tuan-Hua David Ho – Academia Sinica Regulation of root architecture is essential for maintaining plant growth under adverse environment. A synthetic abscisic acid (ABA)/stress-inducible promoter was designed to control the expression of a late embryogenesis abundant protein (LEA; HVA1) in transgenic rice. The background of HVA1 is low but highly inducible by ABA, salt, dehydration and cold. HVA1 was highly accumulated in root apical meristem (RAM) and lateral root primordia (LRP) after ABA/stress treatments, leading to enhanced root system expansion. Water use efficiency (WUE) and biomass also increased in transgenic rice, likely due to the maintenance of normal cell functions and metabolic activities conferred by HVA1 which is capable of stabilizing proteins, under osmotic stress. HVA1 promotes lateral root (LR) initiation, elongation and

emergence and primary root (PR) elongation via an auxin-dependent process, particularly by enhancing asymmetrical accumulation of auxin in LRP founder cells and RAM, even under ABA/stress-suppressive conditions. This is the first demonstration that ectopic expression of a LEA protein leads to enhanced LR development due to asymmetric accumulation of auxin. We also demonstrate a successful application of an inducible promoter in regulating the spatial and temporal expression of HVA1 for improving root architecture and multiple stress tolerance without yield penalty.

100-018-Z Next Generation Weed Control System in Cotton Rita Varagona – Monsanto Company Clayton Larue – Monsanto Company, Marianne Malven – Monsanto Company, Sherri Leclere – Monsanto Company, Ron Brinker – Monsanto Company, Paul Feng – Monsanto Company, Ty Fowler – Monsanto Company Next generation weed control systems in cotton is needed to help manage difficult broadleaf and glyphosate-resistant weeds. U.S. farmers have used dicamba on 237 million acres in the past 10 years. Engineering dicamba-tolerant crops would provide an additional tool for farmers to use in their weed control systems. The gene for dicamba monooxygenase (DMO) was isolated from a soil bacterium (Herman et al. 2005) and used as the starting material to make dicamba-tolerant cotton. Stacked with the phosphinothricin acetyl transferase gene (PAT, Wohllenben et al., 1988), cotton plants were tested to determine the expression elements necessary to make the plants fully tolerant to sprays with dicamba and glufosinate. To make a complete product, this vector stack has been deployed as a trait stack with genes for glyphosate-tolerance and insect-control genes. Extensive testing shows that the DMO enzyme deployed as a transgene that targets DMO to the plastid, conveyed in a vector stack, can provide excellent tolerance to the dicamba herbicide. This presentation will discuss the development of the Bollgard II® XtendFlexTM Cotton product.

100-019-Z Obtaining of Asexual Hybrid Varieties of Drought Tolerant Maize Kenny Alejandra Agreda-Laguna – Centro de Investigación y de Estudios Avanzados del IPN Roberto Ruiz-Medrano – Centro de Investigación y de Estudios Avanzados del IPN, Maria Eugenia Hidalgo-Lara – Centro de Investigación y de Estudios Avanzados del IPN, Ma. Del Carmen Montes-Horcasitas – Centro de Investigación y de Estudios Avanzados del IPN, Jesus Hinojosa-Moya - UPAEPE , Beatriz Xoconostle-Cazares – Centro de Investigación y de Estudios Avanzados del IPN The accumulation of the disaccharide trehalose may provide drought tolerance in plants. We silenced trehalose degradation by the silencing the trehalasa encoding gene, employing antisense RNA in maize. Adults plants were tested in open field trials, displaying drought and cold tolerance. In addition to this characteristic, it is desirable to improve maize production by maintaining hybrid vigor (heterozygous) in this important cultivar. For this purpose, we expressed the modified Cenh3, which encodes a specific protein of kinetochore, involved in the chromosome migration during meiosis and mitosis division. It is hypothesized that this modification gives the corn’s ability to maintain maternal genotype and the genetic background of drought tolerance. We employed vectors that contain the Cenh3 sequence with a dominant mutation (without N-terminal) under the regulation of promoter CaMV-35S and other vector regulated by the tissue-specific promoter knotted1. With this constructs the embryogenic maize callus Hi-II and L1 (H99XB73) were transformed by means of biobalistics, where it has obtained a transformation efficiency of 1.5%. We then employed ddPCR for evaluated the number of copies and is found that only have a copy on each plant. Expression levels were significantly decreased compared to normal controls. Cell

cycle and nuclear DNA content was assessed by flow cytometry, where it was observed that plants do not present alterations in the cell cycle or DNA content. At the present is being performed FISH and evaluating the F2 of genetically modified plants.

100-020-Y The O, R and M Loci and the Genetic Model for Color Inheritance in the Spathe of Anthurium David Gopaulchan – The University of the West Indies, St. Augustine, Trinidad and Tobago Adrian Lennon – The University of the West Indies, St. Augustine, Trinidad and Tobago, Pathmanathan Umaharan – Cocoa Research Centre, Trinidad and Tobago Anthurium andraeanum (Hort.) is a tropical ornamental valued for its brightly colored cut-flowers which comprises of a modified leaf (spathe) and an inflorescence (spadix). The genetic model for color inheritance suggests three loci, O, R and M determine spathe color. To evaluate the genetic model, the expression of the anthocyanin biosynthetic genes, CHS, F3H, DFR, ANS and F3’H were determined in cultivars representing different states of the O, R and M loci and correlated to anthocyanin content and spathe color. The results showed that the expression of F3H and ANS were controlled by the R locus and the expression of DFR was controlled by the O locus and the M locus was implicated as encoding F3’H. White cultivars which were in the homozygous recessive state for O or R or both, exhibited reduced expression of the anthocyanin genes. Evidence suggests the R locus may encode a R2R3-MYB transcription factor (AaMyb1), a putative regulator of the pathway. The model could not account for the differences in color among the red and pink spathes or differences in shade intensity among pinks. To identify the mechanisms responsible, genetically defined red and pink spathed cultivars with varying shade intensities were analyzed. Color intensity correlated strongly with anthocyanin abundance. Additionally, red spathes accumulated anthocyanin throughout development whereas pinks displayed temporal variations in accumulation. Anthocyanin abundance mirrored the expression of F3’H but did not correspond with the expression of any other gene assayed. The study demonstrated that earlier and higher the rate of F3’H expression during spathe development, the greater the accumulation of anthocyanins. Together these suggest that F3’H expression is a key control point in the regulation of anthocyanin biosynthesis in anthurium and plays a major role in influencing shade intensity. It is proposed that shade intensity is primarily controlled through transcription factors regulating F3’H.

100-021-Y Controlling Lepidoptera Pests of Cotton Through the Expression of the Hybrid Gene Coding for the Deltaendotoxin of Bacillus Thuringiensis Laura Sofia Castillo-Ortega – CINVESTAV IPN Beatriz Xoconostle-Cazares – CINVESTAV IPN, Roberto Ruiz-Medrano – CINVESTAV IPN, Jesús Hinojosa-Moya – BUAP The cotton is an economically important plant; the fruit is used in the manufacture of textile fibers, while seeds are employed in the manufacture of oils and waxes. During plant growth is attacked by lepidopteran insects, among other pests. Their control consists in the application of chemical agents; however, these compounds reduce the fruit quality and are toxic to the environment. Bacillus thuringiensis is widely used to control insect pests, both as a bacterial formulate as well as via genetic engineering of plants overexpressing the delta-endotoxin-encoded gene. When the protein is ingested by the insects, it is activated in the midgut by proteolysis, then provoking membrane pores and the insect death by sepsis. A new hybrid gene was generated employing two Bt genes of berliner and kustaki varieties (Cry1Aa1 and Cry1Ba1), thus producing an active, toxic delta-endotoxin. The gene encoding the insecticide was cloned in a plant expression vector for further

expression in cotton. The characteristics of the GM plants will be presented in terms to control lepidopteran pests in cotton

100-022-Z RICE BIG GRAIN 2, a Novel Plant Specific Gene Promotes Cell Proliferation and Controls Organ Size in Rice Ching-Yi Liao – Academia Sinica Shuen-Fang Lo, Meng‐Yuan Rao, Tuan-Hua Ho, Su-May Yu Breeding of high-yielding rice is crucial for meeting the food demand of increasing world population. Using the forward genetic approach, we identify a T-DNA activation tagged mutant exhibiting big grain phenotype. RICE BIG GRAIN 2 (RBG2), a novel gene encodes an unknown function protein which is conserved in Angiosperms, is validated responsible for the big grain phenotype. Elevated RBG2 expression causes pleiotropic effects in transgenic rice, including increase in plant height, panicle length, grain size, and tiller angle. RBG2 is expressed preferentially in shoot and young tissues, and mainly localized in periphery plasma membrane. RBG2 was found to accelerate cell proliferation that contributes to increased organ size in transgenic rice. Expression of genes involved in cell cycle and plant hormone regulation are altered in RBG2 transgenic rice. RBG2 contains six conserved domains with unknown function. Loss-of-function mutants of these domains, except domain 5, each moderately affects the function of RBG2. Our findings suggest that RBG2 acts as a novel positive regulator in grain development, thus a potential target for molecular breeding.

100-023-Z Conservation of an Endangered Species Dinosya Mira Through Tissue Culture Sardar Farooq – Sultan Qaboos University Hajer Al Balushi – Sultan Qaboos University Dinosya mira is a perennial herb of the family Primulaceae. It is an endangered species endemic to Oman. It is found at an approximate altitude of 1600-2800 m in the Hajar Mountains and Al Jabal al Akhdar Mountains. It grows on rocky slopes on which water flows temporarily during rains. It is used in the folklore medicine locally. There is not much literature published on the biology and tissue culture of Dinosya mira which are essential for conservation. Present study aims to develop a reproducible micropropagation protocol for micropropagation of this plant. Nodal explants of Dinosya mira were cultured on Murashige and Skoog medium supplemented with various concentrations of plant growth regulators, such as Benzyl adenine (BA), (Kinetin KN) Naphthalene acetic acid, (NAA) and their combinations. Among different combinations used, medium supplemented with 16 μM BA, 2 μM NAA resulted in higher rate of adventitious shoots per explant. Addition of adenine sulphate yielded improved numbers of shoot buds from nodal explants. For rooting, half the strength of MS medium with reduced amount of sugar was used. Higher frequency of rooting was achieved with 0.5 μM NAA and MS medium without PGRs. The rooted plantlets were transplanted into pots containing a mixture of vermiculite + peat moss (1: 1) at 70-80% relative humidity and 28°C -30°C for hardening. 35% of in vitroraised plantlets survived under field conditions.

100-024-Y Mesoporous Silica Nanoparticles as a Biomolecule Delivery System in Plants Dequan Sun – Deakin University Hashmath Hussain, Zhifeng Yi, James Rookes, Lingxue Kong, David Cahill Monodispersed mesoporous silica nanoparticles (MSNs) of 20 nm diameter with interconnected 2.58 nm pores were synthesized and functionalized for the application as ideal carriers in agrochemical delivery system. The translocation

and wide distribution of MSNs in plants were determined. Enhancement of seed germination, biomass, total protein and chlorophyll contents, and photosynthetic activity was observed in wheat and lupin seedlings following treatments with 500 and 1000 mg L-1 MSNs. These findings demonstrated that MSNs used at concentration up to 2000 mg L-1 are not phytotoxic and their uptake may favor plant growth. The controlled release of two key phytohormones salicylic acid (SA) and abscisic acid (ABA) was mediated by using a novel decanethiol gatekeeper system grafted onto mesoporous silica nanoparticles (MSNs). The decanethiol was conjugated only to the external surfaces of the MSNs through glutathione (GSH)-cleavable disulfide linkages and the introduction of a process to assemble gatekeepers only on the outer surface so that the mesopore area can be maintained for high cargo loading. In planta experiments showed that the release of SA from decanethiol gated MSNs by GSH induced sustained expression of the plant defence gene PR-1 up to seven days after introduction, while free SA caused an early peak in PR-1 expression which steadily decreased after three days. Similarly, compared with direct application of ABA, the release of entrapped ABA with decanethiol gated MSNs enhanced the expression of ABA-induced gene AtGALK2 and promoted Arabidopsis drought resistance. This study showed the potential use of MSNs in plants as a controlled agrochemical delivery system.

100-025-Y Overexpression of ZmGA20ox cDNA in Switchgrass Increases Biomass Production and Alters Plant Morphology and Lignin Content Phat Do – University of Missouri Hyeyoung Lee, Xiaoyan Yin, Zhanyuan Zhang There are several constraints in utilizing switchgrass for biofuel production. Of these, a low biomass yield and inefficient conversion of lignocellulose to ethanol are two important constraints that are gaining significant attentions from plant breeding, genetics and molecular biologists. Genetic engineering could improve switchgrass biomass yields by modulating plant growth regulator levels. Overexpression of gibberellin 20-oxidase (GA20ox), a key enzyme in gibberellin biosynthesis, was reported as an effective strategy to increase biomass yields in certain plant species. In this present report we overexpressed the GA20ox cDNA from Zea mays which was driven by constitutive 35S promoter with omega enhancer sequence. The GA20ox overexpression cassette was introduced to switchgrass genome through Agrobacterium-mediated T-DNA transformation at an efficient transformation frequency using hygromycin selection. The present of GA20ox gene and selectable marker hptII in transgenic plants was confirmed by PCR and antibiotic screen (leaf painting) using hygromycin-B. The integration and expression levels of GA20ox gene in transgenic T0 plants were analyzed using Southern blot and qRT-PCR. Under the greenhouse conditions, transgenic plants showed increased biomass as indicated by the increased number of tillers, longer leaves, internodes, and dry weights as compared to wildtype controls. In addition, the overexpression of GA20ox affected the expression of important genes coding for key enzymes in lignin biosynthesis. Consequently, lignin contents of transgenic plants were altered to varying degrees. Progeny segregation analysis of primary GA20-transgenic plants will be conducted and the levels of active gibberellin as well as biomass production will be analyzed using transgenic T1 plants.

100-026-Z Genetic Engineering of the Xa10 Promoter for Broad-spectrum and Durable Resistance to Xanthomonas Oryzae Pv. Oryzae Xuan Zeng – Temasek Life Sciences Laboratory Dongsheng Tian – Temasek Life Sciences Laboratory, Keyu Gu – Temasek Life Sciences Laboratory, Zhiyun Zhou, Yanchang Luo – Temasek Life Sciences Laboratory, Frank White – Kansas State University, Zhongchao Yin – Temasek Life Sciences Laboratory, Xiaobei Yang Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into

plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10E5. Xa10E5 has an EBE-amended promoter containing five tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10E5 was generated in the cultivar Nipponbare. The Xa10E5 gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10E5 gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10E5 and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight.

100-027-Z Syringin Production from Suspension Cells of Snow Lotus Li-Fen Huang – Yuan Ze University Ying-Kai Chu – Yuan Ze University Snow lotus (Saussurea involucrate) is traditional Chinese herbal medicine that contains plant secondary metabolites to treat illness. Syringin, extracted from snow lotus, is anti-hypersensitivity and anti-inflammatory for autoimmune disease. Due to over-exploitation of snow lotus plants which naturally grow in 4000 to 4500 meter-high mountains in China, making an insufficient supply of wild snow lotus. By plant tissue culture, plants are able to regenerate from undifferenticated callus. In this study, we established an efficient system to get the regenerated snow lotus. In addition, fast growth of snow lotussuspension cells was set up. Suspension cells of snow lotus under different culture conditions were oven-dried and then syringing contents were measured from cell extracts. Our current result showed that suspension cells of snow lotus were able to obtain Syringin under combination of different ratio of hormones under either light or dark condition. Bioreactor was then applied to scale up suspension cells of snow lotus. The cells were dried by either oven-drying process or freeze-dry, and the result showed that the freeze-dry method had almost 7 folds higher Syringin content than the oven dry method. In this study, we offered a cryopreservation method for suspension cells of snow lotus.

100-028-Y Sweet Potato Leaf Curl Virus: Coat Protein Gene Expression in Escherichia Coli and Product Identification by Mass Spectometry Norimoto Murai – Louisiana State University Dina Lida Gutierrez Reynoso, Rodrigo Valverde Sweet potato is one of the first natural GMO, genetically modified 8,000 years ago by Agrobacterium rhizogenes (Kyndt et al. 2015). A section of 10 kbp long DNA (Transferred-DNA or T-DNA) of the Ri (Root-inducing) plasmid was transferred to the plant genome by A. rhizogenes and has been maintained in all 291 hexaploid sweet potato cultivars from the world. The maintenance and expression of two T-DNA genes for tryptophan-2-monooxygenease (iaaM) and for indole-3acetamide (iaaH) are of a functional significance since they involve in the production of indole-3-acetic acid, a major plant growth hormone auxin. Leaf curl virus diseases have been reported in sweet potato throughout the world. One of the causal agents is Sweet potato leaf curl virus (SPLCV) which belongs to the genus Begomovirus (family Geminiviridae). Although SPLCV does not cause symptoms on Beauregard, the most predominant sweet potato cultivar in the US, it can reduce the yield up to 26

% (Clark and Hoy, 2006). Serological detection of SPLCV is not currently available due to the difficulties in obtaining purified virions that can be used as antigen for antiserum production. In attempts to obtain the coat protein (CP) of SPLCV for antibody production, primers were designed to amplify the CP gene. This gene was cloned into the expression vector pMAL-c2E as a fusion protein with maltose-binding protein, and transformed into Escherichia coli strainXL1-Blue. After gene induction, a fusion protein of 72 kDa was purified by amylose affinity chromatography. The yield of the purified fusion protein was approximately 200 µg/liter of bacterial culture. Digestion with enterokinase cleaved the fusion protein into a 42.5 kDa maltose-binding protein and a 29.4 kDa protein. The latter protein was identified by mass spectrometry analysis as the coat protein of Sweet potato leaf curl virus (SPLCV).

100-029-Y Transcriptional Response of Sugarcane During Meristem Culture David Hallahan – E.I. DuPont de Nemours & Company Thaya Ganzke, Joseph Lamendola, Hongxian He, Corban Rivera, Mary Beatty, Gina Zastrow-Hayes, Kevin Hayes, Nancy Rizzo Efficient meristematic tissue culture of sugarcane is key to commercial bulk-up and introduction of new varieties. Traditionally, sugarcane mericloning has been conducted on semi-solid medium, with considerable involvement of skilled labor. We have developed an efficient mericloning system involving initiation of cytokinin-induced meristem proliferation on semi-solid medium followed by temporary immersion bioreactor culture. We then employed nextgeneration sequencing techniques to better understand the response of sugarcane to these different culture regimes. Firstly, RNA prepared from cane stalk apices was subjected to Illumina sequencing to generate a BLAST-searchable transcriptome database. 170 x 106 reads (average length 88bp) were assembled into 100,828 contigs (40.9% > 1kbp) using the Oases short-read assembler. BLAST analysis identified several sugarcane orthologs of known meristem-specific genes. Illumina RNA-seq analysis of transcripts in different tissues from different tissue culture regimes was conducted, and the data aligned to the transcriptome assembly. Analysis of these data using Arrayviewer allowed identification of genes differentially expressed 1) between meristematic and non-meristematic tissue, 2) between different tissue culture regimes (semi-solid and liquid media) and 3) over time in culture in the two different regimes. Expression profiling of key meristematic genes and the general utility of transcriptomic analysis in tissue culture will be discussed.

100-030-Z Food Safety Assessment of Drought-resistant Rice Simyung Lee – NAAS, RDA Sunwoo Oh, Yunsoo Yeo, Hyunsuk Cho This study was to evaluate the food safety assessments of the inserted gene product that Methionine-Sreductase(MsrB2) from pepper. Bioinfomatical research of MsrB2 was performed. Deduced amino acid sequences of MsrB2 was compared to the protein database, known or suspected to be allergen or toxin to which determine if it has significant sequence identity. These proteins do not share overall sequence homology with any known allergenic and toxic proteins. MsrB2 protein was purified as His-tag fusion protein. For the future studies, MsrB2 specific antibody was produced. And the substantial equivalence, purified protein was sequenced by Edman degradation. The amino acid sequences of purified protein were the same as deduced amino acid sequences exclude N-terminal Histidine. For the internal sequences of MsrB2 amino acid sequence, we performed MALDI-TOF Mass. The results of MALDI-TOF Mass was compared Mascot Database and confirmed the sequence coverage was 52%. These results mean bacterially produced MsrB2 was the same with inserted gene product.For the possibility of allergen, we examined in vitro digestion by simulated gastric fluid(SGF) and simulated intestinal fluid(SIF), MsrB2 protein was digested within 15 seconds. And

glycosylation of GM rice expressed MsrB2 was not glycoprotein. We will perform more experiments for confirming food safety assessment.

100-031-Z Overexpression of SHI Genes from Brassica Rapa Induce Growth Retardation in Chrysanthemum Eun Jung Suh – National Academy of Agricultural Science, RDA Joon ki Hong – National Academy of Agricultural Science, RDA, Yeon-Hee Lee – National Academy of Agricultural Science, RDA, Seong-Kon Lee – National Academy of Agricultural Science, RDA, Kyung Hwan – National Academy of Agricultural Science, RDA, Hye Jin Yoon – National Academy of Agricultural Science, RDA In horticultural plant production, compact plant is an essential target of breeding. Generally, growth retardation with various chemical growth regulator is accomplished but these compound are hazardous to environment and human. We isolated 4 types of SHI (Short Internodes) related genes from Brassica and confirmed their growth retardation in Arabidopsis and Brassica. To try the potential of SHI genes in ornamental plant chrysanthemum, we introduced four 35SBrSHI genes constructs operated by 35S CaMV promoter to spray type chrysanthemum. After acclimation of transgenic plants, we re-planted each cutting from grown-up chrysanthemum to eliminate the effect of tissue culture. Plant height was reduced approximately 14-25% in 4 SHI transformed plants although several plants had a long vegetative growth. We also isolated 1432 bp 5’ UTR region of chrysanthemum actin gene and fused to 4 SHI genes as a substitute for 35S CaMV promoter. When four ChA-BrSHI constructs were introduced to the same spray chrysanthemum, the ChA promoter had higher activity than 35S CaMV promoter up to two-fold reduction. Especially, transgenic plants containing SRS 7(Short related genes 7) and SRS-gene(same sequence of SRS7 including genomic region) were showed growth retardation 50.0% and 54.1% under the control of ChA promoter. Additionally, ChA-BrSHI constructs containing four SHI genes were transferred into pot-mum chrysanthemum. The 4 genes of transgenic plants showed decrease of plant height to 40% on average compared to non-transgenic plant. These findings suggest that BrSHI genes could be useful tools for growth retardation and modification of plant height, and this approach could substitute the use of chemical growth retardants in horticulture.

100-032-Y Exploiting Dosage Sensitivity of the Florigen Pathway Optimizes Tomato Architecture and Yield Soon Ju Park – Wonkwang university Ke Jiang – Cold Spring Harbor Laboratory, Lior Tal – Weizmann Institute of Science, Yoav Yichie – The Hebrew University, Oron Gar – The Hebrew University, Dani Zamir – The Hebrew University, Yuval Eshed – Weizmann Institute of Science, Zachery Lippman – Cold Spring Harbor Laboratory Improving crop productivity is a major challenge in modern agriculture. Here we select tomato mutations with plant architectures that greatly improve yield. Determinate tomato plants are compact because of a mutation in SELF PRUNING (SP), encoding an antagonist of the flowering hormone florigen. Heterozygosity for mutations in the florigen gene SINGLE FLOWER TRUSS (SFT) partially relieves sp-imposed determinacy and increases yield. By screening for other suppressors of determinacy, we isolated a weak sft allele and two mutations in SUPPRESSOR OF SP (SSP), which abolish determinate growth and allow new flower clusters (inflorescences) to develop continuously. The SSP mutations disrupt a critical motif in a bZIPtranscription factor required for assembling a ‘florigen activation complex’. Combining selected alleles of ssp and sft in heterozygous statesresults in a continuous range of shoot architectures, providing architectural optimums that translate to significant increases in yield. Using mutations that tweak the universal florigen pathway to tailor shoot architecture, flower production, and yield offers a toolkit for customized crop breeding.

100-033-Y The Validation of Methods Based on Polymerase Chain Reaction for the Detection of Newly Approved GM Events in Korea Yu Jihn Kwon – Gyeongin Regional Food and Drug Administration So Young Chung, Mi Ran Kim, Dong Hyuk Seo, Ji Hyup Baek, Jeongyun Cho, Chul Joo Lim, Sun Ok Choi In this study, we validated event specific qualitative and quantitative detection methods for the newly developed genetically modified (GM) crops in order to strengthen the label management for GM foods. Two GM events [one soybean (DAS-81419-2), one maizes (MON87411)] were selected for the qualitative detection methods and four GM event [two soybean (DAS-81419-2, DAS-44406-6), two maize (DP004114-3, MON87411)] was selected for the quantitative detection method. For the validation of a qualitative detection method, polymerase chain reaction (PCR) were tested for specificity, sensitivity and repeatability. As a result, the limit of detection (LOD) of qualitative detection methods for DAS-81419-2 and MON87411 are ranged from 0.05 to 0.01% and showed specificity to other crops or other GM events. Quantitative detection method for four GM event was validated according to the CODEX guideline and satisfied the performance requirements. This study may imply that detection methods can be established for the GMO analysis.

100-034-Z A Facile Method for Quantifying Volatile Metabolites in Raspberry Fruit Stephen Brockman – University of Minnesota Jayanti Suresh – University of Minnesota, Adrian Hegeman – University of Minnesota Raspberries (Rubus idaeus) are a fruit crop widely grown for their pleasing flavor and potential health benefits. Raspberries are capable of synthesizing hundreds of chemically diverse volatile metabolites including terpenes, apocarotenoids, lactones, alkenes, furanones, and phenolic compounds. However, the composition of metabolites produced in a given plant is heavily influenced by environmental factors such as temperature and moisture. As a result a single genotype may produce multiple distinct volatile profiles. Investigating the interaction between environmental factors and aroma volatile production requires methodology capable of detecting and quantifying the diverse range of volatiles produced in raspberry fruit. Gas chromatography mass spectrometry (GC-MS) is an ideal platform for such an analysis, but previous attempts at this approach have been hampered by the chemical complexity of the fruit volatiles. To overcome this challenge, we have developed an extraction protocol and accompanying GC-MS method utilizing a DB5MS UI column capable of detecting and quantifying, in a single analysis, over a dozen volatiles in raspberry fruit that are known to be important for flavor and aroma. Metabolite quantification is accomplished by adding a stable isotope labeled internal standard at the beginning of the extraction. The metabolites that can be detected include α-ionone, βionone, (Z)-3-hexenol, linalool, benzyl alcohol, as well as raspberry ketone. It has also been previously demonstrated that their concentrations are highly variable between and within genotypes. In addition to elucidating the interaction between environmental factors and volatile production, this methodology can provide a useful tool for investigating numerous aspects of fundamental and applied raspberry biology. These include evaluating postharvest physiology, facilitating gene annotation, and high throughput screening of breeding populations.

100-035-Z Grain Drydown in Maize - Native Variation and Transgenic Approach Dongsheng Feng – DuPont Pioneer Joanie Phillips – DuPont Pioneer, Colleen Jeffrey – DuPont Pioneer, Weiqing Zeng – DuPont Pioneer, Xiaomu Niu – DuPont Pioneer

Enhanced grainfill and faster grain dry-down are essential objectives of maize breeding programs. To better understand these traits, we surveyed 33 inbred lines for their drydown variation, and tested four genes for their impact on grain moisture content. Significant variations of drydown rates were found among the inbred lines. Through manipulation of gene expression, the four genes (ZM-MATE9, ZM-VRS1, ZM-GPCNAC-1 and ZM-SGR1) were showed to play a role in grainfill, senescence and drydown rates.

100-036-Y Complex Metabolic Engineering of Camelina for Advanced Biofuels and Industrial Feedstocks Tara Nazarenus – UNL Tadele Kumssa – UNL, Patrick Rädler – UNL, Sandra Erdozáin Salón, Edgar Cahoon – UNL Camelina sativa (camelina or false flax) is an emerging Brassicaceae oilseed crop in the North American Great Plains and US Pacific Northwest. Growing interest in camelina is largely due to its potential for biodiesel production in geographic areas with sub-optimal rainfall and soil fertility. Because it is not grown for food use in the US, camelina can serve as a production platform for biofuels as well as for high value industrial feedstocks. Camelina has emerged as an especially attractive platform because it can be rapidly transformed by Agrobacterium with technical ease. This capability has enabled complex metabolic engineering of novel oil-related traits. A major limitation of camelina oil for biofuels is its low oxidative stability arising from a high content of the polyunsaturated fatty acids (PUFAs) linoleic acid (18:2) and linolenic acid (18:3) content. To improve the oxidative stability of camelina oil, we have devised a number of metabolic engineering strategies to generate oils enriched in the more oxidatively stable oleic acid (18:1) and low in PUFAs. Research has also been directed at stacking the high oleic acid trait with gene combinations aimed at increasing total seed oil content. Progress to date from greenhouse and multi-year field trials of genetically enhanced camelina lines will be presented. The value of the high oleic acid trait as a background for the production of novel oil functionalities will also be discussed.

100-037-Y Using RNA Interference to Improve Soybean Seed Traits Michelle Folta – University of Missouri Muruganantham Mookkan – University of Missouri, Joann De Tar – University of Missouri, Kristin Bilyeu – USDAAgricultural Research Service, Zhanyuan Zhang – University of Missouri Soybean (Glycine max) is the number one oil and protein crop in the United States, but the seed contains several antinutritional factors that are toxic to both humans and livestock. RNA interference technology has become an increasingly popular technique in gene silencing because it allows for both temporal and spatial targeting of specific genes. The objective of this research is to use RNA-mediated gene silencing to down-regulate the soybean gene raffinose synthase 2 (RS2), to reduce total raffinose content in mature seed. Raffinose is a trisaccharide that is indigestible to humans and monogastric animals, and as monogastric animals are the largest consumers of soy products, reducing raffinose levels would improve the nutritional quality of soybean. An RNAi construct targeting RS2 was designed, cloned, and transformed to the soybean genome via Agrobecterium infection. Resulting plants were analyzed for the presence and number of copies of the transgene by PCR and southern blot. The efficiency of mRNA silencing was confirmed by realtime quantitative PCR. Total raffinose content was determined by HPLC analysis. Transgenic plant lines were recovered that exhibited dramatically reduced levels of raffinose in mature seed, and these lines will be further analyzed for other phenotypes such as development and yield. Finally, a chicken feeding assay will determine if lower levels of raffinose in soy meal correspond with improved animal growth. This research will validate both the effectiveness and specificity of RNA interference, and thus the technology could be applied to many other important genes in crop plants.

100-038-Z The Impact of Increased Plastid Size on Plant Growth and Development Aleel Grennan – University of Illinois Zhengxiang Ge, Timothy Wertin, Mayandi Sivaguru, Mayu Sugikawa, Samuel Boctor, Thomas Clemente, Donald Ort Sorghum (Sorghum bicolor), a C4 grass, is among the most productive plants in agriculture, making it an ideal candidate for a biofuel feedstock. In spite of this, sorghum’s high photosynthetic rates still falls short of the modeled theoretical C4 NADP-ME maximum. Modeling has shown that increases in potential yield can be achieved by increasing the photosynthetic conversion efficiency of intercepted solar energy. One approach we are taking to achieve this improvement is to alter the light environment within the leaf by changing chloroplast size. Using Arabidopsis thaliana as a model system, we have shown that increasing chloroplast size alters the light environment within the leaf thus allowing light to reach more cell layers. Although there is no significant change in photosynthetic rates, there is an apparent positive impact on growth. Arabidopsis plants with enlarged chloroplasts set seed earlier and invested more resources into inflorescences. When we moved this trait into sorghum, we also observe similar changes in the internal light environment within the leaf (i.e. more light within a leaf). A detailed greenhouse study has been started to compare growth and potential developmental differences between the parental sorghum line and chloroplast size mutant. In addition to growth parameters, we will be measuring photosynthetic rate, chlorophyll content and leaf optical properties.

100-039-Z Rapid and Hormone-free Agrobacterium Rhizogenes-mediated Transformation in Rubber Producing Dandelions Taraxacum Kok-saghyz and T. Brevicorniculatum Yingxiao Zhang – The Ohio State University Brian Iaffaldano – The Ohio State University, Wenshuang Xie – The Ohio State University, Joshua Blakeslee – The Ohio State University, Katrina Cornish – The Ohio State University Taraxacum kok-saghyz (TK) and T. brevicorniculatum (TB) are rubber-producing dandelion species under development as potential crops and model systems of rubber biosynthesis. A rapid and hormone-free transformation system was developed for these two species. Here we show that root fragments can regenerate entire plants on half strength Murashige and Skoog medium without iterative hormone treatments or manual manipulations. After root fragments were inoculated with Agrobacterium rhizogenes harboring kanamycin resistance genes encoding neomycin phosphotransferase II (nptII), as well as green or cyan florescent proteins, non-composite transgenic plants were obtained within 8 weeks. On average, transformation efficiency (number of transgenic plants/number of root fragments) was 24.7% and 15.7% for TK and TB, respectively; about seven independent transgenic events were generated per starting plant for TK and four for TB. Overall, this high efficiency transformation method provides a rapid and simple system for these two dandelions to yield viable transgenic seeds in as little as 20 weeks. Protocols developed in this study allow introduction of genes of interest to facilitate the improvement of these rubber producing plants into domestic crops and provide an avenue to explore rubber biosynthesis and gene functions.

100-040-Y Improved Breeding Efficiency for Cold Induced Sweetening in Potatoes by the Use of Biochemical Markers Sanjay Gupta – University of Minnesota Potato breeders can be expected to make increasing use of biochemical markers in their programs. With respect to cold induced sweetening (CIS), I have developed biochemical markers that predict the storability and processing quality of

potato selections and parents. By identifying appropriate parents, progeny can be obtained with a much higher frequency of desirable sugar levels and chip color. In my studies, potato clones containing A-II isozymes of UGPase, reduced vacuolar acid invertase (VAcInv) enzyme and inhibitor protein express resistance to CIS. Forty families representing parents from various CIS resistance classes were established. Parents and progeny were characterized and the heritability of these biochemical factors determined following 6 months of storage at 5.5 °C. Progeny from parents containing 1.0 or less units of VAcInv enzyme activity with low invertase inhibitor protein and presence of A-II isozymes of UGPase demonstrated more than 93% of potato clones with desirable sugar levels and chip color. Whereas, progeny from parents representing more than one unit of VAcInv enzyme activity demonstrated only 18% clones with desirable reducing sugar level and chip color. High levels of VAcInv enzyme activity mask the effect of A-II isozyme of UGPase.

100-041-Y Insights from Above: A First Look at Aerial Phenotyping for Crop Improvement in Wheat Margaret Krause – Cornell University Michael Gore – Cornell University, Mark Sorrells – Cornell University A main objective for crop improvement through plant breeding is to be able to predict crop performance from genotypes and phenotypes. While rapid advancements in DNA sequencing technologies have driven the cost of genotyping down, methods for quantifying plant phenotpes have not experienced the same level of improvement. Aerial phenotyping opens up the possibility to non-destructively and rapidly quantify traits at multiple time points over the course of the growing season. Aerial images of winter wheat breeding lines planted in 1.5x3 meter plots at three locations in Ithaca, NY, were collected by an airplane equipped with a near infrared converted camera at six time points during the 2015 growing season. The green normalized difference vegetation index (GNDVI), which is correlated with biomass and grain yield, was calculated for each plot using the near infrared and green spectral bands. Because GNDVI has a covariance structure with grain yield, the objective is to incorporate this longitudinal phenotype into a multivariate genomic selection model for predicting grain yield.

100-042-Z Novel Diagnostic Alleles of CBF-A12 and CBF-A15 Genes Are Associated with the Cold Tolerance Response in Winter Wheat Marwa Sanad – Washington State University Jie Zhu – Washington State University, Kimberly Campbell – USDA-ARS C-repeat binding factors (CBFs) regulate the expression of the cold responsive genes in many plant species. The linked CBF-A12 and CBF-A15 genes have high transcription levels in frost tolerant winter wheat. Our objectives were to: 1) Develop Kompetitive allelic specific PCR (KASP) assay; and validate 10 SNPs and 2 indels of CBF-A12 and CBF-A15 genes between the tolerant and the susceptible haplotypes; 2) Annotate the effective and consistent diagnostic SNPs. Two key mutations (insertion\deletion) of CBF-A12\CBF-A15 genes, respectively, were validated using 892 genotypes of winter wheat and showed to be associated with the survival changes of the haplotypes. The short insertion (2bp) of CBF-A12 is located 41bp upstream of the start codon in the susceptible haplotype. Although it is calculated to be a nonsense mutation for protein structure, it might influence the binding process. The 9bp deletion in the susceptible haplotype of the CBF-A15 gene was found in the open reading frame. The deletion resulted in an encoding protein with 3 deleted serine residues. The missing residues (SSS) fall within the beta sandwich-like proteins (SPs). Additionally, the mutation caused the (SWTSSSSSLPSGDGM) motif that belongs to the serine/threonine kinase and the phosphoserine/ threonine binding groups to be defective. Two KASP assays have been designed to diagnose the tolerant and susceptible alleles of CBF-A12 and CBF-A15 genes in winter bread wheat.

100-043-Z Transient Expression of a Chimeric Antimicrobial Peptide in Phloem of Mexican Lime Plants (Citrus Aurantifolia) Miguel Guerra-Lupián – Center for Research and Advanced Studies of the National Polytechnic Institute. Roberto Toscano-Morales – Center for Research and Advanced Studies of the National Polytechnic Institute, José Ramírez-Pool – Center for Research and Advanced Studies of the National Polytechnic Institute, Roberto Ruiz-Medrano – Center for Research and Advanced Studies of the National Polytechnic Institute, Beatriz Xoconostle-Cazares – Center for Research and Advanced Studies of the National Polytechnic Institute The causal agent of Huanglongbing or Citrus Greening is the alpha-proteobacteria, Candidatus Liberibacter asiaticus(CLa), which is restricted to the phloem and is vectored by the Asian citrus psyllid Diaphorina citri Kuwayama. CLa multiplies primarily in trees and mainly distributed in stem and leaf midrib. Control strategies have focused on insect vector control by insecticides application, and by removing symptomatic plants to reduce sources of inoculum. The main problem of these strategies is that symptoms only appear after months or even years the plant acquired the infection and by that time active transmission already occurred. In this work, we tested different methods for antimicrobial protein transient expression on citrus plants using a gene coding for an antimicrobial peptide under the control of the CaMV35S promoter, linked to vascular mobile proteins and GFP to track its accumulation pattern. For this purpose, agroinfiltration of different tissues were performed. Molecular analysis including PCR, qPCR, Western blotting and confocal microscopy were carried out to confirm the genetic expression as well as the correct folding of the marker protein.

100-044-Y QTL Underlying Androgenic Haploid Production in Pepper Theresa Hill – University of California Davis Allen Van Deynze – University of California Davis, Khalis Afnan Abdul Rahman – Erasmus Mundus Master Programme in Evoluntionary Biology The use of doubled haploids (DH) to accelerate homozygous line production is a valuable tool in plant breeding programs. A limitation to the utilization of DHs in pepper (Capsicum annuum) breeding is the variability and unreliability among genotypes of plant regeneration from microspore culture. A classical genetics approach, recombinant inbred lines (RILs) derived from a cross between regeneration-responsive and regeneration-unresponsive lines, was used to determine if haploid regeneration from microspores is a quantitative trait controlled by genetic loci. Transcriptome sequencing of the RIL parental lines was used to develop 250 SNP assays that were used to genotype 200 F5 RILs. Thus far, phenotyping of 2 F6 individuals (2 reps) from each of the 200 RILs indicate two QTL, one controlling embryogenesis and one controlling plantlet regeneration. The embryogenesis QTL showed a positive effect on the phenotype coming from the regeneration-responsive line, while the regeneration-unresponsive line contributed to plant regeneration at the second QTL. This is consistent with heterosis that was observed among the F1 and the transgressive segregation observed in the RIL population.

Applied: Genome Editing 100-045-Y CRISPR/Cas9- and TALEN-mediated Mutagenesis in Maize SiNian Char – Iowa State University Erica Unger-Wallace – Iowa State University, Hartinio Nahampun – Iowa State University, Bronwyn Frame – Iowa State

University, Sarah A. Briggs – Iowa State University, Marcy Main – Iowa State University, Martin H. Spalding – Iowa State University, Blake Meyers – University of Delaware, Virginia Walbot – Stanford University, Erik Vollbrecht – Iowa State University, Kan Wang – Iowa State University, Bing Yang – Iowa State University Technologies based on nucleases like modified Cas9/sgRNA (Clustered Regularly Interspaced Short Palindromic Repeat single guideRNA/CRISPR associated protein Cas9) and custom-engineered TALENs (transcription activator-like effector nucleases) have been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This presentation describes application of these two technologies to generate heritable genome modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (9 mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. Cas9/sgRNA constructs have also been applied for mutagenesis of two Argonaute genes in maize. Transgenic lines of T0 generation carrying site-specific mutations were produced at frequency ranging from 50 to 80%. The on-going characterization of the inheritability of mutations will also be presented. Our results demonstrate that TALENs and Cas9/sgRNA are effective toolboxes for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

100-046-Z A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation Yiping Qi – East Carolina University Levi Lowder – East Carolina University, Nicholas Baltes – University of Minnesota, Yong Zhang – University of Electronic Science and Technology of China, Daniel Voytas – University of Minnesota The relative ease, speed and biological scope of CRISPR/Cas9-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or “multiplexing”, which is a significant advantage of CRISPR/Cas9 versus other genome editing systems. In order to streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 T-DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco, Arabidopsis and rice, demonstrating its utility for basic and applied plant research.

100-047-Z Generating a Mutant Library of Stress-responsive Rice MAP Kinases via CRISPR-Cas9 Mediated Multiplex Genome Editing Bastian Minkenberg – The Pennsylvania State University Kabin Xie – The Pennsylvania State University, Yinong Yang – The Pennsylvania State University Mitogen-activated protein kinases (MPKs) phosphorylate downstream components like transcription factors to regulate gene expression and cellular responses. While the Arabidopsis MPKs MPK3, MPK4, and MPK6 have been well characterized for their functions in biotic and abiotic stress response, not much is known about their rice orthologues

OsMPK1, OsMPK2, OsMPK5 and OsMPK6. Currently, the functional studies of rice MPKs are mainly based on transgene overexpression and RNA interference analysis. A more detailed functional analysis of OsMPKs is still missing due to the lack of available rice mutants. In order to fill this knowledge gap, we exploited the endogenous tRNA processing machinery to simultaneously produce up to eight single guide RNAs for CRISPR-Cas9 mediated multiplex targeted mutagenesis of the aforementioned stress-responsive OsMPKs. A library of rice mutants carrying single, double, or quadruple mutations of OsMPKs has been successfully generated. DNA analyses revealed that the precise mutation at these OsMPKs mainly resulted from small indels or deletion of a chromosomal fragment between two targeted sites. Genetic analyses demonstrated that these mutations could be faithfully inherited into next generations. In addition, the T-DNA fragment carrying Cas9 and the hygromycin-resistance gene was segregated and successfully removed to produce transgene-free OsMPK mutants. Therefore, this genetic resource is expected to facilitate the functional characterization of OsMPKs and enhance our understanding of MAP kinase pathways and their roles in regulating rice growth and development as well as biotic and abiotic stress tolerance.

100-048-Y CRISPR-Cas Nuclease Mutagenesis for Genetic Containment of genetically Engineered Forest Trees Estefania Elorriaga – Oregon State University Amy Klocko – Oregon State University, Cathleen Ma – Oregon State University, Steve Strauss – Oregon State University Genetically engineered (GE) trees have been produced that show a wide variety of improved characteristics, including rapid growth rate, strong pest resistance, and improved processing for pulp and energy. However, gene flow from GE trees into feral or wild populations are significant obstacles to their use as a result of regulatory, public perception, and ecological concerns. Because many trees are vegetatively propagated, inducing complete sexual sterility is a powerful means to reduce these concerns. Loss-of-function mutations in essential floral genes has led to complete or nearly complete sterility in many plant species, however such mutations are rare and generally recessive, thus are very difficult to use via conventional tree breeding. The recent development of efficient systems for site-directed mutagenesis, particularly the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated Cas system, appears to be a powerful tool for generating homozygous loss-of-function mutations in many species. Compared to other sterility methods, the mutations it induces should be highly predictable and stable, as reversion will be extremely rare or impossible (e.g., with deletions of essential parts of coding regions). We are testing the mutation efficiency of six CRISPR-Cas nucleases targeting two essential floral genes in poplar (Populus). The targets are the poplar orthologs LEAFY and AGAMOUS—well studied genes that are essential for both male- and female-fertility. The nucleases have been stably transformed into poplar and hundreds of independent transformation events are now being analyzed for the rate and types of mutations. Preliminary results indicate one of the LEAFY CRISPR Cas nucleases has a biallelic mutation efficiency of ~45%. Results from study of the full population, and preliminary results from studies of off-target mutagenesis, will be presented.

100-049-Y The Generation of Targeted Mutants Identified by Genome Wide Association Studies in Medicago Truncatula Shaun Curtin – University of Minnesota Chanlan Chun, Junqi Liu, Sarah Hoerth, Joseph Guhlin, Justin Anderson, Peng Zhou, Paul Atkins, Nick Baltes, Aaron Hummel, Tomas Cermak, Colby Starker, Ryan Morrow, Roxanne Denny, Daniel Voytas, Robert Stupar, Peter Tiffin, Michael Sadowsky, Nevin Young Recent progress in the functional genomics of Medicago has been hindered by available mutagenesis platforms. This is particularly pertinent as plant researchers explore the biological function of genes identified by RNA-seq or genomewide association studies (GWAS). Here we demonstrate a suite of mutagenesis platforms including; Tnt1 retro-

transposons, hairpin constructs, TAL-effector nucleases (TALENs) and CRISPR (RNA-guided)/Cas9 nucleases to engineer nodulation mutant candidates in Medicago. We have used the genetic transformation of Tnt1, hairpins, TALENs and CRISPR/Cas9 to knock-out/down single copy and duplicated genes and show disruption of nodulation in some of these mutant plants. These developments demonstrate the utility of these mutagenesis platforms for the identification of novel nodulation related genes in Medicago.

100-050-Z miRNA Maturation and Target Transcript Knock-down Are Severely Disrupted in Soybean dcl1 Double Mutants Shaun Curtin – University of Minnesota Jean-Michel Michno, Benjamin Campbell, Javier Gil-Humanes, Sandra Mathioni, Ryan Donohue, Michael Kantar, Andrew Eamens, Blake Meyers, Daniel Voytas, Robert Stupar Small non-protein-coding micro RNAs (miRNAs), typically 21- or 22 nucleotides in length, are present in most eukaryotes and are central effectors of RNA silencing and regulators of gene expression. DICER-LIKE1 (DCL1) proteins, a highly conserved family of RNaseIII-like endonucleases, are a core machinery protein component of each plant-specific miRNA pathway. We used zinc-finger nucleases to generate non-transgenic single and double mutants of the putative soybean Dcl1 homologs, Dcl1a and Dcl1b, and confirm their role as the miRNA-processing Dcl protein. We show that the single dcl1 mutants did not exhibit strong morphological or molecular phenotypes. The dcl1a/dcl1b double mutants, however, exhibited strong morphological and molecular phenotypes, including reduced seed size, aborted seedling development, and reduced miRNA processing. Furthermore, protein encoding genes targeted by miRNAs were shown to have increasingly perturbed transcription states in the double mutants compared to the single mutants. These findings suggest that the two paralogous Dcl1 genes are functionally redundant. 100-051-Z Towards Improved Water Use and Nitrogen use Efficiency in Lettuce: Mapping QTL Associated with Nitrogen and Water use Efficiency. You Youngsook – Cal Poly Pomona Danielle Ellis – Cal Poly Pomona, David Still – Cal Poly Pomona Lettuce crops are intensively managed, requiring a dependable source of high quality water and relatively high nitrogen fertilizer inputs. Competing demands for a diminishing water supply and environmental concerns over nitrogen fertilizers has increased the pressure to use these resources more efficiently. The goal of this research was to determine if genetic variation exists in lettuce for water use efficiency (WUE) and nitrogen use efficiency (NUE) and if so, simultaneously map quantitative trait loci associated with these traits. A lettuce recombinant inbred line (RIL) population consisting of 152 families was grown in fields under a control or deficit water/nitrogen regime. The control treatment consisted of irrigation treatments to replace 130% of reference crop evapotranspiration (ETo) and 100% nitrogen (168 kg/ha) and a stress treatment consisting of replacing water at 75% of ETo and providing nitrogen at 50% of control levels (84 kg/ha). Carbon isotope discrimination was used as a proxy for WUE; NUE and nitrogen metabolism was measured by 14N isotope discrimination and biomass accumulated per nitrogen applied, respectively. At market maturity the fresh weight of each RIL family was obtained, and leaf carbon and nitrogen content obtained through the isotope analyses. Three major QTL associated with 13C isotope discrimination (WUE) were mapped, with the largest effect accounting for 24% of the observed variation. A single QTL was associated with nitrogen content and another QTL associated with 14N isotope discrimination (nitrogen metabolism). Each of these QTL accounted for 17% of the variation observed. Fresh weight did not differ between the two treatments in one year, while in a second year head weight averaged across all genotypes was greater in the deficit irrigation/low nitrogen treatment. Environmental variation for WUE and NUE is significant, especially NUE. Future work will identify specific genes that contribute to NUE and WUE.

GENES & GENOMES - Zone 200 Genes & Genomes: Genetics 200-001-Y Assessment of the Genetic Diversity of Papaya (Carica Papaya) Cultivars in Puerto Rico Dianiris Luciano-Rosario – University of Puerto Rico-Mayaguez Dimuth Siritunga – University of Puerto Rico-Mayaguez Native to Central America, papaya (Carica papaya) is now one of the most cultivated fruit crop in tropical and subtropical areas. In 2012, the Caribbean produced 1,010,467 tons of papaya achieving the 7th most produced commodity in the area. Though having only 84 commercial papaya farms in Puerto Rico, papaya plays a culturally important role and many people cultivate it for its own consumption. Up to now, studies on papaya in Puerto Rico have been limited to morphological and agronomical properties. Papaya in Puerto Rico is poorly understood and there is a need to properly assess its diversity especially since most are cultivated and maintained by farmers. In this project we will study the genetic diversity of papaya cultivars in Puerto Rico using 20 simple sequence repeats (SSR) markers. The samples for this project consist of 14 known cultivars from the USDA germplasm collection and 100 unknown accessions from different municipalities of Puerto Rico. DNA extracted from leaves are being amplified using SSR primers and electrophoresed in a polyacrylamide gel prior to scoring of the bands and subsequent statistical analysis. This project expands our knowledge of papaya in Puerto Rico and has the potential to address problems such as losses by the ringspot virus.

200-002-Z Genetic Resources of a Model Legume Medicago Truncatula Jiangqi Wen – The Noble Foundation Xiaofei Cheng – The Noble Foundation, Kiran Mysore – The Noble Foundation, Pascal Ratet – ISV-CNRS, Michael Udvardi – The Noble Foundation Medicago truncatula is a model legume species for genetics, genomics, and functional genomics studies. It has been utilized for researches in many areas of plant biology, including plant physiology, nutrition, metabolism, growth and development, and plant-microbial and plant-environmental interactions. To better facilitate the characterization of gene functions in M. truncatula and other legume species, we have generated more than 21,700 Tnt1 retrotransposon insertion lines which encompass approximately 500,000 insertions in the genome of M. truncatula, at The Samuel Roberts Noble Foundation over the last 10 years. To make the maximal utilization of the mutant resources, two reverse genetics platforms have been established: one is the searchable web-based database (http://medicagomutant.noble.org/mutant/), which currently contains more than 220,000 Tnt1-flanking sequence tags (FSTs) mapped to pseudo-chromosomes and we expect to have more than 300,000 FSTs available by the end of 2015; the other is the PCRbased reverse screening, which provides screening service for genes-of-interest in genomic DNA pools of insertion lines. Over the past six years, more than 1,000 genes have been screened by the PCR approach. One or more Tnt1 insertions have been successfully identified in 85% of these genes, ranging from ~0.5 to 22 kb with genomic or cDNA sequences. So far, we have distributed more than 5500 Tnt1 lines to scientists in 24 countries and more than 40 papers resulting from Tnt1 mutants have been published.

200-003-Z Insights into the Relationship Between Structural Diversity and Transcriptional Diversity in Maize Candice Hirsch – University of Minnesota Cory Hirsch – University of Minnesota, Alex Brohammer – University of Minnesota, Megan Bowman – Michigan State

University, Kevin Childs – Michigan State University, Ilya Soifer – NRGENE LTD, Omer Barad – NRGENE LTD, C. Robin Buell – Michigan State University, Natalia de Leon – University of Wisconsin, Shawn Kaeppler – University of Wisconsin, Mark Mikel – University of Illinois Maize is a species with extensive sequence diversity. To further understand the maize pan genome, we have generated a comprehensive de novo assembly of the inbred line PH207 to complement the existing B73 reference genome assembly. B73 is an important founder line of the Stiff Stalk pool, while PH207 is an important founder of the Iodent Non-Stiff Stalk pool, both of which have been critical components of U.S. temperature maize germplasm. The PH207 assembly contains 132,022 scaffolds with an N50 of approximately 630 kb and a total assembly size of 2.1 Gb. Alignment of RNAseq reads from diverse tissues as well as conserved eukaryotic genes mapping approach (CEGMA) indicated that the gene space is well represented and comparable to the representation present in the B73 reference assembly. Comparative analysis between the B73 and PH207 genome assemblies revealed thousands of genotype specific genes and extensive expansion/contraction of gene families between the two genotypes, consistent with previous estimates based on transcriptome assemblies across 503 diverse inbred lines. We have also deeply resequenced 35 maize inbred lines and surveyed the genomic content of these lines. Interestingly, core genes that were present in all individuals had higher average expression levels across 80 tissues throughout development and were expressed in nearly all tissues, while genes present in a subset of the individuals showed more tissue/condition specific expression and on average had lower expression levels. Additionally, extensive genome content variation between heterotic groups was observed in the set of 35 resequenced inbred lines. A comparative analysis between the genomes and transcriptomes of diverse maize inbred lines and the role these differences may play in heterosis will be presented.

200-004-Y Regional Mutagenesis in Maize Using a Two Component Ac/Ds Tagging Platform Thomas Brutnell – Danforth Plant Science Center Kevin Ahern – Boyce Thompson Institute, Anthony Studer – Danforth Plant Science Center, Quan Zhang – Danforth Plant Science Center, Sarit Weissmann – Danforth Plant Science Center, Rachel Mertz – Danforth Plant Science Center, Time Anderson – Danforth Plant Science Center, Pinghua Li – Shandong Agricultural University, Jon Duvick – Iowa State University, Erik Vollbrecht – Iowa State University Transposon-based mutagenesis has served as a foundation for maize genetics since the beginning of the 20th century. The advent of next-generation sequencing technology now promises to greatly accelerate the physical placement of transposon to reference genomes and thus enable both forward and reverse-genetic strategies for genome-wide mutagenesis maize. To date, we have mapped over 2,000 Ds insertion to the maize B73 reference genome using DNA sequences flanking multiple independent Ds insertions that have been maintained in a W22 genetic background. However, over 15% of flanking Ds insertions sites could not be accurately placed on the B73 genome. Here we describe a draft de novo W22 genome assembly generated from short-read Illumina sequencing. The quality of the genome is exceptionally high, enabling the placement of multiple Ds insertions to the genome. To demonstrate the utility of Ds in regional mutagenesis strategies, I will detail 10 examples in which local Ds insertions (less than 200 kb from the target locus) were used as donor loci to create multiple alleles of target loci, including several genes necessary for C4 photosynthetic development. Through these pilot mutagenesis programs, we are able to define metrics for successful regional mutagenesis in maize and demonstrate the power of Ds mutagenesis in creating loss of function alleles of tandemly duplicated genes, creating dozens of allelic variants in any given target and the value of utilizing a uniform genetic background for performing detailed histological and biochemical analyses of Ds-tagged mutants.

200-005-Y Genetic Diversity Study of a Core Collection of Brassica Napus Accessions Based on Genotyping by Sequencing Mukhlesur Rahman – North Dakota State University M. Michalak de Jiménez – North Dakota State University, Sujan Mamidi – North Dakota State University, Phillip McClean – North Dakota State University North Dakota is the leader in canola acreage and production with over 83% of U.S. acreage and produces about 84% of all U.S. canola (1.02 million acres and 1.7 billion pounds with a value of $364 million – 5 yr. average from 2009-2013; USDA-NASS). It is crucial to study and preserve genetic diversity in canola since the diversity is the only source of resistance to different stresses as well as various agronomically important traits. Studies that describe the genetic variation in canola populations are limited in USA. The germplasm-based studies help to understand the genetic variation and marker-trait associations that can have applications for marker assisted selection. Therefore, this study was conducted assessing the genetic diversity, population structure and linkage disequilibrium (LD) of canola core collection and its future utility in association mapping studies. A total of367 canola germplasms originated from 27 countries were genotyped using GBS Illumina pipeline. The GBS reads were mapped to the reference genome of Brassica napus. A total of 42,575 high quality polymorphic SNPs were identified. Of these SNPs, 20,543 were found on genome A and 21,624 on genome C. Three subpopulations were estimated using a subset of 12,908 markers based on LD. The individuals of each of these subpopulations were belonging to all geographical types with no specific distribution.

200-006-Z High-throughput Image Analysis Identifies Naturally-varying Genetic Loci Controlling Root Gravitropism in Maize Seedlings Takeshi Yoshihara – University of Wisconsin-Madison Nathan Miller – University of Wisconsin-Madison, Ii-Youp Kwak – University of Wisconsin-Madison, Hannah Myles – University of Wisconsin-Madison, Logan Johnson – University of Wisconsin-Madison, Shawn Kaeppler – University of Wisconsin-Madison, Karl Broman – University of Wisconsin-Madison, Edgar Spalding – University of Wisconsin-Madison We developed a high throughput pipeline for measuring maize root gravitropism at 3-min intervals by combining digital imaging devices, agar plates for seedling culture, and computerized image analysis. With this system we measured individual lines making up the intermated B73 x Mo17 (IBM) recombinant inbred population and the Wisconsin Diversity (WIDIV) panel. With 300-400 lines per population, there was sufficient statistical power in the data to support either QTL analysis or GWAS at each point in the 3-h time course. The gravitropism response typically displayed a lag phase, a period of curvature development including overshooting in some genotypes, and a plateau phase. Different groups of genes were predicted to function during these different phases, an expectation supported by the QTL results obtained with the IBM data set. QTL appeared and disappeared during the time course. To narrow down the lists of candidate genes within the large confidence intervals, a reciprocal Basic Local Alignment Search Tool (BLAST) approach was used to identify related genes also underlying QTL identified in an analogous study of Arabidopsis seedling roots (Moore et al. 2013. Genetics 195: 1077). This method of leveraging two related data sets greatly narrowed the list of candidates to 5 genes. One of the candidates encodes a phosphatidylinositol 4-phosphate 5-kinases (PIP5K) known to be involved in clathrin mediated PIN recycling and thus in gravitropism in Arabidopsis. GWAS using the WIDIV results also showed interesting candidate genes becoming significant in a time-dependent manner, but not during the plateau phase. These candidate genes are under investigation.

200-007-Z Dissecting Quantitative Regulation of Root Growth Using Systems Genetics Wolfgang Busch – Gregor Mendel Institute Santosh Satbhai – Gregor Mendel Institute, Radka Slovak – Gregor Mendel Institute, Takehiko Ogura – Gregor Mendel

Institute A fundamental question in biology is how the genome of an organism gives rise to its phenotype. In plants, growth and development are of particular interest in regard to the phenotype- genotype problem as much of plant form and function, including biomass, root foraging ability, and resistance to many abiotic stresses, are related to growth and development. An excellent experimental system to approach the genotype to phenotype challenge in the context of growth and development is the root of Arabidopsis thaliana, for which unique resources exist. These include cell-type specific transcriptome data, high resolution genotyping data along with the full genome sequences of more than a thousand inbred, phenotypically diverse wild strains, and high quality genomic-scale gene network models based on a large number of system-biology-type experimental datasets. Using natural variation of root traits between hundreds of Arabidopsis accessions, we study how this phenotypical variation is genetically determined and which genes, networks, and biological pathways lead to differences in root growth and architecture. For this, we use custom phenotyping pipelines that enable us to capture quantitative root phenotypes of a very large number of individuals, genome wide association studies (GWAS) to identify the associated loci in the genome, and systems-biology driven approaches to identify the gene networks and pathways that provide the molecular and cellular context within which the underlying genes quantitatively regulate root growth. Using these approaches, we have recently identified and characterized multiple novel regulators and regulatory modules of epistatically interacting genes that shape root growth. Overall, using this systems-genetics approach enables us to approach the genotype to phenotype question at the level of genetic networks, significantly advancing our comprehension of how complex biological traits are modulated by different genotypes.

200-008-Y Next-generation Sequencing and Forward Genetics Identifies Novel Genes Required for Symbiotic Nitrogen Fixation in Medicago Truncatula Rebecca Dickstein – University of North Texas Vijaykumar Veerappan – University of North Texas, Khem Kadel – University of North Texas, Mehul Jani – University of North Texas, Naudin Alexis – University of North Texas, Kirankumar Mysore – Samuel Roberts Noble Foundation, Jiangqi Wen – Samuel Roberts Noble Foundation, Rajeev Azad – University of North Texas When bioavailable nitrogen (N) is limiting, legumes enter into a relationship with compatible rhizobia, forming new root organs called nodules. Within nodules, rhizobia reduce atmospheric N to ammonia and exchange it for reduced carbon from the plant host, giving legumes an N source. Although more than a few plant genes essential to establishing and maintaining the nodule and symbiotic N fixation (SNF) have been characterized, many more await identification. In M. truncatula, large-scale mutagenesis with the tobacco retrotransposon Tnt1 has almost saturated the genome and produced many mutants with SNF defects. Tnt1 mutagenesis produces 10-100 new Tnt1 inserts per mutagenesis and primarily targets coding regions. Individual mutant’s Tnt1 insertion sites have been investigated by sequencing TAIL-PCR products obtained with Tnt1-specific primers. While this works well for reverse genetics, it is not efficient for forward genetics. We screened for and obtained Tnt1 mutants with N fixation defective (Fix-) nodules. In a pilot study, we studied ten different mutants. Back-crossed plants segregated 3:1 (WT:mutant) in the F2 generation, demonstrating single gene, recessive inheritance. DNA from F2 mutant segregants was subjected to TAIL-PCR, and Illumina genome resequencing (GRS) to 30X coverage each. One mutant’s defect, in an already-characterized gene, was identified via TAILPCR and co-segregation of the mutant allele with the phenotype in F2 plants. In five mutants, GRS identified candidate mutant alleles that co-segregated perfectly with the defective phenotype, including two known and three novel genes. In two mutants, GRS yielded molecular markers genetically linked to the mutation. For two mutants, the GRS data is currently being analyzed. Thus, our data indicate that genome re-sequencing is a powerful tool when coupled with

forward genetics for defective gene identification in M. truncatula Tnt1 mutants. This work was supported by NSF IOS1127155 to K. Mysore and R. Dickstein.

200-009-Y Molecular Basis of the Ability for Short Days to Substitute for Vernalization in the Temperate Grass, Brachypodium Distachyon Daniel Woods – UW-Madison Richard Amasino – UW-Madison Proper timing of flowering is essential for reproductive success in plants and is a major determinant of biomass yield.A key adaptation to seasonal variation in temperate climates is the evolution of a vernalization requirement.Vernalization is the process by which competence to flower is achieved after prolonged exposure to winter cold.In addition to vernalization prolonged exposure to non-inductive short days (SD) followed by a shift into inductive long days (LD) has the ability to substitute for the vernalization response in a range of flowering plant families.However, unlike vernalization, little is known about the molecular nature of the SD substitution for vernalization phenomenon.We are using the small temperate grass, Brachypodium distachyon as a model to study flowering in the grasses.Here, we report the characterization of the SD substitution response across 60 diverse Brachypodium accessions, and have begun to assess the genetic basis of flowering in populations derived from crosses between accessions that vary in their ability for SD to substitute for vernalization.An F2 cross between a SD responsive and SD non-responsive accession reveal that the ability for SD to substitute for vernalization is controlled by a single large effect locus which we have fine mapped to an FT-like (FTL) paralog.Remarkably, all SD non-responsive accessions share the same non-synonomous change within a conserved residue in the 3rd exon that is not present in any of the SD responsive accessions.FTL is dynamically regulated by the photoperiod and is expressed only in SD.Furthermore, a key vernalization gene VERNALIZATION 2 (VRN2) is responsible for the LD repression of FTL as overexpression of VRN2 in SD results in nearly undetectable expression levels of FTL and knock-down of VRN2 in LD results in FTL induction.The cloning of FTL provides the first molecular insight into the SD substsitution for vernalization phenomenon in plants.

200-010-Z Quantitative Growth Measurement of Individual Mutants in Pooled Culture of the Green Alga Chlamydomonas to Identify Mutants Deficient in Photosynthesis and Photoprotection Ru Zhang – Carnegie Institution for Science Leif Pallesen – Carnegie Institution for Science, Luke Mackinder – Carnegie Institution for Science, Nina Ivanova – Carnegie Institution for Science, Weronika Patena – Carnegie Institution for Science, Xiaobo Li – Carnegie Institution for Science, Rebecca Yue – Carnegie Institution for Science, Arthur Grossman – Carnegie Institution for Science, Martin Jonikas – Carnegie Institution for Science The eukaryotic, unicellular green alga Chlamydomonas reinhardtii has immense potential as a functional genomic platform to study photosynthesis because: (1) its photosynthetic apparatus is similar to land plants; (2) mutants deficient in photosynthesis can be maintained in the dark on fixed carbon - acetate; (3) vegetative cells are haploid, so mutant phenotypes are visible immediately; (4) it grows fast with a doubling time of 6–8 hours. The Chlamydomonas mutants deficient in photosynthesis were usually identified by eye on plates with an acetate-requiring phenotype. These traditional methods have led to many important discoveries of photosynthesis but with challenges: the genotyping was low-throughput and the phenotyping was not quantitative. We have developed a high throughput genotyping and quantitative phenotyping tool, ChlaMmeSeq (Chlamydomonas MmeI-based insertion site Sequencing). ChlaMmeSeq employs a restriction enzyme (MmeI) based strategy to yield a unique 20 bp genomic sequence flanking the insertion cassette in each mutant. The 20 bp flanking sequences are used to map random insertion sites of tens of thousands of

Chlamydomonas mutants in one pool simultaneously. By using ChlaMmeSeq, we generated an indexed insertional mutant library of Chlamydomonas. Each mutant in the library has a mapped insertion site and a unique 20 bp genomic flanking sequence, serving as a tag to track the growth rate of each mutant in pooled cultures. We can detect a 20% reduction of growth rate after 7 generations, and a 10% reduction after 14 generations. These tools enable highthroughput and quantitative pooled screens to identify mutants deficient in photosynthesis and photoprotection. Mutants identified can be picked directly from the mutant library based on unique flanking sequences for further characterization. These tools would transform our understanding of photosynthesis and they can also be used for screens under other stresses, e.g. high temperature, nutrient deficiency, or hypoxia/anoxia.

200-011-Z Discovery of the ‘Most Famous Wheat Gene’ Ph1 (Pairing Homoeologous 1): Light at the End of the Tunnel Ramanjot Kaur – Washington State University Kanwardeep Singh – Washington State University, Ragupathi Nagarajan – Washington State University, Amita Mohan – Washington State University, Kulvinder Gill – Washington State University Instead of being a diploid with two sets of chromosomes like humans wheat is a polyploid, with seven sets of six related chromosomes. Starting in 1958, just five years after the discovery of DNA’s double-helix structure, researchers suspected that a specific gene, ‘Ph1’ controls the orderly pairing behavior of wheat by differentiating the related (homoeologs) from identical chromosomes (homologs) thus making it fertile. In the absence of this gene there is a total chaos in the nucleus resulting into multivalent formation leading to sterility. Although the gene was identified back then but wasn’t cloned yet. Correcting the misidentification of the gene reported in the journal Nature in 2006, our recent results (PNAS 2014) have conclusively shown that the gene we have identified (C-Ph1) is indeed the Ph1 as silencing of the gene results in a phenotype similar to that of Ph1 gene mutations including homoeologous chromosome pairing, multivalent formation, and disrupted chromosome alignment on the metaphase I plate. Although with highly conserved DNA sequence, the homoeologs showed dramatically different structure and expression pattern suggesting multiple functions of the Ph1 gene explaining the previous observations and hypotheses associated with the gene function. The unique expression pattern of the 5B copy at metaphase I along with the disruption of chromosome alignment along the metaphasic plate in C-Ph1 silenced plants support the suggested Ph1 gene function via centromere-microtubule interaction. Furthermore, stable RNAi silencing of the gene in Arabidopsis also showed centromere clustering and misalignment similar to that of the wheat RNAi silenced plants thus strengthening the above-mentioned fact. This gene also has orthologs in other diploids and ancient polyploids such as rice, maize, barley, and Brachypodium suggesting functional conservation across plant species.

200-012-Y Generation of Paternal Dihaploids in Tall Fescue Bryan Kindiger – USDA-ARS Within the Lolium-Festuca genome complex there is a need for modern breeding approaches to facilitate the rapid development of improved germplasm or cultivars. Traditional recurrent or mass-selection methods for population or synthetic development are labor- intensive and time- consuming. The recent development of dihaploid (DH) inducer lines of annual ryegrass (Lolium perenne L. subsp. multiflorum (Lam.) Husnot (syn. Lolium multiflorum Lam.) that exhibit genome loss when hybridized with tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum (Schreb.) Darbysh.) can provide an alternative breeding approach. Hybridizations between the inducer lines and tall fescue result in F1 hybrids which will loose either their ryegrass or tall fescue genomes. This behavior results in the occasional generation of both ryegrass and tall fescue DH lines through parthenogenic embryo formation within the F1 inflorescence.

200-013-Y Association Mapping of Quantitative Trait Loci for Tillering Traits in Barley Allison Haaning – University of Minnesota Kevin Smith – University of Minnesota, Gary Muehlbauer – University of Minnesota The majority of grain yield in barley comes from inflorescences that develop on lateral stems called tillers. Despite the fundamental relationship between tiller production, or tillering, and yield, the underlying genetics are not well understood. In this study, the global genetic diversity of barley represented in the National Small Grains Core Collection (NSGCC) was exploited to maximize detection of quantitative trait loci (QTL) associated with tillering. A subset of spring two-row and six-row lines was selected by including the most genetically diverse lines from the NSGCC. Lines were genotyped with 4500 SNP markers. Tillering traits were measured for each line, including average tillers per plant, measured weekly from two weeks until eight weeks past emergence; average productive tillers per plant, and average tiller mortality. Heading date, plant height, and percentage of plants lodged were also measured. Extensive phenotypic variation was observed for all traits. As expected, two-row lines tillered more than six-row lines on average, but, despite this, one-quarter of all six-row lines tillered more than the two-row average. As expected, the week that tillering stopped was positively correlated with heading date, and average tiller number was also positively correlated with heading date. Genome-wide association mapping was used to detect a QTL on chromosome 2H associated with tillering traits that overlapped the Photoperiod-H1 (Ppd-H1) locus, which controls photoperiod sensitivity. Including the significant SNPs in Ppd-H1 as covariates in the mixed linear model enabled detection of a second QTL on chromosome 7H that overlaps a locus encoding a putative AUX/IAA protein. Additional analyses to detect tillering QTL and traits correlated with tillering are ongoing and will result in a better understanding of tillering genetics, which could lead to improvement of yield and other agronomic traits in barley.

200-014-Z A Meta-analysis of Nitrogen-use Efficiency Genes in Barley Julia Wong – University of Alberta Tao Su – University of Alberta, Julia Wong – University of Alberta, Allen Good – University of Alberta A meta-analysis of nitrogen-use efficiency genes in barley Over the past half century, the use of synthetic nitrogen fertilizers has resulted in increased crop yields, but with profound impacts on the surrounding air and water ecosystems. Consequently, there has been a strong push to reduce the amount of N fertilizer by maximizing the nitrogen-use efficiency (NUE) of crop plants. One approach to improving NUE in plants uses a combination of traditional breeding and concurrent quantitative trait loci (QTL) mapping, allowing for the use of marker-assisted selection for key regions of the chromosome that segregate with NUE-associated traits. Subsequent characterization of candidate genes followed by cloning of these genes/alleles will facilitate the genetic engineering of plants with increased NUE. On the basis of gene structure and expression data, we have identified and mapped candidate genes that exhibit potential roles in improving NUE in barley. We then performed a meta-analysis of QTLs associated with NUE in field trials and further analyzed the map-location data to narrow the search for candidate genes. These results provide a platform for an understanding of the diversity of gene families and their physiological significance to NUE in crop plants.

200-015-Z Molecular Marker Development and Genetic Diversity Exploration by RNA-seq in Platycodon Grandiflorum Hyun Jung Kim – Cheonan Yonam College Jungsu Jung – Andong National University, Myung-Shin Kim – Seoul National University, Je Min Lee – Kyungpook

National University, Doil Choi – Seoul National University, Inhwa Yeam – Andong National University Platycodon grandiflorum, generally known as the bellflower or balloon flower, is the only species in the Platycodon genus of the Campanulaceae family. Platycodon plants have been traditionally used as a medicinal crop in East Asia for their antiphlogistic, antitussive, and expectorant properties. Despite these practical uses, marker-assisted selection and molecular breeding in platycodons have lagged due to the lack of genetic information on this genus. In this study, we performed RNA-seq analysis of three platycodon accessions to develop molecular markers and explore genetic diversity. First, genic simple sequence repeats (SSRs) were retrieved and compared among these three accessions to ; dinucleotide motifs were the most abundant repeats (39–40%) followed by tri (25–31%), tetra (1.5–1.9%), and penta (0.3–1.0%) repeats. The result of in silico SSR analysis three SSR markers were detected and showed possibility to distinguish three platycodon accessions. After several filtering procedures, 180 single nucleotide polymorphisms (SNPs) were used to design 40 cleaved amplified polymorphic sequence (CAPS) markers. Twelve of these PCR-based markers were validated as highly polymorphic and utilized to investigate genetic diversity in 21 platycodon accessions collected from various regions of South Korea. Collectively, the 12 markers yielded 35 alleles, with an average of 3 alleles per locus. Polymorphism information content (PIC) values ranged from 0.087 to 0.693, averaging 0.373 per locus. Since platycodon genetics have not been actively studied, the sequence information and the DNA markers generated from our research have the potential to contribute to further genetic improvements, genomic studies, and gene discovery in this genus.

200-016-Y Identifying Genes Important for Soybean Shoot Architecture Traits Using a Fast Neutron Mutagenized Population Suma Sreekanta – University of Minnesota Fengli Fu – University of Minnesota, Benjamin Campbell – University of Minnesota, Anna Hofstad – University of Minnesota, Adrian Stec – University of Minnesota, Jeffrey Roessler – University of Minnesota, Robert Stupar – University of Minnesota, Gary Muehlbauer – University of Minnesota Soybean (Glycine max) genetic diversity has been reduced due to domestication and selective breeding. Therefore, most soybean cultivars grown today are genetically very similar. Mutagenesis is one approach to introduce genetic diversity in plants. We used fast neutron mutagenesis to create a large mutagenized population of soybean individuals in the M92220 background that are altered in qualitative and quantitative traits. Changes in shoot architecture are of particular interest to us since altering shoot architecture could enhance light perception as well as harvest index, resulting in increased yield. We screened the population for shoot architecture phenotypes and identified mutants that show dwarf stature, reduced branch angle, shorter petiole and reduced leaf angle. Our aim is to identify the loci responsible for the different phenotypes by mapping and eventually cloning the underlying gene(s). We are using a complementary approach of whole genome sequencing based bulk segregant analysis and array Comparative Genomics Hybridization (aCGH) to identify region(s) of the genome controlling these mutant phenotypes. Using this approach we identified regions harboring mutations that result in plants with reduced branch angle, reduced stature, short petiole and leaf chlorosis. Experiments are underway to fine map the regions identified as well as identify specific genes that are determinants of these traits.

200-017-Y Enhancement and Suppression of Barley (Hordeum Vulgare L.) Low-tillering Mutants by Eligulum-a, a Previously Uncharacterized Plant Gene - Axillary Bud versus Ligule Development Ron Okagaki – University of Minnesota Hatice Bilgic – University of Minnesota, Allison Haaning – University of Minnesota, Arnis Druka – The James Hutton Institute, Micha Bauer – The James Hutton Institute, Gary Muehlbauer – University of Minnesota

Using a suppressor screen we identified a new gene in the vegetative axillary bud development pathway that, although conserved in land plants, does not correspond to previously described mutants. Forward genetic screens have identified numerous genes controlling axillary meristem development and the resulting branches or tillers, and these genes are largely conserved between monocots and eudicots. To identify additional genes, we screened for suppressors of the barley low-tillering mutant uniculm2; axillary buds rarely develop in uniculm2 plants. Two uniculm2 suppressor mutations were recovered that resulted in axillary bud and tiller development in the cul2 mutant background; the two suppressors were allelic. The suppressor mutant, in the wild-type background resembled the eligulum-a mutant, altering ligule, stem, and spike development. Eligulum-a was also allelic to the suppressor mutations. A candidate gene was identified by RNAseq, and confirmed by resequencing the original two suppressor mutations and three eligulum-a mutant alleles. In situ hybridization experiments found that Eligulum-a was mainly expressed in young leaves and leaf primordia, primarily around vascular tissue, the leaf margin, and in epidermal tissue. It was not expressed in meristems. The predicted polypeptide contained a Hermes-type RNaseH-like domain and probably a BED-type zinc finger. Our results suggest a complicated relationship between genetic control of axillary meristem and ligule development.

200-018-Z Fast Neutron Induced Structural Rearrangements at a Soybean NAP1 Locus Result in Gnarled Trichomes Benjamin Campbell – University of Minnesota Anna Hofstad – University of Minnesota, Suma Sreekanta – University of Minnesota, Fengli Fu – University of Minnesota, Thomas Kono – University of Minnesota, Jamie O’Rourke – Iowa State University, Carroll Vance – University of Minnesota, Gary Muehlbauer – University of Minnesota, Robert Stupar – University of Minnesota A soybean (Glycine max (L.) Merr.) gnarled trichome mutant was identified in a fast neutron mutant population. The gnarled trichome phenotype is characterized by stunted trichomes as compared to wild-type. A population was developed to map the causative mutation using whole genome sequencing-based Bulk Segregant Analysis (BSA). A single round of BSA using bulks of 50 F2 individuals identified an interval on chromosome 20 that co-segregates with the phenotype. In this interval, array Comparative Genomic Hybridization identified a 2kb deletion in Glyma.20G019300, the soybean ortholog of Arabidopsis NAP1. Further sequencing of the fast neutron mutant revealed a second deletion (1kb), a translocation, and an inversion all within the Glyma.20G019300 gene. In Arabidopsis, the NAP1 gene is an important regulator of actin nucleation during trichome morphogenesis. PCR based genotyping of 121 F3 individuals from different F2:3 families revealed that the mutant and wild-type GmNAP1 alleles perfectly co-segregate with their respective phenotypes. GmNAP1 functionally complemented an Arabidopsis nap1 mutant (grl-4) suggesting that GmNAP1 is essential for proper trichome development and that the soybean gnarled phenotype resulted from complex structural rearrangements at the GmNAP1 locus.

200-019-Z Genetic Resources for Fragaria Vesca: Understanding Developmental Processes and Physiological Responses in Strawberry Janet Slovin – USDA-ARS Numerous genomic resources are available for the Rosaceae model plant, the diploid strawberry F. vesca, including inbred lines, a sequenced genome, and comprehensive transcriptome data for flower and early fruit development. Currently, few morphological or physiological genetic traits such as runnering, leaf color, and fruit color have been described and mapped. EMS mutagenesis was used to generate M2 populations for two inbred cultivars, Hawaii 4 (H4F7-3) for which there is genome sequence, and YW5AF7, which does not produce runners. Both lines have yellow

fruit, and can cycle from seed to seed in 3-4 months in a 10 cm pot. A catalog of existing phenotypic mutants will be presented, together with more extensive characterization of several of them. A recessive, single gene, mutation in YW5AF7 resulted in runners forming at every node, and elongation of the crown. Two different 2 year old mutant plants of H4F7-3 have not produced runners. Selfed progeny from these plants also do not produce runners. Crosses between these two plants (Or703 and Y064) and backcrosses to wild-type have been made to examine the genetics of the runnerless trait. Additional mutants being characterized include: palemale, which has pale yellow anthers, produces no pollen, and retains petals after the flower has opened independent of fertilization status; incomplete male, which has been found in progeny from both lines, is missing the inner whorl of anthers; petiolulong, which has highly elongated petiolules (the stems at the base of each leaflet); floppy, which has thin, weak, petioles and narrow light green leaflets; and green petal, which has irregularly shaped green and white petals. We are also screening M2 seedlings for increased thermotolerance and for resistance to auxin. We are currently testing one thermotolerant line for ability to produce completely fertile flowers at moderately elevated temperatures.

200-020-Y Complexity of Whole Chromosome Evolution in Lupins Karolina Susek – Institute of Plant Genetics, Polish Academy of Sciences Wojciech Bielski – Institute of Plant Genetics, Polish Academy of Sciences, Katarzyna Wyrwa – Institute of Plant Genetics, Polish Academy of Sciences, Bogdan Wolko – Institute of Plant Genetics, Polish Academy of Sciences, Barbara Naganowska – Institute of Plant Genetics, Polish Academy of Sciences The legume family comprises three subfamilies: Mimosoideae, Caesalpinoideae and Papilionideae and is remarkable for its evolutionary diversification and global distribution. The genus Lupinus belongs to the Papilionoideae with about 275 species divided into two groups according to geographical distribution: ‘Old World’ lupins (OWL) and ‘New World’ lupins (NWL). The complex organisation and evolution of the Lupinus genomes, most striking in the OWL group, is demonstrated by a high diversity in chromosome number (2n=32 – 52), and wide ranges in basic chromosome number (x=6 – 9, 13) and genome sizes (0.97 pg/2C – 2.68 pg/2C). To track the lupin genome evolution regarding chromosomal variation we have used BAC clones as cytogenetic chromosome markers for L. angustifolius (2n=40) – the reference species among OWL. The integration of L. angustifolius linkage groups and chromosome maps has enabled us to establish the set of BACs applicable as probes for fluorescence in situ hybridisation (BAC-FISH). This allowed us to identify chromosome rearrangements among analyzed species. With the aim of understanding lupin chromosomal relationships, the 30 BACs were mapped by FISH in both crops: L. albus (2n=50), L. luteus (2n=52) and wild species: L. atlanticus (2n=38) L. cosentinii (2n=32), L. cryptanthus (2n=40), L. digitatus (2n=36), L. micranthus (2n=52) and L. pilosus (2n=42). The results suggest complex and dynamic chromosome evolution within the genus, possibly involving chromosome breakage and/or fusions/fissions, as well as the polyploidisation event(s). We have identified the L. angustifolius-derived BACs giving unique signal, dispersed signals (various patterns) or not detected in chosen lupins. The outcomes of integrated chromosomal evolutionary research are pioneering for lupins and along with further sequence analyses will provide the answers for key questions in their phylogeny including pattern of evolution event(s). Acknowledgements: This work was performed with the financial support of the National Science Centre, Poland (grant no. 2011/03/B/NZ2/01420).

200-021-Y Development of Automated DNA and RNA Plant Extraction Kits Using the New Maxwell® RSC Platform Lyndsey Jager – Promega

Samantha Lewis – Promega, Brad Hook – Promega, Adam Blatter – Promega, Chris Moreland – Promega, Cris Cowan – Promega The wide diversity among plant taxa makes it difficult to find universal and robust solutions for extracting DNA and RNA. Poor and inconsistent extraction methods can carry over secondary compounds such as phenols and carbohydrates resulting in inaccurate quantification and inhibition of downstream amplification analyses. With this challenge in mind, Promega has developed an automated system that provides consistent DNA and RNA extraction from a variety of plant tissue samples. The DNA extraction protocol was specifically optimized for purifying high molecular weight DNA, balancing the need for maximizing DNA yield per sample while minimizing multiple types of qPCR and next-generation sequencing inhibitors. Likewise, the RNA extraction protocol was optimized to prevent degradation of RNA, improve purity, minimize RT-qPCR inhibitors, and maximize yield. Both the RNA and DNA Plant kits require a minimal amount of sample input for the purification of high quality nucleic acids, compatible with all common downstream amplificationbased assays. While optimizing these protocols, we also discovered some advantages and pitfalls of various quantification methods that apply specifically to plant samples. In some species, absorbance peaks in the 225-245 nm region cause a drastic overestimation of the UV-based concentration. As a consequence, performance of downstream applications may suffer due to incorrect input volumes. We show that fluorescent dye-based quantification methods more accurately estimate nucleic acid concentration and correlate well with qPCR quantification.

200-022-Z Quantitative Genetic Analysis of Tiller Angle as a Target for Improved Radiation Interception efficiency in C4 Crops Mark Holmes – University of Illinois Darshi Banan, Rachel Paul, Max Feldman, Hannah Schlake, Andrew Leakey – University of Illinois, Carl R Woese Institute for Genomic Biology Increasing yield is an important target in the development of lignocellulosic biofuel feedstocks. Changes in plant architecture through the production of tillers can alter light interception (εi), an important component of yield. This study aims to use Setaria as a model C4 grass system to identify tillering quantitative trait loci (QTL) that change plant architecture in response to light. The light environment was altered by planting Setaria in either a high or low planting density treatment. Tiller spread angle (the angle created from the two outermost tillers of an individual plant) and tiller spread plasticity (the change in tiller spread angle between treatments) were both measured. QTL analysis results identified regions of genetic control for tiller spread angle and spread plasticity. Also, results show that the traits tiller spread angle and tiller spread plasticity are under separate genetic control. QTL results from this study offer a starting point for further research in identifying candidate genes that control variable tiller response to light.

Genes & Genomes: Epigenetics 200-023-Z Genetic and Epigenetic Variation in Crop Legume Species Scott Jackson – University of Georgia Kyung Do Kim – University of Georgia, Moaine El Baidouri – University of Georgia, Carolina Chavarro – University of Georgia, Marc Libault – University of Oklahoma Legumes are important sources of protein for human consumption, forage for animals and through association with soilborne microbes provide for fixation of atmospheric nitrogen for sustainable agriculture. In the past several years there has been a proliferation of legume genome sequences which provide an opportunity to explore what separates legumes

from other plant groups and how polyploidy, domestication and selection have molded crop legume genomes. Soybean (Glycine max) and common bean (Phaseolus vulgaris) share a polyploidy (WGD) event, approximately 56 million years ago (MYA), followed by a Glycine-specific polyploidy, ~10 MYA. Due to the independent WGD and relatively slow process of diploidization, 75% of soybean genes are present in more than one copy, as compared to 43% in common bean. Cytosine methylation is an epigenetic mark that plays an important role in the regulation of genes and transposable elements (TEs). We used whole-genome bisulfite sequencing to produce nucleotide resolution methylomes for soybean and common bean. We found that within the gene body, CG methylation was positively correlated with gene expression, and non-CG methylation negatively associated with gene expression. CG body-methylated genes were most abundant in WGD genes, which were, on average, more highly expressed than single-copy genes and had slower evolutionary rates than unmethylated genes, suggesting that WGD genes evolve more slowly than single copy genes. In addition, diverged methylation patterns in non-CG contexts between paralogs were found to be mostly due to TEs in or near genes, suggesting a possible role for TEs and non-CG methylation in regulating gene expression post polyploidy.

200-024-Y A Pumpkin Phloem SMALL RNA-BINDING PROTEIN1-ribonucleoprotein Complex Functions in Delivery of Small RNAs to Sink Tissues Byung-Kook Ham – University of California-Davis Gang Li – University of California-Davis, Weitao Jia – University of California-Davis, Zhangjun Fei – Boyce Thompson Institute for Plant Research, Cornell University, Julie A. Leary – University of California-Davis, William J. Lucas – University of California-Davis Phloem delivery of RNA, in the form of ribonucleoprotein (RNP) complexes, participates in systemic control over developmental and defense-related processes. Cucurbita maxima PHLOEM SMALL-RNA BINDING PROTEIN1 (CmPSRP1) functions in cell-to-cell trafficking of small RNA (sRNA) and is present in pumpkin phloem sap. Here, we further characterize the role of CmPSRP1 in sRNA complex formation and long-distance delivery of its sRNA cargo. Phloempurified CmPSRP1 is phosphorylated at four Ser residues, but this posttranslational modification is not required either for sRNA binding or entry into the sieve tube system. However, these phosphoserine residues are essential for assembly of a CmPSRP1-based sRNP complex, present in phloem sap as a 334 kDa homodimer that carries predominantly 24-nt RNAs. A phloem sap Cm_PHLOEM SMALL-RNA BINDING PROTEIN KINASE1 was identified that specifically phosphorylated PSRP1, consistent with assembly of the PSRP1-sRNP complex within the sieve tube system. Gel mobilityshift and RNase-protection assays revealed that the PSRP1-sRNP complex confers enhanced binding affinity and stability for cargo sRNAs. Phloem translocation studies established that stabilized PSRP1-sRNP complexes permit systemic delivery of sRNA to the plant apex. These findings provide insight into the nature of phloem sRNP complexes that likely mediate delivery of sRNA associated with systemic gene silencing in plants.

200-025-Y MSH1-Derived Epigenetic Breeding Potential in Tomato Xiaodong Yang – University of Nebraska-Lincoln Hardik Kundariya – University of Nebraska-Lincoln, Sally Mackenzie – University of Nebraska-Lincoln We report here the induction of non-genetic developmental reprogramming (MSH1-dr) in the tomato variety ‘Rutgers’ by silencing the single nuclear gene MutS Homolog 1 (MSH1). Crossing these transgene-null, developmentally altered plants to their isogenic wild-type counterpart gave rise to heritable enhanced growth vigor and increased yield performance in subsequent generations tested under both greenhouse and field conditions. Selected F4 populations showed up to 35% increases in mean yield after two rounds of selection. Our results show that the enhanced growth vigor is responsive to selection and reverted to wild-type levels upon treatment with the methylation inhibitor 5-

azacytidine, suggesting influence of epigenetic factors in the growth vigor. Similar growth vigor can also be obtained by grafting these MSH1-dr plants onto wildtype scions, implying that the important determinants in the process are mobile. Transcriptome data from MSH1-dr plants show heightened expression of stress responsive genes. We are conducting cross-comparative analysis of transcriptome behavior from tomato and Arabidopsis to shed light on the network of genes modulated by MSH1 and yield candidate genes involved in developmental reprogramming phenomenon. Currently, we are also testing the possibility of using MSH1-dr in grafts to both determinate and indeterminate commercial varieties of tomato for enhanced growth vigor. Our data provide compelling evidence for the feasibility of epigenetic breeding in tomato.

200-026-Z Genetic Variation for Retrotransposon Derived Small RNAs in Maize Bosen Zhang – University of Illinois at Urbana-Champaign Stephen Moose – University of Illinois at Urbana-Champaign Retrotransposons comprise a significant proportion of eukaryotic genomes and although typically silent, when expressed they can regulate gene expression in a variety of ways. The majority of the maize genome consists of long terminal repeat (LTR)-retrotransposons that produce a large and diverse source of small interfering RNAs (LTR-siRNAs). Deep sequencing of small RNAs from the seedling shoot apex of 36 diverse maize inbred lines demonstrates that the accumulation patterns of LTR-siRNAs exhibit both a strong genetic component and dramatic genetic diversity. The majority of LTR-siRNAs are produced from a small group of high copy number families that expanded within the maize genome 0.5-1.5 million years ago and are found in all genotypes surveyed; however, the total abundance of 21-22-nt and 23-24-nt LTR-siRNAs for these families varies among genotypes. Other LTR-retrotransposon families produce siRNAs in a subset of genotypes, with examples of some families where siRNAs are only found in one or few inbred lines. We discovered that among a number of factors known to influence population structure among the inbred lines surveyed, divergence in LTR-siRNA profiles was most prominent among genotypes representing populations artificially isolated to exploit hybrid vigor. Quantitative RT-PCR analyses on some of the maize inbred lines and three genetic pedigrees constructed by B73, Mo17 and PH207 showed that mRNA expression level, to some extent, positively related with siRNA abundance in some retrotransposon families, and the mRNA variations existed in those F2 populations were also observed within their further progenies F4 and F6. Our results indicate that LTR-siRNAs contribute another component to regulatory diversity in complex genomes such as maize.

200-027-Z Parent-of-origin Effect Mutants Regulating Endosperm Cellular Development in the Maize Seed Fang Bai – University of Florida Mary Daliberti – University of Florida, Miaoyun Xu – Chinese Academy of Agricultural Sciences, Alyssa Bagadion – University of Florida, Jeff Gustin – University of Florida, Yubing Li – University of Florida, John Baier – University of Florida, Yubing Li – University of Florida, Chi-Wah Tseung – University of Florida, Matthew Evans – Carnegie Institution for Science, Stanford, A. Mark Settles – University of Florida Genomic imprinting in plants is an epigenetic phenomenon by which a subset of genes is expressed in a parent-oforigin–dependent manner. Although many maize imprinted genes have been identified through transcriptome analysis, imprinted genes with developmental functions in the maize seed have not been identified. We screened 178 rough endosperm (rgh) mutants for parent-of-origin effects using reciprocal crosses to inbred parents. Six maternal rough endosperm (mre) and three paternal rough endosperm (pre) mutants were identified. Characterization of the maternaleffect isolates shows a range of seed defects with several mutants showing embryo defects in addition to the endosperm phenotype. The pre mutants show a high frequency of embryo abortion leading to low oil levels in mature

kernels. Developmental sections of pre1 indicate that embryos abort at a globular stage, while the starchy endosperm shows cell differentiation defects. Embryo abortion is less frequent in mre1, mre2, mre3, and mre*-1014 suggesting these loci control seed size. Developmental sections of mre1 suggest these mutants show a general delay in endosperm development with smaller starchy endosperm cells, delayed basal endosperm transfer cell layer development, and delayed accumulation of starch granules. The mre2 mutant shows multiple starchy cell differentiation defects, while mre3 mutants have an endosperm phenotype consistent with reduced sink strength. Molecular mapping experiments identified four loci on chromosomes 4, 6, and 10. Additional mutant alleles obtained from the UniformMu reverse genetics resources appear to identify mre1, mre3, and pre1 as imprinted genes in maize.

200-028-Y Exploring Mechanisms and Conservation of Gene Imprinting in an Outcrossing Crucifer, Arabidopsis Lyrata Maja Klosinska – Whitehead Institute for Biomedical Research Colette Picard – Computational and Systems Biology Graduate Program, MIT; Whitehead Institute for Biomedical Research, Mary Gehring – Whitehead Institute for Biomedical Research Gene imprinting evolved independently in mammals and flowering plants. Imprinted genes are expressed primarily from one allele in a parent of origin dependent manner, leaving the other allele repressed. In flowering plants gene imprinting occurs in the seed nutritive tissue, the endosperm, which is the product of fertilization of the central cell by a sperm cell. According to the kinship or parental conflict theory of imprinting, the mother (and maternally expressed imprinted genes) promotes equal resource distribution to all her offspring, while the father (and paternally expressed imprinted genes) tries to extract as many resources as possible for his own particular offspring. Gene imprinting has been extensively studied in the model crucifer Arabidopsis thaliana, which is a selfing plant that might lack strong selective pressures to maintain imprinted gene expression. Arabidosis lyrata is a closely related outcrossing perennial with a sequenced and annotated genome. Gene imprinting in A. thaliana has been found to be associated with differential DNA methylation of parental genes, and often with transposable elements. A. lyrata has been shown to possess both more DNA methylation and TEs in its euchromatic regions, perhaps providing additional potential for imprinted gene evolution, in addition to stronger selection for imprinting due to a greater parental conflict caused by outcrossing. We set out to discover imprinted genes in A. lyrata through performing crosses between the sequenced strain MN47 and plants from a population in Karhumaki, Russia. We used RNA-seq data to identify over 150,000 SNPs between these populations. SNPs and expression data were subsequently used to detect imprinting. Among the detected genes we observed some overlap with A. thaliana, as well as genes conserved also in maize and rice, such as paternally expressed YUC10. The newest results will be presented.

200-029-Y FRIGIDA: Its Crystal Structure and as a Biochemical Activator of the Histone Methyltransferase EARLY FLOWERING in SHORT DAYS Kyung-gi Hyun – KAIST Ji-joon Song – KAIST The transition from vegetative to reproductive growth is a major developmental switch in the life cycle of flowering plants. FLOWERING LOCUS C (FLC), a MADS-box transcription factor, acts as a major repressor of this transition. While vernalization and the autonomous pathway repress FLC expression, FRIGIDA (FRI) activates FLC transcription and confers vernalization requirement to winter-annual type Arabidopsis accessions for their rapid flowering. Despite this key role of

FRI in flowering regulation, little is known for its biochemical and structural characteristics of FRI. Here, we report a crystal structure of FRI core domain that adopts a novel extended helical bundle. Based on sequence conservation and structural analysis, we identified a highly conserved surface with positive charges on FRI, and the mutating this surface resulted in loss of FRI activity in vivo. We also mapped naturally-occurring non-synonymous mutations on the structure, providing a structural basis of non-functional alleles of FRI. Furthermore, we show that FRI directly stimulates the Histone H3 Lys36 methyltransferase activity of EARLY FLOWERING IN SHORT DAYS (EFS) in vitro. Our study reports the structure of FRI for the first time and provides structural and biochemical frameworks for the investigation of the molecular mechanisms of flowering time regulation.

200-030-Z A Neutrality Test for DNA Methylation Using Single Methylation Polymorphisms Chuanzhu Fan – Wayne State University Jun Wang – Wayne State University Inheritable epigenetic mutations (epimutations) can contribute to transmittable phenotypic variation. Thus, epimutations can be subject to natural selection and impact the fitness and evolution of organisms. Based on the framework of the modified Tajima’s D test for DNA mutations, we developed a neutrality test with the statistic “Dm ”to detect selection forces on DNA methylation mutations using single methylation polymorphisms (SMPs). With computer simulation and empirical data analysis, we compared the Dm test to the original and modified Tajima’s D tests and demonstrated that the Dm test is suitable for detecting selection on epimutations and outperforms original/modified Tajima’s D tests. Due to the higher resetting rate of epimutations, the interpretation of Dm on epimutations and Tajima’s D test on DNA mutations could be different in inferring natural selection. Analyses using simulated and empiricalgenome-widepolymorphism data suggested that genes under genetic and epigenetic selections behaved differently. We applied the Dm test to recently originated Arabidopsis, and showed that newly evolved genes contain higher level of rare epialleles, suggesting epimutation may play a role in origination and evolution of genes and genomes. Overall, we demonstrate the utility of the Dm test to detect whether the loci are under selection regarding DNA methylation. Our analytical metrics and methodology could contribute to our understanding of evolutionary processes of genes and genomes in the field of epigenetics. The Perl script for the “Dm” test is available at http://fanlab.wayne.edu/.

200-031-Z H4K16 Acetylation Exhibits a Species-specific Distribution Pattern and Acts in a Combinatorial Manner with H3K23 Acetylation on Gene Expression in Plants Dean Sanders – University of Wisconsin-Madison Li Lu, Xuehua Zhong, Xiangsong Chen, Shuiming Qian Histone (de)acetylation is a key epigenetic gene regulatory mechanism that plays critical roles in many biological processes. Acetylation of histone H4 lysine 16 (H4K16ac) is implicated in chromatin dynamics, transcriptional activation, replication, aging, and dosage compensation. However, its biological function and relationship with transcription and other epigenetic modifications are completely unknown in plants. Here, we generated a genome-wide high-resolution map of H4K16ac localization in the model plants Arabidopsis thaliana and Oryza sativa. As comparison, we also determined the global distribution pattern of H3 lysine 23 acetylation (H3K23ac) in both plants. Our data show that H4K16ac and H3K23ac co-localize in both the Arabidopsis and rice genomes and are preferentially enriched at euchromatic regions, particularly around transcription start sites of highly expressed genes. Unlike animal H4K16ac which often has distinct functions from the other acetylation marks, plant H4K16ac appears to act in a combinatorial manner with H3K23ac on transcriptional activation. These data suggest that there may be species-specific functions of

H4K16ac. Our data also suggests a potential role of H4K16ac in plant development and stress responses. Together, this study provides a number of novel findings that have significant implications for the future understanding of histone acetylation in plant developmental and environmental regulation.

200-032-Y Histone Acetyltransferase of the Creb Binding Protein Family1 (HAC1; At1g79000) Plays a Role in Developmental, Age-Induced Leaf Senescence Judy Brusslan – CSULB Keykhosrow Keymanesh – CSULB, Emon Perl – CSULB, Ana Rus Alvarez-Canterbury – CSULB, Judy Brusslan – CSULB Leaf senescence is the final nutritive stage of leaf development in which the organ that provided sugars to the growing plant undergoes a controlled degradation process that recycles much of the nitrogen incorporated into the protein-rich photosynthetic complexes. These nutrients are transported to the reproductive and storage structures of the plant and are the largest contributor to seed nitrogen. We have shown that the breadth of histone acetylation is positively correlated to changes in gene expression during leaf senescence (Plant Physiology doi:10.1104/pp.114.252999) and screened histone acetyltransferase and deacetylase T-DNA insertion lines for senescence phenotypes. Two hac1 alleles (At1g79000) which encodes a histone acetyltransferase in the CREB binding protein family displayed darker green leaves in older plants. Although hac1 mutants flowered slightly later than wildtype, the darker green color was retained for up to 80 days, over a month after flowering. Rubisco activity was estimated using the initial slopes of the ACi curves and found to be increased in mutant leaf 10 of 50 day old plants. Total chlorophyll and protein levels were increased by 30% in mutant leaves 11-13 of 61 day old plants. These leaves also showed increased expression (1.5 to 3-fold) of genes encoding the three subunits of Mg-chelatase, which catalyzes the first committed step in chlorophyll biosynthesis, as well as numerous chlorophyll-binding Lhcb1, Lhcb2 and Lhca proteins. The WRKY75 gene, a transcription factor with increased expression throughout senescence, had decreased (5-fold) expression in the hac1 alleles. These data suggest that loss of function hac1 mutants have delayed age-related senescence. Currently, RNA-seq and ChIP-seq datasets are being generated to determine which genes show a change in histone acetylation and gene expression with the goal of identifying HAC1 targets that contribute to the delayed senescence phenotype.

Genes & Genomes: Comparative Genomics, Domestication 200-033-Y Loci Involved in Domestication of Potato as Revealed Through Whole Genome Sequencing C Robin Buell – Michigan State University Michael Hardigan – Michigan State University, Emily Crisovan – Michigan State University, Krystle Wiegert-Rininger – Michigan State University, David Douches – Michigan State University Potato is the world’s most important non-cereal food crop and is gaining importance in developing countries as a food source due to its high yield potential and adaptability. Cultivated potato (Solanum tuberosum) is a heterozygous autopolyploid (2n=4x=48) that was domesticated in the Andes from wild Solanum species over 10,000 years ago. Comparison of allele frequencies between a 74 plant introduction Solanum sect. Petota Diversity Panel representing 25 wild Solanum species and a cultivated tetraploid diversity panel of 213 clones genotyped with a 8303 single nucleotide polymorphism (SNP) marker array revealed numerous loci with diverged allele frequencies including genes involved in carbohydrate metabolism and tuber development, consistent with phenotypic features of potato domestication and improvement. To further dissect loci important in domestication of potato, we generated whole genome sequence from 20 wild species, 20 cultivated landraces, and 20 elite cultivars of potato. To provide a historical perspective of potato

breeding we included two cultivars generated in the 19th century. We are currently using whole genome scans to identify loci associated with domestication of potato as well as loci selected in development of elite processing cultivars.

200-034-Z Population Genomics of Allopolyploid Wheat Adaptation Eduard Akhunov – Kansas State University Katherine Jordan – Kansas State University, Shichen Wang – Kansas State University, Yanni Lun – Kansas State University, Alina Akhunova – Kansas State University, Luther Talbert – Montana State University, Matthew Hayden – Department of Environment and Primary Domesticated crops experience strong human-mediated selection aimed at developing varieties adapted to local environmental conditions. To investigate the impact of selection on variants distributed among homoeologous wheat genomes and to build a foundation for understanding genotype-phenotype relationships, we performed populationscale re-sequencing of a diverse panel of wheat lines. A panel was selected to capture the genetic diversity of the major global wheat growing regions and span several levels of improvement ranging from wild ancestors to landraces and cultivars. We find contrasting patterns of variation and linkage disequilibrium among the wheat genomes; this, in addition to demographic factors, could be explained by differences in the effect of directional selection on duplicated homoeologs. We found evidence of a small population bottleneck during transition from landraces to cultivars. The majority of the selected alleles were present at low frequency in local populations suggesting either weak selection pressure or temporal variation in the targets of directional selection. Only a small fraction of the homoeologous regions harboring selected variants overlapped among the wheat genomes in any given wheat line. Our data suggest that directional selection in wheat rarely acted on multiple parallel advantageous mutations across homoeologous regions, likely indicating that a fitness benefit could be obtained by a mutation at any one of the homoeologs. Additional advantageous variants in other homoelogs probably either contributed little benefit, or were unavailable in populations subjected to directional selection. Our study highlights the importance of allopolyploidy in the evolution of wheat’s adaptive potential by increasing the likelihood of beneficial allele recovery and broadening the set of possible selection targets.

200-035-Z Browsing and Comparing Genomes Using the Gramene Browser Joshua Stein – Cold Spring Harbor Laboratory Banita Tamot – Cold Spring Harbor Laboratory, Marcela Monaco – Cold Spring Harbor Laboratory, Kapeel Chougule – Cold Spring Harbor Laboratory, Dan Bolser – European Bioinformatics Institute, Paul Kersey – European Bioinformatics Institute Learn the benefits of using Gramene (http://www.gramene.org) to accelerate your research goals. We currently host browsers for over 40 complete reference genomes, each displaying value-added annotations, gene-trees, and whole genome alignments. In the last year we added the basal angiosperm, Amborella trichopoda, the crops Brassica oleracea, Prunus persica, and Theobroma cacao, five rice-related species in the Oryza and Leersia genera, and the unicellular alga Ostreococcus lucimarinus. These build upon a foundation that includes rice, maize, sorghum, diploid and hexaploid wheats, barley, Brachypodium, and banana. As well, we host soybean, Arabidopsis, Brassicas, potato, tomato, grapevine, and several lower plants. Evolutionary histories are provided in phylogenetic gene trees that classify orthologous and paralogous relationships as speciation and duplication events. Orthologous genes inform synteny maps that enable interspecies browsing across ancestral regions. In addition, genome browsers from multiple species can be viewed simultaneously, with links showing homologous gene and whole-genome alignment mappings. SNP and structural diversity data, available for eleven species, are displayed in the context of gene annotation, along with the

consequence of variation on transcript structure (e.g. Missense variant). For most studies users can drill-down to individual genotypes of each accession within the study’s diversity panel. Learn how visual displays can be downloaded as high-resolution, publication-ready, image files, along with the underlying supporting data in conventional crosscompatible formats. In addition our browser platform, Gramene produces and hosts metabolic pathway databases and visualization tools. The Plant Reactome (http://plantreactome.gramene.org/) hosts over 200 pathways curated in rice and inferred in thirty-three additional plant species by orthology projection. Complementing these resources is our BioMart data-mining interface, which enables complex queries of annotation, homology and variation data, and serves as an additional gateway into the genome browsers. Gramene is supported by an NSF grant IOS-1127112.

200-036-Y Candidate Genes Conferring Reniform Nematode Resistance in Two Upland Cotton Interspecific Introgression Lines Dinum Perera – Mississippi State University Chuan-Yu Hsu – Mississippi State University, Mark Arick II – Mississippi State University, Daniel Peterson – Mississippi State University Upland cotton (Gossypium hirsutum L.), the world’s leading natural textile fiber crop, accounts for more than 95% of the annual cotton crop worldwide. Reniform nematode (Rotylenchulus reniformis; RN) has been an emerging threat to the U.S. cotton industry and has supplanted root-knot nematode as the major nematode pest of cotton in Mississippi, Louisiana, and Alabama. Although, natural resistance to RN has not been observed in upland cotton, resistance to RN has been identified in other Gossypium species including Gossypium barbadense. The G. barbadense line with the highest known RN resistance is GB713. RN resistant upland cotton introgression lines, M713 Ren1 (Ren1) and M713 Ren2 (Ren2), derived from an interspecific cross between Sure-Grow 747 (upland cotton line) and GB713, resulted in >90% reduced infection. Resistance in Ren1 and Ren2 is conditioned by three RN resistance quantitative trait loci from the GB713 parent. The objectives of our research are identification and characterization of genes involved in RN resistance in Ren1 and Ren2 and to explore candidate gene expression patterns in response to RN infection. DNA libraries, constructed from genomic DNA of Sure-Grow 747, GB713, Ren1, and Ren2, were sequenced to depths of 35x using an Illumina HiSeq and assembled using ABySS. Regions of sequences shared by GB713 and the two introgression lines (i.e., Ren 1 and Ren2) but not found in Sure-Grow 747 were annotated using the MAKER-P annotation pipeline. Analyses resulted in discovery of fifty-nine candidate RN-resistance genes. Quantitative reverse transcription PCR will be utilized to identify those candidate genes that show changes in expression levels in response to RN infection. Information on the genes conferring RN resistance should facilitate efficient production of RN-resistant upland cotton lines while illuminating molecular pathways underlying RN resistance that can be exploited in RN control in cotton and other species.

200-037-Y Understanding the Evolution of the F-box Gene Familiy Across the Plant Kingdom Anandkumar Surendrarao – Plant Biology Graduate Group, UC Davis Ravi Patel – Cornell University, Layal Al-Ait – Georg-August-University, Douglas Cook – Dept. of Plant Pathology, UC Davis All cellular proteins need to be turned over. A major mechanism to achieve this is by F-box proteins (FBPs) targeting them to the 26S proteasomal machinery for degradation. As opposed to animal genomes, the genomes of plant species can code for > 10X as many FBP loci. For example, the Medicago genome contains 1429 FBP genes, whereas the larger soybean and maize genomes contain only 504 and 366 genes respectively. To explain these observations, we have split our analyses into a. investigating the mechanisms of birth and death of these loci, and b. analyzing the selection schemes that operate upon these loci.

The contribution of pseudogenization to the extinction, and of transposon-mediated events to the genesis of these FBD loci will be discussed. We hypothesize that the variability in the rates of these two biological processes across plant species contributes to the differential birth and death of members in the F-box gene family, partly explaining the observed differences in FBP gene numbers across plants. Genome-wide epigenomic re-programming of F-box genes has been reported in A. thaliana. We propose a model wherein lineage-specific loci in the accessory genome are preferentially silenced, thereby constituting a ’hidden’ genomic cache. Under this model, the genomic innovations in this cache are activated during stresses such as pathogen attack or adverse climactic conditions. The congruence between this proposed model and observations regarding the selection schemes under which these FBD encoding loci are operating will be also discussed. Ours is the first known study that links both the molecular mechanisms and the selection constraints that are responsible for the large and size variant F-box gene families across plant species. Furthermore, our analyses pipeline can be extrapolated to study other species, as well as to analyze evolution of other gene families.

200-038-Z A Platform for Soybean Molecular Breeding Based on Variation Blocks Yul-Ho Kim – Highland Agriculture Research Center, NICS, RDA Hyang-Mi Park – National Institute of Crop Science, RDA, Sunghoon Lee – Theragen Bio Institute, Yu-Young Lee – Department of Central Area, NICS, RDA, Su Jeong Kim – Highland Agriculture Research Center, NICS, RDA, Whang-Bae Sohn – Highland Agriculture Research Center, NICS, RDA, Su-Young Hong – Highland Agriculture Research Center, NICS, RDA, Jeong Hwan Nam – Highland Agriculture Research Center, NICS, RDA, Kibum Kweon – Highland Agriculture Research Center, NICS, RDA, Jin-Cheol Jeong – Highland Agriculture Research Center, NICS, RDA Soybean is one of the most widely planted oilseed crops in the world. Much effort has been expended to find agronomically important loci and improve soybean traits. However, the complexity of genome, such as genome duplication, limits the utility of genome-wide association studies and linkage analyses to identify genes that regulate important traits. We propose the variation block method, a three-step process for recombination block detection and comparison. The first step is to detect variations by comparing short-read DNA sequences of the cultivar to a reference genome of the target crop. Next, sequence blocks with variation patterns are examined and defined. The boundaries between the variation-containing sequence blocks are regarded as recombination sites. All the assumed recombination sites in the cultivar set are used to split the genomes, and the resulting sequence regions are named as variation blocks. The practicality of this approach was demonstrated by the identification of a putative locus determining soybean hilum color. We suggest that the variation block method is an efficient genomics method for recombination block-level comparison of crop genomes. We expect that this method holds the prospect of developing crop genomics by bringing genomics technology to the field of crop breeding.

200-039-Z Cross-species Studies of Nodulation-related Signaling Peptides Diana Trujillo – University of Minnesota Nevin Young – University of Minnesota, Kevin Silverstein – University of Minnesota Symbiotic nitrogen fixation in legume nodules requires an interplay of signaling processes between legume hosts and their rhizobial symbiotic partners. The rapid evolution of nodulation-related signaling peptide families among legume lineages, including expansion or contraction, can lead to novel functional traits such as differences in infection efficiency

or nodule viability. The primary goal of this study was to identify lineage-specific expansions (LSEs) of nodulation-related small secreted peptides in a systematic way. Nodule RNA-seq data was obtained for the IRLC legumes Pisum sativum, Trifolium pratense, Medicago truncatula, and Melilotus alba, as well as for Glycine max. Assembled transcripts were filtered to select for short open reading frames that contained signal peptides, and peptides were assigned into groups based on sequence similarity. Clustered peptides were used to run HMM searches of each group against legumes with available genome sequences, in order to expand the pool of genes of interest. A final set of clustered genes was then used to identify potential LSEs defined as either: 1) unequal gene family size between legume species or 2) gene families with notably different subsets of nodule-specific expression across legumes species. Candidate gene families were annotated through a consensus of information obtained from InterProScan, HMM searches against the UniRef90 database, and BLAST searches against the annotated M. truncatula proteome. In addition to previously described LSEs (such as Nodule-specific Cysteine-Rich peptides and nodule-specific Glycine-Rich Peptides), our study discovered novel gene families with nodule-specific expression that are unique to specific Trifoleae and Phaseoleae lineages.

200-040-Y A Reference Genetic Map of Korean Radish (Raphanus Sativus) by Whole-genome Resequencing of F2 Mapping Population Hee-Ju Yu – The Catholic University of Korea Jeong-Hwan Mun – Myongi University, Hee Chung – The Catholic University of Korea, Won-Hyong Chung – Korea Research Institute of Bioscience and Biotechnology, Mijin Oh – National Academy of Agricultural Science, Rural Development AdmiAdministration, Young-Min Jeong – The Catholic University, Namshin Kim – Korea Research Institute of Bioscience and Biotechnology, Byung Ohg Ahn – National Academy of Agricultural Science, Beom-Seok Park – National Academy of Agricultural Science, Suhyoung Park – National Institute of Horticultural and Herbal Science, Ki-Byung Lim – Kyungpook Nationa University, Yoon-Jung Hwang – Sahmyook University, Hee-Ju Yu – The Catholic University of Korea Radish (Raphanus sativus L.) is a member of the Brassicaceae family and a close relative of Brassica species. It has long been used as an edible root vegetable and is one of the key plants in the seed industry, especially in East Asia. In this study, to anchor genome sequence assemblies of R. sativus cv. WK10039, we constructed a reference genetic map using whole-genome resequencing of 93 individuals of the F2 mapping population (WK10039 X WK10024). High-confidence genetic markers were developed based on an Illumina 100-bp paired-end sequence of ~83 Gb of parental lines and ~591 Gb of the mapping population. High-stringency sequence analysis of the reads mapped to 344 Mb of genome sequence scaffolds identified a total of 16,282 SNPs and 150 PCR-based markers. As a result, a high-density genetic map was constructed from the analysis of 2,637 markers spanning 1,538 cM with 1,000 unique framework loci. The genetic markers integrated 295 Mb of genome sequences into the cytogenetically defined chromosome arms. Comparative analysis of the chromosome-anchored sequences with Arabidopsis thaliana and Brassica rapa revealed triplicated subgenomes of R. sativus and high similarity to R. sativus and B. rapa gene space. Therefore, the genetic map developed in this study will serve as a fundamental resource for genetic and breeding studies in R. sativus.

200-041-Y Q-type 2-fingered C2H2 Transcription Factor Proteins from the Solanum Tuberosum Group Phujera Genome Susan Lawrence – USDA-ARS Nicole Novak Several of the Q-type 2-fingered C2H2 transcription factors (TF) from Arabidopsis play a crucial role in the abiotic stress response. For example, over-expression of the Arabidopsis TF Zat7 confers an increase in salt tolerance in Arabidopsis. In silico analysis of the C2H2 TFs of the Arabidopsis genome has identified 176 proteins containing C2H2 motifs with eighteen Q-type 2-fingered proteins. Q-type C2H2 TFs are so named because they contain an invariant QALGGH as part

of their zinc finger motif. Recently we have identified two 2-fingered C2H2 TFs from potato induced upon insect herbivory. The potato genome was queried to identify similar proteins finding a total of 28. In most cases conserved motifs were identified including zinc-finger domains, L-box, nuclear localization signals (NLS), and the EAR motif required for transcriptional repression in Zat7. Several whole transcriptome studies in potato were used to determine where and when these genes are expressed. Comparing the potato protein sequences with similar sequences from the tomato genome demonstrates that many of these genes maintained similar map locations and evolved prior to the separation of these species.

Genes & Genomes: Molecular Evolution 200-042-Z The Genetics and Evolution of Reinforcement Robin Hopkins – Harvard University Understanding the genetic basis of speciation is a long-standing goal in evolutionary biology. Specifically, determining the genetic variation causing reproductive isolation between emerging species will provide insight into how the process of speciation occurs. Reinforcement, the process in which reduced hybrid fitness generates selection for the evolution of reproductive isolation can be an important mechanism through which pre-zygotic reproductive isolation evolves. Yet, there is very little known about the genetics of reinforcement. Reinforcement has caused the evolution of flower color in the native Texas wildflower Phlox drummondii. This change in flower color increases reproductive isolation with the sympatric sister species Phlox cuspidata and thus contributes to speciation in Phlox. The variation in flower color is caused by two, independently segregating, genetic loci. Both loci involve cis-regulatory changes in genes that are part of the anthocyanin biosynthesis pathway. The genetic basis of flower color variation in this system has informed extensive research on the mechanisms of reinforcement, and the strength of selection causing reinforcement. This work demonstrates how knowledge of the genetic basis of a trait facilitates field experiments, mathematical modeling, and population genetics to better understand the evolution of reproductive isolation.

200-043-Z Elucidating Mechanisms and Dynamics of Reproductive Isolation in Wild Tomato Species Patricia Bedinger – Colorado State University You Soon Baek – Colorado State University, Amanda Broz – Colorado State University, Suzanne Royer – Colorado State University, April Randle – Colorado State University, Roger Chetelat – University of California Davis, Alejandro TovarMendez – University of Missouri Columbia, Bruce McClure – University of Missouri Columbia Interspecific Reproductive Barriers (IRBs) prevent hybridization between species, including wild tomato species (Solanum sect. Lycopersicon). Using interspecific crosses between accessions from nine different sympatric sites in Peru, we documented prezygotic IRBs including active pollen tube rejection, as well as a major postzygotic IRB leading to defective seed development. The active rejection of interspecific pollen tubes within styles often follows the SI x SC rule: crosses between females of self-incompatible (SI) species and males from self-compatible (SC) species fail, while the reciprocal crosses succeed, a phenomenon called unilateral incompatibility (UI). Using the diverse array of mating systems within the tomato clade, we tested the generality of the SI × SC rule, examining pollen tube growth in crosses between all 13 clade members. We found that pistils of SI species always reject pollen of SC species. However, pistils of some SC species and SC populations of SI species, while unable to reject their own pollen, still rejected interspecific pollen. Thus, an intact SI system is apparently sufficient for the rejection of interspecific pollen, but partial SI systems, or redundant non-SI systems, can also contribute to UI pollen rejection. In order to further explore the relationship between SI and UI, we investigated the dynamics of mating system and IRBs at the species margins of the wild tomato

Solanum habrochaites. At the southern species margin, a specific S-RNase allele encoding in a low-activity protein is associated with the loss of SI. In this case, pistil IRBs seem to be unaffected by the loss of SI. At the northern species margin, the loss of SI is associated with the loss of S-RNase expression in at least two apparently independent events. In this region, IRBs are weakened, and in some cases are even lost, as mutations accumulate after the initial loss of SI.

200-044-Y Characterization of BTB E3 Ubiquitin-Ligase Gene Families in Lower Plants and Algae Katie Plamann – University of Wisconsin-Eau Claire Derek Gingerich – University of Wisconsin-Eau Claire Ubiquitylation, the attachment of ubiquitin to proteins to mark for degradation by proteasomes, is crucial for proper organism function. One family of complexes that play a role in this process is the BTB/Cullin 3/RBX E3 ubiquitin-protein ligases, which catalyze attachment of ubiquitin to target proteins. The BTB (Bric-a-Brac, Tramtrack, Broad Complex) domain-containing proteins are the target-adapters, binding to the proteins to be ubiquitylated via motifs appended to the BTB domain. Genes encoding BTB proteins have been identified in wide range of eukaryotic organisms (including fungi, protists, animals, and plants) but the BTB gene families in different groups show great variability in size, complexity, and composition. In land plant genomes thus far studied BTB gene families are large (~75-150 members) and complicated (with multiple subtypes based on the presence of a diverse set of target-binding motifs). We are interested in when the particular BTB family composition seen in the higher plants may have arisen in evolution. To help answer this question we are currently working to characterize this family in lower plants (including the nonvascular moss Physcomitrella patens and the seedless vascular lycophyte Selaginella moellendorffii) and in the green algal species Ostreococcus lucimarinus, Chlamydomonas reinhardtii, and Volvox carteri. Our analyses thus far have shown that the family in Physcomitrella is large (65 members), with many of the same BTB types found in higher plants. In contrast, while the family in Chlamydomonas is also large (>65 members), most BTB types in this alga are not seen in land plants and vice-versa. In contrast to the land plants and to Chlamydomonas, the genome of the tiny phytoplankton Ostreococcus lucimarinus contains only two BTB protein-encoding genes. Collectively these data show that there have been dramatic changes in both the size and composition of this E3 ubiquitin-ligase gene family during Viridiplantae evolution.

200-045-Y Impacts of norgDNA on the Nuclear Genomes of 11 Oryza Species Christos Noutsos – Cold Spring Harbor Laboratory Jshua Stein – Cold Spring Harbor Laboratory, Yeisoo Yu – Arizona Genomics Institute, Andrew Olson – Cold Spring Harbor Laboratory, Rod Wing – Arizona Genomics Institute, Doreen Ware – USDA/Cold Spring Harbor Laboratory The endosymbiotic origin of plastids and mitochondria led to the massive relocation of genes from proto-organelles to host in early eukaryote evolution. DNA insertion from organellar to nuclear genomes is an ongoing process giving rise to nuclear organelle DNA (norgDNA) in plants and animals. Though norgDNA’s are widely documented in plant genomes, significance to function and evolution is not well understood. Because norgDNA has high turnover rates, a powerful approach to studying their dynamics and impact is to perform comparative genomics among closely related species. The Oryza genus includes two cultivated species of rice and over twenty wild species. Recent species divergence provides a time resolution that is ideal for studying rapid processes in genome evolution. We annotated norgDNA’s in complete reference assemblies of eleven species, O. sativa ssp. japonica, O. sativa ssp. indica, O. glaberrima (African rice), O. punctata, O. brachyantha, O. barthii, O. nivara, O. rufipogon, O. meridionalis, O. glumaepatula, and the outgroup species, Leersia perrieri. We found that norgDNA constitutes as much as ~2 Mb of nuclear sequence, depending on species, with norgDNA ranging up to 60Kb. Although plastid-derived norgDNA generally harbored plastid coding regions,

there was no obvious bias of source sequence within the plastid genome. About 2600 norgDNA’s altered the structure of resident nuclear genes by inserting into intron or exon sequences. Comparison of norgDNA insertion sites among the eleven genomes showed that the vast majority occurred as unique events within individual species, and that very few were retained over time. These observations suggest that norgDNA’s in general have neutral or deleterious effects on species fitness. By the same token, some very rare cases of norgDNA conservation throughout the Oryza clade suggest that novel, potentially advantageous functions may have arisen from this dynamic process.

200-046-Z Loss of Conserved Non-Coding Sequences Upstream of Paralogous Genes Leads to Reduced Transcript Levels Robert Hoffmann – University of Copenhagen Michael Palmgren – University of Copenhagen Although conserved non-coding sequences (CNSs) in the Arabidopsis thaliana genome have been identified by comparative genomic analyses with other plant species, the function of most of these CNSs remains elusive. Our genomic analysis of paralogous genes resulting from a whole genome duplication showed that the number of 5’ upstream CNSs is correlated with increasing expression divergence between two paralogs in A. thaliana. This correlation was only found for the paralog with reduced transcript levels. We also found that the number of 5’ upstream CNSs does not generally correlate with transcript levels. Together, these findings suggest that the loss of 5’ upstream CNSs leads to reduced gene expression, and assigns a function to CNSs as transcriptional enhancers. Moreover, a gene ontology (GO) term enrichment analysis showed that paralogs with similar expression levels were enriched in GO terms that differed from those enriched in paralogs with different expression levels. These results indicate that for paralogous genes the retention and loss of CNSs that function as transcriptional enhancers, and hence gene expression levels, are dependent on whether the proteins the genes encode are dosage sensitive.

200-047-Z Evolution of the β-amylase Gene Family in Land Plants: Amplification and Specialization of Function Jonathan Monroe – James Madison University Amanda Storm – James Madison University, Catherine Torres – James Madison University Plants use starch for diurnal or seasonal glucose storage. Starch degradation in living cells requires a variety of enzymes but β-amylase carries out most of the hydrolysis, producing maltose that is exported from plastids. There are nine βamylase (BAM) genes in Arabidopsis. Three encode non-plastidic proteins; one that is active and cytosolic (BAM5), and two that are nuclear transcription factors (BAM7 and -8). We are using purified proteins and T-DNA mutants lacking multiple BAMs to characterize the plastid-localized members of the family. Only three of the six plastidic BAMs have significant catalytic activity. We found that catalytic BAM1 is thermally adapted to work best during the day whereas catalytic BAM3 is thermally adapted to work best at night, both in young leaves. BAM6 is also catalytic and appears to function late in leaf development. The remaining three plastid-localized forms (BAM2, -4 and -9) are catalytically inactive and probably play regulatory roles. We are also using a phylogenetic approach to characterize the evolution of the gene family in land plants. Sequenced plant genomes all contain at least one member from both the BAM1/3 clade and one member from either the BAM2 clade or the BAM4/9 clade, suggesting a potential requirement for pairing of active and regulatory BAMs. Green algae contain at least two BAM genes and these align with BAM1/3 and the regulatory BAM9. The BAM9-like genes may have evolved in two separate events as they are found in green algae and flowering plants but not in seedless plants or gymnosperms. The regulatory BAM2 clade appears to have arisen in non-vascular plants, whereas the regulatory BAM4 clade probably arose in early seed plants. It is interesting that catalytic BAM6 appears to be restricted to the Brassicaceae. These complementary techniques provide a new perspective on how BAM proteins participate in starch metabolism.

200-048-Y Pollen Tube Attractants as Potential Sexual Isolating Barriers in Wild Tomato Species (Solanum Sect. Lycopersicon) Leonie Moyle – Indiana University Cathleen P. Jewell Chemoattraction between male and female gametophytes and gametes is essential for normal sexual reproduction, whereas misrecognition of gamete chemical cues can disrupt interspecific mating events; the resulting post-mating prezygotic (“gametic”) isolation is a potentially powerful, though relatively poorly understood reproductive isolating barrier between species. Here we demonstrate that active pollen tube-ovule recognition occurs within species in the wild tomato clade--consistent with other plant and animal systems--and can also act as a strong postmating, prezygotic isolating barrier between some species. Using an in vitro assay that allows pollen tube-ovule interactions between pollen tubes and ovules to be observed directly, we quantified gamete recognition both within and between four tomato species (Solanum lycopersicum, S. pimpinellifolium, S. arcanum, and S. pennellii). In conspecific assays, growing pollen tubes reorient toward and make contact with conspecific ovules, indicative of an active behavioral reaction by pollen tubes in response to an ovule-secreted chemoattractant. In vitro tests of heterospecific pollen tube-ovule interactions between species that are very closely related (S. lycopersicum and S. pimpinellifolium), indicate equally high recognition and no evidence for isolation. In contrast, in vitro pollen tube-ovule interactions between more distantly related species (diverged < 2 MY) show a reduction in pollen tube recognition of heterospecific ovules, consistent with the evolution of partial isolation. These data indicate that pollen tube-ovule recognition can act as a postmating, prezygotic reproductive isolating barrier in the wild tomato clade, including among species that co-occur and coflower in nature. Analysis of gene expression in mature ovules identifies two cysteine-rich peptides as candidate ovule-secreted chemoattractants, both of which have fixed non-synonymous sequence differences between species showing reduced chemoattraction. These candidates are the basis of ongoing studies to understand the molecular basis and evolutionary dynamics of divergent heterospecific pollen tube-ovule signaling across the wild tomato clade.

200-049-Y Breakdown of Self-incompatibility in Solanum Habrochaites Is Associated with Loss of Function in Pollen Factors ui1.1 and ui6.1 Dragomira Markova – University of California, Davis Jennifer Paterson – Monsanto, Roger Chetelat – University of California, Davis Self-incompatibility (SI) is a widespread genetic mechanism in hermaphroditic plants that allows for the recognition and rejection of closely related pollen to prevent inbreeding. The breakdown of SI to self-compatibility (SC) through mutation occurs frequently, presumably driven by reproductive assurance under conditions where pollen from compatible mates is limiting. While the molecular mechanisms of SI have been well studied, relatively little is known about the specific genes involved in SI to SC transitions, the order in which pistil and pollen SI factors are mutated, or the evolutionary constraints and dynamics governing this process. The objective of the present study was to determine whether SI to SC transitions in normally SI tomato species involved mutations in the pollen factors ui1.1 and/or ui6.1 (Cul1). We hypothesized that pollen mutations would fail to transmit unless pistils were already lacked S-RNase activity, and thus would be rare and most likely to occur only on the geographic margins (i.e. indicating well-established SC populations). We evaluated SC strains of S. habrochaites by crossing onto a pistil tester stock that requires both pollen factors for a compatible reaction. Control crosses onto reference stocks of ui1.1 or ui6.1 were used to assign the mutations to one or both loci. The geographic distribution of pollen mutations discovered S. habrochaites suggests they likely reinforce but do not initiate SI to SC transitions.

200-050-Z Natural Variation of Transactivation Activities of Three Cold Responsible Transcription Factors and Their Correlation with Habitat Temperature in Populations of Arabidopsis Thaliana Yihao Shi – Peking University, Tianshu Sun – Peking University, Juqing Kang – Shaanxi Normal University, Hongya Gu – Peking University Arabidopsis thaliana is a short-lived annual plant flowering in early spring, and is distributed worldwide in diverse geographic regions especially in the Northern Hemisphere. Temperature is one of the major environmental factors which play a fundamental role in plants’ survival and distribution range. There have been many studies in recent years on transcriptional, post-transcriptional and post-translational regulation of genes critical for cold acclimation and tolerance. It has been found that a group of C-repeat binding factors genes (CBF) playing an important role in cold acclimation and tolerance. Our present study indicates that transactivation activity of CBFs differentiate among accessions all over the world. The analysis also shows that both the transactivation activities and expression levels of different CBF3 haplotypes have a significant negative correlation with the average temperature of habitats where the populations were collected. Then, we set up a new index called Integrated Function Parameter (IFP) - the product of the measurements of transactivation activity and expression level. We studied on the correlation between IFP and local temperature among all accessions under study, and found that CBF3 still was the gene being most significantly correlated to habitat temperature. These results indicated that CBF3 may contribute more in Arabidopsis thaliana’s adaptation to local temperature compared with CBF1 and CBF2. It also indicates that three CBFs may be under different selection pressures. The artificial point mutations were made to mimic those observed in CBF3 haplotypes with low transactivation activity. We could find some single amino acid mutation sites led to a significant decrease of transactivation activities compared to those with higher transactivation activities. Moreover, The G53R mutation of CBF3 observed in CQtlx haplotype resulted in the total loss of transactivation activity. It might be due to the introduction of a side chain in R53 which disrupted its folding configuration.

200-051-Z Divergence in Thermotolerance and Transcriptional Activity in the Arabidopsis Thaliana Populations Along the Yangtze River Tianshu Sun – Peking University Yihao Shi – Peking University, Xuefei Wang, Hongya Gu Natural populations of Arabidopsis thaliana are widely distributed in the north hemisphere, most of them within the latitude 30 degrees north (30° N). However, many populations along the Yangtze River, China are south of 30° N, at the southeastern edge of its global distribution range. Thermotolerance tests are designed for these populations by exposing them to a lethally high temperature (45℃, 75 minutes) and measuring survival rate afterwards. Indeed natural populations along the Yangtze River have higher thermotolerance than Col and the populations from populations of northwestern China. Using RNA-seq, we found that the transcriptional responses of these populations were differentiated at genome level under heat shock stress (37℃，3 hours). In total, about 2000-3000 genes are differentially regulated during heat stress, including many transcription factors, among them, 231 upregulated transcription factors that are shared by all populations and only 44 transcription factors are shared by the populations along the Yangtze River. We hypothesized that an expression change of the early transcriptional regulators may be crucial for plant overall thermotolearance and these genes might be responsible for the divergence in thermotolerance between the populations in northwestern China and along the Yangtze River.

Genes & Genomes: Gene Regulation and Molecular Biology 200-052-Y Identification of Proteins Required for Modification of Organelle Transcripts by RNA Editing Xiaowen Shi – Cornell University Maureen Hanson – Cornell University, Stephane Bentolila – Cornell University Post-transcriptional C-to-U RNA editing occurs in chloroplasts and mitochondrial transcripts. This process is essential for proper expression of many chloroplast and mitochondrial genes that have accumulated T to C mutations. Otherwise, these unedited transcripts would produce non-functional proteins that could have a detrimental effect on some critical processes such as photosynthesis and respiration, sometimes leading to lethality. Editing is carried out by a small RNA/protein complex called the editosome, whose composition is beginning to be unraveled. Recognition of the C target is mediated by pentatricopeptide repeat (PPR) motif-containing proteins that specifically recognize a cis-element near the edited nucleotide. Members of the Arabidopsis RNA-editing factor interacting protein (RIP) family and ORRM1 (Organelle RRM protein 1) have been recently identified as essential components of the RNA editing apparatus. ORRM1, a chloroplast editing factor, belongs to a distinct clade of RNA Recognition Motif (RRM)-containing proteins, most of which are predicted to be organelle-targeted. We subjected additional members of the ORRM clade to insertional mutagenesis and virus-induced gene silencing. Extent of editing at mitochondrial C targets in silenced and mutated tissue was compared to wild-type. We report the identification of members of the ORRM family as factors essential for efficient mitochondrial RNA editing. Yeast-two-hybrid assays with these mitochondrial editing factors revealed their interaction with other known components of the RNA editosome. This study reveals a previously unknown role of plant RRM proteins as mitochondrial editing factors. The identification of a new family of mitochondrial editing factors further expands our knowledge of the composition of the editosome. Thus, it paves the way for future genetic engineering of plants through deliberate post-transcriptional modification of gene expression.

200-053-Y The OsSPL16-GW7 Regulatory Module Determines Grain Shape by Changing Cell Division Patterns and Simultaneously Improves Grain Quality and Yield in Rice Xiangdong Fu – Institute of Genetics and Developmental Biology, Chinese Academy of Sciences Shaokui Wang – Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shan Li – Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Qian Liu – Institute of Genetics and Developmental Biology, Chinese Academy of Sciences The deployment of heterosis in the form of hybrid rice varieties has boosted grain yield, but grain quality improvement still remains a challenge. Here we show that a rice grain quality quantitative trait locus qGW7 reflects allelic variation for GW7, a gene encoding a TONNEAU1-recruiting motif protein with similarity to C-terminal motifs of human centrosomal protein CAP350. The up-regulation of GW7 was correlated with the production of more slender grains as results of increased cell division in the longitudinal and decreased cell division in the transverse direction. OsSPL16/GW8, a SBPdomain transcription factor which regulates grain width, binded directly to the GW7 promoter and repressed its expression. The presence of a semidominant GW7TFA allele from tropical japonica rice was associated with higher grain quality without yield penalty imposed by the Basmati gw8 allele. The manipulation of OsSPL16-GW7 module thus represents a novel strategy to simultaneously improve rice yield and grain quality.

200-054-Z Mining Trichome Regulating Genes in Watermelon Using Genome Wide Association Mapping and Functional Genomic Approach Thangasamy Saminathan – West Virginia State University

Abiodun Bodunrin – West Virginia State University, Padma Nimmakayala – West Virginia State University, Umesh Reddy – West Virginia State University Narrow genetic diversity is associated with susceptibility to a large number of diseases and pests in watermelon (Citrullus lanatus). Trichomes, hair-like structures found on leaves and stems, produce, store and secrete large amounts of different secondary metabolites becoming commercially important as natural pesticides. In this study, we took the advantage of whole genome information, availability of single nucleotide polymorphism (SNP) information and trichome variation in 170 accessions collected from America, Africa, Asia and Europe regions. Trichomes from pedicels of four-leaf stage seedlings were photographed using motorized Leica Z16APO microscope with multiple Z-sections. The high quality individual pictures from three replications of each accession were further uploaded into Trichomenet and ImageJ softwares to count trichome density and trichome length respectively in a unit area. These two phenotypic data were further uploaded into SNP & Variation Suite (SVS v8.1.5) for GWA mapping to find out closely associated SNP markers for trichome length and density. SNPs with high p-values and log2>3.0 were only chosen. Of 23,693 SNPs, 11,485 were filtered with a MAF of ≥ 0.01, a call rate of 90%, and facilitated to identify 11,000 SNPs with 0.05 minor allele frequency with the call rate of >85%. The data of both the trichome traits segregated in a normal distribution indicating presence of poly genes controlling these traits. From our study, top 30 SNPs are located in promoter, intron and exon region. SNPs on exon region with nonsynonymous mutations are important. Those identified genes are SCL1 (SCARECROW-LIKE 1), Myb family transcription factor, unfertilized embryo (UNE10), AGO1 (ARGONAUTE 1), etc. Our Scanning electron microscope data showed that, unlike Arabidopsis, watermelon contains both glandular and non-glandular unbranched trichomes. In order to functionally validate, we would like to overexpress watermelon genes in Arabidopsis Landsberg erecta.

200-055-Z Transcriptome-wide Identification of RNA Targets of SR45 Uncovers Its Novel Roles in RNA Processing Denghui Xing – Colorado State University Yajun Wang – Colorado State University, Mike Hamilton – Colorado State Univerisity, Asa Ben-Hur – Colorado State University, Anireddy Reddy – Colorado State University Arabidopsis SR45, an SR-like RNA-binding protein, is involved in regulating multiple developmental processes and stress responses. Genetic, biochemical and cell biological studies indicate that SR45 is a splicing regulator. In vivo and in vitro studies have shown that SR45 interacts with multiple spliceosomal proteins. However, thus far no in vivo RNA targets of SR45 have been identified. To understand the role of SR45 in regulated splicing and other RNA processing events it is crucial to identify its in vivo RNA targets. Using RNA immunoprecipitation followed by sequencing (RIP-seq), we identified >3000 Arabidopsis SR45-associated RNAs (SARs). The sequence analysis of the SARs indicated that at least four RNA motifs mediate the association of SR45 with its targets. Gene ontology analysis of SARs has revealed overrepresentation of genes in abscisic acid (ABA) signaling pathway, suggesting a role of SR45 in ABA response, which was verified experimentally. While most SARs are derived from intron-containing genes, unexpectedly 380 SARs were from intronless genes. Analysis of expression of SARs suggested that SR45 differentially regulates intronless and introncontaining SARs. Our results uncovered an unexpected role of SR45 in the processing of intronless mRNAs and that the expression of ABA signaling genes is massively regulated at the post-splicing level. This is the first study to report genome-wide identification of a spliceosomal RNA binding protein-associated RNAs in plants.

200-056-Y microRNA160 Regulation of Auxin-Cytokinin Balance During Soybean Root Nodule Development Spencer Schreier – South Dakota State University Narasimha Nizampatnam – South Dakota State University, Suresh Damodaran – South Dakota State University, Senthil

Subramanian – South Dakota State University Legume nodules result from coordinated interactions between the plant and nitrogen-fixing rhizobia bacteria. The phytohormone cytokinin promotes nodule formation. Recent findings suggest that the phytohormone auxin can inhibit nodule formation. We show that microRNA160 (miR160) is a key signaling element that regulates auxin-cytokinin balance during nodule development in soybean. miR160 acts by suppressing the levels of ARF10/16/17 family of repressor ARF transcription factors. We identified through qPCR assays and a fluorescence miRNA sensor that miR160 levels are low in emerging nodules and high in mature nodules. Over-expression of miR160 (reduced levels of repressor ARFs) led to enhanced sensitivity to auxin and reduced sensitivity to cytokinin. These roots had significantly reduced nodule formation suggesting that low miR160 levels favor cytokinin activity to promote nodule formation. In support of this hypothesis, exogenous cytokinin rescued nodule formation in miR160 over-expressing roots. Suppression of miR160 levels using a short tandem target mimic (STTM160) resulted in reduced sensitivity to auxin and enhanced sensitivity to cytokinin. In contrast to miR160 over-expressing roots, STTM160 roots had increased nodule formation, but nodule maturation was significantly delayed. This suggested that high miR160 activity favors auxin activity to promote nodule maturation. In agreement, exogenous auxin restored proper nodule formation and maturation in STTM160 roots. Therefore, miR160 dictates appropriate sensitivities to auxin and cytokinin to direct proper nodule formation and maturation. This is the first report of a microRNA dictating appropriate balance between auxin and cytokinin in plants.

200-057-Y An Extensive Small RNA-mediated Regulatory Network in Developing Fibers of Upland Cotton (Gossypium Hirsutum L.) Zhixin Xie – Texas Tech University Arnab Ghosh – Texas Tech University, Forrest Bao – Texas Tech University, Jianyong Wu – Texas Tech University, Xiaopeng Qi – Texas Tech University, Gengxiang Jia – Texas Tech University, Christian Bezboruah – Texas Tech University, Jane Dever – Texas A&M AgriLi Cotton is the most important source of natural spinnable fiber and the top value-added crop. Cotton fibers also serve as a unique single-celled platform for studying cellulose synthesis and cell wall biogenesis. To explore the regulatory role of small RNAs (sRNAs) in fiber development, we took next generation sequencing (NGS) approaches for genome-wide discovery and analysis of sRNAs and their regulatory targets in upland cotton. Extensive profiling by sRNA-seq across a set of twelve tissue samples including developing fibers revealed highly informative spatial and temporal patterns of sRNA expression in cotton. Reliable identification of sRNA regulatory targets was achieved through computational prediction in combination with degradome sequencing. A subset of known plant microRNAs (miRNA) were found in high abundance in both fiber and non-fiber samples. Significantly, several conserved miRNAs and trans-acting siRNAs targeting auxin signaling components were enriched in early stage fibers, which is consistent with an important regulatory role of sRNAs in fiber initiation and elongation. Locus-specific, fiber-enriched miRNAs known to be involved in regulation of lignin biosynthesis were also identified. In addition, multiple 21-nt sRNAs derived from a diverse set of protein coding transcripts were uniquely present in the developing fiber. Intriguingly, novel, lineage-specific miRNAs that are expressed in allotetraploid G. hirsutum and G. barbadense, but absent in the presumed ancestral diploid species G. arboreum and G. raimondii were also identified, including one that appeared to be specifically expressed in developing fiber. These likely represent evolutionary younger miRNAs resulting from the genome merging events occurred during allotetraploid formation within the Gossypium lineage. Finally, the large comprehensive datasets generated in this work provided valuable resources that shall facilitate functional dissection of the extensive sRNAmediated regulatory networks emerged in developing cotton fiber, which shall ultimately contribute to understanding the regulatory innovations occurred during evolution and domestication of cotton.

200-058-Z Application of Short Tandem Target Mimics (STTMs) for Deciphering Functions and Regulatory Networks of MicroRNAs in Crop Plants Sachin Teotia – Michigan Technological University Guiliang Tang – Michigan Technological University microRNAs are small non-coding RNAs that suppress the expression of their complementary target genes at the posttranscriptional level through either cleavage and/or translational inhibition. miRNAs play key roles in controlling plant development and defense against biotic and abiotic stresses. Hundreds of miRNAs have been identified from different plant species and are awaiting functional analysis. Despite underlying importance of regulatory roles of miRNAs, a robust methodical way to study miRNA functions has not been devised due to established redundancy among different members of a miRNA family and lack of tools to effectively knock down miRNA levels. Recently, Short Tandem Target Mimic (STTM) technology, that can target specific miRNAs of interest for destruction without affecting other miRNAs, has been developed. In this study, we have applied STTM technology in a few major crops and generated a large population of STTM miRNA-knockdown transgenic collections both as a resource and materials for functional genomics studies of crop miRNAs. STTM transgenic plants of rice, tomato, soybean and Arabidopsis, targeting some conserved and functionally important miRNAs, are characterized and presented in detail. One such study found that STTM transgenic plants targeting miR165/166 exhibit quite similar phenotype across different species, which includes deformed and curled leaves, short stature and reduced seed set. Deep sequencing analyses of the above STTM transgenic plants targeting different miRNAs is being done. Functional conservation and diversification of miRNA-regulated networks across different species is being studied.

200-059-Z Transcriptional Profiling and Coexpression Analysis of the Chlamydomonas Cell Cycle Ian Blaby – University of California Matt Zones – Donald Danforth Plant Science Center, Sabeeha Merchant – University of California, James Umen – Donald Danforth Plant Science Center Chlamydomonas reinhardtii is a premier organism for studying the molecular genetics of a number of biological processes, including photosynthesis, micronutrient homeostasis and flagella biosynthesis. One particular advantage of the organism is the high degree of culture synchrony that can be achieved when grown phototrophically in a diurnal cycle. We have employed RNA-Seq to analyze gene expression of highly resolved samples taken at 1h or 30min intervals throughout two 12hr light/12hr dark cell cycles. The transcript abundance for over 80% of predicted genes demonstrated diurnal cycling, of which 12,482 loci could be grouped into 6 major clusters and 18 sub-clusters with distinct expression profiles that reveal global temporal progression of gene expression. We have comprehensively analyzed both cluster data and individual gene expression profiles across each of the 56 transcriptomes comprising the dataset. Our data reveal coordinated gene expression patterns of genes encoding subunits of the photosynthetic and mitochondrial electron transport chains, central carbon metabolism, flagella and basal body formation and cell cycle progression. Furthermore, we have leveraged gene co-expression to i) predict pathway participation of enzyme isoforms, ii) identified genes likely to be involved in specific processes by comparing absence of expression in the cell cycle vs. expression in other datasets and iii) recognition of a group of genes whose transcript abundance remains similar in all presently available Chlamydomonas transcriptomes.

200-060-Y The Fate of Non-stop Polyadenylated RNAs in Arabidopsis Laura de Lorenzo – University of Kentucky Arthur G. Hunt – University of Kentucky Posttranscriptional regulation of mRNA plays an important role in gene expression. One mode of control that is accomplished through RNA processing is the alternative polyadenylation (APA). Polyadenylation can occur within 3’UTR, 5’-UTR, introns and open reading frames. Among them, coding region-APA (CDS-APA) is the least understood. Such events should usually lead to transcripts that lack a translation termination codon, and affected mRNAs should be subject to rapid degradation via the nonstop decay mechanism. In Arabidopsis, the paradoxical outcomes of polyadenylation within coding regions raises questions as to if CDS-APA is linked with non-stop decay. In this context, we conducted a study to analyze the stability of CDS-APA-associated RNAs using a genome-scale approach (PAT-seq, Poly(A)-Tag coupled to high-through-sequencing). To explore the genome-wide effects of APA on mRNA stability, we prepared PAT libraries from Arabidopsis plants treated with cordycepin to block transcription. Global poly(A) profiles changes were observed in plants treated with cordycepin. Additionally, a significant number of genes had unstable CDS-isoforms compared with the number of genes with unstable 3’-UTR-isoforms. These results suggest that CDS-APA-associated mRNAs are less stable than mRNAs that end at 3’-UTR poly(A) sites. Regions flanking CDS-APA sites show reduced U content, although within 30 nt there was an A+U enrichment, suggestive of possible cis-elements. The role of CDS-APA is biologically relevant; this is reflected in the observation that genes related to development, biogenesis and transcription are over-represented in the list affected by CDS-APA. These results support the hypothesis that CDS-APA utilization is a mechanism of negative regulation. Motifs associated with increased or decreased stability of alternative isoforms are being investigated. Also, the translation of non-stop mRNAs, and the activity and degradation of the translated proteins, will be studied in the future. These studies will shed light into a novel mode of gene regulation in plants.

200-061-Y Tight, Inducible Regulation of Plant Immune Responses by Combining Promoter and Suicide Exon Elements Tania L. Gonzalez – University of California, Berkeley Yan Liang – Lawrence Berkeley National Laboratory, Bao N. Nguyen – University of California, Berkeley, Brian J. Staskawicz – University of California, Berkeley, Dominique Loque – Lawrence Berkeley Nationational Laboratory, Ming Hammond – University of California, Berkeley Inducible promoters alone are insufficient regulation for many toxic transgenes, resulting in unwanted background phenotypes such as the hypersensitive response (HR) associated with effector-triggered immunity. We have engineered HyP5SM, a plant-derived alternatively spliced cassette which can be inserted directly and tracelessly into a variety of open reading frames to inducibly regulate protein expression in dicot plants. HyP5SM takes advantage of a gene regulation strategy common in nature – alternative splicing coupled to nonsense-mediated decay – to produce “default off, inducible on” gene expression. Here, we demonstrate that HyP5SM can regulate the HR phenotype, a defensive programmed cell death response initiated by disease resistance plants upon detection of specific pathogen effector proteins. We combine the dexamethasone inducible promoter and the HyP5SM cassette exon to regulate pathogen effector proteins. The inducible promoter alone result in leaky effector protein and HR, but HyP5SM renders the leaky transcript non-productive, thus eliminating leaky protein detected by Western blot or leaky HR phenotype. Furthermore, plants inducibly recover both effector protein expression and the HR phenotype. We have tested this with Bs2/AvrBs2and RPP1/ATR1Δ51-dependent hypersensitive response pathways, in Nicotiana benthamiana and Nicotiana tabacum, respectively. We also show that Arabidopsis thaliana plants transgenic for these resistance/effector gene pairs are viable, healthy, and can complete their full life cycle (seed-to-seed), unless the HR immune phenotype is induced with

dexamethasone. The alternatively spliced HyP5SM cassette can be generally applied to regulate other genes in dicot plants, and may be utilized with conditional, constitutive, or native promoters. Currently, we are also engineering new sequence diverse variants of HyP5SM for multi-gene regulation in transgenic plants.

200-062-Z Role of SR45a in Alternative Splicing and Stress Response April Estrada – UNC Charlotte Nowlan Freese – UNC Charlotte, Ann Loraine – UNC Charlotte Most protein-coding genes in higher eukaryotes contain introns, regions of the primary transcript that are removed during pre-mRNA splicing. Thanks to alternative splicing, one gene can produce multiple mRNA species encoding proteins with different but related functions. Alternative splicing is believed to affect more than 20% of all multi-exon genes in plant species. In plants, cold, heat, and water deprivation stresses activate expression and differential alternative splicing of many genes involved in splicing, suggesting that stresses remodel the splicing machinery to enable correct splicing under stress. An alternative view is that remodeling the splicing machinery enables expression of alternative splice forms that are better adapted to stresses. To investigate the role of alternative splicing in stress adaptation, we used RNA-Seq to survey gene expression and splicing in plants undergoing heat or drought stresses. This identified a large number of “super splicers,” genes with highly variable splicing patterns. One such “super splicer” was SR45A, an SR-like protein involved in splicing that produces at least four alternative splicing variants that change in relative abundance under heat stress. To study SR45a function, we characterized an SR45a T-DNA insertion line with reduced expression and altered splicing. Mutants had fewer inflorescence structures and lower first nodes with rosette shaped leaves in place of cauline leaves. Application of daily, low-level heat stress increased severity of the phenotype. These defects suggest the SR45a protein plays a role in meristem function. RNA-Seq analysis of mutant and wild-type plants found that genes with functions related to jasmonic acid, auxin biosynthesis, stress responses, and meristem development were disrupted in the heat-stressed mutant in comparison to wild type controls. These results indicate that SR45a plays a role in both development and stress responses in Arabidopsis.

200-063-Z Atlas of Alternative Polyadenylation in Both Japonica and Indica Rice: Impacts on Developmental Gene Expression Regulations Q. Quinn Li – Xiamen University Haihui Hu – Xiamen University, Dewei Yang – Fujian Acad Ag Sci, Xiaohui Wu – Xiamen University, Guoli Ji – Xiamen University, Liuyin Ma – Xiamen University, Wenyue Su – Xiamen University, Xinfu Ye – Fujian Acad Ag Sci Successful responses of a plant to its environment require precise tuning of gene expression. Alternative polyadenylation (APA), where a gene may use any of its poly(A) sites to produce a defined 3’-end, has been recognized as a regulatory fine tuning mechanism in eukaryotes. To elucidate such a mechanism in important crop rice, we have begun to reveal the whole genome APA atlas in both indica and japonica rice. Using a protocol that we developed to tally the junctions of poly(A) tails and their attached 3’-UTR, poly(A)-tag sequencing (PAT-seq), we compared 14 different tissues/developmental stages in both rice subspecies, including different stages of the root and leaf, and different tissues like flower parts (glume, caryopsis, mature pollen, anther, pistil), embryo, endosperm, and imbibed seeds, distinct profiles of APA have emerged. Some genes have numerous poly(A) sites that are used in different tissue types and developmental stages. Their roles in gene expression can be drawn from their APA patterns, resulting in truncated proteins to altered mRNA length to attract or avoid miRNA targeting etc. Significantly more effective APAs are found during embryo and seed development. Poly(A) sites located in the intergenic regions are also potentially indicative of differential transcription activities. GO analyses revealed that particular groups of genes responsive to environmental

stimuli, biotic and abiotic stresses are more prone to have APA, indicative of their potential functions. Comparison between two subspecies of rice revealed some specific APAs that are distinct for each. The poly(A) site information of the rice genes is also useful to annotate the ends of genes in the rice genome.

200-064-Y A Comparative Study of Plant Responses Under Excess or Deficient Phosphate (Pi) Regime Reveals Novel Ethylene Responsive Players Involved in Excess Phosphate Response (EPiR) Phenotype Devesh Shukla – WKU Claire Rinehart – WKU, Shivendra Sahi – WKU Phosphorus is an essential macronutrient element, but some time causes problems like eutrophication of water bodies if present in excess. Unlike the enormous molecular and morphophysiological information available in plants regarding phosphate (Pi) deficiency (P0), we have a little and fragmented information about the effect of excess Pi on plants, which is indeed essential for its remediation. Here, we have carried out a detailed investigation of plant responses under excess phosphate in a concentration dependent manner. We show that plasticity of root system architecture (RSA) significantly modulated by the varying supply of excess Pi. Excess 20 mM Pi (P20) supply turns RSA shallow, and reduces primary root growth and root apical meristematic activity. Whereas, the length of lateral root and root hair is increased and shoot area is not changed. Although, the developmental programing is different in comparison to P0, because the primary root growth is inhibited indeterminately by P20. Elemental composition analysis shows the events of cross talk with Fe and Zn as the level of these elements is depleted. These characters point towards the ethylene related responses. Furthermore, transcriptomic investigation of excess phosphate response (EPiR), revealed a significant modulation in genes related to ethylene biosynthesis and signaling, Fe deficiency response, and root development. Several ethylene responsive factors (ERF012, ERF013, ERF003, ERF022) and defensin like genes, possessing a gibberellin regulated domain (GASA like) were significantly induced by P20. In addition, relatively a larger number of genes share upregulation by P20 and P0 (interaction pattern: 1, 1), indicating operation of a coherent signaling. Overall, this study dissected the mechanism of plant responses under excess Pi and identified new genes involved in phosphorus homeostasis.

200-065-Y Development and Testing of a New Biosensor for Real-time in Vivo Measurement of the Cytosolic Secondary Messenger cAMP in Plants Hsuan Chou – University of Connecticut 3’-5’-cyclic adenosine monophosphate (cAMP) is a secondary messenger generated from ATP by adenylyl cyclase (AC) in animals. No canonical AC has been yet identified in plants. cAMP levels in plants (measured on a whole-tissue basis) are very low when compared to animals. However,studies suggest that cAMP in plants is functional and associated with signaling. Published data only show plant cAMP level in vitro; this involves tissue destruction and determination of cAMP as an average quantity across the entire volume fo the cell. The pGloSensor™-22F cAMP plasmid is a biosensor that encodes a cAMP binding domain fused to a mutated form of Photinus pyralis luciferase. Upon binding to cAMP, conformational changes occur which reconstitute luciferase structure, and promote large increases in light output (in animal HEK293 cellsused to test the reporter). We have subcloned the coding region of this cAMP reporter plasmid into a plant expression vector for stable expression. In transgenic GloSensor plant, we are able to detect a 4-fold change that lasted for couple seconds when given lipophilic-cAMP and forskolin. Forskolin activates adenylyl cyclase and allows conversion of ATP into cAMP. Lipophilic-cAMP and forskolin-dependent cAMP elevation both occurred in a dosedependent manner. Control (non-transformed) plants showed no response to forskolin of dibutyrl-cAMP. From theses preliminary results, we conclude that (1) the GloSensor™-22F cAMP coding region can be expressed under a 35S

promoter in the Arabidopsis plant and used as a biosensor to measure cytosolic cAMP level change. (2) The biosensor is sensitive enough to detect endogenous cAMP level change, which is very low in Arabidopsis. Once the system is fully established, this new developed tool can be used to test biotic or abiotic stimulus that are likely to cause cAMP level changes in Arabidopsis.

200-066-Z Transcription Profile of Vascular Development in Setaria Viridis, a Model for C4 Panicoid Grasses Julia Lambret-Frotte – UFRJ-DDPSC Marcio Alves-Ferreira – UFRJ, Thomas Brutnell – DDPSC One of the distinguishing features of most C4 plants is a high vein density associated with Kranz anatomy. In A. thaliana, the HDZipIII family of transcription factors has been associated with early steps in vascular patterning. Expression of the HDZipIII family member, AtHB8, is associated with the specification of procambial initials. To explore the process of vascular initiation in a C4 grass, we first conducted a deep phylogentic analysis of the homeobox gene family in the angiosperms including members of S. viridis, maize, rice, P. trichocarpa and A. thaliana. This analysis led to the identification of a monophyletic clade containing AtHB8 orthologs from all monocot species. Expression analysis of the S. viridis ortholog, SvHB8, was performed using in situ hybridization and confirmed the early pattern of procambial-specific expression observed in A. thaliana. An approximately 2 kb region of the promoter was then used to develop a promoter-YFP fusion to follow early steps in vascular patterning in a C4 specie. This construct was introduced into S. viridis and several independently transformed lines are now being characterized. Laser capture microdissection is also being performed to isolate procambial cell files for use in RNAseq analysis. This survey should help to define suites of transcriptional regulators, including SvHB8, and potential targets the drive vascular cell differentiations. A deeper understanding of the factors that drive vascular development may identify avenues for engineering C4 traits into C3 crops including rice and wheat.

200-067-Z Loss of Function of the (E)-ß-caryophyllene Synthase in Most American Maize Lines Is Due to a Nonfunctional Promoter Claudia Schaff – MLU Halle Annett Richter – MLU Halle, Jörg Degenhardt – MLU Halle The production of volatiles, especially terpenes, is essential for the indirect defense of plants. Maize roots attacked belowground by the larvae of the coleopteran Diabrotica virgifera virgifera, emit the insect-induced plant signal, (E)-ßcaryophyllene, which strongly attracts an entomopathogenic nematode. Most European maize lines and their wild ancestor, teosinte, release (E)-ß-caryophyllene in response to this maize pest. However, most North American maize lines are not able to respond to the larvae attack and may have lost this important defense signal during the breeding process. Recently, we identified the terpene synthase 23 (TPS23), which is responsible for the production of the defense compound (E)-ß-caryophyllene. In our effort to study the molecular regulation of tps23 between European and American maize lines, we used Nested Association Mapping (NAM) and Genome Wide Association Study (GWAS). The variation of volatile production of the 5000 inbred lines within the NAM population enabled us to identify a significant QTL which is responsible for the production of the sesquiterpene (E)-ß-caryophyllene. Further calculation using GWAS resulted in a SNP within the proximity of tps23. Comparison of the open reading frames of tps23 from (E)-ß-caryophyllene-producing maize lines and non-producing lines indicated that the QTL does not correspond to the structural gene itself.

Herbivore induced expression analysis of the possible regulatory factors gave no significant conclusion. We therefore focused our investigations on the promoter of the (E)-ß-caryophyllene synthase using a heterologous expression system in Arabidopsis thaliana. The results indicate that the loss of (E)-ß-caryophyllene production after herbivory in most American maize lines is due to sequence alterations within the promoter of the (E)-ß-caryophyllene synthase.

200-068-Y Young Leaf Chlorosis 2 Encodes the Stroma-localized Heme Oxygenase 2 Which Is Required for Normal Tetrapyrrole Biosynthesis in Rice Qingzhu Li – The University of Hong Kong Fuyuan Zhu In plants, heme oxygenases (HOs) are classified into the subfamilies HO1 and HO2. HO1 are highly conserved plastid enzymes required for synthesizing the chromophore in phytochromes which mediate a number of light-regulated responses. However, the physiological and biochemical functions of HO2, which are distantly related to HO1, are not well understood, especially in crop plants. From a population of 60Coγ-irradiated rice mutants, we identified the ylc2 (young leaf chlorosis 2) mutant which displays a chlorosis phenotype in seedlings with substantially reduced chlorophyll content. Normal leaf pigmentation is gradually restored in older plants while newly emerged leaves remain yellow. Transmission electron microscopy further revealed defective chloroplast structures in the ylc2 seedlings. Map-based cloning located the OsYLC2 gene on chromosome 3 and it encodes the OsHO2 protein. The gene identification was confirmed by complementation and T-DNA mutant analyses. Subcellular localization and chloroplast fractionation experiments indicated that OsHO2 resides in the stroma. However, recombinant enzyme assay demonstrated that OsHO2 is not a functional HO enzyme. Analysis of tetrapyrrole metabolites revealed the reduced levels of most chlorophyll and phytochromobilin precursors in the ylc2 mutant. On the other hand, elevated accumulation of 5aminolevulinic acid and Mg-protoporphyrin IX was observed. These unique metabolite changes are accompanied by consistent changes in the expression levels of the corresponding tetrapyrrole biosynthesis genes. Taken together, our work suggests that OsHO2 has a potential regulatory role for tetrapyrrole biosynthesis in rice.

200-069-Y Oligosaccharyltransferase That Contains Different STT3 Subunits Distinctly Influences the Stability of Nglycoprotein in Arabidopsis Thaliana Guanting Niu – Nanjing University Zhi Hong – Nanjing University Asparagine (Asn) linked-glycosylation is one of the most important protein modifications, which is catalyzed in the ER lumen by Oligosaccharyltransferase (OST) via transferring a high-mannose type oligosaccharide to nascent polypeptides in eukaryotic cells. OST is an octomer and in which Stt3p is the most conserved subunit and plays a role in peptide substrate catalysis in yeast. Plant and mammal genomes encode two STT3p homologs, named STT3a and STT3b. Extensive studies have demonstrated that OST with different STT3 isoform had significantly different enzymatic properties in vitro. Defects of Arabidopsis STT3a caused a salt sensitive phenotype, showing swelling root tip growth and more lateral roots. Comparably, no obvious growth aberrant was observed in STT3b knockout mutation. In order to figure out the STT3 isoforms functional difference on plant growth, we conduct the experiments to assess their express pattern and substrate glycan selection. We report here both STT3a and STT3b have similar expression pattern and they can transfer complete and incomplete oligosaccharides to specific substrate polypeptides as well. Underglycosylation caused by STT3a depletion strongly promotes the progress of unfolded protein degradation. Our deep investigation suggested that OST with STT3a or STT3b probably had different tendency on the N-glycosylation sites according to the band shift in immunoblotting. Taken together, our results suggest that STT3 subunits can be partially redundant but

distinctly modulate the degree of N-glycosylation to ensure the stability of protein. Assays for the further molecular mechanisms are in progress.

200-070-Z Three R2R3-MYB Transcription Factors Regulate Distinct Floral Pigmentation Patterning in Phalaenopsis Spp Chia-Chi Hsu – Nationa Cheng Kung University You-Yi Chen, Wen-Chieh Tsai, Wen-Huei Chen, Hong-Hwa Chen Orchidaceae are well known for their fascinating floral morphologic features, specialized pollination, and distinctive ecological strategies. With their long-lasting flowers of various colors and pigmentation patterning, Phalaenopsis spp. have become important ornamental plants worldwide. In this study, we identified three R2R3-MYB transcription factors, PeMYB2, PeMYB11, and PeMYB12. Their expression profiles were concomitant with red color formation in the flowers of Phalaenopsis spp. Transient assay of overexpression of the three PeMYBs verified that PeMYB2 resulted in anthocyanin accumulation, and these PeMYBs could activate the expression of three downstream structural genes, PeF3H5, PeDFR1, and PeANS3. In addition, these three PeMYBs participated in the distinct pigmentation patterning in a single flower, as revealed by virus-induced gene silencing. In the sepals/petals, silencing of PeMYB2, PeMYB11, and PeMYB12 resulted in the loss of the full-red pigmentation, red spots and venation patterns, respectively. Moreover, different pigmentation patterning was regulated by PeMYBs in the sepals/petals and lip. PeMYB11 was responsive to the red spots in the callus of the lip and PeMYB12 participated in the full pigmentation in the central lobe of the lip. The differential pigmentation patterning was validated by RNA in situ hybridisation and in six cultivars with different color patterns. The combined expression of these three PeMYBs in different ratios leads to a wealth of complicated floral pigmentation patterning in Phalaenopsis spp..

200-071-Z Identification of a Sucrose Responsive Cis Element in the MYB Transcription Factor, PAP1 (Production of Anthocyanin Pigment-1), in Arabidopsis Thaliana Ruth Watson – Colorado State University Alyssa McKenzie – Colorado State University, Bettina Broeckling – Colorado State University, Daniel Bush – Colorado State University The MYB transcription factor, PAP1 (Production of Anthocyanin Pigment-1), is known to be up-regulated in response to sucrose. We previously identified a 90 bp region necessary for sucrose induction of PAP1, that when deleted abolished the sucrose response. To determine whether this 90bp region was sufficient for a sucrose response we generated transgenic Arabidopsis plants with 1-3 copies of the 90 bp region upstream of the CaMV35S minimal promoter and luciferase reporter gene. Additionally, in order to determine if the spatial context of the 90 bp region was important we cloned 1-3 copies downstream of the luciferase reporter gene. Transgenic seedlings were analyzed for sucrose response by treating with water or 90mM sucrose and measuring luciferase activity from plant extracts. Our results from the luciferase assays showed that the 90bp sequence was sufficient for a sucrose response, the response was additive, and that the spatial context of the sequence was important. Furthermore, we used a bioinformatics approach to identify two potential cis elements within this 90 bp sequence. Twenty-six genes in Arabidopsis were identified as having one or both of these potential elements. RT-PCR was used to determine if there was a change in gene expression in these genes in response to sucrose treatment in Col-0 seedlings. Unfortunately, RT-PCR did not show a significant sucrose response in any of the 26 identified genes. Future work is focused on further defining the sucrose response element and identification of transcription factors that bind to the element.

200-072-Y Activation of the TOR Pathway via Tap46 in Plant Growth Enhancement in Arabidopsis Hee-Kyung Ahn – Yonsei University Chang Sook Ahn, Hee-Kyung Ahn, Hyun-Sook Pai Tap46 is one of the major regulatory subunits of protein phosphatase 2A (PP2A), mediating signals from the Target of Rapamycin (TOR) pathway to plant growth and development. We have generated overexpression lines of Tap46 and elucidated the molecular mechanism of its phenotype. Overexpressin of Tap46 in Arabidopsis under the regulation of 35S CaMV promoter resulted in overall growth stimulation with larger organs. Tap46 overexpresion also led to increased seed size and viability. Growth stimulation was also observed in inducible Tap46 overexpression Arabidopsis lines. Leaf enlargement was mostly due to increased cell sizes, revealed by kinematic analysis. Nitrate-assimiliating enzyme activies were enhanced in these Tap46 overexpression lines. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and coimmunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity.

200-073-Y ATL9, an Arabidopsis RING Zinc Finger Protein, Is Involved in Basal Resistance to Fungal Pathogens Tingwei Guo – Biological Sciences, University of Alabama Fengyan Deng – College of Life Sciences, Northwest A&F University, Katrina Ramonell – Biological Sciences, University of Alabama Our research using the model plant Arabidopsis thaliana is focused on unraveling the signal transduction pathways involved in elicitor-mediated plant defense, particularly defense pathways involved in resistance to fungal pathogens. We have isolated a group of related genes, the ATL family, which appear to play a direct role in defense against fungal pathogens. Research in our lab has shown that ATL proteins can be induced by chitin and they are involved in basal resistance to fungal pathogens. ATL9, an Arabidopsis RING zinc finger protein, is an E3 ubiquitin ligase that can be induced by chitin and is involved in basal resistance to the biotrophic fungal pathogen, Golovinomyces cichoracearum (G. cichoracearum). In order to understand the expression and regulation of ATL9, we studied the expression pattern of ATL9 and the functions of its different protein domains. Using a pATL9:GUS transgenic Arabidopsis line we found that ATL9 is expressed in different tissues in Arabidopsis at various developmental stages and that GUS activity was induced rapidly upon wounding. Previous research in our lab also showed that ATL9 is a short lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. Protein prediction software indicated that ATL9 contains two transmembrane domains (TM), a RING zinc-finger domain, and a PEST domain. Data from confocal microscopy and western analysis indicate that both the PEST domain and the RING domain have effects on ATL9 degradation. To study the importance of these domains in ATL9’s function, we constructed a series of deletion mutants and generated transgenic Arabidopsis plants. As expected, transgenic Arabidopsis containing the deletion constructs showed that both the RING and TM domains are important to its resistance phenotype against G. cichoracearum. Interestingly, the PEST domain was also shown to be significant for resistance to fungal pathogens.

200-074-Z Increased Photosynthetic Activity and Starch Metabolism in the Allopolyploid Arabidopsis Suecica Jacie Ihinger – University of Puget Sound Erik Solhaug – University of Puget Sound, Andreas Madlung – University of Puget Sound Allopolyploidization is an event in which the offspring of hybridized species have two or more genomes. Arabidopsis suecica is one such allopolyploid that arose from one hybridization event between A. arenosa and A. thaliana 12,000 300,000 years ago. This hybrid offspring displays increased fitness and vigor. Previous studies in our lab have examined this hybrid vigor phenomenon and found that the allopolyploid offspring demonstrates a higher rate of photosynthesis and increased production of starch under high light conditions when compared to its parent species. Quantitative realtime PCR (qPCR) was performed to examine the gene expression of photosynthesis and starch degradation genes between A. suecica and its parent species to determine if the phenotypic differences observed and increased plant vigor can be explained by transcriptomic changes. The differences found could help our understanding of hybrid vigor in allopolyploids.

200-075-Z Annotation of Root and Haustorial Small RNAs from the Parasitic Flowering Plant Triphysaria Versicolor Saima Shahid – Pennsylvania State University Loren Honaas – Pennsylvania State University, Eric Wafula – Pennsylvania State University, Claude Depamphilis – Pennsylvania State University, Michael Axtell – Pennsylvania State University Triphysaria versicolor (Yellow owl’s clover) is a facultative root-parasitic plant with a broad host range of more than 27 families of flowering plants, including economically important crops such as Zea mays, Medicago truncatula and even model species such as Arabidopsis thaliana. Triphysaria belongs to a larger family of parasitic flowering plants known as Orobanchaceae, which is characterized by formation of haustorium (modified root structure) upon recognition of host. The haustorium plays a significant role in establishment and maintenance of host-parasite relationship as it acts as a physiological bridge for water and nutrient uptake from host plant. Furthermore, haustorium-facilitated bi-directional movement of transgenic siRNAs (short interfering RNAs) between Triphysaria and its hosts has been previously reported. In this study, we analyzed high-throughput small RNA-seq data (four libraries from Triphysaria roots and two libraries from haustoria attached to host M. truncatula) in an attempt to annotate endogenous small RNAs in a parasitic plant species for the first time. In order to improve identification and annotation quality of parasitic plant-specific small RNAs, we utilized unassembled genomic DNA reads as well as a comprehensive transcriptome assembly from Triphysaria as reference sequences. Our de novo small RNA discovery analysis using ShortStack revealed 9 conserved as well as 33 Triphysaria-specific novel microRNA genes. We also identified thousands of siRNA-like small RNA clusters in both unassembled genomic reads and annotated transcriptome of Triphysaria. Evidence of such expressed siRNA-like genes in the Triphysaria haustoria suggests potential involvement of RNA-silencing pathways in host-parasite interaction. In summary, these results provide a valuable resource for further characterization of small RNA pathways in parasitic flowering plants.

200-076-Y The Overexpression of OsNAC9 Alters Root Architecture of Rice Plants Enhancing Drought Resistance and Grain Yield Under Field Conditions Mark Christian Felipe Redillas – Seoul National University Jin Seo Jeong, Youn Shic Kim, Harin Jung, Seung Woon Bang, Yang Do Choi, Sun-Hwa Ha, Christophe Reuzeau, Ju-Kon Kim Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice

NAC domain family. Root-specific (RCc3) and constitutive (GOS2) promoters were used to overexpress OsNAC9. Field evaluations showed grain yields of RCc3:OsNAC9 and GOS2:OsNAC9 plants were increased by 13-18% and 13-32% under normal conditions, respectively. Under drought, RCc3:OsNAC9 plants showed an increased grain yield of 28-72%, while GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The root aerenchyma in RCc3:OsNAC9 were enlarged to a greater extent than in GOS2:OsNAC9 and wild type suggesting its importance in drought tolerance. Microarray experiments identified 40 genes up-regulated in roots of transgenic lines that included 9-cis-epoxycarotenoid dioxygenase, an ABA biosynthesis gene; calcium-transporting ATPase, a component of the Ca2+ signaling pathway involved in cortical cell death and aerenchyma formation; cinnamoyl CoA reductase 1, agene involved in lignin biosynthesis; and wall associated kinases¸ genes involved in cell elongation and morphogenesis. Also, o-methyltransferase, a gene necessary for barrier formation, was upregulated only in the RCc3:OsNAC9 roots. Such target genes regulated in roots of OsNAC9 transgenic plants may account for the altered root architecture conferring drought tolerance to plants.

200-077-Y The Regulation of Gene Expression by PSEUDO-RESPONSE REGULATORS Eva M Farre Farre – Michigan State University Tiffany Liu – Michigan State University, Linsey Newton – Michigan State University Circadian regulated pseudo-response regulators (PRRs) are key components of the plant circadian clock. They are transcription regulators that play partially redundant roles not only in the maintenance of circadian rhythms but also in the regulation of several physiological processes. We have identified PRR9 target genes using chromatin inmunopredicipitation and compared the binding sites among PRR9, PRR7, PRR5 and TOC1 (TIMING OF CAB EXPRESSION 1)/PRR1. PRR9 and PRR7 contained the highest overlap of target genes and TOC1 shared the fewest number of binding sites with other PRRs. We observed that PRR binding sites correlated with low nucleosome occupancy and DNaseI hypersensitive sites, indicating that they encompass regulatory regions. G-box like motives were enriched in the PRR binding regions and we showed that in the PRR9 and CCA1 promoters these motives are necessary for mediating regulation by PRRs.

200-078-Z Quantitative Amplification of Cleaved Ends (qACE) to Assay MiRNA-directed Target Cleavage Sen Subramanian – South Dakota State University Suresh Damodaran, Sajag Adhikari microRNA (miRNA) regulation is crucial to achieve precise spatio-temporal expression patterns of their target genes. This makes it crucial to determine the levels of cleavage of a particular target mRNA in different tissues and under different conditions. We developed a quantitative PCR method “quantitative Amplification of Cleaved Ends (qACE)” to assay levels of specific cleavage products in order to determine the extent of miRNA regulation for a specific target gene. qACE uses cDNA generated from adapter-ligated RNA molecules and relies on a carefully designed fusion primer that spans the adapter-cleaved RNA junction in qPCR to specifically amplify and quantify cleaved products. The levels of full-length transcripts can also be assayed in the same cDNA preparation using primers that span across the miRNA cleavage site. We used qACE to demonstrate that soybean roots over-expressing miR164 had increased levels of target cleavage and that miRNA deficient Arabidopsis thaliana hen1-1 mutants had reduced levels of target cleavage. We used qACE to discover that different levels of cleavage by miR164 in nodule vs. adjacent root tissue contributed to nodule-specific expression of NAC1 transcription factors in soybean. These experiments show that qACE can be used to discover and demonstrate differential cleavage by miRNAs to achieve specific spatio-temporal expression of target genes in plants.

200-079-Z Development of Marker-free Transgenic Rice Expressing Wheat Storage Protein, Glu-Dx5 Soo Kwon Park – National Institute of Crop Science, Rural Development Administration So Hyeon Baek – National Institute of Crop Science, Rural Development Administration, Dool Yi Kim – National Institute of Crop Science, Rural Development Administration, Myoung Ryoul Park – National Institute of Crop Science, Rural Development Administration, Na Ra Lee – National Institute of Crop Science, Rural Development Administration, Jung Kying Moon – National Institute of Crop Science, Rural Development Administration, Kyong Soon Shin – National Institute of Crop Science, Rural Development Administration, Su Kyong Jeon – National Institute of Crop Science, Rural Development Administration, Eun Jae Kim – National Institute of Crop Science, Rural Development Administration, Sun Lim Kim – National Institute of Crop Science, Rural Development Administration Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present study, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated cotransformation method. The Dx5’s own promoter was used for protein expression. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 270 hygromycin-resistant transformants, we obtained 27 transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 gene into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Protein expression of the Dx5 was analyzed with Western blot using polyclonal antibody recognising x-type of glutenin subunits in T1 seeds. It was suggested that the protein-processing system was conserved between rice and wheat. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation.

200-080-Y Transcritional Analysis of Cysteine Proteases in Soybean Root Nodules Experiencing Premature Senescence Due to Water Deficit Magdeleen Cilliers – University of Pretoria Riekert van Heerden – South African Sugarcane Research Institute (SASRI), Barend Vorster – University of Pretoria Cysteine proteases found in soybean’s (Glycine Max) crown nodules, are involved in premature senescence due to water deficit conditions. Water deficit stress in soybean affects plant growth and development as well as the lifespan and nitrogen fixation ability of these root nodules. The expression of papain-like and legumain-like genes have been investigated under different water stress conditions ranging from 60%, 40% and 30% vermiculite water content respectively. The severity of the stress conditions were evaluated by measuring leaf and nodule water potential indicating a significant difference in the water content of these two organs at 30% vermiculite water content. A transcriptome analysis, which was validated by quantitative PCR, was used as a gene discovery technique. Twenty nine papain-like cysteine proteases were found to be expressed in crown nodule tissue. Five of these papain-like proteases showed to be induced by water deficit over the increasing stress treatments and seven showed an increase in expression until 40% vermiculite water content but showed a decrease in expression from 40% to 30%. There were also two papainlike proteases that were only expressed in plants affected by water deficit conditions. Nine of the above mentioned proteases showed a 2-fold and higher increase in expression from control plants to water deficit stressed plants. Eight legumain-like proteases were expressed in the root nodules. Legumain-like cysteine proteases showed to be induced by senescence as well as water deficit stress. Compared to natural senescence, the expression of legumain-like cysteine

proteases in water deficit conditions did not show a fold change of above 2 over the different treatments as seen in natural senescence. While legumain-like cysteine proteases involvement during premature senescence is clear, they don’t seem to be the main protease group responsible for the initiation of premature senescence.

200-081-Y Regulation of Class III Homeodomain Leucine Zipper Transcriptional Factor During Soybean Nodule Development Suresh Damodaran – South Dakota State University Senthil Subramanian – South Dakota State University Class III homeodomain leucine zipper (HD-ZIP III) proteins are a group of plant-specific transcriptional factors (TF) that play key roles in plant development. Spatio temporal activity of HD-ZIP III proteins is tightly regulated by posttranscriptional and post-translational mechanism to achieve proper plant development. While these genes are expressed in soybean nodules, their functional roles or how their activity is regulated are not known. We examined posttranscriptional and post-translational regulation of two GmHD-ZIP IIIs (GmHD-ZIPIII-1 & GmHD-ZIPIII-2) during soybean nodule development. The mRNAs of both these HD-ZIP IIIs were found to be regulated by miR166 based on RLM-5’RACE assays. Analysis of a miR166 sensor using confocal microscopy of mature nodule tissues indicated that miR166 might limit the expression of HD-ZIP III to the ‘base’ of the nodules in the nodule parenchyma. Interestingly over-expression of miR166 using CvMv promoter showed reduction in nodule formation. Experiments are in progress to determine the effect of suppressing endogenous miR166 using short tandem target mimic. HD-ZIP III protein activity is modulated through hetero-dimerization with the small leucine zipper protein, ZPR. GmZPR3d was identified to have highest interaction levels with GmHD-ZIP III-1 & 2 based on yeast 2 hybrid assays. Analysis of GmZPR3d promoter:tdTomato indicated that the gene was expressed in the nodule parenchyma closer to the infection zone in mature nodules. However, over- or mis-expression of GmZPR3d did not show any visible phenotype in nodule development. Experiments are in progress to determine the effect of suppression of GmZPR3d expression using RNAi. Our results indicate that HDZIP IIIs are actively regulated by post-transcriptional and post-translational mechanisms during soybean nodule development. Determining the roles of these proteins and spatio temporal regulation of their activity will promote our understanding of symbiotic nodule development.

200-082-Z OR Confers High Level of Carotenoid Accumulation via Regulating Phytoene Synthase and Chromoplast Biogenesis Hui Yuan – Cornell University Katherine Owsiany, T E Sheeja, Yongxi Li, Xiangjun Zhou, Ralf Welsch, Caroline Rodriguez, Noam Chayut, Yong Yang, Theodore Thannhauser, Mandayam Parthasarathy, Qiang Xu, Xiuxin Deng, Zhangjun Fei, Ari Schaffer, Nurit Katzir, Joseph Burger, Yaakov Tadmor, Li Li Carotenoids are a diverse group of pigments widely distributed in nature and play critical roles in plant development and human health. Despite significant progress in our understanding of carotenogenesis in plants, the overall control mechanisms remain largely unknown. Our previous studies identify an Orange (OR) gene as a pivotal regulator of carotenoid accumulation in both cauliflower and melon. OR is ubiquitous and highly conserved among plant species. Here we demonstrate that alteration of a single amino acid in a wild type OR changes its functionality to gain a greatly enhanced ability in promoting carotenoid accumulation in Arabidopsis. Expression of the OR variants (ORMUT) does not greatly affect carotenogenic gene expression as well as plant normal growth. ORMUT like wild type OR protein was found to physically interact with phytoene synthase (PSY) in plastids to posttranscriptionally regulate PSY protein level. Moreover, ORMUT specifically promotes the differentiation of membranous chromoplasts in the Arabidopsis calli, which

shared similar structures as those found in the orange curd cauliflower mutant. The unique ability of ORMUT in mediating chromoplast biogenesis along with its capacity in regulating PSY is responsible for the ORMUT-induced massive carotenoid accumulation. Our findings draw insights into the mechanisms underlying OR regulated carotenoid accumulation and demonstrate the effectiveness to generate functional OR as powerful genetic tools for carotenoid enrichment in any crop species.

200-083-Z Seabuckthorn Transcriptome: Elucidating Putative Genes Involved in Carotenoid Biosynthesis Parul Jain – Saskatoon Research Centre, Agriculture and Agri-Food Canada Isobel Parkin – Saskatoon Research Centre, Agriculture and Agri-Food Canada, Raju Soolanayakanahally – Saskatoon Research Centre, Agriculture and Agri-Food Canada, William Schroeder – Agroforestry Development Centre Seabuckthorn (Hippophae spp.), indigenous to Eurasia is a member of the Elaeagnaceae family. It is a hardy, woody, dioecious shrub bearing yellow to orange berries rich in total antioxidants, flavonoids, amino acids, fatty acids and vitamins (C, A, E, and K). It has been used for therapeutic and pharmaceutical purposes for centuries in Europe and Asia. Seabuckthorn was introduced to the Canadian prairies from Siberia and used as a shelterbelt species to control soil erosion from the 1970s. Since then, it has adapted and integrated well into prairie agriculture systems via farm diversification with small fruit production in shelterbelts and increased interest in wildlife habitat planting. Carotenoid content and profile in berries of Saskatchewan grown species was found to be rich in β-carotene and lycopene. Given its medicinal, nutritional and environmental importance, very limited tissue specific genome and transcriptome information is available in public databases to date. In an ongoing study to understand the molecular mechanism associated with biosynthesis of carotenoids, we obtained more than 170 million paired-end sequence reads from RNA extracted from pulp and skin from different developmental stages of seabuckthorn berries using the Illumina platform. De novo assembly, comparative transcript profiling and qRT-PCR are being used to identify and confirm differentially expressed genes of potential relevance in regulating carotenoid content during berry development. This study will provide significant resources for breeding strategies to establish seabuckthorn as new high value agriculture crop in a rapidly emerging functional food and nutraceutical industry in Canada.

200-084-Y Functional Characterization of the Flowering Time Genes VERNALIZATION1 and VERNALIZATION2 in the Temperate Grass Brachypodium Distachyon Reveals Similarities and Differences Compared to the Crown Pooids Wheat and Barley Yinxin Dong – UW- Madison, Northwest A&F University Daniel Woods – UW- Madison, Richard Amasino – UW- Madison In temperate climates, flowering is often coordinated with seasonal environmental cues such as temperature and photoperiod. Vernalization provides plants the competency to flower only after exposure to winter thus preventing flowering from occurring prior to winter. In wheat and barley, three genes VERNALIZATON 1 (VRN1), VERNALIZATION 2 (VRN2) and FT form a regulatory loop that is critical for flowering. Prior to cold, VRN2 represses FT; however, during cold VRN1 expression increases repressing VRN2, thus allowing FT to be activated by the long day photoperiod pathway to begin the transition from vegetative to reproductive growth. To date this regulatory loop and the functional characterization of VRN1, VRN2 and FT have not been extensively studied outside of the crown pooids. Here we utilized gene knock down and overexpression of these critical genes in Brachypodium to test if this regulatory loop is conserved more broadly in pooid grasses. We found that the overall functions of these genes are conserved (FT and VRN1 promote flowering and VRN2 represses flowering); however, the specifics of the feedback loop are not conserved. For example,

knockdown of VRN1 does not effect the expression of VRN2 before, during or after cold exposure, which is not consistent with the model from wheat and barley. Thus the VRN1-VRN2 part of the regulatory loop is not conserved throughout pooids and thus some aspects of the vernalization regulatory system evolved appear to have evolved specifically in the crown pooids.

200-085-Y The PRC2 Histone Methyltransferase CURLY LEAF Is Required for Epigenetic Silencing of VRN1 in Brachypodium Distachyon Aaron Lomax – University of Wisconsin – Madison Daniel Woods – University of Wisconsin – Madison, Richard Amasino – University of Wisconsin – Madison, Yinxin Dong – University of Wisconsin – Madison To ensure proper timing of flowering many plants require prolonged exposure to cold to acquire the competence to flower. The process by which cold exposure results in competence is known as vernalization. After vernalization is complete, many species also require the lengthening photoperiods of the spring season to flower. In Arabidopsis thaliana vernalization leads to the stable repression of the floral repressor FLOWERING LOCUS C (FLC) by an increase of trimethylation on lysine 27 of histone H3 (H3K27me3) at FLC by the Polycomb Repression Complex (PRC2). Conversely, vernalization in pooids is associated with the stable induction of a floral promoter, VERNALIZATION 1 (VRN1). To further explore flowering pathways in pooids, we performed forward genetic screens for rapid flowering plants in Brachypodium distachyon. We have identified recessive alleles of CURLY LEAF (CLF), a histone methyltransferase in PRC2, and show that CLF is required for the proper regulation of flowering. Plants lacking CLF flower extremely early and are smaller in stature compared to wild-type plants. We show that non-vernalized clf mutants have increased expression of VRN1 and reduced levels of H3K27me3 at VRN1 compared to wild-type plants. Moreover, vernalization leads to the reduction of H3K27me3 at VRN1 in wild-type plants while remaining unchanged in clf plants. Interestingly, loss of VRN1 expression in clf mutants does not suppress the early-flowering phenotypes indicating that other misexpressed genes are likely to be the cause of the early-flowering phenotype. This suggests that PRC2 is required for the maintenance of the epigenetic off state of VRN1 prior to vernalization.

200-086-Z The RNA Structurome and Abiotic Stress Response Sarah Assmann – Penn State University Zhao Su – Penn State University, Laura Ritchey – Penn State University, Yin Tang – Penn State University, Philip Bevilacqua – Penn State University RNA molecules can exhibit complex and dynamic secondary and tertiary structures that are integral to the regulatory mechanisms involved in the control of gene expression. It has been well-documented for individual RNAs that refolding can occur in response to changes in the prevailing physico-chemical environment. Until recently, however, methods were not available to globally assess RNA structures in vivo. We developed a method, Structure-seq1, which couples chemical probing of RNA structure with next-generation sequencing, allowing assessment of RNA structure on a transcriptome-wide scale in living tissue. Application of this method to etiolated Arabidopsis thaliana seedlings yielded information at the individual nucleotide level on structures of over 10,000 mRNAs. Our meta-analysis of the Arabidopsis RNA structurome suggests that mRNAs encoding proteins involved in basic biological functions tend to have more stable structures. Conversely, mRNAs of proteins involved in abiotic stress response exhibit characteristics related to structural plasticity. We hypothesize that these mRNAs may change their fold in response to environmental signals, with

consequent effects on function or stability. We are currently working toward applying Structure-seq to the major crop species, Oryza sativa (rice), which will allow further tests of this hypothesis. 1Ding, Y., Tang, Y., Kwok, C-K, Zhang, Y., Bevilacqua, P.C.,Assmann, S.M. (2014) In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features.Nature. doi: 10.1038/nature12756.

200-087-Z Characterizing Arabidopsis Mutants Lacking Specific Non-Coding RNAs Michael Yuhas – Wilkes University Laura Bauman – Wilkes University, Bryant Morocho – Wilkes University, Catherin Morocho – Wilkes University, Jephte Akakpo – WIlkes University, Xing Wang Deng – Yale University, William Terzaghi – Wilkes University Non-coding RNAs are biologically-functional RNAs which do not encode proteins. Surprisingly, 98% of the human genome that is transcribed into RNA does not encode proteins (ncRNA), and it has been found that ncRNAs participate in many fundamental processes such as regulation of transcription and translation. In collaboration with the labs of Dr. Xing-Wang Deng at Yale and Peking Universities, we have studied the functions of ncRNA 50 to 300 nt long. Dr. Deng provided us with the coordinates of ncRNAs in this size range identified by RNA-seq, and we screened the T-DNA Express database for insertions within 200 bp of the DNA encoding these ncRNAs. We identified nearly 500 DNA insertions in or near loci encoding novel ncRNA, and focused on 33 that affected loci encoding ncRNA that were more than 1000 bp from the nearest annotated protein-coding gene. We ordered seeds for these lines, and studied their phenotypes. Here we report the phenotypes observed for two of these mutant lines. Mutant line 121071 had more biomass and increased anthocyanin concentrations in comparison to wild type when grown on 3 and 5% glucose. Mutant line 024279 had delayed growth but eventually grew much larger than wild type. We have also observed numerous changes in germination and morphology which we are presently quantifying.

200-088-Y Exploring Translational Landscape of Arabidopsis Using Ribosome Profiling Polly Hsu – Duke University Larry Wu – North Carolina State University, Lorenzo Calviello – Max Delbrück Center, Uwe Ohler – Max Delbrück Center, Philip Benfey – Duke University Translation is the final step of the central dogma, yet little is known about how and to what extent gene expression is controlled in translational level. We exploit ribosome profiling or RiboSeq (deep sequencing of ribosome footprints) to explore translational landscape of Arabidopsis shoots and roots. In parallel, we performed an RNAseq from the same samples to dissect gene regulation in transcription and translation levels. Compared to the RNAseq reads, the RiboSeq reads are enriched in coding regions (CDS) as expected, while sparse in untranslated regions (UTRs). In addition, RiboSeq reads demonstrate a remarkable three-nucleotide periodicity, consistent with movement of ribosomes shifting one codon each time. Surprisingly, our RiboSeq reads are also mapped to genomic features that are annotated as non-coding RNAs, suggesting these transcripts might function as a repository of small peptides, which were recently reported in yeast, Drosophilla and zebrafish. We are currently investigating whether these novel proteins (peptides) are stably present in vivo using mass spectrometry analysis. Our data will provide insight into gene regulatory features in translation levels, help improve genome annotations and uncover novel proteins in Arabidopsis.

200-089-Y Light Regulation of Arabidopsis PIRIN1 Indicates Plant and Human Pirin functions Yang Chen – University of Illinois at Chicago

Carlos Montero – University of Illinois at Chicago, Benjamin Okkema – University of Illinois at Chicago, Danielle OrozcoNunnelly – University of Illinois at Chicago, DurreShahwar Muhammad – University of Illinois at Chicago, Ashley Williams – University of Illinois at Chicago, Thomas Ronan – University of Illinois at Chicago, Katherine Warpeha – University of Illinois at Chicago Pirins are cupin-fold proteins that are highly conserved, from prokaryotes to humans. Pirins were identified as transcriptional activators, and possess endogenous quercetinase activity (cleaves quercetin), although in plants they have received little study. Our lab recently published findings which indicate that Pirin1 (PRN1) in Arabidopsis thaliana is critical for seedlings to orient and respond to light in the seed-to-seedling transition. The pool of flavonoids in the seedling is regulated by PRN1 activity, and is tissue specific. Based on genetic studies of mutant and transgenic (complementation and overexpressors) seedlings, PRN1 directs organ and overall hypocotyl growth of the young seedling. Human pirin (hPir) was transformed into prn1 Arabidopsis mutants. We evaluated plant-specific and non-plant specific responses / activities for ability of hPir to rescue defects of prn1. We report that some plant specific light responses can be partially rescued by hPir, revealing information of how PRN1 may function in plants.

200-090-Z ADT Family Regulation in the Seed to Seedling Transition of Arabidopsis Thaliana Huini Wu – University of Illinois at Chicago Yang Chen – University of Illinois at Chicago, DurreShahwar Muhammad – University of Illinois at Chicago, Bethany Huot – Michigan State University, Jennifer Baek – University of Illinois at Chicago, Sookyung Oh – Michigan State University, Beronda Montgomery – Michigan State University, Katherine Warpeha – University of Illinois at Chicago Phenylalanine (Phe) is an amino acid used in a variety of plant compounds, including proteins and phenylpropanoid secondary metabolites. The seed-to-seedling transition is dependent on organic seed reserves and changes in the external environment into which the seedling emerges, including available nutrients and light. We investigated the impact of arogenate dehydratases (ADT) on photoregulation and hormone-dependent aspects of germination and seedling establishment in Arabidopsis thaliana. The ADT gene family has profound impacts on the root and shoot development, indicated by specific ADT genes. adt3, adt5, and adt6 mutants have distinct cotyledon expansion, hypocotyl growth, and root development responses to light conditions, indicating that ADT3, ADT5, and ADT6 may be important in seedling establishment. Tissue-specific and light-dependent expression of ADT genes and disruptions in the regulation of hypocotyl elongation in monochromatic light conditions also support an interaction between photoreceptors and the regulation of Phe pools during seedling establishment, confirmed by transgenic expression studies. The germination and de-etiolation phenotypes of adt mutants indicate that ADT5 and ADT6 in particular play important roles in coordinating phenylalanine supply in response to hormone and light signals in early seedling development.

200-091-Z The Cause of Epinastic Cotyledons of OsEMF2 Transgenic Arabidopsis Dhondup Lhamo – UC Berkeley Mao-Sen Liu – Institute of Plant and Microbial Biology, Li Pu – UC Berkeley, L.-F. Chen – Institute of Plant and Microbial Biology, Zinmay Sung – UC Berkeley In Arabidopsis, EMBRYONIC FLOWER2 (EMF2) is required for the development of vegetative leaves by repressing flowering genes. Homozygous emf2 mutants skipped rosette development and exhibited abnormal seedling phenotypes, such as short hypocotyl, and oval-shaped, petioleless cotyledons. These mutant phenotypes were rescued by the introduction of a rice EMF2 gene (OsEMF2) expressed under the CAMV’s 35S promoter. However, OsEMF2 transgenic

Arabidopsis showed severe epinasty, in which the cotyledons folded 180° under themselves. The previous studies showed excessive stomata formation, and modified stomatal and vein patterns, revealing several candidate genes that regulate auxin homeostasis. The present study evaluates the expression pattern of auxin receptor genes such as TIR1, AFB 1, 2, and 3, and auxin biosynthetic genes such as YUC1 and 2 using quantitative RT-PCR. The results indicate altered gene expression. By targeting these genes with Auxinole, the potent inhibitor of TIR1/AFB, the epinastic cotyledons of OsEMF2 transgenic Arabidopsis are rescued to normal cotyledons. Also, a slight change in endogenous auxin concentration in the transgenic plants is observed using liquid chromatography-mass spectrometry.

200-092-Y Studies of CAF1 and CCR4 Families in Oryza Sativa Chung-An Lu – National Central University Wei-Lun Chou, Jhen-Cheng Fang, Yue-Lin Chung Deadenylation, also called poly(A) tail shortening, is the first rate-limiting step in the general cytoplasmic mRNA degradation in eukaryotic cells. In yeast and human, the CCR4-NOT complex containing two key components, CCR4 and CAF1, is a major player in deadenylation. However, little is known for their major roles in rice (Oryza sativa). In our study, we found that the rice genome contains a number of CAF1 homologs, but only two CCR4 homologs, OsCCR4a and OsCCR4b. Both OsCCR4a and OsCCR4b lack the LRR domain which is required for interaction with CAF1 in yeast and human. We demonstrated that the most of OsCAF1s are able to interact with OsCCR4a and OsCCR4b. Consideration of OsCAF1s and OsCCR4s in deadenylase activity, expression patterns and subcellular localizations, the functional redundancies among OsCAF1s and OsCCR4s may be extensive, but individual OsCAF1s and OsCCR4s may play specific roles in rice.

200-093-Y Characterizing lncRNA Precursors of PhasiRNAs in Maize Anthers Sandra Mathioni – University of Delaware Atul Kakrana – University of Delaware, Parth Patel – University of Delaware, Reza Hammond – University of Delaware, Blake Meyers – University of Delaware Plant long noncoding RNAs (lncRNAs) are involved in various roles in a wide range of biological processes. In maize anthers, a set of lncRNAs is processed into two classes of phased, secondary siRNAs (phasiRNAs), the pre-meiotic (21-nt) and meiotic (24-nt) phasiRNAs. Both classes are highly enriched in anthers. However, little is known about the structure of the precursor transcripts or genomic loci that are transcribed into the lncRNAs precursors (PHAS loci) of phasiRNAs. In order to investigate the PHAS loci in maize anthers, we took advantage of the long read cDNA sequencing from a PacBio instrument, and we sequenced the transcriptome of 1.5 mm anthers, a stage with a high abundance of 24-nt meiotic phasiRNAs. The cDNA transcripts were fractionated into four size ranges (0.8-2.0, 2.0-3.0, 3.0-5.0, and >5.0 Kb) and SMRTbell libraries were prepared. PacBio sequencing of 16 SMRT cells rendered approximately 530,000 full-length nonchimeric reads. Further analysis are underway of transcripts, structure of PHAS precursors and their isoforms, and those data will be presented.

200-094-Z Analysis of Pollen-specific Alternative Splicing in Arabidopsis Thaliana via RNA-seq and the Integrated Genome Browser Nowlan Freese – University of North Carolina at Charlotte April Estrada – University of North Carolina at Charlotte, David Norris – University of North Carolina at Charlotte, Mason

Meyer – University of North Carolina at Charlotte, Tarun Mall – University of North Carolina at Charlotte, Maryam Ishka – University of Nevada, Jeffrey Harper – University of Nevada, Ann Loraine – University of North Carolina at Charlotte Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In plants, at least 20% of multi-exon genes are alternatively spliced. These differences in splicing can have important functional consequences during stress response and development. However, the extent and significance of tissue-specific splicing is not well understood, partly due to difficulty in isolating cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts. To better understand the differences in alternative splicing between various tissues, we compared RNA-seq data from Arabidopsis thaliana pollen and leaves using the Integrated Genome Browser, a freely available program designed for deep analysis of individual genes as well as genome-scale visualization of splicing and gene expression. We identified approximately 30 genes with differential abundance of splice site use between pollen and leaves. We found that for most genes, the details of splicing were rarely perfectly captured by large-scale computational analysis. Instead, deeper understanding required interactive visualization of each gene individually by using the Integrated Genome Browser. Results are freely available from http://www.bitbucket.org/lorainelab/pollenas.

200-095-Z RNA-seq Analysis of Cytokinin-regulated Gene Expression in Rice Ivory Blakley – UNC-Charlotte Tracy Raines, Yu-Chang Tsai, Jennifer Conrad, Jose Franco-Zorrilla, Roberto Solano, G. Eric Schaller, Joseph Kieber, Ann Loraine The plant hormone cytokinin activates transcriptional cascades important for development and the response to biotic and abiotic stresses. Most of what is known regarding cytokinin-regulated gene expression comes from studies of the model dicotyledonous plant Arabidopsis thaliana. To expand the understanding of the cytokinin-regulated transcriptome, we analyzed gene expression in response to cytokinin in roots and shoots of the monocotyledonous plant rice. We identified over 4,700 and 2,400 genes differentially expressed (DE) in response to cytokinin in roots and shoots respectively, with more than 600 DE in both tissues. There were many similarities to cytokinin transcriptome studies in Arabidopsis, including an up-regulation of genes that act to dampen cytokinin function (e.g. type-A RRs, cytokinin oxidases, cytokinin glucosyl transferases). Using a protein-binding microarray, we identified OsRR22 binding sites and found that these sites were over-represented in the promoter regions of up regulated genes and under-represented among down regulated genes, indicating that, similar to its Arabidopsis counterpart ARR10, OsRR22 is primarily a positive regulator of cytokinin-regulated transcription. One substantial difference in the response of rice to cytokinin is a much more prominent regulation of genes involved in the response to biotic stress, including the induction of a large number of WRKY transcription factors. Consistent with this, there is a substantial overlap in the genes regulated by cytokinin and those differentially expressed in response to pathogen infection and BTH (an SA agonist) treatment in rice, suggesting that cytokinin plays an integral role in gene regulation in response to pathogens in rice.

200-096-Y New Insights into Mycorrhizal Interactions Enabled by ChIP-Seq and SWATH Proteomics Geetika Trivedi – University of Alabama in Huntsville Leland Cseke – University of Alabama in Huntsville, Avinash Sreedasyam – HudsonAlpha Institute of Biotechnology, Stephen Barnes – University of Alabama at Birmingham, Helen Kim – University of Alabama at Birmingham, Landon Wilson – University of Alabama at Birmingham, Peter Larsen – Argonne National Laboratory, Sarah Zerbs – Argonne National Laboratory, Frank Collart – Argonne National Laboratory, Geetika Trivedi – University of Alabama

Our lab has been a pioneer and innovator in developing and applying protein based methods to build a predictive understanding of the molecular mechanisms underlying symbiotic plant-fungal associations called ectomycorrhiza (ECM). Such interactions play an essential part in the regulation of soil nutrient cycling and subsequent carbon management in a wide range of plant species. However, the complexity and variation of such systems in natural environments has hindered the accurate assessment of the factors that signal, establish and maintain these interactions. Consequently, our project employs a controlled laboratory Populus tremuloides X Laccaria bicolor ECM model system to explore nutrient utilization and carbon management under nitrogen limiting conditions. RNA sequencing (RNA-Seq) and ChIP sequencing (ChIP-Seq) were employed to produce global transcript abundance data along with increased resolution on transcriptional regulation. ChIP-Seq analyses of eight transcription factors, (TFs) having roles in mycorrhiza-specific carbon and nitrogen metabolism has lead to the identification of new TF targets as well as novel motifs in the promoter regions of these target genes. As transcript expression levels do not always correlate well with protein expression levels, we have also developed quantitative nanoLC-ESI-SWATH-MS to fill the gap in our understanding of how protein concentrations correlate to transcript levels. The SWATH-MS proteomics technology successfully detected and quantified the differential regulation of thousands of proteins under nitrogen limiting conditions during interaction with Laccaria. Thus, poplar-specific ChIP-Seq and SWATH-MS technologies have been successfully implemented to identify novel biochemical and regulatory pathways that also implicate specific protein expression and post-translational modifications as playing important roles in the molecular mechanisms of how plants, including trees, respond to changes in environmental nutrient conditions. Integration of this multi-level information will lead to development of gene regulatory networks and system-scale models that are predictive of the molecular mechanisms that control plant carbon management and allocation.

200-097-Y Investigating Roles for the Tomato 14-3-3 Gene Family in Effector-Triggered Immunity Through the Immune Receptor Tm2-2 Carina Sandoval– California State University Fullerton Jennifer Spencer – California State University Fullerton, Soha Sobhanian – California State University Fullerton, Melanie Sacco – California State University Fullerton Effector-triggered immunity (ETI) is a specific form of plant immunity involving recognition of pathogen effectors by host immune receptors. ETI often results in a type of programmed cell death known as the hypersensitive response (HR). 143-3 proteins are conserved throughout eukaryotes and have functional diversity, including roles in defense against pathogens. Several 14-3-3 isoforms have been shown to interact with immune receptors and other proteins to regulate their functions in signaling pathways. Solanum lycopersicum (tomato) possesses twelve 14-3-3 gene family members; however, potential roles for most 14-3-3 family members in plant immunity remain unknown. We are interested in understanding the contributions of different 14-3-3 gene family members to ETI, primarily through the S. lycopersicum immune receptor Tm2-2, which confers durable resistance against the Tomato mosaic virus (ToMV). Tm2-2 mediates HR through recognition of the 30kDa movement protein (MP) from the Tobamoviruses ToMV and Tobacco mosaic virus (TMV). Co-immunoprecipitation suggested an interaction between the Tm2-2 coiled-coil domain and the Nicotiana benthamiana homolog of tobacco NtFTTa-1 (Nb14-3-3a-1). To investigate the functional role of 14-3-3 proteins in immune receptor-mediated HR, we have performed co-expression assays in N. benthamiana by agroinfiltration to express Tm2-2 and MP, or other immune receptor-effector combinations, either with 14-3-3 constructs or in the presence of AICAR, a known inhibitor of 14-3-3s. HR induction was inhibited by AICAR treatment and by over-expression of one 14-3-3 isoform, while over-expression of three other 14-3-3 isoforms enhanced HR. Mutagenesis of selected 143-3 proteins to produce dominant-negative constructs reversed the phenotypes seen for expression of the corresponding wild-type 14-3-3 proteins. Similar assays are currently being performed using autoactive immune

receptor constructs. These studies could determine whether 14-3-3s enhance or inhibit immune receptor activation during recognition of the effector or downstream during signaling.

200-098-Z GNRF Suppresses Floral Transition and Secondary Wall Synthesis in Brachypodium Distachyon Samuel Hazen – University of Massachusetts Pubudu Handakumbura – University of Massachusetts, Kathryn Brow – University of Massachusetts, Sandra RomeroGamboa – University of Massachusetts, Scott Lee – University of Massachusetts, Ian Whitney – University of Massachusetts, Kangmei Zhao – University of Oklahoma, Laura Bartley – University of Oklahoma,, Henry Preast – The Donald Danforth Plant Science Center, Todd Mockler – The Donald Danforth Plant Science Center, Marion Dalmais – INRA, Eddy Blondet – INRA Several NAC transcription factors have been shown to play crucial roles in secondary cell wall biosynthesis and overall plant growth and development. Brachypodium distachyon GRASS NAC REPRESSOR OF FLOWERING (GNRF) was selected for functional characterization as it is highly expressed in stems, mirroring an expression pattern similar to that of characterized secondary cell wall regulators such as Arabidopsis thaliana NST1, SND1 and SND2. Mutants (GNRF-OE) were generated by constitutively over expressing GNRF full-length coding region under the maize ubiquitin promoter. A homozygous mutant allele (gnrf-1) harboring a non-synonymous point mutation was isolated from a TILLING mutant collection. Surprisingly, over-expression mutants failed to flower and could not transition into the reproductive stage. Conversely the gnrf-1 mutants flowered significantly earlier than control plants. Transcription profiling using a microarray revealed a fifty-fold reduction in two MADS flowering time pathway genes, consistent with the persistent vegetative growth phenotype. Three genes involved in cellulose biosynthesis namely BdCESA4/7/8 and two genes associated with lignin biosynthesis namely, BdCAD1 and BdCOMT4 and a gene with a predicted role in xylan biosynthesis BdGT47-1 were analyzed for changes in transcript abundance. In most cases, these genes were significantly downregulated in GNRF-OE and up-regulated in gnrf-1 stems. Chemical and histological analysis of stems revealed reduced lignin content compared to the controls where as no significant change was observed between the gnrf-1 and control samples. Taken together, these data suggest that GNRF functions as a pleiotropic repressor regulating cell walls and flowering in B. distachyon.

200-099-Z Shining Light on the Role of Conserved CCT-domain Proteins in Flowering in Sorghum Bicolor Manon Doucet – Centenary College of Louisiana Daryl Morishige – Texas A&M University, John Mullet – Texas A&M University, Rebecca Murphy – Centenary College of Louisiana One of the major goals of plant science today is to translate the information gleaned from model systems into crop systems in an applied way. The expression of CCT domain proteins, like CONSTANS and PRR37, is one of the major determinants of flowering time, and flowering time in turn is an important characteristic that can be modulated to optimize grain, sugar, or biomass production in Sorghum bicolor and other crop plants. Therefore, understanding the most basic molecular switch that controls reproductive transition is an important part of optimizing crop production. The mechanism by which these proteins contribute to reproductive growth is not well understood, though it has been demonstrated in Arabidopsis that proteins in this family may interact to form a complex that regulates transcription for important flowering genes. To gain initial understanding of how these proteins may operate in crop plants, analysis of protein-protein interactions between important CCT domain proteins was performed for proteins in Sorghum bicolor using an in vitro translation system to express select tagged CCT-proteins for protein interaction assays.

200-100-Y Analysis of Concurrently Expressed CCT-domain Proteins During the Floral Transition in Sorghum Bicolor Melissa Traver – Centenary College of Louisiana Andrea Lemus – Centenary College of Louisiana, Morgan Navalance – Centenary College of Louisiana, Daryl Morishige – Texas A&M University, John Mullet – Texas A&M University, Rebecca Murphy – Centenary College of Louisiana The transition to flowering requires the precise coordination between external stimuli and internal signals. Once this switch has been triggered, the meristem architecture must be rearranged to produce reproductive organs. This process, from the point at which initial floral signals are received until flowering commences, is accompanied by changes in gene expression. In Sorghum bicolor, much of this change is facilitated by CCT-domain proteins, like PRR37, Ghd7, and CONSTANS. These types of proteins have been shown to assemble into heterotrimeric complexes on distal enhancer elements in Arabidopsis, and influence chromatin structure to regulate gene expression. Additionally, the CCT-domain protein family contains many members, allowing for an immense amount of variation in the composition of these complexes. To gain understanding about how these proteins may be acting in crop plants, the comparative expression studies have been performed in Sorghum bicolor to allow insight into which CCT-proteins are co-expressed, and therefore available for complex formation at certain points in the floral transition.

200-101-Y Gene Association Network Approach to Investigate Xylan Biosynthesis and Function in Developing Rice Endosperm Yuli Hu – Ohio University Nan Jiang, Honglei Zhang, Ahmed Faik Xylans are the most abundant plant hemicellulosic polysaccharides on Earth and play an important role in plant growth and the integrity of plant cell walls. They have a variety of applications that answer our well being and needs in food, feed, and energy. All xylans have a β-(1,4)-linked d-xylose (Xyl) backbone that can be substituted with α-(1,2)-dglucuronic acid (GlcA) residues and/or l-arabinofuranosyl (Araf) residues at C-2 and/or C-3 positions of Xyl residues. Despite the importance of xylans and their well-studied structures, their biosynthetic pathways and regulation are not well understood in grasses. Using wheat as a model system, we succeeded in purifying and characterizing a xylan synthase complex (named TaXPol-1). The central core of this complex is formed of two glycosyltransferases (GTs) from the GT43 and GT47 families, and is sufficient and necessary for Xyl and GlcA incorporation into xylan-like products. In this work, we are combining the advances gained in xylan synthesis in wheat with gene association network (GAN) approach in rice endosperm to discover new genes involved in the process and the regulatory genes that control xylan synthetic process. A GAN has two linked entities: nodes and edges. While nodes consist of 10 genes that may encode for proteins of the same multi-protein complex or enzymes catalyzing different steps of the same metabolic pathway, the edges between nodes represent regulatory interactions including protein-protein and DNA-protein interactions. This GAN includes several GTs, transcription factors, and and nucleotide sugar transporters (NSTs). We are currently focusing on validating protein-protein interactions, subcellular localization, and enzyme activity of rice GT proteins. We will use a genetic approach to investigate the physiological role of xylan synthesis during rice endosperm development.

200-102-Z Over 10,000 New Maize Mutants Added to the UniformMu Public Resource: Now 67,000 Total Mu Insertions with 42% Genome Coverage Jonathan Saunders – University of Florida Charles Hunter, Shan Wu, Masa Suzuki, Maria Angélica Sanclemente, Peng Liu, Wayne Avigne, Carson Andorf, Marty

Sachs, Don McCarty, Karen Koch Over 10,000 new mutants have been added to the UniformMu reverse genetics resource in release 7, bringing the total to over 67,000 germinal transposon insertions. These are available in 11,140 independent seed stocks. Close to half of the maize filtered gene set (42%) is represented by at least one Mutator insertion allele, and 65% of these genes have two or more mutant alleles. UniformMu insertion lines can be searched by gene sequence or by GRMZM maize-gene identifiers, and can also be browsed at MaizeGDB.org. Seeds can be requested free of charge through this site or directly from the Maize Genetics Cooperation Stock Center. The UniformMu population was created by introgressing a Mutator transposase into a W22 inbred background to provide a uniform phenotypic background for mutant comparisons. Each UniformMu line carries an average of 10 unique, germinal insertions. These lines are provided as segregating F3 material in a “Mu-off”, stable state with a mutable bronze1 allele that serves as a visual marker for activity of the MuDR transposase. Methods and tips for users are available at MaizeGDB.org, including information on how to request seed stocks and effectively test genotype-phenotype cosegregation.

200-103-Z Knockdown of Arabidopsis CTD-phosphatase-like 4 Causes 3’-extension of snRNA and Alters Shoot and Root Organogenesis Capabilities Akihito Fukudome – Texas A&M Univerisity Emre Aksoy – Texas A&M University, In Sil Jeong – Texas A&M University, Hisashi Koiwa – Texas A&M University Phosphoregulation on carboxy-terminal domain of RNA polymerase II (pol II-CTD) governs transcription of both proteincoding and non-protein coding genes. CTD phosphatases in animal and yeast have been studied extensively, but characterization of those in plants is still in its infancy. In Arabidopsis thaliana, CTD phosphatase-like 4 (CPL4) is the major CTD phosphatase that interacts with and dephosphorylates pol II. In the present study, we show that knockdown of CPL4 (CPL4RNAi) results in 3’-extension of snRNA transcripts in A. thaliana. RT-qPCR analysis revealed that upregulation of AT1G61280, the highest up-regulated gene in the CPL4RNAi microarray, is coupled with 3’-extension from upstream AT1G61275, which encodes U12 snRNA. The subsequent RNAseq analysis identified that many pol II-dependent snRNAs such as U1, U2 and U4 exhibit 3’-extension in the CPL4RNAi, indicating that CPL4 regulates 3’-end formation of snRNA transcripts. CPL4RNAi lines over-accumulate U1 snRNA and show enhanced GT-AG splicing in some transcripts. It has been shown in Arabidopsis that transcriptional regulation of snRNA and spliceosome activity is important for development and organogenesis. Consistently, root explants of CPL4RNAi lines exhibit more vigorous shoot organogenesis than wild-type root explants in vitro. The CPL4RNAi but not wild-type root explants can regenerate shoot in the presence of lower concentration of cytokinin. Also, CPL4RNAi lines show abnormal lateral root primordia development upon induction by exogenous IBA. These results imply that CPL4 affects hormonal regulation of development and organogenesis through 3’-end formation of snRNA transcripts.

Genes & Genomes: Bioinformatics 200-104-Y How to Scale Data, Science, and People Using Biological Cyberinfrastructure Jason Williams – iPlant/CSHL The iPlant – iPlant Collaborative At all levels of scientific investigation, valuable opportunities are realized by having the ability to scale questions; singlegene investigations and individual phenotypes are enriched by scaling to explore genomic and environmental contexts. Students, researchers, and educators also enjoy benefits when they can operate along a continuum that allows them to

scale collaborations and more effectively connect. In the context of biology, cyberinfrastructure facilitates these possibilities by scaling data, science, and people. This talk highlights cyberinfrastructure (CI) developed by the iPlant Collaborative and features research examples (such as analysis of high-throughput sequence data in RNA-Seq) that leverage CI to address problems of size and scope that would otherwise be intractable to address. Attendees new to iPlant will get an overview of available resources and familiar users will enjoy previews of new and upcoming functionalities. Importantly, we will highlight how increased community-building efforts will give users greater abilities shape and customize functionalities in ways that are specific to shared research objectives. The iPlant Collaborative (www.iplantcollaborative.org) develops a comprehensive cyberinfrastructure for the storage, sharing, and analyses of large datasets – from genomes to phenotype data, and beyond. iPlant offers easy-to-use tools that cover a variety of genotype-phenotype related analyses (e.g. genome assembly, annotation, RNA-Seq, GWAS, image analysis, etc.) in a platform that accommodates every level of user - from “bench-biologist” to bioinformatician. Computational resources include generous storage allocations as well as access to high-performance and cloud computing. iPlant platforms are extensible and customizable via application programming interfaces (APIs), RESTful services, and web-based systems for data access, tool integration, and analysis. Training and online learning materials make collaboration and people central to the CI. Funded by the National Science Foundation (#DBI-0735191), iPlant is driven by and freely available to the community.

200-105-Y Genetic Diversity Analysis of Gossypium Arboreum Using Genotyping-by-Sequencing Ruijuan Li – USDA-ARS John E. Erpelding – USDA-ARS The Gossypium arboreum germplasm collection is an important source of resistance to reniform nematode (Rotylenchulus reniformis), which is a serious root pathogen of upland cotton in the United States. With the aim of introgressing reniform nematode resistance from G. arboreum germplasm lines into upland cotton, knowledge of the genetic diversity of the G. arboreum collection is necessary. In this study, genotyping-by-sequencing was conducted. More than 18,000 unique single nucleotide polymorphic (SNP) sequences were captured from 373 G. arboreum accessions. Population structure and phylogenetic analyses were performed on further filtered SNP data to evaluate genetic diversity. Result obtained from this study will aid the utilization of the G. aroboreum collection in detection of quantitative trait loci associated with reniform nematode resistance and other genome wide association studies.

200-106-Z New Insights on Modification of Cell Walls of Bioenergy Grasses from Global Gene Expression During Maize Stem Development Bryan Penning – Purdue University Jacob Shreve – Agronomy Department, Purdue University, Chris Dugard – Department of Botany and Plant Pathology, Purdue University, John Klimek – Department of Botany and Plant Pathology, Purdue University, Tania Shiga – Department of Botany and Plant Pathology, Purdue University, Phillip San Miguel – Purdue University, Jyothi Thimmapuram – Purdue University, Nicholas Carpita – Purdue University, Maureen McCann – Purdue University We documented global gene expression in rind tissues of maize stem during internode development to obtain a comprehensive inventory of the genes involved in secondary wall synthesis in grass species. Through a collection of eighteen samplings from early elongation, biomass accumulation, and lignification stages, we established co-expression

patterns of members of gene families involved in secondary wall development in grasses. In comparison with genes previously implicated in secondary wall development in the dicot Arabidopsis, only one-third of the genes have putative orthologous relationships. In many instances the closest maize homologs of the Arabidopsis genes were not the gene family members expressed during secondary wall deposition, indicating substantial neofunctionalization in maize. Although the same gene family members were expressed in both inbreds B73 and Mo17, we found substantial variation in the level of expression, with 30% of the entire genome exhibiting at least a 5-fold difference at any stage of stem development. Using de novo promoter sequence analysis combined with hierarchical clustering, we found several novel promoter motifs common to genes co-expressed, revealing several new candidate genes linked to secondary wall formation. The results of this study, when combined with genome-wide association studies in populations that represent the majority of maize genetic variation, will provide useful target genes for modification of cell-wall compositional and architectural traits more suitable for conversion of grass feedstocks to biofuels and bioproducts. Supported by the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DESC0000997

200-107-Z Identification of Aberrant Arabidopsis Brassinosteroids Receptors BRI1 Recognized by Endoplasmic Reticulum Quality Control Tianshu Chen – Nanjing University Jian Zhang – Nanjing University, Yi Cao – Nanjing University , Zhi Hong – Nanjing University In eukaryotes, endoplasmic reticulum quality control (ERQC) system which consists of various chaperones and lectins, exquisitely regulates and scrutinizes protein folding to ensure the functional quality of the end protein products, with the recognition of the unfolded peptide/misfolded protein as the initially important step. Although the components of the ERQC recognition system have been widely studied, the basic information about the general recognition motif of ERQC on the misfolded proteins still remains exclusive. Defects of ERQC components usually led to mouse embryonic lethality, making it difficult to conduct such research in vivo. The cloning and functional analysis of Arabidopsis brassinosteroids receptor bri1-9 suppressor genes uncovered a similar ERQC system in plant cell and bri1-9 is a structurally imperfect but functionally competent BRI1 variant recognized by ERQC. Recently, we found that bri1-9 had a lower thermodynamic stability than that of the plasma-membrane-localized BRI1 variants in a numerical investigation, suggesting the energetic variation might be an ERQC recognition factor. In the current study, we took BRI1 as a model protein, employing biophysical computation and biochemical assay, to investigate the features of the misfolded proteins recognized by the plant ERQC and its relevance to the thermodynamic changes of proteins.

200-108-Y MaizeGDB and 85 Years of Collaborative Research Ethalinda Cannon – Iowa State University In 1930, the first edition of the Maize Newsletter was printed by Ralph Emerson and George Beadle. Its purpose was to inform the nascent maize research community about stocks, genetic maps, naming standards, meetings. In 1991, what had started as a single sheet of paper became an Internet resource, MaizeDB, through the foresight of Ed Coe. In 2003, MaizeGDB was established, combining MaizeDB’s genetic data with molecular and genomic data that was rapidly

becoming vitally important. This presentation will look at the history of data sharing within the maize community, its current state, and will consider the data challenges facing all plant researchers now and in the near future.

200-109-Y Araport, the Community-Extensible Portal for Arabidopsis Data Integration Jason Miller – J. Craig Venter Institute Sergio Contrino – Cambridge Systems Biology Centre, Chia-Yi Cheng – J. Craig Venter Institute, Rion Dooley – Texas Advanced Computing Center, Erik Ferlanti – J. Craig Venter Institute, Matthew Hanlon – Texas Advanced Computing Center, Maria Kim – J. Craig Venter Institute, Vivek Krishnakumar – J. Craig Venter Institute, Stephen Mock – Texas Advanced Computing Center, Walter Moreira – Texas Advanced Computing Center, Benjamin Rosen – USDA, Seth Schobel – J. Craig Venter Institute, Joseph Stubbs – Texas Advances Computing Center, Julie Sullivan – Cambridge Systems Biology Centre, Jason Miller – J. Craig Venter Institute, Matthew Vaughn – Texas Advanced Computing Center, Chris Town – J. Craig Venter Institute The Arabidopsis Information Portal (Araport) is an online resource for the plant genomics community. Centered on Arabidopsis thaliana, the portal integrates gene annotation and related data with the high-quality reference genome sequence. The portal hosts several web applications including JBrowse and ThaleMine. The latter is an instance of the InterMine data mining tool that integrates diverse data types and supports sophisticated query and analysis tools as well as data-rich report pages on individual genes and proteins. Araport apps combine data from TAIR, UniProt, PubMed, BAR, EPIC-CoGe, IntAct, Atted II, 1001 Genomes, and other sources. Designed for sustainable growth, Araport makes substantial use of third-party web services to query remote databases and integrate results on demand. Araport exposes its own data via web services supported by documentation and a shared vocabulary. Some of its web services re-expose others to provide on-the-fly data integration or data transformations that increase utility. Araport hosts small applications, or “science apps”, that consume web services and expose visual or computational analysis. Araport invites the community to contribute additional web services and science apps. The portal exposes its internal developer tools to community developers and also provides tutorials, training, and consulting. Araport offers community developers easy access to automatic indexing, caching, security, logging, scaling, styling, and documentation generation. All software is open-source. Araport provides a model for sustainable growth of model organism resources with reliance on federated data, real-time integration, an open architecture, and community-contributed software.

200-110-Z The Federated Plant Database Initiative for the Legumes Steven Cannon – USDA-ARS The "Legume Federation" is an NSF project to foster data standards, distributed development, and comparative analysis, via gene families and shared phenotypes, to support research across the legume family – and to support robust agriculture for a world that is significantly "legume-fed." Participating Genomic Data Portals (GDPs) currently include, but are not limited to MedicagoGenome (http://medicagogenome.org), SoyBase (http://soybase.org), PeanutBase (http://peanutbase.org), the Legume Information System (http://legumeinfo.org), KnowPulse (http://knowpulse.usask.ca), and the Cool Season Food Legume Database (http://www.coolseasonfoodlegume.org). The goals of the Legume Federation include 1) sharing knowledge, development efforts, and data sets across all legume crops; 2) defining standards for data formats, metadata, Web service protocols and ontology use; 3) establishing an open repository for data exchange; 4) encouraging the use of common, open-source model organism database tools. Clear standards and formats, with templates and tools for data collection and submission, should enable broader participation. Although a major focus of the project is on methods for distributed development, we emphasize that the fundamental mission is to enable improved agricultural productivity for this important group of crop plants by

integrating genetic, genomic, and phenotypic data across species to enable identification of common molecular bases for important traits.

200-111-Z TAIR Sets Down New Roots Tanya Berardini – The Arabidopsis Information Resource, Phoenix Bioinformatics Donghui Li, Leonore Reiser, Bob Muller, Eva Huala, Yarik Mezheritsky, Emily Strait, Qian Li, Andrey Vetushko The Arabidopsis Information Resource, in its new home at the non-profit Phoenix Bioinformatics, continues to provide the plant biology research community with up-to-date functional information about Arabidopsis genes gleaned from the continually growing, rich research literature. We will give an overview of the different features of the resource that take advantage of the extracted experimental data, specifically from the viewpoint of the non-Arabidopsis researcher looking for information about this valuable model organism and how that pertains to their organism of interest. There will also be a brief discussion of the curation process, giving researchers insight into how the data in TAIR is organized, interconnected, and maintained.

200-112-Y Evolutionary Relationship of Trichothecene Production in the Hypocreales Fungi Sean O’Mara – University of Minnesota Kathryn Bushley – University of Minnesota The Hypocreales is a diverse order of fungi which includes: Fusarium graminearum, which produces the trichothecene deoxynivalenol (DON) and is the causative agent of Fusarium Head Blight (FHB) in wheat; Claviceps purpurea, which produces ergotamine and subsequently the syndrome St. Anthony’s Fire; and Beauveria bassiana, an arthropod parasite and bio-control agent. The present research aims to understand the evolutionary relationship of trichothecene production in the Hypocreales. Computational and laboratory methods are being employed to analyze the existence of trichothecene producing genes in various members of this order. Preliminary results suggest the existence of the genetic tools necessary for trichothecene production in fungi not known to produce these compounds.

200-113-Y A Smorgasbord of Apps from the BAR for Araport Sylva Donaldson – University of Toronto Asher Pasha, Nicholas Provart The Bio-Analytic Resource (BAR), a widely used plant bioinformatics resource, has partnered with Araport to contribute science apps to this growing online community resource. In 2013 the BAR was funded by Genome Canada to contribute seven data and visualization apps to Araport by building on highly used BAR tools. Currently, both the eFP Browser and the Arabidopsis Interaction Viewer are available on Araport. We are working towards making new tools available shortly. A new version of our zoomable user interface (ZUI) will allow the seamless viewing of large data sets across several levels of biological organization. Users can upload their own data to be viewed in the ZUI, or can view data that is available at Araport or from the BAR. User testing at plant conferences has contributed greatly to the development of this ZUI. Gene Slider, a new sequence logo-based data visualization tool will allow the user to easily observe 90,000 conserved non-coding regions across 10 species from the Brassicaceae.

200-114-Z Highly Dynamic Expansions of Antimicrobial Loci Among Medicago Truncatula Accessions Previously Missed by Short-read Assemblies Are Revealed by Inclusion of SMRT Sequencing Kevin Silverstein – University of Minneosta Jason Miller – J. Craig Venter Institute, Peng Zhou – University of Minneosta, Joann Mudge – National Center for Genome Resources, Thiru Ramaraj – National Center for Genome Resources, Brian Walenz – National Biodefense Analysis and Countermeasures Center, Peer Tiffin – University of Minnesota, Nevin Young – University of Minnesota, Kevin Silverstein – University of Minnesota In order to deconvolute signatures of selection in multi-copy gene families within populations, highly contiguous genome assemblies are needed. This is due to the fact that many large receptor and secreted peptide family genes involved in adaptation appear in tandem and segmentally duplicated physical clusters of 100-250 Kb. We have developed a novel hybrid assembly pipeline called ALPACA that utilizes both Illumina and PacBio sequencing technologies to create contiguous, accurate assemblies with contig N50s in the 100-250 Kb range. We have applied this algorithm to assemble three accessions of the model legume, Medicago truncatula. Genome level comparisons among these three accessions and to the high-quality Sanger-based reference, A17, reveal a high level of structural and copy number variation, especially affecting genes encoding small cysteine-rich peptides. In some cases large lineage-specific expansions have occurred in two or fewer accessions. Most lineage-specific tandem expansions were artifactually collapsed into single-copy representatives in corresponding short-read-only assemblies.

200-115-Z Host Plant Survey of Invasive Species Japanese Beetle (Popillia Japonica) by Using DNA Subway and DNA Barcode with matK and rbcL Markers Brent Gustafson – North Park University Mary Ellis – North Park University, Ter-yun Lin – North Park University DNA Barcoding is a new approach for identifying a wide spectrum of species with a simple protocol. To evaluate the application of DNA barcoding in field study, a survey was done on host plant identification of the invasive species Japanese Beetle, Popillia japonica which has been known to damage plants all over the United States, especially the Eastern and Midwestern states. In order to eliminate geographic factors that affect host plant selection, Chicago Botanical Garden was chosen as the survey site due to its diverse plants species within close vicinity. During the first stage of the project, 50 plants were determined as host plants and 5 were considered non-host plants. These 55 samples were subject to subsequent DNA sequencing on chloroplast DNA barcoding markers, rbcL and matK. An on-line analytical tool, DNA Subway, was used to identify species with barcode database as well as perform a phylogenetic analysis. Among 55 plants, 46 were successfully amplified with rbcL and 36 with matK primers. A total of 44 plants across 19 families were successfully sequenced, 37 for rbcL and 18 for matK. . Phylogenetic analysis does not support a close co-evolution relation. How beetles choose their host plants is to be determined. DNA Barcode of Life provided a useful resource for species identification. DNA Subway also serves as an amiable tool for database search and phylogeny analysis. This study has established a straightforward model for large-scaled surveys on invasive species using both matK and rbcL regions of chloroplast DNA. It may be applied to other invasive species.

200-116-Y Advancements in Small RNA-seq Alignment Methods for Ambiguously Mapped Reads Nathan Johnson – Penn State University Michael Axtell – Penn State University The advent of cheap and ubiquitous sequencing has lead to extensive repositories of small RNA-seq data, providing a strong basis for small RNA research in many common crops and model organisms. Despite this wealth of data, annotations of small RNAs are desperately lacking, as the majority are comprised of well-defined classes, such as microRNAs, which make up only a small fraction of the total small RNA transcripts. A crucial challenge in small RNA annotation is determining where to assign transcripts that map to multiple genomic locations. These ambiguous reads can represent a large proportion of the transcripts in a small RNA-seq experiment, particularly so in non-model organisms, making their accurate placement essentially important. To overcome this difficulty, we are in the process of refining several strategies which make use of uniquely mapping reads as an a priori predictor of small RNA loci, providing a basis for the locational origin of multi-mapping reads. These approaches have demonstrated high-reproducibility and allow for better sensitivity than non-inclusive methods. To quantify the precision of these methods, we have implemented simulations of small RNA-seq libraries in which the origin of a simulated transcript is traceable, allowing for measurement of mis-mapping. These simulations were generated using publicly available microRNA annotations, in addition to real small RNA-seq libraries as a template for loci assignment, leading to similar characteristics compared to authentic libraries. Using the simulation, these methods showed improved scores in combined precision and sensitivity compared to less directed methods of multi-mapper assignment. Developing a technique to reproducibly and accurately assign multi-mapping small RNA-seq reads represents a significant technical hurdle, but must be solved to fully exploit the power of next-generation sequencing and advance our understanding of this form of genetic regulation.

200-117-Y Bioinformatic Discovery of Short Open Reading Frame (sORF) Type Genes from 43 Species in the Plant Kingdom Anandkumar Surendrarao – UC Davis Ravi Patel – Cornell University, Douglas Cook – UC Davis With the advent of new and powerful sequencing technologies, and software for sequence assembly and annotation, the number of novel genes that are being discovered has also increased. However, a disproportionately miniscule number of genes of the short Open Reading Frame (sORF) category, that encode peptides or proteins in the 10-100 amino acid size range have been identified by bioinformatics methods, and even fewer have been functionally characterized. This limitation in sORF identification is primarily due to the fact that distinguishing sORF predictions from false positives is difficult for any gene prediction software available today. For this reason, genome annotation projects typically underpredict a large fraction of sORF type genes. As a consequence of their under-estimation, the roles of sORFs in plant biology are poorly understood. This is despite famous examplars such as CLAVATA, and legume ENODs. To ameliorate this problem, we have performed a comprehensive bioinformatic analysis to identify sORF loci from 43 Viridiplantae species across a wide taxonomic range. Loci initially identified using the sORF Finder Pipeline were subject to additional filters such as the potential to code for Pfam protein domains or non-coding RNAs. These and other filtering steps generated a set of high-confidence sORF genes. Both lineage-specific and conserved sORF genes could be observed across different taxonomic scales. Interestingly, the numbers of high-confidence sORFs loci are not correlated to genome size or other genome parameters tested.

The work we have done is not only the most comprehensive identification and analysis of sORFs across the Plant Kingdom, but importantly can be used as an sORF identification pipeline to discover sORF genes across not just Plantae, but also Animalia and Eubacteria. The addition of sORF loci to genome annotations can only serve to improve and accelerate our understanding of plant biology.

200-118-Z iPTMnet: An Integrated Resource for Post-Translational Modification (PTM) Knowledge Discovery Jung-Youn Lee – University of Delaware Karen E. Ross – Center for Bioinformatics and Computational Biology, Cecilia Arighi – Center for Bioinformatics and Computational Biology, Mengxi Lv – Center for Bioinformatics and Computational Biology, Jung-Youn Lee – Center for Bioinformatics and Computational Biology, Department of Plant and Soil Sciences, Delaware Biotechnology Institute, University of Delaware, Cathy H. Wu – Center for Bioinformatics and Computational Biology, While post-translational modifications (PTMs), such as phosphorylation, ubiquitination, and glycosylation, play a pivotal role in numerous biological processes in plants, as in all eukaryotes, critical gaps remain in the current research framework for studying plant PTMs. At present, the majority of the curated plant phosphorylation data is housed in multiple databases that provide substrate and site information for several plant species; however, kinase-substrate relationships and PTM-form specific functional information is lacking. Consequently, we are constructing iPTMnet, a bioinformatics resource that will enable plant-centric and cross-taxon exploration of PTMs, including PTM-enzyme substrate relationships, and link PTM-forms with their functions in their biological context. The underlying iPTMnet database integrates information from curated databases and knowledge mined from the scientific literature. With the phosphorylation-focused text mining tools, RLIMS-P and eFIP, we are systematically extracting information on plant phosphorylation events and their functional consequences from the literature, leveraging the Protein Ontology (PRO) and Gene Ontology (GO) for the formal representation of modified protein forms and their annotations, respectively. The iPTMnet website (http://proteininformationresource.org/iPTMnet) allows users to query proteins of interest and view kinase, substrate, and site information as well as phosphorylation networks of kinase-substrate relationships and phospho-form specific protein-protein interactions. We successfully used a combined text and data mining approach to construct a phosphorylation network for the Arabidopsis brassinosteroid signaling pathway. iPTMnet is being expanded to include other PTMs and tools to view PTM conservation across species.

200-119-Z Simultaneous Gene Annotation of 54 Brachypodium Distachyon Genomes Shengqiang Shu – Joint Genome Institute Sean Gordon – Joint Genome Institute, Bruno Moreira – Spanish National Research Council, David Des Marais – Harvard University, David Goodstein – Joint Genome Institute, John Vogel – Joint Genome Institute, Daniel Rokhsa – Joint Genome Institute A reference genome with its gene annotation is a good starting point but the reference genome sequences do not represent the genomic diversity of an entire species nor is the reference genome gene set representing the gene repertoire of the species. Leveraging JGI plant gene annotation pipeline, a reentrant system, we simultaneously annotated gene structure from genomes of 54 Brachypodium distachyon highly diverse inbred lines and the pangenome, and found 24,368 genes not present in the reference genome. These additional genes represent the dispensable set of genes for this species.

SYSTEMS BIOLOGY - Zone 300 Systems, Synthetic, and Computational Biology 300-001-Y Identification of Transcriptional Regulators and Corresponding DNA Motifs for Secondary Cell Wall Biosynthesis in Grasses Kangmei Zhao – University of Oklahoma Laura Bartley – University of Oklahoma Cell wall deconstruction inefficiency is a key problem for biofuel production from cellulosic biomass. Relatively few studies have examined secondary cell wall (SCW) regulators in grasses, which may have diverged from dicots in terms of regulatory mechanisms as they have differences in cell wall composition and patterning. To map the regulatory cascade in grasses using rice as the study system, we built a high quality cell wall network with rice homologs of known Arabidopsis genes that encode cell wall-related transcription factors, lignin biosynthesis genes and grass-diverged cell wall crosslinking enzymes called acyltransferases. The high genome coverage and sharing of gene ontology terms among the connected genes suggest the superiority of the combined network compared to the original networks. We observed novel interactions between known cell wall-related transcription factors and acyltransferase within the network, some of which are supported by reverse genetics. Moreover, we predicted additional putative cell wall regulators (e.g., MYB13.a), which we were able to demonstrate stimulate expression of cell wall biosynthesis genes based on a protoplast transient assay. To further understand SCW regulatory mechanisms, we employed de novo identification of putative cell wall promoter motifs in the network using the criteria of conservation between rice and Brachypodium syntenic orthologs and enrichment near the transcription start site. This analysis revealed a known cell wall-related motif, the AC element, and several putative novel motifs. We hypothesize that the cell wall related R2R3 MYB proteins may recognize more than one similar binding site. This study facilitates transfer of understanding of regulatory mechanisms among species to enable control of secondary cell wall biosynthesis in grasses for improved fuel, feed, and fiber production.

300-002-Z Systems Genetics in Brassica Rapa Reveals Genotype to Phenotype Connections RJ Cody Markelz – University of California Davis Robert Baker – University of Wyoming, Upendra Devisetty – University of California-Davis, Michael Covington – University of California Davis, Nan An – Kansas State, Stephan Welch – Kansas State, Cynthia Weinig – University of Wyoming, Julin Maloof – University of California Davis Connecting genotype to phenotype is a challenge that must be overcome if we are to breed crops that can produce enough food, fuel and fiber on existing arable land. Part of this challenge is wading through vast amounts of data to make biologically coherent models describing genotypic effects across molecular, physiological, and developmental scales. We have recently constructed a saturated genetic map ( < 1 cM resolution) from SNPs derived from RNA-seq data for a recombinant inbred line population of Brassica rapa. Concurrently, we collated all the published phenotypes from this population spanning field, greenhouse, and chamber studies. The new genetic map improves resolution of QTL locations for important agronomic traits such as flowering time, plant height, and leaf size. The RNA-seq data also allowed us to map both cis and trans expression QTLs that co-located with physiological QTL and narrow down candidate genes in physiological QTL regions. For example, there is a QTL peak on chromosome A10 for the highthroughput spectral imaging Normalized Phaeophytinization Index (NPQI). Of the 48 candidate genes in this QTL region, there is a cytochrome P450 gene with a strong cis-eQTL peak at the same marker. This cytochrome P450 gene codes for a protein that regulates a metabolite whose absorption peak is in a spectral region detected by NPQI. Through our

systems genetics model we can narrow down candidate genes for important physiological phenotypes and generate specific molecular hypotheses for targeted follow-up work.

300-003-Z Open-source Tools for High-throughput Plant Phenotyping Noah Fahlgren – Donald Danforth Plant Science Center Malia Gehan – Donald Danforth Plant Science Center, James Carrington – Donald Danforth Plant Science Center Demand for food, fuel, and other plant products is expected to increase dramatically over the next century. At the same time, environmental considerations require that increases in agricultural output must occur using less water, land, fertilizer, and other inputs per unit of yield. One strategy to sustainably increase productivity is to develop new crops and cultivars that use resources more efficiently. While decreasing DNA sequencing costs has enabled rapid genetic screening of crop germplasm, only recently has the development of robotic imaging platforms and low-cost sensors led to major improvements in phenotyping throughput. Here we present PlantCV, an open-source framework for analyzing high-throughput plant phenotyping data. We demonstrate the utility of PlantCV and high-throughput phenotyping by analyzing the phenotypic diversity of a population of Camelina sativa natural accessions using the Bellwether Phenotyping Platform at the Donald Danforth Plant Science Center. C. sativa is an oilseed crop from the family Brassicaceae that is an emerging source of oil for fuel and is also being developed as a production platform for highvalue compounds. Analysis of images taken daily for five weeks was used to measure natural diversity in above ground biomass, growth rates, days to flowering, and other traits. Additional analysis of seed phenotypes including yield per plant, seed size, and oil content was used to identify C. sativa accessions that could enhance breeding efforts. Although PlantCV was developed for the LemnaTec-based Bellwether Phenotyping Platform, we demonstrate that PlantCV can also be applied to low-cost phenotyping solutions and encourage community input in future development.

300-004-Y A Proteomic Strategy to Discover Protein Complex Composition and Dynamics in Arabidopsis Uma Aryal – Purdue University Donglai Chen – Purdue University, Jun Xie – Purdue University, Mark Hall – Purdue University, Daniel Szymanski – Purdue University Knowledge about protein complex composition and dynamics is valuable because it can reveal how cells respond to signals or adapt to different physiological conditions, and carry out complicated biochemical tasks. For example, by forming complexes at the mitochondrial surfaces, glycolytic enzymes can channel metabolites from the cytoplasm to the mitochondria. Sequentially assembled protein complexes precisely execute mechanical tasks like chromosome segregation, vesicle budding, and long distance intracellular transport. A given eukaryotic cell contains hundreds if not thousands of protein complexes; therefore, there is a strong need to develop new methods to discover and analyze this complexity. To date, affinity purification-mass spectrometry (AP-MS) and yeast-two-hybrid (Y2H) are the most widely used methods for high throughput analysis of protein complexes. However, these methods are not suitable for many plant species that are refractory to transformation or genome-wide cloning of open reading frames. To overcome these problems, we recently developed a new method that combines size exclusion chromatography (SEC) with quantitative MS to analyze thousands of proteins leading to the discovery of hundreds of novel protein complexes (Aryal et al., 2014, Plant Cell). We have recently expanded this technique by combining SEC with an orthogonal ion exchange chromatography (IEX) separation to generate abundance profiles of thousands of proteins using MS1 extracted ion chromatograms (XICs). These abundance profiles were subjected to clustering analysis to identify proteins that cofractionate, and are putative subunits of native complexes. Our update will include validation of identified complexes using known protein complexes, and global changes in protein oligomerization in response to dark treatment.

300-005-Y The DOE Systems Biology Knowledgebase: Progress Toward a System for Collaborative and Reproducible Inference and Modeling of Biological Function in Plant Research Robert Cottingham – Oak Ridge National Laboratory Meghan Drake, Priya Ranjan, Samuel Seaver, Sunita Kumari, Doreen Ware, Dave Weston, Adam Arkin, Sergei Maslov, Rick Stevens, The KBase Team The U.S. Department of Energy Systems Biology Knowledgebase (KBase, http://kbase.us) integrates commonly used core tools, reference and experimental data, and overlays them with new capabilities for visualization, exploration, and predictive analysis designed to accelerate our understanding of microbes, plants and their communities. KBase offers open access to quality-controlled data and high-performance modeling and simulation tools that enable researchers to build new knowledge, interpret missing information necessary for predictive modeling, test hypotheses, design experiments, and share findings such that they can be reproduced and extended by others. KBase is being built to integrate a growing collection of plant resources and analytical services including plant genomes such as poplar and Arabidopsis that are integrated with phenotype experiments, gene expression profiles, regulatory, interaction, and metabolic networks. These data sources can be used as input to KBase analysis tools to build models and generate new hypotheses such as metabolic reconstruction and flux balance analyses based on transcriptomes. In addition, userfurnished data can be uploaded, analyzed using high-performance bioinformatics tools, and overlaid visually and analytically on KBase-provided data. As the project matures, partnerships with plant resources such as iPlant and Gramene/Ensembl will lead to a broader research platform for predictive plant and microbial biology.

300-006-Z Plant Two-Hybrid (P2H) System Detects Protein-Protein Interactions in Nicotiana Benthamiana Tan Tri V. Nguyen – California State University - Fullerton Benjamin Smyth – California State University – Fullerton, Melanie Sacco – California State University – Fullerton Protein-protein interactions are critical for understanding the functions of proteins within plant cells. Discovery of novel plant protein-protein interactions has been facilitated by the high throughput yeast two-hybrid (Y2H) system. One issue with this system is potential misfolding of proteins from plants when expressed in yeast due to organismal differences. We have developed a plant two-hybrid system (P2H) for expression in plants based on Y2H and the chemically-inducible chimeric transcription factor GVG, which induces expression from the 6×Gal4 upstream activating sequence and minimal 35S promoter (6×G4UAS35S) in the presence of the synthetic glucocorticoid Dexamethasone (DEX). Our system uses a bait protein fused to the GAL1 DNA-binding domain and a prey protein fused to the VP16 transactivation domain, as well as the hormone binding domain of the rat glucocorticoid receptor (GR) to translocate the protein complex to the nucleus in the presence of DEX. The Solanum lycopersicum autoactive resistance gene SLR4, whichinduces a rapid, hypersensitive response-like cell death in Nicotiana benthamiana,has been cloned under control of the 6xG4UAS35S promoter as the reporter gene. When bait and prey proteins, as well as the reporter construct, were transiently coexpressed into N. benthamiana using agroinfiltration, cell death was observed in patches expressing together the potato proteins Rx and RanGap2 or the N. benthamiana SGT1 protein (NbSGT1) and N. tabacum RAR1 (NtRAR1) in leaves treated with DEX. Control bait and prey agroinfiltrations showed no cell death. These results demonstrate proof-ofconcept for our P2H system for two pairs of proteins previously shown to interact. The P2H provides an assay to visualize cytoplasmic protein-protein interactions within the plant N. benthamiana at the macro level for testing protein-

protein interactions.Further development of the clones will be important to optimize them for more widespread use and testing their potential use for screening of prey libraries in planta.

300-007-Z Phenotype Discovery Enabled by a Machine Vision Pipeline for Maize Seed and Seedling Traits Nathan Miller – University of Wisconsin-Madison Jeff Gustin – University of Florida, Takeshi Yoshihara – University of Wisconsin-Madison, John Baier – University of Florida, Bessie Splitt – University of Wisconsin-Madison, Edgar Spalding – University of Wisconsin-Madison, Mark Settles – University of Wisconsin-Madison Establishing predictive relationships between seed and seedling traits benefits basic and applied plant biology research. To discover phenotype relationships between successive life-cycle stages, high dimensional data sets were acquired noninvasively by a machine-vision pipeline. Over 7,000 seeds representing diverse maize (Zea mays) inbred lines including the 27 Nested Association Mapping (NAM) population parents, 422 members of the Wisconsin Diversity Panel, 162 recombinant inbreds from a B73 x NC350 cross, and 13 kernel composition mutants. Individual kernels were weighed, subjected to near infrared reflectance (NIR) spectroscopy, 3D shape analysis, and then germinated to quantify primary root growth rate and gravitropism with automated image analysis. Kernel height, width, and depth correlated with NIR spectral features representing oil, starch, protein, density, and air space. Position on the cob was tracked for NAM population parents, revealing a kernel NIR phenotype related to density of seed set on the cob. Trends in kernel composition relative to the major axis of the cob were found for a subset of genotypes. Statistical modeling showed that a kernel NIR spectrum could predict strong versus weak gravitropism of the subsequently produced primary root. At different times during the three hour gravitropism time course, kernel weight, density, and oil correlated as highly as r=0.23 with root tip angle. This work shows how a longitudinal phenotype pipeline can discover predictive relationships between life-cycle stages and define new quantitative phenotypes.

BIOCHEMISTRY - Zone 400 Biochemistry and Metabolism 400-001-Y Exogenous Polyamine-induced Modulation of Antioxidant Defense and Glyoxalase System to Confer Salt Stress Tolerance in Mung Bean (Vigna Radiata) Kamrun Nahar – Kagawa University Mirza Hasanuzzaman – Sher-e-Bangla Agricultural University, Mahabub Alam – Biotecnology Group, Masayuki Fujita – Kagawa University Salinity is one of the major limiting factors for crop productivity and a great threat to agriculture worldwide. Polyamines (PAs) are low molecular mass aliphatic amines and organic polycations having diversified physiological roles enhancing stress tolerances including salinity. Effects of salt stress (200 mM NaCl, 48 h) on mung bean (Vigna radiata cv. BARI Mung-2) seedlings and roles of exogenous PAs [putrescine, Put (0.2 mM); spermidine, Spd (0.2 mM) and spermine, Spm (0.2 mM)] in alleviating salt injury were investigated. Salt stress caused severe oxidative stress as indicated by histochemical detection of H2O2 and O2•– in mung bean leaves, increased lipid peroxidation, H2O2 content, O2•– generation rate and lipoxygenase (LOX) activity. Salinity induced methyglyoxal (MG) toxicity is clearly evident. Salinity decreased chlorophyll (chl) and leaf relative water content (RWC); whereas increased proline level. Decrease in ascorbate (AsA), increases in reduced glutathione (GSH), oxidized glutathione (GSSG) and GSH/GSSG ratio were noticed.

Salt stress differentially regulated enzymes of antioxidant and glyoxalase system. In contrast to salt stress, exogenous application of PAs with salt stress increased chl and RWC, PAs positively regulated antioxidant and glyoxalase system. Application of PAs enhanced the activities of antioxidant enzymes (superoxide dismutase, SOD; ascorbate peroxidase, APX; monodehydroascorbate reductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione reductase, GR; catalase, CAT; glutathione S-transferase, GST; glutathione peroxidase, GPX) and glyoxalase enzymes (glyoxalase I, Gly I and glyoxalase II, Gly II) which reduced salt-induced oxidative stress and MG toxicity of mung bean seedlings. Among different PAs, Spm showed better results in enhancing salt stress tolerance.

400-002-Z Relative Tolerance of Different Varieties of Mustard and Rapeseed to Cadmium Toxicity Jubayer-Al- Mahmud – Kagawa University Mirza Hasanuzzaman – Sher-e-Bangla Agricultural University, Kamrun Nahar – Kagawa University, Anisur Rahman – Kagawa University, Masayuki Fujita – Kagawa University To investigate the phytoaccumulation abilities of different Brassica species, B. napus, B. campestris and B. juncea seedlings were exposed to different levels of cadmium (Cd) stress (0.25 mM and 0.5 mM CdCl2 on 8-d-old seedlings) for 3 days. Cd stress at any level reduced fresh and dry weight, leaf relative water content (RWC), and chlorophyll (chl) content in all species where B. jucea showed the least damage effects. Cd increased proline (Pro), malondialdehyde (MDA) and H2O2 contents and lipoxygenase (LOX) activity in all species. Ascorbate (AsA) content decreased in B. napus and B. campestris under severe stress but decreased in B. juncea under both levels of stress. Cd stress increased glutathione (GSH) content in B. juncea under both level of stress, in B. napus under mild stress and in B. campestris under severe stress. But Cd increased glutathione disulfide (GSSG) content and decreased GSH/GSSG ratio in all the species. Cd increased the activities of ascorbate peroxidase (APX), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPX) in all species; increased catalase (CAT) activity only in B. juncea, increased monodehydroascorbate reductase (MDHAR) activity in B. juncea and B. napus but decreased the activities of dehydroascorbate reductase (DHAR) in B. campestris and B. napus under both levels of stress and in B. juncea under severe stress. Cd increased glyoxalase I (Gly I) and decreased glyoxalase II (Gly II) activities in all species. Compared to control, Cd exposure resulted in accumulation of Cd in root and shoot tissue of Brassica seedlings showing the highest accumulation in B. juncea where root tissue accumulated a higher Cd content. Considering the Cd accumulation capacity, growth and physiological attributes B. juncea is the most tolerant species to Cd toxicity.

400-003-Z Phosphorylation Is an On/Off Switch for 5-Hydroxyconiferaldehyde O-Methyltransferase Enzyme Activity in Monolignol Biosynthesis of Populus Trichocarpa Jack Wang – Northeast Forestry University Ling Chuang – Hao Chen, Ying-Chung Lin – Guan-Zheng Qu, David C . Muddiman – Vincent L. Chiang Although phosphorylation has long been known to be an important regulatory modification of proteins, no unequivocal evidence has been presented to show functional control by protein phosphorylation for the monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation mediated regulation of enzyme activity for 5hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem differentiating xylem (SDX) of Populus trichocarpa. Purified phosphorylated recombinant PtrAldOMT2 exhibited essentially no enzyme activity, compared to the unphosphorylated recombinant PtrAldOMT2. Endogenous PtrAldOMT2 enzyme activity in SDX protein extracts was significantly reduced by protein phosphorylation. Protein extracts of P. trichocarpa SDX mediated the phosphorylation of both endogenous and recombinant PtrAldOMT2. PtrAldOMT2 phosphorylation was reversible, and dephosphorylation restored the inhibited PtrAldOMT2 activity back to

the levels of the unmodified proteins. Global shotgun proteomic analysis of phosphopeptide enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 mono-phosphorylation at Ser123 or Ser125 residues in vivo. Phosphorylation site-directed mutagenesis of Ser123 or Ser125 verified PtrAldOMT2 phosphorylation site locations and confirmed the functional significance of these sites for the O-methylation activity. The PtrAldOMT2 Ser123 phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of AldOMTs have either Ser123 or Ser125. PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in fiber than in vessel cells. The phosphorylation of PtrAldOMT2 is likely to have an important regulatory role in monolignol biosynthesis of P. trichocarpa.

400-004-Y The ORANGE Proteins Are the Major Posttranscriptional Regulators of Phytoene Synthase in Governing Carotenoid Biosynthesis Li Li – USDA-ARS Xiangjun Zhou – Cornell University, Ralf Welsch – University of Freiburg Carotenoids are the most widely distributed group of pigments. Phytoene synthase (PSY) is the rate-limiting enzyme in the carotenoid biosynthetic pathway and its activity profoundly affects carotenoid content in plants. Despite of its importance in carotenoid biosynthesis, not much is known about the mechanisms underlying its posttranscriptional regulation. The ORANGE (OR) protein represents a key regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis to confer carotenoid accumulation in plants. By using co-immunoprecipitation and mass spectrometry analysis, we identified PSY as an OR-interacting protein. Both in vitro and in vivo interaction assays provide evidence for the direct interaction between PSY and OR family proteins. OR and PSY interact through the N-terminal region of OR protein in plastids, the organelles where carotenoids are synthesized. Such interaction exerts no effect on PSY gene expression, but positively mediates PSY protein level, enzyme activity, and carotenoid content. Overexpression of AtOR in Arabidopsis significantly increases the amount of enzymatically active PSY, whereas an ator ator-like double mutant shows a dramatically reduced PSY level. Carotenoid content exhibits a correlated change with OR-mediated PSY levels. These results demonstrate that the OR proteins are the major posttranscriptional regulators of PSY and reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants.

400-005-Y Function and Regulation of SnRK1 Complex in Tomato Dongyin Su – Texas A&M University Tim Devarenne – Texas A&M University In resistant plant-pathogen interactions, programmed cell death (PCD) is triggered by the recognition of a pathogen effector protein by a plant resistance (R) protein. Adi3, a Ser/Thr protein kinase, was characterized as a protein downstream of the tomato (Solanum lycopersicum; Sl) R protein Pto and functions to suppress cell death. Previous research in our lab shows that recognition of the Pseudomonas syringae AvrPto effector protein by Pto releases Adi3 cell death suppression, thus PCD occurs. The α-subunit of the SnRK1 protein complex was identified as an Adi3 interacting protein. It was confirmed that Adi3 interacts with both α-subunits (SnRK1.1 and SnRK1.2), all four β-subunits (Gal83, Tau1, Tau2, Sip1), as well as the γ-subunit (Snf4). Our studies have shown that Adi3 phosphorylates the Gal83 β-subunit to control SnRK1 complex kinase activity, as well as its cellular localization. Moreover, in vitro co-IP and kinase assays show that the each β-subunit interacts differently with the α-subunit as well as Adi3. Taking into consideration that βsubunits control substrate specificity for the complex, these differential interactions may play a role in regulation of SnRK1 complex activities, and thus regulate diverse aspects of metabolism during the Adi3-regulated PCD response to pathogens. The tomato homolog of Arabidopsis SnRK Activating kinase (AtSnAK), SlSnAK, significantly activates SnRK1.1.

However, SlSnAK only slightly increases SnRK1.2 kinase activity, indicating the two α-subunits may be regulated differently. Interestingly, SnRK1.2 seems to inhibit SnRK1.1 kinase activity in vitro. It has also been shown in Arabidopsis that the two α-subunits react to phosphate starvation differently. These observations could be an indication that the two α-subunits have different roles in metabolism regulation.

400-006-Z A Novel Protein-targeting Mechanism Is Important for Starch Biosynthesis in Plant Chloroplasts David Seung – ETH Zurich Sebastian Soyk – ETH Zurich, Mario Coiro – ETH Zurich, Benjamin Maier – ETH Zurich, Simona Eicke – ETH Zurich, Samuel Zeeman – ETH Zurich The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers, and was the only protein known to be required for amylose biosynthesis. We now present evidence that another protein, PROTEIN TARGETING TO STARCH (PTST), is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an Nterminal coiled coil domain and a C-terminal carbohydrate binding module (CBM), and the physiological function of the protein was previously unknown. We found that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of starch granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, the CBM domain of PTST, which mediates its interaction with starch granules, is required for correct GBSS localisation. Fluorescently-tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.

400-007-Z 14-3-3 Protein Mediates Plant Oil Biosynthesis Through Interaction with AtWRI1 Wei Ma – Department of Plant Biology, Michigan State University Que Kong – Department of Biochemistry and Molecular Biology, Michigan State University, Jenny Mantyla – Department of Biochemistry and Molecular Biology, Michigan State University, Yang Yang – Department of Biochemistry and Molecular Biology, Michigan State University, John Ohlrogge – Department of Biochemistry and Molecular Biology, Michigan State University, Christoph Benning – Department of Biochemistry and Molecular Biology, Michigan State University Plant 14-3-3 proteins are a family of phosphopeptide-binding proteins which are involved in numerous physiological processes including plant primary metabolism. However, little is known regarding the role of 14-3-3s in plant lipid metabolism. WRINKLED1 (WRI1) is an important transcription factor that regulates fatty acid and oil biosynthesis in the plant kingdom. At present, critical AtWRI1-interacting partners are not well characterized. Here, our in silico analysis indicated that the AtWRI1 protein contains putative 14-3-3 binding motifs. Further experimental evidence showed that multiple 14-3-3 proteins were able to interact with AtWRI1 both in yeast-two-hybrid assays and by bimolecular fluorescence complementation in planta. Co-expression of a 14-3-3 isoform with AtWRI1 in transient assays led to

increased oil biosynthesis in leaves compared to AtWRI1 alone. Stable transgenic plants overexpressing a 14-3-3 isoform displayed increased oil content in seeds compared to wild-type (WT). Our data suggests that the binding of 14-3-3 proteins enhanced the transcriptional activity of AtWRI1. Deletion and site-directed mutagenesis assays are underway to identify the critical site(s) which determine(s) the binding of a 14-3-3 isoform with AtWRI1 protein. Taken together, our evidence identified the transcription factor AtWRI1 as a new client of 14-3-3s and demonstrates a new function of 14-3-3 proteins in plant oil biosynthesis through the interaction with AtWRI1.

400-008-Y The Arabidopsis QQS Orphan Gene Modulates Carbon Allocation Across Species Ling Li – Iowa State University Wenguang Zheng – Iowa State University, Dallas Jones – Iowa State University, Buyun Tang – Iowa State University, Huaxun Ye – Iowa State University, Yanhai Yin – Iowa State University, Eve Wurtele – Iowa State University Sizable minorities of protein-coding genes from every sequenced genome are unique to the species. Little is known about the functional significance of these species-specific orphan genes that are often associated with stress responses/species-specific traits or regulatory patterns. The Arabidopsis thaliana QQS-orphan-gene modulates carbon allocation to protein and starch in Arabidopsis1, 2. Ectopic expression of QQS increases protein content in leaf and seed of soybean2, in multiple high- and low-protein lines of soybean, and in rice3. There are indications that multiple epigenetic pathways may regulate QQS expression. QQS alters its transcript level under stresses and in mutants of genes involved in all sorts of stresses, suggesting that QQS may integrate primary metabolism and environmental perturbations, adjusting adaption to abiotic and biotic stresses4. The QQS protein binds to a transcriptional regulator in Arabidopsis and its soybean and rice homologs3. Overexpression of this QQS interactor in Arabidopsis mimics QQSoverexpression phenotype, increasing protein content and decreasing carbohydrate3. Taken together, the data begin to reveal the skeleton of a previously undefined network in which QQS participates and indicate QQS exerts its effect via an interaction with this transcription factor conserved across eukaryotic species. Deficiency in dietary protein is globally one of the most severe health problems; this insight provides a new strategy to modulate protein levels in crop species. Our research not only presents QQS as a model plant orphan gene regulating plant metabolism, but also illustrates an example of how basic research in Arabidopsis be applied in agriculture. References 1

Li et al, Plant J (2009).

2

Li et al, Plant Biotech J (2015).

3

Li et al, (2015 submitted).

4

Arendsee et al, Trends Plant Sci (2014).

400-009-Y Exogenous Calcium Alleviates Cadmium Induced Oxidative Stress in Rice (Oryza Sativa L.) Seedling by Improving Antioxidant Defense and Glyoxalase System Anisur Rahman – Kagawa University Mohammad Golam Mostofa – Kagawa University, Mirza Hasanuzzaman – Sher-e-Bangla Agricultural University, Masayuki Fujita – Kagawa University The present study was undertaken to investigate the regulatory role of exogenous application of calcium (Ca) in

enhancing the antioxidant defense and glyoxalase systems in mitigating cadmium (Cd) stress in rice. Hydroponically grown fourteen days old rice (Oryza sativa L. cv. BRRI dhan29) seedlings were exposed to 0.25 mM and 0.5 mM CdCl2 alone and in combination with 2.5 mM CaCl2 for three days. Exposure of Cd caused chlorosis, necrosis, leaf rolling symptoms, and growth inhibition. Higher concentration of Cd in growth medium rendered higher Cd accumulation which induced oxidative stress through overproduction of reactive oxygen sprcies (ROS) by disrupting antioxidant defense system. Toxic metal Cd increased methylglyoxal toxicity. Ca supplementation in Cd-treated growth medium reduced Cd uptake by competitive potential. Application of Ca also significantly increased AsA content, activities of superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in antioxidant system, increased glyoxalase I (Gly I) and glyoxalase II (Gly II) activities in glyoxalase system in both levesl of Cd-exposed rice seedlings. Exogenous Ca regulates antioxidant defense and glyoxalase system which reversed overproduced ROS and detoxified methylglyoxal which helped to reduce Cd toxicity.

400-010-Z Decreasing Transcriptional Repressor Activity to Increase Metabolic Flux Through Targeted Pathways Susan Gibson – University of Minnesota Chun Yao Li – University of Minnesota, Brianna Lillo – University of Minnesota, Jacqueline Shanks – Iowa State University, Le Zhao – Iowa State University Catharanthus roseus (L.) G. Don (Madagascar periwinkle) produces a large number of terpenoid indole alkaloids (TIAs), several of which are valuable pharmaceuticals. For example, vinblastine and vincristine are used as chemotherapeutic agents in the treatment of lymphoma and leukemia. Unfortunately C. roseus, which remains the source of these chemicals for the pharmaceutical industry, produces TIAs in only trace amounts, making these drugs very expensive. To increase TIA production in C. roseus, an improved understanding of the regulation of the TIA pathway is needed. Initial efforts focused on increasing expression of the TIA transcriptional activators ORCA2, ORCA3 and CrBPF1. The results of these experiments indicate that increased expression of these transcriptional activators results in significant alterations in the transcript levels of several TIA biosynthetic genes and TIA metabolites. However, overexpression of these transcriptional activators also results in increased expression of TIA transcriptional repressors. As a result, the increases in TIA biosynthetic gene transcript and TIA metabolite levels are typically of limited magnitude and duration. Therefore, our current hypothesis is that the most effective way to increase TIA metabolite levels is by decreasing expression of TIA transcriptional repressors. To test this hypothesis, we have generated transgenic hairy root cultures carrying RNAi constructs designed to allow inducible repression of either the ZCT1, ZCT2 and ZCT3, or the GBF1 and GBF2, TIA transcriptional repressors. Following induction of these RNAi constructs, transcript levels of the ZCTs or GBFs declined 24 fold within 24 hours. Preliminary results indicate that decreased expression of the ZCTs results in altered transcript levels of several TIA biosynthetic and regulatory genes. The results of our work suggest that decreasing expression of repressors may be a more effective means of increasing metabolite production by some biosynthetic pathways than increasing expression of activators.

400-011-Z Biochemical and Phenotypic Characterization of Gibberellin Production in Rhizobia Ryan Nett – Iowa State University Xuan Lu – Iowa State University, Reuben Peters – Iowa State University Gibberellins (GAs) are crucial growth and development hormones in plants, but can also be produced by some plantassociated fungi and bacteria. GA biosynthesis in plants and fungi has been extensively studied and fully elucidated, and it is widely held that these kingdoms convergently evolved the metabolic pathways for GA production. However, GA

biosynthesis in bacteria has remained uncharacterized. Many rhizobia, the nitrogen-fixing symbionts of legumes, (e.g. Bradyrhizobium japonicum USDA110 and Sinorhizobium fredii NGR234) contain a putative gibberellin A biosynthesis operon (GA operon), and previously, it had been shown that a subset of genes in the operon are capable of producing ent-kaurene, the olefin precursor of the GAs. Additionally, feeding studies with B. japonicum USDA110 bacteroids isolated from soybean nodules indicate that the final product is most likely GA9. Through heterologous expression and substrate feeding with GC-MS analysis, we have identified the function of the remaining genes within the GA operon in S. fredii NGR234, and thus identified the pathway by which GA is synthesized in the GA operon-containing rhizobia. In particular, we have characterized the functions of the three cytochrome P450s, the ferredoxin, and the short-chain alcohol dehydrogenase/reductase found in the operon, which act together to produce GA9 from ent-kaurene. Our results also provide strong evidence that the rhizobia have convergently evolved the ability to synthesize GAs independently of plants or fungi. As this operon is only expressed during nodulation, we have also been investigating how rhizobial GA may impact symbiosis with the legume host. Specifically, we have found that knocking out function of the GA operon in B. japonicum USDA110 leads to decreased viability of bacteroids within soybean root nodules. Thus, rhizobia-produced GA seems to provide a selective advantage in symbiosis, particularly for bacteroid propagation following release from nodules into the soil.

400-012-Y Elucidating Regulatory Factors Controlling the Accumulation of Phenylalanine-derived Flavor Volatiles in Tomato Fruits Zhongyuan Liu – Horticultural Sciences Department, University of Florida Denise Tieman – Horticultural Sciences Department, University of Florida, Harry Klee – Horticultural Sciences Department, University of Florida Several of the most important flavor volatiles are derived from phenylalanine (Phe). These volatiles are major contributors to tomato flavor as well as flavor and aroma of many other fruits and flowers. We have studied the regulatory genetic and molecular factors controlling accumulation of important Phe-derived benzenoidphenylpropanoid flavor volatiles. Four introgression lines (ILs) of cultivated Solanum lycopersicum (M82) that contain different, defined chromosomal segments derived from the wild tomato relative, S. pennellii (IL4-3, IL5-2, IL8-2-1, IL11-41) were analyzed for variation in levels of benzenoid-phenylpropanoid volatile compounds. In addition, transgenic plants with over-expression of key controlling factors for benzenoids-phenylpropanoids biosynthesis, AroG*, a feedbackinsensitive DAHP synthase from E. coli, AADC1B, an aromatic amino acid decarboxylase, and ODO1, a Myb transcription factor, were also included as tools to further boost production of secondary metabolites associated with aroma/flavor. We combined multiple traits into the same background and evaluated the effects of these combinations on flavorassociated volatile composition. Volatiles produced from these fruits were analyzed by gas-chromatography/mass spectrometry. Our results indicated that (1) IL4-3 and IL8-2-1 are QTLs stimulating phenylpropanoid volatiles synthesis while IL5-2 and IL11-4-1 mainly promote benzenoid volatiles formation; (2) the combination of AroG with IL8-2-1 generates fruits with higher amount of desired flavor volatiles. This study emphasizes the synergy of combining metabolic engineering and genetic approaches to enhance the production of specialized metabolites in tomato fruits.

400-013-Y Overexpression of Proton-pumping Pyrophosphatases Stimulates Phloem Loading and Long-distance Transport in Arabidopsis Umesh Yadav – Department of Biological Sciences, University of North Texas Aswad Khadilkar – Department of Biological Sciences, University of North Texas, Carolina Salazar – Department of Biological Sciences, University of North Texas, Vladimir Shulaev – Department of Biological Sciences, University of North Texas, Julio Paez-Valencia – Department of Biological Sciences, University of North Texas, Roberto Gaxiola – Department

of Biological Sciences, University of North Texas, Brian Ayre – Department of Biological Sciences, University of North Texas Phloem loading and transport are essential for partitioning of assimilates from source to sink tissues. Overexpression of Arabidopsis type I proton-pumping pyrophosphatases (H+-PPases) enhances plant growth, nutrient acquisition, and abiotic stress responses in various important crops. However, functions of H+-PPases in phloem loading and transport are largely unknown. Genetic and biochemical studies suggest that Arabidopsis vacuolar H+-PPase (AVP1) participates in synthesis of pyrophosphate (PPi) when it is localized at the plasma membrane and in hydrolysis of PPi when it is localized at the vacuolar membrane. H+-PPases localized to the plasma membrane of the sieve element/companion cell complex acts as a PPi synthase, utilizing the proton motive force (PMF) to regulate and maintain cytosolic PPi levels required for efficient sucrose respiration. This in turn generates ATP for the maintenance of the PMF required for phloem loading. The objectives of this project are to investigate whether overexpression of AVP1 improves plant growth by altering carbon utilization, allocation, phloem loading and long-distance transport. AVP1 overexpressing transgenic lines were created with constitutive (CaMV 35S) and companion cell-specific promoters. Plants overexpressing AVP1 have significantly enhanced shoot and root biomass, indicating higher accumulation of photoassimilates and rates of photosynthesis. Targeted analysis of primary metabolites using enzymatic assays showed that there are few changes in the steady-state level of primary carbon metabolites. However, radiolabelling experiments with 14C showed that AVP1 overexpressing lines have significant increases in phloem loading and transport. In conclusion, these results support a role for AVP1 as a PPi synthase contributing to increasing phloem loading and transport in Arabidopsis.

400-014-Z Novel Approach for Visualization of Nitrate and Oligopeptide Transport Processes in Vivo Cheng-Hsun Ho – Department of Plant Biology, Carnegie institute for Science Wolf Frommer – Department of Plant Biology, Carnegie institute for Science Revolutionary new technologies, namely in the areas of DNA sequencing and imaging, continue to impact new discoveries in plant science and beyond. For decades we have been able to determine properties of enzymes and transporters in vitro or in heterologous systems, analyze their regulation at the transcriptional level, use GFP reporters to obtain insights into cellular and subcellular localization, and measure ion and metabolite levels with unprecedented precision using mass spectrometry. However, we lack key information on location and dynamics of the substrates of the enzymes and transporters, and on the regulation of the proteins in their cellular environment. Such information can now be obtained by transitioning from in vitro to in vivo biochemistry using biosensors. In this study, we genetically encoded fluorescent protein-based sensors for nitrate ion, oligopeptides to monitor the activity of transporters in vivo and provide highly resolved spatial and temporal information. We converted the dual-affinity nitrate transceptor and related oligopeptide transporters into fluorescence activity sensors. Substrate addition to yeast expressing transporter fusions with a pair of fluorescent proteins triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. The new technology is applicable in plant and medical research.

400-015-Z Compartmentation of Key Enzymes Reveals a New Putrescine Pathway in Arabidopsis and Soybeans Paul Morris – Bowling Green State University Jigar Patel – Bowling Green State University, Lingxiao Ge – Bowling Green State University, Sheaza Ahmed – Bowling Green State University, Menaka Ariyaratne – Bowling Green State University, Vipaporn Phuntumart – Bowling Green State University, Andrea Kalinosk – University of Toledo

Metabolic pathways can be localized to a single organelle or distributed across several cellular compartments. Here we show that the chloroplasts of A. thaliana and soybeans contain both an Arginine decarboxylase, and an arginase/agmatinase. These two enzymes combine to synthesize putrescine from arginine. Since the sequences of plant arginases show conservation of key residues and the predicted 3D structure of plant agmatinases overlaps the crystal structure of the enzyme from Deinococcus radiodurans, we suggest that these enzymes can synthesize putrescine, whenever they have access to the substrate agmatine. Finally we show that synthesis of putrescine by ornithine decarboxylase in soybeans and rice takes place in the ER. Notably, this pathway is spatially isolated from the chloroplast pathway and the urea cycle. Thus A. thaliana has two, and soybeans have three separate pathways for the synthesis of putrescine. Thus the differential expression of the two Arginine decarboxylase genes in A. thaliana indicates that the two pathways have different roles in responding to biotic or abiotic stress and developmental signals.

400-016-Y Defining the Roles of CRTIL (Carotenoid Isomerase-like) Enzymes in Carotenoid Metabolism and Cold Tolerance Nazia Nisar – The Australian National University Ryan Patrick McQuinn, Dawar Hussain, Christopher Cazzonelli, Barry Pogson Carotenoids are essential pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and serve indispensable functions in plants providing health benefits for humans and animals. Although, carotenoid biosynthetic pathway appears to be fairly complete but the proposed existence for additional isomerases (e.g. neoxanthin and ciscarotenes) has opened new venues to study carotenoid pathway. We focused on characterizing novel enzymes in Arabidopsis (AtCRTIL1 and 2), sharing considerable protein homology toCarotenoid Isomerase (CRTISO). CRTISO catalyses cis to trans conversion of lycopene and is considered one of the rate limiting enzyme regulating the flux through carotenoid biosynthesis pathway. Comparative genomics strategy was employed for functional characterization of CRTIL1 and 2 in Arabidopsis and cyanobacteria to answer a question whether, these enzymes are required for carotenoid metabolism, if indeed they act in carotenoid pathway at all? AtCRTIL1 localized in chloroplast and conserved in oxygenic photosynthetic cyanobacteria as well as in algae, lower plants and higher plants. Over-expression and knockout lines generated for CRTIL1 in Arabidopsis and targeted gene inactivation of CRTIL1 in Synechococcus PCC 7942 are being analyzed by using different physiological and biochemical approaches. The results of the expression and functional analysis of CRTIL1 will be presented. Finally, a novel role of CRTIL1 in mediating cold stress tolerance response is reported suggesting that CRTIL1 perhaps has acquired a specialized function in cold tolerance in addition to its possible yet an unknown function in carotenoid metabolism.

400-017-Y New Roles for Carotenoids and Carotenoid-derived Signals in Regulating Cold Tolerance and Plant Development Barry Pogson – Australian National University Chloroplast-nuclear retrograde signaling pathways have been viewed as a means for bi-lateral communication between organelles and nuclei, ignoring the potential for interaction with processes that regulate plant form and function. Likewise, carotenoids are metabolic compounds that are essential for photosynthesis and also serve as precursors for strigolactones and ABA, yet roles for carotenoids beyound these core processes have received much less attention. In recent years evidence is accumulating that there are additional carotenoid derivatives that function in processes regulating chloroplast gene expression, leaf development and the signaling of oxidative stress. This presentation will cover recent work in our group and that of our collaborators that has revealed roles for novel apocarotenoids in regulating gene expression, leaf shape and plant development (for example see Avendaño-Vázquez et al 2014, The Plant Cell) and new roles for carotenoids and their derivatives in cold tolerance of cyanobacteria and Arabidopsis.

400-018-Z Refinement of Maize Genome-scale Metabolic Models for Maize Leaf, Embryo, and Endosperm with Biochemical Evidence and Transcript Profiles Samuel Seaver – Argonne National Laboratory Louis Bradbury – York College – CUNY, Oceane Frelin – University of Florida, Raphy Zarecki – Tel Aviv University, Eytan Ruppin – University of Maryland, Andrew Hanson – University of Florida, Christopher Henry – Argonne National Laboratory There is a growing demand for the effective metabolic and genetic of plants, for the benefit of human society by numerous means, but this is nevertheless a difficult task due to the layers of complex biological networks affecting plant growth. The publication of genome-scale metabolic reconstructions is a direct response to this problem, where researchers can explore a plant’s ability to biosynthesize biomass in silico. There are challenges in the generation and utility of these reconstructions, and we address two such challenges n our work presented here. All hypotheses that a researcher may generate from metabolic reconstructions in silico are dependent on the accuracy with which genes have been mapped to biochemical reactions. Errors in these mappings lead to an inflated number of reactions and possible generation of unreliable metabolic phenotype predictions. Here we introduce a new evidencebased genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the genereaction associations included within our model. Furthermore, we present a new approach for refinement of our reconstructions to predict active genes based on transcript profiles generated from different cells, tissues, and organs. This method includes a minimal set of reactions associated with low expression genes to enable activity in a maximal number of reactions associated with high expression genes. We apply this approach to construct an organ-specific model for the maize leaf, and tissue-specific models for maize embryo and endosperm cells. We validate our models using fluxomics data for the endosperm and embryo, demonstrating an improved capacity of our models to fit the available fluxomics data. All models are publicly available via the DOE Systems Biology Knowledgebase and PlantSEED, and our new method is generally applicable for analysis transcript profiles from any plant, paving the way for further in silico studies with a wide variety of plant genomes.

400-019-Z Ornithine – a Small Molecule with Big Roles in Nitrogen and Carbon Metabolism in Plants Subhash Minocha – University of New Hampshire Rajtilak Majumdar – USDA/ARS, Boubker Barchi – UNH, Rakesh Minocha – USDA Forest Service, Maegan Gagne – UNH, Swathi Turlapati – UNH, Stephanie Long – USDA Forest Service, NRS After the twenty amino acids that make up proteins in all living organisms, ornithine perhaps occupies the most critical position among the non-protein amino acids. It sits at the crossroads of inter-conversions of glutamate and arginine on one hand, and the production of proline, polyamines, GABA and several alkaloids on the other; all products of great significance to plants. In a comprehensive study involving constitutive and inducible metabolic engineering of the polyamine biosynthetic enzymes (via the use of a transgenic ornithine decarboxylase gene) in poplar (Populus nigra x maximowiczii) and Arabidopsis thaliana, in combination with the techniques of radio-labeled substrates, quantitative PCR, metabolomics and transcriptomics, we learned that ornithine, which is present in relatively small quantities (vs. other amino acids) in the cells, not only regulates the polyamine biosynthetic pathway but also plays an important role in regulating the flux of nitrogen from glutamate to arginine, proline and GABA. The metabolic effects of altering the

Glu-Orn-Arg-Pro-putrescine flux go far beyond the biosynthesis of polyamines and the associated amino acids. We hypothesize that ornithine not only regulates its own biosynthesis (presumably by acting as a sensory molecule) but it also regulates the biosynthesis and accumulation of glutamate (the primary product of nitrogen assimilation) in the cells. An understanding of the regulation of ornithine metabolism should help us in metabolic engineering of plants for stress tolerance (via manipulation of the proline, GABA, and the polyamine pathway), nutritional improvement (cellular contents of important amino acids – arginine and citrulline among others), and to enhance nitrogen assimilation and consequently increased carbon sequestration for use in increased biomass production.

400-020-Y Chasing Dynamics of Amino Acids Using Genetically Encoded Sensors Sakiko Okumoto – Virginia Tech Julien Besnard, Rejane Pratelli – Virginia Tech, Chengsong Zhao – Virginia Tech, Guillaume Pilot – Virginia Tech, Sakiko Okumoto – Virginia Tech From detailed analyses of enzymes and transporters that are involved in nitrogen metabolism, as well as recent highresolution transcriptomics using cell-sorting and laser capture microdissection techniques, it is becoming more and more clear that expression and regulation of genes involved in the metabolism and transport of nitrogen is cell- or tissuespecific. It is therefore well conceivable that the resulting metabolite fluxes are drastically different between cell types. However, the analysis of metabolites at a single cell level has so far been challenging. Genetically encoded sensors are powerful tools to detect changes in metabolite flux that happen in very limited spatial and temporal scale. Recent data suggest that such sensors can be used to estimate the steady state metabolite level in a cell, as well as to study the mechanism of transport. We successfully developed sensors for two amino acids, glutamine and glutamate, that are key compounds in nitrogen assimilation and transport, and also might act as signaling molecules for nitrogen availability. Our lab has developed sensors can be used for the detection of transport of these amino acids in vivo. The current technique for the analysis of in vivo fluxes using genetically encoded sensors will be discussed.

400-021-Y Putative Chloroplast Inner Membrane Protein BASS6 Is Involved in Photorespiratory Metabolism Paul F South – USDA-ARS Photosynthesis Research Unit, Institute for Genomic Biology, University of Illinois Berkley J Walker – USDA-ARS Photosynthesis Research Unit, Institute for Genomic Biology, University of Illinois, Donald R. Ort – USDA-ARS Photosynthesis Research Unit, Institute for Genomic Biology, University of Illinois In C3 plants roughly 25% of Rubisco reactions is the fixation of oxygen instead of carbon dioxide resulting in the conversion of RuBP to one molecule of phosphoglycerate and one molecule of glycolate. C3 plants recover the carbon from oxygenic photosynthesis through the C2 photorespiratory pathway. The C2 pathway is highly compartmentalized involving the chloroplast, peroxisome, and mitochondria. Though the soluble enzymes involved in photorespiration are well characterized only a few transporters in photorespiration have been identified such as the glycolate glycerate transporter PLGG1. The difficulty in identifying transporters in photorespiration could be due to transporters playing multiple roles in central metabolism or possible redundancies in the transport processes. To identify additional transporters involved in the C2 photorespiratory pathway we selected known and putative chloroplast inner membrane proteins and characterized T-DNA insertion lines in Arabidopsis for defects in photorespiration utilizing a fluorescence based screen. Our results identified the Na / Bile acid symporter BASS6 a putative chloroplast inner membrane protein of unknown function. The Arabidopsis bass6-1 T-DNA plants exhibit a classic photorespiratory phenotype (lethal or slow growth) that is rescued at elevated CO2 concentrations, as well as show reduced photosynthetic rates. Double knockout Arabidopsis line bass6-1plgg1-1 resulted in an additive growth defect and further reductions in photosynthetic rates

compared to either bass6-1 or plgg1-1 single mutation. In addition, metabolomics analysis and genetic complementation in yeast suggests that BASS6 is involved in the flux of photorespiratory intermediates. We show that BASS6 is involved in photorespiratory metabolism possibly through redundant or currently unknown transport processes. Identifying transporters involved in photorespiration can provide insight into how plants maintain efficient transport of metabolites and co-factors involved in the refixation of carbon, abiotic stress response and amino acid metabolism.

400-022-Z Function of Amino Acid Permeases (AAPs) in Rice (Oryza Sativa) and the Liverwort Marchantia Polymorpha Margaret Taylor – University of Minnesota Anke Reinders – University of Minnesota, John Ward – University of Minnesota The amino acid permeases (AAPs) are a family of proton-coupled amino acid transporters that are ubiquitous in land plants. To date, the study of AAP function has largely been limited to eudicots. Here we report the functional characterization of AAPs in two new plant clades: OsAAP1, OsAAP3, OsAAP7, and OsAAP16 in rice (Oryza sativa), a monocot, and MpAAP9A and MpAAP10A in Marchantia polymorpha, a liverwort. The OsAAPs and MpAAPs were expressed in Xenopus oocytes and analyzed by electrophysiology. They show K0.5 values for proteinogenic amino acids similar to K0.5 values previously reported for Arabidopsis AAPs. Some of the OsAAPs and MpAAPs show different substrate specificity compared to the broad substrate specificity of the Arabidopsis AAPs. OsAAP3 and MpAAP9A have high transport rates for lysine and arginine while excluding the aromatic amino acids. MpAAP10A transports cysteine more than previously characterized AAPs. The use of GFP fusions to localize rice AAPs was problematic: N-terminal fusions did not express well and C-terminal fusions position GFP in the apoplast where fluorescence is quenched due to low pH. Therefore, we localized two of the rice AAPs to the plasma membrane using C-terminal fusions to the eGFP-RFP tandem fluorescent protein pHusion.

400-023-Z Integrated Metabolomics and Transcriptomics Reveal Enhanced Specialized Metabolism in Medicago Truncatula Root Border Cells Bonnie Watson – The Samuel Roberts Noble Foundation Mohamed Bedair – Monsanto, Ewa Urbanczyk-Wochniak – Monsanto, David Huhman – The Samuel Roberts Noble Foundation, Dong Sik Yang – Samsung Advanced Institute of Technology (SAIT), Stacy Allen – The Samuel Rob The root tips of many plants produce thousands of specialized cells which are separate from the root but remain appressed to it until released by exposure to water. These “border cells” provide a biotic boundary important in plant defense and plant-symbiont interactions. Integrated metabolomics and transcriptomics studies of Medicago truncatula seedling border cells and root tips revealed substantial metabolic differences between these distinct and spatially segregated root regions. Large increases in oxylipin-pathway lipoxygenases and auxin-responsive transcript levels in border cells corresponded to observed differences in phytohormone and volatile levels compared to adjacent root tips. Microscopic examinations of border cells revealed the presence of significant starch deposits which serve as critical energy and carbon reserves. Most primary metabolism transcripts were decreased in border cells while many flavonoidand triterpenoid-related metabolite and transcript levels were dramatically increased. Metabolic resources normally destined for growth and development were redirected towards accumulation of specialized metabolites in border cells, resulting in constitutively elevated defense and signaling compounds needed to protect the delicate root cap and recruit beneficial microbes. Elevated levels of the rhizobial signaling compound 7,4’-dihydroxyflavone (DHF) were further increased in border cells of roots exposed to Phymatotrichopsis omnivora, and the value of DHF as an antimicrobial compound was demonstrated using in-vitro growth inhibition assays. The cumulative data provide strong evidence that

primary and secondary metabolism are differentially programmed in border cells relative to root tips and implicate a more prominent mechanistic role for border cells in plant-microbe signaling, defense and interactions than previously envisioned.

400-024-Y 'Omics' Analysis Reveal Profound Physiological Effects of Arogenate Dehydratase Modulation in Arabidopsis Thaliana Joaquim Marques – Washington State University Tetsuro Ito – Gifu Pharmaceutical University, Ricarda Hoehner – Washington State University, Helmut Kirchhoff – Washington State University, Laurence Davin – Washington State University, Norman Lewis – Washington State University A fundamental class of metabolites for plant life is the phenylpropanoid. This biosynthetic pathway was crucial for plant evolution, being in great part responsible for the adaptation of plants to the terrestrial environment. Its main product is the polymer lignin, the second most abundant biopolymer in the planet. Besides this polymer, the phenylpropanoid pathway also produces a great variety of small molecules with many roles in plant physiology. Our group has developed A. thaliana lines with single and multiple arogenate dehydratase (ADT) isoenzymes knockouts producing plant lines with reduced lignin content. Interested in determining possible unforeseen physiological effects of ADT modulation in A. thaliana, we decided to employ an integrated “holistic” analysis of these transgenic plants, using the considerable technological capabilities at our disposal. In an attempt to characterize the systemic effects of our manipulation we performed a comprehensive, untargeted metabolomic profiling, coupled with a comprehensive photosynthetic characterization. The former was done by means of UPLC-ESI-TOF analysis of hydro-alcoholic extracts and the latter by using the phenomics facilities at our disposal. The metabolomic profiles generated were then analyzed by means of specialized software leading to the identification of several affected metabolites, phenylalanine-derived and also unrelated compounds. The unforeseen effects included reduced content of glucosinolates, phenolics and apocarotenoids woth potential repercussions on plant physiology and fitness. Besides the metabolomic analysis, photosynthetic parameters characterized in our phenomics facility were significantly altered. Although the specific mechanism underlying the effects are still being investigated, differences observed underscore the systemic effects from modulation of this crucial metabolic step, with repercussions back to the photosynthetic apparatus. This research is part of a larger efford in engineering plants for improved properties and is an example of the necessary multi-disciplinary and systemic approach to plant investigation that has recently become a reality due to current technological advances.

400-025-Y Arabidopsis AMINO ACID PERMEASE 8: The Missing Link in Source to Sink Partitioning of Nitrogen James Patrick Santiago – Washington State University Mechthild – Washington State University Nitrogen (N) partitioning to developing vegetative and reproductive sink organs is important for plant growth and yield. In Arabidopsis, N assimilation and synthesis of amino acids mainly occur in photosynthetically active source leaves. While some of the newly produced amino acids are used in leaf metabolism or are transiently stored, a large amount is translocated in the phloem from the source leaves to sinks (e.g. growing leaves or fruits). Transporter function in phloem loading of amino acids has long been postulated, however the molecular mechanism had yet to be discovered. We found that the broad-specific Arabidopsis AMINO ACID PERMEASE8 (AAP8) is expressed in the minor veins of source leaves and we hypothesized that AAP8 is involved in phloem loading of amino acids. Using a combination of molecular, biochemical, physiological, and phenotypic approaches, AAP8 T-DNA insertion lines were analyzed. Our studies showed reduced phloem amino acid content in the knock-out plants, and especially of glutamine, the dominant long distance N

transport form in Arabidopsis. Source leaf feeding with 14C-labeled glutamine confirmed a decrease in source to sink transport in the mutants. Metabolite analyses revealed alterations in source leaf N and carbon metabolism, which was corroborated by expression analyses and photosynthesis measurements. The consequence of the knock-out of AAP8 was reduced sink development. Taken together, our results demonstrate that AAP8 is not only essential for phloem loading and sink supply of N, but that its function also affects source leaf physiology.

400-026-Z Canopy Position Has a Profound Effect on Soybean Seed Composition Ivan Baxter – USDA-ARS, Donalf Danforth Plant Science Center Kunzhi Li – Kunming University of Science and Technology, Randall Nelson – USDA-ARS, Alexander Ulanov – University of Illinois at Urbana-Champaign, Steve Huber – USDA-ARS While much thought has been given to the variation of seed composition in crop plants driven by the location of the field, very little attention has been paid to the variation in seed composition within the plant canopy. Soybeans are valued for their protein and oil content, but when used for human nutrition the content of minerals such as iron is also critically important. Although soybean seeds appear homogenous, the composition of mature seeds varies depending on the position on the main stem where the pods developed. We investigated the effect of canopy position on the organic and mineral composition of soybeans from 14 cultivars grown in Urbana, IL, across several years. Seed produced at the top of the canopy had higher protein and lower oil concentrations compared to seeds from the bottom of the canopy. Metabolomic analysis suggested that supply of amino acids, in particular asparagine, to developing seeds may control the storage protein accumulation gradient. While the concentrations of some minerals did not vary with canopy position, iron concentration was generally 20% higher in seeds from the bottom of the canopy. The oil/protein and elemental gradients appear to be driven by different causes. Soy food products (soy flour, milk and okara) made from seeds from the top or bottom of the canopy reflected the difference in total seed iron concentration. Canopy position effects on seed iron concentration have been observed with diverse germplasm grown in the USA as well as cultivars grown in Africa. Knowledge of these canopy position effects could have immediate application for human health and nutrition in countries such as Africa where iron is limiting in the diet, and identifies potential targets for future improvement in soybean composition.

400-027-Z The P450-type Carotene Hydroxylase from Red Alga Xing-Qi Huang – Nanjing University Li-En Yang – Nanjing University, Yu Hang – Nanjing University, Yin-Yin Deng – Changshu Institute of Technology, Qin-Qin Lu – Institute of Fisheries of Jiangsu Province, Shan Lu – Nanjing University Carotene hydroxylases catalyze the hydroxylation of α- and β-carotene hydrocarbons into xanthophylls. In red algae, βcarotene is a ubiquitously distributed carotenoid, and hydroxylated carotenoids such as zeaxanthin and lutein are also found. However, no enzyme with carotene hydroxylase activity had been previously identified in red algae. Here, we report the isolation of a gene encoding a cytochrome P450-type carotene hydroxylase (PuCHY1) from Porphyra umbilicalis, a red alga with an ancient origin. Sequence comparisons found PuCHY1 belongs to the CYP97B subfamily, which has members from different photosynthetic organisms ranging from red algae to land plants. Functional complementation in Escherichia coli suggested that PuCHY1 catalyzed the conversion from β-carotene to zeaxanthin. When we overexpressed PuCHY1 in the Arabidopsis thaliana chy2 mutant, pigment analysis showed a significant accumulation of hydroxylated carotenoids, including neoxanthin, violaxanthin, and lutein in the leaves of transgenic plants. These results confirmed a β-hydroxylation activity of PuCHY1, and also suggested a possible ϵ-hydroxylation

function. The pigment profile and gene expression analyses of the algal thallus under high-light stress suggested that P. umbilicalis is unlikely to operate a partial xanthophyll cycle for photoprotection.

400-028-Y ‘C4’ Metabolism in the Vasculature: relationships Between PEP Carboxykinase Activity and Xylem Nitrogen Recycling and Hydraulic Conductivity in Rice Leaves Richard Leegood – University of Sheffield Karen Bailey – University of Sheffield Measurements of amino acids in the guttation fluid and in the xylem exudates of cut leaves from intact plants provide evidence of the remarkable efficiency with which these nitrogenous compounds are reabsorbed from the xylem sap. This could be achieved by mechanisms involving intercellular transport and/or metabolism. Developmental changes in transcripts and protein showed that transcripts for PEP carboxykinase (PEPCK) increased from the base to the leaf tip, and were markedly increased by supplying asparagine. Supplying amino acids also increased the amounts of protein of PEPCK and, to a lesser extent, of PPDK. PEPCK is present in the hydathodes, stomata and vascular parenchyma of rice leaves. Evidence for the role of PEPCK was obtained by using an activation-tagged rice line that had an increase in PEPCK activity and by using 3-mercaptopicolinic acid, a specific inhibitor of PEPCK, to show that activation of PEPCK resulted in a decrease in N in the guttation fluid and xylem sap and that inhibition of PEPCK resulted in an increase in N in the guttation fluid and xylem sap. Furthermore, increasing PEPCK decreased the volume of xylem sap or guttation fluid, whereas decreasing PEPCK increased the volume of xylem sap or guttation fluid. These findings suggest the following hypotheses, that (a) both metabolism and transport are involved in xylem recycling and (b) excess N is the signal involved in modulating xylem hydraulic conductivity, whether in the roots or shoots. Thus feeding N or inhibiting PEPCK increases hydraulic conductivity, perhaps via nutrient regulation of water-transporting aquaporins. Water relations, nitrogen recycling and vascular metabolism and transport are thus intimately linked.

400-029-Y Mutations in AtKIN11 and AtKIN10 and Their Effects on Protein Structures and Glucose Sensing Vanya Aggarwal – St. Bonaventure University Rijo Maracheril, Xiao-Ning Zhang – St. Bonaventure University SR45 is a serine-arginine rich protein that serves as a splicing activator for spliceosome assembly in Arabidopsis thaliana, and is conserved across kingdoms. A null mutant, sr45-1, has been shown to have late flowering, altered leaf and flower morphology, smaller plant size, and delayed root growth. It also shows hypersensitivity to 3% glucose treatment, as demonstrated by slower root growth. A mutation (atkin11-2) in an AMP-activated kinase homologous gene AtKIN11 has been found to be an enhancer of sr45-1. The sr45-1;atkin11-2 double mutant demonstrates higher levels of sterility as well as stronger hypersensitivity to glucose, when compared to the sr45-1 and atkin11-2 single mutants, and the wild type. In AtKIN11, a point mutation in the 1456th nucleotide changes a guanine to adenine, causing a missense mutation at a highly conserved alanine195 (A195) to a threonine195 (T195). Predicted protein structures suggest that A195 to T195 mutation could inhibit phosphorylation at threonine176, which is required for the AtKIN11 activity. Root length measurements and downstream ASN1 expression levels suggest synergy between AtKIN11 and SR45. AtKIN10 is homologous to AtKIN11, and its mutant, atkin10-1, also shows hypersensitivity to glucose. This is a point mutation at the 1301st nucleotide changing a cytosine to a thymine, causing a missense mutation changing the plantspecific serine152 (S152) to a phenylalanine152 (F152). Predicted protein structures suggest that this mutation does not have an impact at the catalytic site. Root length measurements suggest that SR45 and AtKIN10 function in the same pathway for glucose sensing.

To confirm the effects seen with the predicted protein structures, we created a series of mutant constructs. Our next step is to study the effect of these mutations for in vitro and in vivo studies.

400-030-Z Characterization of Nitrogen-Efficient Phenotype in Arabidopsis Thaliana Engineered with Alanine Aminotransferase Variants Lai To – University of Alberta Chandra McAllister – University of Alberta, Allen Good – University of Alberta Alanine aminotransferase (AlaAT) is an enzyme that converts alanine and 2-oxoglutarate to glutamate and pyruvate, and therefore is at a key junction of both N and C metabolism in plants. It has been shown that over-expression of barley AlaAT (HvAlaAT) driven by a tissue-specific root promoter, results in a significant increase in both biomass and seed weight in rice and canola. While the importance of HvAlaAT in increasing nitrogen use efficiency (NUE) in crop plants has been well characterized, a comparative examination of the AlaAT enzymes from other sources, and their impact on plant NUE, has not been explored. To address the impact of AlaAT variants on NUE, AlaAT’s from diverse sources were transformed into both a wild type Arabidopsis thaliana Col-O background and an alaat1;2 knockout background and assessed under differing N conditions. Plants expressing different AlaAT’s showed alterations in growth on various N sources and concentrations, as well as differences in biomass and uptake of amino acids between control and transgenic plants, as well as between plants expressing different AlaAT enzymes. This study suggests that AlaAT variants can have homologue-specific effects on plant NUE. The characterization of these homologues will aid in the development of transgenic crop plants with improved NUE.

400-031-Z Starch Accumulation in Leaf Sheath Regulated by CRCT Is a Key Factor of Photosynthetic Acclimation Under Elevated CO2 in Rice Ryutaro Morita – Kobe University Tomoko Hatanaka, Shuji Misoo, Hiroshi Fukayama Photosynthetic rates of most C3 plants are stimulated by elevated CO2. However, prolonged exposure to CO2 enrichment often reduces the initial stimulatory effect of photosynthesis and sometimes decreases the photosynthesis. These photosynthetic acclimations are likely attributed to secondary responses related to increased carbohydrate accumulation in leaves. Therefore, some species which has large sink for carbohydrate accumulation did not show the reduction of photosynthesis under elevated CO2. In rice, the leaf sheath can act as a temporary sink for photoassimilates, mainly as starch. In our previous work, we analyzed the function of CO2-responsive CCT domain containing protein, designed CRCT, which is up-regulated under elevated CO2 condition in rice. Our results suggest that CRCT is a positive regulator of starch accumulation in leaf sheath, regulating coordinated expression of starch synthesis genes in response to the levels of photoassimilates. In this study, the effects of starch accumulation in leaf sheath on the photosynthetic rate of rice were analyzed using CRCT transgenic rice grown under ambient (38 Pa) or elevated (100 Pa) CO2. The overexpress or RNAi knockdown of CRCT did not cause large effects on the photosynthetic rates grown under ambient CO2. In contrast, the photosynthetic rates of overexpression lines were enhanced and those of RNAi lines were decreased compared with non-transgenic rice grown under elevated CO2. Although the soluble sugar and starch contents of leaf blade were unchanged in overexpression lines, these were increased in RNAi lines grown under elevated CO2. These results suggest that sink capacity of leaf sheath regulated by CRCT affects the carbohydrate contents and then photosynthetic rate in leaf blade of rice grown under elevated CO2.

400-032-Y Overexpression of Pyruvate Orthophosphate Dikinase (PPDK) Contributes to Higher Photosynthetic Rates in Sugarcane (Saccharum Officinarum) Nikhil Jaikumar Steve Long, Fredy Altpeter, Ratna Karan, Steve Moose Pyruvate orthophosphate dikinase (PPDK) is a critical enzyme in C4 photosynthesis, as it regenerates the substrate (PEP) for the initial carbon-fixing step in the pathway. In spite of the very high photosynthetic rates already displayed by many C4 species, some theoretical modeling work suggests that their photosynthetic apparatus may still not be optimized for the relatively high-CO2 modern environment, and that in particular they may be limited by PPDK. We sought to overcome this control point by introducing extra copies of genes for one of two PPDK isoforms from Miscanthus x giganteus into the related species sugarcane. Transformation was carried out via biolistic methods and confirmed by ELISA. Plants from each event of interest were clonally reproduced and gene expression quantified by qRT-PCR. Photosynthetic rate at 400 ppm CO2 and 28o C was quantified at 2-3 time points in two separate experiments. Photosynthetic rates were on average 12% higher in the transgenic plants than in the wild type, and was correlated with level of gene expression. Introduction of the PPDK gene caused large increases (2.5 - 4.5 fold) over wild type in the number of PPDK transcripts. Increases in photosynthetic rate were due to a combination of stomatal and biochemical influences (in particular, higher rates of PEP regeneration as inferred from A/Ci curves). Our results confirm that PPDK is an important control point for C4 photosynthesis and that overexpressing this gene is a productive strategy to push the envelope of C4 photosynthesis even higher. This could have important implications for photosynthetic traits and productivity in some of the world’s most important food, fuel and fodder crops.

400-033-Y Dissecting the Effects of Induced Amino Acid Perturbation in Plants Shi Yu – Virginia Tech Delasa Aghamirzaie – Virginia Tech, Ruth Grene – Virginia Tech, Guillaume Pilot – Virginia Tech Nitrogen status in plants is tightly regulated to fit developmental needs and rapid changes in the environment. Amino acids constitute the major organic form of transported nitrogen in plants, fundamental elements for protein synthesis, and precursors of many plant secondary metabolites, such as lignin, hormones, and flavonoids. Furthermore, amino acid metabolism lies at the crossroad of carbon and nitrogen metabolism. The Arabidopsis gdu1-1D mutant secretes glutamine from hydathodes, a phenotype caused by the overexpression of Glutamine Dumper1 (GDU1). GDU1 is a small transmembrane protein presents only in higher plants. The gdu1-1D mutant shows a pleiotropic phenotype: perturbed amino acid metabolism, such as increased amino acid content in leaves, tolerance to exogenous toxic concentrations of amino acids, elevated amino acid export, and stress/defense responses, such as enhanced ROS production, hypersensitive cell death, and smaller rosettes. To better elucidate the biological processes leading to the complex gdu11D phenotype, we employed a dexamethasone-inducible system to trigger the expression of GDU1 in Arabidopsis. We probed the early events triggered by the perturbation of amino acid homeostasis in the induced plants using RNAseq analysis and metabolomics. Since overexpression of other members in the GDU family leads to a similar phenotype as gdu1-1D, we compared RNAseq data obtained from plants overexpressing different members of the GDU family. We found common biological processes shared by these mutants and biological processes unique to each member. Our results showed that GDU1 overexpression first increases amino acid export, which is followed by amino acid imbalance and stress responses. The study shed light on how amino acid imbalance interacts with various plant signaling pathways and stress responses.

400-034-Z Application of Mathematical Modeling to Develop Traits Targeting C4 Carbon Assimilation Esteban Bortiri - Syngenta Laura Potter, Josh Cohn, Michael Nuccio Plant genetic engineering depends on the identification of genes or leads, that when manipulated, confer the intended trait. Traits include improvement in crop performance by increasing resistance to pest pressure, conferring resistance to herbicides and improving agronomic traits like drought tolerance and nitrogen use efficiency. We used a C4 carbon assimilation mathematical model to identify 3 and 4 gene combinations that when up-regulated are predicted to improve photosynthetic output, under the hypothesis that enhancing photoassimilation will increase yield. Transgenic maize that contain a 3- or 4-gene trait were constructed and analyzed. Results show that variable degrees of gene upregulation were achieved. Physiological analysis shows that some events do display increased photoassimilation as predicted by the model. These results indicate that our computational approach was effective for complex trait lead generation.

400-035-Z Biosynthesis of Granatane Alkaloids in Punica Granatum Benjamin Chavez – Texas Tech University Kaylene De Vries – Texas Tech University, Kenneth Galloway – Texas Tech University, Christin Fellenberg – Texas Tech University, John D’Auria – Texas Tech University The granatane and tropane alkaloids are structurally similar classes of bicyclic alkaloids which have an uneven distribution across flowering plant families. Specifically granatane alkaloids are found in the pomegranate plant (Punica granatum) where alkaloids are thought to be biosynthesized in the root and accumulate in the bark. It has been previously shown in the D’Auria lab that tropane alkaloid biosynthesis has evolved independently in different plant families. We are therefore interested in testing the hypothesis that the structurally similar granatane alkaloids also have an independent origin. A homology based in silico search was performed on a P. granatum transcriptome database in order to identify genes thought to be involved in alkaloid biosynthesis. Gene sequences used for this approach originated from known tropane alkaloid biosynthetic enzymes either from E. coca or members of the Solanaceae family. Several putative biosynthetic enzymes were successfully identified during this initial BLAST (Basic Local Alignment Search Tool) search. The genes encoding these enzymes were evaluated to determine whether or not they existed as Open Reading Frames (ORFs) and primers were designed to isolate them from pomegranate tissue. Ongoing studies include the heterologous expression of these enzymes in a host such as E. coli or yeast. Furthermore, these enzymes will then be characterized via enzyme assay for their biochemical activities.

400-036-Y Essential Role of Choline/ethanolamine kinase4 (CEK4) in Phospholipid Biosynthesis and Embryo Development in Arabidopsis Thaliana Yuki Nakamura – Institute of Plant and Microbial Biology, Academia Sinica Ying-Chen Lin – Institute of Plant and Microbial Biology, Academia Sinica, Yu-chi Liu – Institute of Plant and Microbial Biology, Academia Sinica Phospholipids are highly conserved and essential components of biological membranes. The major phospholipids, phosphatidylethanolamine and phosphatidylcholine (PtdCho), are synthesized by the transfer of the phosphoethanolamine or phosphocholine polar head group, respectively, to the diacylglycerol backbone. The metabolism of the polar head group characterizing each phospholipid class is poorly understood; thus, the biosynthetic pathway of major phospholipids remains elusive in Arabidopsis thaliana. The choline/ethanolamine kinase (CEK) family

catalyzes the initial steps of phospholipid biosynthesis. Here, we analyzed the function of the four CEK family members present in A. thaliana. Knocking out of CEK4 resulted in defective embryo development, which was complemented by transformation of genomic CEK4. Reciprocal genetic crossing suggested that CEK4 knockout causes embryonic lethality, and microscopy analysis of the aborted embryos revealed developmental arrest after the heart stage, with no defect being found in the pollen. CEK4 is preferentially expressed in the vasculature, organ boundaries, and mature embryos, and CEK4 was mainly localized to the plasma membrane. Overexpression of CEK4 in wild-type A. thaliana increased the levels of PtdCho in seedlings and mature siliques, and also of major membrane lipids in seedlings and triacylglycerol in mature siliques. CEK4 may be the plasma membrane-localized isoform of the CEK family involved in the rate-limiting step of PtdCho biosynthesis and appears to be required for embryo development in A. thaliana.

400-037-Y Bacterial and Plant HAD Enzymes Catalyze a Missing Phosphatase Step in Thiamin Diphosphate Biosynthesis Ghulam Hasnain – University of Florida Sanja Roje – Washing¬ton State University, Na Sa – Washington State University, Rémi Zallot – University of Florida, Michael Ziemak – University of Florida, Valérie Crécy-Lagard – University of Florida, Jesse Gregory III – University of Florida, Andrew Hanson – University of Florida Thiamin diphosphate (ThDP), the active form of thiamin (vitamin B1), is an essential cofactor. In plants and microorganisms the thiazole and pyrimidine moieties of thiamin are synthesized separately and then coupled together to form thiamin monophosphate (ThMP). Most bacteria convert ThMP directly to ThDP via a specific kinase. However, plants, yeast and some bacteria first dephosphorylate ThMP to give thiamin, and then pyrophosphorylate the thiamin to give ThDP. The gene for the dephosphorylation step (ThMPase) is missing in all organisms. Com­par­ative genomic analysis uncovered bacterial HAD phosphatase family genes (from subfamilies 1A and 1B) that clust­er on the chro­m­o­­some with, or are fused to, thiamin syn­th­esis genes, and are thus candidates for the missing ThMPase. Three typical can­did­ates (from Anaerotruncus colihom­inis, Dorea longicatena, and Syn­trophomonas wolfei) were shown to have efficient in vivo ThMP­ase activity by express­ing them in an Escherichia coli strain engineered to require an active ThMPase for growth. In vitro ass­ays con­firm­ed that these candidates all preferred ThMP to any of 45 other phosphate ester substrates tested. An Arab­idop­s­is ThMPase homolog (At4g29530) of unknown function whose ex­pr­­ess­ion pat­tern and com­partmentation fit with a role in ThDP syn­thesis was shown to have in vivo ThMP­ase activity in E. coli and to prefer ThMP to any other substrate tested. However, insertional in­act­iv­ation of the At4g29530 gene did not affect growth or the levels of thiamin or its phosphates, in­dic­­ating that Arabidopsis has at least one other ThMP­ase gene. The maize ortholog of At4g­29530 (GRM­ZM­2G­­035134) was also shown to have ThMPase activity. These data identify HAD genes that spec­i­fy­ the elusive ThMP­ase activity, indicate that ThMPases are substratespecific rather than gen­eral phos­ph­atases, and suggest that different evolutionary lineages have recruited ThMP­ases independ­ently from different branches of the HAD family.

400-038-Z DUF89: A Novel Family of Metal-dependent Phosphatases Implicated in Metabolite Damage-control Lili Huang – University of Florda Anna Khusnutdinova – University of Toronto, Greg Brown – University of Toronto, Xiaohui Xu – University of Toronto, Boguslaw Nocek – Argonne National Laboratory, Andrzej Joachimiak – Argonne National Laboratory, Alexei Savchenko – University of Toronto, Rémi Za – University of Florda, Kelly Balmant – University of Florda, Michael Ziemak – University of Florda, John Shanklin – University of Florda, Valerie Crecy-Lagard – University of Florda, Jesse Gregory – University of Florda, Alexander Yakunin – University of Toronto , Andrew Hanson – University of Florda The DUF89 (Pfam01937) protein family is widely distributed but its function is unknown. Signature motifs define three

DUF89 subfamilies: subfamily I has a CxxC motif plus a third con­served cysteine, and occurs in archaea and bacteria; subfamily II has the motif RTxK and occurs in bacteria, animals, and fungi; and subfamily III has the motif EGMGR and occurs in plants and animals as a standalone protein or as a fusion with panto­thenate kinase (PanK). Purified proteins from each subfamily showed metal-dependent phos­ph­at­ase activity against diverse phosphometabolites including, for PanK DUF89 domains, phospho­pan­te­theine and its oxidized (S-sulfonate or sulfonate) forms. The crystal struct­ure of the yeast subfamily II protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near the conserved signature residues Asp254 and Asn255. That PanK DUF89 domains hydrolyze canonical and oxidatively dam­ag­ed PanK reaction products shows that they can limit harmful buildup of normal and abnormal CoA precursors; genomic evidence suggests a comparable role for DUF89 subfamily I in purine biosynthesis.

400-039-Z GIGANTEA Influences Carbon Export from Leaves in Arabidopsis Thaliana Eva Farre – Michigan State University Sean Weise – Michigan State University, Jesica Reemmer – Michigan State University, Rebecca Piasecki – Michigan State University The circadian clock is involved in the accumulation of transitory starch in leaves. GIGANTEA (GI) is a protein involved in multiple physiological processes including circadian rhythms and photoperiod control of flowering time. It has been proposed that GI could mediate the effect of the circadian clock on starch accumulation. We have analyzed the amount of starch in gi alleles displaying different circadian and flowering time defects. Our results indicate that the role of GI in starch accumulation is independent of its function in the circadian central oscillator. We also observed that the starch excess phenotype could be rescued by expressing GI in the vascular tissue. In addition, starch accumulation in gi alleles was related to a decrease in carbon partitioned to the roots and a reduction of cell wall ingrowths in the phloem parenchyma cells. Based on these results we propose that GI regulates carbon export from the leaves.

400-040-Y Reverse Genetic Studies of Sirtuins Assessing Protein Lysine Acylation in Arabidopsis Thaliana Under Abiotic Stresses Aleksandar Radakovic – University of Minnesota Dana Freund – University of Minnesota, Jerry Cohen – University of Minnesota, Adrian Hegeman – University of Minnesota Sirtuins are a family of highly conserved NAD+-dependent deacylating enzymes found throughout the cell. Lysine acetylation and other acylations are post translational modifications that occur on proteins with diverse functions. In mammals, there are 7 sirtuins that are implicated in a wide variety of cellular processes such as aging, transcription, and cellular metabolism. Lysine acylations longer than acetylation and their removal by sirtuins in mammals are novel findings; these acylations have been identified on histones as well as proteins involved in energy metabolism. Mammalian studies of sirtuins have described protein structures, subcellular localization, enzyme activity, protein targets and functions; however, studies of plant sirtuins are lacking. Presently, there are two sirtuin genes identified in Arabidopsis thaliana - SIRT1 and SIRT2. SIRT2 has been recently described as a mitochondrial localized enzyme with deacetylase activity. In our studies, we investigate the roles of SIRT1 and SIRT2 on global protein lysine acylation levels in Arabidopsis thaliana by analyzing homozygous TDNA insertion lines of SIRT1, SIRT2, and the double knockout. Specific lysine acyl antibodies will be used for western blot analyses including lysine acetylation, propionylation, butyrylation, crotonylation, malonylation, succinylation, and glutarylation. Currently, we are assessing the effects of abiotic stresses on lysine acylation levels in the sirtuin lines and the visible morphological phenotypes. The following abiotic stress conditions will be tested: heat (45℃, 5h), cold (3℃, 3h), and drought (desiccation, 2h). Our goal is to determine the

extent of lysine acylation in plants and measure changes during abiotic stresses to better elucidate and understand sirtuins’ regulatory roles.

400-041-Y Metabolic Characterization of Arabidopsis Thaliana Sirtuins Dana Freund – University of Minnesota Jerry Cohen – University of Minnesota, Adrian Hegeman – University of Minnesota Sirtuins from the Sir2 (silent information regulator 2) gene family are highly conserved, from prokaryotes to eukaryotes. Sirtuins are nicotinamide adenine dinucleotide (NAD+)–dependent protein deacetylase enzymes that have recently been described as general deacylases. Sirtuins target diverse protein substrates that regulate a broad range of cellular metabolism, especially central carbon metabolism. Lysine acylation is a readily reversible post-translational modification that can alter enzyme activity and protein function. Lysine acylation as a regulator of metabolism is a fairly new discovery and little is known about sirtuins and lysine acylation in plants beyond histone proteins. There are two Sir2 family genes, SIRT1 and SIRT2, in the reference plant Arabidopsis thaliana. Arabidopsis SIRT2 has deacetylase activity and is localized to the mitochondria. Here we explore the role sirtuins have on the regulation of acylation in Arabidopsis Col-0, SIRT1 and SIRT2 single and double knockout mutants by evaluating protein lysine acylation and metabolic profiles. Western blot analyses, using an anti-acetyl-lysine antibody to investigate global differences in lysine acetylation in the SIRT mutants compared to Col-0, are underway. Thus far, data indicate a large number of extranuclear proteins are acetylated in all genotypes. In mammals there are seven sirtuins and SIRT5 has high activity for the removal of lysine malonylation, succinylation, and glutarylation. Currently, we are investigating these novel lysine acylation modifications in Arabidopsis. To increase our understanding of the regulation of metabolism by sirtuins, a metabolomics comparison of the genotypes was also conducted. Principal component analysis (PCA) illustrates a separation of Col-0 and the sirtuin mutant metabolic profiles, suggesting sirtuins have a role in regulating plant metabolism. As photosynthetic autotrophs, plants are likely to have unique acylation-mediated metabolic regulation and downstream effects, especially in energy metabolism.

400-042-Z Cell Wall Composition and Digestibility Alterations in Brachypodium Distachyon Achieved Through Reduced Expression of the UDP-arabinopyranose Mutase David Rancour – U.S. Dairy Forage Research Center, USDA-ARS Ronald Hatfield – U.S. Dairy Forage Research Center, USDA-ARS, Jane Marita – U.S. Dairy Forage Research Center, USDAARS, Nicholas Rohr – University of Georgia, Robert Schmitz – University of Georgia Plant cell wall polysaccharide biosynthesis requires nucleotide-activated sugars. The prominent grass cell wall sugars [glucose (Glc), xylose (Xyl), and arabinose (Ara)], are biosynthetically related via the UDP-sugar interconversion pathway. RNA-seq analysis of Brachypodium distachyon UDP-sugar interconversion pathway-encoding genes indicates high gene expression in aerial organs with active cell wall biosynthesis. We sought to generate UDP-sugar interconversion pathway transgenic Brachypodium lines, resulting in cell wall carbohydrate composition changes with improved digestibility while maintaining normal plant stature. Both RNAi-mediated gene-suppression and constitutive gene-expression approaches were performed. Leaf cell walls from 336 T0 transgenic plants with normal appearance were screened for complete carbohydrate composition. RNAi mutants of BdRGP1, a UDP-arabinopyranose mutase, resulted in significant decreases in cell wall Ara content, but with minimal change in plant stature. Five independent RNAi-RGP1 T1 plant lines were used for in-depth analysis of plant cell walls. Real-time PCR analysis indicated that gene expression levels for BdRGP1, BdRGP2 and BdRGP3 were reduced in RNAi-RGP1 plants to 15-20% of controls. Cell wall Ara content was reduced by 23-51% of

control levels, but no changes in Xyl and Glc content were observed. Corresponding decreases in cell wall ferulic acid (FA), ferulic acid-dimers (FA-dimers), and cell wall p-coumarates (pCA) were observed. We demonstrated the cell wall pCA decrease corresponds to Ara-coupled pCA. Xylanase-mediated digestibility of RNAi-RGP1 Brachypodium cell walls resulted in a nearly twofold increase of released total carbohydrate. However, cellulolytic hydrolysis of cell wall material was inhibited in leaves of RNAi-RGP1 mutants. Our results indicated that targeted manipulation of UDP-sugar biosynthesis can result in biomass with substantially altered compositions, but highlighted the complex effect cell wall composition may have on digestibility.

400-043-Z Spectrofluorometric and High Performance Liquid Chromatography Assays to Detect 4-methylthiol-2hydroxybutyrate S-methyltransferase Activity in Ulva Intestinalis, Protein Purification, and Gene Candidate Identification Michelle McLauchlan – Mount Allison University Rohil Dureja – Mount Allison University, Jeffrey Waller – Mount Allison University Dimethylsulfoniopropionate (DMSP) is a secondary metabolite made by many macro- and micro-algae with diverse proposed roles. Once released, DMSP is degraded to dimethylsulfide (DMS), accounting for greater than 40% of atmospheric sulfur production and playing a role in global climate. Despite the importance of both DMSP and DMS, there is surprisingly little research on DMSP synthesis. DMSP is produced in a four-step enzymatic pathway in Chlorophyte algae. My research focuses on the third enzyme of the biosynthetic pathway, an S-adenosylmethionine (SAM) dependent 4-methylthiol-2-hydroxybutyrate (MTHB) S-methyltransferase. This enzyme catalyzes the conversion of MTHB to 4-dimethylsulfonio-2-hydroxybutyrate (DMSHB), the direct chemical precursor to DMSP. In order to detect MTHB S-methyltransferase activity, various coupled spectrofluorometric enzyme assays were developed, none of which were suitable for detecting enzyme activity in crude algal extracts. High performance liquid chromatography (HPLC) enzyme assays were then investigated that directly detect either MTHB and DMSHB or SAM and Sadenosylhomocysteine (SAH). MTHB S-methyltransferase activity was detected using the latter HPLC analysis in crude extracts of Ulva intestinalis. The development of this sensitive and reliable HPLC enzyme assay enabled us to explore protein purification approaches including ammonium sulfate precipitation, and various chromatographic separations via fast protein liquid chromatography (FPLC). The results of these experiments will be presented in addition to the identification of gene candidates. The discovery of the MTHB S-methyltransferase enzyme(s) and the genes that encode them will enhance and enable further studies on DMSP synthesis in U. intestinalis and other DMSP-producing marine species.

400-044-Y Deciphering the Fatty Acid Elongation Pathway in Botryococcus Braunii Race A Dan Browne – Texas A&M University Hnin Mwei – Texas A&M University, Timothy Devarenne – Texas A&M University Sustainability is one of the most pressing issues currently faced by civilization. Petroleum in particular presents a glaring example of an unsustainable energy source. Yet liquid fuels are undeniably the best product yet devised for converting energy to work. Thus the development of renewable, biomass-based, drop-in liquid fuels is a highly attractive proposition. Botryococcus braunii is a colonial green microalga that is renowned for its ability to accumulate liquid hydrocarbons as a significant fraction of its dry weight. There are three morphologically identical sub-species of B. braunii called "races" that are distinguishable only by the type of liquid hydrocarbon accumulated. The "race A" produces very long chain fatty acid (VLCFA)-derived alkenes. The fatty acid elongation pathway, responsible for synthesizing the alkene precursors, has not been thoroughly characterized in B. braunii race A. Using bioinformatics, molecular biology, and biochemistry, the present work will determine which enzymes are critical for generating VLCFA

alkene precursors in race A. The detailed molecular model of fatty acid elongation in race A developed here will serve as a foundation for deeper studies on the regulation of this process and will be useful for anyone who seeks to reconstitute this pathway in a heterologous system.

400-045-Y Incorporation of P-coumarates into the Cell Walls of Alfalfa Changes the Lignin Composition Ronald Hatfield – USDA-ARS Jane Marita – USDA-ARS, Dave Rancour – USDA-ARS In general, monocots can contain a significant amount of an ester-linked p-coumarate (pCA) in their cell walls, but its function is unclear. One hypothesis is that pCA aids in the formation of syringyl-rich regions during lignification. Alfalfa (Medicago sativa), a dicot, is a cultivated perennial forage crop and an important food component in dairy production systems around the world. Unlike many monocots, this dicot does not contain significant amounts of ester-linked pCA in its stem cell walls, but there appear to be measurable amounts present in its leaf cell walls. The pCA incorporated into leaves does not appear to be linked to lignin. Expression of a maize p-coumaroyl:CoA hydroxycinnamyl alcohol transferase (pCAT) in alfalfa was used to test the influence that ester-linked pCA has on lignin composition in alfalfa stem and leaf cell walls and to determine what affect any such changes have on its digestibility. In general, isolated lignin of alfalfa cell walls is guaiacyl-rich, especially in the leaves. If pCA-conjugates help in the formation of syringyl-based lignin, cell walls of pCAT-expressing alfalfa should exhibit both increased ester-linked pCA and sinapyl alcohol content of the lignin. This study revealed that fresh plant tissue and isolated alfalfa leaf and stem cell walls from pCAT-expressing lines had increased amounts of ester-linked pCA after alkaline hydrolysis when compared to controls. Total cell wall lignin amounts were similar even when pCAT expression was high. Gel-state 2D-NMR analysis of cell walls revealed changes in lignin syringyl and guaiacyl content in stems. Therefore, pCAT expression in the dicot, alfalfa, altered the lignin composition and increased the amount of ester-linked pCA on cell walls, especially in stems. This supports the hypothesis that ester-linked pCA on cell walls influences lignin formation and composition, but does not result in increased lignification.

400-046-Z Comparative Metabolomics of Dicot Nectar Composition Elizabeth Chatt – Iowa State University Basil Nikolau A primary function of nectar is to attract mutualistic insect partners such as pollinators and ants to promote out-crossing and provide protection from detrimental herbivores. Our knowledge of the composition, synthesis, and secretion mechanisms of nectar are astonishingly limited. Historically, studies of nectar have focused on quantifying the major sugars: glucose, fructose, and sucrose. In more recent years, it has been shown that nectar contains amino acids, proteins (nectarins), lipids, phenolics, organic acids, terpenoids, flavonoids, minerals, and additional carbohydrates (Bender et al., 2012). Utilization of an “omics” approach including transcriptomics, metabolomics, and proteomics, will expand our knowledge of nectar production. This approach is capable of discovering genes involved in synthesis and secretion of nectar, and can furthermore link those genes to their metabolic output. The current working hypothesis is that synthesis mechanisms of nectar are conserved amongst dicots and nectary locations. This study will sample a broad range of dicots including: 1) Brassica juncea, cv. Southern Giant Curled, 2) Gossypium hirsutum, cv. TM1, 3) Cucurbita maxima, cv. Big Max, 4) Phaseolus lunatus, cv. Willow Leaf, and 5) Nicotiana sp. Solanaceae. Nectar composition from these dicots will be analyzed using GC-GC-MS and LC-MS to complete the metabolomics portion of this study.

400-047-Z Wheat Germ Cell-free Based Approach for Exploring Ubiquitination Mediated by the Arabidopsis RING-type E3 Ligases Abdelaziz Ramadan – Proteo-Science Center, Ehime University Keiichirou Nemoto – Proteo-Science Center, Ehime University, Hirotaka Takahashi – Proteo-Science Center, Ehime University, Motoaki Seki – Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science, Kazuo Shinozaki – Gene Discovery Research Group, RIKEN Center for Sustainable Resource Science , Hiroyuki Takeda – Proteo-Science Center, Ehime University, Tatsuya Sawasaki – Proteo-Science Center, Ehime University The protein ubiquitination is a ubiquitous mechanism in eukaryotes. In Arabidopsis plant ubiquitin modification is mainly mediated by 2 ubiquitin activating enzymes (E1s), 37 ubiquitin conjugating enzymes (E2s) and more than 1300 ubiquitin ligase enzymes (E3s) with about 470 are of the RING-type E3s. Large fraction of these RING E3`s gene products is yet to be characterized in vitro. In our study, we used the RIKEN Arabidopsis full-length cDNA library (RAFL) with the “splitprimer” PCR method and the wheat germ cell-free system for establishing protein libraries of Arabidopsis E2 and RING E3 enzymes. We could express 35 Arabidopsis E2s and about 204 RING proteins, which represents about 95% and 40% of their proteome. Thioester assays using Dithiothreitol (DTT) showed DTT-sensitive ubiquitin conjugation for all E2s expressed. The activity of another 28 RING proteins were evaluated with AtUBC10 and/or a group of different E2s. All the RING E3s tested showed ubiquitin ligase activity. Our current target is how to effectively use the wheat germ-based protein libraries described here for substrate screening and analysis. Combination between “AlphaScreen”; a luminescence-based high-throughput protein-protein interaction screening technology and simple ubiquitination assay based on the wheat germ extract seems to be a promising approach for exploring E3 targets.

400-048-Y Taxonomic Relationships of Korean Native Chrysanthemum Taxa Based on Flavonoid Su Jeong Kim – Highland Agriculture Research Institute, NICS, RDA Tae Joung Ha – Research Policy Bureau R&D Evaluation Division, RDA, Yul Ho Kim – Highland Agriculture Research Institute, NICS, RDA, Hwang Bae Sohn – Highland Agriculture Research Institute, NICS, RDA, Su Young Hong – Highland Agriculture Research Institute, NICS, RDA, Jeong Hwan Nam – Highland Agriculture Research Institute, NICS, RDA, Dong Chil Chang – Highland Agriculture Research Institute, NICS, RDA, Jong Taek Suh – Highland Agriculture Research Institute, NICS, RDA, Jongyun Kim – Highland Agriculture Research Institute, NICS, RDA, Ki Sun Kim – Highland Agriculture Research Institute, NICS, RDA This study was conducted to compare flavonoids of 15 Korean native Chrysanthemum namely: luteolin 7-O-rutinoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside, apigenin, and acacetin-7-O-rutinoside using HPLC/MS. All flavonoids were identified and analyzed. Cluster analysis was performed for five flavonoids where in the dendrogram of relationships was deduced to15 Chrysanthemum taxa and categorized into three groups. Group I mostly had luteolin-7O-glucoside and acacetin-7-O-rutinoside. Different taxa were identified in thisgroup such as C. zawadskii ssp. acutilobum, C. zawadskii ssp. acutilobum var. alpinum, C. zawadskii ssp. acutilobum var. tenuisectum, C. zawadskii ssp. lucidum, C. zawadskii ssp. latilobum var. leiophyllum, C. makinoi, C. zawadskii ssp. coreanum, and C. lineare. Likewise, Group II was distinguished from other groups having higher apigenin contents and had only one taxon, C. indicum var. acuta. Group III had higher luteolin-7-O-rutinoside compared to the other two groups. C. zawadskii ssp. naktongense, C. zawadskii ssp. yezoense, C. zawadskii ssp. latilobum, C. indicum, and C. boreale Were the six taxa identified in this group. Characteristics of Chrysanthemum through cluster analysis, functional group of flavonoid compounds such as luteolin, apigenin, and acacetin showed differences.

400-049-Y Stable Isotope Labeling Systems for Whole Plants for Compound Identification and Metabolic Flux Estimation Erin Jewett – University of Minnesota Nathan Tivendale – University of Minnesota, Adrian Hegeman – University of Minnesota, Jerry Cohen – University of Minnesota Accurate compound identification is a critical part of any metabolomics experiment. It has been shown previously that stable isotope labeling techniques using 13C and 15N can be used to extend the mass range of unique elemental composition assignments in high resolution mass spectrometry (HRMS)-based metabolomics experiments. Use of this labeling information coupled with tandem mass spectrometry (MS/MS) can also help inform the structural elucidation of unknown compounds. These same data also have great utility in the estimation of metabolic flux. Outside of cell culture, however, the labeling of plant tissue using stable isotopes is a non-trivial task. Intact plants assimilate the vast majority of their carbon from atmospheric CO2, which is a much more difficult carbon source to control than those that can be incorporated into a growth medium such as sugars or bicarbonate. Here, two isotopic labeling experimental approaches are described and characterized for the extent of isotopic enrichment. The first is the labeling of the aquatic angiosperm, duckweed (Spirodela polyrhiza), incorporating 13C from glucose and 15N from nitrate in the liquid medium. The second experiment is the labeling of Brassica rapa subsp. chinensis (Chinese cabbage) using 13C from CO2 gas and 15N from nitrate in a hydroponics system. Preliminary experiments show the utility of both of these stable isotope labeling approaches for metabolite identification and flux estimation in intact plants.

400-050-Z Carbon Transport Between Mesophyll and Bundle Sheath Cells in Maize Stéphanie Arrivault – Max Planck Institute of Molecular Plant Physiology Toshihiro Obata, Manuela Guenther, Melanie Hoehne, Alisdair Fernie, Mark Stitt Photosynthesis in C4 plants takes place in the mesophyll (MC) and bundle sheath cells (BSC) with fixation of CO2 in the MC, shuttling of the fixed carbon via four-carbon acids (malate and/or aspartate) to the BSC where CO2 is released by decarboxylation and incorporated into the Calvin-Benson cycle (CBC). By concentrating CO2 at the site of RuBisCO the carboxylation of Ribulose 1-5 bisphosphate (RuBP) is favored over its oxygenation and therefore photosynthesis efficiency is increased. C4 plants are historically classified into three biochemical subtypes based on the major decarboxylation enzyme involved: NADP-ME, NAD-ME and PEPCK subtypes. However multiple lines of evidence suggest that subtypes might coexist in the same leaf. Our goal was to determine which pathways are involved (and to which extent) in maize, a C4 plant classified into the NADP-ME subtype, and to quantify the pool sizes of metabolites directly involved in C4 photosynthesis. For this purpose we supplied maize leaves with 13CO2, quenched them at various time intervals and used MS-based methods to monitor the incorporation of 13C into about 40 metabolites of central carbon metabolism, including malate, aspartate, PEP, pyruvate and CBC intermediates. In addition we determined metabolite gradients between MC and BSC in labelled material.

400-051-Z Identification and Characterization of a Gene Responsible for the Biosynthesis of Glucoraphasatin (4methylthio-3-butenyl Glucosinolate) in White Radish Tomohiro Kakizaki – National Institute of Vegetable and Tea Science Hiroyasu Kitashiba – Tohoku University, Zhongwei Zou – Tohoku University, Feng Li – Tohoku University, Nobuko Fukino – National Institute of Vegetable and Tea Science, Takayoshi Ohara – National Institute of Vegetable and Tea Science, Takeshi Nishio – Tohoku University, Masahiko Ishida – National Institute of Vegetable and Tea Science

Glucosinolates (GSLs) are secondary metabolites in Brassicaceae that play important biological roles in the plant defense system. GSLs are synthesized from amino acids and are divided into three groups (aliphatic, benzenic, and indolic GSLs), according to their precursors. The structure and composition of GSLs depend on plant species, developmental stages, and tissues. Genetic loci controlling structural and quantitative variation have been identified using natural variation among Arabidopsis accessions. White radish (Raphanus sativus L., 2n = 2x = 18) is an important root vegetable that generally contains glucoraphasatin (4-methylthio-3-butenyl glucosinolate), an aliphatic GSL derived from methionine. Because glucoraphasatin is specific to white radish, its biosynthesis pathway remains unclear. In our previous study, we identified a mutant, glucoraphasatin synthase1 (grs1), which lacks glucoraphasatin owing to a recessive mutation at a single locus. Here we identified and characterized GRS1 by map-based cloning. We first genotyped 96 F2 plants from a cross between grs1 and wild-type white radish and mapped the mutation to the end of linkage group 1. Fine mapping narrowed the mutation region to a 23-kb region covered by a single BAC (Bacterial artificial chromosome) clone. Although seven gene models were confined in this region, only a single gene indicated the difference in the gene expression level between grs1 and wild-type in leaves and roots. The results of gene structure analysis and homology search revealed that GRS1 encodes 2-oxoglutarate dependent dioxygenase. The grs1 allele has a large insertion in the first exon causing a null mutation. Complementation analysis using A. thaliana, which contains glucoerucin (4methylthiobutyl glucosinolate), a candidate precursor of glucoraphasatin, indicated that 2-oxoglutarate dependent dioxygenase is involved in the biosynthesis of glucoraphasatin. Thus, this discovery may have important implications for elucidating the biosynthesis of GSLs in white radish.

400-052-Y Functional Analysis of Biotin Carboxyl-carrier Protein-like (BCCPL) Proteins in Arabidopsis Geng Ding – Iowa State University Basil Nikolau – Iowa State University In plants, biotin is covalently bound to three catalytic proteins, homomeric acetyl-CoA carboxylase (hmACCase), heteromeric acetyl-CoA carboxylase (htACCase), and 3-methylcrotonyl-CoA carboxylase (MCCase). In these proteins the biotin-prosthetic group is covalently bound to the side chain of a lysine residue, and the domain that contains this modified lysine residue is called the biotin carboxyl-carrier protein (BCCP) domain. Sequence analysis of the Arabidopsis genome identifies three genes that encode proteins that have high sequence similarity with the BCCP subunit of htACCase, which we term BCCP-like (BCCPL) genes. Immunological, and chromatographic analyses indicate that these BCCPL proteins are not biotinylated, nor are they lipoylated. Confocal microscopic analysis of transgenic plants expressing BCCPLs tagged with a fluorescent protein, demonstrate that all BCCPLs are located in plastids. No phenotypic changes in plant growth and morphology are detectable in T-DNA insertional mutants of the three BCCPL genes. Double mutant lines generated by intermating tested whether the three genes are redundant to each other. Homozygous double mutants of bccpl1/bccpl3 and bccpl2/bccpl3 are viable and readily recovered, however, the double mutant bccpl1/bccpl2 is embryo-lethal. The fact that single mutants of each BCCPL gene are completely viable, while the bccpl1/bccpl2 double mutant is lethal, indicates that these latter two genes in combination encode an essential functionality. Genetic transmission of the male gametes of bccpl1/bccpl2 double mutant is reduced to ~50%. To further define the physiological function of these BCCPL proteins in fatty acid biosynthesis, we are determining the fatty acid content and profiles of seeds of single and double bccpl mutants, and the tertiary organization of BCCPL-containing complexes..

400-053-Y Analysis of Proteome-scale Protein Turnover in Arabidopsis Thaliana Seedlings and Its Application to the Plant Heat Stress Response

Kai-Ting Fan – University of Minnesota Aaron Rendahl – University of Minnesota, Wen-Ping Chen – Yeastern Biotech Co. Ltd, Dana Freund – University of Minnesota, Thomas McGowan – University of Minnesota, William Gray – University of Minnesota, Sanford Weisberg – University of Minnesota, Jerry Cohen – University of Minnesota, Adrian Hegeman – University of Minnesota Protein turnover, the balance between protein synthesis and degradation, is an important aspect of the regulation of cellular processes for organisms as they respond to developmental or environmental cues. Protein turnover in plants, in contrast to that of rapidly growing unicellular organismal cultures, is made more complicated by the high degree of amino acid recycling that results in significant transient isotope incorporation distributions that must be dealt with computationally for high throughput analysis to be practical. An algorithm implemented in R, ProteinTurnover, was developed to calculate protein turnover with transient stable isotope incorporation distributions in a high-throughput automated manner using high resolution mass spectrometry and proteomic analysis of stable isotopically labeled plant material. It extracts isotopic distribution information from raw MS data for large numbers of peptides identified by tandem MS from datasets of either isotopic label dilution or incorporation experiments. Variable isotopic incorporation distributions were modeled by maximum likelihood estimation (MLE) using beta-binomial distributions to deconvolute (1) the natural abundance, (2) newly synthesized, partial labled peptide and (3) fully-labeled peptide distributions. MLE was performed to calculate the distribution abundance proportion of old and newly synthesized peptides. Half-life or turnover rate constant of each peptide and protein was calculated from changes in the distribution abundance proportions using non-linear regression. This algorithm makes it possible to measure the proteome turnover rates of Arabidopsis seedling roots: half-lives of 64, 54, and 133 proteins were measured in enriched soluble, organelle, and microsomal fractions, respectively, using isotopic incorporation and UHPLC-MS/MS. ProteinTurnover was also used to measure changes in proteome turnover in enriched soluble, organelle, and microsomal fraction of Arabidopsis seedling shoots or roots comparing 22°C and 30°C growth conditions. In our study, a total of 468 proteins with significant changes in turnover rate in response to elevated temperature were identified.

400-054-Z Molecular and Biochemical Characterization of Daurichromenic Acid Synthase from Rhododendron Dauricum Miu Iijima – University of Toyama Futoshi Taura – University of Toyama, Hiromichi Kenmoku – Tokushima Bunri University, Hironobu Takahashi – Tokushima Bunri University, Masao Toyota – Tokushima Bunri University, Jung-Bum Lee – University of Toyama, Fumiya Kuro – University of Toyama, Yoshinori Asakawa – Tokushima Bunri University Rhododendron dauricum is distributed in the northern China, and Hokkaido, Japan. This plant produces daurichromenic acid (DCA), a unique meroterpene with highly potent anti-HIV activity. DCA is biosynthesized by an oxidocyclase named DCA synthase via stereoselective oxidative cyclization of grifolic acid. DCA synthase may be applicable to produce DCA easily. The purpose of this study is to clarify structure and function of DCA synthase, and open the way to biotechnological production of DCA. DCA synthase cDNA contained a 1,602-bp open reading frame encoding a 533-amino acid polypeptide. The deduced primary structure showed > 40 % identities with various flavoprotein oxidases such as cannabinoid synthases and berberine bridge enzyme. In addition, the sequence encoded putative N-terminal signal sequence, Asn-glycosylation sites, and RSGGH motif for covalent FAD attachment, suggesting that DCA synthase is a kind of flavoprotein. The recombinant DCA synthase was expressed using a Pichia pastoris expression system, and purified by hydroxyapatite and CM-Toyopearl column chromatographies. The purified protein showed a glycosylated protein band with average molecular mass of 66 kDa on SDS-PAGE. The recombinant enzyme catalyzed the oxidocyclization in the presence of molecular oxygen, and produced (＋)-DCA and hydrogen peroxide. These catalytic properties were similar to those

previously reported for the native enzyme. DCA synthase is the first plant enzyme that catalyzes oxidocyclization of the farnesyl group of a natural product. With respect to the gene expression profile, RT-PCR analysis confirmed that DCA synthase is exclusively expressed in the glandular scales, the secondary metabolite-producing glandular organ attached on the leaves and twigs. DCA might be a defensive component of R.dauricum plant. These results provided new insights into molecular structure and biochemical properties of DCA synthase from R. dauricum.

400-055-Z Detergents Improve the Solubility and Purification of Recombinant Diacylglycerol Acyltransferases from E. Coli and Yeast Heping Cao – USDA-ARS Southern Regional Research Center Kandan Sethumadhavan – USDA-ARS Southern Regional Research Center, Xiaoyu Wu – Jiangxi Agricultural University, Xiaochun Zeng – Yichun University Diacylglycerol acyltransferases (DGATs) are integral membrane proteins responsible for the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Understanding DGATs in plant oil biosynthesis will help to create new oilseed crops with value-added properties. Plants have different forms of DGATs with nonredundant functions in TAG biosynthesis. Three forms of DGATs are identified in tung tree (Vernicia fordii) but DGAT2 has been suggested as the major form of DGATs responsible for the synthesis of tung oil, which contains approximately 80% high-value eleostearic acid in the TAG fraction of tung tree seeds. However, native and recombinant DGATs were only purified in a few reports. The objective of this study was to explore the possibilities of using various detergents to improve the solubility of recombinant DGATs so that soluble protein could be purified for biochemical studies. Recombinant tung tree DGATs contained a maltose binding protein (MBP) and/or a poly-histidine (His) at its N- and C-termini, respectively, were expressed in E. coli and yeast. Recombinant DGATs were solubilized by 7 detergents (Brij 35, CHAPS, NP-40, SDS, Triton X-100, Tween 20 and Tween 80) followed by affinity-based purification. The proteins were barely purified under native conditions by amylose resin, Ni-NTA beads, tandem affinity beads, size exclusion and Mono Q anion exchange chromatography. Addition of detergents to insoluble fraction or soluble extract resulted in improved DGAT solubility. The proteins were significantly purified from detergent-treated protein mixtures by affinity beads. The purified DGATs were used for studying the biochemical properties and enzymatic activity (Supported by the USDA-ARS Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6054-41000-102-00D).

400-056-Y Investigating the Post-embryonic Function of Plastidial NAD-dependent Malate Dehydrogenase in Arabidopsis Tina Schreier – ETH Zurich Martha Stadler – ETH Zurich, Samuel Zeeman – ETH Zurich, Oliver Kötting – ETH Zurich Malate dehydrogenases (MDH) catalyse the reversible interconversion of malate and oxaloacetate, using NAD(H) or NADP(H) as a cofactor. Plant tissues contain multiple isoforms of MDH, which are claimed to play an important role in balancing the availability of redox equivalents between different cellular compartments. Of the nine MDH isoforms in Arabidopsis, only two are plastidial - one is NADP-dependent, while the other is NAD-dependent (pdNAD-MDH). The pdNAD-MDH isoform shows by far the most severe knockout phenotype among all the MDH isoforms. We could show that a pdnad-mdh-null mutation is lethal (Beeler et al, 2014: Plant Physiol. 164: 1175–1190): pdnad-mdh embryos arrest in the globular-to-heart transition stage and therefore, pdNAD-MDH is essential for embryo development in Arabidopsis.

The constitutive silencing line, miR-mdh-1, with strongly reduced pdNAD-MDH levels, is dwarfed, shows a disturbed chloroplast ultrastructure, and exhibits severe pleiotropic effects compared to the wild type. Since these effects are alleviated as the plants grow, we wondered whether they result from developmental defects during embryogenesis, or specifically due to pdNAD-MDH silencing after germination. To exclusively investigate the post-embryonic function of pdNAD-MDH, we expressed pdNAD-MDH under the control of the embryo-specific ABI3 promoter in the pdnad-mdh knockout line. Embryo-specific expression of pdNAD-MDH rescued embryo-lethality. However, the growth of the pABI3a::pdNAD-MDH lines was even more severely reduced than that of miR-mdh-1. Furthermore, these plants failed to develop any chlorophyll, and while they initiated true leaves, they did not develop further and died within a few weeks. These findings suggest that pdNAD-MDH is not exclusively essential for embryogenesis, as it also plays a crucial role for the establishment of mature plants.

400-057-Y Characterization of a Novel Spermine Hydroxycinnamoyl Transferase in Solanum Richardii Fruits Tianbao Yang – USDA-ARS Food Quality Lab Hui Peng, Bruce Whitaker – USDA-ARS, Lingfei Shangguan, Wayne Jurick – USDA-ARS Polyamine hydroxycinnamoyl transferases (HT) catalyze biosynthesis of hydroxycinnamic acid amides (HCAAs)from polyamines and hydroxycinnamic acid. HCAAs are important in plant development and adaptation to environmental changes. They are also beneficial to human health due to their anticarcinogenic and antihypertensive effects. Like most plants, the predominant HCAA in cultivated eggplant fruit (Solanum. melongena) is hydroxycinnamoyl-spermidine (HCSpd). However, wild eggplant fruit (S. richardii) is rich in hydroxycinnamoyl-spermidine (HCSpm). It has been shown that HCSpm confers greater biological and medicinal values. In this study, a novel HT responsible for the biosyntheses of HCSpms was identified in S. richardii, and designated as SrSpmHT (spermine hydroxycinnamoyl transferase). SrSpmHT was highly expressed in both young and mature fruits, while its ortholog (SmSpmHT) in the cultivated eggplant was hardly detected in all tissues including leaves, flowers, young and mature fruits. SrSpmHT has conserved HXXXD and DFGWG domains of BAHD family. Enzyme activity assays showed that SrSpmHT exclusively catalyzed one hydroxycinnamoyl moiety to Spm. As for acyl donor substrate, SrSpmHT prefers caffeoyl CoA, followed by feruloyl-CoA and ρ-coumaroyl-CoA. Molecular docking suggests that Spm is better than Spd to fit to the binding pockets SrSpmHT. These results indicate that SrpmHT is an exclusive spermine HT for the HCSpms. The results provide a platform for metabolic engineering of HCAA pathway to generate HCSpm rich fruits for eggplant and other fruits.

400-058-Z Accurate Retention Prediction in LC-MS by “Retention Projection” and Its Prediction Accuracy in Complex Plant Matrices Nu Wang – University of Minnesota Paul G. Boswell The uncertain of what metabolites are produced at each stage of plant growth becomes a major limitation in any plant resource analysis. LC-MS is widely applied to identify metabolites. Unfortunately, its chromatographic retention information is useless in metabolite identification across labs since it is irreproducible, while the mass information alone is insufficient to identify metabolites such as isomers. “Retention projection” is a novel methodology that makes retention information predictable in RPLC-MS by back calculating the real behaviors of LC instrumentation by using the retention times of well characterized standards. However, RPLC-MS that generally works for nonpolar compounds is insufficient to identify all metabolites. Therefore, we extended retention projection from RPLC-MS to HILIC-MS which is widely used to identify polar compounds.

23 compounds (10 standards and 13 test compounds) that are chemically diverse were selected to back calculate the produced gradient by LC instrument, and measure the prediction accuracy of retention projection in 3 gradients (10 min, 20 min, and 30 min) at 3 flow rates (0.2 ml/min, 0.4 ml/min, and 0.8 ml/min). The overall prediction error of these 13 test compounds by retention projection in HILIC-MS is 0.53%-1.83% and the smallest prediction error is occured in 20 min gradient at 0.4 ml/min flow rate. This high prediction accuracy is compatible with RPLC-MS by the same method and accurate enough compared with other publications (>5% prediction error). To successfully apply retention projection to identify more metabolites in more complicated natural matrices, we measured the overall prediction error of test compounds by RPLC-MS in blood and urine by retention projection. Our preliminary data indicates the background of samples does not affect the accuracy of retention projection at typical concentrations. To thoroughly explore the limits of retention projection in natural samples, we systematically study how plant matrices affect the accuracy of retention projections.

400-059-Z Characterization of Oryza Sativa ACYL ACTIVATING ENZYME3 (OsAAE3) Peter Lambert – Rice University Paul Nakata – USDA/ARS Children’s Nutrition Research Center Oxalate, the smallest of the dicarboxylic acids, is produced in many plants. This acid has been shown to play an important role in both plant physiology and defense, specifically in regards to metal detoxification, calcium regulation, sucking and chewing insect deterrence, and the production of calcium oxalate crystals. The most studied pathway in plants for oxalic acid degradation involves an oxalate oxidase that converts oxalic acid to CO2 and H2O2. This activity has been detected extensively in monocots, including wheat, barley, and rice. Recently, a novel pathway of oxalate metabolism was proposed in Arabidopsis, a plant that lacks oxalate oxidase activity. Acyl activating enzyme (AAE) 3, encoding an oxalyl-CoA synthetase, was proposed to catalyze the first step in this novel pathway of oxalate catabolism. The discovery of AtAAE3 has sparked a search for homologous pathways in other plants including crop plants. This study identifies Oryza sativa ACYL-ACTIVATING ENZYME3 (OsAAE3: Os04g0683700) as a gene encoding an oxalyl-CoA synthetase. This activity is particularly interesting given that rice also possesses the more common oxalate catabolic pathway utilizing oxalate oxidases. To investigate the function of OsAAE3 we have produced a recombinant enzyme that demonstrates activity against oxalate in vitro, exhibiting Michaelis-Menten kinetics with a Km of 98.24 ± 19.94 µM and a Vmax of 6.880 ± 0.3218 µmol/min/mg. Additionally, we have introduced OsAAE3 into Ataae3 null mutants, and found that plants expressing OsAAE3 in the mutant background have a lower concentration of tissue oxalate than the Ataae3 null mutants. This decrease in oxalate is accompanied by a reduction in calcium oxalate crystals seen in the OsAAE3 plants.

400-060-Y Characterization of Cysteine Mutants of Ascorbate Peroxidase from Panicum Virgatum L Paul Twigg – University of Nebraska-Kearney Tara Bjorkland – University of Nebraska-Kearney, Frank Kovacs – University of Nebraska-Kearney, Amanda Glass – University of Nebraska-Kearney, Zachary Porter – University of Nebraska-Kearney Switchgrass (Panicum virgatum L.) has been targeted for development as a bioenergy species. Recently an early version of its genome has been released permitting a route to the cloning and analysis of key proteins. Ascorbate peroxidases (APx) are an important part of the antioxidant defense system of plant cells and present a well-studied model to understand structure-function relationships. Analysis of the genome revealed that switchgrass encodes several cytosolic ascorbate peroxidases with apparent varying levels of tissue expression. We have cloned and expressed in E. coli cells to

obtain purified active protein a major cytosolic ascorbate peroxidase. A homology model has been generated from an ascorbate peroxidase crystal structure with high sequence similarity (>89%). This model was used to predict amino acids that could be mutated in an effort to engineer disulfide bonds into the protein. Two pairs of mutants were attempted but only one was expressible (M36C/A100C). Characterization of Pvi-APx (M36C/A100C) has been carried out using circular dichroism and variable temperature ascorbate assays. Our studies indicate that this double mutation reduces both activity and thermal stability. Further studies were done on mutants of wildtype APX where normally occurring cysteines were replaced and no significant impact on activity was observed.

400-061-Y Examining the Role of BAM6: A Largely Uncharacterized, Plastidic β-amylase in Arabidopsis Amanda Storm – James Madison University Catherine Torres – James Madison University, Megan Hines – James Madison University, Jonathan Monroe – James Madison University β-amylase proteins are one the principle enzyme families involved in starch degradation in plants. In Arabidopsis there are nine β-amylase (BAM) genes; however, only four of them have been shown to encode proteins capable of catalyzing starch degradation. One of these catalytic BAMs, BAM5, is localized to the cytoplasm and is found primarily in sieve elements. BAM1 and BAM3 are the most well-studied of the catalytic BAMs. They are chloroplast-localized and provide the principal β-amylase activity for starch breakdown during the day and night, respectively. Little is known about the remaining catalytic BAM, BAM6, which is also located in the chloroplast. We are using a combination of techniques to study the properties and potential function of this lesser known BAM. Recombinant BAM6 is being used to determine catalytic properties such as pH and temperature optima and kinetic parameters. Plants lacking different combinations of catalytic BAMs have been generated to study the role of BAM6 in vivo. A phenotype was observed where leaves from older, flowering plants (>8-week-old) lacking both BAM6 and BAM3 break down less starch than leaves lacking BAM3 alone. This phenotype is not seen in leaves of younger (5-week-old) plants, so it appears that BAM6 may have an agedependent function. An additional finding was that a triple knockout plant lacking BAM5, -1, and -3 broke down more starch and had higher total β-amylase activity than a double mutant lacking only BAM5 and -3. An increase in BAM6 mRNA was also detected in the triple mutant indicating that BAM6 is being induced and is capable of partially compensating for loss of the two primary starch degradation enzymes. Intriguingly, this increase in β-amylase activity is only detected if the cytoplasmic BAM5 is absent as a similar effect is not seen in the double mutant lacking BAM1 and -3.

400-062-Z CrBPF1 Functions in Regulation of Terpenoid Indole Alkaloid Biosynthetic and Regulatory Genes Guy Sander – University of Minnesota Chun Yao Li – University of Minnesota, Alex Leopold – University of Minnesota, Jacqueline Shanks – Iowa State University, Susan Gibson – University of Minnesota Terpenoid indole alkaloid (TIA) biosynthesis in Catharanthus roseus is a complex and highly regulated process. Understanding the biochemistry and regulation of the TIA pathway is of particular interest as it may allow the engineering of plants to accumulate higher levels of pharmaceutically important alkaloids. Towards this end, we generated a transgenic C. roseus hairy root line that overexpresses the CrBPF1 transcriptional activator under the control of a β-estradiol inducible promoter. CrBPF1 is a MYB-like protein that was previously postulated to help regulate the expression of the TIA biosynthetic gene STR. However, the role of CrBPF1 in regulation of the TIA and related pathways had not been previously characterized. In this study, transcriptional profiling revealed that overexpression of CrBPF1 results in increased transcript levels for genes from both the indole and terpenoid biosynthetic pathways that provide precursors for TIA biosynthesis, as well as for genes in the TIA biosynthetic pathway. In addition, overexpression

of CrBPF1 causes increases in the transcript levels for 10 out of 12 genes postulated to act as transcriptional regulators of genes from the TIA and TIA feeder pathways. Interestingly, overexpression of CrBPF1 causes increased transcript levels for both TIA transcriptional activators and repressors. Despite the fact that CrBPF1 overexpression affects transcript levels of a large percentage of TIA biosynthetic and regulatory genes, CrBPF1 overexpression has only very modest effects on the levels of the seven TIA metabolites analyzed. This finding may be due, at least in part, to the up-regulation of both transcriptional activators and repressors in response to CrBPF1 overexpression, suggesting that CrBPF1 may serve as a “fine-tune” regulator for TIA biosynthesis, acting to help regulate the timing and amplitude of TIA gene expression.

400-063-Z The Sucrose Synthase Isozyme RcSUS1, Phosphorylated in Vivo on Ser-11 by the Ca2+-dependent Protein Kinase RcCDPK2, Provides the Sucrolytic Activity Necessary for Rapid Triacylglycerol Biosynthesis in Developing Castor Oilseeds Eric Fedosejevs – Queen’s University Sheng Ying – Queen’s University, Yi-Min She – Shanghai Center for Plant Stress Biology, Erin Anderson – University of Guelph, Robert Mullen – University of Guelph, William Plaxton – Queen’s University Sucrose synthase (SUS) catalyzes the UDP-dependent conversion of sucrose into UDP-glucose and fructose, and is an increasingly important target for crop improvement via metabolic engineering. However, the pathways and control of sugar metabolism in oilseeds are poorly understood. Our recent research with castor oilseed (Ricinus communis) has established that: (i) the SUS isozyme RcSUS1 is the dominant sucrolytic enzyme in the triacylglycerol-rich endosperm of the developing seed, (ii) dynamic and high stoichiometric in vivo phosphorylation of RcSUS1 occurs at a conserved Ser11 residue and appears to protect RcSUS1 from proteolysis, and (iii) RcSUS1 is phosphorylated on Ser-11 by a Ca2+dependent RcSUS1-kinase during seed development (Fedosejevs et al. 2014 J Biol Chem). We have identified the RcSUS1-kinase as RcCDPK2 via purification by FPLC followed by LTQ-FT MS/MS. Recombinant RcCDPK2 expressed in E. coli catalyzes the rapid Ca2+-dependent phosphorylation of dephospho-RcSUS1 at Ser-11. Based on transient expression of 35S:RcCDPK2-GFP in tobacco suspension cells, RcCDPK2 appears to be primarily cytosolic, as with RcSUS1. However, microsomal isolation studies suggest that, similar to RcSUS1, approximately 17% of RcSUS1-kinase activity is membraneassociated. RcCDPK2 expression and RcSUS1-kinase activity are highest during early seed development, when RcSUS1 phosphorylation at Ser-11 is also at its peak. Interestingly, while removal of sucrose supply via fruit excision rapidly decreases RcSUS1 Ser-11 phosphorylation, it has no noticeable effect on total RcSUS1-kinase activity, despite halving the proportion of RcSUS1-kinase activity that is membrane-associated. Studies are ongoing to further characterize the purified native RcSUS1-kinase by examining its substrate specificity and its regulation by metabolite effectors. Together, our research aims to elucidate the role of RcCDPK2 in maintaining the high levels of RcSUS1 necessary for rapid triacylglycerol biosynthesis in developing castor oilseeds.

400-064-Y Discovery of a Novel Family of Cytokinin Biosynthetic Enzymes in Endophytic Fungi William Beeson – Dow Agrosciences Endophytic fungi are important symbionts of many plant species. They are responsible for the production of potent secondary metabolites and can confer beneficial traits to their hosts, such as drought tolerance and stand persistence in tall fescue. Although much is known about the toxic compounds produced by endophytic fungi, little is known about how these organisms confer beneficial agronomic traits to their host. In this work, experiments documenting the discovery of a novel family of cytokinin biosynthetic enzymes, termed cytokinin synthases, are reported. Cytokinin

synthases are bifunctional enzymes composed of an N-terminal isopentenyl transferase-like domain and a C-terminal phosphoribosyl hydrolase-like domain. They catalyze the formation of isopentenyl adenine directly from nucleotide donors and dimethylallyl pyrophosphate. Cytokinin synthases are present in many species of endophytic fungi, especially in the Clavicipitaceae family. An analysis of recently published transcriptomic studies suggests that the endophyte, Epichloe festucae, expresses cytokinin synthases under conditions of stress while inside its host. A model for how these enzymes may confer beneficial traits to their plant hosts will be presented.

400-065-Y Functional Characterization of the M-type Rice Thioredoxin Protein and Its Interaction Proteins Jung Ro Lee – National Institute of Ecology Hae-Ryong Song – National Institute of Ecology Although subcellular localization and substrate specificity of thioredoxin isoforms have been characterized, there is little information on the functions of m-type plant thioredoxin proteins or their interaction targets. Here, we describe the functional characterization of an Oryza sativa thioredoxin m isoform (OsTrxm). We undertook yeast two-hybrid screening of Oryza sativa leaf library using OsTrxm as a bait and found three in vivo interaction proteins, Pex14 and two Pex5 protein variants. Furthermore, the N-terminal domain of OsTrxm was sufficient for interaction with these peroxisome proteins and two active cysteine residues of OsTrxm were important for the interactions. To verify whether OsTrxm and the target proteins can be co-localized, we examined subcellular localization of OsTrxm and the peroxin proteins fused with GFP or RFP signals in Arabidopsis protoplast cells. OsTrxm was localized in the cytosol as well as chloroplasts, suggesting that OsTrxm can be interacted with the target proteins in the cytosol. Our results indicate that OsTrxm may play important roles in peroxisome biogenesis as well as the redox regulation of chloroplast target proteins.

400-066-Z Development of Multiplex PCR Methods for Detection of LM Crop in Nature Environment Wonkyun Choi – National Institute of Ecology Jung Ro Lee – National Institute of Ecology, Hae-Ryong Song – National Institute of Ecology To improve insects and herbicide resistance, abiotic stress tolerance, nutritional quality, genetically modified (GM) crops have been developed. Self-sufficiency rate of grain in Korea has been decreased and ten million tons of LM crops were imported for FFP in 2014. Over the years, LM crop was found in nature environment because of the unintentional release. Moreover, the potential risk of gene flow, foreign gene transfer from GM corps to its related species or wild native species, has been cited and argued as an environmental concern. Recently, the multiplex PCR method has been developed as simultaneous detection tools to recognize quickly and efficiently LM events. In this research, the multiplex PCR method was developed to detect fifteen individual LM crop events which had approved to use in Korea. The eventspecific primers were designed for grouped LM events and their genomic DNAs were used as templates for simultaneously characterization specific LM events. Furthermore, 13 suspicious LM events obtained from natural environmental monitoring were used to confirm detection efficiency of our multiplex PCR assay and event specific PCR products were amplified from all samples. As a results of LMO monitoring in 2013, 21 LM events(18 sites) were escaped to nature environment. The results indicate that it is a suitable method for the identification of LM events, and considered to be used as a basis for the scientific management and safety surveillance against unintentional release of LM crops.

400-067-Z Starch and Sucrose Metabolic Pathways in Chinese Chestnut Seeds Heping Cao – USDA-ARS Lin Zhang – Central South University of Forestry and Technology, De-Yi Yuan – Central South University of Forestry and Technology, Xiaofeng Tan – Central South University of Forestry and Technology Chinese chestnut (Castanea mollissima) provides a good source of carbohydrates as food and feed, but little is known about starch and sucrose metabolism in the seeds. The objectives of this study were to determine seed composition profiles and identify genes involved in starch and sucrose metabolic pathways. Seed component analysis showed that starch was the major composition and rapidly accumulated during seed endosperm development. Amylopectin was approximately three-fold of amylose content in chestnut starch. Illumina platform-based transcriptome sequencing generated 56,671 unigenes in two cDNA libraries from seed endosperms collected at 45 and 75 days after flowering (DAF). 1,537 unigenes showed expression differences ≥ 2-fold in which 570 unigenes were upregulated and 967 unigenes were down-regulated in the two stages of seeds. We identified 152 unigenes involved in starch and sucrose metabolism, including one for glycogenin glucosyltransferase, four for adenylate transporter (brittle1–type), three for ADP-glucose pyrophosphorylase (AGP, not brittle2- or shrunken2-type), eight for starch synthase (SS), two for starch branching enzyme, five for starch debranching enzyme, eleven for sucrose synthase, and three for sucrose-phosphate synthase. Among them, 58 unigenes showed a ≥ 2-fold expression difference between the 45 and 75 DAF seeds including 11 up- and 47 down-regulated unigenes. The expression of 21 unigenes putatively coding for major enzymes in starch and sucrose metabolism was validated by qPCR using RNA from five seed stages. Expression profiles and correlation analysis indicated that the mRNA levels of AGP (large and small subunits), granule-bound SS2, soluble SS1 and SS4 were well-correlated with starch accumulation in the seeds. This study suggests that starch biosynthesis pathway in Chinese chestnut is similar to that of potato tube/Arabidopsis leaves and differs from that of maize endosperm. The information provides valuable metabolite and genetic resources for future research in starch and sucrose metabolism in Chinese chestnut tree.

400-068-Y New Facettes of the Flexible Interplay Between Energy Production and Consumption by Challenging the Chloroplast Redox-poise Renate Scheibe – University of Osnabrueck Vera Linke – University of Osnabrueck, Jennifer Selinski – University of Osnabrueck, Guy Hanke – University of Osnabrueck During photosynthesis, reducing equivalents are generated as required for reductive processes, mainly in the form of NADPH and of reduced ferredoxin. At the same time, the proton gradient leads to the production of ATP. Since supply and demand will vary constantly, in particular under fluctuating light, the chloroplast is equipped with various poising systems, such as the malate valve for export of excess reducing equivalents [Scheibe, 2004]. Plants with decreased expression of NADP-malate dehydrogenase (NADP-MDH) and increased expression of ferredoxin-NADPH reductase (FNR) were employed to further investigate the regulatory mechanisms that can balance the fluxes through the ATP and NADPH pools for continuous metabolic functions. In particular, the roles of thylakoid-bound and soluble FNR were investigated by expressing maize FNR isoforms in an Arabidopsis NADP-MDH-knockout background [Hebbelmann et al., 2012; Twachtmann et al., 2014]. From transcript analyses of the various electron-consuming reactions it is possible to extend the systems used for poising to other assimilatory processes such as N-assimilation and to NADH-dependent reactions including the “dark malate valve” [Selinski & Scheibe, 2014]. A hypothesis is presented that comprises various basic metabolic pathways as well as protective systems operating to maintain redox-homeostasis during photosynthesis under variable conditions.

References: Scheibe, R. (2004): Physiol. Plant. 120, 21-26 Selinski, J. & Scheibe, R. (2014): Plant Signal. Behav. 9(7), e29057 Twachtmann, M. et al. (2014): Plant Cell 24, 2979-2991 Hebbelmann, I. et al. (2012): J. Exp. Bot. 63, 1445-1459

400-069-Y Antimicrobial and Phytotoxic Activity of Organic Extract from Different Species of Pleroutus Mushrooms Against Lemna Minor (Duck Weed) Salma Bibi – Texas A&M University Asma Noshad – The University of Agriculture, Peshawar, Mudassar Iqbal – The University of Agriculture, Peshawar The antimicrobial and phytotoxicity of organic extracts of three macro-fungi Pleurotus ostreatus, Pleurotus florida and Pleurotus citrinopileatus was carried out to assess their potential as possible green pesticides in agriculture sector. The fruiting body and mycillium as natural sample whereas fermentation filtrate was used as a lab culture of all above species of mashrooms were extracted with ethyl acetate. The experimental results showed that all the three extracts from fruiting body, mycillium and fermentations filtrate of Pleurotus ostreatus were effective against the fungal isolates (Pinicillium, Rhisopus stolonifer and Alternaria alternate) and bacterial isolates (Ervinia caratovora and Xanthomonas) while the mycillial extract of P. ostreatus showed no antibacterial activity against the tested isolates i-e Ervinia and Xanthomonas. All the three extracts of P. florida showed significant antifungal activity, whereas 0% antifungal amd antibacterial activities were shown by the mycillial extract of P. Florida. Similarly the phytoxicity of all extracts was assessed against very sensitive aquatic weed (Lemna minor). The results showed a potent phytotoxity of fruiting bodies extract of P. ostreatus with 14.05 µg mL-1 LC50 with nearly 70% mortality of L. minor after 72 hours. The fruit bodies and fermentation filtrate extract of P. citrinopileatus showed 80% mortality of Lemna minor after 72 hour exposure at 300 µg mL-1 concentration. The results of Lemna minor mortality were both time and concentration dependent.

Biochemistry: Specialized Metabolites 400-070-Z Investigating the Biological Function of Diterpenoids in Rice Plant Defense Xuan Lu – Iowa State University Meimei Xu – Iowa State University, , Bing Yang – Iowa State University Among the various responses of plants to microbial infection is the deployment of an arsenal of antibiotic natural products often termed phytoalexins. While these have been identified on the basis of their antibiotic activity in vitro, their sheer numbers begs the question of relevance. The vast majority of phytoalexins from the important crop plant rice (Oryza sativa) are diterpenoids, whose biosynthesis proceeds via either ent- or syn- copalyl diphosphate (CPP) intermediates, and which were isolated on the basis of their antibiotic activity against the fungal blast pathogen Magnaporthe oryzae. While rice encodes for a single syn-CPP synthase, Os-CPS4, its genome contains two ent-CPP synthase, Os-CPS1, which seems to be solely responsible for gibberellin phytohormone biosynthesis, and Os-CPS2, inducible transcription of which suggests a role in the production of phytoalexins. However, cps4 rice plants do not exhibit increased susceptibility to M. oryzae, instead exhibiting decreased allelopathy. Here, cps2 rice plants are found to exhibit increased susceptibility to not only M. oryzae, but also the bacterial leaf blight pathogen Xanthomonas oryzae

pv. oryzae (Xoo). In addition, cps4 plants are found to exhibit decreased, rather than increased, susceptibility to both M. oryzae and Xoo. This seems to arise from redirected metabolic flux, with blockage of syn-CPP production leading to increased levels of the effective ent-CPP derived phytoalexins. These results not only establish the relevance of Os-CPS2 dependent diterpenoid phytoalexins to rice plant defense against both fungal and bacterial pathogens, but also some insight into the evolution of flux balance between related branches in more specialized metabolism.

400-071-Z Genetics of Cannabinoid Accumulation in the Glandular Trichomes of Cannabis Sativa M. David Marks – University of Minnesota George Weiblen – University of Minnesota, Jonathan Wenger – University of Minnesota, Kathleen Craft – Concordia University Chicago, Mahmoud Elsohly – University of Mississippi, Zlatko Mehmedic – University of Mississippi, Erin Treiber – University of Minnesota Cultivars of Cannabis sativa are grouped into either marijuana or hemp categories on the basis of their accumulation of either tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA), respectively. These compounds accumulate in the glandular trichomes located on the bracts of both types. Hemp cultivars have long been used for the durable fibers they produce and for their oil seeds, while the psychoactive drug containing marijuana cultivars have been used recreationally, religiously, and medicinally. The molecular genetic mechanisms controlling cannabinoid inheritance are not understood. To gain a better understanding of these mechanisms hemp and marijuana lines were crossed and F1 plants were induced to self. An analysis of the resulting F2 population has led to the creation of the first linkage map for Cannabis and to the identification of QTLs controlling drug content. The analyses also suggest a history of gene duplications that likely played key roles in the diversification of the two types of cultivars.

400-072-Y Plants as Synthetic Biology Platforms Toni M Kutchan – Donald Danford Plant Science Center Many plant-derived compounds of high value for industrial or pharmaceutical applications originate from plant species that are not amenable to cultivation. Biotechnological production in low-input organisms is an attractive alternative. Microbial production systems such as Escherichia coli and Saccharomyces cerevisiae are well-established biotechnological platforms that can often be successfully engineered to produce phytochemicals. In addition to microbes, plant cell cultures have been exploited as potential biotechnological production platforms for phytochemicals. Despite the advantages of such cell-based production systems over native low-yield producers, a common drawback is the requirement for specialized fermentation facilities, energy input and a continuous supply of macro- and micronutrients. Whereas production of pharmacological proteins in plants has advanced significantly, bioengineering of low-input crop plants for the production of small molecules remains under-explored. Camelina sativa is an emerging low-input, non-foodstuff Brassicaceae oilseed crop that grows on marginal land or can be used as a rotation crop on fallow land. Historically cultivated in Europe, it is now grown in northwestern regions of the United States and Canada and is considered a practical agronomic oilseed crop. Furthermore, camelina oil has been successfully tested by commercial airlines as a hydrotreated, renewable jet fuel. Camelina is genetically similar to the model dicot arabidopsis and is likewise genetically transformed by floral dip. We explore whether camelina can successfully be refactored to produce and store novel compounds in seed. As proof-of-concept, we have produced the cyclic monoterpene hydrocarbon (4S)-limonene and the bicyclic sesquiterpene hydrocarbon (+)-δ-cadinene, which have potential biofuel and industrial solvent applications, in camelina seed.

400-073-Y Investigating (di)terpene Synthase Mechanisms in Vascular Plants Meirong Jia – Iowa State University Reuben Peters – Iowa State University The labdane-related diterpenoids (LRDs) are a large super-family of natural products that play important roles in plant defense. LRD biosynthesis generally depends on gene duplication and neo-functionalization of the ent-kaurene synthases (KSs) found in all vascular plants for gibberellin hormone metabolism. Accordingly, these enzymes are termed KS-like (KSLs), and they have become a model system for investigation of terpene synthases, which form moderately sized gene families in all tracheophytes. The derivation of KSLs from the ancestral KSs requires alteration of the bicyclization and/or rearrangement necessary in formation of ent-kaurene. For example, four recently characterized KS(L)s from the dicot plant Ricinus communis, namely RcKS1, and RcKSL2, 3 & 4, share over 60% identity in protein sequence, and carry out distinct reactions that nonetheless are closely related to that catalyzed by KSs. Accordingly, we are investigating the enzymatic basis for these subtle variations in catalysis, using site-directed mutagenesis of not only RcKS(L)1-4, but a number of other KSLs as well. Currently, on the basis of structural comparison and sequence alignment a residue in the RcKS(L)s critical for secondary cyclization has been identified. Additional studies of the mechanistic basis for this effect, and other potentially important active site residues are underway. Accordingly, we expect to present identification of key residues playing important roles in the neo-functionalization of KSLs to yield altered products for the biosynthesis of more specialized labdane-related diterpenoid natural products.

400-074-Z Polyamine Transporters as Modulators of Plant Developmental Responses Menaka Ariyaratne – Bowling Green State University Paul Morris – Bowling Green State University Polyamines are essential compounds that are present in all living organisms. In plants, elevated levels of polyamines are seen in response to stresses such as chilling, drought, heat, heavy metals, and temperature. Furthermore transgenic manipulation of polyamine biosynthesis has clearly demonstrated that increased polyamine levels activate hormoneresponsive pathways that enable tissues to respond to exogenous environmental signals. To test the hypothesis that the regulation of polyamine transport represents an additional mechanism that cells can use to generate localized changed in polyamines, we altered the expression of genes that are responsible for polyamine transport in Arabidopsi. A family of polyamine uptake transporters (PUTs) and a family of bidirectional polyamine/amino acid exchangers (BATs) was used in this study. Alterations in the expression of these genes showed tissue-specific responses such as a delay in flowering, delay in senescence, and vascular tissue development. HPLC analyses of plant tissues showed that alteration of gene expression was correlated with changes in spermidine and spermidine conjugates. Analyses of polyamine levels in knockout lines, WT and transgenic plants overexpressing polyamine transporters demonstrate that these localized changes in polyamine levels can cause alterations in the developmental-response pathways. There are multiple genetic mechanisms that account for the phenotypic diversity of species across their geographical ranges. Here we have shown that mere changes in the expression of polyamine transporters, can account for some of the developmental diversity that is present in nature.

400-075-Z Transorganellar Complementation Functionally Demonstrates a New Interface for the Synthesis of Non-polar Metabolites by Membrane Spanning Pathways Dean DellaPenna – Michigan State University Payam Mehrshashi – Michigan State University, Casey Johnny – Michigan State University

Plastids are subcellular factories that participate in the synthesis of a bewildering array of compounds, often by initiating biosynthetic pathways that are subsequently completed in other organelles. Such organelle-spanning pathways require extensive exchange of metabolites with the extraplastidic environment, which for polar metabolites, is handled by dozens of well-characterized envelope membrane transporters. However, for the many thousands of plastid-synthesized nonpolar compounds synthesized by membrane-spanning pathways, such transporters have remained elusive. This talk will highlight recent data from an approach we have termed transorganellar complementation that functionally demonstrates enzymes in one organelle can directly access nonpolar metabolites from a companion organelle. We propose a mechanism, based on hemifused-membranes at plastid:ER contact sites, that allows enzymes in one organelle direct, transporter-independent access to a range of nonpolar compounds in both organelle membranes. Such an interface would facilitate interorganellar metabolism, allow for allosteric regulation between organelles and allow the rapid evolution of membrane-spanning pathways for the thousands of nonpolar metabolites in the plant kingdom to be uncoupled from coevolution with nonpolar metabolite transporters.

400-076-Y Exploration on Biologically Active Secondary Metabolites from Eastern Himalayan Mosses Sumedha Roy Chowdhuri – University of Calcutta Jayashree Datta, Mousumi Poddar-Sarkar – University of Calcutta The Eastern Himalayan Biodiversity Hotspot (EHBH) is a plethora of mosses (division- bryophyta), which is still unexplored. Our main focus is to tap such a treasure that can facilitate natural product research with special emphasis on secondary metabolite profiling and antioxidant assay. Since, mosses usually grow in diverse climatic conditions; it is for their survival that they need to biosynthesize secondary metabolites to fight against biotic or abiotic stresses. Mosses of six families, comprising a total of ten identified species characterized. These are Thuidium sparsifolium(Mitt.) A. Jaeger’ - Thuidiaceae, Wijkia lepida (Mitt.) H.A. Crum - Sematophyllaceae, Campylopus introflexus (Hedw.) Brid. Dicranales, Scopelophila ligulata (Spruce) Spruce-Pottiaceae, Brachymenium capitulatum (Mitt.) Paris-Bryaceae, Hyophila involuta (Hook.) A. Jaeger-Pottiaceae, Bartramia ithyphylla Brid.-Bartramiaceae, Hymenostylium recurvirostrePottiaceae, Barbula horricormis Gangulee- Pottiaceae, Barbula constricta(Mitt.)-Pottiaceae.Phenolic acids such as Gallic acid, 4-Hydroxy benzoic acid, Vanillic acid, Syringic acid, p- Coumaric acid, Ferulic acid, Caffeic acid and Jasmonic acid, flavonoids e.g. Rutin, Myricetin and Quercetin and alcohols like Guaicol, p-Cresol, o-Cresol and 3,4 – Xylenol were identified by HPLC and LCMS using C18 column and maintaining a concentration gradient for mobile phase-methanol and water. Antioxidant assay was performed spectrophotometrically using DPPH. Each species was unique in showcasing a diverse secondary metabolite profile. It is interesting to note that Barbula constricta(Mitt.), collected from ice-laden rock surface, is the only species with high accumulation of Jasmonic acids (JA). Therefore, not only shikimic acid pathway but also JA pathway was activated for engineering metabolic system under freezing condition. The antioxidant property was not directly correlated with the high amount of phenolic acid and flavonoid in these mosses. The high altitude species were represented by the members of family pottiaceae, concluding that selection pressure influencing the diminution of hierarchical diversity, therefore, shades a light on conservation of the mosses of EHBH.

400-077-Y CYP93G1 Is a Flavone Synthase II Which Channels Flavanones to the Biosynthesis of Tricin O-linked Conjugates in Rice Pui Ying Lam – The University of Hong Kong Clive Lo Flavones are a major class of flavonoids with a wide range of physiological functions in plants. They are constitutively accumulated as C-glycosides and O-linked conjugates in vegetative tissues of grasses. It has long been presumed that

the two structural modifications of flavones occur through independent metabolic routes. Previously we reported that CYP93G2 functions as a flavanone 2-hydroxylase (F2H) which provides 2-hydroxyflavanones for C-glycosylation in rice. Flavone C-glycosides are subsequently formed by dehydratase activity on 2-hydroxyflavanone C-glycosides. On the other hand, O-linked modifications were proposed to proceed after the flavone nucleus is generated. In this study, we demonstrate that CYP93G1, the closest homolog of CYP93G2 in rice, is a bona fide flavone synthase II (FNSII) which catalyzes the direct conversion of flavanones to flavones. In recombinant enzyme assays, CYP93G1 desaturated naringenin and eriodictyol to apigenin and luteolin, respectively. Consistently, transgenic expression of CYP93G1 in Arabidopsis thaliana resulted in the accumulation of different flavone O-glycosides which are not naturally present in Cruciferous plants. Metabolite analysis of a rice CYP93G1 insertion mutant further demonstrated the preferential depletion of tricin O-linked flavanolignans and glycosides. By contrast, redirection of metabolic flow to biosynthesis of flavone C-glycosides was observed. Our findings established that CYP93G1 is a key branch point enzyme channeling flavanones to the biosynthesis of tricin O-linked conjugates in rice. Functional diversification of F2H and FNSII in the cytochrome P450 CYP93G subfamily may represent a lineage-specific event leading to the prevalent co-occurrence of flavone C- and O-linked derivatives in grasses today.

400-078-Z Tomato Hairless Is Involved in Epidermal Hair and Stem Development, and Modulates the Production of Trichome-Borne Metabolites Jin-Ho Kang – Seoul National University Marcelo Campos – Michigan State University, Starla Zemelis – Michigan State University, Jameel Al-Haddad – Michigan State University, Frank Telewski – Michgian State University, Federica Brandizzi – Michigan State Universitye, A. Daniel Jones – Michigan State University, Gregg Howe – Michigan State University Trichomes are specialized epidermal structures that function as physical and chemical deterrents against arthropod herbivores. We previously observed that the tomato (Solanum lycopersicum) trichome mutant hairless (hl) has distorted trichomes and produces reduced levels of trichome-derived compounds, including terpenoids and flavonoids implicated in anti-insect defense. Here, we demonstrate that Hl encodes an Arabidopsis homolog of PIROGI (PIR), which is a component of the WAVE complex that mediates polymerization of actin filaments. The SlPIR gene is comprised of 30 exons spanning a 42 kb region on the chromosome 11. hl plants contain a ~3-kb deletion that encompasses the last exon of the genes, resulting in a C-terminally truncated form of SlPIR. Expression of a wild-type SlPIR cDNA in the hl mutant background restored normal trichome development and accumulation of trichome-derived defense metabolites. We further found that stem tissue of the hl mutant is more brittle than wild type. These defective mechanical properties were rescued in the PIR-complemented transgenic lines. Our results establish a function for PIR in tomato trichome and stem development, and suggest that the actin-cytoskeleton network plays a role in the production of trichome-borne defense compounds.

400-079-Z Genetic Analysis of the CYP72A Enzymes Contributing to Stress Defense in Flowering Plants Leeann Thornton – The College of New Jersey The diversification of plant secondary metabolites provides the arsenal necessary for adaptation to life on land and survival in the face of competition and attack. Cytochrome P450 monooxygenases (CYPs) are enzymes that have been implicated in many aspects of secondary metabolism, but much is still unknown about the biochemical capabilities of this large class of enzymes. Plant genome sequencing has revealed the presence of thousands of CYP genes with an average of about 300 genes per plant. Whole genome comparisons and microarray data allow CYPs to be organized based on gene structure, protein sequence, and similarities in expression patterns. These data provide insight into the

CYP diversity in plants, but little is known about the connections between sequence similarity and biochemical function within large groups of closely related CYPs. The CYP72A subfamily appears to have members in all angiosperms and provides the potential for a variety of biochemical functions in each plant species. This work examines the evolutionary relationships within the CYP72A subfamily as the framework for predicting functional diversity in the substrate binding sites of each enzyme. Our phylogenetic analysis shows relationships between CYP72A sequences from 34 angiosperm species. Our data support the hypothesis that all CYP72As are derived from a common ancestor prior to the monocot/eudicot split. We used the phylogenetic tree to predict biochemical potential within the CYP72A subfamily and to make predictions for functions specific to smaller clades of CYP72A enzymes. We are beginning to explore individual contributions of CYP72A enzymes in the model plants Arabidopsis and maize. These data contribute to a better understanding of the metabolic potential of the CYP72A subfamily in all plants.

400-080-Y Studying Resveratrol and Piceid Production by Japanese Knotweed Matthew Yatison – Wilkes University David Pupaza – Wilkes University, Abigail Mikolon – Wilkes University, Jared Luchetta – Wilkes University, Shaiva Shah – Wilkes University, David Hontz – Wilkes University, Lauren Gunn – Wilkes University, Gregory McFarlane – Wilkes University, Kathryn Margavage – Wilkes University, Kenneth Klemow – Wilkes University, Donald Mencer – Wilkes University, William Terzaghi – Wilkes University Resveratrol is a natural compound produced by plants that has been shown to extend the lifespan of various organisms, and has been shown to be produced by plants in response to stresses including wounding, drought and pathogen attack. Resveratrol and its glycosylated derivative, piceid, are made by local populations of Japanese knotweed (Fallopia japonica). We tested the hypothesis that resveratrol and piceid synthesis would be stimulated by nutrient deprivation. We first grew thirty clones derived from a single rhizome collected from a site in Kingston, PA, and then split them into ten treatments with three ramets per treatment. Each group of clones was grown in perlite in a 25 liter planter box, and watered with either distilled water, a complete nutrient solution, or nutrient solution lacking one of the following: N, P, K, S, Mg, Ca, Fe, or micronutrients. We sampled the leaves weekly, extracting resveratrol and piceid with 80% Ethanol, and analyzing the extracts by reverse-phase HPLC on C18 columns. Resveratrol and piceid peaks were identified by comparison with the elution times of known standards and by ESI-MS. We found that piceid is 3- to 5–fold more abundant than resveratrol on a molar basis, but did not detect any significant differences in resveratrol and piceid contents between treatments.

400-081-Y Metabolic Profiling Reveals Metabotype of DUF642 Mutants and Identifies Associated Pathways in Arabidopsis Thaliana Shanjida Khan – University of Alberta Michael Deyholos – University of British Columbia The DUF642 domain is mainly plant specific. In Arabidopsis thaliana there are 10 proteins that contain DUF642 domains. We studied two of these A. thaliana genes: AT5G25460 (DGR2) and AT5G11420 (DUFB). Both proteins are expressed in all vital organs suggested an important physiological role in growth and development of Arabidopsis. Based on structural homology using the models generated by I-TASSER, DGR2 and DUFB are predicted to be involved in primary metabolic process with carbohydrate binding and hydrolase activities. In vivo subcellular localization study indicated that DGR2 localizes in the trans-Golgi network. Metabolic profiling of the T-DNA insertion mutants, RNAi and overexpression plants of DGR2 and DUFB was performed by gas chromatography/mass spectrometry. PCA analysis indicated that many compounds contributed to distinguishing between the genotype including amino acids, sugars and other organic compounds, which could be regarded as potential biomarkers to elucidate the functions of DUF642 genes. Metabolic

profiling revealed that overexpressed plants of DUFB had an increase in fucose and decrease in xylose whereas overexpressed plants of DGR2 were increased in xylose and decreased in galactose. Significant decreased in malate, fumarate and succinate were observed in the mutant lines. The pathway enrichment analysis revealed that TCA cycle is one of the pathways perturbed by both genes. Amino acid metabolism made up the majority of affected pathways by DGR2 and DUFB mutants. Our data illustrated the power of metabolic profiling in finding gene metabolic phenotype to elucidate function and provide a broader assessment of pathways perturbation following the genetically diverged plants.

400-082-Z A Zebra-band Phenotype in Maize Can Be Suppressed in Constant Light, and Results from Mutation of a PPOXlike Gene (protophorphyrinogen Oxidase IX-like) for Porphyrin Biosynthesis Jonathan Saunders – University of Florida Nicole Traugh – University of Florida, Charles Hunter – University of Florida, Alex Boucher – University of Florida, Mark Lubkowitz – University of Florida, David Braun – University of Florida, Karen Koch – University of Florida A zebra-band phenotype was identified in a maize population of transposon-tagged mutants (UniformMu, searchable by sequence at MaizeGDB.org). Genotype-phenotype analysis of an F2 family showed that the zebra stripes co-segregated with a single Mu insertion in the second exon of a Protoporphyrinogen oxidase IX (PPOX)-like gene. The association was confirmed by presence of the phenotype in two additional alleles from the UniformMu population as well as by allelism tests between the three lines. Although roles of PPOX have been studied in Arabidopsis and elsewhere, the effects of its mutation have not been previously linked to zebra-striping. The PPOX enzyme catalyzes the last step in common for both heme and chlorophyll biosynthesis. If the reaction is defective, then toxic, photosensitive precursors accumulate. In maize, our analyses indicate that a PPOX deficiency leads to wide, non-green bands that form perpendicular to the long axis of the first 5 leaves. Each of the cross-bands arises from a series of closely-packed, parallel, non-green stripes associated with major vascular bundles. The whitest stripes within the cross-bands often include narrow, open gaps where cells have died. The phenotype is most pronounced in field-grown plants (high light), but persists under low-light intensities. Cross-bands of this mutant are unique among diurnal-banding phenotypes in maize due to their combination of developmental timing, degree of severity, and pronounced enhancement near major vascular bundles. Light-dark shift experiments alter band patterns, however, continuous light suppresses zebra-banding and leads to solidgreen leaves. Results are consistent with the accumulation of phytotoxic intermediates in darkness, but also indicate the involvement of light- and clock-regulation. The zebra-banding mutant described here will facilitate further investigations into roles and regulation of porphyrin biosynthesis.

400-083-Z Molecular Cloning and Characterization of Two Novel Sesquiterpene Synthase Genes from Ginkgo Biloba L Zhiqiang Pan – USDA-ARS, NPURU Iffat Parveen – School of Pharmacy, University of Mississippi, Scott Baerson – USDA-ARS, NPURU Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including flavonoids, diterpene lactones, terpenoids and polysaccharides accumulating in leaf tissues. Relatively few genes associated with these biosynthetic pathways from Ginkgo have been characterized to date, due in large part to the initial lack of publicly-available genome and transcriptome data. By mining a data set comprised of 64,057 expressed sequences derived from leaves of G. biloba (Lin et al. 2011), 16 candidate ESTs potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoids compounds were found, two of which were identified as putative terpene synthases (TPS). Here, we describe the results for the cloning and functional

characterization of GbTPS1 and GbTPS2, which were identified as farnescene synthase and bisabolene synthase, respectively.

Biochemistry: BioEnergy 400-084-Y Small-angle X-ray Scattering and X-ray Crystallography Reveals the Structure of the Catalytic Domain and Plant Conserved Region of a Plant Cellulose Synthase Phillip Rushton – Purdue University Anna Olek – Purdue University, Catherine Rayon – University of Picardie Jules Verne, Hyung-Rae Kim – Purdue University, Ciesielski Peter – National Renewable Energy Laboratory, John Badger – DeltaG Technologies, Lake Paul – Purdue University, Michael Crowley – National Renewable Energy Laboratory, Michael Himmel – National Renewable Energy Laboratory, Daisuke Kihara – Purdue University, Lee Makowski Northeastern University, Cynthia Stauffacher – Purdue University, Nicholas Carpita – Purdue University The processive plant cellulose synthase (CesA) polymerizes a chain of (1→4)-β-D-glucan polysaccharides and forms the cellulose microfibril that is the fundamental rigid scaffolding of the plant cell wall. A recombinantly expressed catalytic domain (CatD) of a rice secondary cell wall OsCesA8 specifically binds a radiolabeled UDP-glucose substrate. Size exclusion chromatography (SEC) and analytical ultracentrifugation (AUC) shows a reversible homo-dimerization sensitive to protein concentration and thiol reducing agents. Small angle X-ray scattering (SAXS) enabled prediction of the molecular envelopes for both the monomer and dimer. The OsCesA8 CatD monomer is composed of two differential lobes that extend from a central core and the dimer shows an interaction utilizing one of the lobes. Molecular docking of the OsCesA8 CatD model within the SAXS data shows that the OsCesA8 CatD fits well into the core and gives a likely orientation of the P-CR and CSR based on size predictions. Recombinant truncations of only the OsCesA8 P-CR purifies well as a monomer and shows distinct α-helical secondary structure by circular dichroism analysis. The OsCesA8 P-CR was crystallized and structure solved to 2.4Å resolution revealing a coiled-coiled domain containing an ordered loop connecting α-helices, with a small α-helix in the center of the loop. The P-CR could be involved in cellulose synthase complex (CSC) protein-protein interactions, where it may be possible for the coiled-coil connector loop to act as a flexible region that enables or modifies binding. SAXS data was collected on the OsCesA8 P-CR and a molecular envelop was modeled. The P-CR crystal structure fit this molecular envelop and the CatD molecular envelop as predicted by optimal orientation. Supported by the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DESC0000997.

400-085-Y The Cell Wall Arabinose-deficient Arabidopsis Thaliana Mutant mur5 Encodes a Defective Allele of Reversibly Glycosylated Polypeptide2 Nicholas Carpita – Purdue University Christopher K. Dugard – USDA-ARS, Rachel A. Mertz – Cornell University, Christopher Hart – Univ. Connecticut, Matheus R. Benatti – Purdue University, Anna T. Olek – Purdue University, Catherine Rayon – University of Picardie-Jules Verne, Davide Mercadante – HITS, Maureen McCann – Purdue University, Wolf-Dieter Reiter – University of Connecticut Traditional marker-based mapping and next-generation sequencing were used to determine that the Arabidopsis thaliana low cell-wall arabinose mutant mur5 encodes a defective allele of Reversibly Glycosylated Polypeptide2 (RGP2). Marker analysis of thirteen F2 confirmed mutant progeny from a recombinant mapping population gave a rough map

position on the upper arm of chromosome 5, and deep sequencing of DNA from these thirteen gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of Reversibly Glycosylated Protein2 (RGP2), a gene that encodes a UDP-arabinose mutase that interconverts UDParabinopyranose and UDP-arabinofuranose, exhibited the low cell-wall arabinose phenotype. Identity of mur5 and two SALK insertional alleles was confirmed by allelism and overexpression of wild-type RGP2 cDNA placed under the control of the 35S promoter in the three alleles. The level of recovery of wild-type cell-wall arabinose levels was correlated with the relative expression of the RGP2 transgene, although other mechanisms impose a limit on the relative arabinofuranosyl abundance in cell-wall polysaccharides. The mur5 mutation results in conversion of Cys257 to Tyr257 within a conserved hydrophobic cluster, well downstream from the catalytic amino acids. Homology modeling of RGP2 places Cys257 in proximity to Cys252at the exterior of the protein, where formation of a disulfide bond could impart structural stability to a dimer interface. Supported by the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Award Number DESC0000997

400-086-Z Modifying Lignin Content to Improve Sorghum for Bioenergy Scott Sattler – USDA-ARS Erin Scully – USDA-ARS, Thomas Clemente – University of Nebraska-Lincoln, Deanna Funnell-Harris – USDA-ARS Modifying lignin content and composition are major targets for bioenergy feedstock improvement for both cellulosic and thermal bioenergy conversion. Sorghum (Sorghum bicolor) is currently being developed as a dedicated bio-energy feedstock. To alter lignin content for thermal bioenergy conversion processes including pyrolysis and direction combustion of biomass, a series of transgenic events ectopically expressing phenylpropanoid biosynthetic genes and a putative pathway regulator were developed. Higher lignin content is more desirable for thermal conversion, because lignin has higher energy contents than cellulose or hemicellulose cell wall components. A sorghum Myb transcription factor (TF) was identified as a potential regulator of the lignin biosynthetic pathway based on similarity to known pathway regulators from other plant species. The events overexpressing this TF have elevated levels of proteins in the lignin biosynthetic pathway, increased levels of phenolic compounds and increased energy content. This result indicates this TF is sufficient to induce phenylpropanoid synthesis in sorghum. The development of these experimental lines with altered phenylpropanoid metabolism will lead to a greater understanding of how these modifications impact plant fitness and affect bioenergy conversion processes in sorghum and other bioenergy grasses.

400-087-Z Tracking the Rate of Turnover of Different Protein Types in Plants: a New Frontier in Understanding the Cost of Protein Synthesis to Plant Growth Harvey Millar – The University of Western Australia Clark Nelson – The University of Western Australia, Lei Li – The University of Western Australia, Richard Jacoby – The University of Western Australia, Ralitza Alexova – The University of Western Australia Proteomic studies focus almost exclusively on the proteins that are changing in abundance in response to genotype, development or treatments in order to find biological insights. This requires statistically significant changes in the total accumulated protein pool size to determine that ‘anything has occurred’. Analysing protein synthesis and degradation rates with progressive stable isotope labelling provides a new window on the control of protein abundance and the energy expended in maintaining the steady-state protein across genotypes, development and environments. With this

approach we can determine the ‘relative age’ of the proteins that we see and define the energetic effort employed by the cell to build or maintain particular activities. We are using progressive 15N labelling of plant cells from nitrate and ammonia salts and modelling heavy isotope incorporation to calculate the rate at which proteins are turning over. We have developed pipelines to undertake these studies for nearly a thousand proteins in Arabidopsis cells, Arabidopsis leaves and in whole Barley plants through the use of hydroponics. Projects assessing the impact of plant growth, leaf age, phosphate limitation and groundwater salinity on protein turnover changes in plants will be discussed. Rapidly turnover proteins in thiamine and chlorophyll biosynthesis and changes in the turnover rates of Calvin cycle enzymes will be highlighted. Through combining such labelling with separation of protein complex and subcomplexes by native electrophoresis, we can observe the in vivo turnover rate of protein complex assembly intermediates which has identified new assembly pathways of respiratory complexes in plants. Combined there approaches provide new avenues for peptide mass spectrometry to answer a wide range of questions in plant biology, and allows researchers to assess the cost of environmental factors on protein turnover and plant growth efficiency.

400-088-Y Structural Basis for β-(1,4)xylosyltranferase and α-(1,2)glucuronyltranferase Activities of TaXPol-1, a Xylan Polymerase from Wheat Yunyi Feng – Ohio University Nan Jiang – Ohio University, Ahmed Faik – Ohio University The characterization of a wheat xylan synthase (named TaXPol-1) in our lab represents a seminal advance in understanding xylan biosynthesis. TaXPol-1 has a central core formed of two glycosyltransferases (GTs) from the GT43 and GT47 families (named XS-P1 and XS-P2, respectively). Both GTs are sufficient and necessary for the synergistic incorporation of xylose and glucuronic acid into xylan-like products. To understand the molecular basis of donor/acceptor specificities and the catalytic mechanism during the biosynthetic process, we used bioinformatics programs to generate 3D models for XS-P1 and XS-P2. The overall quality of these models was extensively analyzed and refined before further analysis. While XS-P2 structure has two “Rossmann-like” domains, which is a characteristic of GTB-like fold with a retaining mechanism, XS-P1 structure has characteristics of GT-A-like fold with an inverting mechanism. XS-P2 structure revealed eight conserved residues at the UDP-sugar binding site located in a loop between β-4 strand and α-4 helix at the C-terminal domain, and seems to lack a DxD motif. However, XS-P2 contained a conserved Glu residue (Glu296) that is considered as a signature for a retaining mechanism. The central region of XS-P1 revealed three long conserved peptide sequences located on β-1, β-2, and β-4 strands in the UDP-sugar binding site, which encompasses the [DDxN] motif involved in coordinating the binding of Mn2+ and UDP. XS-P1 has also a conserved Glu420 that can act as a catalytic base that deprotonates the acceptor group, which is compatible with an inverting mechanism. In addition, XS-P1 structure has two loops around the catalytic domain that are absent in other structures, suggesting a possible role in the synergistic mechanism. A combination of site-directed mutagenesis and heterologous co-expression in Pichia pastoris is used to investigate the role of these conserved residues in xylan biosynthesis.

400-089-Y Characterization of TaXPol-1, a Wheat Xylan Synthase Complex, Reveals New Insights on Enzyme Activities and Trafficking of the Complex Nan Jiang – Ohio University Richard Wiemels – Ohio University, Ahmed Faik – Ohio University It is believed that Golgi-localized, multi-protein complexes are involved in biosynthesis of plant hemicelluloses. We describe the characterization of the first hemicellulose synthase complex involved in xylan biosynthesis in wheat (Xylan Polymerase-1, TaXPol-1). Xylan is the most abundant plant hemicellulosic polysaccharide on Earth and plays an

important role in the integrity of cell walls. It consists of β-(1,4)-linked d-xylose backbone that can be substituted with α(1,2)-d-glucuronic acid residues and/or l-arabinofuranosyl residues at C-2 and/or C-3 positions of xylose residues. A xylan synthase activity was immuno-purified from etiolated wheat seedlings, and its protein composition determined by proteomics analysis. Four glycosyltransferases (GTs): a member of the CAZy GT43 family (named XS-P1), a member of the GT47 family (named XS-P2), and two members of the GT75 family (named XS-P3 and XS-P4) and a germin-like protein (TaGLP) belonging to cupin family were identified. Heterologous co-expression of XS-P1, XS-P2, XS-P3, XS-P4, and TaGLP in Pichia pastoris cells resulted in the reconstitution of xylan synthase activity in micosomes from transgenic lines. Our results showed that (i) XS-P1 and XS-P2 form a central core that is necessary and sufficient for synergistic incorporation of xylose and glucuronic acid, but requires XS-P3 and/or XS-P4 (putative mutases) for synergistic incorporation of xylose and arabinose, and (ii) TaGLP was needed for optimal xylan synthase activity. Furthermore, radiolabeled products generated by microsomes have xylan characteristics. Using bimolecular fluorescence complementation (BiFC), we showed that XS-P1 interacts with XS-P2, XS-P3, XS-P4, and TaGLP, and these proteins assemble into a complex in the ER before export to the trans-Golgi. Importantly, ER export of TaXPol-1 requires that XSP1 and XS-P2 interact. Furthermore, immuno-gold labeling electron microscopy analysis, using anti-XS-P1 and anti-XS-P2 antibodies, confirmed that most of the label was localized to the ER and the trans-Golgi.

400-090-Z Comparative Transcriptome Analysis for Metabolic Engineering of Oil in Biomass Crops Aruna Kilaru – East Tennessee State University John B. Ohlrogge – Department of Plant Biology, Michigan State University Triacylglycerols (TAGs) are stored in variable amounts in diverse tissues of several plant species. Using comparative transcriptomics approach, we analyzed tissue- and species-specific regulation and biosynthesis of TAG in plants. In both seed and nonseed oil-rich tissues, irrespective of the species, an increased expression was noted for genes mostly associated with hexose metabolism in plastids, relative to cytosol, which is likely associated with the need for higher pyruvate flux directed toward plastid fatty acid synthesis. A corresponding increase in expression for plastidial fatty acid synthesis genes was also noted but not for TAG assembly genes. Additionally, WRINKLED1 (WRI1), a regulatory element typically associated with seed oil biosynthesis, was also highly expressed in oil-rich nonseed tissues of oil palm, a monocot and avocado, a basal angiosperm. Together these data suggest a ubiquitous role for WRI1 and that a major point of regulation of oil biosynthesis in oil-rich tissues most likely occurs at the level of source and not sink. Transcriptomics also revealed that multiple acyltransferases that participate in rate-limiting step in TAG synthesis might be active concomitantly in tissues to achieve high levels of TAG accumulation. Although transcripts for proteins such as oleosins were not expressed in nonseed tissues, novel lipid-droplet associated proteins were expressed, which are presumed to stabilize nonseed TAG. Overall, a comparative transcriptome analysis in diverse plants and tissues revealed several distinct and conserved features for storage oil biosynthesis and identified key genes necessary for metabolic engineering to increase oil accumulation in nonseed perennial crops for bioenergy.

400-091-Z Engineering Carbon Concentration Mechanisms (CCM) in C3 Plants Sowmya Surbamanian – New Mexico Consortium/Los Alamos National Laboratory Fangfang Ma – Donald Danforth Plant Science Center, Natalia Friedland – New Mexico Consortium, Lara Jazmin – Vanderbilt University, Malathy Krishnamurthy – Los Alamos National Laboratory, David Hanson – University of New Mexico, Jamey Young – Vanderbilt University, Douglas Allen – Donald Danforth Plant Science Center, Richard Sayre – New Mexico Consortium/Los Alamos National Laboratory Improving global crop productivity through sustainable methods is a shared goal pursued by research labs all over the

world. One of the methods most commonly sought by researchers to increase crop productivity is by markedly improving the CO2 fixation rates by the enzyme, Rubisco. In nature, aquatic organisms have achieved elevated CO2 fixation rates using an energy-dependent Carbon Concentration Mechanism (CCM) to increase the concentration of CO2 near the site of carbon fixation by Rubisco. C3 plants however, do not have a functional CCM. In our current work, we sought to augment CO2 concentration near Rubisco by introducing carbonic anhydrase (CA) in the chloroplast stroma. Preliminary results on T3 lines in Arabidopsis thaliana show improved photosynthetic rates and bio-mass productivity when expressing a bacterial CA (BCA) but not a eukaryotic CA having a similar kcat. Moreover, modeling studies suggest that while the carboxylation efficiency was increased by 30 % on fresh leaf weight basis, this factor alone could not account for differential rates of photosynthesis. Metabolic flux analyses indicated that both fixed carbon and oxygen flux through the Calvin cycle were accelerated in BCA transgenics. Efforts are also underway to understand the effect of the CA transgene in Camelina sativa and Solanum tuberosum. The merits of introducing a CCM in C3 plants will be discussed based on these results.

400-092-Y Targeted Metabolic Engineering of Camelina Sativa to Synthesize High Levels of Industrially Useful Lipids Timothy Durrett – Kansas State University Sunil Bansal – Kansas State University, Jinjie Liu – Michigan State University, Jillian Silva – University of Nebraska – Lincoln, Ed Cahoon – University of Nebraska – Lincoln, Mike Pollard – Michigan State University, John Ohlrogge – Michigan State University Vegetable oils have long been used as sources of energy and chemical industry feedstocks. However, metabolic engineering to improve the chemical and physical properties of plant oils for these application has proven difficult. Through the expression of a novel acyltransferase and the downregulation of competing reactions, we have been able to transgenically engineer Camelina sativa plants with the highest accumulation of unusual oil achieved so far. EaDAcT, a diacylglycerol acyltransferase (DGAT) with sn-3 acetyltransferase activity synthesizes the abundant 3-acetyl1,2-diacylglycerols (acetyl-TAGs) found in the seeds of Euonymus alatus. Expression of EaDAcT in Camelina sativa, combined with the RNAi-mediated suppression of endogenous TAG biosynthesis, led to acetyl-TAG levels as high as 85 mol% in the best transgenic lines. These high acetyl-TAG levels led to an overall increase in the moles of oil produced, were stable across multiple generations and did not affect seed viability. The sn-3 acetate group means that acetyl-TAGs possess a lower viscosity and improved cold temperature properties compared to other vegetable oils. To expand the utility of acetyl-TAGs, we have combined the expression of EaDAcT with the synthesis of unusual fatty acids. For example, acetyl-TAGs with medium chain fatty acids (MCFA) at the sn-1/2 positions are predicted to possess further reductions in viscosity, potentially allowing their use as an improved straight vegetable oil (SVO) biofuel. These acetyl-TAGs could also be useful as biodegradable lubricants with improved oxidative stability and cold temperature performance. Expression of EaDAcT combined with suppression of endogenous DGAT1 activity in a Camelina background previously engineered to synthesize MCFA resulted in acetyl-TAG levels as high as 70 mol%. Importantly, electrospray ionization mass-spectrometry (ESI-MS) revealed that MCFA were incorporated in acetylTAGs. Future field growth of these lines will produce large quantities of MCFA acetyl-TAGs for product testing.

400-093-Y Generating a Promising System for Directed Evolution of Efficient and Continuous Production of Hydrogen from Water Using the Model Green Alga Chlamydomonas Reinhardtii Thummala Chandrasekhar – Tempe University Andrey Kanygin – Tempe University, Kevin E. Redding – Tempe University

Fossil fuel reserves are finite and it is only a matter of time when they run out. Moreover, climate change may necessitate the production of renewable fuels that do not contribute to increases of atmospheric CO2. Most of these alternate energy sources depend on sunlight in one way or another. Biological production of hydrogen (H2), a clean and efficient fuel, from green algae using light has been researched by several groups, but there is still no system for efficient, continuous production of H2. We are using Chlamydomonas reinhardtii mutants that lack the PsaC subunit of Photosystem I (PSI), containing the terminal FeS clusters of PSI, as a platform in which to introduce a chimeric gene fusing HYDA (FeFe-hydrogenase gene) with psaC. If functional, the PSI-hydrogenase hybrid will use light to oxidize plastocyanin and produce H2. We have constructed libraries with different junctions between PsaC and the hydrogenase, as the best way to link them together is not obvious. After introduction of the psaC-HYDA fusion gene into the chloroplast genome by bioballistic transformation, transformants were selected by virtue of an antibiotic resistance marker (aadA) inserted near the psaC locus. The sequence of the chimeric gene in selected transformants was determined. Some of the psaC-HYDA fusion genes are able to partially complement the light-sensitivity of the psaC mutation, which is due to low PSI levels. This indicates that the PsaC domain should have folded properly and been coassembled with the PSI core. Preliminary measurements of H2 by gas chromatography show enhanced H2 production of hydrogen in some transformants, indicating that the hydrogenase domain should be folded and properly assembled in those strains as well. This system may be useful for the biological hydrogen production in future.

400-094-Z Peroxide Effects Lipid Accumulation in Chlamydomonas Reinhardtii Rachel Wemhoff – University of Nebraska-Kearney Ashleigh Teten – University of Nebraska-Kearney, Isabella Gomez – University of Nebraska-Kearney, Paul Twigg – University of Nebraska-Kearney Microalgae are utilized for the production of biodiesel with the intent to find a renewable fuel source that may reduce the consumption of fossil fuels. The purpose of this study was to determine whether Chlamydomonas reinhardtii cultures treated with hydrogen peroxide would accumulate a significantly different amount of lipids when compared to a nitrogen-deprived control. Two separate experiments were performed using different treatments of hydrogen peroxide. Nile red staining was utilized to quantify lipid accumulation in each sample using a fluorometer. Results of the initial study indicated that peroxide treatment was not a useful method for inducing lipid accumulation in microalgae. However, it was likely that the hydrogen peroxide concentrations used were not high enough to show an effect. A second trial was done using 5 different peroxide treatments to create a concentration curve. Results from the second experiment showed that treatments with 5mM and 10mM of hydrogen peroxide resulted in significantly more lipids than nitrogen-deprived controls on days 1, 2, and 3. These results indicate that hydrogen peroxide induces lipid accumulation more quickly than nitrogen deprivation, but is not as effective in the long term. This project was funded by the NSF-EPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094).

400-095-Z Tracking Transcription Factor Expression Levels Under Nitrogen Depletion in Chlorella Sorokiniana Paul Twigg – University of Nebraska-Kearney Corey Willicott – University of Nebraska-Kearney, Kelsie Musil – University of Nebraska-Kearney, Ashleigh Teten – University of Nebraska-Kearney Chlorella sorokiniana is a green micralga this is being developed as a possible production strain for use in biofuel production. Use of this alga is however hindered by a lack of genetic resources. Members of our group have been building scaffolded genomes and transcriptomes for this alga under various sets of environmental growth conditions. In

this project, we approach one aspect of this issue by developing resources for qRT-PCR for Chlorella sorokiniana. Using a draft genome of this alga, we developed and tested positive and negative control TaqMan assays. We also developed and tested a TaqMan assay for an analog of the Chlamydomonas reinhardtii transcription factor RLS7. This transcription factor is related to the RegA protein of Volvox carteri and is a putative transcription repressor with a SAND domain. We will present our TaqMan qRT-PCR data for the expression of these the above controls and transcripts over a six day time course of nitrogen deprivation. This project was funded by the NSF-EPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094).

400-096-Y Comparative Biochemical Analysis of Diverse CELLULOSE SYNTHASE-LIKE a Proteins Aaron Liepman – Eastern Michigan University Mannan polysaccharides are non-cellulosic carbohydrates with multiple functions in plants, including cell wall structure and energy storage. Among various plants, the composition of mannans differs. Some plants synthesize mannans with main chains consisting purely of 1,4-β-linked mannosyl residues while other plants synthesize glucomannans with main chains consisting of a combination of 1,4-β-linked mannosyl and glucosyl residues. Members of the CELLULOSE SYNTHASE-LIKE A (CSLA) protein family have been implicated in the biosynthesis of mannan polysaccharides in plants, however little is known about factors that govern mannan composition. To explore the factors influencing product structure, recombinant CSLA proteins from various plants, including representatives from plants that produce pure mannans and plants that produce glucomannans, have been expressed in Pichia pastoris, and these proteins have been biochemically characterized. Results of these analyses are presented.

400-097-Y Suppression of SpKRP1 Increases Leaf Biomass Through Alternating Leaf Shapes and Delaying Flowering Time in Solanum Pennellii Shuxin Ren – Virginia State University Sarah Weeda, Laban Rutto, Thomas McKnight S. pennellii is a wild relative of cultivated tomato (S. lycopersicum) native to arid regions of Peru. One factor facilitating survival in arid conditions is the secretion of 2,3,4 tri-O-acylated glucose esters (glucolipids) that coat the whole leaves. Like glycolipids produced in oil crops, these glucolipids can be used as biofuels for industry and transportation use. About 25% of energies are used by S. pennellii to synthesize these unique esters. The lengths of hydrocarbons in these glucolipids fall within the range of gasoline (C4-C12) with almost half are C10-C12. Therefore, S. pennellii is a potential source for bio-gasoline production. However, the leaf biomass of S. pennellii is relatively less comparing to cultivated tomatoes. It was reported that inhibition of KRP1, a cyclin-dependent kinase inhibitor, could increase about 10-20% of leaf biomass in tomato and Arabidopsis. In this study, we successfully cloned the homolog of tomato KRP1 (LeKRP1) gene from S. pennellii (designated as SpKRP1). Sequence analysis indicated that SpKRP1 shared high identity with LeKRP1 with only one nucleotide change, causing amino acid change at position 77 from Asparagine to Serine. Transgenic S pennellii lines carrying the RNAi construct of SpKRP1 gene were successfully generated through Agrobacteria-mediated transformation system. When SpKRP1 being suppressed, it dramatically altered leaf shape and size. Mature transgenic leaves showed cup-shape or more round-like comparing to the control wild-type lines. Furthermore, the transgenic S. pennellii lines significantly delayed flowering time for at least two months under greenhouse condition. Leaf biomass analysis indicated that transgenic lines drastically increased leaf biomass production with ~28% more produced. Effect of SpKRP1 suppression on glucolipids production in S. pennellii is under investigation.

400-098-Z Inducible Extreme Expression of Cellulases in Poplar Charleson Poovaiah – Syracuse University Yao Xiao – Syracuse Universtiy, Heather Coleman – Syracuse University Cost of enzymes is one of the major limitations in the economical production of biofuels from lignocellulosic biomass. These enzymes break down cellulose in the biomass into glucose for subsequent fermentation into ethanol. Production of enzymes in planta will decrease the amount of enzymes added to the biomass thereby reducing the cost. Cellulolytic enzymes when produced in plants can have negative effects on the growth of the plants. In Plant Activation (INPACT) technology allows for very high inducible expression of recombinant proteins in planta upon induction. INPACT uses the rolling circle replication of Gemini virus to produce very high levels of gene amplification and protein production upon induction. INPACT also uses split gene technology to prevent gene expression before activation. To test INPACT technology in poplar, we are currently using GUS reporter system to evaluate expression in leaf and developing xylem. INPACT will then be used to express cellulases from three major groups of enzymes, endoglucanases, exoglucanases and β-glucosidases in poplar with constitutive and tissue specific promoters. Cellulases from thermophilic organisms have been plant codon optimized and synthesized with a synthetic intron (syntron) and apoplastic transit peptide. These cellulases are being evaluated in yeast for correct splicing of the syntron. The construct harboring the alcohol inducible promoter driving the production of the replication initiation protein (Rep) which allows the induction of the INPACT system has been successfully transformed into poplar and a mother plant selected based on Rep/RepA gene expression and plant growth before and after alcohol treatment. Mother lines with the highest Rep gene expression after induction, with no expression prior to induction, have been transformed with the split gene cassette to assess the INPACT system in poplar. The transgenic poplar trees with the complete INPACT system are being regenerated.

400-099-Z Enhanced Lipid Production in Microalga via BnDGAT2 Engineering Shashi Kumar – International Center for Genetic Engineering and Biotechnology Irshad Ahmad, Henry Daniell To enhance the lipid, green alga Chlamydomonas reinhardtii was genetically engineered with the heterogeneous diacylglycerol acyltransferase (DGAT2) of Brassica napus and enhanced green fluorescent (eGFP) enzymes. Transgenes integration and expression were confirmed by PCR, Southern blot and Western blot. The main constituent for producing fatty acid methyl esters (FAME) via transesterification, which is neutral lipid yield was observed doubled in transformed alga compared to the control wildtype. The composition of FAME was altered in transformed alga. Saturated fatty acids were decreased about 7% while unsaturated fatty acids were increased in same proportion. The polyunsaturated αlinolenic acid, an essential omega-3 fatty acid (PUFA) was enhanced about 12%. The Nile-Red staining has reaffirmed the large amount of lipid globules in transformed alga comparatively to non-transformed. To assess the vitality of transgenic alga for long period, after 10th generation cell line that was maintained in liquid and cryopreserved state has shown highest lipid production comparatively to the sister line that was maintained over 128th generation on solid medium. The stability of the integrated transgenes and resistance to hygromycin in different transformation events were ascertained periodically. The overexpression of DGAT2 has altered the fatty acids profile in transformed alga and results of the present study substantiate a valuable strategy of genetic manipulation for enhancing polyunsaturated fatty acid and neutral lipids for the biofuel application.

400-100-Y Characterization of Some Useful Traits in Sweet Sorghum for Bioenergy Production Ming Li Wang – PGRCU/USDA-ARS Brandon Tonnis – PGRCU/USDA-ARS, David Pinnow – PGRCU/USDA-ARS, Gloria Burow – PSGDU/USDA-ARS, Zhanguo Xin

– PSGDU/USDA-ARS, Hugo Cuevas – TARS/USDA-ARS, Gillian Eggleston – SRRC/USDA-ARS, Jianming Yu – Iowa State University, Gary Pederson – PGRCU/ASDA-ARS Multiple yearly harvests can increase crop productivity but the crop may encounter different environmental challenges (such as early-spring cold or late-fall frost) depending on cultivation zones. Sweet sorghum as a feedstock may be planted early to get a double harvest or be rotated with sugarcane in the same year for bioenergy production in certain Southeast regions of the U.S. For double harvests of sweet sorghum or rotation of sweet sorghum with sugarcane within the same year, we screened sweet sorghum accessions within the USDA germplasm collection for early-spring cold tolerance, double cutting, and late-fall frost tolerance. We identified a few sweet sorghum accessions with good tolerance to early-spring cold, suitable for double cutting, and good tolerance to late-fall frost, respectively. The identified superior accessions will be tested in different locations. Accessions confirmed for cold/frost tolerance and double cutting can be potentially used as parents for developing new cultivars or hybrids in breeding programs.

400-101-Y Comparative Transcriptome Analysis of Camelina Sativa to Reveal Genes Associated with Triacylglycerol Biosynthesis Hesham M. Abdullah – University of Massachusetts Amherst Danny Schnell – University of Massachusetts Amherst, Om Parkash Dhankher – University of Massachusetts Camelina sativa is an emerging non-food oilseed crop and one of the dedicated plants designed for biofuel and biodiesel applications. Such a valuable plant is suggested for plant breeding programs for higher quality seeds and increased oil yields. To achieve this, a better understanding of the biosynthesis of Triacylglycerol (TAG) and other lipid components at the molecular level is required. Here, we applied Illumina high-throughput transcriptome sequencing (RNA-Seq) for camelina seeds at different stages of development to identify the genes/gene networks expression involved in TAG synthesis and turnover. RNA-Seq data analysis revealed an approximate of 57,854 and 57,973 genes,\ in Camelina seeds at 10-14 DAF and at 16-21 DAF, respectively, were shown to be actively expressed (RPKM ≥ 0.1). Of these, 7932 genes showed temporal and differential gene expression during the seed development (log2 fold change ≥ 1.5 or ≤ -1.5 and Pvalue ≤ 0.05). The differentially expressed genes (DEGs) were annotated, and were found to be involved in distinct functional categories and metabolic pathways. Furthermore, RNA-Seq data were validated by performing quantitativereal time PCR for 13 selected candidate genes associated with TAG biosynthesis. Our results showed a strong correlation to expression abundance measured using both qPCR and RNA-Seq approaches. This work is highly useful towards understanding the regulation of TAG-related genes during Camelina seed development, and how transcriptome profiling can be contributed to metabolome profiling to identifying the rate-limiting components in TAG biosynthesis. Thus, this work is effective in providing precise selection for the candidate genes to create improved Camelina varieties, producing greater oil yield and better composition.

SIGNAL TRANSDUCTION - Zone 500 500-001-Y MMF1, a New Phytochrome-interacting Protein, Regulates the Photoperiodic Control of Hypocotyl Elongation in Arabidopsis He Huang – Donald Danforth Plant Science Center Rebecca Nolan – Donald Danforth Plant Science Center, Jessica Goldsworthy – Michigan State University, Allison Tielking – Mary Institute and Saint Louis Country Day School, Tom Liu – Ladue Horton Watkins High School, Sophie Alvarez – Donald Danforth Plant Science Center, Smitri Nusinow – Donald Danforth Plant Science Center Plants alter growth programs to respond to changing environmental conditions, such as seasonal changes in day length. This plasticity is the result of the integration of the circadian clock with specific signaling pathways. However, the components that mediate the photoperiodic control of growth are poorly understood. Using affinity purification and

mass spectrometry (AP-MS), we discovered a new protein that is associated with both clock and light signaling factors, named MASS SPEC IDENTIFIED MODULATING GROWTH FACTOR 1 (MMF1). MMF1 is a conserved, plant-specific, nuclearlocalized protein whose levels cycle with a peak at dusk. mmf1 mutant seedlings have elongated hypocotyls compared to wild type, in a day-length specific manner. Conversely, constitutive overexpression of MMF1 shortens hypocotyls, regardless of day length. Affinity purification of MMF1 co-precipitates the circadian-clock associated Evening Complex (ELF4-ELF3-LUX, EC), phytochromes, and the COP1-SPA complex. Biochemical and molecular assays demonstrate that MMF1 binds directly to phytochrome B (phyB). AP-MS using tagged MMF1 in various mutant backgrounds defines the in-vivo dependence of phyB, but not the Evening Complex, for recruiting MMF1 to other clock and light signaling components of the EC-phytochromes-COP1 interactome. Our MMF1 AP-MS analysis in the phyB-9 mutant background also demonstrates the in-vivo association of MMF1 with other phytochromes (phyA, D and E) in the absence of phyB. Furthermore, the interaction between MMF1 and phyB is light sensitive, suggesting that MMF1 may potentiate red light signaling. In support of this idea, manipulating MMF1 levels alters hypocotyl elongation in response to red light and modulates the expression of genes known to be downstream of the phytochrome signaling pathways. Taken together, our results define MMF1 as a new phytochrome-binding factor that participates in growth regulation under photoperiodic conditions. Targeting MMF1 for manipulation could lead to improved growth responses to changing environmental conditions.

500-002-Z Identification of Regions in the Receiver Domain of the ETHYLENE RESPONSE1 (ETR1) Ethylene Receptor of Arabidopsis Thaliana Important for Sub-functionalization Arkadipta Bakshi – University of Tennessee Rebecca Wilson – University of Tennessee, Heejung Kim – University of Gothenburg, Sai Keerthana Wuppalapati – University of Tennessee, Brad Binder – University of Tennessee Previous studies have shown that the five ethylene receptor isoforms in Arabidopsis have both overlapping and nonoverlapping roles in regulating diverse responses. ETR1, ETR2, and EIN4 control normal growth recovery after removal of ethylene via an ETR1 His kinase-dependent mechanism and phosphotransfer through any of the receptor receiver domains. A full length ETR1 is necessary and sufficient for ethylene stimulated nutations via a His kinase-independent mechanism and specifically requires the receiver domain of ETR1. Additionally, we recently reported that ETR1 and ETR2 have contrasting roles in the control of Arabidopsis seed germination during salt stress, where ETR1 inhibits germination and ETR2 stimulates germination. These examples of ETR1 functional divergence require the ETR1 receiver domain. To explore this requirement in more detail, we made point mutations in the receiver domain of ETR1. To target specific amino acid residues, we chose non-conserved amino acids based on sequence alignments of the receiver domains of ETR1, ETR2, and EIN4. We also used homology modeling to determine regions with structural divergence. These mutant transgenes were transformed into receptor-deficient plants and the ability of the transgenes to rescue several phenotypes determined. Our results show that one subset of amino acids is important for the regulation of ethylenestimulated nutations and a different subset is important for the regulation of germination under salt stress. Based on the crystal structure of the ETR1 receiver domain, we found that these two subsets of amino acids are on opposite surfaces of the receiver domain. We are now exploring the mechanisms by which these regions of the ETR1 receiver domain control functional divergence.

500-003-Z Purine Nucleotide-regulated Calcium Signalling in Stress and Development Julia Davies – University of Cambridge Extracellular ATP is now recognised as a regulator of development and stress responses. In roots it is involved in

elongative growth (including root hairs), gravitropism and mechanosensing. Salinity stress and exogenous ABA cause its accumulation by Arabidopsis roots. Extracellular ATP signalling operates through the elevation of cytosolic free calcium in Arabidopsis and the pathway involves the plasma membrane DORN1 receptor [1] and the RBOHC NADPH oxidase [2]. We have shown previously that extracellular ATP can elevate cytosolic free calcium by activating plasma membrane calcium channels but their genetic identity has yet to be established. Here we show that ATP-regulated channel activity in Arabidopsis root epidermal plasma membrane requires annexin 1 (AtANN1). This is a Ca2+-binding protein capable of conditional association with or insertion into membranes to form a Ca2+-permeable conductance operating in root elongation and response to salinity stress [3,4]. The ATP-activated root epidermal plasma membrane conductance is absent from the Atann1 mutant and the free cytosolic calcium response is impaired. In addition to root developmental responses to salinity [4], this has consequences for root mechanosensing. - Science 343: 290-294. 2. Plant Journal 58: 903-913. 3. Plant Cell 24: 1522-15334. Plant Physiology163:253-262.

500-004-Y Genetic Regulators of Arabidopsis Circadian Oscillations of Cytosolic-free Calcium Timothy Hearn – University of Cambridge Alex Webb – University of Cambridge The Arabidopsis thaliana circadian clock is a biological oscillator composed of multiple feedback loops that enhances plant fitness and survival. We previously discovered that circadian oscillations in the concentration of cytosolic free [Ca2+]cyt are driven by circadian oscillations in the concentration of cyclic ADP-ribose (cADPR), a Ca2+ agonist that releases Ca2+ from the ER and vacuole. Nicotinamide, the by-product of cADPR synthesis, inhibits ADPRcyclase activity, abolishing circadian oscillations of cADPR and [Ca2+]cyt. Nicotinamide also increases the period of other circadian oscillations such as regulation of the CAB2 promoter. This lead us to propose that cADPR-mediated circadian oscillations of [Ca2+]cyt form a signalling loop that regulates the circadian oscillator. (Dodd et al., 2007 Science 318, 1789 -1792). There is no enzyme with similarity to known ADPR cyclase in plants, making reverse genetic analysis impossible. We screened an EMS population of Arabidopsis for altered responses of the circadian oscillator to nicotinamide. We identified sin1 (insensitive to nicotinamide1; no change in period) and son1 (over sensitive to nicotinamide1; very long period in the presence of nicotinamide). sin1 and son1 are not classical period mutants, having wild-type circadian period in the absence of nicotinamide. sin1 and son1 are instead nicotinamide-response mutants affected in their ability to dynamically adjust circadian period. sin1 and son1 have profound Ca2+ signalling defects, supporting our hypothesis that the effects of nicotinamide on the circadian oscillator are linked to Ca2+ signalling. son1 has arrhythmic and high [Ca2+]cyt in constant light and is over-sensitive to NO whereas sin1 has arrhythmic and low [Ca2+]cyt in constant light and is hyposensitive to NO. Our results indicate that son1 negatively regulates cADPR signalling. We have identified the first genetic components in cADPR signalling in plants, and show that our mutations in this pathway can affect cADPR signalling over both circadian and short time periods.

500-005-Y Regulation of PSEUDORESPONSE REGULATOR7 by Photosynthetically Derived Sugars in Arabidopsis Thaliana Alexander Frank – University of Cambridge Jelena Kusakina – University of Leeds, Fiona E Belbin – University of Bristol, Anupama Chembath – Newcastle University, Michael J Haydon – University of York, Antony N Dodd – University of Bristol, Alex AR Webb – University of Cambridge The circadian clock is a complex mechanism by which plants tell the time, and which is important for photosynthesis, growth, survival as well as competitive advantage. Recently, we demonstrated that endogenous sucrose derived from photosynthesis provides an input into the Arabidopsis circadian clock through regulation of the core circadian clock gene

PSEUDORESPONSE REGULATOR7 (PRR7). I am investigating the mechanisms by which sucrose inhibits the expression of PRR7. Using a reverse genetics approach I have identified a potential pathway by which sugar signals regulate the activity of PRR7 and the speed of the circadian clock.

500-006-Z Visualization of Real-time Cytosolic Ca2+ Elevation Using a Novel Optogenetic Ca2+ Sensor Yi Ma – University of Connecticut Gerald Berkowitz – University of Connecticut Ca2+ is a ubiquitous secondary messenger involved in numerous plant signaling pathways. Ligand binding to a cognate receptor triggers a cytosolic Ca2+ spike required for initiation of downstream signaling. Detection of cytosolic Ca2+ in plants has used Genetically Encoded Ca2+ Indicators (GECIs). GCaMP, a GECI generated from the fusion of circularly permutated GFP, calmodulin (CaM), an2+d the CaM binding peptide M13. GCaMP5 has improved background fluorescence, dynamic range and signal-to-noise ratio. GCaMP has been recently fused with cell membrane ion pumps to examine localized Ca2+ generation in animal cell membrane microdomains. We tested the efficacy of GCaMP5 in the plant system driven by 35S or Ubiquitin10 promoter. At the basal cytosolic Ca2+ level, little GCaMP signal is detected. Ligand addition causes bright GCaMP5-dependent fluorescence. We observed Ca2+ induced fluorescence in protoplasts transiently expressing GCaMP5 in response to various stimuli. Laser Scanning Confocal Microscopy showed that flg22 and Pep3 induced Ca2+ elevation is localized at the plasma membrane and cell periphery. A Ca2+ signal was also detected in the nucleus. Prior studies with GECIs in plants have generally not distinguished Ca2+ spikes localized at the cell membrane in contrast to a general rise in cytosolic Ca2+. Transgenic plants expressing GCaMP will also be examined to detect the origin (i.e. plasmamembrane or endomembrane) of Ca2+ spikes induced by different ligands. We are also constructing GCaMP-receptor fusion proteins to investigate whether Ca2+ generation occurs in localized receptor microdomains. GCaMP differs from Yellow Cameleon (YC) assays in that the GCaMP signal relies on single-protein fluorescence instead of Förster resonance energy transfer (FRET), which makes experimental conditions easier to manipulate. Our work suggests GCaMP may be developed as another useful and important new tool for live-cell Ca2+ detection in plants.

500-007-Z SUGAR INSENSITIVE8, Part of a Mechanism Linking Sugar Signaling and Carbon Partitioning? Erik Solhaug – University of Minnesota Chunyao Li Li – University of Minnesota, Yadong Huang – University of Minnesota, Ling Li – Iowa State University, Eve Wurtele – Iowa State University, Susan I. Gibson – University of Minnesota Some soluble sugars, in addition to being important for metabolism, also act as signaling molecules. The Arabidopsis SUGAR INSENSITIVE8 (SIS8) is a putative mitogen activated protein kinase kinase kinase (MAP3K) that functions in the sugar-response network. Loss-of-function mutations in SIS8 display a sugar-resistant seedling developmental phenotype. Unlike some other sugar-response mutants, sis8 mutants exhibit a wild-type response in all phytohormone response assays conducted to date. In addition to being defective in sugar response, the sis8 mutants also exhibit reduced leaf starch levels. The results of transcriptional profiling experiments indicate that SIS8 affects expression of QQS, a negative regulator of starch metabolism, suggesting that SIS8 and QQS may form part of a pathway linking sugar response and carbon partitioning. Current efforts are focused on further elucidating the role of SIS8 in these processes and identifying additional proteins acting in sugar response and regulation of carbon partitioning. Towards this end, preliminary data suggesting that SIS8 may affect seed composition in addition to starch levels are being investigated. We are also characterizing sugar

response and carbon partitioning in lines carrying mutations in MAP3Ks with sequences similar to SIS8, MAP2Ks, and genes identified using SIS8 as bait in a yeast 2-hybrid screen. To date, mutations in one of the genes identified via the yeast two-hybrid screen, UDP-GLUCOSYLTRANSFERASE72E1 (UGT72E1), have been shown to cause a sugar-resistant phenotype. The results of bimolecular fluorescence complementation experiments confirm that SIS8 and UGT72E1 interact and are predominantly localized to the nucleus. Ultimately, the goal of these experiments is to increase our understanding of sugar response, including the possible role of sugar response in regulation of carbon partitioning.

500-008-Y Development and Testing of a New Biosensor for Real-time in Vivo Measurement of the Cytosolic Secondary Messenger cAMP in Plants Hsuan Chou – University of Connecticut Gerald Berkowitz – University of Connecticut 3’-5’-cyclic adenosine monophosphate (cAMP) is a secondary messenger generated from ATP by adenylyl cyclase (AC) in animals. No canonical AC has been yet identified in plants. cAMP levels in plants (measured on a whole-tissue basis) are very low when compared to animals. However,studies suggest that cAMP in plants is functional and associated with signaling. Published data only show plant cAMP level in vitro; this involves tissue destruction and determination of cAMP as an average quantity across the entire volume fo the cell. The pGloSensor™-22F cAMP plasmid is a biosensor that encodes a cAMP binding domain fused to a mutated form of Photinus pyralis luciferase. Upon binding to cAMP, conformational changes occur which reconstitute luciferase structure, and promote large increases in light output (in animal HEK293 cellsused to test the reporter).We have subcloned the coding region of this cAMP reporter plasmid into a plant expression vector for stable expression. In transgenic GloSensor plant, we are able to detect a 4-fold change that lasted for couple seconds when given lipophilic-cAMP and forskolin. Forskolin activates adenylyl cyclase and allows conversion of ATP into cAMP. Lipophilic-cAMP and forskolin-dependent cAMP elevation both occurred in a dosedependent manner. Control (non-transformed) plants showed no response to forskolin of dibutyrl-cAMP. From theses preliminary results, we conclude that (1) the GloSensor™-22F cAMP coding region can be expressed under a 35S promoter in the Arabidopsis plant and used as a biosensor to measure cytosolic cAMP level change. (2) The biosensor is sensitive enough to detect endogenous cAMP level change, which is very low in Arabidopsis. Once the system is fully established, this new developed tool can be used to test biotic or abiotic stimulus that are likely to cause cAMP level changes in Arabidopsis.

500-009-Y Calcium-Dependent Signalling in Plant Development and Environmental Stress Responses Tina Romeis – FU Berlin Heike Seybold – FU Berlin, Susanne Matschi – FU Berlin and The Sainsbury Laboratory, Guido Durian – FU Berlin and University Turku, Roman Lassig – FU Berlin and IPB Halle, Tiziana Guerra – FU Berlin, Katharina Hake – FU Berlin Ca2+-activated signaling pathways have long been recognized as a prerequisite in the onset of early intracellular signalling to mount respective plant responses mediating abiotic stress tolerance or pathogen resistance but also to developmental processes and advances in calcium imaging technologies reveal increasing insight into the dynamics of stress/growth specific and spatially restricted calcium concentration changes. Consequently, calcium sensors and among them in particular calcium-regulated protein kinases have been postulated as potential decoders that sense and translate induced changes in Ca2+ into further downstream signaling events. With respect to innate immune signaling, members of the calcium-dependent protein kinase (CDPK) gene families have been identified as positive regulators for both local early and late systemic defence responses. CDPK proteins become biochemically activated via posttranslational activation upon stimulation, and constitutive CDPK signalling has been correlated to both, rapid plasma membrane-mediated changes such as NADPH oxidase-mediated ROS production as well as to transcriptional

reprogramming depending on the activity of transcription factors. With respect to shoot development a first member of the CDPK gene family has been identified displaying a negative regulatory function evident at the transition from the vegetative to the generative growth phase. Plant stress-induced as well as developmental phenotypes are dependent on alterations in phytohormone signalling. Our data provide evidence for overlapping but also distinct roles for CDPK isoforms as calcium sensors coordinating stress responses as well as plant growth and development.

500-010-Z Proteomic Analysis of Phosphoproteins Involved in ABA Signaling in Arabidopsis Jiaxu Li – Mississippi State University Jie Song, Hanno Steen Abscisic acid is an important hormone regulating many aspects of plant growth and development as well as stress responses. Reversible protein phosphorylation plays a central role in mediating ABA-regulated physiological responses. Arabidopsis SNF1-related protein kinases 2 (SnRK2s) are thought to be positive regulators in ABA signaling pathway. However, the target proteins of these kinases are still largely unknown. The objective of this project is to identify the target phosphoproteins of SnRK2-2 and SnRK2-3. To analyze phosphoproteins regulated by SnRK2-2 and SnRK2-3, proteins from rice leaves (wild type and srk2-2 or srk2-3 knockout mutants) treated with abscisic acid were separated by two-dimensional gel electrophoresis and probed with phosphoamino acid-specific antibodies, and identified by tandem mass spectrometry. Ten phosphoproteins differentially regulated by abscisic acid were identified. Possible role of these phosphoproteins in abscisic acid signaling will be discussed.

500-011-Z Rate Motifs Tune Aux/IAA Degradation Dynamics Britney Moss – University of Washington Maia Sebek – University of Washington, Amy Lanctot – University of Washington, Amber Hageman – University of Washington, Guseman Jessica – University of Washington, Jennifer Nemhauser – University of Washington Ubiquitin-mediated protein degradation is a common feature in many plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases, SCFTIR1/AFBs, facilitates ubiquitination of Aux/IAA repressor proteins in the presence of the hormone auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation. We have used synthetic auxin degradation assays in yeast to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA repressor degradation. The presence of these “rate motifs” is conserved in phylogenetically-distant members of the Arabidopsis thaliana Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic Arabidopsis thaliana plants expressing an IAA28 with a rate motif deletion resembled plants expressing IAA28 degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses.

500-012-Y Biochemical Analysis of Arabidopsis Glutaredoxin C2-catalyzed S-glutathionylation of BRI1-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1) Kyle Bender – University of Illinois at Urbana-Champaign Xuejun Wang – Washington University in St. Louis, George Cheng – University of Illinois at Urbana Champaign, Hyoung Kim – Korea Institute of Science and Technology, Raymond Zielinski – University of Illinois at Urbana Champaign, Steven

Huber – USDA-ARS Post translational modification (PTM) of proteins is widely recognized as a critical mechanism of signal transduction in eukaryotic organisms. Among PTMs, reversible phosphorylation catalyzed by protein kinases is the best characterized; other PTMs, including redox modifications, remain less well understood, particularly in plant systems. In a yeast-twohybrid screen, we identified a glutaredoxin, AtGRXC2, as an interacting partner of the leucine-rich repeat receptor-like kinase (LRR-RLK) BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR-LIKE KINASE 1 (BAK1) and the interaction was confirmed in vitro using recombinant BAK1 cytoplasmic domain and AtGRXC2. Glutaredoxins are implicated in both the formation and reduction of protein mixed disulfides and so we tested whether BAK1 could be Sglutathionylated in vitro. We found that, at high concentrations of glutathione (GSH or GSSG), BAK1 could be spontaneously glutathionylated, and that recombinant AtGRXC2 could enhance BAK1 glutathionylation at low concentrations of GSSG. Interestingly, BAK1 but neither BRI1 nor FLS2 could be glutathionylated in vitro, suggesting that redox modification may be specific to BAK1 and perhaps other SERKs. The effect of glutathionylation was to inhibit BAK1 kinase activity as determined in peptide kinase assays. Using MALDI-TOF mass spectrometry and various Cys-toSer directed mutants, we demonstrated that Cys353, Cys374, and Cys408 were the sites of glutathionylation on BAK1. Mutation of Cys residues typically resulted in loss of kinase activity indicating the importance of Cys residues in BAK1 structure/function. The C408S directed mutant retained full kinase activity and remained sensitive to peroxide and glutathione, suggesting that Cys408, while capable of being glutathionylated is not the only site contributing to redox inhibition of BAK1. Collectively, our data reveal redox status as a potential new regulatory mechanism for LRR-RLKs in plants. Understanding the role of BAK1 redox regulation and glutathionylation in brassinosteroid versus immunity signaling is the focus of current studies. Supported by NSF IOS grant number 1354094.

500-013-Y Possible Involvement of PP2C-D for Dephosphorylation of the Plasma Membrane H+-ATPase in Guard Cells of Arabidopsis Thaliana Hodaka Sugimoto – Nagoya University Yohei Takahashi, Yuki Hayashi, Koji Takahashi, Shinichiro Inoue, Mee Park, William Gray, Toshinori Kinoshita Plasma membrane H+-ATPase mediates stomatal opening in response to blue light, and blue light induces activation of the H+-ATPase through the phosphorylation of a penultimate threonine (Thr) of H+-ATPase in stomatal guard cells. In vitro biochemical investigation showed that Mg2+ dependent, membrane localized protein phosphatase dephosphorylates the phosphorylated penultimate Thr of H+-ATPase. More recently, it has been demonstrated that type 2C protein phosphatase clade D (PP2C-D) dephosphorylates the phosphorylated penultimate Thr of H+-ATPase in Arabidopsis hypocotyl. However, it is still unclear whether the PP2C-Ds function in stomatal movements through dephosphorylation of the H+-ATPase in guard cells. Transient gene expression experiment using Arabidopsis mesophyll cell protoplasts showed that all members of the Arabidopsis PP2C-D family dephosphorylate the endogenous H+-ATPase. Moreover, they are located around plasma membrane. In silico analysis shows that one of the PP2C-D isoform(PP2C-Di) is strongly expressed in guard cells. A recombinant PP2C-Di directly dephosphorylates the H+-ATPase in vitro. Then, we performed genetic analysis of PP2C-Di using knockout mutants. Stomata in pp2c-di mutants closed more slowly than wild type (WT) when they were transferred to the dark condition. Stomatal apertures in PP2C-Di overexpressing plants driven by guard cell specific promoter GC1 were small under light condition compared with WT. Taken together, these results suggest that PP2C-Di mediates stomatal closure through dephosphorylation of the H+-ATPase in guard cells.

500-014-Z Identification and Characterization of SnRK1 Complexes Present in Leaves of Arabidopsis Thaliana Patricia Coello – National Autonomous University of Mexico Alejandra Avila, Ana Karen Ruiz, Ricardo Trejo, Juan Pablo Salazar, Jose Luis Maya, Eleazar Martinez-Barajas SnRK1 is a heterotrimeric protein kinase complex involved in the maintenance of energy homeostasis at the cellular and whole plant level. It is activated by starvation and energy-depleting stress condition turning on energy-producing catabolic processes and limiting energy-consuming anabolic metabolism. During phosphate (Pi) starvation, plants have severe reduction in ATP levels eliciting a complex morphological, physiological and biochemical/metabolic adaptations known as Phosphate Starvation Responses (PSR). In Pi starvation condition, SnRK1 and SnRK2 protein kinase increase in activity and separation by gel filtration of control and Pi deficient leaf extracts identified high molecular weight fractions containing different subunits, suggesting the presence of several complexes that might have a function in promoting adaptation to Pi deprivation. We co-expressed different recombinant subunits in E. coli and in this work, we described the preferential association among them as well as the catalytic activity of heterotrimeric complexes against specific substrates, such as PHR1 and npG3PDH. This work was supported by DGAPA (IN211513), PAIP 5000-9126.

500-015-Z Characterization of Multiple Chitin-induced Defense Response Genes in Arabidopsis Justin Ray – University of Alabama Xin Yang, Katrina Ramonell Our research using the model plant Arabidopsis thaliana is focused on unraveling the signal transduction pathways involved in elicitor-mediated plant defense, particularly defense pathways involved in resistance to fungal pathogens. Our studies center on the response to the fungal elicitor chitin, which has been shown to induce a strong defense response in numerous plant species. We have isolated a group of related genes, the CRLK (Chitin Receptor-Like Kinase) family, which appear to play a direct role in defense against fungal pathogens. Sequence alignment of these genes reveals orthologs in a variety of crop species (corn, wheat, rice, etc.). Of the seven genes in the CRLK family, certain genes appear to strongly affect the susceptibility of A. thaliana to fungal pathogens in both positive and negative ways. Our goal is to characterize these genes and elucidate the pathways activated and regulated by them.

500-016-Y Plasma Membrane H+-ATPase in the Charophytic Alga Koji Takahashi –Nagoya University Koichi Hori – JST CREST, Tokyo Tech, Kunika Ohtaka – Tokyo Tech, Hiroyuki Ohta – JST CREST, Tokyo Tech, Toshinori Kinoshita – ITbM, Nagoya University Plasma membrane H+-ATPases, which create electrochemical potential gradient across the plasma membrane by utilizing the energy of ATP hydrolysis in plants, are composed of a functional single polypeptide containing 10 transmembrane domains and three cytosolic regions. The C-terminal cytosolic region containing the regulatory domains serves as an autoinhibitory region in the H+-ATPase from vascular plants. The catalytic activity of the H+-ATPase is thought to be regulated through the conformational change occurred by the phosphorylation of the penultimate threonine in the C-terminus and the subsequent interaction of 14-3-3 protein to the phosphorylation site. In the green alga, such regulatory domains and the penultimate threonine have not been observed in their H+-ATPases. We previously reported that both vascular plant-type and green alga-type H+-ATPases are observed in the bryophytes,

suggesting that the vascular plant-type H+-ATPases would appear during the evolutionary transition from an aquatic to a terrestrial habitat. In this study, we focused on the Charophyta that is the closest living relatives of land plants, and have found that the vascular plant-type H+-ATPase is observed in an aero-terrestrial charophytic alga and the penultimate threonine in the C-terminus is phosphorylated in response to physiological stimuli. These results suggest that the H+ATPases having the penultimate threonine in their autoinhibitory C-terminal region and the regulatory mechanism of the H+-ATPase phosphorylation functioned in the earliest plants which were adapted on the terrestrial habitat.

500-017-Y The Activated Prf Resistance Protein Stabilizes the NAC1 Transcription Factor to Trigger Defense Signaling Joanna Kud – University of Idaho Xiangli Niu – Hefei University of Technology, Min Miao – Hefei University of Technology, Yongsheng Liu – Hefei University of Technology, Fangming Xiao – University of Idaho In tomato, resistance to bacterial speck disease, caused by Pseudomonas syringae pv. tomato (Pst), is determined by the Prf, a NB-LRR type resistance protein that remains inactive in the absence of Pst. Although the activation of Prf by the Pst effectors AvrPto/AvrPtoB is well-demonstrated, the transmission of the defense signal from the activated Prf to downstream cellular defense events is poorly understood. We have recently reported that a defense-related tomato NAC1 (NAM, ATAF1.2, CUC2) transcription factor plays a positive role in the Prf-mediated disease resistance, as shown by quick induction of the NAC1 gene in response to Pst infection and the silencing of NAC1 resulting in attenuation of resistance to Pst. Moreover, the stability on NAC1 protein is tightly regulated through ubiquitin-proteasome mediated degradation. Our current research focuses on investigation of the functional mechanisms that link NAC1 to the Prfmediated defense signaling. We have found that the autoactive PrfD1416V mutant, but not the wild type Prf, can stabilize NAC1 in vivo. Significantly, the stabilization of NAC1 appears to be fulfilled through the physical interaction between PrfD1416V and NAC1, which has been determined by the co-immunoprecipitation assay. Several NB-LRR resistance proteins have been shown to interact with transcription factors thus implying the control of transcriptional reprogramming by direct manipulation of transcription factors during defense responses may be the major activity of R proteins, however the role of such interaction and the mechanistic basis of NB-LRR protein-mediated manipulation of transcription factor is still unknown. Our results support a hypothesis that upon activation by pathogen effector(s), activated Prf undergoes conformational change, which allows it to interact with NAC1 transcription factor, thereby stabilizing it presumably by interfering with the ubiquitination of NAC1.

500-018-Z Transcriptomics Analysis of Oryza Sativa L. Overexpressing the Calcium Sensor OsCam1-1 Teerapong Buaboocha – Chulalongkorn University Worawat Yuenyong – Chulalongkorn University, Mayura Thongchuang – Chulalongkorn University, Aumnart Chinpongpanich – Chulalongkorn University, Supachitra Chadchawan – Chulalongkorn University, Luca Comai – University of California, Davis In plants, Ca2+ signals have been implicated in transducing the initial signals from various environmental changes, which must be correctly perceived and discriminated so as to elicit the correct subsequent cellular response, a task performed by the Ca2+-modulated proteins. The overexpression of the Ca2+-modulated calmodulin gene, OsCam1-1 was previously shown to confer a better salt-tolerant ability to the transgenic rice Oryza sativa L. ‘KDML105’. Here, transcriptome profiles of the three-week old transgenic rice seedlings grown under normal and salt stress condition (150 mM NaCl) for four hours was generated and compared with those of the wild type by RNA Seq. Overall, 1,677 genes were found differentially expressed between the wild type and the transgenic rice grown under salt stress. The comparative transcriptome profiling revealed differentially expressed genes in several cellular processes including

glycolysis, tricarboxylic acid cycle, glyoxylate cycle, sucrose/starch metabolism, and transcription. Expression of selected genes involved in these processes was confirmed by real-time RT-PCR. Gene Ontology Enrichment analysis has shown that the OsCam1-1 overexpression highly affects the carbohydrate metabolism under salt stress condition.

500-019-Z An Improved FRET-based Sensor for Calmodulin Activation Ray Zielinski – University of Illinois Kyle Bender – University of Illinois, Steven Huber – University of Illinois A wide array of stimuli trigger changes in intracellular Ca2+ concentration ([Ca2+i]). Considerable evidence supports the idea that characteristic patterns of change in [Ca2+i] (Ca2+ signatures) occur in response to different stimuli; much less is known about how specific Ca2+ signatures are transduced. Calmodulin (CaM), one of the primary Ca2+ sensor proteins, has been implicated as a transducer of [Ca2+i] changes into various cellular responses. We seek to determine whether CaM activation signatures are generated that correspond with specific Ca2+ signatures. CaM sensors, consisting of fusions of cyan fluorescent protein: the CaM-binding domain of myosin light chain kinase: yellow fluorescent protein, have been used in animal cells to monitor changes in CaM activation. They have not been used successfully in plant cells, however. We optimized this sensor by substituting monomeric Turquoise2 and Venus fluorescent proteins for the cyan and yellow versions, respectively, and by incorporating a series of deletions to mTurquoise2 and mVenus. As a result, both the photostability and the dynamic range of the sensor are improved. Potential complications to deploying and interpreting the results of a CaM sensor in vivo include: (1) possible contribution of the large number of CaM-like proteins (CMLs) expressed in plants; and (2) depletion of the pool of CaM and disruption in cellular regulation. Assays comparing the binding of CaM and CML8, which shares 80% sequence similarity, indicate that the sensor is more responsive to CaM and that the CaM sensor does not interact with CMLs more divergent from CaM than CML8. Current efforts are directed toward expressing the CaM sensor in Arabidopsis protoplasts to determine the optimum promoter strength that will facilitate imaging while minimally perturbing of the pool of CaM. Supported by NSF IOS1354094.

500-020-Y Role of Protein Phosphatase 2A in Root Growth Under Salt Stress Estelle Hrabak – University of New Hampshire Megan Thompson – University of New Hampshire, Kristen Getchell – University of New Hampshire, Enhua Wang – University of New Hampshire Salt stress activates signaling networks that are regulated by phosphorylation. Protein Phosphatase 2A is involved in salt stress responses in Arabidopsis thaliana. The PP2A holoenzyme is a heterotrimer consisting of a scaffolding A subunit, a regulatory B subunit, and a catalytic C subunit. Multiple isoforms of each subunit are encoded in the Arabidopsis genome and all A and C subunit genes are ubiquitously expressed with high expression near root tips. When grown on an agar surface, seedlings with mutations in a subset of A and C subunit genes displayed root skewing or curling in response to sodium salts. Sodium-dependent alterations in root growth occurred rapidly following exposure to salt stress. The sodium-induced root skewing was phenocopied by treatment with a phosphatase inhibitor. Epitope-tagged A and C subunits were co-purified from roots, indicating in planta association of the subunits involved in salt stress responses. These results indicate that Protein Phosphatase 2A is involved in maintaining normal root growth under sodium stress.

500-021-Y Recruitment of PLANT U-BOX13 and the PI4Kβ1/β2 by the Small GTPase RabA4B Plays Important Roles During SA-Mediated Plant Defense Signaling in Arabidopsis Gwangbae Bak – University of Michigan Vincenzo Antignani, Amy Klocko, Suma Chandrasekaran, Taylor Dunivin, Erik Nielsen – University of Michigan Protection against microbial pathogens involves the activation of cellular immune responses in eukaryotes, and this cellular immunity likely involves changes in subcellular membrane trafficking. In eukaryotes, members of the Rab GTPase family of small monomeric regulatory GTPases play prominent roles in the regulation of membrane trafficking. We previously showed that RabA4B is recruited to vesicles that emerge from trans-Golgi network (TGN) compartments and regulates polarized membrane trafficking in plant cells. As part of this regulation, RabA4B recruits the closely-related phosphatidylinositol 4-kinases (PI4K) PI4Kβ1 and PI4Kβ2 lipid kinases. Here we identify a second Arabidopsis thaliana RabA4B interacting protein, PLANT U-BOX13 (PUB13), which has recently been identified to play important roles in salicylic acid (SA)-mediated defense signaling. We show that PUB13 interacts with RabA4B through amino-terminal domains, and with phosphatidylinositol 4-phosphate (PI-4P) through a carboxyl-terminal armadillo domain. Further, we demonstrate that a functional fluorescent PUB13 fusion protein (YFP-PUB13) localizes to TGN and Golgi compartments, and that PUB13, PI4Kβ1, and PI4Kβ2 are negative regulators of SA-mediated induction of pathogenesis-related gene expression. Taken together, these results highlight a role for RabA4B and PI-4P in SA-dependent defense responses.

500-022-Z Vac14 Is a Scaffold Protein Involved in PI(3,5)P2 Biosynthesis and May Regulate Auxin Distribution and Osmotic Stress Response in Arabidopsis Kelly Stecker – UW-Madison Michael Sussman – UW-Madison Inositol phospholipids are important signaling molecules found in all organisms. Phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) is a low abundant, poorly understood inositol phospholipid involved in defining vacuole morphology and regulating vesicle transport in eukaryotes. PI(3,5)P2 signaling is involved in the initial osmotic stress response in yeast and mammalian cells where lipid levels rapidly and transiently increase following dehydration. PI(3,5)P2 metabolism is tightly controlled by a protein complex held together by the scaffolding protein Vac14. Both the lipid kinase and lipid phosphatase responsible for PI(3,5)P2 biosynthesis and catabolism are bound by Vac14 in a single complex. Despite the nearly universal importance of this scaffolding protein in eukaryotes, the role of Vac14 in plant systems has not been explored. We recently discovered that Vac14 is rapidly and transiently phosphorylated in response to osmotic stress in Arabidopsis, indicating that the PI(3,5)P2 biosynthesis complex maybe regulated during the initial response to reduced water availability. This evidence supports the hypothesis that PI(3,5)P2 signaling plays an important role during plant osmotic stress response. Interestingly, overexpression of Vac14 leads to severe shoot gravitropic phenotypes, suggesting a defect in polar auxin distribution. In summary, our data indicates that Vac14 is an important regulatory protein involved in stress response and plant growth and provides insight into the function of PI(3,5)P2 in plants.

500-023-Z Multiple Calmodulin Binding Sites Reveal Complex Regulation of Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL 12 (AtCNGC12) Function in Programmed Cell Death Thomas DeFalco – University of Toronto Huda Abdel-Hamid – University of Toronto, Christopher Marshall – University of Toronto, Kim Munro – Queen’s

University, Hong-Gu Kang – Texas State University, Wolfgang Moeder – University of Toronto, Mitsuhiko Ikura – University of Toronto, Wayne Snedden – Queen’s University, Keiko Yoshioka – University of Toronto Cyclic nucleotide-gated ion channels (CNGCs) are non-selective cation channels present as multi-member gene families in plants, where various CNGC isoforms have been implicated in diverse signaling pathways, including immunity. CNGCs are thought to function primarily as Ca2+ channels in plants, and it has also been hypothesized that like their mammalian orthologs, plant CNGCs are regulated by the conserved Ca2+ sensor calmodulin (CaM). However, little is known regarding plant CNGC regulation or structure-function, particularly in contrast to the well-studied mammalian CNGC family. Most plant CNGCs are predicted to share a CaM-binding domain (CaMBD) in their cytosolic C-terminus, which overlaps with the cyclic nucleotide-binding domain (CNBD). However, the Arabidopsis isoform AtCNGC12, a positive regulator of plant defense, shares low sequence conservation at this region. Our results show that AtCNGC12 possesses a novel domain organization featuring multiple CaMBDs arranged at both the cytosolic N- and C-termini of the channel, which do not overlap with the CNBD. Extensive biophysical characterization of these CaMBDs revealed that two sites are Ca2+-dependent and bind CaM with high but differing affinities, while a third site is a conserved IQ motif that binds CaM differentially depending on the presence of Ca2+. Physiological analyses revealed that mutations in individual CaMBDs of AtCNGC12 can trigger or suppress the induction of programmed cell death (PCD), providing the first in planta evidence of CNGC regulation by CaM. Thus our data demonstrate CaM differentially binds multiple sites in AtCNGC12 to coordinate both channel function and regulation, indicating a complex and previously unexplored mode of Ca2+-dependent regulation of CNGCs. Emerging evidence that changes the current view of plant CNGC regulation by CaM will be discussed.

500-024-Y A Tomato Universal Stress Protein Involved in Oxidative Stress Resistance Is a Novel Phosphorylation Target of the CIPK Family Olga del Pozo – Consejo Superior de Invstigaciones Científicas (CSIC) Emilio Gutierrez-Beltran – Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Invstigaciones Científicas (CSIC)/Universidad de Sevilla, Fernando de la Torre – Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior de Invstigacion In plants, calcineurin B-like interacting protein kinases (CIPKs) decode calcium signals upon interaction with the calcium sensors calcineurin B like proteins (CBLs) into phosphorylation events that result into adaptation mainly to environmental stresses. So far, few phosphorylation targets of CIPKs are known and therefore the molecular mechanisms underlying their downstream output responses are not fully understood. Tomato (Solanum lycopersicum) Cipk6 regulates immune and susceptible PCD responses in immunity transforming Ca2+ signals into ROS signaling. To investigate SlCipk6-induced molecular mechanisms and identify putative substrates, a yeast two hybrid (Y2H) approach was carried on and a protein was identified that contained an Universal stress protein (Usp) domain present in bacteria, protozoa and plants, which we named Cip29. Cip29 was found to be an ATP-binding protein that formed homodimers in planta. SlCipk6 and Cip29 interacted using co-immunoprecipitation and bimolecular fluorescence complementation (BiFC) assays in N. benthamiana leaves,and the complex was localized in the cytosol. SlCipk6 phosphorylated Cip29 in vitro, thus defining a novel target for the CIPK family. Cip29 transcript rapidly accumulated in response to different environmental stress condition. Heterologous Cip29 overexpression in yeast conferred resistance to highly toxic LiCl, whereas Cip29 expression in Escherichia coli (E. coli) UspA mutant restored bacterial viability in response to H2O2 treatment. We identified a novel phosphorylation target for the CIPK family, which provides protection against the toxic effect of LiCl in yeast and to H2O2 in bacteria, suggesting that its stress protection-role has been evolutionary conserved from bacteria through plants.

500-025-Y PLDα1 Is a Key Component and Regulator of G-protein Mediated ABA Signaling Swarup Roy Choudhury – Donald Danforth Plant Science Center Sona Pandey Heterotrimeric G-proteins comprised of Gα, Gβ and Gγ proteins are important signal transducers in all eukaryotes. In plants, G-proteins are involved in the regulation of multiple biotic and abiotic stresses as well as many developmental processes, even though their repertoire is significantly limited compared to that in the metazoan systems. One Gα, one Gβ and three Gγ proteins represent the heterotrimeric G-protein complex in Arabidopsis and a single regulatory protein, RGS, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of the signaling components and the range of processes they regulate is rather intriguing. We now present evidence that phospholipase Dα1 protein is a key component and regulator of G-protein complex, especially during ABA signaling. We also show that the same G-protein subunits and their regulators exhibit distinct biochemical and genetic interactions depending on the specific signal. Such biochemical plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine-tune the plants’ response to constantly changing environment.

500-026-Z Unraveling the Molecular Mechanism Ensuring Ca2+ Signaling Specificity in Guard Cell ABA Signal Transduction Shintaro Munemasa – Okayama University Benjamin Brandt – University of California, San Diego, Cun Wang – University of California, San Diego, Desiree Nguyen – University of California, San Diego, Julian Schroeder – University of California, San Diego The universal second messenger, Ca2+ is involved in a broad array of physiological processes in plants. A key question is how Ca2+ signaling specificity is achieved in plant cells. The drought-inducible phytohormone abscisic acid (ABA) triggers stomatal closure, thus reducing excess transpirational water loss. It has been demonstrated for over 20 years that cytosolic Ca2+ ([Ca2+]cyt) plays a key role in guard cell ABA signaling. Recent research has shown that ABA enhances (primes) the guard cell Ca2+ sensitivity providing a mechanism that enables stimuli-specific Ca2+ responses, but the underlying genetic and biochemical mechanisms remain unknown. It has been shown that ABA employs both [Ca2+]cytindependent and [Ca2+]cyt-dependent pathways. However, the underlying molecular details remain unclear as well. Recent ABA-signal transduction pathway reconstitution in Xenopus oocytes showed that the S-type anion channel protein SLAC1 can be activated by co-expression of either the Ca2+-independent protein kinase OST1 or the Ca2+dependent protein kinase (CPK6). Therefore, the Ca2+-independent and Ca2+-dependent branches of ABA signal transduction are presently considered to be independent. Here we provide biochemical, electrophysiological, and cell signaling evidence that unravels molecular mechanisms explaining how plant cells decode stimuli-specific Ca2+ signals and reveal that the Ca2+-dependent and Ca2+-independent ABA signaling mechanisms are interdependent and can form a fined-tuned and robust signal transduction network.

500-027-Z Understanding Redox Signaling in Plants Through Identification and Functional Characterization of Redoxsensitive Proteins Yiji Xia – Hong Kong Baptist University Yimin Li, Pei Liu, Huan Zhong, Shuying Pan, Hailei Zhang, Shoudong Zhang Reactive oxygen species (ROS) have increasingly been recognized as important regulators in various physiological and

developmental pathways. ROS signaling is mediated largely through actions on redox-sensitive proteins that undergo oxidative modifications in response to perturbation of cellular redox states. We have developed gel-based and gel-free (OxiTRAQ) quantitative redox proteomics methods to identify Arabidopsis proteins whose thiols underwent oxidative modifications in response to treatments of ROS, salicylate, flagellin, and the bacterial pathogen Pseudomonas syringae. These redox-sensitive proteins are involved in a variety of biological processes including chromatin remodeling and transcription, mRNA processing, post-translational modifications, and primary and secondary metabolism. A redoxsensitive transcription factor has been found to play an important role in redox sensing and regulation of oxidative stress responsive genes and in tolerance to abiotic stresses. Our progresses in developing redox proteomics methods and in characterizing the role of the redox-sensitive transcription factor in redox signaling and stress responses will be presented.

HORMONE BIOLOGY Zone 600 600-001-Y Identification of Jasmonic Acid and Functional Characterization of Allene Oxide Cyclase in the Model Lycophyte Selaginella Moellendorffii Putri Pratiwi – Hokkaido University Tomohiro Takahashi – Hokkaido University, Hideyuki Matsuura – Hokkaido University, Kosaku Takahashi – Hokkaido University Plants began colonizing the land approximately 430 million years ago and have been adapting to unique environmental stresses ever since. Jasmonic acid (JA) is a known regulator of defense responses and numerous other processes related to plant development and stress adaptation. JA is a naturally occurring phytohormone that so far is ubiquitously produced in vascular plants only. The detailed functions of JA have been well studied in seed plant. Currently, we are investigating the evolutionary history of the role of JA as a mediator to regulate the defense responses against wound stress using Selaginella moellendorffii, a model lycophyte that is taxonomically positioned between bryophyte and euphylophyte. Here, we present the first evidence of the presence of JA in S. moellendorffii as a basal vascular plants. Applying exogenous JA significantly inhibited the growth of this plant. Moreover, a putative homolog of allene oxide cyclase (AOC), designated as SmAOC1, was also identified in this plant. AOC is a crucial enzyme to establish the formation of the basic structure of jasmonates. SmAOC1 is 738 bp long, encoding a 245 amino acid protein with a molecular mass of 26.4 kDa. The amino acid sequence of SmAOC1 shares high similiarity with those of AOCs from other plants. SmAOC1 was actively involved in the formation of cis-(+)-OPDA and located in chloroplast. In response to wound stress, the expression level of SmAOC1 coincided with the accumulation of OPDA and JA, peaking at 2 h after treatment. The expression of SmAOC1, which was also activated by the application of exogenous JA, implies that feedback regulation occurs in this plant in a similar manner to those of vascular plants. Thus, JA is assumed to be an important signal not only in response to stresses, but also acts as a growth regulator in S. moellendorffii.

600-002-Z Investigating Auxin Signaling Through AUXIN RESPONSE FACTOR (ARF) Interactions David Korasick – Washington University Joseph Jez – Washington University, Lucia Strader – Washington University In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain. This domain provides basic and acidic faces of the protein that contribute to front-to-back

electrostatic protein-protein interactions capable of high-order multimerization. Calorimetric and NMR analyses of ARF7 PB1 domain interactions reveal key residues on the basic face that form a two-pronged structural interaction motif at the PB1 interface. Two decentralized interaction hot spots on the acidic protein face coordinate around these two basic prongs to stabilize PB1 interactions. In vivo studies suggest the importance of this PB1 binding interaction in both ARFARF and ARF-Aux/IAA interactions. Further, in planta analysis of the role of this domain in auxin signaling also suggests a multimerization requirement for ARF protein repression. Taken together, these biophysical and biological analyses of suggest both the ability and the importance of ARF and Aux/IAA protein multimerization, leading to a refined auxin signaling model.

600-003-Z Understanding the Role of HR Auxin Response Adam Seroka – Washington University in St. Louis Lucia Strader – Washington University in St. Louis Auxin is the primary plant hormone regulating growth and development and is critical for altering growth under varying environmental conditions, including dark responses. Many genes in Arabidopsis thaliana encode for proteins required for auxin response in a variety of ways. However, many of these genes have yet to be identified. To identify additional factors required for auxin response, I screened for EMS mutants displaying auxin resistance in hypocotyl elongation. One mutant identified was HYPOCOTYL RESISTANT 23 (HR23). Using whole genome re-sequencing, I identified 6 potential causative mutations for HR23. To determine which of these mutations is causative, I identified insertional alleles in each gene. I will examine these for altered auxin response. In addition, I will use these mutants to pinpoint the causative mutation. Although the hypocotyl elongation assay remains an effective method in identifying novel auxin related genes, the molecular mechanisms that govern hypocotyl elongation in general are poorly understood. Therefore, we identified multiple in Arabidopsis genes that are involved in light signaling, cell elongation, and auxin response that may act downstream of factors identified in our screen. I am creating reporter constructs for these to visualize the expression of these genes to elucidate the molecular mechanisms of hypocotyl elongation in vivo. These studies will uncover factors required for auxin response and will broaden our understanding of how auxin governs growth and development in young plants.

600-004-Y Molecular Basis for ENOYL-COA HYDRATASE2 and INDOLE-3-BUTYRIC ACID RESPONSE10 Roles in IBA-to-IAA Conversion Samantha Powers – Washington University in St. Louis David Korasick – Washington University in St. Louis, Lucia Strader – Washington University in St. Louis, Joseph Jez – Washington University in St. Louis In Arabidopsis thaliana, conversion of the auxin precursor indole-3-butyric acid (IBA) to the active auxin indole-3-acetic acid (IAA) is necessary for proper growth and development. IBA-to-IAA conversion occurs by peroxisomal β-oxidation. ENOYL-COA HYDRATASE2 (ECH2) and INDOLE-3-BUTYRIC ACID RESPONSE10 (IBR10) are proteins likely involved in the IBA-to-IAA conversion pathway and mutants of ech2 and ibr10 are resistant to the effects of IBA, likely due to this conversion defect. Despite the importance of these proteins, the exact reactions in the multistep IBA-to-IAA pathway catalyzed by ECH2 and IBR10 remain unknown. We have determined the structure of ECH2 and IBR10 using X-ray crystallography. Consistent with the proposed function of these proteins in peroxisomal β-oxidation of IBA-to-IAA, ECH2 and IBR10 fold into proteins containing domains typically having either thioesterase or enoyl-coA hydratase activity. Using the ECH2 and IBR10 crystal structures, we will model potential substrates in the binding pockets to determine possible roles for these proteins in the IBA-to-IAA conversion pathway. By uncovering and analyzing these structures, we

hope to better understand the molecular mechanism of ECH2 and IBR10 and the role these proteins play in IBA-to-IAA conversion.

600-005-Y Identification and Characterization of the Polarly-localized TRANSPORTER of IBA1 Lucia Strader – Washington University in St Louis Marta Michniewicz – Washington University in St Louis Levels of auxin, which regulate both cell division and cell elongation in plant development, are controlled by synthesis, inactivation, transport, and the use of storage forms. Conversion of the auxin precursor indole-3-butyric acid (IBA) to the active auxin indole-3-acetic acid (IAA) contributes to an array of seedling development events, including cotyledon expansion, root hair elongation, lateral root formation, apical hook formation, and root meristem maintenance. Although IBA transport mechanisms appear to be independent of IAA transport mechanisms, carriers required for IBA movement across the plasma membrane remain largely unidentified. The ATP- binding cassette (ABC) transporter ABCG36 is necessary for IBA efflux and abcg36 mutants are hypersensitive to the effects of IBA on root elongation inhibition. To identify IBA uptake carriers, we performed an abcg36 suppressor screen, hypothesizing that decreasing IBA uptake would suppress the abcg36 hypersensitivity resulting from blocked IBA efflux. In this screen, we identified a mutation in the founding member of an uncharacterized Major Facilitator Superfamily clade of transporters, which we named TRANSPORTER OF IBA1 (TOB1). Intriguingly, TOB1 displays a unique polar localization within several tissues, and its expression is coordinated with sites of IBA-to-IAA conversion, suggesting that TOB1 plays roles in regulating conversion of IBA to active auxin. Our characterization of tob1 reveals the complex nature of regulating levels of IBA and IBA-derived auxin within the cell to drive developmental processes.

600-006-Z Gibberellin-DELLA Signaling in Arbuscular Mycorrhizal Symbiosis Daniela Floss – Boyce Thompson Institute for Plant Research Karen Gomez – Boyce Thompson Institute for Plant Research, Kishor Bhattarai – Boyce Thompson Institute for Plant Research, Hee-Jin Park – Boyce Thompson Institute for Plant Research, Maria Harrison – Boyce Thompson Institute for Plant Research The majority of land plants are able to form mutualistic interactions with arbuscular mycorrhizal (AM) fungi. The association develops in the root and the mutualism is based on a bidirectional nutrient exchange: mineral nutrients are provided by the AM fungi to the plants and in turn, the fungi receive carbohydrates. Symbiotic nutrient exchange occurs inside root cortex cells, where the fungus establishes highly branched, tree-shaped hyphae called arbuscules. Research in our lab focuses on the molecular mechanisms underlying development of the Medicago truncatula/Glomus versiforme AM symbiosis. Recently, we discovered that DELLA proteins, which are repressors of gibberellic acid (GA) signaling, positively regulate arbuscule formation (1). However, the precise mechanism of their function in coordinating GA signaling and gene expression is still unknown. To dissect the molecular mechanisms underlying DELLA-mediated regulation of arbuscule formation, we identified several DELLA-interacting transcription factors in yeast-two hybrid screens and confirmed their interactions by co-immunoprecipitation experiments in planta. Insertion mutants in these transcription factors were obtained and analysis of their AM symbiosis phenotypes indicates at least one of the transcription factors has a role in AM symbiosis.Additionally, we have used targeted transcription profiling with della mutants to identify several DELLA downstream target genes. Recent progress on GA-DELLA signaling in AM symbiosis will be discussed.

References: (1) Floss et al.: DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis. PNAS 110, E5025-5034 (2013)

600-007-Z Electrophysiological Studies of ABCB4 and PIN2 Ion Channel Activities Stephen Des Lauriers – University of Wisconsin-Madison Edgar Spalding – University of Wisconsin-Madison The polar movement of auxin through tissues is known to require PIN and ABCB membrane proteins. The prevailing view is that both types of proteins contribute to the phenomenon by transporting auxin across the plasma membrane. Whether or not these proteins directly transport auxin is a critical point and therefore requires more direct evidence than can be provided by the auxin retention assays used to date. Our approach has been to subject mammalian cells engineered to express Arabidopsis ABCB4, PIN2, or ABCB4 and PIN2 to current-voltage analysis with the whole-cell patch clamp technique. CsCl-based electrolytes were used to suppress endogenous potassium channel activity. Cells expressing ABCB4 or PIN2 displayed ionic currents that were carried more by chloride than cesium. Co-expressing PIN2 and ABCB4 increased anion selectivity of the currents but did not produce any new or emergent properties. The effects of auxin were investigated by introducing IAA- to the cytoplasmic side of the membrane. Micromolar auxin increased ABCB4 channel activity. Millimolar auxin increased the channel activity for PIN2 in some conditions but inhibited ABCB4. Critically, no auxin transport by ABCB4, PIN2, or ABCB4 and PIN2 could be detected using a rigorous thermodynamic criterion (auxin gradient effect on the reversal potential of the current-voltage curve). Possible interpretations include 1) ABCB4 and PIN2 do not directly transport the auxin anion 2) the true functions of ABCB4 and PIN2 are not expressed in mammalian cells, and 3) the method is not sensitive enough to detect a minor auxin transport activity. Arguing against the second possibility is that a channel blocker known as NPPB completely blocked ABCB4 and PIN2 activity, and it blocked polar auxin transport in seedlings. If the first possibility is true, polar auxin transport apparently relies on ABCB and PIN proteins for channel activities that do not include auxin transport.

600-008-Y Polar Auxin Transport Mediated by ABCB19 Modulates Arabidopsis Hypocotyl Elongation via CCS52A2dependent Effects on the Nuclear Endocycle Guosheng Wu – Botany Department, University of Wisconsin Jacqueline Carville – University of Wisconsin, Edgar Spalding – University of Wisconsin Elongation of the seedling hypocotyl pushes the shoot-producing meristem out of the soil shortly after seed germination by rapid cell expansion. Mutation of the Arabidopsis thaliana ABCB19 gene greatly impairs auxin transport to the hypocotyl elongation zone, limiting growth rate during its period of fastest elongation. High ectopic expression of a cyclin B1;1-based reporter of mitosis in abcb19 hypocotyls was observed in the growth zone of abcb19 hypocotyls. Extra mitotic events were not observed but the endoreplication variant of the cell cycle was slowed in abcb19 hypocotyls. Reduced expression of CCS52A2, an activator of the anaphase promoting complex that targets D-box-containing cyclins for degradation, explained the endocycle defect and the aberrant cyclin B1;1 reporter signal in abcb19 hypocotyls. Nanomolar levels of exogenous auxin rescued the low CCS52A2 expression, the cyclin B1;1 reporter signal, the endocycle defect, and the hypocotyl growth rate phenotype of abcb19 seedlings. Mutation of CCS52A2 produced an abcb19-like growth rate defect that was not rescued by nanomolar auxin, consistent with the overall conclusion that auxin delivered by ABCB19-mediated transport maintains CCS52A2 levels, which regulates mitotic cyclin/CDK complex

levels, which determines entry into the endocycle, which is shown to bear a quantitative relationship with hypocotyl growth during a critical growth stage in the plant life cycle.

600-009-Y Elucidating the Function of the Ethylene Receptor Family in Root Growth and Development Justin Watkins – Wake Forest University, Brad Binder – University of Tennessee, Gloria Muday – Wake Forest University In Arabidopsis thaliana, ethylene signaling controls a diversity of growth and developmental processes through the actions of 5 receptors: ETR1, ETR2, ERS1, ERS2, and EIN4. Ethylene receptors are inactivated in the presence of ethylene, so null mutations in several receptor isoforms results in constitutive ethylene responses. Gain-of-function mutants, like etr1-3, are unable to bind ethylene and show dominant ethylene insensitivity. Although the five receptors have overlapping roles, the function of these receptors is not entirely redundant. By analyzing the developmental and transcriptional phenotypes of null receptor mutantsand the gain-of-function mutant, etr1-3, this project asks which ethylene receptors regulate root growth and development and control gene expression of downstream targets of ethylene signaling. Ethylene inhibits lateral root formation in WT seedlings, but not in etr1-3. The etr1-6 null mutant forms fewer lateral roots than WT and is insensitive to ethylene treatment, while etr2-3 and ein4-4 show a wild-type ethylene response. etr1-6 also shows enhanced root hair initiation and elongation compared to WT, suggesting a role for ETR1 in lateral root and root hair formation. We are examining the effects of the receptor nulls on transcriptional targets, which include genes encoding enzymes of flavonoid biosynthesis and auxin transport proteins using qRT-PCR. The accumulation of transcripts encoding CHS, a flavonoid biosynthetic enzyme, is increased by ethylene, as are the levels of flavonoid metabolites in roots. The levels of CHS are also increased in etr1-6 and etr2-3, consistent with ETR1 controlling flavonoid synthesis.These experiments have uncovered important roles linked to the ETR1 receptor in modulating root growth and gene expression changes in response to ethylene. Experiments with double and triple receptor null mutants are underway to ask if the other receptors have roles in the regulation of root growth and development beyond the activity of ETR1. (Supported by NSF Grant IOS-0820717).

600-010-Z OPDA Is Required for Nectary Development and Function Independent of the JA and COI1 Signaling Pathway in Arabidopsis Peter Klinkenberg – University of Minnesota Anthony Schmitt – University of Minnesota, Mengyuan Jia – University of Minnesota, Clay Carter – University of Minnesota Floral nectar is a reward presented by flowers to attract pollinators. While much is known about the chemical make-up of nectar, little is known about the mechanisms regulating its production. Previous studies suggested jasmonic acid (JA) and its receptor, COI1, are required for nectar production. We further examined the involvement of the octadecanoic biosynthetic and JA response pathways on nectary development and function in Arabidopsis. Our results indicate the octadecanoic pathway is required for nectar secretion, but that it is independent of both the production of JA and COI1. While early octadecanoic synthesis pathway mutants, such as aos-2, did not produce nectar, the downstream synthesis mutant opr3 developed flowers with actively secreting nectaries. opr3 does not synthesize JA in elongating anthers and filaments, but rather accumulates 12-oxo phytodienoic acid (OPDA). Similarly, flowers of the JA-receptor mutant coi1-1 produced nectar, which is contrary to findings in other species. However, a JA-responsive transcription factor, MYB21, was found to be required for nectar production through regulation of nectary-specific genes. Finally, a potential crosstalk between JA and auxin signaling pathways was investigated because auxin is known to strongly regulate nectary function. aos-2 and coi1-1 displayed no auxin response in nectaries, unlike those of wild-type flowers, and also displayed altered sugar metabolism. Cumulatively, these results indicate that a JA- and COI1-independent pathway mediated by

OPDA is required for proper nectary maturation and function. OPDA is known to have strong impacts on defense and developmental signaling, independent of being converted into JA and interactions with COI1. Future studies will investigate the mechanism by which OPDA regulates nectar production.

600-011-Z Gibberellins Negatively Regulate Nectar Production via Cross-talk with Auxin-response Pathways Lisa Wiesen – University of Minnesota Ricci Bender – University of Minnesota, Clay Carter – University of Minnesota Gibberellins (GA) are well known for their roles in regulating stem elongation and seed germination, but less understood is the role of GA in regulating floral maturation. We recently identified GA 2-OXIDASE 6 (GA2OX6) as being highly expressed in the actively secreting nectaries of Arabidopsis thaliana, but at low levels in other tissues. GA2OX6 was previously demonstrated to inactivate bioactive GA. Multiple independent ga2ox6 mutants displayed reduced levels of nectar production, which suggests that elevated levels of active GA negatively regulate nectar production. Indeed, exogenous treatment with paclobutrazol, a GA synthesis inhibitor, restored wild-type levels of nectar in ga2ox6. Similarly, spindly (spy) mutants, which are also predicted to have an increased GA signaling response, displayed reduced levels of nectar production, further supporting the hypothesis that GA negatively regulates nectar production. Wild-type flowers also displayed an intense auxin response in actively secreting nectaries, whereas ga2ox6 and spy mutants had strongly reduced DR5-dependent signal in nectaries. This suggests significant crosstalk occurs between GA and auxin signaling pathways in the regulation of nectar production. Furthermore, expression of the nectary-enriched auxin transporter PIN6 was reduced in both ga2ox6 and spy, which may contribute to the observed decrease in auxin signaling found in those mutants. PIN6 has been shown to be essential for auxin-dependent responses in nectaries and to positively correlate with nectar production. These results suggest that GA negatively regulates nectar production at least in part by affecting auxin signaling and auxin homeostasis.

600-012-Y MNC1 Negatively Regulates the Auxin Response in the Nectaries of Arabidopsis Max Napolitano – University of Minnesota Mengyuan Jia – University of Minnesota, Clay Carter – University of Minnesota The molecular mechanisms regulating nectar secretion have largely been unexplored despite the central role of animalmediated pollination on species diversification and agricultural output. Here, we demonstrate that MEDIAN NECTARY CUPIN 1 (MNC1; At1g74820) substantially contributes to such a process in Arabidopsis thaliana. MNC1 was found to be highly expressed solely in the median nectaries of Arabidopsis flowers, and its expression negatively affected both nectar production and nectary morphology through control of the nectary auxin response. Overexpression of MNC1 led to a strong decrease in the auxin response in nectaries, whereas mnc1 mutants had a significant increase nectary auxin response. Additional experiments indicate that MNC1 and the auxin transporter PIN6 act together to control auxin homeostasis in nectary cells. Current studies are focusing on the biochemical mechanisms by which MNC1 modulates the auxin response pathway in nectaries. For example, MNC1 is in the same family as several known auxin binding proteins and preliminary results suggest MNC1 does bind auxin.

600-013-Y Cytokinin Response Factor 6 Is a Key Regulator of Cytokinin and Oxidative Stress Aaron Rashotte – Auburn University Paul Zwack – Auburn University

Cytokinin Response Factor 6 (CRF6) is a cytokinin responsive AP2/ERF family transcription factor from Arabidopsis. Downstream of cytokinin signaling, CRF6 plays a key role in the inhibition of dark-induced senescence. CRF6 expression is also induced by oxidative stress, in a manner that may be independent of cytokinin. In support of this notion, CRF6 was found to be a direct transcriptional target of ANAC transcription factors involved in retrograde signaling in response to mitochondrial dysfunction. Here we present evidence that CRF6 functions in oxidative stress tolerance. To better understand the specific mechanisms or processes regulated by CRF6, we sought to identify its transcriptional targets in response to both cytokinin and oxidative stress. To this end, WT and cfr6 mutant plants were treated with either cytokinin or hydrogen peroxide and transcriptome level changes were identified by microarray analysis. Comparison of differentially expressed genes in mutant and WT background in response to each treatment facilitated the identification of CRF6-dependent transcripts. For both treatments, a much larger proportion of such transcripts were repressed than were induced. Moreover, many of the repressed genes were also found to have decreased expression in 35S:CRF6 overexpressing plants. Together these findings suggest that CRF6 functions largely as a transcriptional repressor. Interestingly among the hydrogen peroxide repressed – CRF6 dependent transcripts was a set of six cytokinin related genes: ARR6, ARR9, ARR11, AHP1, LOG7, and ABCG14. As cytokinin signaling is antagonistic toward tolerance of several forms of abiotic stress that lead to the production of reactive oxygen species, CRF6 may act to attenuate cytokinin response by repression of these and/or other genes involved in its metabolism, transport and signal transduction pathways.

600-014-Z Inhibition of Cell Division at the Arabidopsis Root Apical Meristem by Cytokinin and Ethylene Ian Street – Dartmouth College G. Eric Schaller – Dartmouth College, Kieber Joeseph – UNC Chapel Hill, Mathews Dennis – Unviersity of New Hampshire, Yan Zubo – Dartmouth College, Aleena Ramzan – Dartmouth College, Samina Shakeel – Quaid-i-azam University, Pakistan The root system of plants plays a critical role in plant growth and survival, with root growth being dependent on both cell division and cell elongation. Not surprisingly, multiple phytohormones interact to control root growth, including the cytokinin and ethylene. Prior studies have defined a role for cytokinin in inhibiting both cell division and elongation, and a role for ethylene that is restricted to inhibiting cell elongation. To better understand the control of cell division at the root apical meristem, we have employed genetic approaches making use of mutants affecting cytokinin and ethylene signaling. For characterization of cytokinin responses, we examined mutants affecting a key family of transcription factors, the type-B response regulators. Examination of loss-of-function mutants define the relative contributions of this family in regulating root cell division in response to cytokinin; examination of a hypersensitive gain-of-function mutant demonstrates the extremes to which cell division can be hormonally manipulated at the root tip. For characterization of ethylene responses, we employed ethylene-insensitive and constitutive ethylene-response mutants. Our results uncover a substantial and previously uncharacterized role for ethylene in inhibiting root cell division that primarily operates through the classical ethylene receptor à CTR1 à EIN2 à EIN3/EIL signaling pathway. Because cytokinin induces ethylene biosynthesis, we also examined the role of ethylene in mediating the effects of cytokinin in inhibiting cell division at the root meristem. Our results indicate that ethylene signaling contributes to but is not essential for cytokinin’s ability to inhibit cell division. In conclusion, our results define both independent and overlapping roles for cytokinin and ethylene in the control of cell division at the root apical meristem.

600-015-Z The HSP90-SGT1 Chaperone System Integrates Environmental and Internal Signals Through Regulating Stability of Auxin Receptors

Renhou Wang – HHMI, University of California San Diego Yi Zhang – University of California San Diego, Martin Kieffer – University of Leeds, Hong Yu – UCSD, UCLA, Stefan Kepinski – University of Leeds, Mark Estelle – HHMI, University of California San Diego Recent studies have revealed that a mild increase in environmental temperature stimulates plant growth through promoting bio-synthesis of plant hormone auxin. However, little is known about the role of other factors in this process. In this report we show that temperature increase stimulates the accumulation of auxin receptors TIR1 and AFBs, which causes hypersensitivity of seedlings to auxin and thus leads to plant growth. Further, the molecular chaperone-cochaperone system HSP90-SGT1 is required for the temperature-regulated plant growth and auxin signaling through modulating TIR1 stability. Inhibition of HSP90 activity results in degradation of TIR1 and defects in a variety of temperature/auxin-mediated growth processes. In addition, we show that HSP90 and SGT1 interacts and form a complex with TIR1, providing the molecular basis for how HSP90 and SGT1 integrate the environmental temperature signal into auxin signaling pathway to regulate plant growth in the changing environment.

600-016-Y Transcriptional Logic of the Abscisic Acid Response Network Liang Song – Salk Institute Shao-shan Carol Huang – Salk Institute, Aaron Wise – Carnegie Mellon University, Ziv Bar-Joseph – Carnegie Mellon University, Joseph Ecker – Salk Institute The sessile nature of land plants requires them to constantly integrate multiple environmental cues and balance between growth and stress responses. Plants have developed sophisticated signaling networks over hundreds of million years of evolution, largely by utilizing nine major phytohormones. To elucidate the hormone signaling network for abscisic acid (ABA)-related transcription factors (TFs), we epitope tagged over 20 ABA related TFs and reintroduced to Arabidopsis with their native genomic context using recombineering. Binding sites of ABA-responsive TFs were profiled at a genome-wide scale by ChIP-seq in the presence and absence of ABA. The genomics locations of thousands of binding sites result from these experiments were combined with time series RNA-seq data to identify the hierarchy of TF networks involved in the initiation and maintenance of ABA responses. These data reveal a major role for transcriptional feedback regulation in ABA signaling. We also found that highly dynamic multi-TF targets are excellent predictors of known and novel ABA network components. Using these deep data sets, we identified new genes related to seed germination and ABA sensitivity.

600-017-Y Cytokinin: Beyond Two Component Signaling Joseph Kieber – UNC Tracy Raines – UNC, Carly Sacks – UNC, Christian Burr – UNC Cytokinins are N6-substituted adenine derivatives that have been implicated a wide variety of plant growth and development processes. A basic framework for cytokinin signal transduction has emerged that is similar to twocomponent phosphorelays, which rely on the transfer of phosphates between alternating histidine and aspartic acid residues. Cytokinins are perceived by a family of histidine kinase receptors (AHKs), which, following binding of cytokinin, transfer a phosphoryl group to the histidine phosphotransfer proteins (AHPs), which in turn donate the phosphate to the response regulators proteins (ARRs) thereby regulating their activity. The ARRs fall into two groups, the type-A and typeB ARRs, which act as negative and positive elements in cytokinin signaling respectively. Two-component elements are partially functionally redundant in mediating the response to cytokinin and in various roles in regulating plant growth and development.

We are characterizing the mechanism underlying cytokinin perception and signaling in both Arabidopsis and rice, and are exploring how this two-component signaling pathway modulates the many processes regulated by cytokinin. We have characterized the cytokinin-regulated transcriptional network, as well as other outputs of the cytokinin twocomponent signaling pathway including proteins that interact with type-A ARRs. We have characterized the role of several of these outputs in cytokinin function, focusing on transcription factors. We continue to explore the roles of cytokinin two-component signaling elements in plant growth and development, including roles in the development of the female gametophyte and in root growth. Finally, we have begun to characterize two-component function in the monocot rice.

600-018-Z ABI4 Mediates the Antagonistic Effect Between Abscisic Acid and Gibberellins Crosstalk in Arabidopsis Kai Shu Wenyu Yang – Sichuan Agricultural University Qi Xie – Institute of Genetics and Developmental Biology, CAS Abscisic acid (ABA) and gibberellins (GA) are plant hormones which antagonistically regulate numerous physiological processes, and their optimal balance is essential for normal plant development. However, the molecular mechanism underlying ABA and GA antagonism still needs to be determined. Here, we report that ABA- INSENSITIVE4 (ABI4) is a central factor which regulates GA/ABA homeostasis and antagonism in post-germination stages. ABI4 over-expression in Arabidopsis (OE-ABI4) leads to the additional developmental defects, including plant height decrease and poor seed production. The transcription of a key ABA biosynthetic gene, NCED6, and of a key GA catabolic gene, GA2ox7, is significantly enhancedby ABI4 over-expression, and reduced by ABI4 absence. ABI4 directly binds to the NCED6 and GA2ox7 promoters, and mutation in these two genes partially rescues the dwarf phenotype of ABI4 overexpressing plants. Consistently, ABI4 overexpressing seedlings have a lower GA/ABA ratio compared to the wild type. We further show that ABA induces GA2ox7 transcription while GA represses NCED6 expression in an ABI4-dependent manner; and that ABA stabilizes the ABI4 protein, whereas GA promotes its degradation. Taken together, these results propose that ABA and GA antagonize each other by oppositely regulating ABI4 transcript and protein levels.

600-019-Z S-nitrosylation of RLF1 Mediates Auxin-cytokinin Interaction During Lateral Root Development in Rice Aizhen Sun – Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Jikai Li, Xiaoling Shang, Fang-Qing Guo Auxin and cytokinin are known to act antagonistically in regulating lateral root (LR) development. However, the regulatory signaling mechanism of LR development by these two signaling molecules is still largely unknown. By analyzing the expression profiles of rice roots treated with IAA, we characterized an IAA-responsive gene, encoding a putative DNA-binding protein and named it as RLF1 (reduced lateral root formation 1) since the number of lateral roots was significantly reduced in the rlf1 mutant compared with wild-type plants. Interestingly, RLF1 was found to be highly expressed in LR primordium and emerged LR, implying that RLF1 may be a key regulator of auxin–dependent LR development. It is known that IAA treatment induces nitric oxide (NO) accumulation in roots. By performing biotin switch assay, we found that RLF1 could be S-nitrosylated by NO in vitro. Furthermore, S-nitrosylation of RLF1promoted its binding affinity to the promoter region of COG1, which encodes a cytokinin glucosyltransferase in rice that plays important roles in regulating cytokinin levels by conjugation. The generated COG1 RNAi lines showed reduced LR formation, while overexpression lines of COG1 enhanced the LR densities. It is implied that NO-dependent Snitrosylation of RLF1 initiated by auxin might act on cytokinin metabolism, thereby leading to the regulation of LR

development. In summary, this study delineates a putative NO-RLF1-COG1 signaling cascade that regulates auxincytokinin interaction during LR development in rice.

600-020-Y Characterization of the Roles of SPINDLY, SECRET AGENT and the DELLA Protein, PROCERA, in Gibberellin Signaling in Solanum Lycopersicum Vai Lor – University of Minnesota Neil Olszewski Gibberellin (GA) signaling is well characterized in Arabidopsis thaliana. Studies have demonstrated that DELLA proteins negatively regulate GA signaling. Loss of DELLA function results in plants that are tall; while gain of DELLA function results in plants that are short. SPINDLY (SPY) is another negative regulator of GA signaling. Similar to DELLA loss of function, loss of SPY function (spy) mutants are tall plants. SPY is an O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) predicted to affect DELLA protein activity via O-GlcNAc modification. SECRET AGENT (SEC) is another OGT. Unlike spy mutants, SEC loss of function (sec) mutants exhibits only subtle mutant phenotypes. SEC has been demonstrated to O-GlcNAc modify DELLA. Tomato has a SPY-like gene (SlSPY) and two SEC-like genes (SlSECs). In this study, we are testing the hypothesis that SlSPY and SlSECs are involved in the regulation of GA signaling in tomato. The microRNA-induced gene silencing (MIGS) system was used to repress SlSPY and SlSEC expression. Stable transgenic lines were generated and the SlSPY_MIGS lines were tall while the SlSEC_MIGS lines were short. Bimolecular fluorescence complementation assays were conducted; which demonstrated that SlSPY and SlSEC can interact with DELLA proteins. These results suggests that SlSPY negatively and SlSEC positively regulate GA signaling, possibly through DELLA proteins.

600-021-Y The Purine Metabolite Allantoin Can Activate the MYC2-modulated JA-signaling Pathway in an ABAdependent Manner Hiroshi Takagi – Hiroshima University Yasuhiro Ishiga – University of Tsukuba, Mayumi Egusa – Tottori University, Shunsuke Watanabe –Hiroshima University, Tomokazu Konishi – Akita Prefectural University, Nobuhiro Akiyoshi – Hiroshima University, Takakazu Matsuura – Okayama University, Takashi Hirayama – Okayama University, Hiroshi Shimada – Hiroshima University, Hironori Kaminaka – Tottori University, Atsushi Sakamoto – Hiroshima University Allantoin is a metabolic intermediate of purine catabolism that often accumulates in various plants subjected to stress conditions. Recently, we showed that this metabolite activates abscisic acid (ABA) production, thereby stimulating ABA signaling and enhancing abiotic stress tolerance in seedlings of Arabidopsis knockout mutants (aln) of the ALLANTOINASE gene. Interestingly, microarray analysis of an aln mutant (aln-1) revealed not only upregulation of ABAresponsive genes, but also that of genes involved in jasmonic acid (JA) responses, in a manner likely modulated by MYC2 that plays as a master regulator in JA signaling. Consistent with this, the aln-1 mutant increased the levels of JA and its bioactive isoleucine conjugates, and displayed enhanced responses to mechanical wounding and exogenous methyl jasmonate. Moreover, two allelic mutants, aln-1 and aln-2, demonstrated high susceptibility to Erwinia carotovora EC1 and Pseudomonas syringae pv. tomato DC3000, probably reflecting the fact that MYC2 suppresses the defense against these pathogens. Exogenously administered allantoin elicited the expression of JA-responsive genes including MYC2 in wild-type plants, supporting that allantoin might be responsible for the observed JA-related aln phenotypes. To gain mechanistic insights into how allantoin activates the JA signaling pathway, the responses to exogenous allantoin were tested in JA-deficient (jar1-1), JA-insensitive (myc2) and ABA-deficient (aba2-1 or bglu18) single mutants. In addition, genetic interactions in the responses to endogenously accumulated allantoin were examined between aln and JA- or ABA-deficient mutations (aln-1/jar1-1 and aln-1/bglu18). The results of these experiments indicate that, depending on

an intact ABA production pathway, allantoin can activate a MYC2-branch of the JA signaling pathway. Overall, these studies provide novel evidence for the links between purine metabolism and stress hormone homeostasis and signaling, and highlight the importance of the metabolite allantoin in these interactions.

600-022-Z Gibberellin Promotes Shoot Branching in the Perennial Woody Plant Jatropha Curcas Zeng-Fu Xu Jun Ni, Congcong Gao, Mao-Sheng Chen, Bang-Zhen Pan, Kaiqin Ye Strigolactone (SL), auxin, and cytokinin (CK) interact to regulate shoot branching. CK has long been considered to be the only key phytohormone to promote lateral bud development. Here we report that gibberellin (GA) also acts as a positive regulator in controlling shoot branching in the woody plant Jatropha curcas. We show that GA and CK synergistically promote lateral bud outgrowth, and that both hormones influence the expression of putative branching regulators, J. curcas BRANCHED1 and BRANCHED2,which are key transcription factors maintaining bud dormancy. Moreover, treatment with paclobutrazol, an inhibitor of de novo GA biosynthesis, significantly reduced the promotion of bud outgrowth by CK, suggesting that GA is required for CK-mediated axillary bud development. We further show that GA and CK treatments evoked a shared transcriptomic response in axillary buds, which provides molecular support for the physiological observation that GA and CK collaboratively promote bud outgrowth. In addition, auxin and SL, two types of plant hormones involved in the repression of shoot branching, acted antagonistically to both GA and CK in the control of lateral bud outgrowth. Consistent with this, the expression of JcMAX2, a J. curcas homolog of Arabidopsis MORE AXILLARY GROWTH 2 encoding an F-box protein in the SL signaling pathway, was repressed by GA and CK treatment. We also provide physiological evidences that GA induces shoot branching in other trees, such as papaya and mulberry, indicating a more complicated regulating network occurs in the control of shoot branching in perennial woody plants.

600-023-Z Fine Tuning of the ABA Core Signaling Pathway by mRNA Stability Control Gustavo Duarte - UNICAMP João Guilherme Portugal Vieira, Lucas Eduardo Costa Canesin, Sandra Pelletier, Jean-Pierre Renou, Renato Vicentini, Michel Vincentz Plants have developed a set of mechanisms to counteract adverse environmental conditions, hence ensuring their development and propagation. The efficiency of these processes relies on the integration of hormone biosynthesis, activation of stress-responsive pathways and on a balanced use of the available energy. Abscisic acid (ABA) is one of the major plant hormones involved in abiotic stress responses, but also plays a role in plant development. Kinetics of ABApromoted changes in gene expression reveal that as part of a negative feedback regulatory mechanism, the mRNA levels of ABA receptors of the PYR/PYL family (PYL4-6) are promptly repressed upon ABA treatment while the expression of protein phosphatases 2Cs genes (PP2Cs ABI1, ABI2, HAB1, HAI1 and HAI2) and some SNF1-related protein kinases 2 (SnRK2.6 and SnRK2.7), both of which are part of the core of the ABA signaling pathway, go through an activation period followed by a stabilization phase. Evaluation of the stability of these transcripts also indicates that post-transcriptional regulations contribute to this ABA-promoted responses, possibly reflecting a requirement for fast and fine adjustment of the ABA signaling pathway to control ABA-mediated responses homeostasis. These later results raises the question of which would be the mechanisms involved in such regulation.

600-024-Y Genetic Interaction of HY5 and IBR5 Links Light and Hormone Signaling Pathways Thilanka Jayaweera – Texas State University Yuting Hou – Texas State University, Jason DiGiovanni – Texas State University, Jeffrey Hall – Texas State University,

Lauren Minter – Texas State University, Nihal Dharmasiri – Texas State University Light and the plant hormone, auxin are two of the major determinants of plant growth and development. Several proteins involved in the interaction between these two signaling pathways have been reported. HY5 is one of the bZIP family transcription factors that are implicated in photomorphogenesis as well as in auxin signaling. HY5 is involved in auxin signaling mainly through the regulation of auxin related gene expression. Here we report the identification of genetic interaction between HY5 and IBR5, a dual specificity phosphatase involved in auxin signaling. Our studies reveal that IBR5 is involved in light mediated growth responses in Arabidopsis and HY5 is genetically epistatic to IBR5. Conversely, IBR5 acts in an epistatic manner to HY5 in regulating auxin related growth responses such as primary root growth. Interestingly, in many developmental processes such as lateral root, apical hook, leaf development and hormonal responses, HY5 and IBR5 seem to be interacting in a complex manner. In silico analysis suggests that the IBR5 promoter contains a putative HY5 binding site and HY5 interacts with promoter of IBR5 in vitro. Moreover, quantitative RT-PCR analyses indicate that IBR5 is over-expressed in the hy5-1 mutant background. Therefore, it is possible that HY5 directly binds to the IBR5 promoter to suppress its expression. In summary, this study demonstrates the possible regulation of IBR5 expression by HY5 and their genetic interaction, linking light and hormone signaling pathways.

600-025-Y ABA-HYPERSENSITIVE BTB/POZ PROTEIN 1 Is Negatively Involved in ABA-mediated Cellular Signaling Through the Repression of ABI5/DPBF Subfamily Genes Hani Kim – Pusan National University Soon-Hee Kim – Pusan National University, Dong Hye Seo – Yonsei University, Woo Taek Kim – Yonsei University, JaeHoon Lee – Pusan National University To elucidate the contribution of Cullin3-RING E3 ligase (CRL3) to ABA-mediated cellular responses, we tried to find a substrate receptor of CRL3 which is involved in ABA signaling. One gene named AHT1 (ABA-HYPERSENSITIVE BTB/POZ PROTEIN 1) was upregulated more than 2.5 times by exogenous ABA application, and the loss of AHT1 leads to the retardation of germination process, not to the inhibition of root growth. The expression of AHT1 was dramatically increased in dry seeds which have high level of ABA, alluding its important role in both seed stage and ABA signaling. High expression of AHT1 in dry seeds was inhibited by the loss of ABI1, ABI3 and SRK genes, indicating the expression of AHT1 is dependent on ABA signaling. Among various bZIP transcription factors participating in ABA signaling, the losses of ABI5, AREB1, EEL and DPBF2 resulted in the reduction of AHT1 expression, showing these four transcription factors mainly play an important role of ABA-induced AHT1 expression. While knock-out of AHT1 gene did not affect the expression pattern of NCED3, ABI2 and SRKs, it led to the hyperinduction of DPBF genes such as ABI5, EEL, AREB3, which are mainly expressed in seeds, and downstream genes such as RD29B and Em1 in ABA signaling. Therefore, it shows that AHT1 negatively regulates ABA-mediate cellular response by repressing the expression of genes encoding a subset of bZIP transcription factors, especially related to ABI5/DPBF subfamily. Since the expression of AHT1 is also dependent on most of ABI5/DPBF transcription factors, AHT1 is expected to be involved in negative feedback regulation of ABA signaling through its linkage with ABI5/DPBF group, providing a process to fine-tune the homeostasis of ABA signaling.

600-026-Z The Effect of Hormones and Other Growth Regulators on the Accumulation of Anthocyanins in Wildtype and Mutant Arabidopsis Seedlings Valerie Sponsel – University of Texas at San Antonio Amanda Graham The levels of anthocyanins in plants are regulated by many factors including light, sugars, nitrate, phosphate, and several

plant hormones. These pigments can have protective effects against biotic and abiotic stress. Arabidopsis plants expressing mutations at the BIG locus are semi-dwarf, have reduced apical dominance and altered polar transport of the hormone, auxin. We have shown that ten-day old seedlings expressing the tir3 allele of BIG have elevated levels of jasmonic acid (JA) and its isoleucine conjugate. These seedlings, which were grown in aseptic liquid culture, also show higher anthocyanin accumulation than wildtype seedlings. This undergraduate research project tested the effects of auxin, auxin transport inhibitors, gibberellin A4, the GA biosynthesis inhibitor paclobutrazol (PAC), and JA on anthocyanin levels in WT and tir3 seedlings. All hormones and growth regulators altered anthocyanin accumulation in WT and/or tir3 seedlings in some way. JA enhanced anthocyanin accumulation in both WT and tir3 seedlings whereas PAC, at the concentrations tested, enhanced anthocyanin levels in WT seedlings only. Conversely GA4 reduced anthocyanin levels in tir3 but was without significant effect in WT seedlings. The two auxin transport inhibitors tested (1naphthoxyacetic acid, NOA, and naphthylphthalamic acid, NPA) reduced anthocyanin levels in tir3 seedlings. The results are discussed in relation to what we know about the level of endogenous hormones in WT and tir3 seedlings and the complex network of signals that control anthocyanin production in Arabidopsis.

600-027-Z Characterization of Cytokinin Response Factors 1 and 2 in Response to Abiotic Stress Erika Keshishian – Auburn University Aaron Rashotte – Auburn University Cytokinin is a plant hormone known to be involved in growth and development, regulating such processes as response to light, maintenance of apical dominance, and onset of senescence. A sub-family of the AP2/ERF transcription factors called Cytokinin Response Factors (CRFs) has been shown to be a side branch of the cytokinin signaling pathway and is conserved across all land plants. In addition to playing a role in the canonical cytokinin processes, CRFs seem to be linked to several different abiotic stress responses. In Arabidopsis, CRFs can be divided into four distinct phylogenetic clades that appear to have differing functional roles in cytokinin signaling and stress responses, including Clade I CRFs: CRF1 and CRF2. CRF1 and CRF2 are transcriptionally cytokinin-inducible genes that we have also shown are regulated in the presence of high salt and osmotic stress conditions. Although many cytokinin signaling pathway genes including the CRFs are functionally redundant, we find differences in stress responsiveness between CRF1 and CRF2. Detailed examination of seedling growth and developmental parameters, as well as germination findings under different salt, osmotic, and cytokinin treatments will be presented. In addition, qPCR and promoter::GUS expression of both CRF1 and CRF2 in tissues will be shown in response to cytokinin and abiotic stress treatments, indicating unique profiles and potential different functional roles of these genes.

600-028-Y JASMONATE HYPERSENSITIVE 3: A Novel Component of the JA Signaling Pathway in Arabidopsis Thaliana Gregory Harrison – Washington University in St. Louis Kwi Mi Chung, Agnes Demianski, Neva Laurie-Berry, Shuo Zhang, Barbara Kunkel Jasmonic acid (JA) is a hormone important in plant development and defense. To improve our understanding of the pathways regulating JA responsiveness in Arabidopsis we performed a screen for mutants with enhanced sensitivity to JA in a root inhibition assay. Here we describe the characterization of a JA-hypersensitive mutant (jah3-1) isolated in this screen. To further investigate JA sensitivity in jah3-1, we measured the expression of the JA-responsive genes LOX2 and VSP2, as well as the JA- and ethylene-responsive gene PDF1.2a during treatment with JA. LOX2 and VSP2 expression appears to be hyper-responsive to JA in jah3-1, consistent with the hypersensitive phenotype. In contrast, JAresponsiveness of PDF1.2a is decreased in jah3-1. Genetic mapping and DNA sequencing suggested that the mutation responsible for the jah3-1 phenotype is a nonsense mutation in the gene At4g16530, which we have designated

JASMONATE HYPERSENSITIVE 3 (JAH3). This gene has not been previously identified as being involved in JA signaling. The identity of the JAH3 gene was confirmed by complementation of the root growth phenotype by a genomic clone carrying the WT At4g16530 region. Analysis of JAH3 gene expression led us to question the predicted structure of the gene, which is annotated in TAIR as having three exons. Complementation experiments and transcript start site mapping indicate that the first exon may not be included in the JAH3 gene. Thus we propose a new gene structure for JAH3 that is consistent with our results. To determine if jah3-1 is JA hypersensitive due to elevated JA, we monitored endogenous JA levels. The mutant does not accumulate elevated JA, and indeed JA biosynthesis genes are downregulated. Double mutant analysis indicates the jah3-1 phenotype is dependent on COI1and MYC2. Based on these results we propose that JAH3 is a negative regulator of JA signaling.

600-029-Y Loss of GSNOR1 Function Leads to Compromised Auxin Signaling and Polar Auxin Transport Jianzhong Liu – Zhejiang Normal University Ya-Fei Shi – Zhejiang Normal University, Da-li Wang – Zhejiang Normal University, Chao Wang – Zhejiang Normal University, Jerry Cohen – University of Minnesota, Jianwei Pan – Zhejiang Normal University, Barbara Baker – University of California, Berkeley Cross talk between phytohormones nitric oxide (NO) and auxin has been implicated in the control of plant growth and development. Two recent reports indicate that NO promoted auxin signaling but inhibited auxin transport probably through S-nitrosylation. However, genetic evidence for the effect of S-nitrosylation on auxin physiology has been lacking. In this study we used a genetic approach to understand the broader role of S-nitrosylation in auxin physiology in Arabidopsis. We compared auxin signaling and transport in Col-0 and gsnor1-3, a loss of function GSNOR1 mutant defective in protein de-nitrosylation. Our results showed that auxin signaling was impaired in the gsnor1-3 mutant as revealed by significantly reduced DR5-GUS/DR5-GFP accumulation and compromised degradation of AXR3NT-GUS, a useful reporter in interrogating auxin mediated degradation of Aux/IAA by auxin receptors. In addition, polar auxin transport was compromised in gsnor1-3, which was correlated with universally reduced levels of PIN or GFP-PIN proteins in the roots of the mutant in a transcription- and 26S proteasome degradation-independent manner. Our results suggest that S-nitrosylation and GSNOR1-mediated de-nitrosylation contribute to auxin physiology and the impaired auxin signaling and compromised auxin transport are responsible for the auxin related morphological phenotypes displayed by the gsnor1-3 mutant.

600-030-Z The Role of Oryza Sativa Pseudo-Histidine Phosphotransfer Protein in Cytokinin Signal Transduction Yu-Chang Tsai – National Taiwan University Chia-Yun Lee – National Taiwan University In Arabidopsis cytokinin signaling system, histidine phosphotransfer proteins (AHP) mediate the transfer of the phosphoryl group from sensor histidine kinases to the response regulators (RR) and regulate the downstream responses. Oryza sativa pseudo-HP (OsPHP) lacks the conserved His residue required for phosphotransfer as authentic-HPs and is thought of as an inhibitor in rice cytokinin signaling. To explore the role of OsPHP in cytokinin signaling and stress response, the phenotypic analyses of Osphp mutants have been undertaken. The Osphp mutants reveal longer root growth in the seedling stage relative to wild type. Expression analysis of AHP downstream RRs are induced in Osphp mutants in response to the exogenous cytokinin treatment. Additional stress responses of Osphp mutants have also been evaluated and the results will be presented.

600-031-Z The Role of DWARF 14 LIKE 2 (DLK2) in Strigolactone Signaling Vilmos Soós – Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences Attila Vegh – Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Ervin Balázs – Agricultural Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Martin Posta – Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Heqiang Huo- UC Davis, Kent Bradford – UC Davis Strigolactones have been found to play important roles in many aspects of plant development. It has been shown that strigolactones are involved in the control of shoot branching, stature, root growth, root hair elongation, germination and light responses. DWARF 14 (D14) and KARRIKIN INSENSITIVE 2 (KAI2) have been found to play important role in strigolactone signaling. The third member of the α/β hydrolase family, DWARF 14 LIKE 2 (DLK2) is structurally very similar to the other DWARF proteins, but no functions have been assigned yet. We analysed the expression of DLK2 in Arabidopsis using constructs expressing GUS and GFP under the control of DLK2 promoter. We found that the length of the promoter and the presence of the intron in the construct strongly influence the expression pattern of DLK2, which can be altered by adding exogenous strigolactones. The RNASEQ analysis of the dlk2 mutant highlighted the importance of DLK2 in light responses. Using dlk2 mutant and DLK2 overexpressing Arabidopsis lines, simple and double DWARF mutant lines (kai2, d14, dlk2/d14, dlk2/kai2, d14/kai2) and triple mutants (dlk2/d14/kai2), we identified the role of DLK2 in plant development and unraveled the interaction network between the three hydrolases. Furthermore, we made efforts to identify the ligand of DLK2 in in vitro assays. DLK2 protein and mutant protein variations lacking amino acid residues essential for ligand perception and hydrolysis were expressed in E. coli. Proteins were incubated with synthetic strigolactone analogues and various strigolactone enatiomer pairs and residues were extracted and subjected to LC-MS. We found that strigolactones are degraded in the presence of wild type DLK2 and not in mutant DLK2 proteins. Acknowledgements: ICGEB Research Fund, Bolyai Fellowship, Hungarian National Research Fund (OTKA) , HAESF

600-032-Y Conversion of indole-3-butyric Acid to indole-3-acetic Acid in Shoot Tissue of Hazelnut and Elm Molly Kreiser – University of Minnesota Chad Giblin – University of Minnesota, Ryan Murphy – University of Minnesota, Gary Johnson – University of Minnesota, Donald Wyse – University of Minnesota, Jerry Cohen – University of Minnesota Indole-3-butyric acid (IBA) is an endogenous compound that appears to regulate both lateral and adventitious root formation in many plant species and is also the auxin most available commercially for application to promote rooting. IBA is converted to indole-3-acetic acid (IAA) by β-oxidation in the peroxisomes. This process has been observed in a number of plant species and has been shown to be critical for normal root development in response to treatment with IBA. We investigated this process in hybrid hazelnuts (­Corylus americana x C. avellana) and American elm (Ulmus americana), in which adventitious rooting is a major bottleneck for vegetative propagation and the efficacy of IBA treatment is highly variable across different cultivars. Using differentially stable isotope labeled IBA and IAA tracer and internal standard, respectively, and using gas chromatography coupled with selected reaction monitoring mass spectrometry, IBA-derived IAA was measured in shoot tissue treated with stable isotope labeled IBA. Variable levels of IBA-to-IAA conversion were observed across different hybrid hazelnut genotypes, which may partially explain differences in rooting ability. In elm, higher levels of IBA-to-IAA conversion were observed in cultivars which formed adventitious roots most easily in softwood stem cutting trials. High rates of root formation is a key trait for establishment of large-scale production systems. Screening for optimal rates of IBA-to-IAA conversion may facilitate

selection against genotypes which respond poorly to exogenous IBA. Such genotypes are difficult to propagate using hormone treatment and thus can be eliminated from further evaluations.

600-033-Y RESPONSE of Amphicarpaea Bracteata (L.), the DAKOTA PEA, to PLANT GROWTH REGULATORS and PHOTOPERIOD Sonja Maki – University of Wisconsin-River Falls Emily Bethke – University of Wisconsin-River Falls Amphicarpaea bracteata (L.) is an edible, vining, woodland legume native to Midwestern and Eastern parts of North America. The plant inhabits woodlands and thickets and is known by several common names including Dakota Pea, Hog Peanut, and Pea Vine. Three different inflorescence morphologies are produced on the plant. The objectives of this study were to investigate the effects of photoperiod on plant growth and development and to determine the effects of a gibberellin biosynthesis inhibitor (Prohexadione-Ca) on plant growth. Aerial seeds were collected from plants found in their native habitat in western Wisconsin. Seeds were imbibed in 100 or 200 ppm Prohexadione-Ca prior to a 3 day cold treatment (4C), sown in a peat-based media, and grown in a greenhouse. Long days (16h) were provided with supplemental HID lighting and short days were provided by an automated short day cloth greenhouse system (9h). Growth data was analyzed with JMP11 (SAS). Seeds treated with 100 ppm Prohexadione-Ca produced plants approximately one-half the normal height growth when compared to the control group. Seeds treated with 200 ppm Prohexadione-Ca produced plants with even less growth. When Amphicarpaea bracteata (L.) is grown under natural long days in the Midwest, it flowers in late July. Prohexadione-Ca treatment affected plant height similarly in short days, however plants flowered early, at the first node, and only one-seeded pods were produced, in contrast to the 3-4 seeded pods produced in upper regions of long day grown plants. These one-seeded pods were not observed on longday grown plants, however more research on plants growing in their native environment is necessary to determine whether this fourth type of flowering and fruiting pattern occurs in nature.

600-034-Z Drugged Plants Talk: Chemical Genetic Dissection of Phytohormonal Crosstalk in Arabidopsis Thaliana Vivek Halder – Max Planck Institute for Plant Breeding Research Erich Kombrink – Max Planck Institute for Plant Breeding Research, Markus Kaiser – Universität Duisburg-Essen, ZMB By employing forward chemical genetic screens in Arabidopsis thaliana, I identified a compound, P486, which may usher novel insights into how pattern recognition receptors (PRR) regulate crosstalk between salicylic acid (SA) and jasmonic acid (JA) signaling. First, for screening chemicals, I developed a highthroughput, in-situ, quantitative GUS (ßglucuronidase) assay applicable to intact GUS-reporter seedlings. This method combines speed, ease and quantification into one-step process and is thus superior to traditional histochemical and other quantitative GUS assays. Principally, appropriately induced GUS-reporter seedlings are incubated at 37°C with a GUS-specific substrate made in lysis buffer. The substrate permeates through plant tissues and interacts with GUS protein to produce a fluorogenic byproduct, which is quantifiable and permits statistical checks for confident hit selection. P486, a rhizobacterial lipopeptide, activates SA signaling in Col-0 plants and at the same time inhibits JA signaling both in MeJA (Methyl JA) induced wild type as well as SA-deficient sid2 plants. However, to activate SA signaling, P486 requires SA-biosynthesis enzyme SID2 and upregulates endogenous SA-levels but act independently of known SA-regulators EDS1, PAD4 and SAG101 and the downstream SA-signal transducer, NPR1. On the other hand, inhibition of JA-signaling happens downstream of COI1 via upregulation of JA-transcriptional repressor, JAZes, as well as interference with MeJA-induced JAZ protein degradation. Further characterization assays revealed that P486 requires certain pattern recognition receptors (PRR) receptors for its activity, which could be due its peptidic moiety that might act as a ligand to the receptor/s. Interestingly, P486 showed

no interference with early defense responses like ROS and MAPK signaling, indicating a novel role of PRRs towards hormonal crosstalk specific to the interacting ligand. Ongoing work with P486 will further confirm its potential role as a chemical tool to study SA-JA crosstalk studies and will promote the importance of chemical genetics in Arabidopsis thaliana.

600-035-Z Distinct Transport Characteristics of indole-3-acetic Acid and Phenylacetic Acid, Two Naturally Occurring Auxins in Plants Hiroyuki Kasahara – RIKEN Center for Sustainable Resource Science Satoko Sugawara – RIKEN Center for Sustainable Resource Science, Kiyoshi Mashiguchi – RIKEN Center for Sustainable Resource Science, Keita Tanaka – RIKEN Center for Sustainable Resource Science, Shojiro Hishiyama – Forestry and Forest Products Research Institute, Tatsuya Sakai – Niigata University, Kousuke Hanada – Kyushu Institute of Technology, Kaori Kinoshita-Tsujimura – Osaka University, Hon Yu – University of California San Diego, Xinhua Dai – University of California San Diego, Yumiko Takebayashi – RIKEN Center for Sustainable Resource Science, Noriko Takeda-Kamiya – RIKEN Center for Sustainable Resource Science, Tatsuo Kakimoto – Osaka University, Hiroshi Kawaide – Tokyo University of Agriculture and Technology, Masahiro Natsume – Tokyo University of Agriculture and Technology, Mark Estelle – University of California San Diego, Yunde Zhao – University of California San Diego, Ken-ichiro Hayashi – Okayama University, Yuji Kamiya – RIKEN Center for Sustainable Resource Science Auxin plays a fundamental role in many aspects of plant growth and development. Indole-3-acetic acid (IAA) is a naturally occurring auxin that possesses the property of polar transport in plants. Phenylacetic acid (PAA) has also been recognized as a natural auxin, but its role in plant growth and development remains to be elucidated. In this study, we demonstrate that IAA and PAA have overlapping regulatory roles but distinct transport characteristics as auxins in plants. We show that PAA is widely distributed in plant kingdom. Although biological activities of PAA are lower than those of IAA, the endogenous amounts of PAA are much higher than those of IAA in various plant tissues in Arabidopsis. PAA and IAA can regulate the same set of auxin responsive genes through the TIR1/AFB-dependent pathway. IAA actively forms concentration gradients in maize coleoptiles in response to gravitropic stimulation, whereas PAA does not, indicating that PAA is not actively transported in a polar manner. The induction of the YUCCA genes remarkably increases PAA metabolite levels in Arabidopsis, suggesting that YUCCA flavin-containing monooxygenases may play a role in PAA biosynthesis. Our results provide new insights into the regulation of plant growth and development by IAA and PAA.

CELL BIOLOGY - Zone 700 Cell Biology: General 700-001-Y Plant Enzyme Assisted – CLARITY Enabling 3D Immunohistochemistry of Whole Plant Tissues William Palmer – University of Newcastle Antony Martin – University of Newcastle, Robert Furbank – Australian National University, Chris Grof – University of Newcastle The use of confocal laser scanning microscopy (CLSM) has been applied to many plant tissues types for a multitude of applications. However, this has been limited by the depth of penetration due to light scattering effects caused by the lipid membranes and cell wall characteristics of plant tissue. To obtain greater optical penetration, tissue is often subjected to harsh ‘clearing’ techniques that remove molecular structures including protein, DNA and RNA. A new technique known as Clear Lipid-exchanged Acrylamide-hybridised Rigid Imaging / Immunostaining / in situ-hybridisation-

compatible Tissue hYdrogel or CLARITY has overcome these limitations in mammalian species by fixation of molecular molecules within a polyacrylamide hydrogel before removal of lipid membranes with SDS (for full details see Chung et al., 2013). Here we show the modified use of this technique in plant tissues using a novel enzymatic degradation of the cell wall components and other optically interfering compounds such as starch, which allows deep optical penetration of leaves, stem and other tissue types using CLSM. Futhermore, immunohistochemistry has been performed showing protein localisation in 3D intact tissue without the need for any sectioning of the material. Plant Enzyme Assisted (PEA)CLARITY is now being used to interrogate the mechanisms of sugar accumulation in stems of Setaria viridis, an emerging C4 model species. Concurrently, PEA-CLARITY is also being used to examine the structure/function and molecular differences between Rice (C3) and Setaria (C4) photosynthesis.

700-002-Z Localization of an Evolutionarily Conserved Protein Proton Pyrophosphatase in Evolutionarily Distant Plants Oryza Sativa and Physcomitrella Patens Kamesh Regmi – Arizona State University Shangji Zhang – Arizona State University, Roberto Gaxiola – Arizona State University, Kendal Hirschi – Baylor College of Medicine Proton Pyrophosphatase (H+-PPase) is a highly evolutionarily conserved protein that is prevalent in the plant kingdom. One of the salient features of H+-PPase expression pattern, at least in vascular plants like Arabidopsis, is its conspicuous localization in both actively dividing cells and the phloem. On one hand, the role of H+-PPase as a Pyrophosphate (PPi) scavenger at the tonoplast of metabolically active cells is well documented. On the other hand, the plasma membrane localization of this protein in the phloem is also recognized, wherein this protein is hypothesized to play a crucial role as a PPi synthase, energizing active sucrose (Suc) loading. Within this wider purview, we have used two evolutionarily distant plants, a vascular monocot Rice and a non-vascular moss Physcomitrella, to analyze the in situ localization patterns of H+-PPase in various tissues of these two model organisms. Were H+-PPases co-opted into a novel functional role in the vascular tissues of higher plants? Or are there evolutionarily conserved roles of this protein that transcend the phylogenetic diversity of land plants? Using light microscopy, we show that H+-PPases are distinctly expressed in the actively growing cells in both Rice and Physcomitrella. As expected, H+-PPases were also localized in the vascular tissues of Rice, but surprisingly, H+-PPases were prominently expressed at the gametophyte-sporophyte junction of Physcomitrella. Upon immunogold labeling, H+-PPases were found to be localized at both the vacuolar and plasma membranes of the sieve element-companion cell complexes of Rice leaves, and transfer cells of the Physcomitrella haustorium.

700-003-Z GENOTOXIC and PHYTOTOXIC EFFECTS of CRUDE OIL-POLLUTED SOILS on Vernonia Amygdalina Del Ndubuisi Nwakanma Emmanuel Ikegwu – Yaba College of Technology Yaba Efe Osaigbovo – Healthplus Pharmacy Limited The general toxicity and phytotoxic effect of crude oil-polluted soils on Vernonia amygdalina root tips were investigated in this study. Stems of V. amygdalina were cultivated in various concentrations (5ml, 10ml, 15ml, 20ml and 25ml) of crude oil -polluted soils with non polluted-soil as control. After two weeks, five root lengths and five shoot lengths of V. amygdalina were measured from each test concentration of crude oil-polluted and control soils respectively, using a well calibrated meter rule. Subsequent measurements were taken per week for another three weeks. Five root tips from each stem of V. amygdalina were harvested and processed using the aceto-orcein squash technique at week two. Two tests (proximate analysis on the experimental plants and soil analysis) were equally carried out. The mitotic index (M.I)

was determined from 500 cells and was 8.80 for the control. The M.I. for crude oil - polluted soils on the root tip cells of V. amygdalina were 4.56 (5ml), 4.47 (10ml), 4.72 (15ml), 6.60 (20ml) and 3.74 (25ml). The mitotic indices observed for the test concentrations of crude oil were found to be lowest at 25ml (3.74). The microscopic studies showed several chromosomal abnormalities which included bridges and fragments, vagrant chromosomes, c-metaphase among others. The treatment on the test plants showed some phytotoxic effects with chlorosis and mottling of leaves being the most prominent. The significance of this study in environmental monitoring and the potential use of Vernonia amygdalina in the phytoremediation of crude oil-polluted environments are discussed.

700-004-Y Division Plane Orientation in Plant Cells Carolyn Rasmussen – University of California, Riverside Pablo Martinez – University of California, Riverside, Claire Stowers – University of Wyoming, Christopher Hoyt – Harvey Mudd College, Tianying Su – Stanford University, Anne Sylvester – University of Wyoming, Zhouxin Shen – University of California, San Diego Plants have three primary methods for establishing a body plan: division, differentiation and expansion. Therefore, understanding cell division, particularly the coordination between cell division and differentiation mediated by correct orientation of the division plane, is crucial to understanding plant development. Although there has been recent progress in modeling some of the mechanical forces that regulate division plane orientation in plants, much less is known about the molecular factors regulating this process. TANGLED (TAN), a cortically localized protein with similarity to the microtubule binding domain of the tumor suppressor Adenoma polyposis coli (APC), promotes proper orientation of the division plane in plant cells. Intriguingly, APC also promotes proper orientation of division planes and localizes to the cell cortex. Live cell imaging was used to analyze the structure and dynamics of division structures in the maize tangled (tan) mutant. Use of temporally regulated expression of TAN-YFP by ubiquitin-mediated proteolysis in maize tan mutants demonstrates that TAN function is most important during early stages of the cell cycle. Together with the TAN interactors identified by yeast-two-hybrid and mass spectrometry, a temporally and spatially regulated division site interactome is proposed to mediate proper orientation of the division plane.

700-005-Y Endocytosis of Plant Receptor Kinases Eugenia Russinova – VIB-UGent Receptor-mediated endocytosis is an integral part of signal transduction, as besides signal attenuation by removing activated receptors and their bound ligands from the cell surface, it allows spatial and temporal regulation of the signaling outputs from the endosomes. However, studying the interplay of endocytosis and signaling of plant receptor kinases is limited by the lack of appropriate tools to follow active receptors and receptor complexes in living cells by fluorescence microscopy. Crucial for the advancement of this research is the development of imaging tools that allow visualization of membrane-associated signaling events at a high spatiotemporal resolution. We recently started developing bioactive fluorescent probes (small molecule and peptide ligands) to visualize endocytosis of different receptor kinases in living Arabidopsis cells. Those tools together with genetic, biochemical and pharmacological analyses revealed differences in endocytosis and in regulation of signaling outputs.

700-006-Z Activation of Autophagy by Unfolded Proteins During Endoplasmic Reticulum Stress Xiaochen Yang – ISU Renu Srivastava – ISU, Stephen Howell – ISU, Diane Bassham – ISU

Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum and is caused by conditions such as heat or endoplasmic reticulum stress agents, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in an inositol-requiring enzyme 1b-dependent manner and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress. The chemical chaperone sodium 4-phenylbutyrate was found to reduce tunicamycin- or dithiothreitol - induced autophagy, but not autophagy caused by unrelated stresses. Similarly, overexpression of binding immunoglobulin protein, a heat shock protein70 molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to require inositol-requiring enzyme 1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heatinduced autophagy is due to endoplasmic reticulum stress. Expression in Arabidopsis of a single constitutively misfolded protein, zeolin, was sufficient to induce autophagy in an inositol-requiring enzyme 1b-dependent manner. Moreover, zeolin co-localized with the autophagic body marker green fluorescent protein-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress.

700-007-Z Dissecting the Requirement of Arabidopsis RanGAP1 Subcellular Targeting and GAP Activity for Its Cellular and Developmental Functions Anna Griffis – The Ohio State University Joanna Boruc – The Ohio State University, Ghent University, Thushani Rodrigo-Peiris – The Ohio State University, Xiao Zhou – The Ohio State University, Bailey Tilford – The Ohio State University, Daniel Van Damme – Ghent University, Iris Meier – The Ohio State RanGAP is the GTPase activating protein of Ran, in vertebrates involved in nucleocytoplasmic transport, spindle organization and post-mitotic nuclear assembly. Unlike vertebrate and yeast RanGAP, plant RanGAP has an N-terminal WPP domain, required for nuclear envelope association and several mitotic locations of Arabidopsis RanGAP1. A double null mutant of the two Arabidopsis RanGAP paralogs is gametophyte lethal. Here, we have created a series of rangap mutants with various reductions in RanGAP levels by combining a RanGAP1 null allele with different RanGAP2 alleles. As RanGAP level decreases, severity of developmental phenotypes increases but nuclear import is unaffected. To dissect whether the GAP activity and/or the subcellular localization of RanGAP are responsible for the observed phenotypes, this series of rangap mutants were transformed with RanGAP1 variants carrying point mutations abolishing the GAP activity and/or the WPP-dependent subcellular localization. The data show that plant development requires the GAP activity of RanGAP and is susceptible to reductions in RanGAP protein level, while the subcellular positioning of RanGAP is dispensable. In addition, our results indicate that nucleocytoplasmic trafficking can tolerate both partial depletion of RanGAP and delocalization of RanGAP form the nuclear envelope.

700-008-Y The SEC14-Nodulin AtSFH1 Patterns Phosphoinositide Distribution to Control Polarized Membrane Growth Marilia K. F. de Campos – University of Tuebingen Gabriel Schaaf – University of Tuebingen Root hairs provide a remarkable plant-soil interface that favors water and nutrient absorption, as well as plant-microbe interactions. Their development relies on fine-tuned molecular events that culminate in the polarized expansion of distinct root epidermal cells (i.e. trichoblasts). Phosphoinositides play a key role in root hair development by establishing signaling foci that localize cellular events, such as cytoskeleton organization and vesicle trafficking, to specific membrane sites. Our recent work in yeast demonstrates that SEC14-like lipid binding proteins act at the interface between

phospholipid homeostasis and membrane trafficking by assisting lipid kinases to overcome an intrinsic inefficiency in recognizing their phospholipid substrates. In particular, yeast SEC14 renders Phosphatidylinositol (PtdIns) vulnerable to PtdIns 4-OH kinase attack during Phosphatidylcholine (PtdCho)-dependent heterotypic phospholipid exchange, generating a PtdIns(4)P pool at trans-Golgi membranes that is crucial for proper vesicle biogenesis. Notably, root hair development relies on AtSFH1, a multidomain protein harboring an N-terminal SEC14 domain and a C-terminal Nlj16-like nodulin domain. We will present evidence that the Nlj16 module exhibits high PtdIns(4,5)P2 binding specificity in vivo and will provide a striking mechanism of how AtSFH1 couples phosphoinositide synthesis with lateral organization of PtdIns(4,5)P2 in membranes.

700-009-Y Characterization of the Tangled-1 Mutant in Maize Pablo Martinez – University of California Riverside Claire Stowers – University of Wyoming, Carolyn Rasmussen – University of California Riverside During the onset of early plant cell division, a structure named the pre-prophase band forms and is thought to be a marker for the orientation of the division plane. Later in the division the phragmoplast will lay down the foundation for the cell plate which ultimately guides the formation of the cell wall to the position indicated by the pre-prophase band. TANGLED-1 mutants in maize exhibit misoriented division planes. Though many of the cells in the tan mutants have atypical shapes and strange orientations, the overall shape of the maize plant is maintained. TAN has been show to localize to the cortical division site at least twice during division and is sustained at this location throughout division. Since tan mutants exhibit misoriented division planes and TAN is localized at the cortical division site, TAN can be used as a candidate for identifying new proteins required for plant cell division as well as division orientation. We are addressing the role that TAN is playing through careful analysis of tan mutants using time-lapse imaging as well astaking more biochemical approaches into characterizing proteins that interact with TAN to construct a pathway of events leading to the formation of these misoriented divisions.

700-010-Z Disruption of Cellulose Synthase Complex Localization and Mobility by Small Molecules Natasha Worden – UC Davis Thomas Wilkop – UC Davis, Victor Esteva Esteve – UC Davis, Richard Jeannotte – UC Davis, Rahul Lathe – Max Plank Institute of Molecular Plant Physiology, Samantha Vernhettes – INRA Centre Versailles-Grignon, Bart Weimer – UC Davis, Glenn Hicks – UC Riverside, Jose Alonso – NC State University, John Labavitch – UC Davis, Staffan Persson – Max Plank Institute of Molecular Plant Physiology, David Ehrhardt – Carnegie Institute for Science, Georgia Drakakaki – UC Davis In order to better understand and manipulate plant cell wall deposition, we need to investigate the endomembrane trafficking processes involved, because of their critical regulatory role on the cell wall. To investigate these processes, we are using chemical genomic screens, a revolutionary approach that involves the use of small molecules, rather than mutations to inactivate proteins. This is particularly useful when studying both subcellular trafficking and cell wall development because both processes often lead to lethal mutants and are difficult to be studied by traditional genetics. Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. In fluorescently labeled hypocotyls, CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and

POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

700-011-Z Division Plane Orientation in Tobacco Cells Using Chemical Screening Lindy Allsman – University of California Riverside Carolyn Rasmussen Division plane orientation of plant cells is key to ensuring proper growth and development. Currently, there is not much information known about molecular mechanisms regarding the orientation of the plant cell division plane. The compelling question is: in order to properly divide the cell, how does the new cell wall form in the correct location? There are many chemical libraries available to assess which chemicals affect division plane orientation. The Spectrum Library, which contains 2,320 compounds with known biological profiles, was used to treat tobacco cells to identify those compounds that alter cell wall orientation. Tobacco cells are used in our chemical screen because they grow well in liquid culture and respond well to cell cycle synchronization treatments. Cells are synchronized, transferred into 96 well plates, treated with compounds and then screened using an automated microscope. My initial analysis identified a limited number of compounds that caused cell wall orientation defects in tobacco cells. These compounds will be assessed for their ability to alter cell wall orientation in whole plants such as maize and then their targets will be identified.

700-012-Y Myosin VIII Links Actin to Microtubules During Cell Division and Polarized Growth Magdalena Bezanilla – University of Massachusetts Amherst Plants have two families of actin-based molecular motors: class VIII and class XI myosins. While much attention has been garnered by class XI myosins, the class VIII myosins, which were the first to be cloned in plants, have remained largely unstudied. In the moss, Physcomitrella patens, we have generated a line that lacks the entire family of class VIII myosins. While viable, this plant has a number of cellular and developmental defects that are beginning to reveal the role of myosin VIII in plant cell division and growth. In the filamentous tissue of moss, myosin VIII null plants have aberrantly positioned cell plates. By imaging a functional copy of Myo8A fused to GFP, we found that during cell division myosin VIII localizes to the ends of microtubules where it helps to guide phragmoplast expansion to the cortical division site. This same localization was also observed in tobacco BY-2 cells, suggesting that myosin VIII may have a similar role in seed plants. Myosin VIII null plants also grow slower than wild type plants. We found that during polarized growth, myosin VIII is found at the ends of cytoplasmic microtubules that are focused behind the growing tip, where there is an accumulation of actin filaments. These results suggest that myosin VIII helps to coordinate the interaction between the microtubule and actin cytoskeletons optimizing rates of polarized growth.

700-013-Y Cell Cycle and Plant Development: Dissection of FBL17 Function(s) in Sporophytic Tissues Pascal Genschik – IBMP CNRS

The cell cycle represents one of the most fundamental cellular events and is key to growth, development and reproduction of all living organisms. In particular, the control of the G1-to-S-phase transition is a key step in cell cycle regulation since cells become typically committed to divide once they replicate their DNA. This step is tightly regulated in all eukaryotes by various mechanisms incorporating intrinsic information such as nutrient status and hormonal signals with extrinsic, environmental conditions. Despite of its importance, little is still known about the molecular mechanisms of the G1-to-S-phase transition in higher plants. The Arabidopsis Cdk1 homolog CDKA;1, which is required for both Sphase and mitosis entry, operates independently of CDKA;1 dephosphorylation indicating that the regulatory wiring of cell cycle regulation is different in plants versus yeast or metazoans. However like in other kingdoms, plant CDKA;1 activity is negatively regulated by cyclin-dependent kinase inhibitors (CKIs). Here we will present novel data on the function of an Arabidopsis F-box protein, called FBL17, representing the major regulator of the G1-to-S-phase transition in plants.

700-014-Z Interrogating Actin Filament Dynamics in Living Epidermal Cells of Arabidopsis Lingyan Cao – Purdue University Jessica L. Henty-Ridilla – Purdue University, Jiejie Li – Purdue University, Christopher J. Staiger – Purdue University The actin cytoskeleton comprises a malleable framework that responds to diverse biotic and abiotic stimuli. Using stateof-art fluorescence microscopy and a robust collection of quantitative imaging tools, we have established a testable model for the dynamic behavior of actin filaments in plant epidermal cells. Two key features, extremely rapid filament elongation and disassembly through prolific severing activity, distinguish this turnover mechanism from textbook models of actin dynamics. However, the molecular mechanisms that underpin initiation of new filaments remain rather unclear. Profilin is an abundant G-actin binding protein that forms a 1:1 complex with actin monomers and suppresses spontaneous filament nucleation in vitro. To test the function of profilin in plants, we are characterizing mutants for the five Arabidopsis PROFILIN (PRF) genes. Here, we report on the contribution of PRF1 to actin organization and dynamics during epidermal cell elongation through directly analyzing the behavior of individual filaments in the cortical array of living Arabidopsis thaliana epidermal cells. We examined two prf1 T-DNA insertion mutants and found that reduced PRF1 levels result in increased overall actin dynamicity but significantly decrease the actin filament elongation rate and maximum filament length. Surprisingly, we found that the frequency of nucleation events was dramatically decreased in prf1 mutants. Specifically, we found a significant reduction in both branched and end nucleation events, but an increase in de novo filament origins. Moreover, we found that inhibition of the nucleation factor, formin, in wild-type plants using a small molecule inhibitor (SMIFH2) reduced fiilament initiation in a dose-dependent manner and phenocopied the prf1 mutants. Formins contain a proline-rich (FH1) domain and this recruits profilin-actin for filament nucleation in vitro. Therefore, we predict that PRF1 contributes to new actin filament initiation in a formin-dependent manner and will further evaluate this hypothesis in living cells.

700-015-Z Patterning Mechanisms of Cytoskeletal and Cell Wall Systems During Morphogenesis Makoto Yanagisawa – Purdue University Chunhua Zhang – University of California, Riverside, Robert Stahelin – University of Notre Dame, Anastasia Desyatova – University of Nebraska-Lincoln, Linda Robles – North Carolina State University, Jose Alonso – North Carolina State University, Daniel Szymanski – Purdue University Both the actin and microtubule cytoskeletons are required for polarized diffuse growth; however, the mechanisms by which they work together to pattern the cell wall and growth are not known. In this study, we used a combination of

finite element computational modeling and multivariate time-lapse imaging to discover functional relationships between the cytoskeleton, cell wall heterogeneity, and the polarization of trichome growth. The data-derived model predicted three key mechanical properties of the cell wall that dictates cell morphogenesis: a highly aligned cellulose microfibrils transverse to the growth axis, a proximo-distal cell wall thickness gradient that enables tip-biased anisotropic diffuse growth, and a tip isotropic zone that mediates tip refinement. These predictions were experimentally validated. In general the function of cortical actin is poorly understood in plant cells, even in trichomes in which mutations in the actin filament nucleating ARP2/3 complex have obvious cell shape defects. A functional live cell probe revealed that ARP2/3 accumulates at the branch apex. Multiple lines of evidence showed that the tip ARP2/3 is active and generates an apical actin meshwork. ARP2/3-genrated actin meshworks control the alignment of actin bundles that organize organelle trafficking at cellular scale. Organized cellular flow is needed to maintain a cell wall thickness gradient. ARP2/3-generated apical actin meshworks may have another role to locally modulate the size and positioning of the tip isotropic zone. ARP2/3 functions within an apical microtubule-depleted zone, and is required to coordinate the size and position of the microtubule-depletion zone during branch tip narrowing. Potential molecular mechanisms by which this specialized cortical domain is assembled will be discussed.

700-016-Y At1g18720, a Gene of Unknown Function, Is Required for Nectar Production in Arabidopsis Anthony Schmitt – University of Minnesota Ricci Bender – University of Minnesota, Metadel Abegaz – University of Minnesota, Clay Carter – University of Minnesota Floral nectar plays an essential role in plant-pollinator interactions. While the function and composition of nectar has been characterized, the molecular mechanisms involved in nectar secretion has remained elusive and is a current focus of research. Here we report that At1g18720, a gene of “unknown function,” plays an essential role in nectar production. Silencing of At1g18720 resulted in flowers that produce no nectar. At1g18720 encodes a small protein (206 amino acids) with four predicted transmembrane domains. GFP fusions suggested that At1g18720 is localized to the plasma membrane, and promoter::GUS analyses demonstrated that the gene is expressed exclusively in the nectaries and anthers of flowers. While the encoded protein contains no conserved domains of known function, At1g18720 shares identity with genes found in many plant species, as well as yeast. Future efforts will be dedicated toward elucidating the mechanisms by which At1g18720 controls nectary function.

700-017-Y Protein Trafficking – the Only Constant Thing Is Change Karin Schumacher Heidelberg University Stefan Scholl – COS, Upendo Lupanga – COS Plants constantly adjust their repertoire of integral membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients and hormones. The importance of regulated secretory and endocytic trafficking is by now well established and the trans-Golgi network/early endosome (TGN/EE) has emerged as the central hub for protein sorting. We have shown previously that activity of the vacuolar H+-ATPase (V-ATPase, VHA) characterized by the presence of subunit VHA-a1 is essential for TGN/EE integrity and function. Although this pointed to an important role of low pH for protein sorting and vesicle trafficking, the mechanistic basis remained to be clarified. In my presentation, I will thus focus on our recent efforts to measure pH in the TGN/EE, to understand how steady localization of the V-ATPase in the dynamic environment of TGN/EE is achieved and, last but not least, to address a potential role the V-ATPase subunit VHA-a1 as a pH-sensor.

700-018-Z Early Identification and Elimination of Non-germline Events in Transgenic Soybean Production Process Geny Anthony – Dow AgroSciences Siva Chennareddy, Dayakar Pareddy, Rodrigo Sarria, Pon Samuel, Toby Cicak, Brandon Bishop, Nolan Shumway In soybean, both somatic embryogenesis and organogenesis methods have been used for transformation. In the case of organogenesis based methods using meristematic target tissue such as cotyledonary node, embryonic axis or split-seed explants, the transgenic plants could either be germline or non-germline (chimeric), depending on the cell layer (L1, L2, L3) that was transformed. Historically, about 50 percent of the plants produced are undesirable non-germline transformation events. The T1 progeny of those plants are screened for heritability via topical application of selection agents in the field or greenhouse or molecular analysis methods. Maintenance of primary transformants (T0 events) until seed production and screening of T1 progeny involves significant use of resources. We developed a simple and efficient method for early identification and elimination of non-germline events based on root phenotype. Similar to germline tissue, roots develop from the deeper layers of meristem (L2 and L3 layers). Hence, development of nontransgenic roots indicated non-germline transformation and such roots were easily distinguished by adding selection to the media during the rooting phase. We used the Agrobacterium-mediated split seed transformation method in combination with the pat (phosphinothricin acetyltransferase) selectable marker gene. Transgenic shoots produced under selection were transferred to ‘Rooting Medium’ containing glufosinate ammonium. In rooting media, nongermline events and ‘escapes’ produced either no roots or unhealthy, brown roots that were visually distinguished and discarded. With this method, up to 90% of non-germline events were eliminated early in the transformation process, thereby saving significant greenhouse and field resources.

700-019-Z AKIN10 and the Regulation of Autophagy in Arabidopsis Thaliana Junmarie Soto-Burgos – Iowa State University Diane Bassham – Iowa State University Autophagy is a degradation process in which cells break down and recycle their cytoplasmic contents when subjected to environmental stress or during development. AKIN10 is a protein kinase and is a component of the SnRK1 (Snf1-related protein kinase 1) complex, which senses changes in energy levels. Its mammalian ortholog, AMPK (AMP-kinase), and yeast ortholog, Snf-1 (Sucrose non-fermenting 1), are involved in the regulation of the autophagy pathway. In mammals AMPK can regulate autophagy in an mTOR (Target of Rapamycin) -dependent or -independent manner. TOR is known to be a negative regulator of autophagy in plants. We therefore hypothesize that AKIN10 may play a role in the regulation of autophagy in Arabidopsis. To test this hypothesis, we obtained transgenic lines overexpressing AKIN10 and determined the effect on autophagy activation. Abiotic stresses (nutrient deficiency, salt, osmotic, oxidative, and ER stress) were used to activate autophagy. While wild-type plants had basal autophagy activity in control conditions, AKIN10 overexpression lines had constitutive autophagy under these conditions. In addition, after stress treatment the overexpression lines showed a reduction in autophagy activity. These results suggest that AKIN10 is a positive regulator of autophagy, but further research is needed to determine its exact role. This research is funded by an Alliance for Graduate Education and the Professoriate (AGEP) Fellowship and by the Walter E. and Helen Park Loomis Fund.

700-020-Y Identifying Plant Programmed Cell Death Regulators Using an Insect Inhibitor of Apoptosis from Spodoptera Frugiperda (SfIAP) Ryan Kessens – University of Wisconsin - Madison Roberta Dollinger – University of Wisconsin – Madison, Mehdi Kabbage – University of Wisconsin – Madison Programmed cell death (PCD) is a conserved fundamental process that is triggered in response to developmental and environmental cues in both animals and plants. However, plants lack obvious homologues of many regulators of apoptosis from animals. This includes caspases, which are cysteine proteases that execute the death signal by targeting specific cellular components necessary for an orderly cellular demise. Caspase activity is kept under tight regulation by the inhibitor of apoptosis (IAP) family of proteins. Members of this family are characterized by a caspase-binding Baculovirus IAP Repeat (BIR) domain and often contain a RING domain with E3 ligase activity. While plants lack clear homologues of caspases and IAPs, heterologous expression of human and insect IAPs in plants has been shown to inhibit cell death caused by a number of abiotic and biotic stresses. Recent studies have shown that expression of an IAP from Spodoptera frugiperda (SfIAP) can inhibit cell death caused by salt, heat, fumonisin B1 (FB1), and Alternaria alternata in transgenic tobacco and tomato. To determine the biochemical context of SfIAP in plants, a yeast two-hybrid screen was conducted using SfIAP as bait to screen a tomato cDNA library. Results revealed that putative interactors of SfIAP were enriched with members of the SQUAMOSA promoter binding protein (SBP) family of plant specific transcription factors. Transient expression assays are being used in tobacco leaves to determine the effect of SfIAP and SBP overexpression on cell death imposed by salt, FB1, and Alternaria solani treatment. We propose that the anti-PCD function of SfIAP in plants may be owed to its targeting and inhibition of SBP family of plant transcription factors.

700-021-Y Development of Physcomitrella Patens Transgenic Marker Lines to Visualize PtdIns(4)P Dynamics Angela Ai – Wellesley College Fabienne Furt – Worcester Polytechnic Institute, Luis Vidali – Worcester Polytechnic Institute, T. Kaye Peterman – Wellesley College Phosphoinositides (PIs) are key signaling lipids that are involved in many cellular processes, such as membrane trafficking and regulation of the actin cytoskeleton. Phosphatidylinositol-4-phosphate (PtdIns(4)P), a member of the PI family, is involved in polarized cellular growth and in the biogenesis of secretory vesicles during tip growth of root hairs and pollen tubes. In Arabidopsis thaliana root hairs, PtdIns(4)P localizes to the plasma membrane, trans-Golgi network and recycling endosomes. However, there is limited work on PtdIns(4)P in non-vascular plants, such as the moss Physcomitrella patens. Due to its susceptibility to reverse genetics and high efficiency of homologous recombination, P. patens has recently emerged as an excellent model system. It is especially suited for tip growth studies because establishment of its structure is dependent on tip growth. To investigate PtdIns(4)P’s dynamic localization in P. patens, we generated fluorescently-tagged lipid binding modules composed of 3xmEGFP fused to the PtdIns(4)P binding pleckstrin homology (PH) domains of OSBP and FAPP1 under the control of the constitutive maize ubiquitin promoter. Additionally, nonbinding mutant constructs, PHOSBP R107E, R108E and PHFAPP1 R18L, have been produced. Preliminary confocal imaging of both lines revealed PtdIns(4)P localization to the plasma membrane and, in the case of lines expressing PHFAPP1, to punctate structures. The non-functional mutants showed non-specific cytoplasmic localization. Interestingly, lines over-expressing PHOSBP-3xmEGFP displayed a wavy growth phenotype, reminiscent of Arabidopsis root hair mutants with disrupted PtdIns(4)P domains, suggesting a dominant negative effect. Further characterization of these moss lines will be valuable in understanding how PtdIns(4)P functions during polarized tip growth.

700-022-Z Arabidopsis Glutamate Receptor Homolog AtGLR3.5 Modulates Cytosolic Ca2+ Level to Counteract Effect of Abscisic Acid in Seed Germination June M. Kwak Chuanli Ju, Dongdong Kong, Aisha Parihar, So Kim, Deashik Cho Seed germination is a critical step in a plant’s life cycle that allows successful propagation and is therefore strictly controlled by endogenous and environmental signals. However, the molecular mechanisms underlying germination control remain elusive. Here, we report that the Arabidopsis glutamate receptor homolog AtGLR3.5 is predominantly expressed in germinating seeds and increases cytosolic Ca2+ concentration ([Ca2+]cyt) that counteracts the effect of abscisic acid (ABA) to promote germination. Repression of AtGLR3.5 impairs [Ca2+]cyt elevation, significantly delays germination, and enhances ABA sensitivity in seeds, whereas overexpression of AtGLR3.5 results in earlier germination and reduced seed sensitivity to ABA. Furthermore, we show that Ca2+ suppresses the expression of ABA INSENSITIVE4 (ABI4), a key transcription factor involved in ABA response in seeds, and that ABI4 plays a fundamental role in modulation of Ca2+-dependent germination. Taken together, our results provide molecular genetic evidence that AtGLR3.5-mediated Ca2+ influx stimulates seed germination by antagonizing the inhibitory effects of ABA through suppression of ABI4.

700-023-Z PDI7, a Novel Membrane-bound Member of the Protein Disulfide Isomerase Family, Localizes to the Cis-Golgi Cisternae and Endoplasmic Reticulum in Arabidopsis Thaliana Christen Yuen – University of Hawaii at Manoa Kristie Matsumoto, Byung-Ho Kang, David Christopher In eukaryotes, members of the protein disulfide isomerase (PDI) family catalyze the formation and isomerization of disulfide bonds to fold nascent secretory proteins in the endoplasmic reticulum (ER). Classical PDIs have a modular domain arrangement, a-b-b’-a’, where a and a’ are thioredoxin-like catalytic domains, and b and b’ are redox-inactive thioredoxin-like fold domains. Plants possess a novel non-classical PDI subfamily, PDI-C, which is absent in animals and yeast. PDI-C isoforms harbor two transmembrane domains (TMDs) separated by a large hydrophilic loop containing a single a-type catalytic domain. In addition, PDI-C isoforms possess the ERGIC-N and COPII-coated Erv conserved domains found in the yeast cargo receptor protein Erv46p, and its mammalian orthlolog ERGIC3. Arabidopsis has three PDI-C genes: PDI7, PDI12, and PDI13. Expression analysis of promoter:β-glucuronidase (GUS) reporter gene fusions indicated that PDI7 is distinctly expressed from PDI12 and PDI13, with prominent expression in hydathodes, the style, and the vasculature of leaves, sepals, and stamen filaments. Transient expression of PDI7:GFP fusions, and immunoelectron microscopy experiments using a PDI7 antibody, revealed that PDI7 localizes to both the ER membrane and the cis Golgi. Electron tomography analysis indicated that PDI7 was specifically associated with the two cis-most Golgi cisternae. The catalytic domain of PDI7 was shown by protease protection experiments to be positioned in the ER/Golgi lumen, while the N- and C-proximal TMDs were integral to the membrane. The presence of both a COPII-coated Erv domain and xKxxtype ER retrieval motif at the C-terminus of PDI7 suggests that the protein may cycle between the ER and Golgi in a manner similar to yeast Erv46p. The prominent accumulation of PDI7 at the cis Golgi indicates a novel function for the PDI-C subfamily outside of the traditional role of PDIs as ER resident enzymes involved in oxidative protein folding.

700-024-Y Trichomes in Genus Scutellaria and Their Possible Roles Brajesh Vaidya – Fort Valley State University Nirmal Joshee – Fort Valley State University

Scutellaria, comprising more than 400 species around the world is one of the largest genera in the family Lamiaceae. There are more than 90 species reported from North America. We have 22 Scutellaria species in our germplasm collection at Fort Valley State University representing major continents. We are involved in studying trichomes diversity in vegetative and reproductive structures of these species. Two species that are found in Georgia, Scutellaria ocmulgee and Scutellaria montana are of significance due to their rarity in wild (S. ocmulgee is in State threatened list of plants and S. montana is in Federal threatened list), high antioxidant capacity, and antitumor activity on glioma cells and tumor reduction in animal models. We present our research on morphohistological details using paraffin sectioning, scanning electron microscopy to study surface morphology in terms of glandular and non-glandular trichomes and fluorescent microscopy for the detection of bioactive compound. Trichomes have been studied and cataloged. These micromorphological features can play important role in identifying adulteration in the herbal supplements and as a taxonomic tool in differentiation closely related speci

700-025-Y Investigating the Role of Arabidopsis Thaliana Neighbor of BRCA1 Gene 1 (NBR1) in Pexophagy Pierce Young – Rice University Bonnie Bartel – Rice University Peroxisomes sequester necessary yet harmful oxidative reactions. Although this sequestration protects the rest of the cell from damage, peroxisomes are susceptible to the reactive oxygen species produced from these reactions, and cells turn over damaged peroxisomes via a form of selective autophagy known as pexophagy. In Arabidopsis thaliana, the LON2 peroxisomal protease prevents pexophagy, and lon2 mutants exhibit peroxisome-related defects due to heightened pexophagy. We are seeking to elucidate the molecular components of pexophagy in plants. During pexophagy in other organisms, selective autophagy receptors recognize and deliver damaged peroxisomes to the autophagy machinery. Neighbor of BRCA1 gene 1 (NBR1) is the only characterized selective autophagy receptor in Arabidopsis thaliana, but although NBR1 acts as the pexophagy receptor in mammalian cells, a role for NBR1 in pexophagy has not been demonstrated in plants. We found that nbr1 mutants, unlike atg mutants, fail to suppress lon22 phenotypes. Furthermore, overexpression of NBR1 fails to phenocopy lon2 mutants. These results indicate that NBR1 is not involved the heightened pexophagy observed in lon2 mutants and suggest that NBR1 is not involved in pexophagy in plants. We are currently conducting a forward genetic screen for plants with pexophagy defects in an attempt to identify plant pexophagy receptors. (This work is supported by the NSF and the Robert A. Welch Foundation.)

700-026-Z ES16 Mediated Recycling Pathway Is Essential for Apical Polarity Establishment and Plant Development Ruixi Li – UC Riverside Glenn Hicks – UC Riverside, Natasha Raikhel – UC Riverside In plant, spatial regulation of the plant hormone indole -3 – acetic acid (IAA, or auxin) is essential for developmental program. The auxin gradient is established and maintained by polarly localized auxin transporters, including PINFORMED (PIN) proteins. Endocytic and recycling pathways are important for the generation of the asymmetries of the PIN proteins. Previous research has implicated that the localization and abundance of basal localized PIN1 is regulated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF – GEF ) GNOM, whereas the mechanism of the maintenance of apical localized PIN proteins is largely unknown. Our study characterized an inhibitor endosidin 16 (ES16), which selectively interferes with the PIN2 apical polarity without altering the polarity of basal proteins. ES16 does not alter trafficking from the ER to the Golgi, but significantly changes the post-Golgi trafficking. Pharmacological evidences indicate that ES16 does not affect endocytosis but inhibits recycling back to the plasma membrane as well as biosynthetic secretion. ES16 changes the auxin distribution pattern in the root, and induces a strong developmental

phenotype, including reduced root length and agravitropic phenotype but shows no significant effect on lateral root initiation. Consistent with its selectivity of interference with apical but not basal localized PIN proteins, ES16 does not alter the subcellular localization of GNOM. None of the ARF – GEF mutants display hypersensitivity or resistance to ES16 treatment. Our data indicate that different endomembrane trafficking systems exist for the maintenance of basal and apical localized PIN proteins and reveal a ES16 mediated recycling / secretion pathway selectively regulates PIN2 apical polarity and is essential for normal plant development.

700-027-Z Development of New Tools for High-Quality Yeast Two-Hybrid Analysis of Crop Interactomes Stéphanie Blachon – Hybrigenics Services SAS Anne-Aymone Grenouilloux – Hybrigenics Services SAS, Vincent Collura – Hybrigenics Services SAS, Thomas Moncion – Hybrigenics Services SAS, Senthil Subramanian – Plant Science Department, South Dakota State University, Guoshen Li – University of Arizona, Ramin Yadegari – University of Arizona, Daniel Grimanelli – Institut de Recherche pour le Devloppement, Prateek Tripathi – USC Fornslife College of Letters, Paul Rushton – Texas A&M University, Etienne Formstecher – Hybrigenics Services, SAS The understanding of protein interaction networks provides crucial insights into the molecular mechanisms of signal transduction, stress responses and developmental processes. Applied to plant science, this will open up the way to improve relevant traits of agronomically important crops and help understand host-pathogen interactions. Yeast twohybrid (Y2H) protein interaction screening has proven instrumental for the analysis of the interactome of crops, mostly thanks to pairwise testing or screening of oligo dT-primed cDNA libraries. However, interaction map completeness has been limited by the use of full-length proteins and C-terminal polypeptide fragments which result in significant false negative rates. To circumvent these limitations, we have used a domain-based strategy to construct highly complex, random primed cDNA libraries from different tissues of Solanum lycopersicum, Oriza sativa, Triticum aestivum and very recently Glycine max and Zea mays. This strategy has been shown to be very successful with over 300 screens performed on libraries from model plants like Arabidopsis thaliana and Nicotinana benthamiana. The complexity of these libraries is greater than 10 million independent fragments in yeast, with an average fragment size of 800 bp. To ensure reproducible and exhaustive Y2H results, these libraries are screened to saturation using an optimized mating procedure, allowing to test on average 83 million interactions per screen (8-fold coverage of the library). As a consequence, multiple, independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score. These libraries have been integrated into our high-throughput yeast two-hybrid platform and are available for screening on a fee-for-service basis. Results from representative screens on these libraries will be presented at the meeting. In addition, we will demonstrate our sophisticated PIMRider software that allows straightforward analysis and navigation inside complex networks combining interaction, genetic and expression data from different sources.

700-028-Y STUCK, a Gene Promoting Cellulose Deposition and Cell Elongation in Arabidopsis Thaliana Christy Moore – Illinois State University Bangxia Suo, Mais Zahde, Viktor Kirik Arabidopsis hypocotyls contain long, narrow cells that elongate along the axis of plant growth, pushing the developing shoot through the soil. Certain cell wall mutants are deficient in the process of elongation, causing their hypocotyls to be

swollen and shorter than those of wild type plants. This phenotype is most noticeable when these mutants are grown in darkness because hypocotyl cells will elongate more in an effort to break through the darkness and find sunlight. My research focuses on the stuck (stu) mutant, which, when grown in darkness, exhibits a smaller and slightly swollen hypocotyl than those of wild type plants. STU was rough mapped to a 2994kb region on chromosome 5. We tested known mutants within our mapping region that have stu-like phenotypes (cobra and procuste1/cesa6) to verify that stu is novel. A conditional radial expansion test revealed that stu does not exhibit a cobra phenotype. A confocal microscopy analysis of progeny from a stu x YFP-tagged CESA6 cross indicated that STU is not CESA6 and that CESA6 appears to be sequestered in Golgis of stu plants. In addition, we measured birefringence, an optical property of crystalline cellulose, in mutant and wild type trichomes, revealing a possible cellulose deficiency. We performed a biochemical cellulose assay to quantify the amount of cellulose in dark-grown mutant and wild type plants, to test this possibility. Through the identification and preliminary characterization of the STU gene, we hope to learn new information regarding the molecular processes important for cell wall building and cell elongation.

700-029-Y Cortical Microtubule Arrays and Plant Hormone Signaling: AXR2 Gates Transverse Microtubule Patterning in Epidermal Hypocotyl Cells Jillian True – Indiana University Andrew Elliott – Indiana University, Sidney Shaw – Indiana University Cortical microtubules (MTs) influence plant cell morphology by patterning the deposition of cellulose into the cell wall. We previously demonstrated that a combined auxin (IAA) and gibberellin (GA4) treatment triggers transverse coalignment of MT arrays in Arabidopsis hypocotyl cells in two distinct phases: I) early reduction of growing MT plus ends and II) later co-alignment progressing from the midzone to the cell’s apical-basal regions. In this study, we seek to identify the specific signaling pathways activating these core phases of transverse MT array organization. To begin, we tested the hypothesis that exogenous hormone treatments elicit a shade-avoidance response in light-grown seedlings. We found that exposing seedlings to dark conditions produced both the Phase I and Phase II responses previously observed with GA/IAA addition. We next asked which individual hormones known to function in hypocotyl extension (IAA, GA, and brassinosteroid (BR)) initiate Phase I and Phase II responses. We found that IAA and BR both led to Phase I and Phase II MT responses similar to dark treatment, whereas GA only triggered Phase II. To elucidate downstream targets of these pathways, we have begun examining mutants in the auxin pathway. We found that axr2-1, a dominant negative allele of the Aux/IAA co-receptor AUXIN RESISTANT 2, effectively prevents both phases of hormone-induced transverse MT patterning when treated with IAA, GA4, or BR. Our results suggest genes expressed downstream of AXR2 are required for transverse MT patterning and for integrating other hormone pathways in this response. Thus, we propose that AXR2 serves as a gatekeeper for hormone-induced transverse MT patterning.

Cell Biology: Plastids and Organelles 700-030-Z Guard Cell Photosynthesis Is Critical for Stomatal Turgor Production, yet Does Not Directly Mediate CO2- and ABA-induced Stomatal Closing Tamar Azoulay-Shemer – University of California, San Diego Axxell Palomares – University of California, San Dieg, Andish Bagheri – University of California, San Diego , Maria Israelsson-Nordstrom – Swedish University of Agriculture Sciences, Cawas B. Engineer – University of California, San Diego, Bastiaan O.R. Bargmann – Cibus US LLC, Aaron B. Stephan – University of California, San Diego, Julian I. Schroeder

– University of California, San Diego Stomata regulate gas exchange between the inter-cellular spaces of leaves and the atmosphere. CO2 levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO2]. [CO2] in leaves regulates stomatal movements. The role of guard-cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll-deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard-cell specific enhancer trap-line. Our data show that more than 90% of guard cells were chlorophyll-deficient. Interestingly, approximately ~ 45% of stomata had an unusual, previously not-described, morphology of thin-shaped chlorophyll-less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole-leaf photosynthetic parameters (PSII, qP, qN, FV’/FM’) were comparable to wild-type plants. Time-resolved intact leaf gas exchange analyses showed a reduction in stomatal conductance and carbon assimilation rates of the transgenic plants. Normalization of CO2 responses showed that stomata of transgenic plants respond to [CO2] shifts. Detailed stomatal aperture measurements of normal kidney-shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO2] elevation and abscisic acid (ABA), while thin-shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO2] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard-cell CO2 and ABA signal transduction are not directly modulated by guard-cell photosynthesis/electron transport. Moreover, the finding that chlorophyll-less stomata cause a “deflated” thin-shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard-cell turgor production.

700-031-Z Roles of MAP KINASE17 in Peroxisome Proliferation and NaCl Response in Arabidopsis Thaliana Elizabeth Frick – Washington University in St. Louis Lucia Strader – Washington University in St. Louis Peroxisomes are organelles found in all eukaryotes, from single-celled yeast to plants to humans, and perform a wide variety of functions. Universally, peroxisomes are the site of fatty acid breakdown through β-oxidation and hydrogen peroxide detoxification. In plants, peroxisomes are responsible for many other essential processes, including synthesis of branched chain amino acids, biotin, some vitamins, auxin and jasmonate, as well as conversion of oils to acetyl-CoA. Although many peroxisome proliferation factors, such as DRP3A, FIS1A, and PEX proteins, have been identified, how the plant integrates competing signals from multiple stimuli to control division is not fully understood. We have identified roles for MAP KINASE17(MPK17) in peroxisome proliferation. The mpk17 mutant displays numerous peroxisome-related phenotypes, including hypersensitivity to indole-3-butyric acid (IBA) and altered peroxisome size and number. In the mpk17 mutant, the transcript level of peroxisome division factor PMD1 is elevated and the pmd1 mutation suppresses mpk17 phenotypes, suggesting that MPK17 effects on peroxisome proliferation are at least partially through regulation of PMD1 levels. In addition to altered peroxisome number under normal growth conditions, mpk17 peroxisomes do not proliferate under NaCl stress. Together, these data suggest a role for MPK17 in repressing peroxisome division under normal growth conditions, then relieving this repression to allow peroxisome division under specific stress conditions.

700-032-Y Utilizing Alloplasmic Wheat to Understand Cytonuclear Crosstalk Marisa Miller – USDA-ARS Katie Liberatore – USDA-ARS, Shahryar Kianian – USDA-ARS During the evolution of both animals and plants, numerous cytoplasmic (i.e. mitochondria and chloroplast) genes migrated to the nuclear genome necessitating tightly coordinated expression of many organellar- and nuclear-encoded

genes. In wheat (Triticum aestivum), the effects of disrupting these native interactions by substituting the cytoplasm of domesticated wheat with that of wild relatives (alloplasmic lines) can range from negative to positive phenotypes (e.g. cytoplasmic male sterility versus yield heterosis and improved stress tolerance, respectively). Pilot studies suggest that sequence changes, increased levels of heteroplasmy (genome variants), and altered organellar gene expression in alloplasmic lines may lead to changes in phenotype, however, the molecular mechanisms responsible for this phenomenon remain elusive. To gain a deeper understanding of how the alloplasmic condition influences organellar genomes and impacts phenotype a series of experiments were performed. Next-generation sequencing and de novo genome assembly of organellar DNA isolated from alloplasmic lines (backcross generation 10 or higher) and euplasmic parents revealed the mitochondria and chloroplast genomic diversity across divergent species. Additionally, comparative analysis of alloplasmics and euplasmic parents uncovered the extent of both single-nucleotide and larger structural variants induced by altered cytonuclear communication. In parallel, the homogeneity of the nuclear genome after backcrossing was verified utilizing a high-density 90,000 single nucleotide polymorphism genotyping array. Ongoing experiments are investigating the mechanisms by which alien cytoplasm from specific wild relatives in the Aegliops and Triticum genera can lead to improvements in phenotype, such as biomass and stress tolerance. Our results suggest that organellar genomes provide a unique resource to advance our understanding of cytonuclear crosstalk and to rapidly improve performance of existing cultivars.

700-033-Y Identification on Putative Substrates of SecY2, a New Translocase in Plant Chloroplasts Yubing LI – University of Florida Jonathan Martin – University of Colorado, Donna Fernandez – University of Wisconsin-Madison, Kenneth Cline – University of Florida The SecA/SecYE translocase (Sec) is the general system for protein transport in prokaryotes. Chloroplasts, which evolved from a prokaryote, consist of a double membrane envelope that encloses an aqueous stroma and an internal thylakoid membrane system. A homologous Sec system in thylakoids transports or integrates a large proportion of thylakoid proteins. Recently, a second Sec system, SecA2/SecY2E2, was identified in the chloroplast envelope. The null secy2/secy2 in Arabidopsis exhibits a severe embryo lethal phenotype. We used inducible RNAi of SecY2 to by-pass the embryo defect. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids. In addition, membrane proteins TIC40, FtsH12, TATC and SecY1 were substantially reduced and an incomplete processing form accumulated. Additional insight into putative SecY2 substrates was achieved by in vitro chloroplast protein import into chloroplasts from secy2 mutant seedlings. Results showed that TIC40 integration and processing were significantly slowed. TATC and FtsH12 had reduced import efficiency, while there were no significant defects observed for SecY1 and SecY2. FtsH12 is a chloroplast inner envelope metallo protease. In Arabidopsis, FtsH12 null mutants are lethal at the embryo stage, i.e. a similar phenotype as null mutants in SecY2. In vitro reconstitution experiments indicate that FtsH12 is imported into the plastid stroma and then integrated into the envelope in reaction that is blocked by the SecA inhibitor sodium azide. These data suggest that FtsH12 is integrated by the SecY2 translocase. Mechanisms of membrane protein integration into the inner envelope membrane are not well understood. SecY2 is the only translocase identified to direct membrane protein integration into the inner envelope from the stroma. Our data not only point to an essential role of SecY2 for inner envelope biogenesis beyond embryo development, but also shed new light on our current understandings of protein trafficking within chloroplasts.

700-034-Z Learning the Language of the Chloroplast: Retrograde Signals That Regulate Stomata and ABA Responsiveness Barry Pogson – Australian National University

The chloroplast can be an environmental sensor for the cell, communicating with the nucleus via retroagrade signals during biogenesis and operation to change the expression of thousands of genes. Recent advances have identified retrograde signals and pathways ranging from carotenoid derivatives, phosphoadenosines such as PAP, tetropyrroles and heme together with reactive oxygen species and proteins that build a communication network to regulate gene expression, RNA turnover and splicing. However, retrograde signaling pathways have largely been viewed as a means for bi-lateral communication between organelles and nuclei, ignoring the potential for interaction with hormone signaling regulating plant form and function. The impact of new findings on the processes by which organelle communication is initiated, transmitted and perceived to regulate not just chloroplast processes, but intersect with hormonal signaling altering physiological responses will be considered. Specifically, genetic manipulation of the retrograde signal, PAP, enables ABA-mediated stomatal closure conferring drought tolerance in wild-type and ABA insensitive mutants; likely via a distinct, XRN mediated transcriptional pathway in guard cells. Genetic manipulation of PAP also enhances ABA sensitivity in seed germination independent of ABI1. Thus, chloroplast-nuclear communication mediated by the SAL1PAP pathway regulates stomata closure and germination.

700-035-Z The HOPS Subunit AtVPS41 Is Involved in Homotypic Vacuole Fusion Jiameng Zheng – North Carolina State University Marcela Rojas-Pierce – North Carolina State University Vacuoles are multifunctional organelles essential for plants. Plant vacuole biogenesis requires homotypic fusion of vacuolar membranes but this process is largely unknown. We previously characterized a new mutant allele of VTI11, itt3, with a novel fragmented vacuole phenotype [Zheng et al. 2014 Mol.Plant Jun;7(6):1026]. VTI11 is a SNARE protein responsible for membrane fusion at pre-vacuolar compartments and vacuoles. The phenotype of vti11/itt3 indicates that VTI11 is required for homotypic vacuole fusion. In addition, we determined that phosphatidylinositol 3-phosphate (PtdIns(3)P), a phosphoinositide that localizes to pre-vacuolar compartments and vacuoles, negatively regulates vacuole fusion. Thus, treatment of itt3 with Wortmannin quickly induces vacuole fusion. Wm also induced vacuole fusion in guard cells where changes in vacuole morphology are important for stomata movements. We hypothesize that PtdIns(3)P functions as a signaling molecule to regulate vacuolar SNARE function and vacuole fusion via the HOPS tethering complex. HOPS (homotypic fusion and vacuolar protein sorting) is a tethering factor that localizes to the vacuole, binds to PtdIns(3)P and interacts with vacuolar SNARE proteins in Saccharomyces cerevisiae. The only member of HOPS known in plants is VACUOLELESS1 (VCL1), and loss of VCL1 function results in lack of vacuoles and embryo lethality. We are currently characterizing a second member of HOPS, AtVPS41, by a reverse genetic approach. A T-DNA insertion line for VPS41 was obtained but no homozygous vps41-1 were recovered from over 40 segregating plants, indicating that homozygous vps41-1 are lethal. A flourescent GFP fusion with the VPS41 protein localizes to the vacuole, supporting a role in regulating vacuole fusion. In addition, BiFC experiments in protoplasts revealed that VPS41 interacts with the vacuolar SNARE SYP22. Together, our data suggests that AtVPS41 is essential for plant survival and may regulate vacuole fusion via interactions with vacuolar SNAREs.

700-036-Y Growth at Elevated Temperature Reduces PEX5 Levels and Ameliorates Peroxisomal Defects of the Arabidopsis pex4-1 Mutant Yun-Ting Kao – Rice University Bonnie Bartel – Rice University Peroxisomes house essential metabolic reactions. For example, fatty acid beta-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into the

peroxisome. Most matrix proteins are delivered to the peroxisome by PEX5, a receptor that forms transient pores that allow fully-folded proteins to translocate across the peroxisomal membrane. After cargo delivery, PEX5 is recycled back to the cytosol in a ubiquitin-dependent manner. A peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling or degradation, respectively. Arabidopsis pex mutants beta-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature rescued the sucrose dependence and reduced PEX5 levels in the pex4-1 mutant. Conversely, overexpressing PEX5 exacerbated pex4-1 defects, suggesting that PEX5 lingering on the peroxisomal membrane when recycling is impaired damages peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature reduced overall PEX5 levels without restoring PEX4 enzymatic function. We found that preventing autophagy in pex4-1 did not restore PEX5 levels. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5 at high temperature. We conclude that growth at elevated temperature activates proteasomal degradation of PEX5 to reduce overall PEX5 levels and ameliorate pex4-1 physiological defects. (This research is supported by the Robert A. Welch Foundation and the National Institutes of Health.)

700-037-Y Characterization of Putative Peroxisomal Proteins in Arabidopsis Thaliana Karen Chanchavac – Spring Arbor University Aaron Wyman – Spring Arbor University Peroxisomes represent understudied components of eukaryotes despite being necessary for the oxidation of long- chain fatty acids, cholesterol synthesis, and maintenance of oxygen radicals. Mammals with dysfunctional or nonfunctional peroxisomes suffer from significant physiological distress. Peroxisomes in plants carry out similar metabolic reactions, have comparable structures, and are generated in equivalent fashions as the onesthose found in mammals. This makes plant peroxisomes a potential model for biomedical studies. I amWe are analyzing genes encoding for proteins of unknown function in Arabidopsis thaliana. The encoded proteins are predicted, based on their primary sequences, to be targeted to peroxisomes. I have utilizedUtilizing PCR-based cloning methods, and havewe’ve successfully moved these coding sequences into various expression vectors, including In planta peroxisomal expression vectors, and are analyzing their in vivo localization in tobacco.. In addition, we’ve I have cloned thecloned the DNA sequences into protein expression vectors and am working towardsare isolating and purifying the recombinant protein for structural and functional characterization. My efforts for cloning the Arabidopsis DNA sequences into in planta expression vector are on their way with the help of the Olsen lab. The next step is to transform into Agrobacterium tumefaciens lines and then eventually into tobacco plants to enable production of YFP-recombinant proteins. I will then be able to confirm if the genes encode for peroxisomal-localized proteins using fluorescence microscopy. This work is the first stage of my senior thesis project, which will also involve genetic and biochemical characterizations of these genes and their encoded proteins. characterization. We’ve also carried out preliminary studies on the impact of T-DNA insertion in these genes on plant growth and development. This work is forming the basis of my undergraduate senior thesis project.

700-038-Z Subcellular Compartmentation of Trehalose Metabolism Implicates a Role for Retrograde Signaling in the Control of Organ Size in Plants Sonia Irigoyen – Texas A&M University Diana Sagiroi – Texas A&M University, Wayne Versaw – Texas A&M University Trehalose (Tre) is a disaccharide found in bacteria, fungi, invertebrates and plants, where it has potent but poorly understood effects on carbon metabolism, development and responses to both biotic and abiotic stress. Tre is present at very low concentrations in plants, and transgenic manipulations suggest that Tre and/or a biosynthetic precursor function as a signaling molecule. Tre biosynthesis occurs in two steps that are highly conserved in plants. First, trehalose 6-phosphate (T6P) is made from UDP-glucose and glucose 6-phosphate by the enzyme T6P synthase (TPS). T6P is then converted to Tre by T6P phosphatase (TPP). In Arabidopsis there are 11 TPS genes, 10 TPP genes, and one trehalase (TRE1) gene. We used GFP fusions to confirm that TPS isoforms are located in the cytosol. In contrast, similar experiments indicated that several of the TPP isoforms are located in plastids. Loss-of-function mutant studies revealed that plastid-localized TPP isoforms affect leaf size through changes in cell proliferation. These results suggest a novel retrograde signaling pathway that coordinates plastid functions with cell division. Efforts to identify the relevant components and mechanisms of this pathway are ongoing and include the construction and analysis of a series of transgenic plants in which the concentrations of T6P and Tre in plastids and the cytosol are manipulated separately within different organs and tissues, including the shoot apical meristem.

700-039-Z Chloroplasts Identified in the Zea Mays (Poaceae) Endothecium Katherine Murphy – Stanford University Rachel Egger – Stanford University, Virginia Walbot – Stanford University Although anthers of maize, rice, and Arabidopsis have been studied intensively using genetic and biochemical analyses in the past 20 years, few updates to the anatomical and ultrastructural description of anthers have been reported. Here we report the presence of chloroplasts in the maize anther endothecium and the expression of photosynthesis-related genes in maize anthers. In doing so, we present the first transmission electron microscopy (TEM) images of the maize anther and redefine the maize anther endothecium that is not sub-epidermal as a new cell type, the interendothecium.

700-040-Y COPI Complex Depletion in Plants Shows Its Role in Plant-specific Functions Hee-Kyung Ahn – Yonsei University Yong Won Kang – Yonsei University, Hye Min Lim – Yonsei University, Hyun-Sook Pai – Yonsei University, Inhwan Hwang – Yonsei University COPI vesicles are essential to the retrograde transport of proteins in the early secretory pathway. The COPI coatomer complex consists of seven subunits, termed α-, β-, β’-, γ-, δ-, ε-, and ζ-COP, in yeast and mammals. Plant genomes have homologs of these subunits, but the essentiality of their cellular functions have hampered the functional characterization of the subunit genes in plants. Here we have employed virus-induced gene silencing (VIGS) and dexamethasone (DEX)-inducible RNAi of the COPI subunit genes to study the in vivo functions of the COPI coatomer complex in plants. The β’-, γ-, and δ-COP subunits localized to the Golgi as GFP-fusion proteins and interacted with each other in the Golgi. Silencing of β’-, γ-, and δ-COP by VIGS resulted in growth arrest and acute plant death in Nicotiana benthamiana, with the affected leaf cells exhibiting morphological markers of programmed cell death. Depletion of the COPI subunits resulted in disruption of the Golgi structure and accumulation of autolysosome-like structures in earlier stages of gene silencing. In tobacco BY-2 cells, DEX-inducible RNAi of β’-COP caused aberrant cell plate formation during

cytokinesis. Collectively, these results suggest that COPI vesicles are essential to plant growth and survival by maintaining the Golgi apparatus and modulating cell plate formation.

700-041-Y Endoplasmic Reticulum-resident Arabidopsis Dolichol Kinase 1, AtDOK1, Is Involved in Protein Glycosylation Kazue Kanehara – Institute of Plant and Microbial Biology, Academia Sinica, Taiwan: Muroran Institute of Technology Yueh Cho – Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, Chao-Yuan Yu – Institute of Plant and Microbial Biology, Academia Sinica, Taiwan The post-translational modifications of protein are well-conserved important mechanisms in eukaryotic cells that control diverse protein functions in various cellular physiological processes. An emerging issue is understanding of functions of glycosylated proteins in plants given the huge number of physiologically important proteins are glycosylated. Dolichol phosphate (Dol-P) serves as a carrier of complex polysaccharides during protein glycosylation including N-linked glycosylation, O-linked glycosylation, and GPI anchoring. Dol-P is synthesized by the phosphorylation of dolichol or the monodephosphorylation of dolichol pyrophosphate (Dol-PP). We previously reported that At3g45040 encodes a protein homologous with Sec59p, a dolichol kinase (DOK1) in Saccharomyces cerevisiae (Kanehara et al., 2015). At3g45040, designated AtDOK1, complemented defects in the growth and N-linked glycosylation of the S. cerevisiae sec59 mutant. The heterozygous plants of two independent T-DNA insertion lines, dok1-1 and dok1-2, showed developmental defects in male and female gametophytes, including an aberrant pollen structure, low pollen viability, and short siliques. Here, we investigated the subcellular localization of AtDOK1 with use of the stable transgenic line, which harbors a plasmid containing the genomic sequence of AtDOK1 fused C-terminally to the Venus fluorescent protein (ProDOK1:DOK1-Ven). The confocal laser scanning microscopy showed that Venus fluorescence was localized to the endoplasmic reticulum (ER). This subcellular localization of AtDOK1 agrees with that of Sec59p in S. cerevisiae. Possible functions of AtDOK1 in protein glycosylation in planta will be discussed. Kanehara et al., (2015) Plant J. 81, 292-303

700-042-Z The Role of EXPO-regulated MATs Distribution in Plant Seedling Growth Yu Jin – The Chinese University of Hong Kong It is generally believed that signal peptide (SP)-dependent protein secretion is achieved via a conventional ER (endoplasmic reticulum)- Golgi- TGN (trans-Golgi network)- PM pathway in the plant endomembrane system. However, one of the exo70 members, Exo70E2, not only locates to the PM, but also presents in a recently discovered organelle EXPO (exocyst-positive-organelle). The methyl donor S-adenosylmethionine is synthesized in plants by MATs and plays an essential role in ethylene biosynthesis and methylation reactions. Our work describes Exo70E2-positive EXPO is responsible for transporting cytosolic MATs to the plasma membrane to mediate degradation of MATs. Phenotypically, the exo70e2 mutant seedlings displayed increased sensitivity to ethylene, as indicated by the shortened hypocotyls. We further prescent evidence that MATs are degraded by proteasome. Taken together, our results suggest a posttranslational regulation mechanism of MATs by which ethylene biosynthesis is affected to regulate plant seedling growth.

700-043-Z Sequence Motifs in Transit Peptides Act as Independent Functional Units and Can Be Transferred to New Sequence Contexts

Dong Wook Lee – POSTECH Seungjin Woo – POSTECH, Kyoung Rok Geem – POSTECH, Bong Soo Choi – POSTECH, Inhwan Hwang – POSTECH A large number of nuclear-encoded proteins are imported into chloroplasts after they are translated in the cytosol. Import is mediated by transit peptides (TPs) at the N-termini of these proteins. TPs contain many small motifs, each of which is critical at a specific step in the process of chloroplast protein import; however, it remains unknown how these motifs are organized to give rise to TPs with diverse sequences. In this study, we generated various hybrid TPs by swapping domains between RbcS (Rubisco small subunit) and Cab (chlorophyll a/b-binding protein), which have highly divergent sequences, and examined the abilities of the resultant TPs to deliver proteins into chloroplasts. Subsequently, we compared the functionality of sequence motifs in the hybrid TPs with those of wild-type TPs. The sequence motifs in the hybrid TPs exhibited three different modes of functionality, depending on their domain composition, as follows: active in both wild-type and hybrid TPs, active in wild-type TPs but inactive in hybrid TPs, and inactive in wild-type TPs but active in hybrid TPs. Moreover, synthetic TPs, in which only three critical motifs from RbcS or Cab TPs were incorporated into an unrelated sequence, were able to deliver clients to chloroplasts, at the comparable efficiency to RbcS TP. Based on these results, we propose that diverse sequence motifs in TPs are independent functional units that interact with specific translocon components at various steps during protein import and can be transferred to new sequence contexts.

700-044-Y Truncated and Full-length TatC (Twin-arginine Translocase C) Target to Different Membranes in Chloroplasts Stacy Anderson – University of Wisconsin-Madison Donna Fernandez – University of Wisconsin-Madison Chloroplasts are the main source of fixed carbon and energy for humans and were initially derived from the endosymbiosis of cyanobacteria. Many chloroplast membrane proteins are synthesized in the cytosol, imported into the stroma, and then post-translationally targeted to the inner envelope or thylakoids. The determinants used in this targeting process, known as conservative sorting, are largely undefined. The targeting of twin-arginine translocase subunit C (TatC) is an interesting case. The protein is targeted to the plasma membrane in bacteria, which is the topological equivalent of the inner envelope in chloroplasts. However, chloroplast TatC is only found in the thylakoids. In this study, we sought to define the region of TatC required for correct targeting. Constructs were generated encoding two, four, or six (full length) transmembrane (TM) domains fused to a fluorescent tag (GFP). These constructs were used to transfect Arabidopsis protoplasts and observed using confocal laser scanning microscopy. This experiment yielded surprising results: TatC with either two or four TM domains localized to the periphery of the chloroplasts and colocalized with inner envelope markers, while TatC with all six TM domains correctly localized to the thylakoids. This suggests that more C-terminal portions of the mature protein contribute to thylakoid targeting. Further experiments will be done to determine whether this region contains specific targeting signal(s). Supported by NSF MCB 1158173 and NSF GRFP under Grant No. DGE-1256259.

700-045-Y Functional Analysis of the Arabidopsis Thaliana GERANYLGERANYL DIPHOSPHATE SYNTHASE Gene Family Nicholas Ruppel – Randolph-Macon College Fernanda Algarin – Randolph-Macon College, Ashley Andersen – Randolph-Macon College, Diana Futrell – RandolphMacon College, Riley Martin – Randolph-Macon College, Aubrey Munoz – Randolph-Macon College, Amy Northrop – Randolph-Macon College, Courtney Stevens – Randolph-Macon College, Martika Williams – Randolph-Macon College, Darron Luesse – Southern Illinois Univeristy

Plant isoprenoids are a diverse class of natural compounds ranging from simple 5-carbon units to longer, more complex metabolites. These compounds encompass a functionally diverse family and are ultimately required for the biosynthesis of chlorophyll, carotenoids, secondary metabolites, hormones, and for the electron acceptors required for photosynthesis and respiration. The twenty-carbon isoprenoid geranylgeranyl diphosphate (GGPP) is synthesized in plastids, mitochondria, and the cytoplasm by homologous GERANYLGERANYL DIPHOSPHATE SYNTHASE (GGPS) proteins. Analysis of the Arabidopsis thaliana genome reveals the presence of ten functional GGPS homologs, and we have previously demonstrated the essential nature of GGPS1 (At4g36810) for chloroplast development and plant growth. Here, the aim of our work was two-fold: 1) to identify the functional relatedness among the plastid-localized GGPS enzymes, and 2) to search for genetic interactions with GGPS1. For the former, we utilized the unique, temperaturesensitive ggps1-1 mutant by placing selected plastid-localized GGPS gene family members behind a 35S overexpressionconstruct to test for complementation of the green/white variegated phenotype. For the latter, we have initiated a suppressor screen using ggps1-1. Our results will contribute to the understanding of regulatory controls placed on isoprenoid biosynthesis and provide a model for future gene family studies where the unique and/or overlapping biological functions are unclear.

700-046-Z Identification of the SecE2 Component of the Chloroplast Inner Envelope Sec2 Translocase Donna Fernandez – University of Wisconsin-Madison Yubing Li – University of Florida, Rajneesh Singhal – University of Wisconsin-Madison, Isaiah Taylor – University of Missouri, Patrick McMinn – University of Wisconsin-Madison, Cullen Vens – University of Wisconsin-Madison, Xien Yu Chua – University of Wisconsin-Madison, Kenneth Cline – University of Wisconsin-Madison Biogenesis of chloroplasts involves a series of protein trafficking events. Nuclear-encoded chloroplast proteins that are synthesized in the cytosol and imported into the stroma may function in other locations within the chloroplasts. Achieving the correct localization requires additional targeting information and protein trafficking, and typically involves systems that are directly homologous to bacterial systems. While the thylakoid-based systems have been studied extensively, much less is known about the systems that reside and function in the inner envelope membrane. One such system, the Sec2 translocase, is homologous to both the thylakoid-based Sec1 translocase and bacterial Sec translocases and may mediate both integration and translocation across the inner envelope. At a minimum, this system is expected to include three components, homologous to SecA, SecE, and SecY in bacteria; but only two, SCY2 and SECA2, were previously identified in Arabidopsis (Skalitzky et al. (2011) Plant Physiol. 155: 354-369). A combination of BLAST searches and protein modeling was used to identify a candidate for the missing component, which was expected to be homologous in structure and function to bacterial SecE. We have used mutant analysis, cellular localization, biochemical fractionations, and protein interaction assays in yeast to establish that this protein is an integral membrane protein of the inner envelope, interacts with the SCY2 component, and is essential for plant growth and development. We conclude that we have successfully identified the SECE2 component of the inner envelope-based Sec2 translocase. Supported by NSF MCB 1158173 (DF) and 1158110 (KC).

700-047-Z Comparative Analysis of Green and White Sectors of Variegated Epipremnum Aureum ‘Marble Queen’ Chiu-Yueh Hung – North Carolina Central University Ying-Hsuan Sun – National Chung Hsing University, Jianjun Chen – University of Florida, Farooqahmed S. Kittur –North Carolina Central University, Jie Qiu – Zhejiang University, Richard J Henny – University of Florida, Longjiang Fan – Zhejiang University, Jiahua Xie – North Carolina Central University Variegated plants ‘Marble Queen’ having both green (MG) and white (MW) sectors within the same leaf provide a

valuable system for physiological study. Apparently, the physiological statuses of green and white cells are very different. Thus when color deficient cells grow/develop side by side with normal green cells, they need to cope with the differences between each other and respond to the environmental conditions more efficiently than cells of a normal leaf. To unveil their communication and interaction mechanisms, we obtained transcriptome data of green and white sectors using NGS technology. Initial comparison results show that there are 848 differentially expressed contigs. Among them, 52.6% were more abundant in MW while 47.4% were more abundant in MG. Contigs abundant in MW were mostly stress induced or related genes which encode products including glutathione S-transferase, heat-shock protein, Rab18, Derlin-2, and temperature-induced lipocalin. Those abundant in MG were mostly involved in the photosystem I and II. Further metabolite analysis of green and white sectors including hormones, carotenoid and chlorophylls was performed. Morphologies of white cells and adjacent green cells were also examined in detail by TEM. In general, the results observed in MW are similar to that in white tissues of Arabidopsis mutant immutans (im). However, the induction of sucrose catabolism and transport observed in white tissues of im did not found in MW, suggesting potential interesting differences between these two white tissues.

700-048-Y Interaction Mapping Between Precursor and Hcf106 or Tha4 During cpTat Transport Nefertiti Muhammad – Miami University Carole Dabney-Smith – Miami University, Debjani Pal – Cleveland Clinic Chloroplast biogenesis requires the import of nuclear-encoded, cytoplasmically synthesized proteins and subsequent localization to its sub-organellar site of action. Roughly half of the proteins found in the thylakoid lumen are transported from the stroma by the chloroplast twin arginine translocation (cpTat) system. The cpTat system is unique in that it is powered by the proton motive force and can transport fully folded substrates without disrupting the membrane. The cpTat system is composed of three membrane proteins, Tha4, Hcf106 and cpTatC. Tha4 oilgomerizes and is predicted to form the point of precursor passage in the membrane. However, Hcf106 likely plays a role as well, potentially as a nucleator for Tha4. How and where the precursor to be translocated engages either Tha4 or Hcf106 has recently been explored. Certain contacts between the N termini of Tha4 and Hcf106 have been detected, but what is unknown is which side of the membrane the substrate (precursor) is on during these interactions with the translocon pore. Here, we studied crosslinking between Hcf106 and the 17 kDa subunit of the oxygen-evolving complex of photosystem II (OE17) to map close proximity interactions under transport conditions. Cysteine mutants in the mature domain of OE17 various regions of Hcf106 were constructed. External protease was used to identify the location of the OE17 complex in either the stroma or thylakoid lumen. The results showed crosslinking interactions between Hcf106 Cys variants and three of the OE17 Cys variants. The protease treatment showed that precursor was interacting with the translocon pore on the stromal side of the membrane, likely immediately prior to transport. Current interaction mapping and previous data indicate that three of the helices in OE17 are oriented towards Hcf106 and the fourth has interactions with Tha4. How this fits into current models will be discussed.

700-049-Y Characterization of Thylakoid Membrane Protein Hcf106 in Arabidopsis Qianqian Ma – Miami University Kirsten Gonzales – Miami University, Carole Dabney-Smith – Miami University To generate primary metabolites through photosynthesis, chloroplasts require thousands of proteins, many assembling into multi-subunit complexes that carry out the core reactions of photosynthesis. To complicate matters, only ~150 proteins are encoded by the plastid genome, while the rest are encoded by the nuclear genome and are synthesized in the cytosol as higher molecular weight precursor proteins contiaining N-terminal “transit peptides” that ensure proper

targeting to the chloroplast. Proteins functioning in the thylakoid or thylakoid lumen contain a bipartite targeting signal with an additional thylakoid targeting signal peptide to direct them into or across the thylakoid membrane. The correct localization of proteins destined to the thylakoid lumen, is critical for the proper functioning of chloroplasts. Two protein translocons located in thylakoid membrane are responsible for the transport of thylakoid lumen precursor proteins. One of these, the cpTat (chloroplast Twin Arginine Translocation) pathway transports about half of thylakoid lumen proteins through the coordinated function of three membrane protein components: Tha4, Hcf106 and cpTatC. Lack of cpTat components could result in inactivation of the cpTat system, disrupting the correct localization of about half of the thylakoid lumen proteins and thus affecting thylakoid biogenesis. Here, a previously unstudied SALK line T-DNA insertional mutant of one cpTat component, Hcf106 (high chlorophyll fluorescence106), in Arabidopsis was characterized by phenotypic and genotypic analyses. We identified one T-DNA insertion line with pale green leaves at the very early stage of development. Interestingly, reduced chlorophyll fluorescence parameter (Fv/Fm) of this line was observed, indicating the mutant plants are under stress due to an altered Hcf106 expression level, in direct contrast to the high chlorophyll fluorescence seen in the maize mutant, which gives the gene its name. The chloroplasts of this mutant line will be observed by electron microscopy for the potential defective thylakoid.

700-050-Z Investigating the Role of the Arabidopsis Cohesin Protein SYN3 in Chloroplasts Ramja Sritharan – Miami University Kendyl Kennon – Miami University, Christopher Makaroff – Miami University, Carole Dabney Smith – Miami University Chromosome cohesion plays a central role in chromosome segregation during mitosis and meiosis. Cohesin complexes contain four essential proteins: SCC3, two long coiled-coil proteins SMC1&3 and an α-kleisin. Arabidopsis has four αkleisin proteins: SYN1, SYN2, SYN3 and SYN4. SYN1 functions in meiosis, whereas SYN2 and SYN4 are essential for mitosis. SYN3 is a large protein (692 AA) where its N and C-terminus bind to SMC1 and SMC3 respectively. SYN3 is essential for megagametogenesis and is enriched in the nucleolus of mitotic and meiotic cells. Previous work clearly established the role of α-kleisins, including SYN3, in the nucleus; however, α-kleisins have not been reported to be localized to organelles. However, we recently identified chloroplast stroma proteins essential for chloroplast development and division, Ftz1-1 and 2-1, as SYN3 interacting proteins through a yeast two-hybrid screen and confirmed the plastid location by immunodetection of SYN3 in isolated and fractionated chloroplasts. Co-IP techniques with SYN3YFP or SYN3-MYC transgenic plants have been used to isolate SYN3 interacting proteins such as FtsZ or other potential interacting partner proteins, thus confirming in vivo the results of the yeast two-hybrid studies. The focus of this project is to unravel the potential roles of SYN3 in chloroplasts. Here we demonstrate import of recombinant SYN3 into intact chloroplasts and localization to the thylakoid.Syn3 import into intact chloroplasts results in the presence of a lower molecular weight protein insensitive to externally added protease, indicating transport. Future works will be done to determine if SYN3 interacts with chloroplast DNA.

700-051-Z Targeting of an Essential β-Barrel Protein to the Chloroplast Outer Membrane Philip Day – University of California at Davis Kentaro Inoue – University of California at Davis During the evolution of chloroplasts, most of the genes from the cyanobacterial endosymbiont migrated to the host nucleus. This process required the evolution of a protein import apparatus at the double-membrane envelope of the endosymbiont. Toc75 is a component of this apparatus that forms the import channel across the outer membrane (OM). Toc75 evolved from an ancestral cyanobacterial protein and coexists with its paralog, OEP80. OEP80’s function is unknown, but it is essential for plant viability and is hypothesized to retain the ancestral function. We are interested in

comparing the targeting of OEP80 and Toc75 to gain insights into the factors leading to their divergence in function. Toc75 is targeted to the chloroplast OM by a bipartite targeting signal that contains a chloroplast import signal and a polyglycine stretch. The polyglycine is required for envelope sorting and is conserved in Toc75 orthologs throughout the Viridiplantae. OEP80 orthologs lack the polyglycine, suggesting its OM sorting was distinct from Toc75 early in chloroplast evolution. Our results using import of radiolabeled OEP80 into isolated chloroplasts suggest that OEP80 has an N-terminal extension that is removed upon import. Interestingly, processed OEP80 is distributed to both the soluble and membrane fractions of the chloroplasts. These data suggest that OEP80 uses a transit peptide to enter the chloroplast where it exists as a soluble intermediate before assembly into the OM. We are currently using in vitro import and various other biochemical assays to i) characterize the size and function of the N-terminal extension of OEP80 and ii) define the mechanism by which OEP80 is sorted to the OM. The outcome of these experiments will help us understand the divergence and early evolution of OEP80 and Toc75. Funding is provided by the NSF Molecular and Cellular Biosciences Program and the UCDavis Department of Plant Sciences.

700-052-Y Sorting of Sec Translocase Component, Scy, to Different Membranes in Chloroplasts Rajneesh Singhal – University of Wisconsin-Madison Donna Fernandez – University of Wisconsin-Madison Most of the proteins that function in chloroplasts are encoded by nuclear genes. Precursor proteins are synthesized in the cytosol and are post-translationally imported into chloroplasts by Toc and Tic translocons in the envelope membranes. Within the chloroplasts, further sorting of imported proteins depends on systems that are homologous to bacterial systems, such as the Sec translocase/integrase system. Chloroplasts contain two distinct Sec systems: the Sec1 system in the thylakoids, and the Sec2 system in the inner envelope. The differential localization of these two systems is likely to contribute in a pivotal way to membrane differentiation. To understand how this differential localization is achieved, we have been studying the targeting of SCY1 and SCY2, the major channel-forming proteins in Sec1 and Sec2 respectively. Both SCY1 and SCY2 are similar in structure to bacterial SecY proteins, which contain ten transmembrane segments, and have conserved amino acids at key functional sites. To assay targeting, we transiently expressed constructs encoding GFP fusions with different portions of the proteins in Arabidopsis leaf protoplasts and visualized the fluorescent proteins by confocal microscopy. We find that either full-length or truncated proteins that include the Nterminal region plus four transmembrane domains are faithfully targeted to the appropriate membrane. SCY1-GFP colocalizes with chlorophyll fluorescence (thylakoids), while SCY2-GFP co-localizes with an inner envelope marker. SCY2GFP is sorted equally well under light or dark conditions, but SCY1-GFP requires light. Under dark conditions, SCY1-GFP piles up in the stroma. When regions of the two proteins were swapped, proteins with only the N-terminal region of SCY1 or only the transmembrane domains of SCY1 were directed to the thylakoids in a light-dependent manner. This suggests that thylakoid targeting is dominant over envelope targeting. We plan further studies to pinpoint the important sequences and reveal a possible targeting mechanism. Supported by NSF MCB 1158173.

700-053-Y Enhancer Screen to Identify IBA Response Mutants Reveals Interaction Between Peroxisome Metabolic Pathways Vanessica Jawahir – University of Missouri-St. Louis Bethany Zolman – University of Missouri St. Louis Plant peroxisomes mediate numerous processes in primary and secondary metabolism crucial for growth and development. Peroxisome functions include inactivation of hydrogen peroxide, fatty acid β-oxidation, the glyoxylate cycle, photorespiration, and production of the phytohormones indole-3-acetic acid (IAA) and jasmonic acid (JA). Indole-

3-butyric acid (IBA) is metabolized into free IAA in a stepwise fashion similar to fatty acid β-oxidation. A screen identified IBA response mutants (ibr) defective in IBA to IAA conversion, including peroxisomal biogenesis and import proteins. ibr3-1 and ibr1-1 were mutagenized and screened for enhanced IBA resistance in hypocotyls. This enhancer screen seeks to identify and define additional factors involved in the function of IBR3 and IBR1, the conversion of IBA to IAA, and general peroxisome function in Arabidopsis thaliana. Here we describe the initial phenotypic characterization of our mutants. The Z377ibr3-1 mutant has IBA-response phenotypes but germinates normally and is sucrose independent, suggesting specificity to IBA metabolism. Causative mutations are being identified through whole genome sequencing complemented with rough mapping data. The enhancing mutation in Z353ibr3-1 was identified in ACX3, a fatty acid βoxidation enzyme. ACX3 and IBR3 are hypothesized to function in the same step of their respective pathways. It is unknown if these enzymes are catalytically redundant or both rely on rate limiting cofactors. Analysis of Z353ibr3 will provide insight into the extent of interaction between these two pathways and the metabolic necessities of peroxisomes.

700-054-Z Characterization of IPI1, a Novel Pentatricopeptide Repeat Protein That Is Essential for Arabidopsis Embryogenesis Tyra McCray – University of Tennessee Rachel Williams, Sarah Hardin, Krzysztof Bobik, Tessa Burch-Smith – University of Tennessee ISE2 PROTEIN INTERACTOR 1 (IPI1) is a nuclear gene encoding a DYW (Asp-Tyr-Trp) pentatricopeptide repeat (PPR) protein required for Arabidopsis embryogenesis. IPI1 was identified in our lab in a protein interaction screen with the chloroplast-localized DEVH RNA helicase INCREASED SIZE EXCLUSION LIMIT 2 (ISE2). Both ISE2 and IPI1 are embryo lethal, and the silencing of IPI1 or ISE2 produces a chlorotic phenotype in mature leaf tissue. Confocal microscopy illustrates that IPI1-YFP, similar to ISE2, localizes to chloroplasts, consistent with its predicted chloroplast N-terminal targeting sequence. Additionally, gene expression data demonstrate that IPI1’s expression is not confined to the embryo, but additionally expressed in mature organs such as leaf and root. Current experiments are aimed towards further characterizing the putative interaction between ISE2 and IPI1 and examining whether IPI1 plays a similar role as ISE2 in RNA metabolism and intercellular trafficking.

700-055-Z Dynamics of Golgi Apparatus Under Sucrose Starvation Moses Abiodun – Kyushu University Ken Matsuoka – Kyushu University A lot of work has been done on the dynamics of Golgi apparatus during starvation but little attention has been paid to the synthesis and/or degradation of the organelle under sucrose starvation. Here, we simultaneously monitored the increase and decrease of both the newly synthesized and pre-existing cis-Golgi stacks under induced sucrose starvation using a photoconvertible fluorescent protein (PFP), monomeric Kikume green red (mKikGR). A type II cis-Golgi localizing prolyl-4-hydroxylase protein was tagged with the PFP and expressed in tobacco bright yellow (BY-2) cells. Transformed cells were exposed to purple light to convert the fluorescence from green to red. A time series analysis of sucrose starved photoconverted logarithmic cells revealed a reduced but continuous synthesis of hydroxylase even at extended time of 48 hours. The degradation rate of the pre-existing Golgi stacks was slower compared to the control. A reduced synthesis of the hydroxylase affected the progression of cell cycle by reducing the cell volume. When the 48 h-starved cells were transferred into normal medium, active synthesis resumed but at a slower rate. The new hydroxylase synthesized during starvation is being analayzed for DNA size and function.

DEVELOPMENT - Zone 800 Development: General 800-001-Y Analysis of the YDA MAPKK Kinase Gene Function Potentially Involved in Phloem Development Angelica Concepcion Martinez-Navarro – CINVESTAV-IPN Santiago Valentin Galván-Gordillo – CINVESTAV-IPN, Byung-Kook Ham – UC-DAVIS, Jorge Luis Ruiz-Salas – CINVESTAVIPN, William J. Lucas – UC-DAVIS, Beatriz Xoconostle-Cázares – CINVESTAV-IPN, Roberto Ruiz-Medrano – CINVESTAV-IPN YDA is a MAPKKK that regulates asymmetry in zygotes and stomata, as well as pedicel length and inflorescence architecture. YDA loss of function suppresses the asymmetric divisions of the zygote, whereas in gain of function results in excessive elongation of the embryo making it difficult for functional studies in post-embryonic stages. On the other hand, during the formation of guard cells, the MAPK pathway negatively regulates asymmetric cell division, avoiding the excessive formation of these cells. This process requires the interaction of YDA with MAPK3/6 and MKK4/5, which are MAP kinases downstream of YDA, and BIN2, which is a brassinosteroid-responsive protein that regulates YDA activity. Analysis of the YDA promoter showed its expression in phloem companion cells, suggesting a possible role for this protein in vascular tissue differentiation. Since some elements of the stomatal MAPK pathway are also involved in development of other tissues, such as pedicels and the zygote, it is possible that during evolution they were recruited for vascular tissue differentiation. We are currently analyzing the role of YDA in vascular tissue differentiation. YDA alleles (yda-1, emb71) display an alteration in vascular bundle shape and, in addition, a reduced number of cambium and phloem cells were observed in inflorescence stems. YDA RNAi knockdown plants exhibited a similar phenotype as reported in the yda alleles, and they had a delay in development. Hormone assays revealed that YDA expression is induced by cytokinins, auxins and abscisic acid.

800-002-Z Insights into the Molecular Basis of the Sunflower Heliotropic Response Through High Resolution RNA-Seq Analysis Hagop Atamian – UC Davis Upendra Devisetty – UC Davis, Stacey Harmer – UC Davis Sunflower’s dynamic motion in response to the trajectory of the sun, named heliotropism, involves both the continual orientation of the apex towards the sun throughout the day and the gradual re-orientation of the apex from west to east at night in anticipation of sunrise. Interestingly, heliotropism in sunflower ceases at anthesis stage of development with flower heads facing east. Previously we demonstrated that the sunflower heliotropic response is mediated by oppositely phased rhythms of growth on each side of the stem and is regulated by the circadian clock. In addition, we provided evidence that the east facing of mature heads is caused by circadian clock gating of light responses and showed that this eastward orientation significantly increases pollinator visits. Now, we have carried out high resolution RNA-Seq analysis on the opposite sides of sunflower stems to better understand the mechanism(s) of sunflower heliotropism at the molecular level. Unlike solar tracking in legumes, which is mediated by pulvini via turgor pressure-based changes in cell shape, not much is known about the mechanism(s) of sunflower heliotropism. Our preliminary analysis found homologs of genes known to be involved in auxin signaling and hypocotyl elongation as well as those encoding cell wall associated proteins to be differentially expressed on opposite sides of the stem at different times of the day. Currently we are in the process of assembling a comprehensive list of genes differentially expressed on opposite sides of the sunflower stem and constructing pathways explaining the heliotropic response.

800-003-Z Control of Vein Formation by an Auxin Signaling Threshold Jason Gardiner – University of Alberta Tyler Donner – University of Alberta, Enrico Scarpella – University of Alberta In both plants and animals, the formation of the different cell types that make up a mature organ or organism is the result of differential expression of genes during organ or embryo development. How genes are differentially expressed during development is thus a fundamental question, the understanding of which is simplified in plants, as—unlike animals—they lack cell movements. In particular, developing leaves are easily accessible and amenable to manipulation, thus offering a convenient system to understand how genes are differentially expressed during plant development. At early stages of leaf development, cells are indistinguishable from one another; however, some of these cells will differentiate into mesophyll cells, while others will differentiate into vein cells. Before any change in shape or size occurs, files of cells that will differentiate into vein cells activate expression of the ARABIDOPSIS THALIANA HOMEOBOX8 (ATHB8) gene. Activation of ATHB8 expression depends on binding of the auxin-signaling transcription factor MONOPTEROS (MP) to an MP-binding site in the ATHB8 promoter. However, ATHB8 is expressed in narrow domains, while MP is expressed ubiquitously. Why not all cells that express MP also express ATHB8 is unknown. We will present and discuss evidence suggesting that ATHB8 expression is restricted to narrow domains by binding of peak levels of ubiquitously expressed MP to a low-affinity MP-binding site in the ATHB8 promoter.

800-004-Y A Mutant with Reduced Rhamnose Synthesis Implicates Rhamnogalacturonan-I in the Control of Cell Expansion and Helical Twisting of Epidermal Cells Adam Saffer – Yale University Vivian Irish – Yale University Cell walls control the shape of plant cells and consist primarily of polysaccharides including cellulose, hemicelluloses, and pectins, but it is unclear how each polysaccharide contributes to the biophysical properties and function of the cell wall. We have isolated an Arabidopsis mutant named dairy queen (dq) that results in reduced cell expansion in conical petal epidermal cells and a left-handed helical twist in both individual cells and whole petals. dq is an allele of RHM1, which encodes an enzyme that synthesizes UDP-L-rhamnose. We have shown that dq substantially reduces levels of the pectic polysaccharide rhamnogalacturonan-I (RG-I) in petals and affects the response of petal development to osmotic stress, suggesting that dq alters the mechanical properties of petal cell walls. Like previously studied rhm1 mutants, dq has abnormal cotyledon pavement cell morphology. Although it has been suggested that the phenotypic effects of rhm1 mutants are caused by accumulation of non-rhamnosylated flavonols, we will present data showing that rhm1 mutant effects on cell morphology are flavonol independent. Rather, flavonols represent an abundant pool of rhamnose and eliminating flavonols rescues rhm1 defects by freeing that rhamnose for other pathways. Our data implicate a novel role for RG-I in the control of cell expansion and morphology in multiple organs. Furthermore, nearly all mutants with left- or right-handed spiral phenotypes affect microtubules and/or cellulose. The left-handed twisting in dq petals raises the intriguing possibility that RG-I restricts the influence of cellulose orientation on cell shape to prevent twisting of plant cells, and we will present ongoing investigations into how RG-I and cellulose interact to control cell morphology.

800-005-Y Constitutive Expression of MiR396 Alters Plant Development and Overcomes Vernalization Requirement for Flowering in Transgenic Creeping Bentgrass Shuangrong Yuan – Clemson University

Zhigang Li – Clemson University, Qian Hu – Clemson University, Hong Luo – Clemson University MicroRNA396 (MiR396) is a conserved microRNA family targeting growth-regulating factors (GRFs). It has been suggested to regulate plant leaf and flower development in annual species. However, the underlying molecular mechanisms of miR396-regulated floral organ development remain elusive. In order to investigate if it has the conserved function in perennial species and to analyze its molecular mechanisms, we generated a miR396 overexpression construct and introduced it into an economically important perennial crop, creeping bentgrass. Transgenic plants display reduced leaf length and width, shortened internodes, less tillers, reduced biomass, and became more creeping in comparison with wild type (WT) controls. In long-day conditions, transgenics flower in four weeks without vernalization, whereas WT controls require 15-week cold treatment. Transgenic spikelet is smaller and has less floralets than WT control. In addition, transgenics show anther dehiscence defects and pollen sterility. During the cold treatment, transgenics exhibit greener and less wilted leaves than WT plants. Four genes from GRF family have been identified as targets of miR396. The expression patterns of the closest orthologs of VRN1, VRN2 and FT are characterized in WT and transgenic plants during short-day, long-day, and cold exposure. In comparison with WT controls, VRN1 in transgenics is induced dramatically during long-day without cold exposure; VRN2 in transgenics is depressed during short-day, longday and cold exposure. Currently, miR396-affected pathways of vernalization, floral organ development, and male sterility are under further investigation via RNA-seq analyses. Our data indicate that miR396 is implicated in multiple plant physiological processes, suggesting its potential in developing new molecular strategies for enhanced crop yields via regulating flowering time.

800-006-Z Arp1, a Novel Protein Involved in Auxin- and Light- regulated Growth and Vascular Development Jie Li – Michigan State University Susanne Hoffmann-Benning – Michigan State University Arp1 a novel protein of unknown function with a DUF538 domain was first identified from rapidly-growing corn coleoptiles (Li et al., 2013). We had shown that Arp1 expression is closely associated with cell and organ expansion growth in above ground tissues. Upon auxin-treatment Arp1 mRNA and protein increase within 30 minutes – prior to measurable growth and to the expression of genes encoding cell wall biosynthetic enzymes, suggesting a possible intracellular signaling function. This is further supported by the finding that transiently-expressed Arp1 is localized in the periphery of the cell as well as in the nucleus. Arabidopsis plants lacking the Arp1 homologue will grow normally under standard conditions, but show delayed growth, and delayed flowering, when germinated in the dark and transferred to light. This growth retardation can be reversed by complementing the Arabidopsis mutant with the corn gene. Overexpression of maize Arp1 in Arabidopsis causes longer hypocotyl length. These findings are consistent with our observation that gene expression is also controlled by light. Using Z.m.Arp1 as bait, we identified several putative interacting proteins in corn, including a homologue of VH1/BRL2, a protein which plays a role in early vascular development. Yeast two hybrid analysis confirmed that Z.m. Arp1 interacts with the cytoplasmic (kinase) domain of Arabidopsis VH1/BRL2. Preliminary data suggest that Z.m.Arp1 expression is also associated with the vascular bundles. Our findings suggest that Arp1 may have a regulatory function in the initial steps of light- and auxin-induced seedling growth, possibly acting in vascular bundle development. We thank undergraduate students Paula Boakye and Zach Johnson for their support during PCR analysis.

800-007-Z The Membrane-structuring Protein TETRASPANIN6 Controls Abscisic Acid (ABA) Responses During Germination in Arabidopsis Marisa Otegui – University of Wisconsin-Madison Xiuling Wang – State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Hannetz Roschzttardtz – University of Wisconsin-Madison, Julio Paez-Valencia – University of Wisconsin-Madison, Kaija Goodman – University of Wisconsin-Madison, Dana Martinez – University of La Plata, Rafael Buono – University of WisconsinMadison Tetraspanins (TETs) are evolutionary-conserved integral proteins that facilitate the assembly of protein networks in membranes. TETs interact with each other generating large networks called ‘TET webs’ that bring together receptors and other structural and signaling components to specific sites of membranes, establishing signaling modules. In Arabidopsis, there are 17 TET proteins that localize to the plasma membrane, plasmodesmata, and endoplasmic reticulum (Boavida et al. 2013. Plant Physiol. 163:696; Fernandez-Calvino et al. 2011. PLoS ONE 6:e18880). Within the Arabidopsis TET family, only TET1 has been functionally characterized; it acts as a general regulator of development and tissue patterning (Cnops et al. 2006. Plant Cell 18:852). We have now found that TET6 controls seed dormancy and ABA responses during germination. The tet6 mutant seeds age prematurely and seedlings germinated from 10-month-old tet6 seeds show sectors of dead tissues or whole dead organs. The tet6 seeds are hyposensitive to exogenous ABA during germination when compared to WT seeds. TET6 interacts with the endoplasmic reticulum-localized FACE2/RCE1 endoprotease involved in the processing of isoprenylated CaaX-box-containing proteins. Protein prenylation acts as a negative regulator of ABA signaling, establishing a functional connection between TET6-FACE2 and ABA responses. Based on the expression and subcellular localization patterns of FACE2 and TET6, ABA-dependent responses in seeds overexpressing FACE2, and the analysis of single face2 and higher order mutants, we conclude that TET6 and FACE2 are part of the same functional protein network and that TET6 acts as a direct negative regulator of FACE2, and indirectly, as a positive regulator of ABA signaling.

800-008-Y The Role of Interacting Transcription Factors in Control of Plant Stem Cell Niches Yun Zhou – California Institute of Technology Eric Engstrom, Zachary Nimchuk, Jose Pruneda-Paz, Paul Tarr, Steve Kay, Elliot Meyerowitz – California Institute of Technology Within shoot apical meristems (SAMs) of Arabidopsis thaliana, a stable pool of stem cells is persistently maintained through a balance between cell division and continuous transition into differentiation states. The molecular network and the intra-/inter- cellular signaling pathways underlying the control of stem cell identity are not, however, fully understood. Using a functional genomic approach, we identified HAIRY MERISTEM (HAM) family transcription factors as working partners of WUSCHEL (WUS), and demonstrated their physical interactions through multiple independent approaches. Molecular genetic analyses showed that WUS and HAM interdependently function in the same pathway in shoot meristem maintenance. HAM proteins regulate expression of genes reported to be directly regulated by WUS, and WUS-HAM interaction is important for transcription activities of their target genes. Moreover, HAM proteins associate in vivo with the target gene promoters, genomic regions similar to those reported to associate with the WUS protein in vivo. Double-labeled live imaging showed that WUS, HAM1, and HAM2 are co-expressed in the same cells in the SAMs, and they are regulated by CLV signaling in a similar manner. In addition, we have shown that the HAM family proteins are conserved interacting co-factors with not only WUS, but also with other WOX proteins, in control of both

root meristems and vascular stem cells. In summary, our current work has uncovered a novel and conserved molecular link between two transcription factor families, which is necessary for plant stem cell activities.

800-009-Y Using Natural Variation and Forward Genetics to Extend Genetic Networks Controlling Maize Inflorescence Architecture Dave Jackson Hannes Claeys – Cold Spring Harbor Lab, Byoung Il Je – Cold Spring Harbor Lab, Brian Dilkes – Purdue University , Andrea Eveland – Danforth Center, Hajime Sakai – 4DuPont Pioneer, Agricultural Biotechnology Major changes in inflorescence architecture have been responsible for the development of agricultural crops. In cereal crops, for example, a reduction in branching and an increase in seeds per inflorescence have greatly enhanced yields. In recent years, several factors controlling maize inflorescence architecture were discovered, such as RAMOSA (RA) genes, which inhibit branching, and FASCIATED EAR (FEA) genes, which control meristem size and kernel row number. In order to better understand how these genes function and to uncover novel regulators, we used EMS mutagenesis to find enhancer mutations of the classical branching mutant ra3, which encodes a trehalose phosphate phosphatase (TPP), and leveraged the power of natural variation to identify modifiers of ra3 and fea2. We have used Next Gen sequencing to identify an EMS-induced ra3 enhancer, and are currently studying the impact of this mutation. In particular, it could help us to distinguish between the 2 hypothetical modes of RA3 action, either as a sugar metabolic enzyme, or as a regulatory protein. In a parallel approach, ra3 and fea2 mutants were crossed to the diverse maize nested association mapping (NAM) inbred lines, and we identified accessions that greatly enhance the severity of each mutant phenotype. A major fea2 enhancer locus from NC350 was mapped using both bulked segregant analysis and screening of F2 populations made using the NAM RILs, and this locus overlaps with a known kernel row number QTL. Fine-mapping is currently underway. Similarly, the ra3 phenotype is enhanced in the Ki11 background, and we are also pursuing its mapping. Combining these approaches, we aim to extend our knowledge of the genetic mechanisms that control inflorescence architecture. Considering the importance of cereals for food and feed production, modulation of these genes holds great agronomic potential.

800-010-Z The SCAR/WAVE Complex Polarizes PAN Receptors and Promotes Division Asymmetry in Maize Laurie Smith – University of California San Diego Michelle Facette – University of California San Diego, Yerk Park – University of California San Diego, Dena Sutimantanapi – University of California San Diego, Heather Cartwright – Carnegie Inst. of Washington Dept. of Plant Biology, Bing Yang – University of California San Diego, Anding Luo – University of Wyoming, Eric Bennet – University of California San Diego, Anne Sylvester – University of Wyoming Specialized cell types and new cell lineages in plants are produced via asymmetric cell division. In maize, stomatal complexes consist of two guard cells each flanked by a subsidiary cell. Subsidiary cells arise via asymmetric divisions of subsidiary mother cells (SMCs), which polarize toward adjacent guard mother cells (GMCs). Previous work showed that two receptor-like kinases (PAN2 and PAN1) and the small GTPase ROP promote mother cell polarity and subsequent division asymmetry in SMCs. PAN proteins become polarized prior to asymmetric cell division, followed by formation of a dense actin patch, nuclear migration, and eventual asymmetric cell division. Loss of function mutations in pan1 and pan2 genes result in aberrant subsidiary cells, due to a failure in SMCs polarization. Mutations in brk1 and brk3 cause similar subsidiary cell formation defects, and thus we set out to establish the role of BRK proteins in SMC polarization. BRK1 and BRK3 are components of the SCAR/WAVE regulatory complex (WRC), which activates the actin-nucleating

ARP2/3 complex. Given the known interactions between BRK, SCAR, actin and ROPs in other organisms, we hypothesized that PAN1-dependent recruitment of ROPs would in turn activate the WRC (BRKs), resulting in actin patch formation. Contrary to predictions of this hypothesis, BRK1 localizes within SMCs at GMC contact sites earlier than PAN1 and PAN2 and does not depend on PANs for its polarized accumulation. Furthermore, polar localization of PAN1 and PAN2 fails in brk1 and brk3 mutants, demonstrating that polarization of PAN LRR-RLKs requires the SCAR complex. These findings demonstrate that SCAR complex subunits function upstream of PANs in SMC polarization and establish the SCAR complex as the earliest acting component of the SMC polarity pathway.

800-011-Z Cotton Architecture in the Balance: the Gossypium Hirsutum SINGLE FLOWER TRUSS and SELF-PRUNING Orthologs Regulate Branching Patterns Roisin McGarry – University of North Texas Yuval Eshed – The Weizmann Institute of Science, Eliezer Lifschitz – Technion - Israel Institute of Technology, Brian Ayre – University of North Texas Plant architecture and the timing and distribution of reproductive structures are fundamental agronomic traits shaped by patterns of determinate and indeterminate growth. Florigen, encoded by SINGLE FLOWER TRUSS (SFT) in tomato and FLOWERING LOCUS T (FT) in Arabidopsis, advances determinate growth while its closely-related antagonist, SELFPRUNING (SP) in tomato and TERMINAL FLOWER 1 in Arabidopsis, maintains indeterminate growth. The ratio of SFT to SP, and their functional homologs in other flowering plants, is proposed to control the patterns of determinate and indeterminate growth and thus plant architecture. Consistent with this, determinate growth habits of several domesticated crops is shown to result from artificial selection at SFT and SP loci in wild progenitors. Domestication of upland cotton (Gossypium hirsutum) converted it from a lanky photoperiodic perennial to a compact day-neutral plant that is managed as an annual row-crop. Despite this domestication, crop management is complicated because cotton maintains robust indeterminate (~vegetative) and asynchronous determinate (~flowering and fruit set) growth throughout development. We demonstrate using transient, virus-based expression systems that G. hirsutum orthologs of SFT and SP profoundly impact cotton architecture. GhSFT encodes the florigenic signal stimulating determinate growth and sympodial branching in both wild photoperiodic and modern day-neutral accessions, but does not influence monopodial growth. GhSP is an essential repressor of determinate growth, and in its absence both monopodial and sympodial meristems immediately terminate with determinate floral structures. Together, GhSFT and GhSP control monopodial and sympodial branching patterns in cotton, and our findings support and extend the SFT/SP balance model postulated in tomato. Our results suggest that selection at the GhSFT and GhSP loci may have influenced domestication of this important crop, and the balance between the activities of these gene products remains an ideal target for continued optimization of cotton agriculture.

800-012-Y A Tale of Two Viruses: Comparing Cotton Leaf Crumple Virus and Tobacco Rattle Virus as Tools to Transiently Manipulate Gene Expression in Cotton Samantha Culpepper – University of North Texas Brian Ayre – University of North Texas, Roisin McGarry – University of North Texas Cotton (Gossypium spp) is the world’s most important fiber crop but research is hampered because it is recalcitrant to stable transformation. Virus-based technologies for transiently manipulating gene expression, by overexpression or by gene silencing, are productive alternatives. We compared the geminivirus Cotton leaf crumple virus (CLCrV) and the RNA virus Tobacco rattle virus (TRV) for manipulating target gene expression in domesticated and wild cotton accessions. TRV effectively and quickly silenced the gene encoding the magnesium chelatase (MgChl) subunit, with all

new growth completely bleached within 5 days of inoculation. Silencing was sustained for one month and the lack of photoautotrophic tissue severely hampered growth. After this time, new tissues were fully green and growth resumed. Silencing MgChl with CLCrV, on the other hand, resulted in chlorotic patches ~12 days post-inoculation, and this spotty silencing continued in all new growth for the life of the plant. When FLOWERING LOCUS T (AtFT) cDNA was delivered with CLCrV to wild photoperiodic cotton, all infected plants flowered early under non-inductive conditions and domesticated day-neutral cotton had more determinate and compressed growth. These phenotypes were not observed among TRV-AtFT-infected plants. RT-qPCR is being used to correlate these phenotypes with MgChl and AtFT expression, and virus titer. These data argue that TRV provides strong but short-lived silencing and is ineffective for gene delivery while CLCrV provides weaker but longer-lived silencing and is an effective tool for gene delivery. These findings are valuable to the cotton community as we continue to investigate the function of target genes to improve crop development.

800-013-Y The Role of Auxin and the Auxin Response Network in Tomato Compound Leaf Development Naomi Ori – Hebrew University Hadas Ben Gera – Hebrew University, Ido Shwartz – Hebrew University Tomato leaves are compound with multiple leaflets, each resembling a simple leaf. Recently, juxtaposition of regions with high auxin response and regions with low auxin response has been shown to be essential for leaflet initiation and separation. An auxin response sensor marks the sites of leaflet initiation in the tomato leaf margin and is absent from adjacent regions. Mutations in the tomato Aux/IAA gene ENTIRE (E) lead to leaf simplification due to expanded auxin response into the entire leaf margin. Downregulation of a family of ARF genes, including ARF10, by overexpression of their negative regulator miR160, also leads to expansion of the auxin signal and leaf simplification. Conversely, overexpression of an auxin resistant form of E, or a miR160 resistant form of ARF10, results in the formation of very narrow leaflets and in the case of ARF10 also to increased leaf complexity. Genetic analysis indicates that E and ARF10 depend on each other for their function in the inhibition of lamina growth and the formation of separate leaflets. Genetic interactions with plants with altered activity of the NAM/CUC gene GOBLET (GOB) suggest that GOB acts downstream of ARF10 and in parallel with E in leaflet specification and separation. To identify mediators of auxin function in leaf and fruit development, we generated a mutant population in the background of the e mutant and screened it separately for suppressors of the e phenotype. This screen identified the t282 mutant, which suppresses the e leaf phenotype. T282 encodes a homolog of the Arabidopsis VRN5 gene, involved in the repression of FLC during the vernalization process. t282 enhances phenotypes of mutants with decreased auxin response, and its phenotype is suppressed by downregulation of GOB. These results reveal a complex network mediating the auxin effect on leaf shape.

800-014-Z CLAUSA Restricts Tomato Leaf Morphogenesis and Negatively Regulates GOBLET Maya Bar – Hebrew University Ori Ben-Herzel – Hebrew University, Hagay Kohay – Hebrew University, Ilana Shtein – Hebrew University of Jerusalem, Naomi Ori – Hebrew University Leaf morphogenesis and differentiation are highly flexible processes. Compound leaves result from a developmental program that has extended morphogenesis as compared with simple leaves. The tomato mutant clausa (clau) possesses an extremely elaborate compound leaf. Here we show that this elaboration is generated by extension of the morphogenetic window, partly via the activity of ectopic meristems present upon the clau leaf. Further, we propose CLAU as a potential negative regulator of the NAM/CUC gene GOBLET (GOB), an important modulator of compound leaf development. GOB expression is upregulated in clau mutants, and its downregulation suppresses the clau phenotype.

GOB expression is also upregulated in the compound leaf mutant lyrate (lyr), and synergistic genetic interaction between clau and lyr suggests that they regulate leaf development and GOB in different pathways. Combined mapbased cloning and RNA sequencing identified the CLA gene as encoding a transcription factor from the MYB family. CLA is expressed in initiating leaflets at the tomato leaf margin. Together, these results uncover a unique capacity for prolonged morphogenetic capacity of the tomato leaf, which is restricted by CLA.

800-015-Z Control of Vein Formation by Plasmodesmata Aperture Linh Nguyen – University of Alberta Enrico Scarpella – University of Alberta Auxin is the only plant signalling molecule known to be able to induce vein formation. The inductive role of auxin on vein formation seems to depend on auxin’s polar transport through plant tissues. In turn, the polarity of auxin transport seems to depend on the asymmetrical localization of auxin transporters of the PIN-FORMED (PIN) family at the plasma membrane of auxin-transporting cells. Consequently, loss of function of all PIN genes should lead to loss of polar auxin transport and thus loss of vein formation. Unexpectedly, however, mutants lacking function of six of the eight PIN genes—pin sextuple mutants—still form veins, and the phenotype of pin sextuple mutants is unchanged by additional mutation in the remaining two PIN genes or in any other known auxin-transporter genes. Moreover, pin sextuple mutants form new veins in response to auxin treatment, suggesting the existence of an unidentified PIN-independent polar-auxin-movement mechanism that controls vein formation. Here I provide evidence consistent with the hypothesis that auxin movement through plasmodesmata (PD) intercellular channels is at the basis of such mechanism. By analyzing vein networks of mutants with reduced PD aperture, I found that reduced PD aperture leads to defects in vein formation. I also found that the severity of vein formation defects is proportional to the decrease in PD aperture. My results suggest a new, unsuspected level of control of vein formation by auxin movement.

800-016-Y The Maize YABBY Transcription Factor Drooping leaf1 and Its Enhancer Drooping leaf2 Regulate Midrib and Carpel Development Josh Strable – Iowa State University Sarah Briggs – Iowa State University, Erik Vollbrecht – Iowa State University A major question in plant biology remains what genetic factors determine grass leaf architecture e.g., leaf length, width, angle (deflection from the culm). Collectively, such morphological traits directly influence canopy structure and light penetration, photoassimilate production, and overall yield. We discovered and characterized a maize mutant with aberrant leaf architecture we named drooping leaf (drl), as leaf blades are midribless. Additionally, gynoecium development is severely compromised: unfused carpels encompass protrusive nucelli, akin to the pleiotropy observed for rice dl mutants. These mutant phenotypes are drastically enhanced by a modifier locus in the Mo17 inbred. We cloned the underlying gene, drl1, and identified its paralogous enhancer, drl2, using positional cloning and generated a second drl1 allele by Ds transposon remobilization. The genes encode the maize CRABS CLAW ortholog, a putative transcriptional regulator with zinc-finger and YABBY domains. Sequence variation at the drl2 locus in Mo17 likely enhances drl1 mutant phenotypes; additional natural variants of drl2 are currently under investigation. In situ hybridizations indicate drl1 and drl2 transcripts are absent from the central domain of the vegetative shoot apical meristem, but are detected in the incipient primordium, young leaf primordia and in reproductive organs. The apolar expression patterns of drl1 and drl2 in developing leaf primordia together with histological analyses suggest that these genes promote differentiation of a specific cell type, the clear cells, in the central midrib. Partial rescue of midrib and carpel phenotypes in drl1-R; drl2-Mo17; Liguleless3-O (Lg3-O) triple mutants reveals Lg3-O is likely epistatic to drl1 and

drl2. In floral tissues, zea agamous1 interacts synergistically with drl1; drl2, as triple mutants develop indeterminate branch-like structures in the axils of bracts, indicating these genes redundantly promote floral meristem determinacy. Our data suggest a conserved mechanism where DRL proteins are essential for two agronomic traits: leaf architecture and grain yield.

800-017-Y Regulatory Pathways of Flowering: Where Do Polyamines Fit In? Sheaza Ahmed – Bowling Green State University Menaka Ariyaratne – Bowling Green State University, Gopala Mulangi – Bowling Green State University The regulation of flowering involves a complex interaction of several genetic pathways in Arabidopsis thaliana. Changes in the levels of polyamines are known to affect both biotic and abiotic stress responses in plants. However no direct evidence for a role of polyamines in the regulation of flowering has yet been demonstrated. Our research has identified a class of highly specific Polyamine Uptake Transporters (PUTs) in plants which are known to import polyamines. Our hypothesis is that altering a specific membrane bound polyamine transporter would affect polyamine homeostasis and will affect the growth and development of a plant as a whole. To test the hypothesis, overexpressed constructs of one of the highly specific polyamine uptake transporter (PUT5) localized in leaf were made. The resulting transgenic plants were compared to the wild type and a mutant of the leaf localized polyamine uptake transporter (PUT5). The transgenic plants showed increase of spermidine and spermidine conjugates in the leaf, resulting in the delay of flowering. Some of the other phenotypic characteristics observed in the transgenic plants relative to wild type were taller plants, thicker stems, bigger leaves and an increase in the number of seed pods and flowers and. All these observations provide the first genetic evidence for a role of polyamine in the regulation of flowering.

800-018-Z Developmental Transitions in Embryogenesis Uncover a New Pathway Regulating Plasmodesmata Transport Anne Runkel – University of California, Berkeley Jacob Brunkard – University of California, Berkeley, Min Xu – Northwest University, Mary Ahern – University of California, Berkeley, Patricia Zambryski – University of California, Berkeley Plasmodesmata (PD), the plasma membrane-lined channels connecting neighboring plant cells, are essential for normal development. A transition occurs at the torpedo stage of embryogenesis in Arabidopsis restricting PD transport (1). The embryo defective mutants ise1, ise2, ise3, and dse1 show disrupted PD transport during this critical developmental period. The ise mutants have increased PD transport while dse1 has reduced PD transport (2, 3, 4). Transcriptionally, the ise1 and ise2 mutants show delayed embryo development, while the dse1 mutant shows increased expression of genes associated with late embryogenesis. We hypothesize that dse1 skips over the normal torpedo stage developmental program and shows precocious restricted PD transport at an earlier developmental stage. Using a genetic approach in Arabidopsis embryos and Virus Induced Gene Silencing (VIGS) of DSE1 in Nicotiana benthamiana leaves, we have uncovered a new pathway regulating PD transport that is crucial throughout plant development. Surprisingly, the ISE and DSE1 proteins are not localized at PD. Instead, ISE1 and ISE3 are localized to the mitochondria, ISE2 is in the chloroplast, and DSE1 is a WD-40 repeat protein found in the nucleus and cytoplasm. Through transcriptome analyses, protein interaction studies, and perturbation of downstream targets of these mutants, we are writing a distinct story on PD regulation. Organelle Nucleus Plasmodesmata Signaling (ONPS) challenges the notion that PD regulation must occur at PD, but rather, many components of the plant cell control PD transport (2, 5, 6). 1. Kim, I., et al. (2002). Development 129: 1261–1272.

2. Burch-Smith, T.M., et al. (2011). PNAS Plus 108: E1451–E1460. 3. Stonebloom, S., et al. (2012). Plant Physiol. 158: 190–199. 4. Xu, M., et al. (2012). PNAS 109: 5098–5103. 5. Burch-Smith, T.M. and Zambryski, P.C. (2012). Annu. Rev. Plant Biol. 63: 239–260. 6. Brunkard, J.O., et al. (2013). Curr. Opin. Plant Biol. 16: 614–620.

800-019-Z RAMOSA1 Interacts with KNOTTED1 and Regulates Meristem Determinacy via Gibberellins During Maize Inflorescence Development Erik Vollbrecht – Iowa State University Xiang Yang – Iowa State University, Joshua Strable – Iowa State University, Kokulapalan Wimalanathan – Iowa State University Inflorescence architecture results from the locations and activities of meristems. In maize, critical developmental regulators and transcriptional networks that control distinct steps in generating this architecture have been identified. A key component regulating branching is the ramosa pathway, in which the ramosa1 (ra1) gene encodes a C2-H2 zinc finger protein with two EAR repression motifs. In ra1 mutants, the inflorescences (the tassel and the ear) become overly branched due to loss of meristem determinacy. We found that GFP-RA1 fusion proteins enter into the nucleus in planta and that a conserved motif in the zinc finger domain serves as the NLS. To elucidate the mechanism of RA1 action we identified RA1-interacting proteins in young ears. Several putative transcription factor proteins including KNOTTED1 (KN1) were identified. The interaction between RA1 and KN1 was confirmed in vitro and in vivo, and mapped onto the domain structure of the two proteins. Tests for a genetic interaction also supported an interaction between RA1 and KN1 in regulating inflorescence branch architecture. KNOX proteins are known to regulate gibberellin (GA) levels in lateral organ initiation, and GA metabolic genes are direct targets of the KN1 and RA1 transcription factors. We confirmed altered transcript levels of gibberellin biosynthesis genes in developing ra1-R mutant inflorescences, and exogenous gibberellic acid 3 (GA3) partially corrected the ra1-R mutant phenotype. These results and additional double mutant studies all indicate a fundamental and complex role for gibberellins in regulating meristem determinacy during maize inflorescence development.

800-020-Y Phe-Net: Development of a Phenylalanine-derived Network Underlying Epidermal Development and Environmental Interaction Katherine Warpeha – University of Illinois at Chicago Alessia Para – Northwestern University, Danielle Orozco-Nunnelly – University of Illinois at Chicago, DurreShahwar Muhammad – University of Illinois at Chicago, Ramis Memishi – University of Illinois at Chicago, Michael Naldrett – Donald Danforth, Sophie Alvarez – Donald Danforth The transition from seed to seedling is a critical period of plant life. Once dormancy has been broken, imbibition ruptures the seed coat and promotes cell enlargement in the embryo. It also initiates metabolism to produce carbon (C) backbones for new structures, and produces energy for the cell cycle and protein synthesis prior to development of the competent chloroplasts. Through the characterization of adt3 phenotype, we demonstrate Phenylalanine (Phe) metabolism in defense preparation, and we reveal a more direct function of Phe in counteracting the oxidative imbalances that can occur in early development and in response to environmental stimuli. In development of the young

seedling, we identified the impact of Phe on epidermal tissue patterning in cotyledons. We found that the defects in cell expansion and proliferation are also accompanied by alterations in organelle development and function. Cuticle structure and/or deposition was also linked to specific phenotypes in development, and persisted in true leaves. The molecular basis of these phenotypes indicated demonstrable links to the adt3 proteome, and revealed additional impact on specific pathways vital during normal transition from seed to seedling.

800-021-Y Sex Related Response of Mercurialis Annua Plants to Stress Conditions Micha Guy – Ben Gurion University Ezra Orlofsky, Giorgi Kozhoridze, Avi Golan-Goldhirsh The annual dioecious species, Mercurialis. Annua, where female and male functions are born on separate individuals, offers an advantageous system for studies on the dimorphic response of these plants to adverse environments. We have observed that female M. annua plants outlive male plants, when grown together under the same conditions. The question addressed in this study was whether this observation is reflected in differential sex related physiological and biochemical traits under normal growth and under salt stress conditions. This was tested by comparing morphological, biochemical (anti-oxidative and detoxification enzymes) and metabolic characteristics of female and male M. annua plants during their development and under senescence and salt stresses. Under control and stress conditions, female plants produced significantly more new reproductive nodes. During plant development, there were no differences between female and male plants in the anti-oxidative enzyme activities, except for significantly higher APX and CAT activities in ‘young’ female plants. Furthermore, there was higher POD (Guaiacolperoxidase) activity in male plants, throughout development. This may be associated with the earlier initiation of senescence in that gender. Taken together, the results presented suggested more responsiveness of female M. annua to the stresses studied. The results presented here provide biochemical support for the suggested “division of labor hypothesis” about evolution of dioecy that female plants direct resources towards reproductive development for securing completion of the reproductive life cycle and seed production.

800-022-Z The Effects of VAL1 Mutation on Arabidopsis Development Erin Friedman – Lynchburg College Plants offer a solution to growing food and energy needs worldwide. As biological producers, seeds represent the lowest cost but highest volume of protein and oils, which are valuable food and chemical sources. To obtain high oil content in seeds, knowledge of the mechanisms that regulate metabolic systems is critical. As identified in a metabolic screen, one gene that controls such processes in Arabidopsis is the transcriptional regulator VPI/ABI3-like 1 (VAL1). During germination, VAL1 has been shown to suppress the accumulation of seed storage compounds. In addition, VAL1 negatively regulates drought stress. However, little is known about the additional roles of VAL1, both in response to environmental stresses and during development. We aim to further characterize val1 mutant seeds with respect to developmental and stress-response phenotypes. We also aim to determine whether the levels of additional metabolites are altered in val1 mutant plants. By further characterizing the roles of VAL1 in metabolic composition and stress response, we can begin to understand the pathways that control these processes.

800-023-Z PeEREBP_PHL1 Is a SHINE-like Transcription Factor Without Complete SHINE Motifs Involved in Lip Cuticle Formation in Phalaenopsis Flowers Hong-Hwa Chen – Department of Life Sciences, National Cheng Kung University Pei-Han Lai – Department of Life Sciences, Wann-Neng Jane – National Cheng Kung University, Mei-Chu Chung – – National Cheng Kung University, Wen-Huei Chen – Institute of Plant and Microbial Biology, Academia Sinica Phalaenopsis orchid hasbecome one of the most popular ornamental plants owing to its elegant floral morphology and long florescence duration. Most orchids represent a highly evolved petal, labellum or lip that offers a landing platform for pollinators. Previously, several highly enriched transcripts were identified in lip by a homemade oligoarray analysis. The expression of a member of AP2/EREBP family, PeEREBP_PHL1 was found to have a 2~3-fold increase as compared to that in sepal or petal. In this study, we further characterized the function of PeEREBP_PHL1 and its relationship to lip development. First, the phylogenetic analysis showed thatPeEREBP_PHL1is close to At5g25190, a SHINE clade homolog in Arabidopsis. The SHINE clade is reported to control cuticle formation, but the At5g25190 lacks intact ‘mm’ and ‘cm’ motifs and does not have SHINE functions. Heterologous overexpression of PeEREBP_PHL1 in Arabidopsis showed phenocopied overexpression of the AtSHNs, with shiny leaves on the adaxial surface, and not only the cuticular folds but also the wax deposition were observed in the epidermis of rosette leaves under cryo scanning electron microscopy. Knockdown expression of PeEREBP_PHL1 revealed morphological alterations in epidermal cells and reductions in cuticular folds in the lip of Phalaenopsis by using a CymMV-based virus-induced gene silencing. Furthermore, the expression of PeEREBP_PHL1 and cell morphology were examined in the P. Big Foot ‘TH.365’ which has a petal-like lip. Significantly reduced expression of PeEREBP_PHL1 (a 96% reduction) in the petal-like lip of P. Big Foot ‘TH.365’. In addition, the cell morphology of the P. Big Foot ‘TH.365’ also shows round conical shape of typical orchid petal epidermis, which lacks cuticular folds on the surface. Consequently, this study demonstrated that the PeEREBP_PHL1 without entire SHINE domains can exert SHINE functions in Arabidopsis, and it provides new insights into transcriptional mechanisms regulating cuticle formation in Phalaenopsis flowers.

800-024-Y Identification of Homozygous Suppressors and Enhancers of the Gravitropism Defective 2 Mutant Kirsten Famiglietti – Southern Connecticut State University Rajkumar Prabhu – Southern Connecticut State University, Leighton Duncan – Southern Connecticut State University, Rebecca Silady – Southern Connecticut State University GRAVITROPISM DEFECTIVE 2 (GRV2) functions in vesicle trafficking, from the pre-vacuolar compartment to the vacuole (Silady et al., 2008). Arabidopsis thaliana plants with mutations in GRV2 exhibit a number of phenotypes, all dealing with defects in asymmetrical growth and vesicular trafficking. The first observable phenotype of grv2 mutants is seen in the two-cell stage of the plant embryo. The grv2 mutants, unlike wildtype, have an enlarged and highly vacuolated cell at the embryo apex (Silady et al., 2008). Despite this abnormality, the embryo is capable of developing and the plant eventually reaches reproductive maturity. Other phenotypic variances from wild type are seen in young grv2 seedlings. The grv2 mutant exhibits a widened apical hook, reduced gravitropic response in hypocotyls, and reduced phototropic response in hypocotyls when compared to wildtype. In adult grv2 mutants there is an obvious defect in shoot gravitropism, as well as a defect in sorting storage proteins into developing seeds. This mis-sorting results in seedling arrest when grv2 mutant seeds are grown without exogenous sucrose. This study aims to identify mutants homozygous for the grv2-1 mutation and also homozygous for one additional mutation that will either enhance or suppress the grv2-1 phenotypes. To date one putative homozygous suppressor has been identified. This mutant suppresses grv2-1 defects in apical hook maintenance, gravitropism, and phototropism, as well as seedling arrest in the absence of sucrose. Future work will focus on mapping and cloning this suppressor and identification of additional enhancers and suppressors of grv2-1.

Silady, R., Ehrhardt, D., Jackson, K., Faulkner, C., Oparka, K., Somerville, C. (2008) The GRV2/RME-8 protein of Arabidopsis functions in the late endocytic pathway and is required for vacuolar membrane flow. The Plant Journal, 53, 29-41.

800-025-Y RNA-Seq Analysis of the Arabidopsis Flower Mutant Ant ail6 Beth Krizek – University of South Carolina Carlton Bequette – University of South Carolina, Ann Loraine – University of North Carolina at Charlotte AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6), two related transcription factors in Arabidopsis thaliana, have partially overlapping roles in several aspects of flower development including floral organ initiation, identity specification, growth and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA-Seq on ant ail6 double mutants. Analyses of genes differentially expressed in the double mutant compared to wild type suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall and spatial regulation of auxin accumulation. We find reduced levels of demethyl-esterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNASeq experiment also reveals cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented GO terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we find that ant ail6 plants have elevated levels of two defense hormones: salicylic acid (SA) and jasmonic acid (JA). Our results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

800-026-Z The Role of the Chromatin Remodeling Protein CHR9 in LFY-dependent Transcription Jeffrey Kovach – Ohio State University Rebecca Lamb – Ohio State University, Matthew Habina There are numerous genes that are necessary for the formation of normal flowers in Arabidopsis thaliana. One of the most important of these genes is LEAFY (LFY). LFY is a floral meristem identity gene which regulates the formation of flowers and reproductive structures. The ectopic expression of LFY results in the formation of flowers where branches would normally form while loss of function lfy mutations results in extra branches before the development of abnormal flowers. The LFY gene encodes a transcription factor which exists in a multiprotein transcriptional complex along with cofactors. The role of these cofactors in regulating LFY-dependent gene expression is an area of interest for research. Previous work in the Lamb lab has identified a potential LFY cofactor, CHR9. The CHR9 gene encodes a protein in the SWI/SNF chromatin remodeler family; its function in reproductive development is unknown. Loss of function chr9 mutants as well as overexpression lines were investigated to gather information about their phenotypes in relation to flower development. Preliminary data suggests that CHR9 is involved in flower development with phenotypes reminiscent of some of the characteristics of lfy mutants. The examination of the expression pattern of CHR9 is currently under way. In addition, chr9; lfy double mutants are being analyzed as well.

800-027-Z On the Way to Protocorm-like-body (PLB) – Somatic Embryogenesis or Organogenesis? Su-Chiung Fang – Academia Sinica Jhun-Chen Chen, Miao-Ju Wei

The efforts to develop efficient tissue culture protocol for propagation of clonal plantlets are instrumental in orchid biotechnology. One of the tissue culture means to produce clonal seedlings is through protocorm-like-body (PLB) propagation. Protocorms are the small spherical tuber-like bodies germinated from orchid seeds. PLBs resemble protocorms structurally but are triggered from explants and/or callus in vitro. It has been suggested that protocorm is extended stage of embryo development and PLB development is therefore referred as somatic embryogenesis. However, the molecular evidences supporting such hypothesis remain scarce. Here, we used RNA-sequencing to monitor the transcriptome dynamics in reproductive tissues of Phalaenopsis aphrodite. Our data showed that the regulatory network controlling genes expressed during seed development is evolutionarily conserved in P. aphrodite. Global comparison of mRNA populations suggested that protocorms and PLBs share the greatest similarity in overall gene expression and therefore possible cellular functions. However, mRNA populations of the protocorm and PLB were largely different from those of zygotic embryonic tissues, indicating regeneration of PLB does not follow the embryogenesis program. Instead, a class I KNOTTED-LIKE HOMEOBOX (KNOX1) gene seems to play an important role in PLB regeneration. Taken together, the data obtained establish a fundamental framework for orchid reproductive development and enable prediction of gene regulatory networks that is required for specialized developmental programs of orchids.

800-028-Y N-Acetylglucosamine-1-P Uridylyltransferase 1 and 2 Are Required for Gametogenesis and Embryo Development in Arabidopsis Thaliana Ya-Huei Chen – Academia Sinica Hwei-Ling Shen – Academia Sinica, Wan-Hsing Cheng – Academia Sinica The N-acetylglucosamine-1-P uridylyltransferases (GlcNAc1pUTs) catalyze the final step of hexosamine biosynthetic pathway (HBP) to produce UDP-N-acetylglucosamine (UDP-GlcNAc), which is an essential amino sugar for glycosylation in prokaryotes and eukaryotes. Two GlcNAc1pUTs, which are encoded by GlcNA.UT1 and GlcNA.UT2, respectively, has been identified in Arabidopsis; however, their biological function in plants remains unknown. Single mutants of glcna.ut1 and glcna.ut2 reveal no obvious phenotype but their homozygous double mutant is lethal, reflecting their functional redundancy and essential role for plant growth. Compared with the wild-type, the mutant plants GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 display shorter siliques and fewer seed settings with impaired pollen viability and degenerate ovules. Genetic analyses further demonstrated that the GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 mutant plants, rather the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants, suffer from the aberrant transmission of (glcna.ut1 glcna.ut2) gametes. Cellular biological analyses indicated that pollen defects in GlcNA.UT1/glcna.ut1 glcna.ut2/glcna.ut2 appear during pollen mitosis I stages and the female gametophytes were arrested during the uninucleate stage. Although the glcna.ut1/glcna.ut1 GlcNA.UT2/glcna.ut2 mutant plants exhibited a normal transmission of (glcna.ut1 glcna.ut2) gametes and gametogenesis, the development of numerous embryos is arrested during the early globular stage. Conclusively, GlcNA.UT1 and GlcNA.UT2 have overlapping functions in Arabidopsis, whereas the GlcNA.UT2 plays a primary role in gametophyte development.

800-029-Y The Role of COW1 Orthologs in Protonemal Development of Physcomitrella Patens Elena Cravens – Wellesley College Fabienne Furt – Worcester Polytechnic Institute, Luis Vidali – Worcester Polytechnic Institute, T. Kaye Peterman – Wellesley College Sec14, a protein first discovered in yeast, has been highly conserved throughout evolution and is present in all eukaryotic organisms. Sec14 functions as a regulator of lipid metabolism and functions in vesicle formation from the

trans-Golgi network. By regulating the biogenesis of phosphatidylcholine and phosophoinositides, Sec14 regulates the lipid composition of vesicles formed from the trans-Golgi network. COW1 is a Sec14-like protein found in the vascular plant Arabidopsis thaliana whose function is crucial in tip growth, a specialized form of polarized cell growth, which results in an outgrowth extending from one end of the cell body. COW1 is expressed in developing root hairs, where it is involved in membrane trafficking and in maintaining phosphoinositide landmarks during tip growth. We chose to investigate the role of COW1 proteins in tip growth in the protonemata of Physcomitrella patens, as the two types of protonemal cells grow exclusively by tip growth. Four COW1 orthologs have been identified in P. patens, PpCOW1a-d. To determine their function, we generated stable transgenic knockout (KO) strains for all 4 orthologs, in which the gene has been disrupted with a selectable marker through homologous recombination, as demonstrated by a PCR genotyping assay. A preliminary analysis of protonemal growth indicated that none of the PpCOW1 genes are essential for tip growth as the plant size for all single KO lines was not significantly different from wild type. However, the Δcow1c and Δcow1d mutants exhibited alterations in overall plant shape and structure suggesting a role for these genes in normal protonemal development.

800-030-Z miR156/SPL, a New Module, Regulates Axillary Bud Development in Switchgrass Jiqing Gou – The Samuel Roberts Noble Foundation Sijia Liu – The Samuel Roberts Noble Foundation, Chunxiang Fu – The Samuel Roberts Noble Foundation, Yuhong Tang – The Samuel Roberts Noble Foundation, Zeng-Yu Wang – The Samuel Roberts Noble Foundation Plant architecture is greatly influenced by branch development. Grasses have two kinds of branches: aerial and basal, both are developed from buds that are formed by axillary meristems. The difference is that the basal branches (tillers) arise from the buds of non-elongated internodes at the base of the main shoot while the aerial branches come from the buds of elongated internodes in the upper part of a stem. Aerial axillary meristems also give rise to spikelets/panicles during reproductive phase. Therefore, axillary bud development plays an essential role in both biomass yield (more vegetative branches) and grain production (more spikelets). Switchgrass has been identified as a dedicated bioenergy crop by the U.S. Department of Energy. Our research found that most switchgrass genotypes can be propagated via node culture, but one genotype-NFCX1 cannot be propagated because it lacks axillary bud. Our research suggests that node culture is directly determined by the formation of axillary buds.Overexpression of miR156 successfully promoted bud development in NFCX1. It further enable the transgenic plants to be successfully propagated via node culture. Microarray analysis indicated that PvSPL4 and PvSPL5 should be the target genes of miR156 that were significantly knocked down by miR156 in the node meristematic tissues. By targeting the conserved domains of PvSPL4 and 5, we developed an RNAi construct that specifically suppressed PvSPL4 and 5 expression without influencing other SPL genes. The PvSPL4-RNAi transgenics demonstrated that PvSPL4&5 are the targets of miR156 and directly regulate axillary bud formation. Additionally, knockdown PvSPL4 & 5 dramatically improved tiller development and biomass yield. Our results suggested that the miR156/SPL module directly regulates axillary bud initiation, this represents both distinct and common regulation mechanisms of aerial and basal axillary buds formation.

800-031-Z Self-directed Control of the Diurnal CONSTANS Dynamics in Arabidopsis Photoperiodic Flowering Mi-Jeong Park – Seoul National University Kyung-Eun Gil – Seoul National University, Young-Ju Kwon – Seoul National University, Pil Joon Seo – Chonbuk National University, Jae-Hoon Jung – University of Cambridge, Chung-Mo Park – Seoul National University The circadian clock control of CONSTANS (CO) transcription and the light regulation of CO stability coordinately regulate photoperiodic flowering by triggering rhythmic expression of the floral integrator FLOWERING LOCUS T (FT). The diurnal

pattern of CO accumulation is modulated sequentially by distinct E3 ubiquitin ligases, such as HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 1 (HOS1) in the morning, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1) in late afternoon, and CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) at night. In particular, CO is stabilized by FKF1 in late afternoon only under long days. Here, we show that CO abundance is not simply regulated by the E3 enzymes in a passive manner but also self-regulated actively through dynamic interactions between two CO isoforms. CO alternative splicing produces two protein variants, the full-size COα and the C-terminally truncated COβ. Notably, COβ, which is resistant to the E3 enzymes, induces the interactions of COα with CO-destabilizing HOS1 and COP1 but inhibits the association of COα with CO-stabilizing FKF1. These observations demonstrate that CO plays an active role in sustaining its diurnal accumulation dynamics in Arabidopsis photoperiodic flowering.

800-032-Y Analysis of Differentially Expressed Genes in Relation to Failure of Free Nuclei Mitosis in Female Sterile Clone of Pinus Tabulaeformis Carr Caixia Zheng – Beijing Forestry University Yang Yao – Beijing Forestry University A sterile female clone of P. tabulaeformis, growing in a Chinese pine seed orchard in Xingcheng, Liaoning in China, was identified. This clone exhibits normal cone growth, however, the female gametophyte (FG) was unable to form due to a failure of mitosis of free nuclei in FG. To understand the underlying molecular mechanism leading to the cessation of mitosis in the clone, suppression subtractive hybridization (SSH) was employed to identify differentially expressed genes in the female fertile and sterile clones’ ovules during the period of free nuclei of FG. Forward and reverse SSH libraries were constructed. A total of 756 clones were randomly selected from the libraries and sequenced, yielding an average length of 286 bp. A total of 430 ESTs (56.9%) showed high similarity to hits in the non-redundant database, and 336 (47.9%) proteins were annotated to GO terms. The expression levels of 11 selected genes were confirmed by qRT-PCR and they were categorized into developmental factors, transcriptional regulators and cell components. The upregulation of genes encoding CKX-like and MYB family-like proteins was detected in the SC, while genes encoding BEL1-like, CDKlike and kinesin-1-like proteins were downregulated. The abnormal expression of these genes may be responsible for the failure of free nuclei mitosis, leading to the abortion of ovulesin the SC. The candidate genes identified here that are related to FG development will be helpful for further studies of the molecular mechanisms of free nuclei mitosis in FG of the Chinese pine.

800-033-Y Allelic Differences in the CAULIFLOWER and APETALA1 Genes Do Not Determine Flowering Phenotype in Brassica Oleracea Hybrids Marilyn Cruz-Alvarez – Florida Gulf Coast University Sandra Londono – Florida Gulf Coast University, Inna Timshina – Florida Gulf Coast University, Daniel Dorado – Florida Gulf Coast University, Franco Migliolo – Florida Gulf Coast University Cauliflower, broccoli, Brussels sprouts, cabbage, kale and kohlrabi are all varieties of the species Brassica oleracea exhibiting very different developmental patterns. These vegetables have been cultivated for centuries and the complete genome of the species has been sequenced recently. However, the genetic differences that lead to their characteristic distinct morphologies and edible parts remain unknown to a large extent. Studies in Arabidopsis thaliana, as well as previous results from a cross between broccoli and cauliflower led to the hypothesis that the lack of the functional proteins encoded by CAULIFLOWER (CAL) and APETALA 1 (AP1), two floral meristem identity genes, was responsible for the floral developmental arrest and formation of the curd in cauliflower. In order to better understand the phenotypic differences and the underlying genetic differences between cauliflower and Rbo, a rapid cycling variety of Brassica

oleracea that exhibits normal floral development, we have produced F1 and F2 hybrids between these two varieties, and analyzed their phenotype and genotype. The hybrids showed an intermediate phenotype between the two varieties. Phenotypic variables such as the length of internodes in the main stem and flowering time were significantly different between the F1 hybrids and the Rbo plants. The F2 generation showed a wide distribution of phenotypes and a lack of correlation between the different phenotypic variables among plants, indicating the contribution of several segregating genes to the phenotypic differences. Our results show that there is no correlation between presence/absence of a functional CAL gene and the morphological and developmental differences between these two varieties. In addition, no differences in the sequences of the AP1a and AP1c alleles have been found so far between Rbo and the cauliflower cultivar used in our studies, suggesting that this gene does not contribute to the flowering arrest in cauliflower either.

800-034-Z Genome Assembly and Investigation into the Chasmogamous / Cleistogamous Mixed Breeding System of Viola Pubescens (Violaceae) Anne Sternberger – Ohio University Harvey Ballard – Ohio University, Sarah Wyatt – Ohio University Flowering plants utilize multiple reproductive strategies, from forced self-pollination to strict cross-pollination. Many members of the genus Viola use a mixed breeding system comprised of both chasmogamous, open flowers that are cross-pollinated and cleistogamous, small mechanically closed flowers that force self-pollination. This research investigates floral genes and their role in the mixed breeding system exploited by many Viola (violet) species. Previous research provides preliminary data, using the Downy yellow violet (Viola pubescens) as a model, that supports hypotheses that floral gene expression differs between the two flower types of species with mixed breeding systems. The genomic DNA of V. pubescens was sequenced, producing millions of raw sequence reads allowing for assemblage of a rough draft of the genome sequence. However, the research stalled because of a lack of molecular tools available in V. pubescens. Further transcriptome data was needed to fill sequence gaps and allow for more complete genomic assemblage. To assemble a more complete genome, two objectives were to be met: 1) obtain transcriptome (RNAseq) data and 2) use those RNAseq data to complete the genome assembly. RNA was extracted from nine tissue types from numerous V. pubescens specimens, available locally at Sells Park, Athens, Ohio. RNA samples were then sent to the Genomics Facility at Michigan State University for sequencing and will be used as a reference to join fragmented genomic DNA reads into contiguous sequences with aid of a bioinformatic pipeline. The assembled genome will provide a novel resource to support the use of V. pubescens as a model organism for additional biological studies, including further investigation into the Viola mixed breeding system.

800-035-Z Molecular Mechanism of Flowering Transition Induced by Short-day in Chrysanthemum Lavandulifolium Silan Dai – Beijing Forestry University Jianxin Fu – Beijing Forestry University, Liwen Yang – Beijing Forestry University, Shuai Qi – Beijing Forestry University Transition from the vegetative state to the onset and development of flowering is regulated by both endogenous and environmental signals. Circadian clock genes and CO-FT model play important roles in flowering transition. Here, we reported the identification of circadian clock genes, CO and FT homologous genes from Chrysanthemum lavandulifolium, an obligate short-day plant, which is a species closely related to chrysanthemums. Expression analysis indicated that the diurnal rhythms of most circadian clock genes continue cycling though the peak levels are dampened and the oscillating periods are changed under continuous light conditions, indicating that these circadian clock genes are synchronized by light. The peak levels of ClGIs and ClFKF1 alter in non-inductive condition (night break and 8 h light/8 h dark), indicating that ClFKF1 itself or the synchronous expression of ClFKF1 and ClGIs might be essential to initiate the flowering of C.

lavandulifolium. The expression levels of ClCOL1 and ClCOL5 are highly increased under SD condition compared to LD condition. ClFT1 is expressed higher under the SD condition than LD condition while ClFT2 reacts against that. The ectopic expression of the ClGI-1 in Arabidopsis results in early flowering, with high expression levels of endogenous CO and FT in transgenic Arabidopsis, indicating that ClGI-1 might activate the expression of CO and FT to induce flowering in C. lavandulifolium. Over-expression of ClCOL1, ClCOL5 and ClFT1 lead to early flowering, while over-expression of ClFT2 suppresses flowering. In summary, when the circadian rhythms form under SD condition, the flower transition of C. lavandulifolium could be completed. The rhythmic expression of circadian clock genes and signal outputs play an important role in maintaining the circadian rhythms of C. lavandulifolium. This research lays a foundation for explaining the molecular mechanism of flowering transition induced by SD in chrysanthemums.

800-036-Y Regulation Mechanism of Anthocyanin Biosynthesis Branch Pathways in Senecio Cruentus He Huang – Beijing Forestry University Xuehua Jin – Beijing Forestry University, Lu Wang – Beijing Forestry University, Silan Dai – Beijing Forestry University Senecio cruentus is a popular potted flower which possesses various colors. HPLC/MS analysis showed that the five S. cruentus cultivars with different flower color accumulated different pigments. Few anthocyanins existed in the yellow and white cultivars and the carotenoids is the main pigments in the yellow cultivar. The pink, blue, and carmine cultivars all contained two anthocyanin compounds responsible for color pigmentation: 26% of total flavonoids for Pg (Pelargonidin) and 2% for Cy (cyanidin) in the pink cultivar; 67% for Dp (delphinidin) and 11% for Cy in the blue cultivar whereas 92% for Cy and 1% for Dp in the carmine cultivar. Transcriptome sequencing of these cultivars indicated that ScF3H, ScDFR and ScANS are the key genes for anthocyanin biosynthesis in S. cruentus and the expression of which showed low levels in the white and yellow cultivars. The competition between F3’H and F3’5’H for the DHK substrates determined the final flower color formation in pink, blue and carmine cultivars while the substrate specificity of 4 ScDFRs were not observed. The relatively low expression of ScF3’H4 and ScF3’5’H as well as the high expression of ScDFR3 which directly catalyzed DHK were responsible for the final formation of Pg in the pink cultivar. The high expression of ScF3’5’H and low expression of ScF3’H4 determined the final formation of Dp in the blue cultivar while the high expression of ScF3’H4 and the low expression of ScF3’5’H determined the final formation of Cy in the carmine cultivar. The expression level of ScF3’5’H was closely related to a 530 bp insertion sequence bp in its promoter which contained the MRE elements for recruiting MYB transcription factors. This study preliminarily analyzed the competition mechanism of the different branches of anthocyanin biosynthesis and the flower coloring mechanism in S. cruentus.

800-037-Y CYP79B2 and CYP79B3 Metabolites Play a Protective Role During Age-related Developmental Leaf Senescence in Arabidopsis Thaliana Renee Crane – California State University, Long Beach Judy Brusslan – California State University, Long Beach During leaf senescence nutrients are mobilized towards newly developing vegetative and reproductive structures. Premature senescence induced by hormone responses to environmental stress results in low crop yield. Auxin regulates nearly all developmental stages, however, its role in senescence remains unclear. RNA-seq data generated by our lab has shown the up-regulation of CYP79B2 and CYP79B3 genes that function as a branch-point between the production of auxin and indole glucosinolates (IGs), small molecules known to function during defense. To investigate the role of CYP79B2/CYP79B3 metabolites in age-dependent leaf senescence in Arabidopsis thaliana we isolated single T-DNA insertion lines disrupting CYP79B2 and CYP79B3. Single mutants were crossed and two independent cyp79b2/cyp79b3 double mutants were isolated. Chlorophyll, protein, and gene expression data suggest that double mutants lacking IGs

display early senescence. Since CYP79B2/CYP79B3 play only a minor role in auxin synthesis, it is likely that the lack of IGs is responsible for the early senescence phenotype and suggest that IGs are playing a protective role during ageinduced developmental leaf senescence. Several IG metabolites are known to scavenge free radicals and so hydrogen peroxide levels were measured using 3,3’-diaminobenzidine (DAB) histochemical staining. Double mutants showed elevated levels of hydrogen peroxide further supporting the protective role played by IGs. Auxin signaling was also tracked using transgenic lines containing the auxin-inducible reporter gene fusions DR5:GUS and DR5:GFP. GUS staining and GFP expression observed at the hydathodes and in the vascular tissue declined during senescence, suggesting a decline in auxin signaling. In addition, down-regulation of auxin biosynthesis and signaling genes was observed in RNAseq data from fully-expanded rosette leaves isolated from progressively older plants. Identifying molecules that slow down the rate of senescence may allow for genetic manipulation to increase nutritional value and crop yield.

800-038-Z Heterosis of XBrassicoraphanus, an Intergeneric Hybrid Between Brassica Rapa and Raphanus Sativus Gibum Yi – Seoul National University Jeong Eun Park – Seoul National University, Hyerang Park – Seoul National University, Jong Hwa Ahn – Seoul National University, Hosub Shin – Seoul National University, Hye Yeon Soh – Seoul National University, Jin Hoe Huh – Seoul National University Heterosis is one of the important biological phenomena remains to be elucidated. Even without systematic understanding of its mechanism, it has been well known that the degree of heterosis is tend to be increasing as the genetic distance between the parents is getting farther. The intergeneric hybrid between Brassica rapa and Raphanus sativus is an extreme example of heterosis because of the parental genetic distance. The hybrids have been recurrently generated by many plant scientist since Segeret, 1826. And the gigantic plant was introduced for its hybrid vigor by Gravatt, 1914. A total of 28 synthetic F1 hybrids were newly obtained from a cross between B. rapa cv. Chiifu and R. sativus cv. WK10039 and both of them were used for the reference genome sequencing for Chinese cabbage and big root radish, respectively. These hybrids showed more or less of hybrid vigor in terms of plant height and number of flowers on a floral stem. Some of the hybrids were over three meters tall. Flowering time of these 28 hybrids were distributed like a normal segregating population mostly spanning from early flowering WK10039 to late flowering Chiifu. However, the plant height of the hybrids was much higher than those of the parents and that was because of longer floral meristem maintaining. These materials also have advantages for NGS based systematic understating for heterosis with the help of the reference genomes.

800-039-Z Hydroxyproline O-arabinosylation Has Distinct Functions Across Plant Taxa Cora MacAlister – University of Michigan Carlos Ortiz-Ramírez, Jörg Becker, José Feijó, Zachary Lippman Across all domains of life, secreted proteins are often targeted for glycosylation, the post-translational addition of sugar moieties. Hydroxyproline O-arabinosylation is a plant specific modification of signaling peptides and cell wall-associated extensins that is catalyzed by the recently identified, highly conserved HPAT enzyme family. In Arabidopsis, two of the three HPATs function redundantly in pollen to promote fertilization. We have found that hpat1 hpat3 pollen tubes are shorter than their wild type counterparts in vitro and in vivo, though they are capable of low rates of fertilization in the absence of competing pollen, suggesting other pollination steps remain functional. Pollen tubes, like root hairs and moss protonemal cells, expand by tip growth and are particularly vulnerable to disruptions in cell wall integrity. To determine the functional significance of this modification in a distantly related tip growing plant, we generated hpat knockout mutants in the moss Physcomitrella patens. Interestingly, in contrast to the shorter pollen tubes of hpat1 hpat3

mutants, Physcomitrella Pphpat mutants produced longer protonemal cells resulting in significantly larger plants. Transcriptional profiling further revealed altered expression of many cell wall associated genes in Pphpat mutants. Unlike Arabidopsis, Physcomitrella lacks canonical extensins, but we have identified a group of secreted extensin-like chimeras which may be targets of HPAT modification. In conclusion, we show that the phenotypic consequence of disrupted hydroxyproline O-arabinosylation vary by species as do the protein targets of this modification.

800-040-Y Investigations of a Possible Enzymatic Switch in Xylan Backbone Synthesis John Tran – Michigan State University Jacob Jensen – Michigan State University, Curtis Wilkerson – Michigan State University Xylans are an important group of plant cell wall hemicelluloses that are abundant in all higher plants. Xylans are key components of the cell walls that enable plants to grow upright by providing mechanical strength, allowing vessel elements to withstand the negative pressure created by transpiration. Although much is known about the structure of xylan, which is characterized by a linear backbone composed of (1-->4)-linked β-D-xylosyl residues with side-chain modifications, less is known about synthesis and elongation of the xylan backbone. Recent studies by us and others have demonstrated xylan:xylosyltransferase activity in vitro by heterologous expressed protein of IRX10 (Jensen et al., 2014) and IRX10-like (Urbanowicz et al., 2014). Between the two, IRX10 showed a lower level of activity than IRX10-like, yet the two proteins are 86% identical. We find this difference in activity intriguing and propose that it likely involves a regulatory mechanism controlling the enzymatic activity of IRX10. Revealing such a mechanism would allow for a better understanding of xylan formation and perhaps explain the role of a number of other proteins which genetic evidence suggest are involved in xylan backbone synthesis. As a first step in such investigations, we wish to identify regions of IRX10 or IRX10-like responsible for the different levels of enzymatic activity by conducting a series of domain-swap experiments between IRX10 and IRX10-like.

800-041-Y Long-Distance Regulation of Stomatal Patterning by CO2 Across Arabidopsis Tissues Miranda Haus – University of Illinois- Urbana Champaign Thomas Jacobs – University of Illinois- Urbana Champaign Stomatal development is environmentally regulated and stomatal patterning changes based on atmospheric carbon dioxide concentrations. The regulation by [CO2] is sensitive enough that mature tissue conveys developmental cues to immature tissue, but the mechanism controlling this change in patterning is unknown. We constructed a doublechamber that subjected rosette and cauline leaves to varying [CO2] to characterize potential stomatal patterning changes associated with a mobile signal between leaf tissue types. The epidermis was scored for stomatal index (SI), proportion of satellite stomata (SatI), and estimated wax quantity. Young rosette tissue adjusts SI based on both the current and previous environment, cauline leaves do not alter stomatal indices under any [CO2] treatment, and leaf epicuticular wax across the entire plant is dependent upon the [CO2] experienced by the young tissue. We also tested if patterning changes are conserved in the next generation by surveying SI and SatI in ungerminated cotyledons. In cotyledon tissue, SI increases when elevated [CO2] is experienced regardless of timing. SatI increases when seeds set in ambient [CO2] and cotyledons are germinated in elevated [CO2], but this does not alter the average distance between stomata across the leaf. The alterations within the stomatal developmental pathway must not occur until after germination, however, because stomatal lineage precursor cells do not differ in ungerminated cotyledons. This report suggests, for the first time, that [CO2] regulation directly affects stomatal pathway specifically through the development of satellite stomata. Cotyledons likely have a related but distinct mechanism for controlling stomatal development in response to [CO2] from parental leaf tissue.

800-042-Z Use of Cytokinin During Seed Germination to Enhance Shoot Organogenesis from Primary Leaves of Young Sunflower (Helianthus Annuus L.) Seedlings Zhifen Zhang – OARDC/The Ohio State University John Finer – OARDC/The Ohio State University For biotechnological improvement of sunflower, an efficient regeneration method using leaf tissue is not yet available. In this report, adventitious shoots were obtained from primary leaves of 7-d-old sunflower seedlings grown on cytokinin-containing medium. Benzylaminopurine (BA), 6-(γ, γ-dimethylallylamino)purine (2iP) and kinetin, each at five different concentrations, were added to a seed germination medium. Primary leaves of seedlings were then excised and incubated on a shoot induction medium (SIM) containing 1.5 mg L-1 BA. If cytokinin was not added to the germination medium, only 25% leaf explants displayed shoot organogenesis after 3-wk culture on SIM, with an average of 3 shoot primordia per leaf. Adding cytokinin to germination medium significantly enhanced shoot organogenesis from leaf tissue, with up to 6-fold increase in the number of shoot primordia per leaf. Shoot-producing tissues were then transferred to an elongation medium containing 0.1 mg L-1 gibberellic acid for shoot development. Shoot primordia from the leaves of seedlings grown with cytokinin tended to be more capable of elongation and further development than those induced from the leaves of seedling grown without cytokinin. The highest number of developed shoots was obtained from the leaves of seedlings germinated on a medium containing 5 mg L-1 BA, with 1.6 developed shoots per explant. Histological analysis of leaves from 7-d-old seedlings treated with 5 mg L-1 BA revealed increased cell division in the adaxial leaf surface tissue of the mid vein. When the detached primary leaves were incubated on SIM, those dividing cells continued to divide and contribute to adventitious shoot formation along the mid vein, suggesting that the cytokinin in germination medium had preconditioned the primary leaves for shoot organogenesis. In vitro micrografting was used to recover whole plants from the elongated shoots with 57% graft survival.

800-043-Z Differences in Wheat Coleoptile Growth Pattern Impact Seedling Emergence Jennifer Yang – Pennsylvania State University Amita Mohan – Washington State University, Jonathan Lynch – Pennsylvania State University, Kulvinder Gill – Washington State University Wheat seedling emergence is an important trait for early stand establishment. Fast emergence and establishment of wheat seedlings helps not only in reducing competition with weeds but also to conserve soil and water evaporation by canopy cover. Furthermore, quick establishment of wheat seedlings correlates well with a robust growth and yield at the later stages of crop development. Wheat seedling emergence is a long-standing issue particularly in drier regions where wheat is planted up to 7 inches deep to tap available soil moisture. More than 5.5 million acres alone in the eastern Washington are under dryland farming with less than 10 inches of annual precipitation. Coleoptile length was previously considered to be the key component determining seeding emergence. However, in a previous study, we screened wheat lines and several short-coleoptile lines were among the best emergers, and vice versa for long-coleoptile lines, which suggests traits besides coleoptile length may significantly impact wheat seedling emergence. In order to identify relevant traits, 12 genotypes, four each from coleoptile length classes of 30-40mm, 70-80mm, and 100-110mm, with contrasting emergence within each class, were selected. Using a laser ablation tomography (LAT) imaging platform, anatomical traits were quantified at apical and sub-apical regions of coleoptiles. Variation in cortical cell size and coleoptile width was observed among genotypes, while cell file arrangements remained generally consistent. Further, there was differential relative expression of expansin among lines contrasting for emergence.

800-044-Y Comparative Proteomic Analysis During the Course of Cotton Fiber Development Hana Mujahid – Mississippi State University Zhaohua Peng – Mississippi State University The distinct stages of cotton fiber development and maturation serve as an excellent single celled model for studying the molecular mechanisms of plant cell elongation, cell wall development, and cellulose biosynthesis. However, this model system of plant cell development is compromised for proteomic studies due to a lack of an efficient protein isolation method in the late stage due to a tough cell wall and abundant phenolic compounds. Here, we compared the quality and quantities of proteins extracted from late stage cotton fibers (25 dpa and 35 dpa) with multiple protein extraction methods and present a comprehensive proteomic study of fiber development following the time course of cotton fiber development. Quantitative analysis identified a number of differentially expressed proteins in four distinct time points of fiber development. Critical proteins involved in cell wall metabolism and regulation, cytoskeleton development, and carbohydrate metabolism among other functional categories in these four developmental stages have been identified. Our studies provided reliable protocols for protein extraction from maturing fiber tissues for mass spectrometry analysis and our results considerably expanded our knowledge of the cotton fiber proteome during development.

Development: Transcriptional Networks 800-045-Y Transcriptional Networks Uncover Multiple Mechanisms for Establishing Tissue-Specific Expression Patterns in the Arabidopsis Root Erin Sparks – Duke University Colleen Drapek – Duke University, Allison Gaudinier – UC Davis, Ning Shen – Duke University, Yongjian Qiu – Duke University, Meng Chen – Duke University, Raluca Gordan – Duke University, Siobhan Brady – UC Davis, Philip Benfey – Duke University In the Arabidopsis thaliana root two transcription factors (TFs), SHORTROOT (SHR) and SCARECROW (SCR), are required for endodermal development. Both TFs show tissue-specific expression patterns, with SHR transcribed in the stele tissue and SCR in the endodermis. SHR protein then moves from the stele into the endodermis to interact with SCR and initiate a feedback loop on SCR expression and promote division of the cortex-endodermal initial daughter into cortex and endodermal cell fates. While this signaling module is well studied, there are still outstanding questions. For example, it is unknown how the low-levels of SCR required for feedback initiation are established. Additionally, no upstream regulators of SHR expression have been identified. To address these questions, we generated a gene regulatory network using enhanced-Yeast-1-Hybrid assays to screen the promoters of 111 TFs against a library of 555 TFs. The resulting gene regulatory network of 874 interactions between 269 TFs provides a resource through which we can address these questions of transcriptional regulation. We validated sub-networks from the broader gene regulatory network to ask how the expression patterns of SHR and SCR are generated. Interactions were validated in planta by assaying SHR and SCR expression in mutant or overexpression lines of upstream TFs by whole root qRT-PCR. Our results suggest that the tissue-specific expression patterns of SHR and SCR are established through two different mechanisms. For SCR, several activators are expressed at low levels across multiple cells types. We propose that these lowly expressed TFs provide the platform to initiate feedback up-regulation in conjunction with SHR. In contrast, SHR expression is established through a combination of broadly expressed activators and specifically expressed repressors. These results highlight two different mechanisms by which combinatorial TF regulation is utilized to generate tissue specific expression patterns.

800-046-Z Transcriptional Regulatory Networks Involved in the Acquisition of Desiccation Tolerance in Arabidopsis Thaliana Seeds Sandra Isabel Gonzalez-Morales – Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y Estudios Avanzados del IPN Ricardo A Chávez Montes – Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y Estudios Avanzados del IPN, Luis Herrera-Estrella – Laboratorio Nacional de Genómica para la Biodiversidad (Langebio), Centro de Investigación y Estudios Avanzados del IPN Desiccation tolerance (DT) allowed seed plants to conquer ecosystems with long periods of limited water availability. This adaptive features allows seeds to remain dried for very long times without losing their ability to germinate. In Arabidopsis, LEC1, LEC2, FUS3 and ABI3 there are transcription factors (TFs), key players in seed maturation including DT. Their mutants lack DT which have eliminated or reduced some components like LEA, heat shock proteins and the accumulation of oligosaccharides. However, there is little information about all the signaling components required to achieve DT and on how transcription factors (TFs) modulate global DT processes. Therefore, the inference of transcriptional networks through insight regarding DT-specific downstream genes of LEC1, LEC2, FUS3 and ABI3 is a challenge for understanding the regulation of this process. Through a comparative analysis of RNA-seq and metabolic profiles of lec1, lec2, fus3 and abi3 mutants as well as their corresponding wild types during the seed desiccation period, we identified expressed genes specifically the ones involved in DT process. These data enabled us to integrate metabolic processes, signaling pathways, and specific TF activity. Additionally, it is shown that reverse engineering of a DT-specific regulatory network reveal transcriptional modules that activate the DT genes in seeds. Notably, two major transcriptional networks were identified related to storage of reserve compounds and cellular protection mechanisms, respectively. Ectopic expression of some TFs identified in these subnetworks are sufficient to activate genes that contribute to DT because it partially rescues the desiccation intolerance phenotype of abi3 mutant, whereas the elimination of this TFs showed a reduced seed desiccation tolerance.

800-047-Z PETAL LOSS, a Trihelix Transcription Factor, Defines Boundaries in Arabidopsis Flowers by Inhibiting Growth Between Developing Sepals David Smyth – Monash University Ruth Kaplan-Levy – Monash University, Aydin Kilinc – Monash University, Edwin Lampugnani – Monash University, Martin O’Brien – Monash University, Tezz Quon – Monash University, Pia Sappl – Monash University The floral template is defined by differential growth of organ primordia and the boundaries that lie between them. In Arabidopsis, boundaries between initiating sepal primordia are reinforced by the trihelix transcription factor PETAL LOSS that specifically reduces cell division in this location. In loss of function ptl mutants, overgrowth in the inter-sepal zone results in its radial enlargement. This overgrowth apparently disrupts the auxin signalling of petal initiation that occurs nearby. On the other hand, gain of function of PTL results in inhibition of growth wherever it is expressed. A yeast twohybrid screen of early inflorescence mRNA with PTL as bait revealed that it interacts with AKIN10, the energy-sensing kinase subunit of SnRK1. This interaction occurs in vitro, and also when the two proteins are transiently expressed in leaves of Nicotiana benthamiana. In the latter system the AKIN10 subunit is preferentially nuclear-localised when interacting with PTL. It may be that lower energy levels occur in inter-sepal zones. These could be sensed by the SnRK1 isomer, which, together with PTL, may then act to retard cell division.

800-048-Y Inflorescence Architecture Traits in Panicoid Grasses: Regulatory Networks and Translational Genomics Andrea Eveland – Donald Danforth Plant Science Center Shuiyi Thames – Donald Danforth Plant Science Center, Mathew Box – Donald Danforth Plant Science Center, Thomas Brutnell – Donald Danforth Plant Science Center, Elizabeth Kellogg – Donald Danforth Plant Science Center Inflorescence architecture is a primary determinant of yield, contributing to seed number and harvesting ability, yet the underlying molecular networks remain largely unexplored in the world’s most important cereal crops. Here, we use a systems-level approach to elucidate the gene networks that modulate inflorescence architecture in maize and other closely related grasses. Our strategy integrates spatiotemporal expression signatures (i.e. mRNA-seq-based profiles) with morphological changes resulting from genetic perturbations that disrupt discrete aspects of inflorescence architecture in maize, i.e. meristem maintenance, meristem size, and axillary meristem determinacy. Through integration of combinatorial ChIP-seq profiles from key regulators of these developmental processes, we identified convergence points in modulation of specific developmental, hormone and signaling networks. We also find that regulatory modules controlling inflorescence architecture have been co-opted in various spatiotemporal contexts and across grass species, and include core transcriptional components, but subtle differences in their regulation. We further leverage our regulatory networks from maize in cross-species network comparisons to other panicoid grass systems, sorghum and Setaria viridis. In these species, we are performing forward genetic screens to identify novel mutants in inflorescence architecture that are not captured in the maize system. For example, we have identified independent bristleless (bsl) mutants in chemical mutagenesis screens of Setaria, which either lack or produce very few bristles (modified branch structures that are characteristic to Setaria species) compared to normal siblings. The bsl mutants appear to be defective in axillary meristem fate, with many more branches producing spikelets, perhaps in place of bristles. The latter phenomenon could translate to increased yield potential. We are characterizing these mutants at the morphological and molecular levels and performing bulk segregant analysis to clone the underlying gene(s). Travel assistance from the ASPB WYITA and funding by NSF-PGRP are gratefully acknowledged.

800-049-Y Uncovering Auxin Transcriptional Networks Controlling Arabidopsis Lateral Root Development Joëlle Muhlemann – Wake Forest University Sheena Gayomba – Wake Forest University, Stacey Lundy – Wake Forest University, Jessica Martin – Wake Forest University, Amy Olex – Virginia Commonwealth University, Gloria Muday – Wake Forest University Auxin controls lateral root (LR) formation and primary root elongation through transcriptional networks that rapidly modulate expression of primary response genes, and more slowly regulate accumulation of transcripts encoding enzymes and signaling molecules that control growth and development. We examined the transcript accumulation profiles in root samples isolated from seedlings treated with the auxin indole-3-acetic acid (IAA) over an extended time course (from 0.5 to 24 hrs). We identified 1246 transcripts whose abundance was significantly changed after IAA treatment with consistent kinetics across 3 replicates. The genes were grouped into clusters with different induction kinetics and annotated functions. Of particular relevance to IAA-induced LR initiation, one cluster contained transcripts whose increases mirrored the timing of the IAA-induced LR initiation and was annotated with cyclins and genes linked to cell division. Among the set of differentially expressed genes, 82 transcripts were predicted to encode transcription factors (TFs). These TFs were spread across diverse clusters, leading to the testable hypothesis that networks of sequentially induced TFs drive induction of proteins linked to cell cycle progression and division that in turn initiate LR formation. The most rapidly induced TF transcript encodes auxin response factor19 (ARF19), which controls IAAdependent initiation and elongation of LRs. Transcripts encoding three slowly induced TFs - ARF4, MYB6, and MYB93 were misexpressed in arf19, suggesting they are localized to an ARF19-regulated subnetwork. Phenotypic analysis of TDNA inserts in these genes revealed altered number of lateral roots. To identify the complete TF network downstream

of ARF19, we performed RNA-seq and found that 26 TFs are ARF19-regulated. The role of these 26 TFs in auxindependent LR initiation and primary root elongation is currently being tested in insertion mutants. These experiments have uncovered novel networks of auxin-regulated TFs that control lateral root development. (Supported by NSF IOS 0820717)

800-050-Z BvMYB1 Protein Directly Activates Betalain Gene Expression by Binding to the Novel BvMYB1 Response Element (MRE) Neda Akhavan – UT of Austin Tony Gonzales – UT of Austin, Alan Lloyd – UT of Austin Sequence-specific protein-DNA interactions are critical for regulating many cellular processes, including transcription, DNA replication, repair, and rearrangement. We characterized anthocyanin MYB-like protein, Beta vulgaris MYB1 (BvMYB1), which regulates the betalain pathway in beets (Hatlestad and Akhavan et al., 2015; Nature Genetics 47:9296). We have identified a novel DNA sequence that is bound by the BvMYB1 transcription factor. BvMYB1 regulates several betalain biosynthetic pathway genes. Initially, though promoter bashing experiment of sequences upstream of BvCYP76AD1 (encoded at the beet R locus, Nature Genetics 44:816-820) and testing with EMSA we worked toward the identification of a highly active BvMYB1 response element (MRE). Second, we have verified several direct BvMYB1 target genes through yeast 1-hybrid and DEX-CHX experiments. These include BvCYP76AD1, BvCYP76AD5, and BvDODA1. We are now trying to identify the nucleotide sequence of the MRE and how it is different from the anthocyanin MYB cisregulatory elements. After identifying this element we will analyze it in promoter-reporter experiments. Finally, we have identified two other betalain MYB regulatory transcription factors with close homology to BvMYB1, BvMYB2 and BvMYB3, which may activate gene expression via the MRE. Through our understanding of interactions between BvMYB1 transcription factors and downstream betalain biosynthetic genes we can better understand how the betalain secondary pigment pathway is regulated.

800-051-Z Direct Roles of SPEECHLESS in the Specification of Stomatal Self-renewing Cells On Sun Lau – Stanford University Kelli Davies – Stanford University, Jessica Chang – Stanford University, Jessika Adrian – Stanford University, Matthew Rowe – Stanford University, Catherine Ballenger – Stanford University, Dominique Bergmann – Stanford University Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.

800-052-Y Proteomic Analysis of Red Light Induced Phytochrome a Response in Solanum Lycopersicum Drew Anderson – University of Puget Sound Andreas Madlung

Phytochromes are a system of light regulated signaling proteins that control a number of plant growth and development process throughout the life of the plant, from germination to fruit development. Tomato (Solanum lycopersicum) is a fully sequenced model organism for angiosperm fruit development and also for the phytochrome system of light receptors. Of the five PHY genes in tomato (A, B1, B2, E, and F), PHYA is the most well studied an is implicated in seedling germination and the perception of long-days in response to far-red light. Here, we use a two pronged approach to investigate the role of PHYA in tomato germination following red-light exposure, a response that has been previously seen (Van Tuinen et al., 1995), but has yet to be investigated through a proteomic approach. We found a number of genes to be differentially regulated between WT and phyA tomato seedlings both before and after exposure to red light. We present new evidence for the the involvement of PHYA in regulating seed development genes, based on an increased expression of seed storage proteins found in both our transcriptomic and proteomic analyses.

800-053-Y Linking Nutrition to Shoot Development Through the TOR Signalling Pathway Victoria Spencer – University of Manchester Minsung Kim – University of Manchester Eukaryotes share a central nutrition sensing signalling pathway, characterised by the master regulator, TARGET OF RAPAMYCIN (TOR). This kinase is crucial for mediating cell growth and metabolism in response to nutrient levels. In plants, the central components of this pathway (TOR, LST8, RAPTOR, FKBP12 and S6K) have been identified and characterised, however, many of their downstream targets remain elusive. By analysing TOR, LST8 and RAPTOR inducible knockdown lines at different developmental stages in Arabidopsis thaliana, this project intends to uncover potential links between TOR signalling and developmental pathways. Using microarrays and network reconstruction, this project investigates whether, over evolutionary time, the TOR pathway has been recruited into plant specific processes in the growing shoot apical meristem.

800-054-Z Gene Expression Changes Associated with Sprout Regulation in Potato Michael Campbell – Penn State Erie Sprout regulation in potato is critical for storage and processing of tubers. While the natural process of dormancy can be utilized to maintain tubers after harvest the application of chemicals such as chlorpropham (CIPC) and 1,4dimethylnaphthalene (DMN) are often used to prolong storage for fresh market and processing. The molecular and physiological mechanisms associated with natural dormancy progression or with tissues treated with growth suppressants such as CIPC and DMN is poorly understood. Using RNA-seq we have established a detailed transcriptional profile in potato tubers that terminate dormancy naturally, are induced to exit dormancy prematurely, or are treated with suppressants such as CIPC and DMN to prolong storage. The termination of dormancy by artificial means was accomplished through the application of the cytokinin analog 1-(α-ethylbenzyl)-3-nitroguanidine (NG) or with the use of bromoethane (BE). Transcriptional changes associated with termination of dormancy using NG or BE appear to occur by two independent mechanisms, one being an induction of cell division and the other a stress-induced suppression of dormancy resulting in meristem growth. Conversely, sprout suppression with CIPC or DMN appears to be largely associated with stress responses. However, DMN has shown to suppress transcripts associated with plastid development and structure, as well as suppression of genes involved with the photosynthetic process, while CIPC suppression is linked to microtubule disruption and a failure for cell division to progress. This work demonstrates that there are multiple molecular avenues controlling dormancy termination and sprout regulation.

800-055-Z Repressor-like MYB Transcription Factors from Poplar Which Downregulate Flavonoid Gene Expression and Suppress Condensed Tannin Biosynthesis Dawei Ma – University of Victoria Peter Constabel – University of Victoria, Kazuko Yoshida In poplar, the R2R3-MYB transcription factor MYB134 positively regulates the accumulation of condensed tannins, end products of the flavonoid pathway and widespread secondary metabolites in trees. Transgenic overexpression of MYB134 leads to enhanced tannin biosynthesis, as well as upregulation of other regulatory genes including several repressor-like MYB transcription factors. In order to understand the regulation of the tannin pathway in poplar, we are carrying out detailed analysis of these MYB repressors. Transactivation assays using transient transformation of poplar cells indicated that several flavonoid gene promoters are negatively regulated by the repressors. We recently characterized the effects of MYB182 as a repressor of the flavonoid pathway in poplar. This MYB repressor affects both the anthocyanin and condensed tannin branch pathway, and which interacts directly with a bHLH cofactor. A second R2R3 MYB repressor appears to have particularly potent effects on flavonoid and phenolic metabolism; poplars overexpressing this MYB had drastically reduced accumulation of tannins, anthocyanins, as well as the salicinoid phenolic glycosides. Current work focuses on characterizing gene expression profiles in these MYB-overexpressing transgenic poplars in order to identify potential targets of this new repressor. The interaction of the repressors with activators and co-factors is being studied using the yeast two-hybrid approach. Ultimately, this work will lead to a deeper understanding of the gene regulatory network that controls synthesis of the condensed tannins in development and plant stress.

800-056-Y Using Time-Course Transcriptome Data to Elucidate Ethylene Receptor and Transcription Factor Networks Controlling Arabidopsis Root Development Alexandria Harkey – Wake Forest University Gloria Muday – Wake Forest University To provide insight into the mechanisms by which ethylene inhibits primary root elongation and lateral root initiation, while stimulating root hair formation, we examined changes in the transcriptome across a time course of treatment in root tissue. We identified 449 transcripts that showed substantial and consistent responses across all 8 time points spanning the 24 hour microarray time course after treatment with the ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC). Some of these transcripts encode ethylene receptors and other members of the ethylene signaling pathway, including 26 transcription factors (TFs), suggesting ethylene regulates synthesis of its own signaling pathway. In addition, transcripts encoding TFs show a variety of expression patterns, including activation or repression at early (0.5-2 hrs), middle (4-8 hrs) and late (12-24 hrs) time points, while transcripts encoding ethylene receptors and other signaling proteins are differentially expressed late in the time course. This suggests a network in which rapidly modulated TFs regulate the expression of more slowly induced TFs and ethylene signaling genes, which in turn modulate the ethylene pathway, and thereby mediate changes in root growth and development. We are testing the temporally predicted network using mutants for ethylene receptors and T-DNA insertion mutants for 22 of 26 TF genes. We are using qRT-PCR to establish relationships by demonstration that mutations in receptors or early TFs alter synthesis of transcripts encoding downstream TFs. We are quantifying root elongation, lateral root development, and root hair formation in TF mutants to identify those with interesting root phenotypes both with and without ACC treatment. Together, these experiments will determine the network of signaling and transcriptional events that control ethylene dependent lateral root growth and development.

This work is supported by the NSF Arabidopsis 2010 grant IOS: 0820717

800-057-Y Direct Regulation of Plant Argonautes by MicroRNA-targeted Transcription Factors J Steen Hoyer – Donald Danforth Plant Science Center Mariah Hassert – Donald Danforth Plant Science Center, Jose Pruneda-Paz – University of California San Diego, Ghislain Breton – University of California San Diego, Steve Kay – University of Southern California, University of California San Diego, James Carrington – Donald Danforth Plant Science Center Argonautes are the central effector proteins of RNA silencing which bind target transcripts in a small RNA-guided manner. Arabidopsis has ten Argonaute proteins, with specialized roles in RNA-directed DNA methylation, posttranscriptional gene silencing, and anti-viral defense. To better understand specialization among Argonautes (AGOs) at the level of transcriptional regulation we tested a library 1541 transcription factors for binding to the promoters of AGO1, AGO10, and AGO7 using yeast 1-hybrid assays. A ranked list of candidate DNA-binding TFs revealed binding of the AGO7 promoter by a number of proteins in two different miRNA-regulated families with known roles in developmental timing and leaf morphology. We are testing the functional significance of these binding sites for AGO7’s role in vegetative phase change and its polar expression pattern. Reverse genetic, transgenic, ChIP, and expression profiling approaches will be used to define the regulatory network involving AGO7, TFs, and the microRNAs that regulate them.

800-058-Z Comparative Network Analysis of C4 Photosynthesis in Grass Species Sarit Weissmann – The Donald Danforth Plant Science Center Henry Priest – The Donald Danforth Plant Science Center, Pinghua Li – The Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Yang Zhang – Center for Plant Science Innocation, University of Nebraska-Lincoln, James Schnable – Center for Plant Science Innovation, University of Nebraska-Lincoln, Todd Mockler – Donald Danforth Plant Science Center, Thomas Brutnell – Donald Danforth Plant Science Center Transcriptional regulatory networks are complex molecular systems that determine differential gene expression in response to environmental or developmental signals. Transcriptional regulation relies on the combinatorial interplay of trans-acting protein complexes, and cis-regulatory sequence elements located in or near target genes. C4 photosynthesis is an example of a complex metabolic pathway that requires special and temporal expression of specific gene combination for its proper function. The C4 pathway coordinates its function between two distinct, specialized leaf cell types, mesophyll (ME) and bundle sheath (BS). The C4 pathway has evolved independently in at least 60 lineages of angiosperms, and at least 17 times in the grasses alone. In the current work, RNA-seq data from three developmental stages of each BS and M cells, were used to analyze transcriptional regulatory networks in three C4 species, Zea mays, Sorghum bicolor, and Setaria viridis. These three species represent two independent events of C4 photosynthesis. Our results show that genes in all three species cluster according to metabolic pathways, modules tend to have cell type specific expression patterns, and that modules in different species tend to include orthologues. Our network comparison indicates changes in connectivity of core photosynthesis genes between species, suggesting alterations in the organization of photosynthetic networks in different origins of C4 species. Understanding transcriptional regulatory networks in plants will promote the understanding of complex metabolic pathways thus allowing us to manipulate them to create better crops.

Development: Root Biology 800-059-Z Characterizing Molecular Mechanisms of Cadaverine Action in Arabidopsis Thaliana Environmental Response Amy Jancewicz – University of Wisconsin-Madison Nicole Gibbs – University of Wisconsin-Madison, Patrick Masson – University of Wisconsin-Madison Polyamines have been implicated in a wide array of cellular processes including signaling, stress response, growth, and gene regulation. Polyamine levels increase upon stress exposure, and may enhance organismal stress tolerance. Despite evidence suggesting its role in root development and stress signal transduction, the genetic pathways of plant response to the diamine cadaverine (cad) have not yet been characterized. Our lab has previously shown that Arabidopsis accessions display natural variation in cad response. To discover genes involved, an M2 EMS population was screened for cadaverine resistant (cdr) seedlings, yielding seven cdr mutants. To focus on those with a response specific to cad, rather than polyamines in general, these cdr mutants have been screened on the common polyamines spermine, spermidine, and putrescine, as well as the polyamine catabolism product, hydrogen peroxide. So far, two mutants show specificity to cadaverine alone, while five remain to be determined. Next-generation sequencing will be used to identify the genes responsible for the cdr mutants’ phenotypes. To further characterize cad response, our group utilized a mass spectrometric approach to detect cad in plant extract. Supporting work done in the ice plant, our preliminary data strongly suggests deuterium-labeled cad is transported from the root tips to the shoots of 6-day-old Arabidopsis seedlings within two hours of application. Going forward, we will address endogenous levels of cad, as well as the transport dynamics and accumulation of deuterium-labeled cad in our cdr mutants. Together, these analyses will yield important clues to the mechanisms by which cad elicits phenotypic changes in plant growth.

800-060-Y Root Hydropatterning: Local Water Availability Acts as a Signal for Lateral Root Initiation Neil Robbins – Stanford University Charlotte Trontin – Carnegie Institution for Science, Department of Plant Biology, Craig Sturrock – University of Nottingham, Malcolm Bennett – University of Nottingham, José Dinneny – Carnegie Institution for Science, Department of Plant Biology Plants grow in heterogeneous environments, and the mechanisms by which they perceive and integrate environmental signals into their growth and development are poorly understood. The root system must be especially sensitive to external stimuli as it navigates through complex soil environments, experiencing micro-scale differences in the distribution of nutrients, moisture, and other factors. To analyze responses to this heterogeneity, we grow roots along the surface of agar media, exposing them to contact with a wet surface or air on either side. Many aspects of development become patterned in response to this asymmetry: lateral roots (LRs) emerge toward the wet surface, while root hairs and aerenchyma are positioned toward air. We have termed this developmental phenomenon hydropatterning. We aim to identify the physical properties of the environment that serve as cues for hydropatterning, the mechanism of perception of these stimuli, and downstream consequences on plant growth. By modifying rates of water uptake by the root, we show that local differences in water availability at the root tip are informative for LR patterning. Transcriptional profiling of manually dissected roots by RNA-seq has been done to identify genetic programs locally regulated by moisture. We have also identified genetic variation in hydropatterning that will be used to identify genes involved in this process and analyze its role in different environments. Through these efforts, we hope to uncover the mechanisms and physiological implications of a novel plant response to water availability, furthering our understanding how root system architecture is shaped by the environment.

800-061-Y Seed-plant Specific CEP Regulatory Peptides Control Arabidopsis Root Development in Response to Nitrogen Limitations and Other Environmental Cues Nijat Imin – The Australian National University Christina Delay – The Australian National University, Michael Djordjevic – The Australian National University Plants - having to process information without brains - have evolved complex intercellular regulatory systems including regulatory peptide signalling to regulate growth, development and responses to their environment. Here, we describe a seed-plant specific regulatory peptide family called CEP (C-terminally Encoded Peptide) that is involved in the modulation of root and shoot development in response to environmental cues, particularly to nitrate starvation and high salt. Upon overexpression or exogenous application of the peptide, there is a strong reduction in the overall size of the root system. Lateral root formation is perturbed at an early stage and primary root growth is dramatically slowed. A T-DNA insertion mutant shows the opposite phenotype, producing a larger root system, particularly under nitrate limitation and salt stress. Our work1-4 suggests a role for this peptide as a negative regulator of root development in seed plants and provides a link between nitrogen demand signaling, developmental programs and environmental stimuli. References: 1

Delay C, Imin N and Djordjevic MA (2013) CEP genes regulate root and shoot development in response to environmental cues and are specific to seed plants. Journal of experimental botany 64 (17), 5383-5394.

2

Ogilvie HA, Imin N and Djordjevic MA (2014) Diversification of the C-TERMINALLY ENCODED PEPTIDE (CEP) gene family in angiosperms, and evolution of plant-family specific CEP genes. BMC genomics 15 (1), 870.

3

BG Bobay, P DiGennaro, E Scholl, N Imin, MA Djordjevic, DM Bird (2013) Solution NMR studies of the plant peptide hormone CEP inform function. FEBS letters 587 (24): 3979-3985.

4

Imin N, Mohd-Radzman NA, Ogilvie HA and Djordjevic MA (2013) The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula 64(17): 5395-5409.

800-062-Z Medicago Truncatula CEP Peptides Negatively Control Lateral Root Formation and Enhance Competency for Root Nodulation Michael Djordjevic – Australian National University Nadiatul Mohd-Radzman – Australian National University, Nijat Imin – Australian National University Small peptide signals, including CEPs (C-terminally encoded peptides), regulate short and long distance cell-to-cell communication and control developmental processes thought previously to be regulated by phytohormones alone. In Medicago truncatula, MtCEP1, which encodes two CEP peptides, regulates lateral organ development in roots and is upregulated by N-limitations or -starvation and elevated CO2 levels1. Under N-limitation, MtCEP1:GUS expression localises in main root and lateral root tips, young nodules and in pericycle and procambium cells. Lateral root development is strongly inhibited by over-expressing MtCEP1 or treating roots with CEP1 peptides at sub µM levels1. Silencing of several MtCEP genes, including MtCEP1, led to significant increases in lateral root formation and suggests a role for CEPs in negatively regulating lateral root formation. Plants with elevated MtCEP1 peptide levels also show a significant increase in root nodule formation which is typified by a widened developmental competency zone for root nodulation. The enhancement of nodulation imparted by elevated MtCEP1 peptide levels is typified by an increase in nodule number and size and more N-fixation results. In addition, the MtCEP1-peptide dependent enhancement of nodulation is partially tolerant to nitrate levels that suppress nodulation1. We have investigated if this is due to known local or systemic

pathways. In order to identify the MtCEP1 peptide species responsible for controlling lateral organ development, we have developed procedures for identifying MtCEP1 peptides in vivo. Our findings show that MtCEP1 peptide activity on lateral organ formation differs according to the peptide’s post-translational modifications2. 1

Imin N, et al. Journal of Experimental Botany 64, 5395-5409 (2013).

2

Mohd-Radzman N, et al. Journal of Experimental Botany. In press (2015).

800-063-Z How Can We Understand Plants as Integrated Systems? Christopher Topp – Danforth Center Plant architecture can be considered from many vantage points: at scales from cells to organs, over the course of development or evolution, and from biophysical, physiological, and ecological perspectives. In all of these ways, our understanding of plant form and function is greatly limited by our ability to study subterranean structures and processes. The limitations to accessing this knowledge are well known – soil is opaque, roots are morphologically complex, and root growth can be heavily influenced by a myriad of environmental factors. Nonetheless, recent technological innovations in imaging science have generated a renewed focus on roots and thus new opportunities to understand the plant as a whole. The Topp Lab is interested in crop root system growth dynamics and function in response to environmental stresses such as drought, rhizosphere interactions, and as a consequence of artificial selection for agronomically important traits such as nitrogen uptake and high plant density. Studying roots requires the development of imaging technologies, computational infrastructure, and statistical methods that can capture and analyze morphologically complex networks over time and at high-throughput. The lab uses several imaging tools (optical, X-ray CT, PET, etc.) along with quantitative genetics and molecular biology to understand the dynamics of root growth and physiology. We aim to understand the relationships among root traits that can be effectively measured both in controlled laboratory environments and in the field, and to identify genes and gene networks that control root, and ultimately whole plant architectural features useful for crop improvement.

800-064-Y A Mechanism Involving Shoot-root Communication Regulates Lateral Root Development in Arabidopsis Under Salt Stress Lina Duan – Carnegie institution for Science Jose Dinneny – Carnegie institution for Science High salinity greatly affects plant root growth and is a widely prevalent stress in agriculture. In a tap root system such as Arabidopsis, lateral roots (LRs) are important components in shaping the whole root system architecture. Previously we showed that salt stress induces a “growth quiescence phase” in post-emergence LRs. This growth suppression can last a few days before the growth rate recovers. Interestingly, the growth quiescence is correlated with sustained abscisic acid (ABA) signaling, reported by proRAB18::GFP, in the post-emergence LRs, and can be rescued in ABA related mutants. However, it is not clear how ABA signaling and LR growth are temporally controlled. Here, we utilize proRAB18::GFP in an EMS-induced mutant screen, and identified presto (prematurely stressed lateral organs), which causes early induction of ABA signaling and paused growth in pre-emerged lateral root primordia. Using mapping by sequencing, PRESTO was found to encode a cyclophillin, which has been well studied as an important regulator of light dependent photosystem assembly, revealing a potential connection between light signaling and salt-regulated lateral root growth.

In summary, this work suggests that lateral root growth dynamics and stress response may involve signaling mechanisms between the root and shoot, which we are currently investigating.

800-065-Y Deletion Analysis of a Class 1 ARF-GAP Reveals Domains Essential for Its Plasma Membrane Localization and Function in Root Hair Polarity Cheol-Min Yoo – Samuel Roberts Noble Foundation Satoshi Naramoto – The University of Tokyo, J. Sparks – Samuel Roberts Noble Foundation, Elison Blancaflor – Samuel Roberts Noble Foundation Our previous studies showed that a class 1 ADP-ribosylation factor (ARF)-GTPase activating protein (GAP) called AGD1 functions in the establishment of root hair polarity in Arabidopsis thaliana. To gain further insight into the molecular mechanisms by which AGD1 modulates polarized root hair growth, we generated an AGD1-GFP construct under the control of the native AGD1 promoter (AGD1pro:: AGD1-GFP). The AGD1pro::AGD1-GFP complemented the root hair defects of the AGD1 mutant indicating that the AGD1 fusion protein is functional. Using live cell confocal microscopy, we found that AGD1-GFP localized to the plasma membrane of initiating root hairs and was expressed predominantly in trichoblasts. In addition to the GAP domain, AGD1 contains a Bin1-Amphiphysin-Rvs167p/Rvs161p (BAR) domain, a Pleckstrin Homology (PH) domain and 2 ankyrin repeats. Deletion analysis revealed specific protein domains that direct AGD1 to the apical plasma membrane of initiating root hairs. The AGD1-GFP fusion with the PH domain deleted localized to the cytosol and failed to complement the root hair phenotype of the AGD1 mutant suggesting that phosphoinositide binding is essential for plasma membrane localization and for its function. GAP domain deletions on the other hand redirected AGD1-GFP to the subapical zone of root hairs and often induced seedlings to produce root hairs with multiple tip initiation sites and branches. Our results suggest that the GAP and PH domains specify AGD1 localization, which is crucial for sustaining polarized root hair growth.

800-066-Z EXPANSIN1-mediated Pericycle Cell Expansion Is Required for Coordinated Asymmetric Cell Division Priya Ramakrishna – University of Nottingham Malcolm Bennett – University of Nottingham, Siobhan Brady – University of California, Davis, Dolf Weijers – Wageningen University, Graham Seymour – University of Nottingham, Ive De Smet – VIB Lateral roots are key contributors to the root system architecture that help with plant anchorage and water and nutrient acquisition. In Arabidopsis and most dicots, lateral roots initiate post-embryonically from a specialised set of xylem pole pericycle cells called ‘founder cells’ through a coordinated series of asymmetric cell divisions that are crucial for lateral root morphogenesis. Very little is known about the molecular components in the pericycle that play a role in mediating these divisions. Recently, Vermeer et al., (F1000 Prime Reports, 2015; Science, 2014) showed that blocking auxin response in the endodermis affects lateral root initiation dramatically as pericycle cells have to expand into the overlying layers prior to undergoing their initial round of division. However, the underlying molecular mechanism in the pericycle is unknown. To investigate underlying molecular mechanisms associated with radial cell expansion in the pericycle, we probed the differential expression of 406 putative cell wall remodelling enzymes in the pericycle during lateral root initiation (De Smet et al., 2008). This analysis identified 42 candidates that potentially are involved in this process, and we retained 29 candidates for which expression is significantly regulated by plant hormones. EXPANSINs, with their characteristic wallloosening activities form an interesting gene family to characterise and we therefore focused on EXPANSIN1 (EXPA1).

In this context, I will be presenting data from loss- and gain-of-function studies and imaging of spatio-temporal expression patterns that provide insights in the role of EXPA1 in the pericycle during lateral root initiation. Additionally, I will report on components of a regulatory network controlling EXPA1 gene expression.

800-067-Z Cell-type Specific Alternative Polyadenylation in Arabidopsis Roots During Development and Under Stress Jingyi Cao – Miami University Quinn Li – Miami University Alternative PolyAdenylation (APA) is defined as the use of more than one polyadenylation site in a gene thus producing different transcripts with potentially different coding sequences and/or regulatory cis-elements. Dynamic APA has been discovered in different tissues, developmental stages and environmental responses in regulating gene expression across animals and plants under certain conditions, such as cell programming, cancer cell development in mammalian cells and Arabidopsis flowering time control and environmental responses. However, the influences of APA during cell differentiation and organogenesis in plants are not understood. Due to its simple organization, Arabidopsis root becomes a tractable system to study plant organogenesis and cell differentiation since the cell types of the root tip can be clearly defined by layers. The available collection of GFP-labeled specific cell types in Arabidopsis root makes it possible to isolate each cell type by fluorescence activated cell sorting (FACS). APA profiles of the transcriptomes of different cell types will be revealed by using a large-scale deep sequencing protocol we developed that specifically targeting the junctions of the 3’-UTR and poly(A) tails. By applying this FACS approach plus the deep sequencing, we are testing a hypothesis that APA contributes to the change in gene expression and thus plays a role in cell differentiation of Arabidopsis root. Some preliminary results of this work will be presented.

800-068-Y Genome-wide Association Studies of Rice (Oryza Sativa) Root Hair Traits Meredith Hanlon – Penn State University Phancita Vejchasarn – Penn State University, Kathleen Brown – Penn State University Phosphorus availability is one of the greatest constraints on agricultural productivity in the world, especially in developing countries. Root hairs, sub-cellular outgrowths of root epidermal cells, increase phosphorus acquisition under limiting conditions with little carbon cost to the plant. Two parameters of root hair growth, length and density, vary naturally within species and have been targeted by breeders for crop improvement. Root hairs have also served as a basic system to understand cell patterning and directional growth, resulting in a strong understanding of the genetic basis of their growth. We have harnessed the immense global diversity of rice to identify genomic regions that control the variation of root hair length and density by performing genome wide association studies (GWAS) on the rice diversity panel 1 (RDP1). After phenotyping 335 accessions, GWAS was performed using 700,000 SNPs dispersed across the genome, with enriched presence in and around genes. We identified many significant (FDR = 0.1) SNPs for root hair density in the subpopulations indica and temperate japonica and strongly associated (p < 0.0001) SNPs for both length and density in multiple subpopulations. Though some associations highlight genes that are known to be involved in root hair formation, we have also found many loci with no previously known relation to the trait, indicating that novel information about the natural variation of a trait can be discovered independently of conventional mutant studies that generally result in the presence or absence of the trait of interest.

800-069-Y Determining the Molecular Mechanisms Underlying Stem Cell Maintenance in the Arabidopsis Thaliana Root Adam Fisher – North Carolina State University Marina Ramos-Pezzotti, Ashley Jones, Ross Sozzani Plants and animals share similarly structured stem cell niches where stem cells divide asymmetrically. Asymmetric cell divisions of stem cells produce daughter cells that can regenerate themselves, and acquire different fates that go on to build all tissues and organs in a multicellular organism. Coordinated control of stem cell divisions is required to ensure proper tissue and organ formation. Understanding the molecular mechanism behind the maintenance of the cells in the stem cell niche of the Arabidopsis root can serve as a guide to understanding the behavior of stem cells in other organisms. Although the factors that maintain the distal columella stem cell layer in the Arabidopsis root are known, the ones that maintain the proximal stem cells that include the vascular initials, the cortex/endodermal initials (CEI), and the epidermal/lateral root cap initials remain largely unknown. A few factors, including SHORT-ROOT (SHR), SCARECROW (SCR), RETINOBLASTOMA-RELATED (RBR), and a D-type cyclin CYCD6;1 are known to be involved in the two successive asymmetric cell divisions of the CEIs. In particular, CYCD6;1 is expressed immediately preceding the asymmetric cell division of the CEI and MARKS A stem cell specific function. Thus, CYCD6;1 can be used as a reporter to assay alternations in stem cell activity. Accordingly, we mutagenized CYCD6;1:GFP expressing plants to identify possible factors involved in the maintenance of the CEI. A total of 2500 seeds were mutagenized and screened the individual M2 plants for morphological (ie. root length and radial patterning defects) and molecular changes (ie changes in CYCD6;1 expression). We selected for F2 bulk segregant analysis and sequencing 5 individual M2 lines with aberrant radial patterning, 12 lines with expanded marker expression in the longitudinal or radial axis or both, and 2 lines with no marker expression. These individual lines represent our candidate genes involved in CEI maintenance.

800-070-Z Introgression Lines Provide Evidence for a Role of Flavonoids in Lateral Root Formation Kathleen DiNapoli – Wake Forest University Gregory Maloney – Novozymes BioAg, Gloria Muday – Wake Forest University Tomato lateral root initiation and elongation is genetically defined and environmentally sensitive. For example, sucrose positively regulates lateral root formation. We used a population of introgression lines (ILs) to identify candidate genes that control lateral root development and its environmental response. Each IL contains a region of the chromosome of Solanum pennelli (Sp) integrated into the genome of Solanum lycopersicum (Sl, cultivar M82). The lateral root phenotype of the 60 ILs in the population and selected sub ILs, was observed in the presence and the absence of sucrose. Lateral root abundance varied widely across the population, with ILs having increased or decreased numbers of lateral roots relative to the parental M82 and showing a range of responses to sucrose. One interesting line, IL 2-5, formed increased numbers of lateral roots relative to M82 and has a reduced response to sucrose. Sub ILs further defined the functional genomic region introgressed into IL 2-5. This region contains genes of flavonoid biosynthesis, including flavonoid 3-hydroxylase (F3H), a gene we previously linked to lateral root initiation in tomato. We have found through qRT-PCR analysis and though open source RNA-seq data sets, that IL 2-5 has reduced expression of the gene encoding F3H. Comparison of the nucleotide sequence of Sp and Sl has indicated that there is a rearrangement in the F3H gene preventing synthesis of a functional enzyme. To determine if flavonoid biosynthesis is altered in this IL, experiments are in progress to quantify flavonoid metabolites by mass spectrometry. To determine whether altered flavonoid biosynthesis in Sp controls root development, we are using the chromosomal location of predicted flavonoid pathway genes to prioritize other interesting ILs. The work was supported by USDA NIFA (Grant 2009-65116-20436) to GKM and a Beckman scholar award to KTD.

Development: Fruit Biology 800-071-Z Domain-specific Transcriptional Profiles from the Arabidopsis Gynoecium Reveal Robust Developmental Mechanisms Required for Ovule Development from the Meristematic Medial Domain Robert Franks – NCSU Gonzalo Villarino – NCSU, Bhupinder Sehra – NCSU, Miguel Flores – NCSU, Eva Sundberg – Swedish University of Agricultural Sciences Within Angiosperms the female reproductive structure, the gynoecium, is critical for reproductive competence. In Arabidopsis thaliana, meristematic regions within the medial domain of the developing gynoecium give rise to ovules, the precursors of the seeds. These meristematic medial domains (also termed carpel margin meristems) provide an excellent system to study basic problems in developmental biology such as patterning, the regulation of cellular differentiation and the control of organ initiation. The molecular genetic mechanisms required for the specification of medial versus lateral positions within the developing gynoecium, as well as the mechanisms that regulate the specialized developmental progression of the meristematic medial domain remain incompletely described. We have set up a FACS-based protoplast sorting system to characterize transcriptional profiles from the meristematic medial domain. Analysis of this data set will be presented including our efforts to identify medial domain enriched transcripts and cis-regulatory elements that enable medial domain-specific expression. This analysis paired with the analysis of mutants that affect the initiation of ovules from the medial domain, allow us to begin to piece together the transcriptional hierarchies that regulate ovule initiation. We will also report on the role of a functionally degenerate pair of transcriptional regulators SEUSS and AINTEGUMENTA during medial domain development. SEUSS and AINTEGUMENTA play key roles in the specification of the medial domain and the subsequent initiation of ovules from this tissue. We present a model wherein SEUSS and AINTEGUMENTA act in concert with PERIANTHIA to condition the proper transition between the floral inflorescence meristem and the gynoecial medial domain. We hope that our efforts to elucidate the transcriptional gene regulatory network that controls ovule initiation and meristematic competence in the Arabidopsis carpel will enable future efforts to regulate seed number and yield in agriculturally relevant species.

800-072-Y The Role of OFPs in Regulating Proximal-distal Patterning of Tomato Fruit Esther van der Knaap – The Ohio State University Shan Wu – The Ohio State Univeristy, Neda Keyhaninejad – The Ohio State University, Hyunjung Kim – The Ohio State University, Yanping Wang – The Ohio State University The final shape and size of plant organs result from coordinated cell proliferation and expansion along different axes. Despite the advances made in recent years, the understanding of how higher order tissue growth is linked to the subcellular events such as cytoskeleton activity is not fully understood. Tomato accessions vary in fruit shape from flat to round to very elongated. The shape of many elongated and pear-shaped tomato varieties is controlled by a naturally occurring premature stop mutation in the OVATE gene, a member of the Ovate Family Proteins (OFPs). Cell morphology analysis demonstrated that the mutation results in elongated shape associated with an altered cell division pattern. Mapping of the suppressors of the ovate (sov) led to the identification of another member of the OFPs, SlOFP20 (Solyc10g076180), as the best candidate gene underlying the sov1 locus. A synergistic interaction was found between ovate and sov1 loci in controlling fruit elongation, which suggests that OVATE and the gene underlying sov1 are involved in the same pathway. Overexpression of OVATE and SlOFP20 in tomato resulted in shortening of fruit and other aerial organs, implying a function as negative regulators of organ elongation, which is consistent with previous findings. Yeast 2 Hybrid (Y2H) experiments showed that OVATE and SlOFP20 interact with Tonneau1 Recruiting Motif (TRM) proteins,

which are a part of a protein complex regulating the formation of preprophase band and organization of cortical microtubule (MT) array. Transient co-expression of OVATE or SlOFP20 with MT-associated SlTRMs in N. benthamiana resulted in relocalization of the OFP-SlTRM complex. This result suggests that OFPs exert their effects through a pathway regulating the dynamic of cytoskeleton. Our findings are starting to shed light on the role of OFPs in proximal-distal patterning of fruit and provide insights into fundamental aspects of plant organ growth.

800-073-Y Genome-scale Insights into Early-stage Fruit Development in Diploid Strawberry Zhongchi Liu – University of Maryland Rachel Shahan – University of Maryland, Chunying Kang – Huazhong Agricultural University, Rui Xia – University of Delaware, Ira Herniter – University of Maryland Fruits represent a key evolutionary innovation for seed disposal. The earliest stage of fruit development is controlled by signals generated during fertilization. Successful fertilization produces signals that promote the decision to proceed with fruit development. This earliest stage is referred to as “fruit set”. Strawberry has traditionally served as a model for the study of fruit set due to its exposed seeds and ease of manipulation. Fragaria vesca is emerging as a better model than the octoploid garden strawberry due to its diploidy and a recently sequenced genome. Auxin produced from the seed was previously shown to induce receptacle fruit in strawberry, but the underlying molecular mechanisms are not known. Using next-generation sequencing, we profiled mRNAs and miRNAs during early stage fruit development. Analysis of auxin and GA biosynthesis genes and their tissue-specific expression revealed that the endosperm and seedcoat may play a more prominent role than the embryo in the synthesis of auxin and GA, the signals required for fruit set. This was supported by auxin measurement in dissected fruit tissues, leading to a model in which phytohormone signals produced in the endosperm and seedcoat coordinate seed, ovary wall, and receptacle fruit development. Functional studies via transgenic plants are being carried out to determine the receptacle fruit-specific genes in strawberry fruit development. Our studies are beginning to reveal the molecular underpinnings of fruit set and early stage fruit development.

800-074-Z The Role of the Auxin Signalling Pathway in Grape Berry Development Sarah Moss – The University of Adelaide, CSIRO Julian Schwerdt – The University of Adelaide, Christine Bottcher – CSIRO, Matthew Tucker – The University of Adelaide, Christopher Davies – CSIRO A better understanding of grape berry development, in particular ripening, is crucial to the continued success of the wine, table grape and dried grape industries in light of the changing climate. Greater knowledge of the mechanisms that regulate fruit development will contribute to the development of new management strategies and planting material. Plant growth regulators (PGRs) control many aspects of plant growth, including fruit development and ripening. In grape some PGRs, such as abscisic acid, promote ripening, and others, such as the auxin indole acetic acid (IAA), promote berry growth but delay ripening. Our knowledge of the processes controlled by IAA and the mechanisms of control is currently incomplete. Auxin signalling in plants is mediated by a Skp, Cullin, F-box (SCF) protein complex that regulates auxin responsive gene expression. Aux/IAA proteins bind to and repress the activity of a family of transcription factors, known as Auxin Response Factors (ARFs). In the presence of auxin, TIR1/AFB proteins (the F-box of the SCF complex) act as auxin receptors, binding Aux/IAAs and targeting them for degradation thereby allowing ARFs to regulate downstream auxin-responsive gene expression. Analysis of the grape genome showed that there are six TIR1/AFBs, 23 Aux/IAAs and 19 ARFs gene family members and in silico comparison with genes from other plant species has suggested possible roles in development. The expression patterns of the grape gene candidates were determined across a grape berry developmental series and a tissue series using quantitative real-time PCR. The results highlight dynamic expression

profiles and suggest that specific auxin signalling components have been recruited during different stages of grape berry development. Investigation of protein-protein interactions using yeasts two-hybrids and bimolecular fluorescent complementation between the various candidates and ex planta PGR treatments has provided insights into of the role of auxins in controlling berry development.

800-075-Z Transcriptional Regulatory Logic Involved in Carpel Development in Arabidopsis Thaliana Bhupinder Sehra – NCSU April Wynn – St. Mary’s College of Maryland, Robert Franks – NCSU In flowering plants the gynoecium (seedpod) is critical for reproductive success. The Arabidopsis gynoecium is comprised of two fused carpel organs. The carpel margin meristem (CMM) located within the medial region of the gynoecium gives rise to ovules that after fertilization become seeds. The mature carpel protects the seeds during maturation until dehiscence occurs allowing for seed dispersal. The carpel is also a complex organ comprised of different structures. During development coordinated regulatory pathways, often co-opted from other development programs within the plant, ensure the correct patterning of the carpel and ultimately the specification of each structure. The transcriptional regulators SEUSS (SEU) and AINTEGUMENTA (ANT) geneticallyinteract in a synergistic manner to specify the CMM, but the transcriptional hierarchy downstream of SEU and ANT remains poorly defined. REPRODUCTIVE MERISTEMS 15 (REM15) encodes a transcription factor expressed in the CMM that is putatively downstream of SEU and ANT. SHATTERPROOF2 (SHP2), a transcription factor also expressed in the CMM, facilitates ovule identity specification, carpel development and fruit dehiscence, and likely functions in a pathway in parallel to SEU and ANT. Regions of high sequence similarity have been identified phylogenetically in the promoter/enhancer regions of these genes and subsequent functional analyses are being carried out in an effort to identify cis-regulatory elements that confer domain or tissue specific expression. In addition, the aim is to functionally define candidate regulatory motifs and to elucidate new regulatory events that affect carpel and seed pod development. Several binding sites specific to MADS domain proteins, transcription factors that help to specify organ identity during plant development, have been found in regions of high sequence similarity and are being functionally tested. In addition, preliminary Chromatin Immunoprecipitation data has shown BASIC PENTATCYSTEINE proteins may repress SHP2 expression via identified consensus sequences that may function as binding sites.

800-076-Y Transcriptome Analysis of Developing Cucumber Fruit Peel Reveals Potential Role of Flavonoid Biosynthesis in Age-related Resistance to Phytophthora Capsici Ben Mansfeld – Michigan State University Marivi Colle – Michigan State University, Mattew Bedewitz – Michigan State University, Rebecca Grumet – Michigan State University Cucumber (Cucumis sativus L.) is susceptible to fruit rot caused by the oomycete pathogen, Phytophthora capsici. Some cultivars exhibit an age-related resistance (ARR), wherein very young fruit are highly susceptible, but become resistant at approximately 12-16 days post pollination (dpp). The transition to resistance is correlated with a transition away from the period of exponential growth and coincides with a transcriptomic shift away from growth towards defense. ARR has been previously described in several pathosystems, however the molecular mechanisms are not fully understood and appear to vary among systems. In cucumber, ARR is associated with fruit peel and potentially could be conferred by

developmentally preformed defenses. Here we utilize RNA-seq analysis to compare transcriptomic differences in fruit peels from genotypes that do and do not exhibit ARR, at susceptible (8dpp) and resistant (16dpp) ages. We identified a group of 65 genes that are uniquely and significantly up-regulated in the resistant 16 dpp fruit of the ARR+ cultivar ‘Vlaspik’. GO-term analysis reveals that this group of genes is highly enriched for secondary metabolite synthesis, and specifically flavonoid biosynthesis; homologs of all but one of the annotated genes in the initial steps in flavonol biosynthesis were up-regulated. Flavonoid phytoalexins have been previously implicated in cucumber leaf antifungal defense, and preliminary analyses indicated that cucumber peels produce methanol-soluble compounds capable of inhibiting growth of P. capsici in vitro. Current analyses of flavonoid content of peels are underway via UPLC-QToF-MS. Other defense related genes were also identified that could be associated with ARR.

800-077-Y Fruit Ripenig Behaviour: Ethylene and the Regulation of Sugar Homeostasis Macarena Farcuh – UC Davis HoYoun Kim – UC Davis, Bosheng Li – UC Davis, Eduardo Blumwald – UC Davis Japanese plums represent the most abundant and variable group among tree species and include most of the freshmarket plums commercialized worldwide. We characterized and compared two Japanese plum cultivars, “Santa Rosa” (SR) and its bud-sport mutant “Sweet Miriam” (SM). These cultivars share the same genetic background but display contrasting ripening behaviors (SR, climacteric and SM, non-climacteric). Both cultivars differ in their sugar metabolism conferring the SM fruits with unusual quality properties (lower glucose and fructose, higher sorbitol and modifications in galactose-metabolism related sugars, etc). The main objective of this research is to characterize the role(s) of ethylene in the regulation of sugar metabolic pathways and its influence in climacteric (‘SR’) and non-climacteric (‘SM’) fruit ripening behaviours. Fruits from each cultivar were harvested at early (S2: pit hardening) and late (S4: fully-ripe) stages of fruit development and assessed using a Systems Biology approach. Transcriptomics, proteomics and metabolomics methodologies, together with targeted gene expression and enzymatic activity assays were analyzed to reveal complex sugar metabolic interrelations and identify differences between the cultivars that could be associated to the observed changes in sugar homeostasis as well as ethylene biosynthesis and signaling. This experimental system provides a unique tool to study metabolic pathways underlying climacteric and non-climacteric fruit ripening behaviors and offers several practical applications. For example, understanding mechanisms that allow fruits to ‘switch’ to a sorbitol-based metabolism would have a great industry impact, since sorbitol is an alternative and healthier natural sweetener to sucrose. In addition, it could also allow the identification of candidate genes for breeding programs focused on fruit quality improvement.

800-078-Z Flower and Gametophyte Development in ‘Campbell Early’ (Vitis Labruscana) and ‘Tamnara’ (V. Spp.) Grapes Hee-Ju Yu – The Catholic University of Korea Bomi Yim – The Catholic University of Korea, Jeong-Hwan Mun – Myongji University, Young-Min Jeong – The Catholic University of Korea, Youn Young Hur – National Institute of Horticultural and Herbal Science, Rural Development Administration Grape is an important crop species cultivated worldwide for fresh and dried fruit, as well as for fermentation to produce wine. Flowers, berries, and seeds are the major yield products in grape. The majority of cultivated varieties of grape have a flower with a single pistil, five stamens, a protective flower cap (calyptra), and a calyx. After fertilization, an individual flower develops into a single berry. Although there are a number of reported studies focusing on berry formation, berry enlargement, and sugar accumulation in grape, the morphological studies of flower, including gametophyte morphogenesis and structural change in floral organs, have not yet been studied in detail. In this study, we

report the characterization of flower development and gametophyte development in the seeded diploid cultivars ‘Campbell Early’ (Vitis labruscana) and ‘Tamnara’ (V. spp.). Male gametophytes were observed from tetrads to mature pollens, and female gametophytes were observed from megaspore mother cells to mature embryo sacs in floral stage 9 of bud to floral stage 13 of open flowers. During floral development stages 9 to 12, there were no major changes in calyx length, whereas the length of the flower cap continuously increased. The flower cap-to-calyx length ratio was 2.0, 3.0, 4.5, and 6.5 at floral stages 9, 10, 11, and 12, respectively. The flower cap-to-calyx length ratio was consistent in the two grape cultivars, suggesting that the ratio is a morphological character representing floral development stage. This study will act as a reference for determining floral development stage of the two grape cultivars. In addition, it will be useful for the determination of optimum time for microspore culture needed to generate doubled haploid lines and appropriate gibberellic acid treatment needed to induce parthenocarpic fruit development in grape.

800-079-Z The Kiss Me Deadly (KMD) Genes Are Involved in Arabidopsis Gynoecium and Fruit Development Ahiko Libertad Lara-Jacobo – CINVESTAV-IPN AL Lara-Jacobo – Laboratorio Nacional de Genómica para la Biodiversidad (Langebio) – CINVESTAV-IPN, H Kim – Dartmouth College, GE Schaller – Dartmouth College, S. de Folter – CINVESTAV-IPN The gynoecium is the female reproductive structure of the flower, which after fertilization mostly converts into a fruit. Many genes have been found to be important for the correct development of the gynoecium and the fruit, however, much less is known about the importance of hormones. One of them is cytokinin, which in general is known to be important for growth, but the precise role during gynoecium development is still not much studied. Cytokinin responsive genes are regulated by the ARABIDOPSIS RESPONSE REGULATORS (ARR) type B. Recently, the KMD family was found to mark the ARR type B proteins for degradation by the proteasome. The overexpression of the KMD genes reflects a similar phenotype as the triple mutant of the ARRs type B (arr1,10,12), which has been reported to have a mayor decrease in cytokinin sensibility. In this project we analyzed the promoter activity of KMD1 and KMD2 during gynoecium and fruit development. Furthermore, we analyzed the morphological phenotypes of constitutive overexpression lines and of the triple knockout mutant. In summary, the findings demonstrate that the KMD family has an effect on the correct gynoecium and fruit development in Arabidopsis thaliana.

800-080-Y Cutin Synthases and the Molecular Basis of Polymeric Cutin Formation: Tomato Fruit as a Model Nicholas Segerson – Cornell Univeristy Trevor Yeats, Laetitia Martin, Jocelyn Rose – Cornell Univeristy The epidermal surfaces of land plants are covered by a hydrophobic cuticle, comprising a matrix of the polyester cutin, which is coated and infiltrated with a range of waxes. The biosynthetic pathway of the cutin matrix precursors is generally understood, but much remains to be learnt about the assembly, modification and fine structure of the extracellular cutin polymer. Genetic characterization of a tomato (Solanum lycopersicum) mutant (cutin deficient 1; cd1), which has a ~95% reduction in fruit polymeric cutin compared with wild type plants, resulted in the identification of a cutin synthase enzyme, which we termed CUS1. This enzyme polymerizes cutin in vitro through transesterification of cutin monomers and is a member of a superfamily of GDSL motif lipase/esterase enzymes. Phylogenetic analysis suggests that CUS1 belongs to a distinct subgroup of the GDSL gene family, which we have termed cutin-synthase like (CUS) genes. Despite the absence of CUS1 activity, cd1 mutant fruits still synthesize appreciable amounts of polymeric cutin, indicating that there are other factors that can mediate its formation. In addition, although native cutin polymers comprise cross-linked chains, NMR analysis of the in vitro synthesized CUS1 product indicated that CUS1 catalyzes the formation of linear cutin oligomers. The mechanism by which the branched structures are formed is not known. Tomato

is predicted to have five CUS enzymes, but to date only the activity of CUS1 has been reported. We have been characterizing the expression and activities of tomato CUS2, which is also expressed in fruit and shares 78% amino acid sequence similarity with CUS1, in order to address the hypothesis that CUS1 and CUS2 act cooperatively to mediate cutin polymerization in vivo.

Development: Seed Biology 800-081-Y Biology of Sesbania Pachycarpa Sensu Auct. and Its Fallow Potentials:Germination Ecology and Biomass Accumulation Kehinde Egberongbe – National Horticultural Research Institute Rasheed Awodoyin – University of Ibadan, Oluseun Olubode – University of Ibadan Large applied nitrogen losses from agriculture contribute immensely to the atmospheric nitrous oxide, a greenhouse gas, thus aggravating the problem of global warming. Exploring the biological nitrogen fixation may reduce inorganic nitrogen input into the farming system. Sesbania pachycarpa, is a woody herbaceous legume plant that freely nodulates and can be used innovatively as green manure in conservation agriculture to fix atmospheric nitrogen into the soil. The fallow potentials of Sesbania pachycarpa were investigated in Ibadan in 2010 and 2011, by studying the germination biology, phenology, rates of biomass accumulation, viability of acid-scarified seeds and effect of depths of sowing on seed germination in two trials and in a completely randomized design. Sesbania pachycarpa seeds were acid-scarified for durations ranging from 0-60 minutes and later up to 120 minutes, seedlings of Sesbania pachycarpa were raised in 21 pots, with three pots randomly selected for the assessment of seedling performance at 2-week intervals for 14 weeks. The study suggests that Sesbania pachycarpa seeds have inherent impervious seed coat dormancy that can be overcome by acid-scarification for at least 40 minutes. The plants were fast growing accumulating 52.10gram/plant and 59.70gram/plant over 14 weeks during the first and second trials respectively. Acid- scarified Sesbania seeds can be stored for 4 weeks before the seeds lose viability. The tolerance of acid treatment for 120 minutes may indicate that the plants will tolerate acid soils and therefore will be an ideal pioneer plant to restore degraded soils.

800-082-Z Phosphorylation of the Transcription Factor TaABF1 During Gibberellin and Abscisic Acid Signaling in Cereal Grains Russell Johnson – Colby College Xi Yang – Colby College, Justin Lutian – Colby College, Greyson Butler – Colby College, Grace Uwase – Colby College, David Chelimo – Colby College The transcription factor TaABF1 plays an important role at the intersection of ABA and GA signaling in cereal grains, as it mediates both inhibition of ABA-suppressed genes (e.g. Amy32b) and stimulation of ABA-induced genes (e.g. HVA1). There is evidence that the kinase PKABA1 can phosphorylate peptide sequences from TaABF1 in vitro and that TaABF1 undergoes phosphorylation in vivo, suggesting that TaABF1 could be regulated by phosphorylation. We investigated whether altering serine residues on TaABF1 would affect its ability to activate HVA1 expression or downregulate Amy32b expression. First, we prepared TaABF1 effector constructs with four serine codons (S35,S36,S113,S115) altered to alanine (not phosphorylatable) or aspartate (phosphomimetic). Introduction of these constructs into aleurone cells by particle bombardment indicated that phosphorylation at one or more of these sites increases the ability of TaABF1 to upregulate HVA1 and to downregulate Amy32b. An S36D single mutant TaABF1 effector was more active than the WT TaABF1, but not as strong as the 4xD mutant. In contrast, analysis of S113A and S113D TaABF1 effector constructs indicated that phosphorylation at this single site greatly reduces the ability of TaABF1 to upregulate HVA1 or to downregulate Amy32b. Thus TaABF1 activity can be either increased or decreased by phosphorylation, depending on

the site of phosphorylation. Interestingly, while mutations of S318 and S322 in the bZIP domain had no effect on the ability of TaABF1 to regulate Amy32b expression, mutation of those residues to either A or D eliminated its ability to activate HVA1. The simplest interpretation is that an intact unphosphorylated serine is required at position 318 and/or 322, and any alteration prevents binding of TaABF1 to ABRE sequences. These results suggest that while TaABF1 activates HVA1 by directly binding to promoter DNA, its activity in downregulating Amy32b does not require direct DNA binding.

800-083-Z Identification of Cell Differentiation Networks in Maize Endosperm Using Laser-capture Microdissection and RNA Sequencing Junpeng Zhan – University of Arizona Dhiraj Thakare – University of Arizona, Chuang Ma – University of Arizona, Alan Lloyd – University of Utah, Neesha Nixon – University of Utah, Angela Arakaki – University of Utah, Kyle Logan – University of Utah, William Burnett – University of Utah, Guosheng Li – University of Arizona, Shanshan Zhang – University of Arizona, Dongfang Wang – University of Arizona, Xiangfeng Wang – University of Arizona, Gary Frews – Universtiy of Utah, Ramin Yadegari – University of Arizona Seed development in angiosperms is initiated by double fertilization during which one of the two haploid sperm cells delivered by the pollen tube fuses with the haploid egg cell to produce a diploid embryo and the other fertilizes the diploid central cell to produce a triploid endosperm. In most flowering plants, endosperm development is characterized by an early, short period of coenocytic growth followed by cellularization and cell differentiation. A later period of mitosis and accumulation of starch and storage proteins characterizes cereal seed development. The storage capacity of endosperm is established in part through the activity of differentiated cell types that mediate uptake of nutrients from the maternal structures and their storage in the inner regions of the endosperm. Therefore, elucidating how cell differentiation is regulated is central to understanding endosperm structure and function. To begin to identify the gene regulatory networks controlling cell differentiation in maize endosperm, we used a coupled laser-capture microdissection and RNA sequencing strategy to comprehensively profile the mRNA populations present in each of the main cell types of the endosperm, as well as the embryo and four maternal compartments of the maize kernel at 8 DAP. Using Weighted Gene Co-expression Network Analysis (WGCNA), we identified gene co-expression modules associated with each of the endosperm cell types, including modules that are enriched for temporally up-regulated genes and/or imprinted genes that we had previously identified. By focusing on the module associated with the basal endosperm transfer layer (BETL), we further characterized a gene regulatory module associated with BETL cell differentiation.

800-084-Y Genome-wide Analyses of Genes Regulated by the Endosperm-specific Maize Transcription Factor Opaque-2 Junpeng Zhan – University of Arizona Guosheng Li – University of Arizona, Chuang Ma – University of Arizona, Xiangfeng Wang – University of Arizona, Ramin Yadegari – University of Arizona The cereal endosperm contains large amounts of carbohydrates and proteins needed for seed germination, and is an important source of human food, animal feed, and feedstock for numerous industrial products including biofuels. An understanding of how the development and function of cereal endosperm is genetically regulated could lead to increased yield of cereal crops, enhanced human nutrition, and improved global economy. However, despite the obvious importance of cereal endosperm, the full extent of the gene regulatory networks that control the storage product synthesis and deposition within cereal endosperm remain largely unknown. The maize bZIP transcription factor Opaque2 (O2) has been shown to regulate a subset of storage-related genes within the endosperm. The orthologs of O2 have

also been identified in other cereals including wheat, barley, and rice with highly conserved regulatory function. To begin to uncover the gene regulatory networks underlying the storage function of maize endosperm, we mapped O2binding sites in vivo using ChIP-Seq performed with endosperm from wild-type B73 in comparison to an o2 mutant (B73o2), and also identified genes up- or down-regulated in the B73 vs. mutant endosperm using RNA-Seq. The identified O2-regulated genes suggested a broad role of O2 in the regulation of the storage protein and starch synthesis, consistent with the previously reported altered expression of storage proteins and starch accumulation in o2 mutant kernels. In addition, using a motif discovery program, we identified putative cis-regulatory motifs of O2 as well as other co-expressed TFs that may co-regulate O2 target genes.

800-085-Y Effects of Nitrate and Karrikin Treatment on Rose Achene Germination Lawrence Davis – K State University Nitrates have been known for a century or more to stimulate germination of some seeds, probably via NO (Hendricks and Taylorson, Pl. Physiol 34:304, 1974). The karrikins, an active ingredient in smoke water, have recently been shown effective for some seeds, including arabidopsis, under some conditions, e.g. freshly harvested seed. In an exhaustive review of the literature (A Century of Rose Germination Studies, at www.rosebreeders.org), from about 65 papers, I found only one brief mention of nitrate or smoke water tested with rose achenes. No details were supplied and there was no positive effect reported. The achene pericarp securely encloses the embryo, and testa which seems to regulate germination primarily through abscisic acid. Dissected seeds with pericarp and testa removed typically show prompt germination on suitable culture media. Nitrate treatment of intact achenes during cold stratification gives a strong positive response. Calcium nitrate at 10 mM (20 mM nitrate) in moist vermiculite is optimum, giving about two months acceleration of germination time for many cultivars(CVs). For some CVs and species it also dramatically increases total percent germination. Potassium or ammonium nitrate is nearly as effective as the calcium salt, but KCl or CaCl2 is not. Tests have been repeated over several years with consistently positive results. For R canina, a notoriously difficult species, germination rates up to 90 % have been obtained with nitrate, compared to 0-25 % with water. The karrikin kar1 was tested at as many as five concentrations up to 1.5 mM. With R canina it had some benefit, about equivalent to five months warm stratification prior to the cold, but at best it was half as good as nitrate. For many CVs, at least one level of kar1 was better than a water control and in some instances better than the nitrate treatment.

800-086-Z Transcriptome Changes Associated with Seed Dormancy and Dormancy Loss in GA-insensitive sly1-2 Mutants of Arabidposis Thaliana Camille Steber – USDA-ARS and Washington State University Sven Nelson – Washington State University Transcript profiling was used to identify gene expression changes associated with the seed dormancy and dormancy loss in the GA-insensitive sleepy1 (sly1-2) mutant of Arabidopsis thaliana. Because there are many genetic mechanisms contributing to seed dormancy, it is difficult to precisely define specific pathways contributing to dormancy and dormancy loss. The sly1-2 mutant exhibits seed dormancy as a consequence of failure to destroy DELLA repressors of seed germination, because SLY1 encodes an F-box protein required for GA-triggered DELLA proteolysis by the ubiquitinproteasome pathway. The sly1-2 seed dormancy phenotype is partially relieved either by the natural dormancy-breaking process of dry after-ripening (51% germination) or by overexpression of the GA receptor GA-INSENSITIVE DWARF1b (GID1b) (74% germination). Neither of these dormancy-breaking processes causes DELLA protein proteolysis. Thus, a

microarray experiment comparing dormant sly1-2 to wild-type Ler, to after-ripened sly1-2, and to sly1-2 rescued by GID1b-overexpression (GID1b-OE) allowed us to ask several specific questions about the regulation of seed dormancy by GA signaling. Only 23 genes showed changes in accumulation with GID1b-OE, whereas 4594 genes showed altered expression with sly1-2 after-ripening over two imbibition timepoints. Thus, a strong change in germination potential si associated with a small change in gene transcript levels. Most of the genes differentially regulated with GID1b-OE showed similar regulation with after-ripening during early imbibiton, but showed the opposite regulation with afterripening during later imbibition. Mechanisms of dormancy loss that require and that do not require DELLA destruction were defined by comparing transcriptome changes associated with sly1-2 after-ripening to those previously defined during wild-type seed after-ripening.

800-087-Z A Proteomic Approach to Map Regulatory Networks Controlling Germination and Seedling Establishment in Therobroma Cacao L Alexandre Noah – University of Yaoundé I Fred Elisma – University of Ottawa, Zhibin Ning – University of Ottawa, Steven Chatfield – University of Toronto, Denis Omokolo – University of Yaoundé I, Nicolas Niemenak – University of Yaoundé I, Daniel Figeys – University of Ottawa The transition from seed to seedling is crucial in the production of Theobroma cacao (chocolate tree). This developmental process depends on successful seed germination for which the underlying regulatory mechanisms are largely uncharacterized in this species. Here, we report a large scale proteomic analysis of the temporal changes in protein complexes and pathways during the transition from seed to seedling in Theobroma cacao. Label free quantitative proteomic analysis was conducted in cotyledons of germinating seeds, roots and shoot apical region (SAR) of T. cacao seedling over 8 time points. From the 1698 proteins identified in cotyledons, 563 showed significant changes in their profile. In roots, 978 proteins were identified with 183 found differentially expressed. While, 981 proteins were identified in SAR and 116 found differentially expressed. Lateral root outgrowth occurred 7 days after imbibition, and appeared to coincide with a key developmental switch reflected in the recruitment of greater number of pathways. Translation, gene silencing complexes, and ubiquitin proteasome system were found as the key regulatory pathways for T. cacao seedling development. Interestingly, this experiment reports for the first time, the implication of flavonoids in lateral root initiation in T. cacao. Lipid and carbohydrate metabolism were found as the primary sources of energy fuelling the establishment of a vigorous cacao seedling. Data revealed a functional transition of cotyledons from storage organs to photosynthetic activity, and supports key roles for abscisic acid, oxylipins, brassinosteroids and polyamines in cacao seedling establishment. Overall, this study emphasizes the utility of these datasets as an essential resource for the understanding of T. cacao developmental biology.

800-088-Y Role of Ascorbic Acid in Modifying Root Architecture, Growth, and Productivity of Arabidopsis Thaliana Noura Kka – School of Life and Environmental Sciences, Deakin University James Rookes – School of Life and Environmental Sciences, Deakin University, David Cahill – School of Life and Environmental Sciences, Deakin University Ascorbic acid (AsA) participates in several aspects of plant growth, development, and productivity. Under optimum growth conditions, plants accumulate variable amounts of AsA in different organs. This project has examined, in Arabidopsis thaliana, the effect of AsA on growth and seed production through analysis of morphological and physiological parameters. The highest levels of AsA were found in flowering buds, premature siliques, leaves, and stems, respectively. In addition, the AsA level correlated positively with concentrations of indole acetic acid (IAA) but inversely with salicylic acid (SA) concentrations. Plants synthesise AsA through six different pathways and primarily through the

galactose pathway. A mutant defective in production of GDP-mannose pyrophosphorylase, vtc1-1, had significantly reduced growth parameters such as root growth rate, root length, number of leaves, leaf area and plant height compared with vtc5-1 (defective in GDP-L-galactose phosphorylase production), dhar1 (defective in mitochondrial dehydroascorbate reductase) apx1 (defective in cytosolic ascorbate peroxidase) and the Col-0 wild type background. The role of AsA as a signal for the promotion of cell expansion was verified in embryonic root cap studies. In addition, AsA promoted cell division by rapid stimulation of differentiation in surrounding cells of the root quiescent centre in Col-0 compared with vtc1-1. A lack of AsA increased the intensity of hydrogen peroxide and superoxide production in roots and also increased the number of secondary roots. Surprisingly the number of flowers and siliques per plant, the number of seeds per silique and seed size were also all affected in vtc1-1, which indicates a key role for AsA in plants.

800-089-Y Mechanisms of ABA-regulated Seed Germination Under Normal Condition and Copper Haoxuan Li – The Chinese University of Hong Kong Nenghui Ye – Shenzhen Research Institute, The Chinese University of Hong Kong Abscisic acid (ABA) and the antagonism between ABA and gibberellin (GA) play a key role in controlling seed germination. Our work showed that unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in embryo. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis. Besides, we also investigated the relationship between Cu and ABA which is the predominant regulator of seed germination. Cu at a concentration of 30 mM effectively inhibited germination of rice caryopsis. ABA content in germinating seeds under copper stress was also higher than control conditions. Quantitative real-time PCR revealed that Cu treatment reduced the expression of OsABA8ox2, a key gene of ABA catabolism in rice seeds. In addition, both malondialdehyde (MDA) and H2O2 contents were increased by Cu stress in germinating seeds. Antioxidant enzyme assays revealed that only catalase activity was reduced by excess Cu, which was consistent with the mRNA profile of OsCATa during seed germination under Cu stress. Together, our results demonstrate that suppression of ABA catabolism and catalase (CAT) activity by excess Cu leads to the inhibition of seed germination of rice.

800-090-Z EMP16 Is Required for Mitochondrial nad2 Intron 4 Cis-splicing and Seed Development in Maize Zhihui Xiu – State Key Lab of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong Feng Sun – Ministry of Education Key Lab of Plant Cell Engineering and Germplasm Innovation, School of Life Sciences, Shandong University, Yun Shen – State Key Lab of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Jianhua Zhang – State Key Lab of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong, Bao-Cai Tan – Ministry of Education Key Lab of Plant Cell Engineering and Germplasm Innovation, School of Life Sciences, Shandong University The mitochondrial genome in higher plants contains a number of group II introns that are required to be precisely spliced before translating into functional proteins, mostly components in the electron transfer chain. Different from the group II introns in prokaryotes, splicing of mitochondrial introns requires the participation of nuclear encoded factors. By analyzing the emp16 mutant in maize, here we report the EMP16 is required for nad2 intron splicing in mitochondria.

Molecular cloning indicates that Emp16 encodes a P-type PPR protein with 11 PPR motifs and is localized in the mitochondrion. The emp16 mutants are arrested in both embryogenesis and endosperm development. The null alleles are completely deficient of nad2 intron 4 cis-splicing, which severely reduces the complex I assembly and its NADH dehydrogenase activity. The mutant showed a dramatic increase in the expression of alternative oxidase 2 (Aox2). These results indicate that the EMP16 is required for the mitochondrial nad2 intron 4 cis-splicing and essential to embryogenesis and endosperm development in maize.

800-091-Z Effect of Gibberellin on the Expression of Abscisic Acid Insensitive 5 and Delay of Germination Genes in Wheat Seeds SeoHyun Jo – University of Manitoba Belay Ayele – University of Manitoba Gibberellins (GAs) and abscisic acid (ABA) are among the classical phytohormones that are involved in the regulation of seed germination and dormancy; GA is known to promote germination while ABA is antagonistic to the GA action and enhances seed dormancy. Among the major ABA signalling components involved in regulating seed germination are theabscisic acid insensitive 5 (ABI5), one of the transcription factors that control the expression of ABA responsive genes, and delay of germination (DOG), which is involved in sugar signalling and stimulates the expression of abscisic acid insensitive 4 (ABI4), another transcription factor in the ABA signalling pathway. To better understand the antagonistic interaction between GA and ABA in regulating wheat seed germination, this study examined the effect of exogenous GA treatment on the expressions of ABI5 and DOG genes during germination. Our data showed that GA does not affect the percentage of germination and the number of lateral roots produced, but it increased the length of shoot and primary root growth of wheat seedlings at the post germination stage. The expression of ABI5 and DOG genes in 12 h imbibed seeds showed over 2-fold decrease in response to GA treatment; however from 12 h to 24 h imbibition the expression of both genes decreased to a very low level and no effect of GA was evident. The results suggest that GA negatively regulate seed dormancy in wheat by repressing the expressions of ABA signalling genes such as ABI5 and DOG, resulting in decreased ABA response and lower seed dormancy.

800-092-Y Expression Patterns of Gibberellin Metabolic Genes During Seed Germination in Wheat Conrad Izydorczyk – University of Manitoba Zhen Yao – University of Manitoba, SeoHyun Jo – University of Manitoba, Belay Ayele – University of Manitoba Seed germination is regulated by several plant hormones among which gibberellins (GA) and abscisic acid play major roles. While abscisic acid delays or inhibits seed germination, GA is known to promote the germination process. To understand the changes in GA biosynthesis and catabolism during wheat seed germination, this study characterized the temporal expression patterns of genes encoding gibberellin biosynthetic (GA20- and GA3-oxidases) and catabolism (GA2-oxidase) enzymes in germinating embryos. Among the two GA20ox genes analyzed, GA20ox1 exhibited greater expression in the embryos of germinating wheat seeds than that of GA20ox2, and this result suggests the importance of GA20ox1 for the completion of wheat seed germination. The GA3ox gene, encoding an enzyme that converts the product of GA20ox, GA20, to bioactive gibberellin showed increased expression as imbibition/germination progressed. The temporal expression pattern of GA3ox shows a close association with that of GA20ox1 indicating the significance of coordinated actions of GA20- and GA3-oxidases to produce sufficient level of bioactive GA to induce embryo expansion. The low expression of GA2ox, a GA inactivating gene, observed especially by the time of germination completion suggests a decrease in GA catabolism so as to maintain bioactive GA at a level required for radicle expansion through the seed coat.

800-093-Y Structure-Function Relationships of SHORT SUSPENSOR Protein Emily Eilbert – University of Georgia Wolfgang Lukowitz – University of Georgia Embryonic patterning ultimately creates shoot and root meristems at opposite poles of the seedling. This polarity can be directly related to the asymmetric division of the zygote, which establishes an apical daughter destined to become the spherical proembryo and a basal daughter that forms the filamentous suspensor. We have shown that YODA MAP kinase signaling is required for zygote elongation and development of a normal suspensor. The pathway is activated by the SHORT SUSPENOR (SSP) gene, which acts after fertilization by paternal effect. The ability of SSP to activate the YDA MAP kinase pathway in the zygote arose by neofunctionalization, following a whole genome duplication at the base of the Brassicaciae family: while its sister gene BSK1 evolved under purifying selection, SSP has diversified rapidly. Furthermore, BSK1 cannot function equivalently in the embryo when expressed under the control of SSP regulatory elements. SSP and BSK1 both encode for dead kinases of the receptor-like family that are anchored in the plasma membrane by Nmyristoylation and contain a short C-terminal tetratricopeptide (TPR) repeat predicted to mediate protein-protein interactions. Our goal is to map the structural determinants of the SSP protein associated with its newly acquired function by testing the ability of BSK1/SSP chimeras to complement the phenotype of ssp mutant embryos. An insertiondeletion event early in the evolution of the SSP clade rearranged the activation loop of the kinase domain, but neither the activation loop nor other parts of the BSK1 kinase domain mediate SSP-specific function. However, we found that chimeric genes containing the BSK1 TPR domain are inactive. This suggests that changes in the TPR domain are responsible for the new functions acquired by the SSP protein. We’ll map these structural changes in more detail and search for SSP-specific binding partners, to address how SSP activates the YDA MAP kinase cascade.

800-094-Z Molecular Features Underlying Germination Associated Starch Catabolism in Wheat Seeds Yuji Yamasaki – University on Manitoba Aihua Liu, Feng Gao, Belay Ayele Seed dormancy is an important trait in wheat as it prevents field sprouting. Dormancy release can be achieved by various treatments including after-ripening, and this process is often accompanied by changes in hormone metabolism, and other germination related and hormone-controlled biological processes such as mobilization of storage reserves. Gibberellins (GA) and abscisic acid (ABA) are among the major plant hormones that are involved in the control of these processes. In order to gain insights into the molecular basis of starch degradation in wheat seeds with respect to dormancy release and germination, and its regulation by GA and ABA, this study performed comparative analysis of the expression of different types of starch catabolic genes between dormant, and after-ripened (with or with no ABA) seeds during imbibition. Our data indicated that after-ripening induced seed germination is associated with transcriptional activation (5- to 10-fold change) of specific genes encoding late maturity high PI (Amy1-1, Amy1-2 and Amy1-3) and other (Amy2/3) α-amylases, α-glucosidase (AGL1), and cytosolic starch-phosphorylase (PHO2), suggesting the significance of these genes in mediating starch degradation during wheat seed germination. The transcriptional activation of these starch catabolic genes is associated with enhanced expressions of the GA biosynthetic GA20ox and GA3ox genes and down-regulation of ABA signaling genes such as ABI5. Treatment of imbibing seeds with ABA caused a delay but not complete inhibition of coleorhiza emergence, and this effect of ABA is associated with repression of the expression of starch catabolism.

800-095-Z Identification of Useful Grain Quality Characteristics in Rice Mutants Using TILLING and Forward Genetics Mira Yoon – National Institute of Crop Science, Rice Research Division Areum Chun – National Institute of Crop Science, Rice Research Division, Thomas Tai – USDA-ARS Rice (Oryza sativa L.) is unique among major cereal crops as the vast majority is used directly for human consumption, usually in the form of whole milled kernels. Climate change and consumer demand pose significant challenges to rice breeders with regard to maintaining and improving various grain traits that influence appearance, eating qualities, and utilization. Our objective is to employ Targeting of Induced Local Lesions in Genomes (TILLING) and forward genetic approaches to identify chemically-induced rice mutants with grains exhibiting novel cooking, eating, and processing qualities. Towards this end, we have recently identified over 60 putative mutations in 8 starch biosynthesis-related genes from a TILLING by sequencing screen of 2,048 Nipponbare M2 individuals. We have also visually evaluated brown rice grains from the M3 generation of approximately 2,000 Kitaake and 1,200 Nipponbare M2 lines. This has resulted in the identification of > 15 putative mutants exhibiting opaque grains. Validation of the putative TILLING mutants and phenotypic characterization of the lines exhibiting altered grain appearance is underway.

Development: Pollen Biology and Mating Systems 800-096-Y A Novel Flow Cytometry-based Method for Evaluation of Pollen Stress Response and Viability Gilad Luria – Bar-Ilan University Eitay Lazar – Bar-Ilan University, Gad Miller – Bar-Ilan University The majority of food supply, which is largely based on seeds, depends on sexual reproduction in flowering plants. Reproductive development is highly sensitive to abiotic stress conditions, particularly high temperatures, with even a single hot day being deleterious to reproductive success. In many plants, pollen tube development and fertilization are often the weakest links. Therefore, it is of great importance to be able to evaluate pollen quality and viability for crop improvement and breeding. Several techniques and protocols are available for the determination of pollen viability, but most of them are inaccurate, laborious and species-dependent. Here, we present two methods for fast and reliable pollen viability assays. The first technique is based on the conversion of the tetrazolium salt, MTT (3-{4,5dimethylithiazolyl-2}-2,5-diphenyl monotetrazolium bromide) into a colorful and insoluble precipitate, formazan, once modified by active cellular enzymes. This stain was proven to distinguish the live pollen of arabidopsis, tobacco and tomato from the dead pollen, unlike other commonly used methods, such as TTC and Alexander stain. Moreover, MTT can differentiate pollen of stress-sensitive ecotypes from pollen of more tolerant ecotypes, for example, between the arabidopsis Columbia-0 and the hypersensitive ecotype, Hilversum-0. Furthermore, we found that H2DCFDA, the reactive-oxygen-species (ROS)-responsive fluorescent dye, reliably distinguishes between live and dead pollen grains. We further employed flow cytometry for estimating large-scale pollen viability experiments. This novel approach enables not only to detect active pollen grains in large population but also to test their redox state in response to stimuli such as stress, hormones and anti-oxidative agents. In summary, our results indicate that the combination of DCF staining and the use of flow cytometry provides an easy, rapid, robust, reliable and versatile method with agricultural applicability as well as potential for basic study of pollen physiology.

800-097-Y Glyoxalase I Is a Target of Brassica Self-incompatibility Response Subramanian Sankaranarayanan – University of Calgary Muhammad Jamshed – University of Calgary, Marcus Samuel – University of Calgary

Self vs non-self recognition is critical for maintaining outcrossing and hybrid vigor in plants. In Brassica species, haplotype specific interaction between the pollen-specific small cysteine-rich secreted protein (SP11/SCR) and stigmaspecific S Receptor kinase SRK is essential for a self-incompatibility (SI) response. Following self-pollination, SCR/SP11 binds to the membrane localized SRK and this receptor-ligand interaction leads to the activation of SRK, triggering a phosphorylation cascade that activates ARC1 E3 ubiquitin ligase, leading to degradation of compatibility factors needed for pollen germination and tube growth. The targets of ARC1 during SI response have mostly remained elusive. Through a proteomics approach, we identified glyoxalase I (GLO1), a metabolic enzyme, to be downregulated following selfincompatible pollination. GLO1 functions in the cell to detoxify methylglyoxal (MG; pyruvate aldehyde), an endogenous cytotoxic compound formed as a byproduct of glycolysis. RNAi-mediated suppression of GLO1 in compatible Brassica napus stigmas was sufficient to compromise the ability of stigmas to accept compatible pollen, while overexpression of GLO1 in self-incompatible Brassica resulted in partial breakdown of SI response. Self-incompatible pollination led to increased MG levels and a concomitant increase in MG-modified proteins including GLO1 in the stigmatic papillary cells. ARC1 was able to efficiently ubiquitinate GLO1 and GLO1 was constitutively upregulated in arc1 stigmas and stigmas treated with proteasomal inhibitor, suggesting that GLO1 is a direct target of ARC1 during SI response. Our findings demonstrate the elegant nature of plants to use a metabolic byproduct to regulate SI response.

800-098-Z ARC1 Is a Downstream Signalling Component of SRK in the Self-incompatibility Pathway in Arabidopsis Spp Emily Indriolo – New Mexico State University Darya Safavian – University of Toronto, Daphne Goring – University of Toronto Flowering plants have many methods to prevent self-fertilization, one method is the ability to reject self-pollen by selfincompatibility. In the Brassicaceae, this process is regulated by a signaling pathway activated by the stigma-specific S Receptor Kinase (SRK), following binding of a pollen-specific ligand, SCR/SP11. In Brassica species, downstream signaling components of the pathway have been identified such as the M Locus Protein Kinase and the ARC1 E3 ubiquitin ligase which targets the Exo70A1 subunit of the exocyst complex. While the functions of SCR/SP11 and SRK are conserved in various Arabidopsis species, the downstream signaling pathway leading to the rejection of self-pollen is less clear. We performed a genomic survey of numerous species in the Brassicaceae and determined that ARC1 is frequently deleted in self-compatible species (even though some species still had a functional SRK), indicating that ARC1 may have a conserved role in self-incompatibility signaling in the Brassicaceae. We identified an A. lyrata ARC1 homologue to Brassica ARC1, and investigated if the role of ARC1 is conserved in regulating pollen rejection in the naturally occurring Arabidopsis lyrata self-incompatibility system. We demonstrated that ARC1 was required for self-incompatibility in A. lyrata and have now shifted focus to testing ARC1 in the artificial A. thaliana self-incompatibility system. As ARC1 is required for self-incompatibility in A. lyrata, it led us to investigate what would happen when ARC1 was expressed in A. thaliana with SRK and SCR/SP11 and we examined the phenotypes of A. thaliana plants expressing all three genes. The expression of SCR/SP11-SRK-ARC1 in A. thaliana resulted in robust self-incompatibility both at the pollen-pistil level and at the cellular level. We are in the process of examining the conservation of the proposed role of ARC1 in other Brassicaceae species to determine what how ARC1 is involved in their self-incompatibility responses.

800-099-Z Aperture Formation on Arabidopsis Pollen Surface Is Regulated Though a Ploidy-dependent Mechanism and Is Guided by the INP1 Protein Anna Dobritsa – Ohio State University Sarah Reeder – Ohio State University

Pollen presents a powerful model for studying how controlled formation and deposition of extracellular structures is achieved. Pollen is surrounded by a complex extracellular structure, pollen wall exine, which assembles into intricate patterns with enormous diversity across species, yet very conserved within a species. Genetic and developmental programs controlling formation of the precise patterns on pollen surface remain a mystery. We will present data on the formation of one specific patterning element on pollen surface – pollen apertures. Apertures are areas on pollen surface, which either lack exine or have decreased exine deposition. They are speciesspecific in number, morphology, and location – Arabidopsis pollen has three equidistant longitudinal apertures. Existence of apertures indicates that, in a given species, certain areas on the pollen surface differ from others and differences are reliably recognized by the exine deposition machinery. Aperture formation in Arabidopsis depends on the novel protein INP1, which localizes to the positions of future apertures at the periphery of microspores and quantitatively controls aperture lengths. It was previously proposed that aperture number and placement are linked to the geometry of microspore tetrads arising via meiotic cytokinesis and to the number of last-contact points between the sister microspores. We now tested this model by analyzing mutants with abnormal number of apertures. We found that contact points per se do not act as aperture number determinants and the correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. Instead, the aperture number is specified via a ploidy-related mechanism. We also found that in the mutants with ectopic apertures, the number and positions of the INP1 localization sites change depending on ploidy and not on the INP1 dosage, indicating that sites for aperture formation are specified before INP1 is brought to them.

800-100-Y Molecular Dialogues Between Pollen and Pistil Mark Johnson – Brown University Alexander Leydon Pollen tubes penetrate the stigma, extend through specialized transmitting tissue, are attracted to ovules, and deliver two sperm to the female gametophyte so one can fuse with the egg to form the embryo and the other with the central cell to form endosperm. This double fertilization process depends on a system of positive and negative cell:cell interactions that optimize fertility for flowering plants. We have defined changes in pollen tube gene expression that orchestrate crucial cellular interactions as the pollen tube grows through the pistil by defining a group of MYB transcription factors that are required for this response. Pollen tubes lacking these three transcription factors are not recognized by the female gametophyte when they arrive inside an ovule; they fail to induce changes in female gametophyte cells and continue growing within the female gametophyte without releasing their cargo of sperm cells. In many respects, pollen tube mutants lacking these MYB transcription factors behave like pollen from a different species – there is a breakdown in cellular recognition required for fertilization. Our goal is to define the critical effector proteins mediating recognition between pollen tube and the female gametophyte. We have made progress via RNA-sequencing of inter-ecotypic cross-pollinations to precisely define patterns of pollen and pistil gene expression during growth of wild type and mutant pollen tubes. We’ve also been able to define a subset of MYB-regulated secreted peptides that are sufficient to restore proper identity to mutant pollen tubes. Our goal is to understand the biochemical function of these peptides that mediate cellular interactions between the gametophytes.

800-101-Y Dynamic Redistribution of MLO Proteins in Synergid Cells During Pollen Tube Reception Sharon A. Kessler – University of Oklahoma Daniel S. Jones – University of Oklahoma, Patrick Day – University of Oklahoma

Synergid cells have a unique role in plant development, their sole purpose is to attract and receive pollen tubes so that double fertilization can occur to produce viable seeds. In Arabidopsis thaliana nortia (nta) mutants, cell-to-cell communication at early stages of pollination is normal, but upon reaching the synergid, the pollen tubes continue to grow instead of bursting to release the sperm, leading to infertility. Both FERONIA, a CrRLK1L family receptor-like kinase, and MULTIPLE RESISTANCE LOCUS-O (MLO) proteins related to NTA have been shown to be involved in fungal invasion of plant epidermal cells, indicating that mechanisms for penetration of plant cells by tip-growing pollen tubes and fungal hyphae may have been conserved over evolution. In both cases, the MLO protein becomes redistributed to the site of interaction with a tip-growing cell (pollen tube or fungal hyphae). FER activity is necessary for the redistribution of NTA protein to the site of pollen tube entry into the synergid, but little else is known about the molecular mechanisms involved in this pollen tube-synergid communication system. Progress toward understanding the mechanism behind pollen tube-triggered redistribution of NTA to the filiform apparatus of synergid cells will be presented.

800-102-Z Myosin Motor Proteins in Pollen Tubes Drive Movements of Specific Organelles and Are Required for Rapid Tip Growth and Full Fertility Andreas Nebenführ – University of Tennessee Stephanie Madison – University of Tennessee, Matthew Buchanan – University of Tennessee, Jeremiah Glass – University of Tennessee, Tarah McClain – University of Tennessee Pollen tubes grow invasively into the female tissues of a flower and function as a delivery vehicle for the immotile sperm cells that are engulfed in their cytoplasm. Rapid tip growth of pollen tubes depends on efficient delivery of secretory vesicles to the apical dome where these vesicles fuse with the plasma membrane to deliver cell wall material to the growing tip. The arrival of secretory vesicles is thought to depend on cytoplasmic streaming, which is the rapid movement of organelles throughout the cell. Organelle movements depend on the action of myosin motors that move actively along the actin cytoskeleton. We tested the prediction that myosin motors are required for pollen tube growth by examining insertional knock-out mutants of five pollen-expressed myosin XI genes. Most single-gene mutants showed very little effect on seed set and only one had reduced fitness in direct competition experiments with wild-type pollen. Simultaneous loss of the closely related MYO11C1 and MYO11C2 myosin genes, on the other hand, resulted in dramatically lower seed set. This reduced fertility could be traced to slower growth of mutant pollen tubes through the female transmitting tissues as well as slower in vitro growth rates. Within mutant pollen tubes, Golgi stacks and peroxisomes moved at greatly reduced rates compared to wild-type, demonstrating that these two myosins play a central role in driving cytoplasmic streaming in pollen tubes. Curiously, the accumulation of secretory vesicles at the tip or the speed of their movements were not affected in the mutant. These results demonstrate that (a) myosin action is required for cytoplasmic streaming in pollen tubes, that (b) pollen myosins are specialized for the movement of only a subset of organelles, and that (c) reduced intracellular movements limit the ability of pollen tubes to grow.

800-103-Z Cation/H+ Exchangers on Dynamic Membranes Affect Male Fertility and Embryo Development Heven Sze – University of Maryland Daniel Czerny – University of Maryland, Senthilkumar Padmanaban – University of Maryland , Salil Chanroj – Burapha University , Kara Levin – University of Maryland The dynamic endomembrane system of plant cells is emerging as a critical coordinator of plant development, signaling and adaptation to stress. However, the molecular basis of membrane trafficking and its impact on plant life are poorly understood. Several predicted cation/H+ exchangers (AtCHX17-19) localized to the prevacuolar compartment and the plasma membrane have roles in K+ and pH homeostasis and affected protein/cargo sorting in yeast, though their roles in

plants were obscure. Homozygous chx17chx18chx19 (triple) mutant was recovered at a reduced frequency, though they showed vegetative growth similar to wild-type. Triple mutant plants showed a 65% reduction in seed set relative to wild-type and double chx mutants. Reciprocal crossing of chx17chx18chx19 mutant with wild-type indicated defect was largely in the male gametophyte. Pollen grain development was unchanged and triple mutant pollen tubes targeted most ovules. Although pollen tubes visualized by aniline blue fluorescence entered ovules, about half of the ovules remained undeveloped suggesting a failure to complete fertilization. Pods from wild-type pistil pollinated with triple mutant pollen contained a mixture of unfertilized ovules, single-fertilized ovules and developing seeds. Single fertilization events were seen as globular embryo only or endosperm only; whereas double fertilization produced an embryo and an endosperm. CHX expression in pollen tube and sperm cell suggested that altered pH and K+ homeostasis in endomembranes of male gametophyte can partially compromise male fertility. When fertilization is successful, selfpollinated triple mutants produced embryos delayed in development, suggesting a role of micropylar endospermexpressed CHX on embryo development. Our results highlight the critical roles of pH and cation homeostasis and membrane trafficking on male fertility, successful fertilization and embryo development. (Supported by DOE BES to hsze)

800-104-Y Does Parent-offspring Conflict Drive the Evolution of Hybrid Seed Lethality in Mimulus? John Willis – Duke University Elen Oneal – Duke University The origin of species involves the evolution of reproductive isolation between divergent populations. The evolution of hybrid sterility or lethality is particularly difficult to understand. What evolutionary mechanisms drive the accumulation of alleles that disrupt development in hybrids but that do not reduce fitness within populations? Parent offspring conflict, where mothers and offspring spar over the degree and length of maternal investment, has been linked to the evolution of epigenetically imprinted genes controlling normal seed endosperm development. Evidence from interploidy crosses in Arabidopsis indicates that dosage-sensitivity of paternally or maternally expressed genes in developing endosperm is critical to normal endosperm development, and hence embryonic survival. However, seed lethality is a common postzygotic barrier between diploids, and theory suggests that in diploids, species divergence in the protein sequence of regulatory elements or their targets, or gene duplication, may lead to the mis-expression of imprinted loci in hybrid seed, which are then prematurely aborted. A test of postzygotic isolation via conflict in diploid taxa requires two things: a strong hybrid seed incompatibility between a pair of primarily outcrossing species and the appropriate genomic tools to identify and characterize candidate loci. We conduct the first such test in the genus Mimulus,using two incompatible species, Mimulus guttatus and M. nudatus. We show that the primary barrier between them is endosperm development failure. With a crossing design that allows identifying the parental contribution of alleles, and thus whether imprinted genes contribute to endosperm failure, we find two major QTL that cumulatively lead to a 3.5% hybrid viability rate. Future crosses which alternate parental sources of alleles, as well as RNA sequencing of endosperm tissue, will demonstrate whether parent-offspring conflict via imprinted expression is responsible for hybrid incompatibility in Mimulus, and shed light on a dynamic that may be driving speciation in flowering plants.

800-105-Y Genetic Analysis of the Regulation of Pollen mRNA Storage in Cytoplasmic Bodies Maria Regina Scarpin – INGEBI - University of Buenos Aires Lorena Sigaut – Centro de Microscopías Avanzadas and Departamento de Física, FCEN, University of Buenos Aires, Lia Pietrasanta – Centro de Microscopías Avanzadas and Departamento de Física, FCEN, University of Buenos Aires, Jorge Muschietti – INGEBI - University of Buenos Aires

Some pollen mRNAs are synthesized during pollen development and accumulated in dehydrated mature pollen grains, being translated only after pollen germination. Our hypothesis is that the regulation of the movement and accumulation of these pre-synthesized pollen mRNAs is mediated by cytoplasmic bodies. To probe this, we analyzed the cellular localization of some defined pollen pre-synthesized mRNAs during pollen development using the MS2-CP system. We used Arabidopsis thaliana which allow us to carry genetic, molecular and developmental analysis of specific expression of pollen genes. We studied stress granules (SGs) and processing bodies (PBs) in Arabidopsis mature pollen as possible cellular structures where these pre-synthesized pollen mRNAs can be stored before being translated. Our results showed the presence of cytoplasmic mRNA aggregates in mature pollen on transgenic lines containing the MS2-CP system. We validated and quantified these mRNA aggregates using a MATLAB automated analysis. Once confirmed, we began to study the colocalization of these mRNA aggregates with stress granules (SGs) and processing bodies (PBs) by analyzing transgenic lines that express both the MS2-CP system and tagged marker proteins from either SGs or PBs. Our results would contribute to understand the mechanisms of transcriptional and translational regulation in Arabidopsis thaliana pollen grains.

800-106-Z Transcriptomic Profiling Reveals Complex Molecular Regulation in Cotton Genic Male Sterile Mutant Yu98-8A Xiaojie Yang – Economic Crop Research Institute, Henan Academy of Agricultural Sciences Weiping Fang – Economic Crop Research Institute, Henan Academy of Agricultural Sciences, Deyi Xie – Economic Crop Research Institute, Henan Academy of Agricultural Sciences, Li Sun – Biology Department, Texas Tech University, Yuanming Zhao – Economic Crop Research Institute, Henan Academy of Agricultural Sciences Although cotton genic male sterility (GMS) plays an important role in the utilization of hybrid vigor, it’s precise molecular mechanism remains unclear. To characterize the molecular events of pollen abortion, transcriptome analysis, combined with histological observations, was conducted in the cotton GMS line, Yu98-8A. A total of 2,412 genes were identified as significant differentially expressed genes (DEGs) before and during the critical pollen abortion stages. Bioinformatics and biochemical analysis showed that the DEGs mainly associated with sugars and starch metabolism, oxidative phosphorylation, and plant endogenous hormones which may play a critical and complicated role in pollen abortion.

800-107-Z A Plastid-localized Pentatricopeptide Repeat Protein Is Required for Pollen Development in Rice Yujun Liu – Zhejiang University; The Chinese University of Hong Kong Xuejiao LIU – Zhejiang University, Wenyi Wang – Zhejiang University, Jianhua Zhang – The Chinese University of Hong Kong, Juming Tu – Zhejiang University Pollen development is a critical stage in the life cycle of rice. Only several fatty acid production or transfer related plastid-localized proteins were reported involved in this process. Most studied players in pollen development are the fertility restorer (Rf) genes, which is required for the development of a functional male gametophyte in plants carrying a mitochondrial CMS gene. In the majority of cases, Rf genes encode mitochondrial-targeted pentatricopeptide repeat (PPR) proteins. Here, we report a plastid-localized PPR protein OsPPR676 surprisingly required for pollen development in rice. Disruption of the OsPPR676 gene leads to pollen sterility and albino lethal seedling phenotype in two different varieties, Nipponbare and Dongjin, respectively. The findings imply that the requirement of OsPPR676 for pollen development is dependent on genetic background in rice. The results of anther RNA in situ hybridization revealed that OsPPR676 transcript express in tapetum, which plays a major role in actively synthesizing and secreting sporopollenin precursors onto the microspore surface for pollen development. Using biomolecular fluorescence complementation (BiFC), we demonstrated that OsPPR676 interacts with Osj10gBTF3, which is a subunit of the nascent polypeptide-

associated complex (NAC) that has been implicated in regulating protein localization during translation. So Osj10gBTF3 may be involved in the subcellular localization process of OsPPR676. We also demonstrate OsPPR676 is necessary for translation of the plastid atpB open reading frame, which is an important subunit of ATP synthase. ATP is essential in the first step of fatty acid de novo synthesis in plastids. In summary, OsPPR676 protein possibly involves in a plastidial pathway for the production of fatty acid that are essential for pollen development in rice.

800-108-Y All 17 S-locus F-box Proteins of Petunia Inflata Involved in Self-Incompatibility Are Each a Component of the SCF Complex Containing Pollen-Specific Cullin1 and Skp1-Like Protein Shu Li – Penn State University Justin Williams – Pennsylvania State University, Penglin Sun – University of California Riverside, Teh-hui Kao – Pennsylvania State University Self-incompatibility (SI) is a reproductive barrier adopted by many flowering plants to prevent inbreeding and promote outcrossing. According to the collaborative non-self recognition model, proposed to explain S-haplotype-specific rejection of pollen by the pistil for Solanaceae type SI, (i) pollen of a given S-haplotype employs multiple S-locus F-box proteins (SLFs) to collectively detoxify S-RNases produced by pistils carrying non-self S-haplotypes to result in crosscompatible pollination; (ii) none of the SLFs can detoxify S-RNase produced by pistils carrying the same S-haplotype, allowing self-S-RNase to degrade pollen tube RNAs to result in self-incompatible pollination. This model predicts that each SLF is a component of an SCF complex, which mediates ubiquitination of S-RNases with which the SLF interacts. To test this hypothesis, we performed co-immunoprecipitation (Co-IP) and mass spectrometry (MS), using pollen extracts of Petunia inflata containing GFP-fused S2-SLF1 (SLF1 of S2-haplotype) as bait, and identified a pollen-specific Cullin1 (PiCUL1-P), a pollen-specific SLF-interacting Skp1-like protein (PiSSK1), and a conventional Rbx1 (PiRBX1) that are in the complex as S2-SLF1. We next used pollen extracts containing FLAG:GFP-fused PiSSK1 for Co-IP/MS, and confirmed the presence of PiCUL1-P, PiRBX1 and SLF1 in the complex. To examine whether all 17 SLFs (SLF1 to SLF17) of S2 and S3 pollen identified by pollen transcriptome analysis are assembled into similar SCF complexes, we supplemented pollen extracts containing FLAG:GFP-fused PiSSK1 with style extracts from S2S3 or S7S13 plants to see whether presence of SLF substrates could facilitate identification of SLFs. All 17 SLFs were found to co-precipitate with the bait. Of 502 F-box proteins (excluding the 17 SLFs) predicted by the pollen transcriptome, only an SLF-like protein, SLFLike1, and 16 F-box proteins having high sequence similarity with SLFs, co-precipitated with the bait. Whether PiCUL1-P and PiSSK1 of the SCF complexes identified in this work specifically function in SI is being investigated.

800-109-Y POI (Partial Outer Integuments) Acts Upstream of INO (Inner No Outer) During Ovule Development Yuan Chen – UC Berkley/PGEC Ting Zou, Sheila McCormick A genetic pathway for ovule development has previously been deduced; many of the genes involved (e.g. BEL1, ANT, ATS, SIN1, INO) encode transcription factors. Here we describe a new mutant that has seed set defects. Because the gene is highly expressed in pollen (as judged by RNA-Seq analysis) we expected a pollen defect, but surprisingly reciprocal crosses showed that the problem is on the female side. Mutant plants produce fewer seeds than wild type and the seeds have an altered shape. We used SEM to examine ovule development. The ovules were pepper-shaped; this was because the outer integument of the mutant stopped growing and flapped down, so we named the gene POI (Partial Outer Integuments). Additionally, we noticed that pollen tubes fanned out near the top of the pistil, rather than growing towards the bottom; thin sections and histochemical staining showed that the development of the transmitting tract was delayed in poi plants, which might also contribute to the reduced seed set. POI encodes a protein of unknown

function which is nuclear-localized but is not obviously a transcription factor. We analyzed the expression levels of BEL1, ANT, ATS, SIN1 in poi, and all were expressed normally, but the expression of INO (INNER NO OUTER) was significantly reduced. Because POI expression was normal in ino, we think that POI acts upstream of INO.

800-110-Z Analyses of the Early Stages of Exine Develpment in Brassica Rapa, Arabidopsis Thaliana, and Three Arabidopsis Pollen Wall Mutants by Scanning Electron Microscopy Heather Owen – University of Wisconsin - Milwaukee Andrew Kirkpatrick – University of Wisconsin - Milwaukee Mature pollen grains have a complex and multilayered protective coating called the pollen wall. The outermost layer of this wall is the exine and the inner basal layer is the intine. The reticulate pattern of the mature exine in Arabidopsis is made up of rods called columellae and connecting arches called tectum. Although all pollen grains have exine, patterning of the exine is highly variable between species. The patterns formed, therefore, are genetically controlled. While it is understood that the pattern is first visible at the tetrad stage of development, while the haploid microspores are still encased in callose, the mechanisms behind construction of the pollen wall remain enigmatic. Contemporary studies predominantly rely on sectioned tissue to elucidate the events involved in proper development. To gain a better understanding of this mechanism and how superficial features change during early developmental time, a comparative analysis of the surface morphologies of model systems Brassica rapa and Arabidopsis thaliana, as well as three Arabidopsis pollen wall mutants, was conducted. The methods developed allowed observation of the entire surface of coenocytic microsporocytes and tetrad-stage microspores with the high resolution and three-dimensional qualities of scanning electron microscopy, providing new information in the characterization of pollen wall development in both normal and mutant plants. Additionaly, it revealed unique features and characteristics of sporopollenin deposition and exine development not readily observed in sectioned tissue nor explicitly described in previous studies.

800-111-Z The Reproductive Biology of Ecuadorean Wild Tomatoes Chris Miller – Colorado State University Amanda Broz – Colorado State University, Pablo Acosta – Universidad Tecnica Particular de Loja, Tania Riofrio – Universidad Tecnica Particular de Loja, Diego Chamba – Universidad Tecnica Particular de Loja, Alejandro Tovar-Mendez – University of Missouri-Columbia, Bruce McClure – University of Missouri-Columbia, Roger Chetlat – University of California Davis, Patricia Bedinger – Colorado State University Tomatoes, as well as numerous other cultivated Solanum species, originated in South America, derived from wild species found in Ecuador, Peru and Chile. The northern margin of Solanum habrochaites, a wild tomato species, is located in southern/central Ecuador. Ecuadorean populations of S. habrochaites were analyzed for a number of reproductive characters, including inflorescence structure, floral morphology, and mating system. We also identified a specific SRNase allele associated with loss of self-incompatibility in some populations. Our data differentiates three groups of S. habrochaites, each with a distinctive set of reproductive characters.

800-112-Y A Low Activity S-RNase Is Correlated with Loss of Self-incompatability at the Southern Margin of the Wild Tomato Species Solanum Habrochaites Amanda Broz – Colorado State University Alejandro Tovar-Mendez – University of Missouri, Columbia, Bruce McClure – University of Missouri, Columbia, Armeda Van Dam – Colorado State University, Patricia Bedinger – Colorado State University

Self-incompatibility is important in preventing inbreeding depression. In many plant families, it has been reported that populations at the species range limits transition from self-incompatible (SI) to self-compatible (SC) mating systems due to mate limitation. The wild tomato species S. habrochaites includes both SI and SC populations over its native range, making it amenable to studying the transition from SI to SC. In Solanaceae and other families, SI has been genetically linked to the S-locus, which harbors S-RNase alleles. Previously, an S-RNase protein with low enzymatic activity (“hab6”) was identified in pistils of the southern-most population of S. habrochaites. We characterized the distribution of the hab-6 allele and the hab-6 protein product in over 100 individuals comprising 17 populations of S. habrochaites from central Peru and performed controlled self-pollinations to determine the reproductive phenotype of the plants. The hab-6 allele and S-RNase protein were detected only in accessions from the southern end of the species range and were uniformly correlated with an SC phenotype. Interestingly, the cDNA sequence of hab-6 S-RNase is 98% identical to an SI functional S. peruvianum S-RNase (SP11). We are currently investigating why the hab-6 S-RNase protein shows low activity both in planta and in vitro. Computer modeling coupled with molecular and biochemical techniques will be utilized to determine which amino acid residues impact hab-6 S-RNase function. These studies will further our understanding of the observed breakdown of self-incompatability at the species margin in S. habrochaites populations.

800-113-Y Pollen-Pistil Interactions in Interspecific Crosses between Members of the Tomato Clade (SOLANUM SECTION LYCOPERSICON) Yousoon Baek – Colorado State University Paul Covey – Colorado State University, Jennifer Petersen – University of California, Davis, Roger Chetelat – University of California, Davis, Bruce McClure – University of Missouri-Columbia, Patricia Bedinger – Colorado State University Interspecific reproductive barriers (IRBs) act to prevent hybridization between species. Postmating prezygotic barriers act during pollen-pistil interactions, with pistils inhibiting interspecific pollen tube growth to prevent hybridization. Pollen tube rejection often occurs in only one direction in crosses between species; i.e. interspecific crosses often display unilateral incompatibility (UI). In the tomato clade, UI generally follows the SI x SC rule, in which pistils of SI species recognize and reject pollen from SC species whereas pistils of SC species are not able to reject pollen from SI species. This suggests that SI mechanisms are involved in the UI system. We tested postmating prezygotic barriers between all 12 members of Solanum Sect Lycopersicon by examining the interspecific pollen-pistil interactions. As expected, the SI x SC rule was followed in crosses between SI and SC species. Intriguingly, UI was observed in crosses between some pairs of SC species. In addition, UI was seen in some crosses between more recently evolved SC populations of SI species and SC species. Thus, although SC species and SC populations of SI species have lost the ability to reject self-pollen, some are able to reject interspecific pollen from SC species, implying that a complete SI system is not necessary to reject interspecific pollen. Results of pollen tube rejection between species reveal a gradation of strength of pistil IRBs, with the strongest IRBs seen in S. pennellii and the weakest in the four red-fruited species which cannot reject pollen from any other species. This is the first comprehensive assessment of pollen-pistil interactions in the tomato clade, and will be useful for functional studies of reproductive barriers in the future.

800-114-Z Arabidopsis Nodulin-26 Like Intrinsic Protein 7; 1 Is Required for Pollen Cell Wall Development Under Boron Limitation Pratyush Routray – University of Tennessee Tian Li, Won Gyu Choi, Daniel Roberts Boron (B) is an essential micronutrient required by plant for its normal development. While excess of B is toxic for

plants, its deficiency leads to several defects in plants growth and development. Arabidopsis Nodulin-26 like intrinsic proteins (NIP) subfamily II members (NIP5;1, NIP6;1, NIP7;1) have been previously shown to possess boric acid channel activity. In the present study, the physiological significance of NIP7; 1 during plant reproductive development was investigated. By using NIP7;1 promoter-β-glucuronidase reporter plants, specific expression in the anthers and tapetal cells of developing flowers (developmental stages 8 to11) was observed. This expression pattern was confirmed by using recombineering plants expressing a YFP fusion protein of NIP7;1 under the control of the native promoter. Investigation of nip7;1 T-DNA insertion mutants showed a boron dependent defect in reproductive development. While wild type and nip7;1 plants showed no apparent differences under normal B concentrations, nip7;1 plants showed a significant reduction in silique size and evidence of aborted seeds when grown under limiting boric acid (0.3 µM). Under limiting B, the pollen of nip7;1 flowers showed abnormalities in shape and a defective and broken exine pattern. The plasma membrane of these defective pollen grains were detached from their cell walls supporting a defect in cell wall development. These studies suggest NIP7;1 functions as a boric acid channel that is required for normal pollen development

800-115-Z HT-proteins Are Involved in Multiple Interspecific Pollen Rejection Mechanisms in the Tomato Clade Alejandro Tovar-Mendez – University of Missouri Lu Lu, Bruce McClure Unilateral interspecific pollen rejection (UI) often follows the SI x SC rule where self-incompatible (SI) species reject pollen from self-compatible (SC) species, but the reciprocal crosses are compatible. The generality of the SI X SC rule is understood to reflect partial mechanistic overlap between interspecific pollen rejection and S-RNase-based SI. For example, both S-RNase and HT-proteins contribute to SI as well as some types of UI. However, other mechanism(s) also contribute to UI. For example, contrary to the SI X SC rule, UI is also sometimes observed in SC X SC crosses in the tomato clade (Solanum sect. Lycopersicon). Three examples are well documented: pollen from cultivated tomato (SC S. lycopersicum) is rejected by SC accessions of S. arcanum LA2157, S. habrochaites LA0407, and SC S. pennellii LA0716 all of which have S-RNase loss-of-function mutations. Thus, these instances of UI are clearly S-RNase-independent. However, each accession retains HT-protein expression and the role of these proteins had not been assessed. We tested this by suppressing HT-protein expression in LA2157, LA0407, and LA0716 and pollinating with S. lycopersicum VF36 pollen. The results showed that HT-suppression in LA0716 has a quantitative effect on S. lycopersicum pollen tube growth although VF36 pollen tubes did not penetrate to the ovary. In contrast, HT suppressed LA2157 and LA0407 plants accepted S. lycopersicum pollen and viable seeds were produced. These results suggest that HT-proteins are involved in at least two interspecific pollen rejection mechanisms: one that requires S-RNase and another that is S-RNaseindependent.

800-116-Y Ion Dynamics in Pollen Tubes: Oscillatory Signatures Underlying Distinct Growth Regimes Maria Teresa Portes – University of Maryland Daniel Santa Cruz Damineli – University of Maryland, José Feijó – University of Maryland Oscillations are known to play a role in diverse processes in plant development, such as stomatal aperture, lateral root formation, root endosymbiosis, and flowering time. In pollen tubes, oscillations involve the coordination of many cellular processes including apical growth, vesicle trafficking, actin cytoskeleton, and ion dynamics, although their precise relationships and biological functions remain unknown. Herein, we investigated the associations of specific oscillatory signatures in extracellular ion fluxes/intracellular ion concentration with changes in growth regimes in pollen tubes of Nicotiana tabacum and different Arabidopsis ecotypes (Col-0, WS, C24), taking advantage of non-invasive techniques

and long-term live-cell imaging to acquire simultaneous measurements. We found that pollen tubes show a remarkable difference in amplitude and frequency of ionic oscillations in growing compared to non-growing regimes in all species analyzed, being such pattern consistent in all ions evaluated (calcium, protons and chloride). Accordingly, growth arrest induction triggered a distinct ionic response characteristic of the species analyzed. These results suggest that tunability of ion flux oscillations can underlie the capacity to modulate growth. Thus, the interspecific and intraspecific differences observed can reveal distinct functions of ion dynamics. We argue that integrated approaches like this can help to elucidate the role of ion oscillations and the transduction mechanism of oscillatory signals in pollen tube growth.

800-117-Y Glutamate Receptors in the Pollen of Arabidopsis Thaliana - On the Calcium Branch Michael Wudick – University of Maryland Maria Portes – University of Maryland, Cláudia Campos – Instituto Gulbenkian de Ciência, José Feijó – University of Maryland The genome of Arabidopis thaliana contains 20 genes homologous to mammalian ionotropic Glutamate Receptors (iGluRs), denominated Glutamate Receptor-like genes (GLRs). Arabidopsis GLRs group into three clades, are able to oligomerize and show a broad, overlapping expression pattern with no obvious preferential tissue expression. Functional redundancy and genetic compensation are therefore likely to occur. Indeed, we found that inactivation of multiple AtGLR genes often only causes mild macroscopic phenotypes. We previously showed that AtGLRs are Ca2+ channels with important implications in plant reproduction. Using Arabidopsis pollen as our model cell system and focusing on pollen-abundant GLRs from all three clades, we were able to further describe a variety of in-vitro phenotypes, ranging from branching pollen tubes, slower tube growth and decreased Ca2+ fluxes. In a different approach, we recently analyzed a group of GLR-interacting proteins. Our data show that these proteins are not only able to alter the sub-cellular localization of GLRs but also modify their electrophysiological properties, thereby establishing a new angle for the characterization of GLRs and other membrane proteins.

800-118-Z Class III Pistil Extension-Like Protein and Regulation of Pollen Tube Growth in Nicotiana Camila Alves – University of Minnesota Andrzej Noyszewski – University of Minessota, Yi-cheng Liu – University of Minnesota, Alan Smith – University of Minnesota The transmitting tract (TT) is the pathway for pollen tube growth from the stigma to the ovules where fertilization occurs. Nicotiana tabacum transmitting tissue facilitates or inhibits pollen tube growth in a species-dependent manner. Ablation of the TT changed the rate pollen tubes from different species grew in N. tabacum styles. Arabinogalactan proteins (AGPs) are found in the TT and are known to be involved in regulation of pollen tube growth. The Class III Pistil Extensin-Like Protein (PELPIII) is a N.tabacum AGP that inhibits pollen tube growth of N. obtusifolia and N. repanda in styles resulting in prezygotic interspecific incompatibility (II). The goal of this research was to understand the function of PELPIII in prezygotic II among Nicotiana spp. My objective was to produce N. tabacum RNAi-PELPIII knockdown lines to eliminate PELPIII accumulation and to overexpress PELPIII. qRT-PCR analysis of PELPIII mRNA in knockdown lines, showed very low levels of PELPIII. The overexpression PELPIII lines had mRNA levels slightly higher than normal N. tabacum plants. Pollen tube growth assays were performed in plants with different levels of PELPIII. There was no significant difference in pollen tube growth of N. kawakamii, N. otophora, N. suaveolens and N. veluntina in the PELPIII

knockdown lines, the PELPIII overexpressing lines or normal N. tabacum plants. Pollen tube growth of N. obtusifolia increased and N. tabacum decreased in N. tabacum PELPIII knockdown lines, however, no difference was observed in overexpression PELPIII lines. These results implicate PELPIII as a regulator of prezygotic II in some species but it can also facilitate self pollen tube growth.

800-119-Z Arabinogalactan Proteins Regulate Pollen Tube Growth and Interspecific Incompatibility in Nicotiana Andrzej Noyszewski – University of Minnesota, Yi-cheng Liu – University of Minnesota, Camila Alves – University of Minnesota, Alan Smith – University of Minnesota The transmitting tract (TT) of Nicotiana tabacum controls pollen tube growth in part by secreting arabinogalactan proteins (AGPs) into the extracellular matrix. The TT AGPs include: the Class III Pistil-Specific Extensin-Like protein (PELPIII) that is essential for prezygotic interspecific incompatibility (II) with N. obtusifolia and N. repanda; the 120 kDa protein (120K) that is required for N. alata self incompatibility and the Transmitting Tissue Specific protein (TTS) that promotes self pollen tube growth in N. tabacum. We hypothesized that AGP polymorphisms among Nicotiana is important for controlling pollen tube growth and prezygotic interspecific incompatibility. The cDNAs of PELPIII, 120K and TTS were sequenced from multiple Nicotiana spp. Conserved and divergent regions and codons under positive selection were identified. The N terminal domain (NTD) has a high level of insertion-deletion (INDEL) polymorphisms among species and among AGPs and was predicted to be an Intrinsically Disordered Region (IDR). It is possible that the high level of polymorphism in the NTD is responsible for variable posttranscriptional modifications that regulate II among Nicotiana spp. The C terminal domain (CTD) was predicted to have a globular structure (highly conserved among species and among TT AGPs) followed by a short IDR. The six cysteines found in the NTD were conserved among species and among TT AGPs. TTS had an overall greater amino acid conservation among species relative to the PELPIII and 120K genes. Different methods detected positively selected codons, between 3 to 8 for PELPIII, 1 to 12 for 120K, and 1 to 2 for TTS. The positively selected codons were mainly located in the IDR of each protein. High conservation of the CTD among all species and the three TT AGPs indicates a common and biologically important function for this domain.

800-120-Y RNA Editing -- Does It Have a Role in Maize S-type Cytoplasmic Male Sterility? Terry Kamps – New Jersey City University Rebecca McGowan – New Jersey City University, Karen C Chamusco – University of Florida, Susan Gabay-Laughnan – University of Illinois at Urbana-Champaign, Christine D Chase – University of Florida Cytoplasmic male sterility (CMS) is maternally inherited inability to shed viable pollen and results from the interaction of nuclear and mitochodrial expressed gene products. Male fertility in the maize CMS-S system is determined by a gametophytically expressed nuclear restorer of fertility (Rf) gene. The known Rf genes include the commercially utilized Rf3 gene and a collection of unlinked restorers, some of which are homozygous lethal (rfl) for maize kernel development. Post-transcriptional modifications of mitochondrial expressed genes have been linked with pollen rescue in several CMS systems. We have profiled transcripts and protein products of genes encoding subunits of ATP synthase in developmentally staged pollen and immature ears (IE) which, except in the case of homozygous rfl genes, lack a phenotype for restorer genes. Transcript and protein accumulation of the atp4, atp6, atp8, and atp9 genes were compared between CMS-S and normal cytotype isogenic lines lacking an Rf gene. In general the quantity of transcripts and proteins were reduced in microspores compared to IE, the reduction being more pronounced in CMS-S microspores. Differences in atp6 and atp1 transcript and protein accumulation that were assayed from restored fully developed pollen from CMS-S cytotypes were correlated with the presence of either the Rf3, rfl1, or a rfv gene. We examined RNA editing, a post-transcriptional feature of plant mitochondrial gene expression to test it as possible mechanism of the Rf

genes in determining protein accumulation of ATP synthase subunits. We report on the editing patterns including identification of bases which are partially edited, from sequence analysis of RT-PCR products for atp4, atp6, atp8, and atp9. Results indicate that RNA editing post-transcriptional modification of these genes is not involved in restoration of male fertility of CMS-S cytotypes. Analysis of RNA editing of atp1 transcripts is in progress.

Development: Space Biology 800-121-Y Arabidopsis in Space: From Microgravity to Mechanical Signaling and Back Simon Gilroy – Department of Botany, University of Wisconsin-Madison Won-Gyu Choi – Department of Botany, University of Wisconsin-Madison, Sarah Swanson – Department of Botany, University of Wiscosin-Madison, Richard Barker – Department of Botany, University of Wiscosin-Madison, Su-Hwa Kim – Department of Botany, University of Wiscosin-Madison In spaceflight, a complex suite of interacting stimuli operate against the background of reduced gravity leading to a poorly understood range of stress responses that have been collectively termed "space syndrome". For example, mechanical signals such as the forces generated the plant’s own weight are disrupted in space, as are the directional cues offered by gravity. Thus, spaceflight offers a unique laboratory in which to ask which plant processes are shaped by gravitational and mechanical forces. In addition characterizing plant growth and development in space is allowing dissection of the pathways that are triggered or suppressed by the spaceflight environment. We have grown Arabidopsis seedlings on the International Space Station as part of two flights (CRS2 and CRS4). These flights included wild-type plants and mutants in elements of touch sensing (tch2/cml24), Ca2+ signaling (cax2) and several ecotypes (Ler, Cvi, Ws, Col_0). We have generated RNAseq datasets from all these lines and compared them to controls grown under identical conditions on the ground. Molecular fingerprints of mechanical and a range of other stress responses such as hypoxia are contained within the transcriptional data. We are using mutants in the space-responsive genes identified from this transciprtomic profiling to perform high throughput phenotypic screening and qPCR analysis of stress markers. This analysis is helping define pathways important for response to mechanical and gravitational cues. It is also revealing a role for hypoxic stress in the suite of responses seen in spaceflight grown plants. This research is supported by NASA NNX12AK79G, NNX13AM50G, NNX14AT25G.

800-122-Z Spaceflight Biology - Growing Insight from a Novel Environment Anna-Lisa Paul – University of Florida Robert Ferl – University of Florida Gravity is one of the fundamental tropic forces that impact plant growth and development, and the dissection of gravityrelated signaling has been a rich source of insights into the metabolic paths plants take as they response to changes in their environment. Disrupting the gravity vector has long been used in the study of plant tropism (e.g. Darwin and Darwin, 1880, The Power of Movement in Plants), but it was not until the access to space in the mid 1960’s that it was possible to actually take gravity out of the equation. Conducting experiments in a microgravity environment has been an appealing idea to many plant biologists, and since those first early days of catching a ride on a satellite (e.g. Biosatellite II) plant experiments have been conducted in the spaceflight environment on virtually every vehicle that can provide access (e.g. Salyut, MIR, Skylab, Space Shuttle, ISS, Tiangong). The insights that these experiments have contributed to our understanding of plant processes are as varied as the experiments themselves, and extend far beyond gravitropism. Plants know that they are in a novel environment, and adjust by employing alternative tactics to guide growth and development than are employed on the ground. These adjustments have been characterized in terms of differential

changes in morphology, metabolism, transcriptome, proteome, cellular structures and more. The presentations in the Space Biology mini-symposium will provide a cross section of current research from several research groups in the field.

800-123-Z Identification of a Novel Protein Involved in Actin-mediated Directional Root Skewing from Ground- and Spacebased Plant Experiments Elison Blancaflor – The Samuel Roberts Noble Foundation J. Alan Sparks – The Samuel Roberts Noble Foundation, Taegun Kwon – The Samuel Roberts Noble Foundation Our previous studies showed that roots treated with the actin disrupting compound Latrunculin B (LatB) had stronger gravitropic responses on Earth. In related experiments with the Biological Research in Canisters (BRIC) hardware on the second to the last flight of the Space Shuttle Discovery (STS-131), we found that knockouts to vegetative actin isoforms in Arabidopsis had more robust directional root skewing in microgravity. Thus, both ground- and microgravity- based experiments point to the actin cytoskeleton as a regulator of differential growth in roots. To gain new insights into the role of actin in directional root growth, we identified mutants that showed altered responses to low doses of LatB. Seedlings of one recessive mutant were hypersensitive to nanomolar concentrations of LatB but not to microtubule inhibitors. Interestingly, the strong root skewing responses triggered by the microtubule stabilizing drug taxol, were dampened in the mutant suggesting that the disrupted gene might be involved in crosstalk between the actin and microtubule cytoskeleton. Whole genome sequencing of the LatB hypersensitive mutant led to the identification of a deletion in a gene encoding a protein of unknown function with conserved tetratricopeptide repeat (TPR) domains. A native promoter-driven green fluorescent protein (GFP) to the TPR-domain containing protein complemented the root skewing phenotypes and the fusion protein colocalized with trans-Golgi Network (TGN) and early endosome markers. Coimmunoprecipitation revealed that a Guanine Nucleotide Exchange Factor (GEF) small GTPase regulator interacted with the TPR protein, and GEF mutants phenocopied the TPRmutants. Interestingly, TPR mutants were defective in exocytosis whereas GEF mutants had altered endocytosis. Taken together, our studies led to the discovery of a novel TPR-domain containing protein that could function at the crossroads of cytoskeletal and membrane trafficking pathways in the control of directional root growth (supported by NASA grants NNX10AF43G and NNX12AM94G).

800-124-Y Plant Growth Strategies Are Remodeled by Spaceflight – Organ Specific Changes in the Transcriptomes and Proteomes of Arabidopsis Thaliana Robert Ferl – University of Florida Anna-Lisa Paul – University of Florida Arabidopsis thaliana plants were grown on the International Space Station within hardware that combined a plant growth habitat with a camera system (the TAGES GFP Imager System - GIS) that can capture images at regular intervals of growth. In the absence of gravity, but the presence of light, spaceflight roots remained strongly negatively phototropic and grew in the opposite direction of the shoot growth; however, cultivars WS and Col-0 displayed two distinct, marked differences in their growth patterns. WS skewed strongly to the right on orbit, while cultivar Col-0 grew virtually without deviation away from the light source. Skewing and waving, thought to be gravity dependent phenomena, occur in spaceflight plants. Changes in the transcriptome of these plants demonstrated organ-specific changes in response to spaceflight. Leaves, hypocotyls, and roots each displayed unique patterns of response, yet many gene functions within the responses are related. Particularly well represented across the dataset were genes associated with cell architecture and growth hormone signaling. As examples, differential expression of genes involved with touch, cell wall remodeling, root hairs, and cell expansion may correlate with spaceflight-associated root skewing, while differential expression of auxin-related and other gravity-signaling genes seemingly correlates with the microgravity of

spaceflight. Proteomic analyses of plants of the same cohort also showed organ-specific patterns of differential representation between spaceflight and ground controls. These results are discussed in terms of emergence understanding of plant adaptations to spaceflight, the roles of gravity in determining root morphology, as well as in the context of deriving multiple omics datasets from a single on-orbit preservation and operations approach.

800-125-Y Transcriptional Regulation of Seedling Development in Microgravity Imara Perera – North Carolina State University Eric Land – North Carolina State University, Heike Sederoff – North Carolina State University Plants are a vital part of human life support systems for long-duration space flight and habitation. However, the space environment is not optimal for plant growth and plants grown in space are subject to many unfamiliar stresses (in addition to the lack of gravity). Understanding the molecular mechanisms by which plants sense and adapt to changes in this environment is essential for generating plants that are better adapted to withstand space flight, microgravity, and adverse conditions. In our spaceflight experiment “Plant Signaling in Microgravity” (carried out on the International Space Station), we compared transcript profiles of wild type and transgenic InsP 5-ptase plants with compromised InsP3 signaling. The transgenic Arabidopsis plants were generated to constitutively express the mammalian type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme that specifically hydrolyzes the lipid-derived second messenger inositol 1,4,5-trisphosphate (InsP3). These transgenic plants exhibit normal growth and morphology; however, their responses to environmental stimuli including gravity and drought are altered. Results from this study have uncovered regulatory mechanisms that are both conserved and altered between the wild type and transgenic seedlings. One conserved result (between wild type and transgenic seedlings) is the up regulation of several cis-natural antisense (cisNAT) genes in microgravity. Cis-NATs produce antisense transcripts at the same genomic locus and these results support the involvement of small regulatory RNAs in gene regulation in microgravity. One significant difference between wild type and transgenic plants is the up regulation of photosynthesis-related genes in InsP-5ptase transgenic roots over wild type specifically under microgravity conditions. These results suggest that some aspects of light signaling maybe misregulated under microgravity and that the InsP3-mediated pathway may play a role in the integration of light and gravity cues in plants.

800-126-Z Using Brachypodium Distachyon to Investigate Monocot Responses to Gravistimulation and Adaptation to Spaceflight Shih-Heng Su – University of Wisconsin-Madison Richard Barker – University of Wisconsin-Madison, Patrick Masson – University of Wisconsin-Madison Gravity, a constant force on earth that provides a cue for plant growth direction, is reduced to negligible levels (microgravity) during spaceflight or on the International Space Station (ISS). Because plants will be essential components of regenerative life-support systems on spacecraft during long-term space-exploration missions, it is essential to better understand their morphological and molecular responses to microgravity, an environment never experienced during evolution on Earth. In the past, most investigations of plant responses to microgravity have been performed using the dicotyledonous model plant, Arabidopsis thaliana. Yet, most crops grown on Earth for food, feed and fiber production are monocotyledons. To better understand the molecular and morphological responses of monocots to gravity and microgravity environments, we initiated experiments with Brachypodium distachyon, a novel genetic model for monocots. Multiple sequenced Brachypodium accessions are available, providing a large source of natural variation that can be mined for loci contributing to specific traits. We first investigated the sensitivity of seedling roots to gravistimulation (reorientation within a 1xg gravity field) and the kinetics of root gravitropic curvature for 39 distinct

Brachypodium accessions. The results showed a large variability between accessions for gravisensitivity (as determined through both the logarithmic and hyperbolic models discussed in Perbal et al., 2002, Physiol Plant 114: 336-342), kinetics of curvature response, and in the development of strong root-tip oscillations during a gravitropic response. This large variability between accessions in important characteristics of the gravitropic response suggests the possibility of using Genome-Wide Association Studies (GWAS) to identify contributing loci. It also provides outstanding material for investigations of the diversity in growth, morphological and molecular responses to microgravity for this monocot. This work is supported by a grant from the National Aeronautics and Space Administration.

800-127-Z Gravity Induces an Asymmetrical Accumulation of Extracellular Nucleotides That Can Alter Polarization in Ceratopteris Spores Ashley Cannon – The University of Texas at Austin Diana Vanegas – The University of Florida, Greg Clark – The University of Texas at Austin, Eric McLamore – The University of Florida, Stan Roux – The University of Texas In many plant cells and tissues, gravity is a dominant factor that directs asymmetric or polarized growth, leading, e.g., to shoots growing up and roots growing down. The Ceratopteris spore is a model system for studying the molecular characteristics of gravity-directed polarization of single cells. In these cells, polarization begins with the entry of calcium through channels along the bottom of the spore, which is essential for the cell’s downward polarization. Recent data have shown that treatment with applied nucleotides or a purinoceptor antagonist can alter gravity-directed cell polarization in Ceratopteris spores. In Arabidopsis, gravity can induce the opening of mechanosensitive channels, these channels can release ATP, and extracellular ATP (eATP) can promote Ca2+ uptake into cells. Collectively, these studies led to the hypothesis that ATP may be released from gravity-activated mechanosensitive channels preferentially at the bottom of vertically-oriented spores, that this release would result in an asymmetrical accumulation of eATP, and that this could activate Ca2+ channels primarily along the bottom of the spore. In order to test this hypothesis, an amperometric ATP biosensor was used to measure the [eATP] at both the bottom and top of germinating spores during their gravity-directed polarization. The [eATP] at the bottom of the spore was on average 8-fold higher than the concentration at the top. Because chemically blocking eATP receptors alters the direction of spore polarization, these data support the hypothesis that the gravity-induced asymmetrical accumulation of eATP can help direct the polarization of Ceratopteris spores. To determine if there is a link between eATP and calcium uptake, spores expressing the yellow cameleon Ca2+ sensor will be used to monitor changes in [Ca2+]cyt when extracellular nucleotides or ATP receptor antagonists are applied.

800-128-Y Plant Gravitropic Signal Transduction: A Network Analysis Leads to Gene Discovery Sarah Wyatt – Ohio University Colin Kruse – Ohio University Gravity plays a fundamental role in plant growth and development. Although a significant body of research has helped define the events of gravity perception, the role of the plant growth regulator auxin, and the mechanisms resulting in the gravity response, the events of signal transduction, those that link the biophysical action of perception to a biochemical signal that results in auxin redistribution, those that regulate the gravitropic effects on plant growth, remain, for the most part, a “black box.” Using a cold affect, dubbed the gravity persistent signal (GPS) response, we developed a mutant screen to specifically identify components of the signal transduction pathway. We have further exploited the GPS response using a multi-faceted approach including gene expression microarrays, proteomics analysis,

and bioinformatics analysis to identified additional genes and biochemical processes. Gene expression data provided the foundation of a regulatory network for gravitropic signaling. Based on these data and related data sets from the literature/repositories, we constructed a gravitropic signaling network for Arabidopsis inflorescence stems. Both a dynamic Bayesian network and a time-lagged correlation coefficient approaches were used. The dynamic Bayesian network added existing information of protein-protein interaction while the time-lagged correlation coefficient allowed incorporation of temporal regulation and thus could incorporate the time-course metric from the data set. Thus the methods complemented each other and provided us with a more comprehensive evaluation of connections. Each method generated a list of possible interactions associated with a statistical significance value. The two networks were then overlaid to generate a more rigorous, intersected network with shared genes and interactions. This network is flexible and can be updated with new data from the original research. The network allows identification of hubs/additional components and processes that are involved in gravitropic signal transduction to provide further hypotheses for testing. Partially supported by NSF IOS #1147087.

800-129-Y A Discovery-based, Proteomic Analysis of Arabidopsis Seedlings Grown in Microgravity Proma Basu – Ohio University Darron Luesse – Southern Illinois University Edwardsville, Sarah Wyatt – Ohio University On Earth plants are constantly exposed to a gravitational field of 1g. Gravity affects a plant in every step of its development. Germinating seedlings orient their radicle and hypocotyl and growing plants position organs at specific Gravitropic Set-point Angles as dictated by the asymmetric distribution of auxin depending on the gravity vector. Hence gravitropism is one of the fundamental growth responses in plants. For any experiment studying the effects of gravity, the most appropriate control is the microgravity of space. This study aims to compare the proteome of three-day-old Arabidopsis seedlings grown in microgravity aboard the International Space Station to seedlings grown on Earth. The discovery proteomics approach focused on the total proteome and the post-translational modifications which may govern protein location and function. For this, about 22,000 Arabidopsis Wild-Type Col-0 seeds were sterilized, plated on twenty two, 60mm petri plates and cold stratified for 16 hours followed by 2 hours of white light treatment. The seeds were then kept in cold stasis at 4oC until spaceflight to ensure germination in microgravity. A similar set of WT Col-0 seeds were prepared and kept on Earth as ground controls for this experiment. After spaceflight, membrane and soluble proteins were extracted, digested and labelled with iTRAQ reagents and analyzed via tandem mass spectrometry. The relative abundance of proteins in the spaceflight samples vs. ground controls will help identify proteins that may be crucial for response to gravity. This work was partially funded by NASA grant # NNX13AM48G to SEW and DRL.

800-130-Z Cell Wall Peroxidases Repressed Under Microgravity Are Required for Arabidopsis Root Hair Development Taegun Kwon – The Samuel Roberts Noble Foundation Alan Sparks, Jin Nakashima, Stacy Allen, Yuhong Tang, Elison Blancaflor Expression of transcripts associated with oxidative stress and cell wall remodeling were reduced in Arabidopsis thaliana seedlings grown in microgravity for two weeks. A majority of genes down-regulated in microgravity were previously shown to be enriched in root hairs. The reduced root hair length observed in seedlings grown in space was consistent with our gene profiling results. Furthermore, mutations in several genes that were down-regulated in microgravity, including two uncharacterized root hair-expressed class III peroxidase genes (PRX44 and PRX57), led to defective polar root hair growth on Earth. Root hairs of PRX44 and PRX57 mutants had ruptured tips, which is a typical phenotype of tip growing cells with defective cell walls and those subjected to stress. Taken together, our results indicate that long term

exposure of plants to microgravity can have a negative impact on tip growth by downregulating genes essential for normal root hair development. Whereas changes in peroxidase gene expression leading to reduced root hair growth in space are actin-independent, phosphoinositide-modulated root hair development could be dependent on an intact actin cytoskeleton. These results have profound implications for plant adaptation to microgravity given the importance of tip growing cells such as root hairs for efficient nutrient capture.

800-131-Z ROS Release and Gravitropism Correlate with Starch Content in Zea Mays L. Stem Pulvini Shelby Hartwell – New College of Florida Amy Clore – New College of Florida The signaling pathway involved in plant gravitropism is not yet fully understood. Grass stems, such as that of Zea mays L., are excellent models for studying the response to gravistimulation because the response is isolated to pulvini. The pulvinus is a disk of tissue apical to the node responsible for cell elongation that causes bending when the stem is displaced from the vertical position. Previous work in our lab pointed to a role for reactive oxygen species (ROS) in the pulvinus gravitropic signaling pathway, likely associated with amyloplasts. ROS was found to rapidly increase, primarily in cells around vascular bundles throughout the response period, eventually concentrating in the lower half of the pulvinus as seen by 72 hours. To further test this hypothesis, in the present study, B73 maize plants were placed in darkness for 7 days to “burn-off” starch present in the plastids. These plants, along with positive controls grown in light conditions, were then gravistimulated for 1 minute or 72 hours and stained for ROS using 3,3’-diaminobenzidine (DAB). Maize plants kept in darkness had measurably less starch present in their pulvini. These results were consistent with amount of bending in response to gravistimulation, as less starch correlated with an attenuated bending response. The results of DAB staining further suggest that starch-filled amyloplasts are necessary for the full extent of ROS release.

BIOTIC INTERACTIONS - Zone 900 Biotic Interactions: Plant-Insect 900-001-Y The Role of Stress-related Transcription Factors in the Response of Arabidopsis Thaliana to Different Insect Herbivores Erin MacNeal Rehrig – Fitchburg State University Heidi Appel – University of Missouri, A. Daniel Jones – Michigan State University, Jack Schultz – University of Missouri Plant responses to insects and wounding involve substantial transcriptional reprogramming that integrates hormonal, metabolic, and physiological events. The ability to respond differentially to various stresses, including wounding, generally involves hormone signaling and trans-acting regulatory factors. Evidence of the importance of transcription factors (TFs) in responses to insects is also accumulating. However, the relationships among hormone signaling, TF activity, and ability to respond specifically to different insects are uncertain. We examined transcriptional and hormonal changes in Arabidopsis thaliana after herbivory by larvae of two lepidopteran species, Spodoptera exigua (Hübner) and Pieris rapae L. over a 24-hour time course. Transcriptional responses to the two insects differed and were frequently weaker or absent in response to the specialist P. rapae. Using microarray analysis and qRT-PCR, we found 141 transcription factors, including many AP2/ERFs (Ethylene Response Factors) and selected defense-related genes, to be differentially regulated in response to the two insect species or wounding. Jasmonic Acid (JA), JA-isoleucine, and ethylene production by Arabidopsis plants increased after attack by both insect species. However, the amounts and timing of ethylene production differed between the two herbivory treatments. Our results support the hypothesis that the different responses to these two insects involve modifications of JA-signaling events and activation of different subsets of ERF transcription factors, resulting in different degrees of divergence from responses to wounding alone.

900-002-Z DAMPs as Multifunctional Mediators of Damaged-self Recognition and Early Resistance Across the Tree of Life Martin Heil – CINVESTAV Irapuato Multicellular organisms suffer injury and serve as hosts for microorganisms. Therefore, they require mechanisms to detect injury and to distinguish the self from the non-self and the harmless non-self (microbial mutualists and commensals) from the detrimental non-self (pathogens). Plants, like all other multicellular organisms, perceive endogenous molecules or their fragments as ’damage-associated molecular patterns’ (DAMPs) when these are released from the disrupted host tissue [1-3]. Seemingly ubiquitous DAMPs are extracellular ATP or extracellular DNA, fragmented cell walls, peptides, and delocalized molecules or fragments of macromolecules that are released when preexisting precursors come into contact with enzymes from which they are separated in the intact cell. These DAMPs enable damaged-self recognition, initiate processes aimed at restoring homeostasis, and prime the adjacent tissues for the perception of the invaders. Knowledge concerning DAMP-mediated immunity in mammals will help to decipher early responses in plants to damage inflicted by herbivores and pathogens. A double-function as signal and direct resistance agent appears to be a common characteristic of DAMPs in both, plants and mammals, and Ca2+- fluxes, membrane depolarization, the liberation of reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) signaling cascades are the ubiquitous molecular mechanisms that act downstream of DAMPs in organisms across the tree of life. Damaged-self recognition is likely to have evolved in all eukaryotic kingdoms, because all organisms must recognize damage without depending on enemy-derived molecules and because responses to the non-self must be directed specifically against detrimental invaders. 1. Heil, M. (2009) Trends Plant Sci. 14, 356-363. 2.

Heil, M., Ibarra-Laclette, E., Adame-Álvarez, R.M., Martínez, O., Ramirez-Chávez, E., Molina-Torres, J., and Herrera-Estrella, L. (2012) PLoS ONE 7, e30537.

3.

Heil, M., and Land, W.G. (2014) Frontiers in Plant Science 5, art. 579.

900-003-Z Caterpillar and Rootworm Feeding Differentially Affects Defense Protein Accumulation in Corn Lina Castano-Duque – The Pennsylvania State University Dawn Luthe – The Pennsylvania State University When corn is attacked by insect herbivores it responds by accumulating a suite of defense proteins. Two defense proteins that are produced in response to foliar feeding by fall armyworm (FAW) are the insecticidal cysteine protease Mir1-CP and ribosome inactivating protein-2 (RIP2). However, there is little information regarding the accumulation of these defense proteins in response to belowground feeding by the western corn rootworm (WCR). Furthermore, the effects of jasmonic acid (JA) and ethylene (ET) on these defense proteins regulations are unknown. In this study we show that the kinetics and levels of mir1 and rip2 transcript accumulation in whorls and roots is different depending on the herbivore attacking the plant. Immunoblot analysis indicated that foliar FAW feeding increased Mir1-CP abundance in both whorls and roots, and root feeding by WCR increased Mir1-CP abundance in these two organ suggesting a systemic response. On the other hand, RIP2 protein abundance increased only in the tissues immediately attacked by FAW or WCR. The effects of blocking JA synthesis or ET perception on Mir1-CP and RIP2 accumulation during FAW or WCR infestation suggests that insecticidal proteins could have different hormonal regulation in whorl and roots depending on the insect attacking the corn plant.

900-004-Y Recognition of Insect Eggs by Arabidopsis Thaliana Andre Schmiesing – University of Lausanne Philippe Reymond Plants activate direct and indirect defenses in response to insect egg deposition. In Arabidopsis thaliana, oviposition by the Large White butterfly Pieris brassicae triggers cellular and molecular changes that are similar to the changes caused by biotrophic pathogens, including salicylic acid (SA) accumulation, local cell death and the expression of early pathogenresponsive genes. Furthermore, perception of egg-derived elicitors was impaired in a lectin receptor-like kinase (LecRLK). These findings point towards a similar mechanism known for the recognition of pathogens through pathogenassociated molecular pattern (PAMP)-triggered immunity (PTI). However, the nature of egg-derived elicitors that trigger the defense response in Arabidopsis after oviposition by Pieris brassicae is unknown. In this study, we used analytical methods (UHPLC-TOFMS, NMR) combined with reporter gene validation to identify two active elicitors inside P. brassicae eggs. The first molecule is a triacylglycerol (TAG) which is a major storage lipid in insect eggs. The second compound is a linalool conjugate and is so far not present in any database. Spodoptera litorallis and Drosophila melanogaster eggs do not contain this molecule, although they are able to trigger defense responses in Arabidopsis, suggesting that plants may be able to discriminate between eggs from generalist and specialist herbivores. Further characterization and validation of these novel elicitors is underway.

900-005-Y Comparitive Transcriptomics of Plant-insect Interactions Sumitha Nallu – University of Chicago Carlos Sahagun – University of Chicago, Marcus Kronforst – University of Chicago, Sumitha Nallu – Carlos Sahagun, Marcus Kronforst Plants and insect herbivores are believed to interact through a coevolutionary process in which both species are engaged in a continuous battle to dominate over one another. However, the underlying molecular genetic mechanisms that mediate these interactions are largely unknown. Previous work on transcriptional changes in the plant/insect system, the model dicot Arabidopisis thaliana and its insect herbivore, the Pieris butterfly indicate that the plants have a significantly varied response to oviposition and herbivory. To generate a comprehensive understanding of the plantinsect dynamics, we are using whole transcriptome sequencing to investigate the changes in gene expression in both herbivores and their host plants over the time course of their interaction. For the study, we selected herbivores from evolutionary divergent butterfly families and their respective host plant species. Our preliminary results indicate varied patterns of response across the different systems. We are further investigating the conserved parts of the defense network across the evolutionary lineages. In addition to expanding our understanding of fundamental evolutionary processes, the results of our study are likely to have important applied consequences. By characterizing the molecular genetic mechanisms mediating host plant defense, and herbivore countermeasures, our work has the potential to identify new targets for the control of agricultural pests.

900-006-Z Code of Mutualism Not Broken by Parasite: Mutualistic Plant-ants Specifically Induce Reward Supply Omar Hernandez Zepeda – Cinvestav Irapuato Omar Fabian Hernandez – Cinvestav Irapuato, Martin Heil – Cinvestav Irapuato Mutualisms can be stabilized against non-reciprocating exploiters when hosts provide rewards only to mutualistic partners. However, the underlying codes can be broken [1]. In the defensive mutualism between Acacia plants and

Pseudomyrmex ants, extrafloral nectar (EFN) presents the only known source of energy for the adult workers [2, 3]. High rates of EFN secretion can be observed on plants that are inhabited by mutualistic P. ferrugineus ants, but not on hosts colonized by the parasitic ant, P. gracilis. Here, we investigate the physiological mechanism that enables this preferential allocation of a reward towards a reciprocating mutualist. In ant removal and replacement experiments under field conditions, EFN secretion increased one day after re-colonization of the plants by the mutualist, but not the parasite. EFN-secretion, which had been considered constitutive in earlier studies [4], turned out to depend on endogenous levels of jasmonic acid (JA) in ant-free plants and could be activated by the application of gut content of mutualistic, but not parasitic ants. Concentrations of linolenic acid, the precursor of JA biosynthesis in plants, were more than 100 times higher in the gut of mutualistic than parasitic ants, and its exogenous application to the extrafloral nectaries induced endogenous JA levels in the nectary, as did also the application of gut content. Furthermore, the induction of EFN flow by gut content was abolished by the concurrent application of inhibitors of JA synthesis. We conclude that mutualistic plant-ants apply a precursor of endogenous JA to the nectaries and thereby continuously induce EFN secretion by their host.

900-007-Z Partner Manipulation Stabilizes a Horizontally Transmitted Mutualism Martin Heil – CINVESTAV Irapuato Mutualisms are considered textbook examples of co-evolved inter-specific interactions. However, mutualisms among non-coevolved species are common and can be stable even in the presence of exploiters. We use ant-plant mutualisms to study how mutualisms can be established and protected from non-reciprocating exploiters. Pseudomyrmex workers that engage in an obligate defensive mutualism with Acacia hosts feed exclusively on the sucrose-free extrafloral nectar (EFN) that is secreted by their hosts [1], a behaviour which links ant energy supply directly to host performance and thus favours reciprocating behaviour at no apparent proximate benefit for the ant [2-4]. We tested the hypothesis that Acacia hosts manipulate this digestive specialisation of their symbionts. Invertase (sucrose hydrolytic) activity in the ant midguts was inhibited by chitinase, a dominant EFN protein. The inhibition occurred quickly in cell-free gut liquids and in native gels and thus likely results from an enzyme–enzyme interaction [5].Preliminary analyses of common motifs in the reactive centre of plant chitinases indicate that most chtinases might readily interact with the substrate-binding motif of disaccharide-hydrolysing enzymes in insects. Partner manipulation acts at the phenotypic level and means that one partner actively controls the phenotype of the other partner to enhance its dependency on host-derived rewards. 1.

Heil, M., Rattke, J., and Boland, W. (2005) Science 308, 560-563.

2.

González-Teuber, M. et al. (2012) PLoS ONE 7, e46598.

3.

Heil, M. (2013) J. Ecol. 101, 684-688.

4.

Heil, M. et al. (2009) PNAS 106, 18091–18096.

5.

Heil, M. et al. (2014) Ecol. Lett. 17, 185-192.

900-008-Y Evaluating the Role of a Putative E3 Ubiquitin Ligase in Resistance Against Defoliating White Marked Tussock Moth Larvae (Orgyia Leucostigma) in Poplar Trees Samuel Bandi – University of North Dakota Steven Ralph – University of North Dakota

A prior genetic screen of activation tagged Populus tremula x P. alba trees identified the mutant E8-16 as resistant towards white marked tussock moth larval feeding and development. Molecular studies on E8-16 demonstrate the gene 10s12800 is overexpressed ~6 fold due to its proximity to the T-DNA. This gene contains a conserved RING-H2 domain common to E3 ligases, suggesting that 10s12800 is part of the ubiquitin-proteasome system involved in protein degradation. E3 ligases regulate protein turnover by tagging target proteins with ubiquitin. To provide independent genetic confirmation that E8-16 insect resistance is due to 10s12800 overexpression, we have cloned the gene into the plant expression vector pART27 behind a 35S promoter and inserted the construct into P. tremula x P. alba plants using Agrobacterium transformation. Transgenic lines will be screened for 10s12800 overexpression and insect resistance using a combination of quantitative PCR, Southern blots and insect bioassays. To provide biochemical confirmation that 10s12800 is an E3 ligase, we are performing an auto-ubiquitination test where successful addition of polyubiquitin will be determined via Western blots using an anti-ubiquitin antibody.

900-009-Y VIH2 Controls Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-dependent Defenses in Arabidopsis Gabriel Schaaf – University of Tuebingen Debabrata Laha – University of Tuebingen, Cristina Azevedo – MRC, UK, Adolfo Saiardi – MRC, UK Diphosphorylated inositol polyphosphates, also referred to as inositol pyrophosphates, are important signaling molecules that regulate critical cellular activities in many eukaryotic organisms. In mammals and fungi, two distinct classes of inositol phosphate kinases mediate their biosynthesis: Kcs1/IP6K- and Vip1/PPIP5K-like proteins. Recent studies in our lab show that PPIP5K homologs are widely distributed in plants and that Arabidopsis Vip1 homologs VIH1 and VIH2 are functional PPIP5K enzymes. We will report a specific induction of the inositol pyrophosphate InsP8 by jasmonate and will provide evidence that steady-state and jasmonate-induced pools of InsP8 in Arabidopsis seedlings depend on VIH2. We will further report a role of VIH2 in regulating jasmonate related defenses by potentiating jasmonate perception. Using in silico docking experiments and radioligand-binding based reconstitution assays we can show high affinity binding of inositol pyrophosphates to the F-box protein COI1–JAZ jasmonate co-receptor complex and will provide evidence that coincidence detection of jasmonate and InsP8 by COI1–JAZ is a critical component in jasmonate-regulated defenses.

900-010-Z Volatile Cues Provide Honest Information for Partner Choice in a Horizontally Transmitted Mutualism María del Rosario Razo-Belmán – Cinvestav Irapuato Martin Heil – Cinvestav Irapuato In horizontally transmitted mutualisms, the capacity to judge the quality of the future partner before the establishment of the mutualisms is fundamental. In ant-plant mutualisms such as the defensive ant-plant mutualism between Acacia host plants and their Pseudomyrmex ant inhabitants, the foundresses are under high selective pressure to quickly identify suitable hosts and decide whether or not to stay before they shed their wings. Because distinct species of Acacia plants provide different amounts of resources (extrafloral nectar, food bodies and nesting space), they represent hosts of different quality. We studied the host-searching behaviour of foundresses of the obligate plant-ant, Pseudomyrmex ferrugineus, to test the hypothesis that they use volatile cues (traits that are inevitably released by an organism as a consequence of its normal physiological processes) rather than ’signals’ (stimuli that are emitted to elicit specific responses in a specific partner for the benefit of the emitter). Using only volatile organic compounds (VOCs), the foundresses judged from a distance on the identity and quality of the future host plant. They preferred Acacia plants without ants over the inhabited plants, intact plants over damaged ones, and - most interestingly - they preferred the high-reward host A. cornigera over the low-reward host, A. hindsii. Low-quality hosts emitted quantitatively and

qualitatively more VOCs than high-quality hosts. Several of these VOCs (Limonene, ß-Linalool, α-Terpineol, α-Terpinene and ß-Pinene) inhibited bacterial phytopathogens and, thus, serve the plant´s direct antimicrobial defence. Due to tradeoffs between direct and indirect defence, high VOC emission rates indicate low ant reward production rates and, thus, a reduced host quality. Cues, rather than signals, can provide partners with reliable information on host quality when these cues indicate traits of vital importance for the host that are causally related to its quality for the partner.

900-011-Z A Proteomics Approach Towards Elucidating Responses to Herbivory and Wounding in the Tomato Chloroplast Oindrila Bhattacharya – University of California Riverside Linda Walling – University of California Riverside, Songqin Pan – University of California Riverside, Pitter Huesgen – University of California Riverside Chloroplasts perceive biotic stressors and developmental cues, and transduce regulatory signals to the nucleus to modulate gene expression thereby enabling coordination in plant responses (retrograde signaling). The tomato chloroplast stroma-localized leucine aminopeptidase (LAP-A) regulates a retrograde signal in response to wounding, herbivory, and methyl jasmonate (MeJA) to positively regulate late wound-response genes. LAP-A performs this function through its aminopeptidase and/or chaperone activities by affecting stability of its substrates. To identify the LAP-Adependent retrograde signal, we are identifying LAP-A’s stromal substrates (LAP-A targets) using two directed, discovery-based proteomics approaches. A high-yield, high-quality protocol for isolating tomato chloroplast stroma has been developed. Changes in the stromal proteome of wild type, LapA-overexpressed and LapA-silenced plants have been seen using nano-LC MS/MS (Orbitrap Elite, Thermo Scientific). Our data show LAP-A-dependent and -independent changes in the stroma. To identify LAP-A peptidase substrates, we are using a methodology called terminal amine isotopic labeling of substrates (TAILS), to assess the LAP-A-dependent changes in the N-termini of stromal proteins (Nterminomics). Briefly, mature N-termini of proteins (and internal lysine residues) of stromal proteins are blocked by dimethylation. After trypsin digestion, internal peptides with free N-terminal amines bind to a high-affinity dendritic polymer, while the blocked N-terminome (recovered by ultrafiltration) is characterized by nano LC-MS/MS. We report the LAP-A-dependent terminome and identify LAP-A stromal substrates, which may itself be or help to generate retrograde signals that control the robust and durable response to herbivory.

900-012-Y Plant Responses to Leaf Vibrations Caused by Insect Chewing Heidi Appel – University of Missouri Reginald Cocroft – University of Missouri, Elizabeth Haswell – Washington University in St. Louis We have recently shown that the vibrations caused by insect feeding can elicit chemical defenses in plants. Arabidopsis thaliana (L.) rosettes pre-treated with the vibrations caused by caterpillar feeding had higher levels of glucosinolate and phenolic defenses when subsequently attacked by Pieris rapae (L.) caterpillars than did untreated plants and plants treated with the vibrations caused by wind or other insect sounds. This response to herbivore-generated vibrations provides an ecological reason for why plants have evolved the ability to respond to acoustic stimuli. We suggest that feeding vibrations represent a new long distance signaling mechanism in plants that complements other known signaling pathways. How plants perceive these mechanical vibrations is not well understood but we report here the role of mechanoreception in the response by screening mutants defective in specific mechanoreceptors. In addition, we describe the features of these complex vibrational signals that are important to plant response.

900-013-Y Darwin’s Peach: Insect Galls as Ectopic Flowers or Fruits Jack Schultz – University of Missouri Heidi Appel – University of Missouri, Wade Dismukes – University of Missouri, Sarah Witiak – Dept. of Biology, Virigina State University, Patrick Edger – Dept. Plant and Microbial Biology, UC Berkeley Insect galls are unique organs formed by plants in response to chemical signals from insects. These organs house and feed the insects. Darwin, among others, noticed that many galls resemble fruits phenotypically. We undertook transcriptional profiling of galls developing on grapevine leaves in response to feeding by phylloxera (Daktulosphaira vitifoliae (Fitch 1855)) using RNA-seq. More than 6,000 genes were differentially expressed in four gall developmental stages compared with developmentally-matched ungalled leaf tissue. Profiles revealed a significant over-representation of reproductive pathway genes, increasing as galls mature. Key genes involved in the vegetative-to-reproductive transition are activated in galls. Quantitative analysis indicates that galls are transcriptionally more similar to flowers and fruits than to the leaves on which they form. Elements of several of the classical flowering pathways are activated in developing galls as are MADS box and other genes responsible for development of floral organs, particularly the carpel. Taken together, the evidence supports the hypothesis that obtaining plant traits needed to feed and house the insect is accomplished by eliciting development of functional and anatomical traits normally used to feed and house the embryo. Phylloxera leaf galls are transcriptionally convergent on flower and fruit organs.

900-014-Z Involvement of Jasmonic Acid in Airborne Infochemical Based Plant-Plant Communications in Tomato Plants Simon Zebelo – Auburn University Henry Fadamiro – Auburn University Volatile organic compounds (VOCs) emitted from plants upon insect herbivory act as airborne signals that enhance direct and indirect defenses in remote parts of the same plant or neighboring plants. Several studies have provided compelling evidence that receiver plants are able to respond to volatile cues from conspecific or interspecific emitter plants, by activating defense-signaling pathways. Jasmonic acid (JA) is a key regulatory component in defense-signaling pathways. However, studies are limited on the role of JA on perception of airborne signals by receiver plants. Here, we tested the hypothesis that VOCs emitted from herbivore damaged tomato plants will trigger defense-signaling pathways in receiver wild-type (WT) plants, but not in jasmonic acid insensitive1-1 (jai1-1) mutants. To test this hypothesis, we compared the expression levels of three defense related enzymes (Phenylalanine ammonia-lyase (PAL), Polyphenol oxidase (PPO) and Lipoxygenase (LOX)) and quantified transcript levels of several defense-related genes in WT and jai1-1 tomato plants after exposing them to VOCs from donor WT tomato plants damaged by Spodoptera exigua caterpillars. The selected defense enzymes and the transcript levels of defense genes were expressed in higher amounts in WT tomato plants than in jai1-1 plants. In addition, we quantified the VOCs emitted by the donor WT tomato plants damaged by S. exigua caterpillars, which included some green leaf volatiles (GLVs), monoterpenes and sesquiterpenes. These results suggest the involvement of JA in perception of airborne infochemicals during plant-plant interaction upon herbivory.

900-015-Z Regulation of Nectar Composition in Day- and Night Flowering Tobacco Plants Kira Tiedge – University of Wuppertal Gertrud Lohaus – University of Wuppertal During the process of co-evolution a major part of the tobacco plants (Genus Nicotiana) have adapted to nocturnal pollinators like hawkmoths or bats and open their flowers in the late evening or at night. The morphological changes due

to the pollinator shift are obvious, e.g. elongated corolla tubes of the flowers matching the extendible probisces of Sphingidae (hawkmoths). A main aspect of this evolutionary process is the intra-floral nectar production of night flowering tobacco plants. Nectar is the main reward for the pollinators of a plant. Therefore it has to satisfy the preferences and needs of these essential visitors. One main question is whether the nectar constituents (sugars, amino acids, proteins, secondary metabolites etc.) are adapted to nocturnal pollinators or if they are more likely determined by other constraints. Nectary tissue and leaf samples of 26 closely related day- and night flowering Nicotiana species were taken in the beginning of the light and the dark period. Nectar was collected in the whole course of a day. All samples were measured via high-performance-liquid-chromatography (HPLC) for the analysis of sugars and amino acids. Other features of the tobacco flowers like corolla tube length and diameter, nectar volume and flower opening time were determined. By means of these findings it will be discussed which factors have the greatest impact on the nectar composition.

900-016-Y Strategies for Whitefly Control in Soybean and Tomato Plants by Genetic Manipulation Jose Andres Galeana-Lopez – CINVESTAV Soybean is the most important oilseed in the world, as seed and by the products obtained from it. It’s a highly nutritious crop regarded for its high protein content and oil compared to its dry weight. Mexico is considered the fourth largest importer of soybeans in the world, after China, the European Union and Japan. In 2009, Mexico imported 3.5 million tons, allocating 98% to the livestock sector. Meanwhile, tomato is the main food product export of Mexico with an average annual value of 899 million USD in 2000-2009; North America is its main market with 95%. Both cultivars bear trichomes on stems and leaves, representing a defense system to herbivory, protection against water stress and the synthesis of secondary metabolites. The whitefly, (Bemisia tabaci) has become one of the most important pests worldwide, with the host crops such as cotton, sunflower, tomatoes, soybeans, eggplant, and others. This fly causes severe damage by sucking sap from the leaves, secreting sugars through droppings (favoring sooty mold) and transmitting viral diseases. (Jose and Usha, 2003). Several authors have been able to identify a correlation between the degree of pubescence in different plant species including tomato and soybean with whitefly oviposition (Morillo et al., 1997. Etore et al., 2012). Likewise it has been reported a relationship between density, length and angle of the trichomes, indicating that the pubescent varieties show a higher rate of oviposition by whitefly, than those who are glabratas. (McAuslane 1996, Lambert 1995). The strategy proposed in this study is the post-transcriptional gene silencing of GL1 orthologs, whose product is associated with the formation of trichomes, generating mutants with different abundance of trichomes from glabrata to pubescent soybean and tomato plants. Genetically modified plants will be challenged to whitefly, correlating trichome density with the presence of B. tabaci adults.

900-017-Y Functional Characterization of Helicoverpa-inducible Pathogenesis-related Protein 4A (PR-4A) from Chickpea Archana Singh – Univerisity of Delhi Indrakant Singh – Deshbandhu College, Univerisity of Delhi, Dipti Jain – University of Delhi-South Campus Benito Juarez Road PR proteins are commonly known to accumulate in plants by biotic and abiotic stresses. A pathogenesis-related protein 4A was identified from a cDNA subtractive library of chickpea -Helicoverpa interaction and named as CaHaPR-4A. In silico analysis of CaHaPR-4b protein indicated the presence of a Barwin domain and a Rare lipoprotein A (RlpA)-like

double-psi beta-barrel (DPBB) domain. CaHaPR-4b protein is a small secretory protein of 16 kD and it belongs to the PR4 protein subgroup II, based on the absence of a hevein domain. Molecular modeling of CaHaPR-4A confirmed the importance of the cysteine residues to maintain the protein structure, and of several conserved amino acids for the catalytic activity. CaHaPR-4b belonged to a small multigene family. CaHaPR-4A RT-qPCR analysis in chickpea plants infested with Helicoverpa armigera showed an increase of expression from initial time points till 24 h. Increase in expression level has also been observed in Methyl Jasmonate and Ethylene treated samples of chickpea. The recombinant CaHaPR-4A protein exhibited RNase, and bivalent ions dependent-DNase activity, but no chitinase activity. Although PR-4 proteins are biochemically chitinases which catalyse the cleavage of bond between C and C of two consecutive N-acetyl-D-glucosamine monomers of chitin, but it is not necessary that all members of PR-4 family show chitinase activity. CaHaPR-4A nuclease activities may be related to the establishment and maintenance of tolerance and also to the PCD mechanism. Therefore, for further characterization, we are studying other actions like antifungal, antibacterial and insecticidal properties of this protein.

900-018-Z Mechanistic Insights into Mode of Action of Rice Allene Oxide Synthase on Hydroxyperoxides: An Intermediate Step in Herbivory-induced Jasmonate Pathway Indrakant Singh - University of Delhi Chetna Tyagi – Unit of Simulation and Informatics, IARI, Archana Singh – Hans Raj College, University of Delhi Various types of oxygenated fatty acids termed ‘oxylipins’ are involved in plant response to herbivory/insect-attack. Oxylipins like jasmonic acid (JA) and green leafy volatiles (GLVs) are formed by the action of enzymes like allene oxide synthase (AOS) while or hydroxyperoxide lyase (HPL) respectively. In this study, we focus on AOS of Oryza sativa sb. Japonica, that interact with 9- and 13- hydroxyperoxides to produce intermediates of jasmonate pathway and compare it with rice HPL that yields GLVs. We attempt to elucidate the interaction pattern by computational docking protocols keeping the Arabidopsis AOS system as the model system. Both 9-hydroxyperoxide and 13-hydroxyperoxide fit into the active site of AOS completely with Phe347, Phe92, Ile463, Val345, and Asn278 being the common interacting residues. Phe347 and Phe92 were mutated with Leucine and docked again with the hydroxyperoxides. The mutant Phe347àLeu347 showed a different mode of action than AOS-hydroxyperoxide complex with Trp413 in direct bonding with the –OOH group of 9-hydroxyperoxide, the loss of Lys88-OOH interaction in 13-hydroxyperoxide and Leu347 not taking part in any, indicating the importance of Phe347 in hydroxyperoxide catalysis. The second mutant Phe92àLeu92 also shows a very different mode of action with 13-hydroxyperoxide but not with 9-hydroxyperoxide. Therefore it can be conclude that Phe347 is more crucial for AOS functionality than Phe92. The aromatic ring of a Phenylalanine residue is important for catalysis and its mutation affects the binding of the two ligands. Another important residue is Asn278 which forms important part of the AOS catalytic site which when mutated renders inactivity to the enzyme as seen in the Arabidopsis model with residue Asn321. Lastly, the interaction of HPL with these two derivatives involves completely different active sites than AOS giving insight into their different modes of action that result in completely different products from same substrates.

900-019-Z Analyses of Plant Defense and Host Suitability of Herbivores Hiroshi Abe – RIKEN BioResource Center Takeshi Shimoda, Yuji Sawada, Shigemi Seo, Masami Yokota Hirai, Ken Tateishi, Soichi Kugimiya, Masatomo Kobayashi We analyzed the function of JA in the plant response to American serpentine leafminers attack, and function of JA dependent plant induced defense to the leafminers resistance and host suitability using Arabidopsis plants. Expression

analyses of marker genes suggested the involvement of JA in the plant response to the leafminer feeding. The induction of marker genes was reduced or canceled in the JA-insensitive coi1-1 mutants. JA contents were increased after the leafminer feeding in WT plants. Arabidopsis WT plants exhibited higher resistance to American serpentine leafminers. We only found very narrow feeding scars in WT plants. However, JA-insensitive coi1-1 mutant showed notably decreased feeding resistance and suffered from huge feeding scars. The leafminer pupas and adults of next generation were only appeared from coi1-1 mutants but not from WT plants, suggesting that loosing JA-dependent plant induced defense convert non-host plants to accessible host plants. We are now searching the plant metabolites to understand the effect of these plant defenses on the host suitability of the leafminers.

Biotic Interactions: Plant-Microbe 900-020-Y Multiple Xanthomonas Euvesicatoria Type Three Secretion Effectors Target 14-3-3 Mediated Immune Signaling in Tomato Zoe Dubrow – Stanford University Jung-Gun Kim – Stanford University, Nejla Ozbaki-Yagan – Stanford University, Mary Beth Mudgett – Stanford University Xanthomonas euvesicatoria (Xcv) is a bacterial pathogen that is the causal agent of bacterial spot disease in tomato, an economically important crop plant. Xcv utilizes a type III secretion (T3S) system, a needle-like structure, to secrete and translocate effector proteins into host cells. These effectors can suppress plant immune responses by binding and manipulating the functions of endogenous tomato proteins. We demonstrated that seven Xcv T3S effectors interact with multiple isoforms of the tomato 14-3-3 (TFT) family of phosphopeptide scaffolding proteins in yeast. Next, we confirmed direct protein-protein interaction between TFT5 and TFT10 and three Xcv effectors –XopE1, XopE2, and XopO – in planta. Mass spectrometry of proteins purified from plant extracts showed that XopO and XopE2 are phosphorylated. XopE2 is phosphorylated at multiple residues including T66, a residue within a putative 14-3-3 Mode 1 binding motif. Importantly, mutation of T66 and S344 in XopE2 reduced XopE2’s binding affinity for TFT5 and TFT10, demonstrating that effector phosphorylation is required for binding to TFTs. In addition, we obtained evidence suggesting that TFT10 is polyubiquitinated when co-expressed with XopE1, XopE2, or XopO and that TFT10 is degraded in a 26S proteasomaldependent manner. These data reveal a possible molecular mechanism of bacterial pathogenesis whereby effectors target TFTs to promote their degradation. Finally, we determined that XopE1, XopE2, and XopO are required for Xcv to inhibit the development of tissue chlorosis in tomato leaves during infection. Taken together, these data support the hypothesis that Xcv employs a subset of T3S effectors to target and manipulate TFT isoforms directly resulting in interference with tomato immune signaling and symptom development.

900-021-Y The Impact of Canna Yellow Streak Virus on Foliar Pigmentation in Canna Species Ravendra Chauhan – Oklahoma State University Jeanmarie Verchot – Oklahoma State University We have recently identified, sequenced and phylogenetically characterized a new potyvirus Canna yellow streak virus (CaYSV) which causes loss of red foliar pigments in red-green canna cultivars. The red pigments present in a number of plant species have been identified as anthocyanins, proanthocyanidins and flavonols. These pigments play important roles in certain physiological and biochemical processes in plants and are crucial for their survival. Due to the nutritional benefits and biological importance of these foliar pigments the biosynthetic pathways have been thoroughly studied in various plant species. Canna is a popular landscape plant in Oklahoma and represent a multi-million dollar industry. The commercially available cultivars are interspecific hybrids selected for their attractive foliage and flowers. But, there is no information about the foliar pigments present in canna and how the virus disease would impact the nutritional and

ornamental value of the crop. To determine if CaYSV acts to alter the gene expression of critical genes in the pigment pathways, the qRT-PCR analysis has been carried out to compare transcript accumulation of four genes involved in flavonoid and proanthocyanidin biosynthesis. The expression of phytoene desaturase (PDS) and chalcone synthase (CHS3) genes have been monitored through qRT-PCR which are known to be involved in photosynthesis. Silencing these genes often leads to photo bleaching and it would be interesting to determine if virus impacts these genes in a manner that can be correlated with disease phenotypes. The expression of MybA1 and quercetin 3-glucoside (Q3-G) genes have also been monitored which is important for proanthocyanidin pigment synthesis and photo-oxidation. Flavonoids and proanthocyanidins have been extracted from healthy and CaYSV-infected wild canna species Canna edulis and hybrid canna varieties ‘Red Futurity’ and ‘Burning Ember’. The concentrations of total chlorophylls, carotenoids, total anthocyanins, total flavonols, and total proanthocyanidins have been determined by spectrophotometer and HPLC analyses.

900-022-Z Pathological ER Stress and the Unfolded Protein Response Activation by Plant Virus Infection Jeanmarie Verchot – Oklahoma State University Alexis Vela Arias – ESPE, Lix Pena – Oklahoma State University Plant viruses encode small membrane binding proteins that are essential for replication or intercellular movement. We have reported that such proteins which have unrelated functions in virus infection converge to activate the UPR to overcome a condition that is singly termed ER stresss. These small membrane binding proteins elicit transcriptional activation of the UPR and components of the ER associated degradation (ERAD) machineries. Virus infection, heat/cold, and tunicamycin are seemingly dissimilar triggers of ER stress that similarly coordinate the action of a common set of molecular chaperones to cope with the demand for increasing protein folding capacity and to mitigate the toxic accumulation of malformed proteins. RNA viruses create a huge biosynthetic burden on the ER and transiently enhance UPR to manipulate the production of protein chaperones and foldases to meet their needs. Abiotic stressors such as heat/cold/tunicamycin cause accumulation of malformed proteins in the ER and triggers the same machinery to ensure efficient maturation and secretion of proteins. We will provide new data describing the interactions of potex and potyviruses with the ER stress machinery.

900-023-Z Immune Receptor-mediated Translation Suppression Functions as an Antiviral Immunity Mechanism in Plants Elizabeth Fontes – Universidade Federal de Vicosa Joao Paulo Machado – Universidade Federal de Vicosa, Cristiane Zirzatto – Universidade Federal de Vicosa, Otavio Brustolini – Universidade Federal de Vicosa, Anesia Santos – Universidade Federal de Vicosa Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus–plant interactions, one of the major mechanisms for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, thus enhancing viral pathogenicity in susceptible hosts. In addition, plants use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effectortriggered immunity in a defense mechanism similar to that employed in non-viral infections. More recently, plants have also been found to use innate pathogen-associated molecular pattern (PAMP)-triggered immunity to limit viral infection. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host global translation suppression to fight viruses. Here, we demonstrate that the constitutive activation of NIK1, a leucine-rich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component RPL10 to the nucleus, where it interacts with a MYB-like protein, LIMYB (L10-interacting Myb domain-containing protein), to fully down-regulate translational machinery genes.

LIMYB overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral mRNA association with polysome fractions and enhanced tolerance to begomovirus. In contrast, the loss of LIMYB function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMYB links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants.

900-024-Y Cooperation and Punishment in the Arbuscular Mycorrhizal Symbiosis: Insight into Nutrient Exchange and Mutualistic Evolutionary Stability Carl Fellbaum – South Dakota State University Jerry A Mensah – South Dakota State University, Adam J Cloos – South Dakota State Uniiversity, Gary E Strahan – United States Department of Agriculture, Agricultural Research Service, Philip E Pfeffer – United States Department of Agriculture, Agricultural Research Service, Emma W Gachomo – Rutgers University, E. Toby Kiers – Institute of Ecological Science, Heike Bucking – Douth Dakota State University The 400 million year old arbuscular mycorrhizal (AM) mutualism is a symbiosis that is formed between the roots of 65% of all land plant species, and an exclusively subterranean fungus. In this mutualistic interaction the fungus transfers nutrients, such as phosphate (P) and nitrogen (N), sulfur and zinc, and in addition provides the host with a higher stress tolerance in exchange for photoassimilates. Previous studies indicated a direct link between the carbon (C) and P exchange in the symbiosis, but it is unknown whether C also acts as a trigger for fungal N transport. It has been suggested that biological market dynamics could contribute to the evolutionary stability in the AM symbiosis. However, in order for these mechanisms to work, the host plant and the fungus must be able to discriminate between partners that differ in the mycorrhizal benefit that they provide. It is unknown whether host plants can, for example, discriminate between co-colonizing fungi on a fine enough scale to reciprocate accordingly. We developed two hypotheses to address these gaps in our knowledge: - Host C has an effect on AM fungal N uptake and transport to the host. - Host plants and AM fungi can discriminate between beneficial and less beneficial partners and reciprocate accordingly. We tested our hypotheses in root organ cultures and whole plant systems at the community, physiological, and molecular level. We demonstrate that host C stimulates fungal N uptake and transfer to the host. We demonstrate that plants and fungi can preferentially allocate resources to partners that provide more benefit. Our data reinforce our hypothesis that biological market theory provides a suitable context for understanding nutrient exchange between partners and the evolutionary stability of the AM symbiosis.

900-025-Y Flagellin-induced Monoubiquitination of BOTRYTIS-INDUCED KINASE1 Regulates Arabidopsis Innate Immunity Xiyu Ma – Texas A&M University Ping He – Texas A&M University, Libo Shan – Texas A&M University Proper and prompt activation of pattern recognition receptors (PRRs) upon microbial infection is essential for hosts to survive. Plant BIK1 family receptor-like cytoplasmic kinases are key immune regulators associated with multiple PRRs, including flagellin receptor complex FLS2-BAK1 in Arabidopsis. Upon flagellin perception, BAK1 directly phosphorylates BIK1, leading to BIK1 dissociation from FLS2-BAK1 complex to relay immune signaling. How BIK1 activation is regulated remains largely elusive. We report here that flagellin perception triggers rapid monoubiquitination of BIK1 and its closest homolog PBL1 in planta. An ubiquitination inhibitor PYR41 suppressed multiple flagellin-induced immune responses

downstream of FLS2-BAK1 complex formation but upstream of BIK1 phosphorylation and dissociation from FLS2-BAK1 complex. Time course and mutational analysis suggest that flagellin-induced BIK1 phosphorylation precedes BIK1 monoubiquitination. Extensive mutational screen of all individual lysine resides in BIK1 revealed that BIK1K204 plays an essential role in flagellin-induced BIK1 monoubiquitination and phosphorylation. Transgenic complementation assays indicate that BIK1K204 is crucial for BIK1-mediated plant immune signaling. Thus, our study provides the genetic evidence that monoubiquitination of a convergent immune regulator in multiple PRR complexes is essential to relay plant immune signaling, and reveal the intertwined regulation of immune sensory complex activation by layered protein phosphorylation and ubiquitination.

900-026-Z The Role of Xanthomonas Type Three Effector Proteins in Host Specificity Rebecca Bart – DDPSC Andrew Mutka – DDPSC, Sarah Fentress – DDPSC, Mark Wilson – DDPSC, Anupama Vijayaraghavan – DDPSC Crop losses lead to food insecurity, especially in poorer communities and in the developing world. Xanthomonads are plant-associated bacteria that cause disease on most important crops. Classification of bacteria at the species level is complicated by a high degree of horizontal gene transfer and limited morphological or behavioral difference. Nonetheless, Xanthomonads have been divided into approximately 27 species based on techniques such as DNA-DNA hybridization and rRNA sequencing. In addition to species level classification, plant pathogenic bacteria are often given a pathovar designation that indicates the plant host from which the bacteria were isolated. While genetically closely related, distinct pathovars within the Xanthomonas genus have distinct host specificities. Here, we present our work to identify and characterize the molecular determants of virulence and pathovar specificity within the Xanthomonas genus. We are combining traditional gene knockout strategies with a comparative genomics approach. Finally, several exciting new phenotyping techniques will be discussed.

900-027-Z Molecular Mechanisms of Switchgrass Growth Promotion by Burkholderia Phytofirmans Strain PsJN Chuansheng Mei – Institute for Advanced Learning and Research/Virginia Tech Scott Lowman – Institute for Advanced Learning and Research, Alejandra Lara-Chavez – Institute for Advanced Learning and Research, Seonhwa Kim-Dura – Institute for Advanced Learning and Research, Yuhong Tang – Noble Foundation/BioEnergy Science Center, Jiyi Zhang – Noble Foundation/BioEnergy Science Center, Michael Udvardi – Noble Foundation/BioEnergy Science Center, Jerzy Nowak – Virginia Tech, Barry Finn – Institute for Advanced Learning and Research/Virginia Tech Switchgrass is a promising bioenergy crop in the US. Improvement of stand establishment and year-to-year stabilization of biomass yields are primary objectives towards the development of a low-input switchgrass feedstock production system. Our earlier investigations of the effect of Burkholderia phytofirmans strain PsJN, a beneficial bacterial endophyte, on switchgrass germplasm demonstrated differential responses between genotypes. PsJN inoculation of the lowland cv. Alamo increased plant root system, shoot length, and biomass yields, whereas it had no beneficial effect on the upland cv. Cave-in-Rock. To understand the underlying gene networks governing plant growth promotion triggered by PsJN, we analyzed gene expression profiles in these two hosts following seedling inoculation. The Affymetrix platform switchgrass EST microarray chip representing 122,972 probe sets, developed by the DOE BioEnergy Science Center, was employed to assess transcript abundance at 0.5, 2, 4 and 8 DAI (days after PsJN inoculation). Approximately 20,000 switchgrass probe sets showed significant responses in either cultivar during the four time points. Switchgrass identifiers were used to map 19,421 genes in MapMan software. Differential gene expression was recorded between cultivars as there were 14,984 and 9691 genes affected by PsJN inoculation in Alamo and Cave-in-Rock, respectively. Early microbe

recognition in both responsive and non-responsive cultivars was similar in gene categories such as redox, signaling and proteolysis. After recognition at 0.5 DAI, gene expression diverged between the responsive and non-responsive cultivars. The responsive cultivar showed a weak defense response, and the relationship became mutual or beneficial. The nonresponsive cultivar maintained a relatively strong defense response throughout our experimental period, which included redox, signaling, proteolysis, and abiotic stress. Genes related to signal molecules and hormones were also differentially expressed as there was an up-regulation of SA, JA and ABA pathways in Cave-in-Rock, which may induce or enhance resistance to PsJN.

900-028-Y Regulatory Mechanisms of Pathogen-Mediated Cellular Stress Signaling in Arabidopsis Karolina Mukhtar – UAB Xiaoyu Liu – UAB, Camilla Koerner – UAB, Xinran Du – UAB, Marie Vollmer – UAB Induction of plant immune responses involves significant transcription reprogramming as well as endoplasmic reticulum (ER)-mediated protein secretion and quality control. To alleviate ER stress and restore homeostasis, the cell activates a complicated signaling network termed Unfolded Protein Response (UPR). Recently, a heat-shock-like transcription factor TBF1 was identified as the major regulator of UPR-related genes in Arabidopsis thaliana. We also demonstrated that TBF1 directly binds onto the TL-1 cis-element, a motif enriched in the promoters of ER-resident genes. In addition, TBF1 transcripts contain two upstream open reading frames (uORFs) that can exert inhibitory effects on translation of the main ORF. We show that the repressive effects of uORFs on TBF1 translation can be alleviated by AtGCN2-mediated phosphorylation of eIF2α (eukaryotic initiation factor 2α). Our results indicate that AtGCN2 is involved in immune signaling pathways mediated by salicylic acid and jasmonic acid. Current research focuses on characterizing the function of AtGCN2 in plant immunity at various developmental stages and its cross-talk with other phytohormones. Another key regulator of UPR, an ER membrane-located IRE1 kinase/endoribonuclease, can perceive the accumulation of malfolded peptides in the ER lumen. Subsequently, it catalyzes unconventional cytoplasmic splicing of an mRNA for bZIP60 transcription factor, giving rise to a transcriptionally active form that promotes the expression of multiple ER stress-responsive genes. Our data indicate that IRE1a-dependent bZIP60 splicing is a cytoprotective, adaptive mechanism, similar to the yeast and human Ire1/bZIP signaling that is involved in the pro-survival branch of the pathway. However, an acute ER stress caused by cell death-triggering stimuli such as avirulent P. syringae infection, can shut off the classic IRE1-bZIP60 signaling and activate the novel, pro-apoptotic branch of the IRE1a signaling pathway. This process is manifested by down-regulation of the bZIP60 transcript levels and initiation of an intracellular death program. Funded by: NSF CAREER (IOS-1350244).

900-029-Y Arabidopsis NATA1 Acetylates Putrescine and Decreases Defense-related Hydrogen Peroxide Accumulation Georg Jander – Boyce Thompson Institute Yann-Ru Lou – Boyce Thompson Institute, Melike Bo – Ege University, Jian Yan – Boyce Thompson Institute Biosynthesis of the polyamines putrescine, spermidine and spermine is induced in response to pathogen infection of plants. Putrescine, which is produced from arginine, serves as a metabolic precursor for longer polyamines, including spermidine and spermine. Polyamine acetylation, which has important regulatory functions in mammalian cells, has been observed in several plant species. Arabidopsis thaliana (Arabidopsis) N-ACETYLTRANSFERASE ACTIVITY1 (NATA1) catalyzes acetylation of putrescine to acetylputrescine and thereby competes with spermidine synthase for a common substrate. NATA1 expression is strongly induced by the plant defense signaling molecule jasmonic acid and coronatine, an effector molecule produced by Pseudomonas syringae strain DC3000. DC3000 growth is reduced in nata1 mutant

Arabidopsis, suggesting a role for NATA1-mediated putrescine acetylation in suppressing antimicrobial defenses. During infection by P. syringae and other plant pathogens, polyamine oxidases use spermidine and spermine as substrates for the production of defense-related hydrogen peroxide. Compared to wildtype Arabidopsis, the response of nata1 mutants to P. syringae infection includes reduced accumulation of acetylputrescine, greater abundance of nonacetylated polyamines, elevated hydrogen peroxide production by polyamine oxidases, and higher expression of genes related to pathogen defense. Together, these results are consistent with a model whereby P. syringae growth on wildtype Arabidopsis is improved in a targeted manner through coronatine-induced putrescine acetylation by NATA1.

900-030-Z Functional Assignment to Positively Selected Sites in the Ralstonia Solanacearum Type III Effector RipG7 Keke Wang – Laboratoire des Interactions Plantes Micro-organismes (LIPM), INRA, Toulouse Philippe Remigi – Laboratoire des Interactions Plantes Micro-organismes (LIPM), INRA, Toulouse, Maria Anisimova – Institute of Applied Simulations, School of Life Science, Ilona Kars – Laboratoire des Interactions Plantes Microorganismes (LIPM), INRA, Toulouse, Favien Lonjon – Laboratoire des Interactions Plantes Micro-organismes (LIPM), INRA, Toulouse, Stephane Genin – Laboratoire des Interactions Plantes Micro-organismes (LIPM), INRA, Toulouse, Nemo Peeters – Laboratoire des Interactions Plantes Micro-organismes (LIPM), INRA, Toulouse The soil-borne pathogen Ralstonia solanacearum causes wide spread bacterial wilt in a broad range of host plants. The main virulence determinants of R. solanacearum that promote pathogen infection and disease progression in plants is the Type III Secretion System (T3SS) and its protein substrates referred to as Type III Effectors (T3Es) that are secreted inside host cells. A set of 32 T3E proteins defines a ‘core’ effector group that is conserved among the known R. solanacearum genomic diversity. The F-box protein RipG7, which is a core T3E, is required for R. solanacearum pathogenesis on Medicago truncatula. Here, we show that eight RipG7 alleles from four phylotypes spanning the whole diversity of R. solanacearum, have different contributions to pathogenicity on M. truncatula. Only RipG7 alleles from phylotype I and phylotype III strains can complement the absence of RipG7 in GMI1000. Nonetheless, RipG7 alleles from phylotype II and phylotype IV can still interact with SKP1/MSKa, which is a core subunit of Skp1-Rbx1-Cul1-F-box protein (SCF) complex and is essential in the recognition and binding of the F-box. In our study, we identified eleven strong positive selection residues in RipG7 protein that 10 out of the 11 sites are located in Leucine-Rich Repeats (LRR) domain, which is usually involved in substrate binding. By studying the functional impact of those 11 sites, we found mutations of several residues resulted in failure of both bacterial colonization and wilting M. truncatula, or reduced bacterial colonization in plant. Our results reveal genetic/functional variation of RipG7 and critical amino acid elements for its virulence. We hypothesize that during host-pathogen coevolution process, positive selection on RipG7 protein level results in adaptive function in diverse host plants.

900-031-Z UDP-D-Glucuronate 4-Epimerases Are Critical for Pectin Abundance and Immunity in Arabidopsis Thaliana Gerit Bethke – University of Minnesota Guangyan Xiong – UC Berkeley, Amanda Thao – University of Minnesota, Fumiaki Katagiri – University of Minnesota, Markus Pauly – UC Berkeley, Jane Glazebrook – University of Minnesota Plant cell walls are important early barriers to infection by pathogens. Primary cell walls in Arabidopsis leaves contain about 50% pectin. About 60% of this pectin is homogalacturonan, a linear polymer of D-galacturonic acid (GalA) residues. UDP-D-glucuronate 4-epimerases (GAE) interconvert UDP-D-glucuronic acid (UDP-GlcA) and UDP-GalA, the monomeric precursor of pectin. Here, we report that expression of GAE1 and GAE6 can be repressed by treatment with the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326) in a manner that depends on central regulators of immunity

including PAD4, EDS1 and PBS3 . Mutant gae1 gae6 plants show increased susceptibility to Pma ES4326 and some isolates of the fungal pathogen Botrytis cinerea. Leaves of gae1 gae6 plants are brittle and reduced in homogalacturonan. Oligogalacturonides (OGs) are released from pectin through the action of pathogen pectinases, and serve as inducers of immune responses. Interestingly, immune signaling in response to treatment with a commercial pectinase, macerozyme, but not OGs, is altered in gae1 gae6. This suggests that OG release is reduced in these plants while recognition of OGs is unaffected. Further, macerozyme- but not OG-induced immunity to Botrytis cinerea strain Gallo1 is abolished in gae1 gae6 plants. Taken together our data suggest that pectin abundance is an important contributor to physical properties of cell walls and to plant immunity.

900-032-Y Decorated Peptides: a Proposed Glycosylase in the Autoregulation of Nodulation Pathway Stephen Nowak – Clemson University Tessema Kassaw – Colorado State University, Benjamin Flanagan – College of Charleston, Elise Schnabel – Clemson University, Julia Frugoli – Clemson University Under nitrogen-deficient conditions, leguminous plants form a symbiotic relationship with nitrogen fixing bacteria in root structures called nodules. Plants optimize nodule number through a systemic signaling pathway known as the Autoregulation of Nodulation (AON). After inoculation, two CLAVATA3/ENDOSPERM SURROUNDING REGON (CLE) genes, MtCLE12 and MtCLE13, are induced in the nodule meristem and regulate nodule number most likely thorough translocation of the CLE peptides to the shoot, where they bind a receptor complex containing the leucine-rich repeat receptor-like kinase SUNN; subsequent signal transduction results in termination of new nodule formation. Several lines of evidence in Arabidopsis and legumes suggest post-translational addition of a tri-arabinose chain to a hydroxyproline in some CLE peptides is necessary for receptor binding. The RDN1 gene in M. truncatula also regulates nodule number and is part of a gene family in all green plants. The orthologous HYDROXYPROLINE ARABINOSYL TRANSFERASE (HPAT) family in Arabidopsis encodes enzymes that add arabinose to hydroxyprolines in short peptides. We identified root phenotypes in an insertion mutant in AtHPAT3 and the double insertion mutant of AtHPAT1 and AtHPAT2, contrary to previously published reports of no observable effects and we provide evidence supporting a model in which RDN1 modifies MtCLE12 but not MtCLE13. We are currently utilizing RNAi to knockdown expression of MtRDN2 and MtRDN3 and observing nodulation and root phenotypes to determine specificity of the RDN enzymes and involvement in AON. This work is supported by NSF IOS#1146014 and the Clemson Creative Inquiry Program.

900-033-Y The SUNN Symbiotic Kinase Participates in Multiple protein:protein Interactions Ashley Crook – Clemson University Elise Schnabel – Clemson University, Brendan Riely – University of California-Davis, Julia Frugoli – Clemson University Autoregulation of nodulation (AON) is a long-distance signaling pathway in legumes which limits the number of nodules formed in the rhizobial-legume symbiosis. In Medicago truncatula, this systemic regulation includes local signaling events resulting in the translocation of small signaling peptides (CLEs) to the shoot, the binding of CLEs to a receptor complex containing the leucine-rich repeat receptor-like kinase SUNN, and subsequent signal transduction resulting in termination of new nodule formation. We explored protein-protein interactions involving SUNN utilizing in vitro protein pull-downs, Y2H assays, and bimolecular fluorescence complementation analyses to deduce potential receptor complexes and signaling components that may play a role in AON. Three G-protein signaling molecules of the Rop guanine exchange factors (MtRop-GEF1, -GEF5 and -GEF6) and MtCLV2, MtRLP1 and MtCRN were tested by cloning into Gateway BiFC vectors and expressing transiently in Nicotiana benthamiana with SUNN in the same vectors. SUNN, CLV2 and CRN were all capable of homodimerization and SUNN interacted with both CLV2 and CRN but not RLP1. Mutation of

CRN resulted in a hypernodulation phenotype and combined with reports in pea and L. japonicus of a hypernodulation phenotype for clv2 mutants, these interactions are likely part of AON signal transduction. RopGEF1 and RopGEF5 also interacted with SUNN in BiFC and Y2H assays. Together the data suggest SUNN signaling involves multiple protein complexes which may provide ligand specificity, and that downstream signaling to halt nodulation involves components of a G-protein signaling cascade. Supported by NSF IOS#1146014 and a Clemson Wade Stackhouse Fellowship to A.C.

900-034-Z Strength Without Stiffness: How the Plant Immune Signaling Network Achieves Both Robustness and Tunability Rachel Hillmer – University of Minnesota Yungil Kim – Johns Hopkins University, Kenichi Tsuda – Max Planck Institute for Plant Breeding Research, Daisuke Igarashi – Ajinomoto Co., Ghanasyam Rallapalli – The Sainsbury Laboratory, Shuta Asai – RIKEN CSRS Plant Immunity Research Group, Hitoshi Sakakibara – Nagoya University, Jonathan Jones – The Sainsbury Laboratory, Chad Meyers – University of Minnesota, Fumiaki Katagiri – University of Minnesota The plant immune signaling network, frequently exposed to attack from faster-evolving pathogens, must be robust against network perturbations. Simultaneously, the network must be tunable to optimize the cost/effect ratios against diverse attackers. We investigated how these seemingly contradictory network properties of robustness and tunability are achieved during Pattern-Triggered Immunity (PTI) in Arabidopsis. A highly predictive, semi-dynamic regression model of PTI was built for a network composed of the salicylate (SA), jasmonate (JA), ethylene (ET) and PAD4 signaling sectors, which form a relative bottleneck in the signal flow. Our model revealed that JA sector inhibition by the ET sector is central to the network robustness and that the pattern of signal inputs to the network tunes the network response. It also suggests tetra-stable states between the JA and PAD4 sectors, which may imply symmetric roles of the JA and PAD4 sectors in immune signaling. We are currently investigating more detailed dynamics of these signal flows, as well as how to learn fundamental rules governing the signal flows, in a data-driven manner. Funding: MCB-0918908 (FK and CLM), IOS-1121425 (FK), and DBI-0953881 (CLM) from NSF, Ajinomoto Co. (FK), the Max Planck Society (KT), Gatsby Foundation (GR and JJ), Japan Society for the Promotion of Science (SA), NIH/NIGMS training grant 2T32GM008347-21A1 (RAH), a UofM Doctoral Dissertation Fellowship (RAH), and four UofM PBS summer fellowships (RAH).

900-035-Z The Bacterial Wilt Disease of Plants: Studies of Bacterial Effector Proteins and Plant Root Responses to Ralstonia Solanacearum Raka Mitra – Carleton College Anne Duncan – Carleton College, Khuaten Maaneb de Macedo – Carleton College, Calvin Phan – Carleton College, Marie Schaedel – Carleton College, Ka Thao – Carleton College Plants are exposed to a variety of pathogenic microorganisms, many of which reside in the soil and impact plant roots. One such root-invading pathogen, Ralstonia solanacearum, is the causal agent of bacterial wilt. This pathogen causes one of the most devastating bacterial diseases of plants worldwide, affecting hundreds of plant species including many major crops such as tomato and potato and the model plants Arabidopsis thaliana and Medicago truncatula. Ralstonia typically infects plants through the root systems and ultimately colonizes the plant vasculature, where it interferes with water transport, resulting in plant wilting and death.

During plant invasion, Ralstonia employs a type III secretion system to deliver an array of effector proteins directly into the plant cell. The role of most of these effectors in bacterial wilt disease has not been explored. For leaf-invading pathogens, effectors typically target and interfere with host defense pathways. As a large number of Ralstonia effectors are not found in other pathogens, studies of these effectors may reveal novel plant pathways that are targeted during pathogen invasion, possibly illuminating novel aspects of plant root-based defenses. We are using two approaches for studying the Ralstonia-plant interaction. First, we are characterizing the suite of conserved effector proteins employed by a variety of Ralstonia strains. Students in the Carleton College Cell Biology Lab class are investigating the localization of these GFP-tagged bacterial effector proteins in planta. We have identified Ralstonia effectors with cytoplasmic, membrane and punctate localization, indicating a variety of host targets. Second, we are studying the Arabidopsis and tomato root transcriptional response to Ralstonia to determine which plant pathways are activated in response to this pathogen. Through these studies, we hope to develop a better understanding of the interface between the bacterial pathogen and plant cell during bacterial wilt disease.

900-036-Y A Vesicle Trafficking ENTH-protein Functions in flg22-signaling by Regulating Plasma Membrane Abundance of Flagellin Sensing 2 (FLS2) Antje Heese – University of Missouri-Columbia Carina Collins – University of Missouri-Columbia, Schweitzer Hall, Lauren Bond – University of Missouri-Columbia, Schweitzer Hall, John Smith – University of Missouri-Columbia, Schweitzer Hall, Divison of Plant Sciences, Jeff Anderson, Daniel Salamango – University of Missouri-Columbia, Schweitzer Hall, Scott Peck – University of Missouri The plasma membrane (PM) serves as a crucial contact point between hosts and potential pathogens. Plant PM proteins are required for many aspects of plant immunity, from microbe perception to pathogen growth restriction. A complex and dynamic vesicular trafficking network (including secretion and endocytosis) is essential to ensure the correct localization and level of host components at the PM necessary for effective immune responses. As such, the pattern recognition receptor Flagellin Sensing 2 (FLS2) needs to be localized to the PM to perceive its ligand flg22 to initiate robust responses. However, few vesicular trafficking components are known with roles in FLS2 trafficking to/from the PM. Here, we used a phosphoproteomic screen to identify an Arabidopsis ENTH-domain protein differentially phosphorylated in response to flg22, thus potentially placing it in the flg22-response pathway. In plants, ENTH-domain proteins appear to function in clathrin-mediated vesicle formation at the Trans-Golgi Network, potentially for delivery of newly synthesized cargo proteins to the vacuole or PM, endocytosed proteins for degradation in the vacuole or recycling to the PM. Two independent enth mutant alleles showed defects in all investigated flg22-responses and were more susceptible to infection of bacterial Pseudomonas syringae strains. Total and microsomal FLS2 protein levels were similar between Col0 and enth mutants. However, utilizing a simplified PM-enrichment methods, we correlated impaired flg22-signaling to reduced FLS2 protein levels at the PM. Our data identified this vesicular trafficking ENTH-protein as a novel positive regulator of innate immunity with roles in regulating correct FLS2 abundance at the PM.

900-037-Y Actin Remodeling During the Innate Immune Response Requires Both Capping Protein and Phosphatidic Acid Christopher Staiger – Purdue University Jiejie Li – Purdue University, Jessica L. Henty-Ridilla – Purdue University, Benjamin H. Staiger – Purdue University

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) initiates both rapid and long-term signaling events in plant cells and is necessary to fend off microbial invaders. Within minutes, innate immune signaling results in changes to cytosolic calcium levels, phospholipid fluxes, and generation of reactive oxygen species. Whether these signaling events impinge on cytoskeletal rearrangements and what are the underlying molecular events remain poorly understood. Using high performance, live-cell imaging approaches we quantified the actin remodeling that occurs during pattern-triggered immunity. Arabidopsis epidermal cells respond to a diverse array of bacterial and fungal MAMPs by transiently increasing actin filament dynamics and significantly elevating the density of filament arrays. Genetic analyses demonstrate that actin remodeling requires perception of MAMPs via known PRRs, including EFR and LYK1/4. To elucidate which actin-binding proteins are involved, we screened a collection of homozygous mutants expressing an actin cytoskeletal reporter; both actin-depolymerizing factor (ADF) and capping protein (CP) are implicated in transducing early signaling events into actin remodeling. Indeed, cp mutants fail to respond to diverse MAMPs and are more susceptible than wild-type plants to the biotrophic bacterial pathogen, Pseudomonas syringae DC3000 as well as the necrotrophic fungus, Alternaria brassicola. Because phospholipase D (PLD) activity and phosphatidic acid (PA) fluxes have been implicated in PTI, and CP is PA-binding protein, we set out to evaluate whether actin remodeling depends on phospholipid fluxes. Exogenous PA treatments mimic the increase in actin filament array density observed during PTI, whereas two chemical inhibitors of PLD abrogate the cytoskeletal response to MAMPs. We therefore propose a model whereby PRR activation results in activation of PLD/PA signaling that inhibits the heterodimeric capping protein and leads to increased actin polymerization.

900-038-Z Investigating the Roles of Auxin During Pseudomonas Syringe Pathogenesis Barbara Kunkel – Washington University Sheri McClerklin – Washington University, Andrew Mutka – Donald Danforth Plant Science Center, Gregory Harrison – Washington University, Anne Zimmerman – Washington University, Cynthia Holland – Washington University, Catherine Perrot-Rechenmann – ISV, Michael Prigge – University of California San Diego, Mark Estelle – University of California San Diego The bacterial pathogen Pseudomonas syringae manipulates plant hormone signaling to promote infection and disease development. Recent observations suggest that P. syringae utilizes several strategies to manipulate auxin physiology in Arabidopsis thaliana to promote pathogenesis, including synthesis of indole-3-acetic acid (IAA), the predominant form of auxin in plants, and production of virulence factors that alter auxin responses in the host. Further, application of exogenous auxin or elevated levels of endogenous auxin within the plant enhance pathogen growth and disease caused by P. syringae strain DC3000 (PstDC3000). Previous studies demonstrated that auxin inhibits host defense responses mediated by the plant defense hormone salicylic acid (SA). However, recent data from our lab indicates that auxin promotes PstDC3000 growth within the plant via a mechanism independent of suppression of SA-mediated defenses. Thus, it appears that auxin plays multiple roles during infection to promote disease. Surprisingly, A. thaliana mutants in which multiple TIR1/AFB F-box auxin co-receptors are mutated do not exhibit reduced susceptibility to PstDC3000, as would be expected if auxin-mediated responses in the host contribute to pathogenesis. Thus, host auxin perception may not be required for normal disease development. This finding raises the question of whether modulation of plant physiology or signaling is the main role for IAA during pathogenesis, and leads us to speculate that a primary mechanism by which auxin promotes P. syringae pathogenesis is through a direct effect on the pathogen. We will report on our progress towards further understanding the roles that auxin plays during P. syringae pathogenesis.

900-039-Z Dissecting Functions of the Turnip Yellows Virus Suppressor of RNA Silencing P0 in Evasion and Elicitation of Plant Defenses

Melanie A. Sacco – California State University, Fullerton Ken-Der Wang – California State University, Fullerton, Tan Tri V. Nguyen – California State University, Fullerton, Mansour Dughbaj – California State University, Fullerton, Kevin Valdez – California State University, Fullerton The Turnip yellows virus (TuYV) protein P0 (P0Tu) is a viral suppressor of RNA silencing (VSR) that targets Argonaute (AGO) proteins for degradation. P0Tu is also recognized in Nicotiana glutinosa accession TW59, in which it elicits hypersensitive response (HR) and resistance to TuYV. We are interested in understanding how P0Tu interacts with the host plant cell as a VSR versus elicitor of HR. To identify amino acids that are critical for these activities, P0Tu has been subjected to serial deletion analysis and site-directed mutagenesis at either several conserved sites or by systematic substitutions with the 6 amino acid sequence NAAIRS along the entire length of the coding sequence. We predicted that the modifications could potentially cause loss of VSR activity in Nicotiana benthamiana and/or loss of HR elicitation in N. glutinosa to dissect these two functions. Mutant clones were co-infiltrated with GFP into the leaves of N. glutinosa accession TW59 and N. benthamiana to observe for suppression of RNA silencing and induction of cell death. Substitution of two conserved central arginine residues with lysine caused P0Tu to elicit more rapid and robust HRs on N. glutinosa. Deletion or mutation of the amino terminus inactivate P0Tu, while modifications within or deletion of the last 22 carboxy-terminal amino acid residues (227-249) resulted in mutants that maintained VSR activity, but had impaired ability to elicit HR on N. glutinosa, suggesting that P0 activities in suppression of silencing and elicitation of HR may be distinct.

900-040-Y bHLH Transcription Factor DH1 Regulates Tomato Growth and Defense Against Xanthomonas Euvesicatoria Ying Sun – Stanford University Jung Gun Kim – Stanford University, Mary Beth Mudgett – Stanford University Plants dynamically regulate gene transcription to respond to and cope with biotic stress. The pathways that regulate gene expression can be hijacked by pathogens and used to promote pathogenesis. How pathogens manipulate plant development and defense pathways that impact the plant’s own growth is largely unknown. Our lab studies Xanthomonas euvesicatoria-tomato interactions to discover host specific-responses to bacterial virulence factors. We have recently identified a tomato basic helix-loop-helix (bHLH) transcription factor (TF) that is highly up regulated during Xanthomonas infection, and have evidence to show how this gene is involved both in immunity and in plant growth. We hypothesize that this bHLH regulates the transcription of genes involved in both tomato defense and growth. We are currently identifying and characterizing proteins that regulate the transcription of the bHLH TF using yeast 1 hybrid and GUS transcription reporter assay. To identify genes regulated by the bHLH TF and characterize it’s phenotype, we have also generated silenced lines of the bHLH TF in tomato. Recent progress in elucidating the function of the bHLH TF will be reported. Through these results we will identify mechanisms that regulate genes involved in plant growth and defense using perturbations caused by Xanthomonas.

900-041-Y Utilizing Wheat Alloplasmic Collections to Investigate the Role of Nuclear-cytoplasmic Interactions in Pathogen Response Katie Liberatore – USDA-ARS Cereal Disease Laboratory Marisa Miller – University of Minnesota, Shahryar Kianian – USDA-ARS Cereal Disease Laboratory Fungal pathogens pose a major worldwide threat to grain quality and yield in cereal crops (e.g. wheat and oats). Organellar genome diversity represents a potential untapped source for improving disease resistance in these important crop species. Wheat has an extensive collection of alloplasmic lines in which the cytoplasms, and therefore the

organelles (i.e. mitochondria and chloroplasts), of domesticated wheat varieties have been replaced with those of wild relatives through extensive backcrossing. Disruption of native nuclear-cytoplasmic interactions in these lines is known to impact a number of agronomic traits (e.g. fertility, biomass, and grain yield), yet little is known about the genetic diversity of organellar genomes from diverse Triticum and Aegilops species, how these genomes change in the alloplasmic condition, and how organelles contribute to disease resistance. Here, we present results of screening for improved resistance to fungal pathogens in wheat alloplasmic lines compared to their progenitor domesticated varieties. Screening with a panel of leaf rust (Puccinia triticina) and stem rust (Puccinia graminis) races, including Ug99 and additional foreign and domestic races, and the Fusarium toxin DON, has identified candidate cytoplasms that may provide improved disease resistance. Outcrossing and introduction of these cytoplasms to modern cultivars is underway to confirm the cytoplasmic effect and to test their robustness in varying genetic backgrounds. In parallel, deep sequencing and de novo genome assembly of both mitochondria and chloroplast genomes from alloplasmic lines and their progenitors has begun to reveal the organellar genetic diversity available in diverse wheat species. Future genomic analyses will further our understanding of the molecular basis for improved resistance under the alloplasmic condition. Combined, these studies will both enhance our fundamental knowledge of nuclear-cytoplasmic crosstalk and reveal novel sources for improving disease resistance in wheat and related crops.

900-042-Z Lectin Nucleotide Phosphohydrolases, a New Family of Lectins That May Function as Co-receptors or Modulators of Chitin-based Oligosaccharide Signaling Events in Plants Marilynn Etzler – University of California Davis Nicholas Roberts – AgResearch Grasslands Research Centre, Gurpreet Kalsi – ITC Limited, India Previous studies have shown that a novel lectin isolated from the roots of the legume, Dolichos biflorus (Vigna unguiculata), is a glycoprotein with both carbohydrate-binding and nucleotide phosphohydrolase activity. This Lectin Nucleotide Phosphohydrolase (LNP) is a peripheral membrane protein localized on the surface of root hairs in the nodulation zone and binds to the lipoligosaccharidic Nod factor signal produced by the rhizobia that initiate the nitrogen-fixing rhizobium-legume symbiosis. Recent transgenic studies showed that antisense inhibition of this LNP in Lotus japonicus knocks out nodulation as well as the mycorrhizal symbiosis of this plant with the fungus Glomus intraradices. The results showed that this LNP functions upstream of calcium signaling in the earliest stage of the signaling pathway common to the initiation of both symbioses. Using a combination of phylogenetic analysis. biochemical, molecular and cellular techniques, we now report the identification and characterization of other LNPs in Dolichos biflorus. A comparison of the structures, carbohydrate- and nucleotide-binding activities of these LNPs and their differential expression and localization in the plant suggests that these different LNPs may have evolved by gene duplication and subsequent divergence to function as co-receptors or modulators of a variety of chitin-based oligosaccharide signaling events found in plants.

900-043-Z Mechanistic Dissection of Novel Rhg1-Mediated SCN Disease Resistance Andrew Bent – University of Wisconsin – Madison Adam Bayless – UW-Madison, Stephen Mosher – UW-Madison, Katelyn Horgan – UW-Madison, Ryan Zapotocny – UWMadison, Patrick McMinn – UW-Madison, Alice Teillet – UW-Madison The Rhg1 locus contributes the most widely used resistance to the most economically damaging disease of soybean, caused by soybean cyst nematode (SCN, Heterodera glycines). Tens of thousands of DNA marker-assisted selection genotypes for Rhg1 are determined and used every year by soybean breeders, but the molecular nature of Rhg1 was not

known. In 2012 we discovered that Rhg1 encodes three tightly linked genes, specifying three completely distinct proteins that each contribute to SCN resistance. Equally surprising, resistance is dependent on copy-number variation of a multi-gene, ~30 kb Rhg1 segment. Most SCN-susceptible soybeans carry a single copy of the locus, but the most widely used resistance “allele” (haplotype) carries ten tandem repeat copies of this ~30 kb genome segment. The three Rhg1 protein types encoded on each copy of this genome segment have not previously been associated with plant disease resistance: a putative alpha-SNAP (vesicle trafficking), a putative amino acid transporter, and a protein lacking predictive annotation other than a domain common in some wound-inducible proteins. We will present our more recent findings regarding: a) functional impacts of the non-canonical variant residues in resistance-associated alphaSNAP proteins, b) variable Rhg1 locus methylation, depending on genotype and infection status, and c) evidence for copy number dose-dependency of Rhg1-mediated resistance for some but not all components of Rhg1-mediated resistance.

900-044-Y Arabidopsis MORC1/CRT1 Interacts with a Wide Range of Putative Chromatin-remodeling Factors Hong Gu Kang – Texas State University Ji Chul Nam – Texas State University, April Bonnard – Texas State University, Dan Klessig – Boyce Thompson Institute, Yogendra Bordiya – Texas State University Arabidopsis MORC1/CRT1, an ATPase, is necessary for a wide range of resistance including effector-triggered immunity mediated by resistance proteins and PAMP (pathogen-associated molecular pattern)-triggered immunity mediated by PAMP recognition receptors. To further characterize the function of MORC1, we identified 14 MORC1-interacting proteins (MIPs) via yeast-2-hybrid screening using MORC1 as bait. Eight MIPs were shown/predicted to be nuclearlocalized. Interestingly, these putative nuclear MIPs include five potential chromatin remodeling/modifying components. MIP12 carries the mobile domain found in chromatin remodeling helicases, transposases, and some retrotransposons; it also exhibits significant homology to copia retrotransposons and a potential chromatin remodeling helicase. Arabidopsis eFP Browser indicates that MIP12 is highly inducible by the flg22 PAMP treatment. MIP5 is a WAVE/WASP protein that promotes the ARP2/3 complex formation, which functions in polymerizing nuclear actin, a component of the SNF chromatin remodeling factor. MIP8 is a cohesin that is part of a SWI6 chromatin remodeling factor and displays homology to DMS3, a known MORC6-associated protein. MIP13 has a SANT domain that interacts with histones. MIP3 is MORC6, suggesting heterodimerization between MORC1 and MORC6. The interaction of these MIPs with other members of the MORC family was assessed by yeast-two-hybrid analysis. Except for an interaction between MORC7 and MIP12, no other interactions were detected, suggesting that MORC1 performs functions independent from the other family members. We have obtained one or two mutant lines for each MIP and characterized their responses to avirulent and virulent Pseudomonas syringae. T-DNA insertion mutants for five MIPs described above displayed significantly altered resistance to the bacterial pathogens. Furthermore, some of these mip mutants when combined with morc1/2, a double knock-out mutant lacking MORC1 and its closest homolog MORC2, showed highly synergistic interactions in the anti-bacterial defense responses, suggesting that MORC1 may regulate plant defenses via its interaction with these MIPs.

900-045-Y Amino Acid Transporter AAP6 Plays an Essential Role in Nitrogen Export from Pea Nodules Matthew Garneau – Waashington State Uniersity Qiumin Tan – Baylor College of Medicine, Mechthild Tegeder – Washington State University The atmosphere provides a large pool of di-nitrogen (N2) that plants can access through a symbiotic relationship with bacteria. Legumes develop root nodules that house rhizobia. These bacteria fix N2 into ammonium, which is reduced to amino acids, and in pea mainly to asparagine. The amino acids then move symplasmically or apoplasmically towards the

nodule vascular bundle for shoot N supply. The nodule vasculature is surrounded by an endodermis containing the Casparian strip that blocks apoplasmic movement towards the xylem. We hypothesized that transporters are needed to load amino acids from the cell wall space into the endodermis or adjacent cortex cells to bypass the apoplasmic blockage. Using heterologous complementation with yeast mutants we isolated a pea amino acid transporter, PsAAP6 that is expressed in nodules. Protein localization experiments resolved that PsAAP6 is targeted to the plasma membrane, and in situ RNA hybridization showed expression of the transporter in the uninfected cortex cells surrounding the nodule vasculature. To analyze the physiological function of PsAAP6, we then used an RNA interference approach in nodulated hairy pea roots. RNA expression analysis showed that PsAAP6 transcripts were strongly reduced in the transgenic AAP6-RNAi nodules. Further, repression of PsAAP6 led to increased levels of nodule asparagine and other amino acids, and to a decrease in root amino acids. In addition, the leaf chlorophyll content was decreased in the AAP6-RNAi plants. These data suggest an essential role of PsAAP6 in amino acid, and especially asparagine export from nodules. Expression studies of nodule genes related to N assimilation and transport as well as analysis of nodule number indicates that PsAAP6 function is not only important for shoot N nutrition but also plays a role in regulating nodule N metabolism and partitioning, and nodule development.

900-046-Z Comparative Transcriptomics Reveals Distinct Hormone Action Pathways in Lateral Roots and Nodules of Soybean Sajag Adhikari – South Dakota State University, Suresh Damodaran – South Dakota State University, Senthil Subramanian – South Dakota State University Leguminous plants such as soybean produce two kinds of root lateral organs; lateral roots and nodules. Unlike shoot lateral organs that develop from the flanks of shoot apical meristem, lateral roots and nodules are formed away from the root apical meristem via dedifferentiation of preexisting cells, pericycle and cortex respectively. Commonalities in some developmental features of lateral root and nodules has led to the speculation that nodules might have adopted the developmental pathways of lateral roots. However, differences in hormonal requirement of these organs indicates that nodules might have adopted developmental pathways distinct from that of lateral roots. To understand the similarities and differences in the development of these organs we dissected lateral root and nodules at emerging and mature stages of development and determined differences in global transcriptome profiles and specifically compared the enrichment of transcription factors, and hormone biosynthesis and signaling components. Results from these analyses strengthen the conclusion that auxin and cytokinin play opposite roles in the development of nodules and lateral roots. In addition, potential orthologs of genes associated with shoot axillary meristem were enriched in nodules, but not in lateral roots. Together, these results indicate that the soybean nodule might be a modified shoot axillary organ.

900-047-Z Traffic Control for Plant Immunity and Pattern Recognition Receptors Hannah Kuhn – The Sainsbury Laboratory Martina Beck – The Sainsbury Laboratory, Sara Ben Khaled – The Sainsbury Laboratory, Gildas Bourdais – The Sainsbury Laboratory, Malick Mbengue – The Sainsbury Laboratory, Michaela Kopischke – The Sainsbury Laboratory, Jelle Postma – The Sainsbury Laboratory, Thomas Spallek – The Sainsbury Laboratory, RIKEN Yokohama Institute, Matthieu Joosten – Wageningen University, Silke Robatzek – The Sainsbury Laboratory In our attempts to understand the full nature of the interactions that occur between a potential pathogen and its host, we are elucidating the transport processes that are engaged by the plant’s immune system. Our main research focus has been how transport processes regulate defence activation. Combining genetic, molecular and biochemical approaches

with cell biology we have comprehensively dissected the subcellular transport pathways dependent upon microbial stimulation. Our studies have revealed that clathrin- and ESCRT-mediated endosomal trafficking is required for plant defence and is important for stomatal immunity. To identify mechanistic and functional elements of transport-regulated immunity, we focus on how the pattern recognition receptors (PRRs), the primary sensors of the plant’s immune system, are transported through the cell. PRRs are receptor kinases and receptor-like proteins that must be presented at the plasma membrane to recognize potentially infectious pathogens and trigger immunity. We found that PRRs representing different protein families are endocytosed in a ligand-induced and BAK1/SERK3 co-receptor dependent manner. Together with the finding that activated PRRs (FLS2, EFR, PEPR1) traffic via a common endosomal pathway, this suggests a role of endocytosis in the regulation of receptor abundance at the plasma membrane triggered by ligand perception. Furthermore, endocytosis of activated FLS2 is mediated by clathrin and involves sorting by the ESCRT machinery. This indicates a link between transport processes involved in defence and PRR trafficking. Understanding these mechanisms is providing novel insights into the regulation of plant immunity. This work is supported by the Gatsby Charitable Foundation and a grant by the European Research Council (ERC).

900-048-Y Screening and Characterization of Plant-growth-promoting Epiphytic Bacteria from Rice (Oryza Sativa) Chokchai Kittiwongwattana – King Mongkut’s Institute of Technology Ladkrabang Epiphytic bacteria have been widely studied for their plant growth promotion. Several plant-growth-promoting mechanisms by bacteria have been previously proposed. Here, we isolated 98 epiphytic bacteria from leaves, stems and roots of rice plants collected from rice fields in Thailand. All isolates were screened for their plant-growth-promoting activities. Thirty-seven isolates were tested positive for phosphate solubilization on national botanical research institute’s phosphate growth and Pikovskaya’s media. Seventy-six isolates were found IAA producers when tested with Salkowski’s reagent while thirteen isolates produced siderophore on chrome azurol agar medium. These candidates for plant-growth-promoting bacteria were further characterized using partial 16S rRNA gene sequences. The results showed that the predominant groups of phosphate-solubilizing, IAA-producing and siderophore-producing bacteria were those that belonged to the genera Enterobacter (29.7%), Sphingomonas (14.5%) and Microbacterium (46.2%), respectively. All positive bacteria were also tested for growth promotion in ten-day-old rice seedlings, and significant increase (>1.5 folds) in dry weight was found in inoculated rice seedlings compared to the non-treated control group. IAA production and phosphate solubilization were found in these isolates suggesting the significance of these activities in plant-growthpromoting mechanisms. Our results also revealed the potential of these isolates for application as biofertilizers in rice farming.

900-049-Y Root-knot Nematodes Induce Pattern-triggered Immunity Marcella Teixeira – University of California, Riverside Hsuan-Chieh Peng – University of California, Riverside, Isgouhi Kaloshian – University of California, Riverside Root-knot nematodes (RKNs; Meloidogyne spp.) are plant parasites with a broad host range causing great losses worldwide. To parasitize their hosts, RKNs establish feeding sites in roots known as giant cells. The majority of work studying plant-RKN interactions in susceptible hosts deal with the establishment of the giant cells and not with early defense responses. Here we show that, similar to microbial pathogens, early defense or pattern-triggered immunity (PTI) also exists against RKN. To investigate the role of PTI against RKN, we infected Arabidopsis Col-0 and bak1-5 mutant with RKN and evaluated nematode attraction, penetration and root galling. Although nematodes were equally attracted to roots of both genotypes, nematode penetration and root galling were significantly higher in bak1-5 roots. Expression of

PTI marker genes, WRKY11, MYB51 and CYP71A12, was induced in wild-type roots after infection with RKN. Although induction of the transcription factors WRKY11 and MYB51 was abolished in bak1-5 mutant, expression of CYP71A12, a cytochrome P450 involved in camalexin biosynthesis, was only attenuated after RKN infection of bak1-5 mutant. In addition, the pad3 mutant, impaired in camalexin production, showed enhanced susceptibility to RKN similar to bak1-5. Furthermore, mutants of BIK1 and RbohD/F, components of PTI recognition complex, were also more susceptible to RKN. Combined, our results indicate the presence of BAK1-dependent and independent PTI against RKN in Arabidopsis.

900-050-Z Identification of Key Regulators of Pathogen Virulence for Sclerotinia Stem Rot in Soybean and Dry Bean Using Next Generation Sequencing Shalu Jain – North Dakota State University Robert Brueggeman – North Dakota State University, Jonathan Richards – North Dakota State University, Chengxiang Qiu – North Dakota State University, Shyam Solanki – North Dakota State University, Layra Aldrich-Wolfe – Concordia College, Jared LeBoldus – North Dakota State University, Berlin Nelson – North Dakota State University Sclerotinia stem rot caused by Sclerotinia sclerotiorium (Lib.) deBary is one of the most devastating soybean and dry bean diseases in the Unites States. Association mapping (AM) analysis using RAD-GBS genotypic data and lesion length phenotypic data collected on dry bean, soybean, canola and sunflower for 115 isolates led to the identification of several marker trait associations (MTAs) within the S. sclerotiorum genome. Ten loci were identified with significant MTAs (log10(p) >3) using the dry bean data and stringent false discovery rate analyses. To further validate these correlations and identify differentially expressed genes, RNAseq analysis was performed using the virulent strain 140MN of S. sclerotiorium. Three days post inoculation, 779 and 571 S. sclerotiorium genes were differentially expressed in dry bean and soybean, respectively, during host pathogen interaction when compared to controls. Six differentially up-regulated S. sclerotiorium genes common to both the dry bean and soybean infected RNAseq libraries aligned with MTAs identified in the AM analysis. The up-regulated genes were predicted to encode three glycosyl hydrolases (cell wall degrading enzymes), a MFS transporter (involved in the transport of fungal toxins), and a LicD domain protein (involved in cell adhesion, decreased choline uptake, and mutants lacking this protein are known to have decreased virulence). The significantly down-regulated gene is similar to a choline dehydrogenase. These six genes currently represent our best candidate virulence genes. Functional analysis of the candidate genes is being conducted using post-transcriptional gene silencing via stable siRNA transformation of the pathogen and host induced gene silencing in Arabidopsis.

900-051-Z Role of Mevalonate Pathway in Plant-microbe Symbiotic Signaling Pathway Muthusubramanian Venkateshwaran – University of Wisconsin Dhileepkumar Jayaraman – University of Wisconsin-Madison, Mireille Chabaud – Institut National de Recherche Agronomique (UMR 441), Centre Nationale de Recherche Scientifique (UMR 2594), Andrea Genre – University of Torino, David Barker – Institut National de Recherche Agronomique (UMR 441), Centre Nationale de Recherche Scientifique (UMR 2594), Jean-Michel Ane – University of Wisconsin-Madison Legumes establish symbiotic associations with both rhizobial bacteria and arbuscular mycorrhizal fungi. Although several genetic components of these symbioses have been identified in model legume Medicago truncatula, it remains unknown how the signals that are perceived by the receptors in the plasma membrane are transduced to nucleus, triggering periodic oscillations in the nuclear calcium concentration, known as ’calcium spiking’, one of the earliest symbiotic signaling events. We previously identified that a co-receptor in legume symbioses, DMI2 (Does not Make Infections 2), localized in the plasma membrane, interacts with HMGR1 (3-hydroxy-3-methylglutaryl coenzyme A reductase 1). HMGR1 is a key regulator in the mevalonate pathway of isoprenoid biosynthesis and is required for legume nodulation. We

hypothesized that the products of mevalonate pathway may act as signals linking the plasma membrane events to the nuclear events, such as calcium spiking that are regulated by the nuclear cation channel DMI1. These calcium signatures are decoded by DMI3, a calcium and calmodulin-dependent protein kinase, which is a central regulator of symbiosesspecific gene expression, culminating in the symbiotic responses. Silencing HMGR1 affects the nuclear calcium spiking and symbiotic gene expression in response to rhizobial Nod factors. Furthermore, the addition of mevalonate, a direct product of HMGR1 activity, triggers nuclear calcium spiking and symbiotic gene expression in both wild-type and HMGR1-silenced roots. In addition, analyses of mevalonate-induced calcium spiking and gene expression in symbiosisdefective mutants have revealed that mevalonate restores the nuclear calcium spiking and gene expression in dmi2 but not in dmi1 mutants, indicating that HMGR1 activity or the products of mevalonate pathway link the plasma membrane and nuclear events. In addition, the induction of calcium spiking by mevalonate in Human Embryonic Kidney 293 cells expressing DMI1 suggests a direct link between mevalonate and the activation of DMI1 to initiate nuclear calcium spiking.

900-052-Y Investigating Cassava Bacterial Blight with Image-based Phenotyping Methods Andrew Mutka – Donald Danforth Plant Science Center Joel Sher, Anupama Vijayaraghavan, Mark Wilson, Rebecca Bart Cassava, a major food crop for over 500 million people worldwide, is threatened by the bacterial pathogen Xanthomonas axonopodis pv. manihotis (Xam). This pathogen causes cassava bacterial blight (CBB), a disease characterized at early stages by symptoms such as water-soaked lesions on leaves and at later stages by leaf wilting and defoliation. Despite the potential devastating effects of CBB, little is known about the molecular mechanisms underlying Xam virulence. As with many other bacterial pathogens, Xam uses a type III secretion system to deliver virulence factors, known as type III effectors (T3Es), into the host cell. Genome sequencing has enabled us to identify T3E repertoires for a diverse set of Xam strains. While T3Es are collectively essential for virulence, investigating the functions of individual T3Es is challenging, since they exhibit functional redundancy with each other. Traditional methods of monitoring pathogen growth in the host allow us to reproducibly measure bacterial abundance during infection, but single T3E knockout mutants often exhibit only subtle effects on pathogen growth, or no effect at all. To expand the range of phenotypes that we are able to measure during plant-pathogen interactions, we are developing imaged-based phenotyping methods that enable us to quantify disease on multiple scales, from symptoms on individual leaves to the effects of infection on overall growth and development. These approaches offer the potential to generate quantitative data on plant disease progression in a high-throughput manner. Additionally, we are using imaging approaches to investigate other aspects of Xam infection, such as its ability to spread in the host. The long-term goals of this study are to develop effective phenotyping tools for disease detection and assessment, and to enhance our understanding of pathogen virulence mechanisms to inform efforts in developing resistant cassava varieties.

900-053-Y Pseudomonas Infection and AtMORC Family Members Influence Chromatin Accessibility in Arabidopsis Yogendra Bordiya – Texas State University Yi Zheng – Boyce Thompson Institute for Plant Research, Ji-Chul Nam – Texas State University, Hyong Woo Choi – Boyce Thompson Institute for Plant Research, Daniel Klessig – Boyce Thompson Institute for Plant Research A genetic screen for components involved inresistance (R) protein-mediated immunity in Arabidopsis led to isolation of crt1 (compromised recognition of TCV). CRT1/MORC1 was shown to be a MORC ATPase/endonuclease that physically interacts with multiple immune components. While MORC1 is mainly located in endosome-like vesicles in the cytoplasm, a subpopulation resides in the nucleus, which increases after infection. The combined findings that MORC1 i) is an

endonuclease, ii) is localized to heterochromatin, and iii) is implicated in epigenetic regulation, including suppression of heterochromatic transposable elements (TEs), suggest that MORC1 has an important nuclear function(s). Thus, we are investigating MORC1’s role in the nucleus. To understand how MORC1’s role in epigenetic gene silencing impacts its function in plant immunity, genome-wide chromatin accessibility was compared between mock- or Pseudomonas syringae pv. tomato (Pst)-inoculated wild type (WT) Arabidopsis and/or the atmorc1/2 double mutant. Differential DNase I hypersensitive sites (dDHSs) detected in atmorc1/2 vs WT comparisons were predominantly associated with heterochromatic transposons. Chromatin immunoprecipitation revealed that AtMORC1 constitutively bound these sites, suggesting that AtMORC1 mediates heterochromatin silencing. By contrast, the dDHSs identified in Pst vs mock comparisons were primarily associated with signal transduction and biotic/abiotic stress-related genes; a smaller but still significant population was also detected in transposons distributed throughout the genome. Pst-induced, transposonassociated dDHSs displayed enhanced MORC1 binding after infection. Such sites were found in the upstream of two defense-associated genes, which showed differential expression between Pst-inoculate atmorc1/2 and WT plants, suggesting AtMORC1 regulates pathogen-induced expression of these transposons and their neighboring genes.

900-054-Z Pseudomonas Syringae Effector HopZ3 Acetylates Multiple Effector-immune Plant Complexes to Suppress Pathogen Resistance Jiyoung Lee – University of Chicago Andrew Manning – University of Chicago, Donald Wolfgeher – University of Chicago, Stephen Kron – University of Chicago, Jean Greenberg – University of Chicago Many bacterial pathogens of both plants and animals use a type III secretion system to deliver their effector proteins into host cells to promote disease. Pseudomonas syringae pv syringae B728a, a pathogen of bean and Nicotiana benthamiana has a strong epiphytic life style. One of the effectors we have characterized in detail is HopZ3, which promote PsyB728a growth in Arabidopsis and tomato. Here we show that HopZ3 interacts with multiple members of plant immune complexes. HopZ3 is an active acetyltransferase that acetylates itself and many but not all HopZ3interacting proteins with which it can form complexes. HopZ3 targets both kinase and kinase targets, suggesting it acts by preventing key phosphorylation events. We will discuss our progresses in mapping acetylation versus phosphorylation sites in immune complex components.

900-055-Z Alternative Splicing Landscapes Modulated During Plant-Virus Interactions in Brachypodium Distachyon Kranthi Kiran Mandadi – Texas A&M AgriLife Research Jesse Pyle, Karen-Beth Scholthof In plants, alternative splicing (AS) influences diverse biological processes including growth, development and response to stress. There is a gap, however, in our knowledge of AS changes occurring during plant-virus interactions. To address this knowledge-gap, we performed RNA-sequencing of the model monocot, Brachypodium distachyon, infected with Panicum mosaic virus (PMV). Using bioinformatics approaches, we identified ~44,443 transcripts. Expression of ~28,900 transcripts in ~18,800 gene loci was ≥2 FPKM, and ~42% of multi-exonic genes were alternatively-spliced. Among the major AS types analyzed, intron-retentions predominated, followed by alternate acceptor, alternative donor and exonskipping. Comparative analysis of the AS landscapes in Brachypodium, rice, maize, sorghum, Arabidopsis, potato, Medicagoand poplar further revealed conserved AS patterns among monocots and dicots. In Brachypodium, we identified previously unknown AS events in ~100 immune-related genes encoding receptor-like kinases, NB-LRR resistance proteins, transcription factors, RNA-silencing and splicing-associated proteins. Cloning and molecular characterization of Bd-SCL33, a serine/arginine-rich (SR) splicing factor, uncovered multiple novel intron-retaining splice-

variants that are developmentally regulated and modulated during virus infection. Strikingly, Bd-SCL33 splicing patterns are conserved during infections of seven additional grass-infecting viruses including PMV+SPMV, Brome mosaic virus, Barley stripe mosaic virus, Maize mild mottle virus, Sorghum yellow banding virus, Wheat streak mosaic virus and Foxtail mosaic virus. Splicing patterns of Bd-SCL33 during development are also strikingly similar to the splicing patterns of a distant Arabidopsis ortholog, suggesting a conserved mechanism of SCL33 splicing between monocots and dicots. Together, this analysis provides new insights into virus-triggered AS landscapes conserved among monocots and dicots.

900-056-Y Towards Fine-mapping and Cloning Zym-0, a Dominant Resistance Gene to Zucchini Yellow Mosaic Virus in Pumpkin Wolfgang Schweiger – BOKU – Universitat fur Bodenkultur Pawinee Innark, Lucia Nocete, Martin Pachner, Stephanie Hembach, Johann Vollmann, Wolfgang Schweiger, Hermann Buerstmayr Varieties of the Cucurbita pepo species, including field pumpkin, summer squash and zucchini are generally highly susceptible to Zucchini Yellow mosaic virus (ZYMV). No resistance sources to the devastating disease are described in the species but several resistance genes have been identified in varieties of the related Cucurbita moschata and Cucurbita menina species . We have employed the C. moschata variety Nigerian Local as the resistance donor parent of the resistance gene Zym-0, for generating several BC3F8 populations in the genetic background of an Austrian Oil Seed Pumpkin variety as the recurrent parent. Oil Seed Pumpkin comprises a set of hull-less seed C. pepo varieties who are widely cultivated in Austria and Central Europe for the production of edible oil. We have developed a fine-tuned phenotyping protocol for assessing ZYMV resistance and scored several populations derived from single BC3F8 seed, which were heterozygous for Zym-0. Zym-0 confers dominant resostance as the heterozygous progeny retains resistance albeit not as strongly as the homozygous Zym-0 plants. We have utilized a closely linked SSR marker to screen for recombinations between marker and trait have selected several dozen plants which will be sequenced and compared to yield the genomic region harboring the resistance gene.

900-057-Y Isolation and Characterization of a Novel Tomato AGC Kinase That Is Dephosphorylated During the Response to Pseudomonas Syringae In-Cheol Yeo – Texas A&M University Timothy Devarenne The tomato protein kinase Adi3 was previously isolated based on interaction with Pto and AvrPto, which are host R protein and Pseudomonas syringae pv. tomato (Pst) effector, respectively. Adi3 belongs to the AGC family of protein kinases and functions in cell death suppression (CDS). The CDS function of Adi3 is mediated through phosphorylation by Pdk1. In the tomato – Pst interaction, Adi3 forms a complex with Pto and AvrPto, which induces the hypersensitive response programmed cell death due to inhibition of Adi3 CDS activity. A Pdk1 phosphorylation site specific α-Adi3 antibody (α-pAdi3) was developed to monitor Adi3 phosphorylation changes in response to Pst. When a tomato leaf was treated with flg22, α-pAdi3 detected one protein and its detection was significantly decreased within 20 minutes and fully recovered after 3 hours, suggesting this protein is dephosphorylated in response to Pst flagellin. However, the αpAdi3 detected protein showed a different molecular weight (~56 kDa) from that of Adi3 (77 kDa). Thus, α-pAdi3 is able to detect another AGC kinase phosphorylated by Pdk1, which is not surprising since this phosphorylation site is highly conserved in AGC kinases. Detection of this new AGC kinase, Novel AGC family Kinase (NAK), by α-pAdi3 was not reduced when the phosphatase inhibitor Na3VO4 was included with flg22 supporting dephosphorylation of NAK in response to flagellin. To identify and characterize NAK, two candidates, called NAK-1 and NAK-2, were selected and

successfully expressed and purified from E. coli. Both showed autophosphorylation kinase activity and their ATP-binding and Pdk1-mediated phosphorylation sites were identified. Both were phosphorylated by Pdk1 and this phosphorylation event was able to be detected by α-pAdi3. To identify the precise NAK that is dephosphorylated in response to flg22, immunoprecipitation and MS analysis are now in progress using tomato protein extracts and α-pAdi3.

900-058-Z Post-Translational Modification by the Type III Effector HopZ3 Modulates Tomato Host Immunity by Acetylation of Bacterial and Plant Proteins Andrew Manning – University of Chicago Jiyoung Lee – University of Chicago, Don Wolfgeher – University of Chicago, Stephen Kron – University of Chicago, Jean Greenberg – University of Chicago Billions of dollars are lost each year to crops infected with bacterial blights. Understanding how crops are infected is pivotal to the development of new strategies to fight pathogenic bacteria. One key virulence factor for bacterial pathogens is the type III secretion system (TTSS). The TTSS allows for the translocation of bacterial effector proteins from the cytoplasm of the bacteria into the cytoplasm of the host organism. These type III effectors have been shown to have a wide array of different mechanisms all in the effort of altering the host immune response to bacterial infection. In this study, we identify HopZ3 as an acetyltransferase with the capability to modify a wide range of host and other bacterial targets. Previous work in our lab has shown that HopZ3 interacts with and modifies components of the RPM1 plant defense complex in Arabidopsis, as well as other bacterial effectors that activate this immune complex. Examining the homologous defense pathway of Arabidopsis in tomato, we find that HopZ3 also interacts with, and acetylates components of the RIPK/PTO/RIN4/AvrPto complex in tomato. Using Pseudomonas syringae pv B728a effector HopZ3, acetylation sites of tomato and bacterial interactors were mapped using mass spectrometry. Interestingly, we show that the acetylation sites coincide with identified residues important for defense signaling, phosphorylation, and protein-protein interactions. Additionally, acetylation sites were found on residues including lysine, serine, threonine, and histidine. Previously unseen, we find that all four residues are enzymatically modified by the same effector. Histidine acetylation has been rarely reported previously as a transient modification, but in this study is a stable and repeatable modification across substrates of HopZ3. Collectively, our data suggests that HopZ3 targets plant host defense in a multifacted mechanism by halting kinase activity, blocking phosphorylation, and interfering with protein-protein interactions.

900-059-Z The Expression of a Glycine Max Homolog of LESION SIMULATING DISEASE1 Results in the Coexpression of NONEXPRESSOR of PR1 (NPR1), a Gene That Functions in Resistance to Root Pathogens Shankar Pant – Mississippi State University Brant McNeece – Mississippi State University, Keshav Sharma – Mississippi State University, Aparna Krishnavajhala – Mississippi State University, Gary Lawrence – Mississippi State University, Vincent Klink – Mississippi State University Experiments show the membrane fusion genes alpha soluble NSF attachment protein (a-SNAP) and syntaxin 31 (GmSYP38) contribute to the ability of Glycine max to defend itself from infection by the plant parasitic nematode Heterodera glycines. The transcriptional activation of the defense genes ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and NONEXPRESSOR OF PR1 (NPR1) that function in salicylic acid (SA) signaling accompanies their expression, implicating the involvement of the antiapoptotic, environmental response gene LESION SIMULATING DISEASE1 (LSD1) in

defense. In tests examining the role of Gm-LSD1–2 in defense, its overexpression results in a 52 to 68% reduction in H. glycines parasitism as measured by the female index. In contrast, RNA interference (RNAi) of Gm-LSD1–2 in the resistant genotype G. max [Peking/PI 548402] results in 3.24–10.42 folds increased ability of H. glycines to parasitize. The results identify that Gm-LSD1–2 functions in the defense response of G. max to H.glycines parasitism. To examine the conserved nature of this defense process that involves NPR1, the G. max NPR1-2 gene was engineered into Gossypium hirsutum, resulting in a 65-75% reduction in gall formation, an 80-85% reduction in egg mass formation, a 70-80% reduction in egg production and an 89-97% reduction in second stage juvenile formation for the giant cell forming nematode Meloidogyne incognita. The results identify a conserved role for NPR1 in the defense of plants to parasitic nematodes.

900-060-Y Investigate the Regulation of Mitogen-activated Protein Kinase Phosphatase 1 (MKP1) in PAMP Signaling and Resistance to Bacteria Lingyan Jiang – University of Missouri-Columbia, Ying Wang – University of Missouri-Columbia, Jeffrey Anderson – University of Missouri-Columbia, Roman Ulm – University of Geneva, Scott Peck – University of Missouri-Columbia The perception of pathogens starts with recognition of conversed pathogen-associated molecular patterns (PAMPs) mediated by surface-localized pattern recognition receptors (PRRs), which initiates a series of intracellular responses that ultimately limit bacterial growth. One of the important PAMP responses is intracellular protein phosphorylation including mitogen-activated protein kinases (MAPKs) cascades. MAP kinase phosphatases (MKPs) are important negative regulators of MAPKs and play a crucial role in ensuring adequate intensity and duration of MAPK activation. Work with non-plant organisms has shown that MAPKs control their own negative regulators, establishing negative feedback loops to attenuate the response. However, in plants, the regulation of MKPs is less understood. Arabidopsis MKP1 was shown to be a negative regulator of innate immune signaling as the mkp1 mutant displayed enhanced PAMP responses and resistance against virulent bacterium Pto DC3000. MKP1 was also shown to be phosphorylated and activated by MPK6 in vitro, suggesting that phosphorylation may be an important mechanism for regulating MKP1. To test this hypothesis, phosphorylation site mutants were generated in the background of mkp1 null mutant. Phosphorylation site mutants were not able to complement the mkp1 phenotype, indicating that phosphorylation of those sites is required for the function of MKP1. One hypothesis is that MKP1 is phosphorylated in response to PAMP treatment and that phosphorylation stabilizes the protein, and this supposition is supported by the result that both PAMP and MG132 treatment increased MKP1 protein level. In addition, to investigate the possible roles of distinct structural subdomains in the function of MKP1, domain truncation mutants were generated. Preliminary results indicate that the gelsolin but not the camodulin binding domains are required for the function of MKP1. Because the gelsolin domain binds to actin that mediates the protein localization, these results suggest that different protein localizations may also contribute to the regulation of MKP1.

900-061-Y Transcriptome Analysis of Arabidopsis Seedlings in Response to Agrobacterium Infection Po-Yuan Shih – Institute of Plant and Microbial Biology, Academia Sinica Erh-Min Lai – Institute of Plant and Microbial Biology, Academia Sinica Agrobacterium tumefaciens is a causal agent of crown gall disease via delivery of T-DNA into plant genome. Besides bacterial effectors and plant proteins participating in T-DNA translocation processes, activation of plant immunity by recognizing Agrobacterium PAMPs (Pathogen Associated Molecular Patterns) such as elongation factor Tu (EF-Tu) also negatively impacts transformation efficiency. To understand the molecular mechanisms underlying how plants respond to Agrobacterium infection in the presence or absence of EF-Tu receptor (EFR), we investigated the gene expression profiles of the Arabidopsis wild type Col-0 and efr-1 upon Agrobacterium infection in early stage (2 and 24 hours), and

then used intact seedling and root explant transformation assays for functional studies. We found that higher transient transformation efficiency was detected in the shoots of efr-1 than its isogenic wild type Col-0 but not the roots of both seedlings, suggesting that EFR plays a major role in shoot immunity to Agrobacterium infection. Transcriptome analysis revealed Agrobacterium responsive genes enriched in hormone responses, defense responses, secondary metabolite biosynthesis and plant cell wall modification. Arabidopsis mutants impaired in selected EFR-responsive genes were characterized for their involvement in Agrobacterium transformation process. Preliminary data have revealed some mutants with altered tumorigenesis and/or transient transformation phenotypes, which suggests their important roles involved in Agrobacterium transformation process.

900-062-Z Inducible Artificial Micro-RNAs to Investigate Functional Redundancy in the Vesicular Trafficking Genes DRP2A and DRP2B in Flagellin-signaling Sean Rogers – University of Missouri Michelle Leslie – University of Missouri, Gary Baisa – University of Wisconsin-Madison, Bret Huisenga – University of Wisconsin-Madison, Sebastian Bednarek – University of Wisconsin-Madison, Antje Heese – University of Missouri Understanding how plants fight bacterial infection is a centerpiece in developing new methods of agriculture to bring forth higher yields of food production. Research in the Heese Lab is focused on the role of vesicular trafficking in plant immune responses to bacterial flagellin, or its active peptide derivative flg22. In the model plant Arabidopsis thaliana, Dynamin-related proteins (DRPs) facilitate trafficking by pinching off vesicles from a membrane. Recently, our lab has discovered that loss of DRP2B alters a subset of flg22-induced immune responses (Smith et al. 2014 PLoS Pathogens 10(12): e1004578). The goal of my project is to investigate potential functional redundancy of DRP2Aand DRP2B, which share 93% amino acid identity,in flg22-induced immune responses. Loss of both DRP2A and DRP2B causes gametophytic lethality (Backues et al. 2010 Plant Cell 22: 3218-31); thus double mutant drp2a drp2b plants cannot be recovered. Therefore, we have designed inducible artificial micro-RNAs (amiRNAs) that target conserved regions of DRP2A and DRP2B to direct mRNA transcript degradation. DRP2 silencing mechanisms are induced by chemical treatment with 17-b-estradiol (E2). The first aim of my project was to identify plant lines homozygous for a single insertion of the DRP2amiRNA construct utilizing hygromycin resistance screening. With the identified homozygous DRP2amiRNA plants, I have established the efficiency of a variety of E2-induction conditions, using quantitative Real Time PCR (qRT-PCR) and immunoblot analyses to assess DRP2A/B mRNAand DRP2 protein levels, respectively. Currently, I am characterizing flg22-induced immune responses in DRP2amiRNA plants to further our understanding of the roles of DRP2A and DRP2B in plant immunity.

900-063-Z Evolution of the Modern Plant Immune Signaling Network Fumiaki Katagiri – University of Minnesota It is crucial for higher plants to have an immune signaling network highly robust against perturbations from pathogen effectors, as microbial pathogens can evolve much faster than plants. When did plants acquire a highly robust immune signaling network? Evolution of important immune regulators identified in Arabidopsis was studied in the green lineage, from green algae to angiosperms. Specifically, times of diversification of immune-related subfamilies from larger gene families were investigated. A common pattern was found for the MPK3/6/4/11 subfamily in the MAP kinase gene family, the PAD4/SAG101 subfamily in the EDS1 gene family, and the CBP60a/g/SARD1 subfamily in the CBP60 gene family. The common pattern was: (1) no immune-related subfamily in the lycophyte and the lower plants, (2) progenitors of the subfamily in gymnosperms, and (3) establishment of the subfamily in angiosperms, including an ancestral angiosperm. These observations suggest that the modern, highly robust immune signaling network started evolving about the time

gymnosperms evolved and was established in an ancestral angiosperm. Note that this notion by no means suggests lack of basic immune mechanisms in lower plants. A whole-genome duplication (epsilon) was proposed to be important in rapid diversification of the angiosperms and may have contributed to establishment of the modern immune signaling network. Spreading into new environments and having close associations with mobile animals are characteristics of angiosperms and expected to increase the chance of encounters with new pathogens. It is tempting to speculate that acquisition of a highly robust immune signaling network by an ancestral angiosperm was one major factor leading to ecological dominance of angiosperms. This work was supported by grants from NSF, MCB-0918908 and IOS-1121425.

900-064-Y Specific Control of Arabidopsis BAK1- and SERK4-regulated Cell Death by Protein Glycosylation Guangyuan Xu – Texas A&M University Marcos de Oliveira, Xiangzong Meng, Xin Chen, Aline Intorne, Abbey Babinat, Gonçalo de Souza Filho, Libo Shan, Ping He Sessile plants have evolved a large number of cell surface-resident receptor-like kinases (RLKs) in sensing and transducing a variety of extrinsic and intrinsic stimuli1,2. Arabidopsis BAK1/SERK family RLKs have diverse functions in plant growth, immunity and cell death control2,3. In contrast to the well-documented roles in plant immunity and hormone brassinosteroid (BR) signaling, the mechanisms underlying BAK1/SERK4-regulated cell death remain enigmatic. Here we developed an innovative genetic screen for no cell death (ncd) of BAK1/SERK4 silenced with virus-induced gene silencing (VIGS) on Arabidopsis knockout collections. NCD1 encodes STT3a, a catalytic subunit of the oligosaccharyltransferase complex for protein N-glycosylation modification4. Interestingly, the stt3a mutant did not suppress the compromised BR signaling and immune responses in bak1/serk4 mutant, suggesting the uncoupled signaling mechanisms of multiple BAK1/SERK4 functions. Systemic investigation of protein glycosylation pathway and ER quality control (ERQC) components revealed distinct and overlapping mechanisms operating BAK1/SERK4- and their interacting protein BIR1-regulated cell death. Furthermore, the bak1/serk4 cell death is independent of SOBIR1, a RLK required for bir1 cell death5. Our study established a novel and highly efficient genetic screen to uncover the mechanisms regulating plant cell death and elucidated that protein N-glycosylation and specific ERQC components are essential to activate bak1/serk4 cell death.

900-065-Y A Functional Study of a Root Knot Nematode IDA-like (IDL) Peptide Putatively Secreted into the Host Plant Joonyup Kim – USDA-BARC Ronghui Yang – USDA-BARC, Caren Chang – University of Maryland-College Park, Mark L. Tucker – USDA-BARC Inflorescence deficient in abscission (IDA) is a gene that is essential to the regulation in Arabidopsis floral organ abscission. The IDA gene encodes a small, secreted peptide that binds to two redundant receptor-like kinases (HAESA and HAESA-like 2), which alters the expression of KNOX transcription factors. IDA-like genes are conserved in every dicot genome and four monocots we have examined. Root-knot nematodes (RKN), Meloidogyne spp., are costly pathogens of many agriculturally important crops. In addition to the plant species, we identified IDA-like (IDL) genes in the genomic sequence for Meloidogyne incognita and M. hapla. Expression of MiIDL1 is very low in RKN eggs and pre-parasitic J2 but rapidly increases after inoculation of tomato roots and then declines at approximately 14 days post inoculation. Five and three prime RACE of RNA from M. incognita infected roots revealed a sequence of 392 nt with a 47 aa open reading frame (ORF) including a putative 28 aa N-terminal signal peptide. Synthetic peptide for the MiIDL1 ORF minus the signal peptide and transgenic Arabidopsis harboring the full-length ORF both complement the delayed abscission phenotype of the ida mutant. Furthermore, application of the synthetic MiIDL1 peptide to Arabidopsis roots promoted root growth that indicates a possible role for the RKN peptide in root development, which could support RKN infection of roots. Transgenic lines of RNAi that over-express MiIDL1 (MiIDL1i) and GUS (GUSi) were generated and tested for host

infection in Arabidopsis. Preliminary results suggest that suppression of the MiIDL1 gene in the nematode delays gall development and the numbers of galls that form. We propose that IDA-like peptides are conserved throughout the plant kingdom and that the nematode MiIDL1 peptide is a novel effector, which functions as a signaling peptide in the host plant.

900-066-Z The Pseudomonas Syringae Type III Effector HopBB1 Modulates Hormone Balance in Plant Immunity Li Yang – UNC Chapel Hill, Paulo Jose Pereira Lima Teixeira – UNC Chapel Hill, Petra Epple – UNC Chapel Hill, Yijian He – UNC Chapel Hill, Omri Finkel – UNC Chapel Hill, Jeffery L. Dangl – Howard Hughes Medical Institute, UNC Chapel Hill Plants regulate phytohormone crosstalk to launch specific defense response against different pathogens. Pathogen virulence factors, called effector proteins, can modulate plant defense through physically interacting with host immune response components. Independently evolved effectors from three kingdoms of life (eubacteria, ascomycete and oomycete) have been found to converge onto a host transcription factor, TCP14, in Arabidopsis. Here, we show that TCP14 represses Jasmonate (JA) signaling before infection. The Pseudomonas syringae (Pst) effector HopBB1 targets at least two repressors of JA response: TCP14 and JAZ3 in planta. HopBB1 disrupts the inhibitory association between JAZ3 and MYC2, a transcriptional activator of JA response. In addition, HopBB1 recruits TCP14 to the SCFCOI1-dependent degradation pathway by connecting it physically to JAZ3. Thus, HopBB1 promotes virulence by de-repressing the JA response. Our findings demonstrate an immune function for TCP14 and provide a mechanistic understanding of how the HopBB1 effector protein fine-tunes TCP14-mediated phytohormone crosstalk.

900-067-Z An Established Mycorrhizal Symbiosis Makes Barrel Medic Plants More Attractive to Pea Aphids Abhinav Maurya – University of Northern Colorado Susana Gomez – University of Northern Colorado The symbiosis between arbuscular mycorrhizal fungi (AMF) and plants is one of the most important rhizospheric interactions. About 80% of terrestrial plants form mutualistic associations with AMF, where plants supply carbon to AMF in exchange for nutrients (phosphorus and nitrogen). As AMF play an important role in nutrition and chemical composition of the plant, this below-ground beneficial interaction has the capability of affecting plant interactions with above-ground insect herbivores such as aphids via the shared host plant, and vice versa. Aphids are major agricultural pests because of their parthenogenetic mode of reproduction, wide host range, ability to transmit viruses, and develop resistance against insecticides. Our research involves barrel medic plants (Medicago truncatula) that are associated with the AM fungus Glomus intraradices and pea aphids (Acyrthosiphon pisum). The research was aimed at quantifying the impacts of tripartite interactions on each organism to find out if the AM fungus increases or decreases the attractiveness of plants to aphids. The effect of aphid feeding on AM fungus root colonization and overall plant fitness was also evaluated. Our results showed that plant interactions with pea aphids and AM fungi operate in both directions: (i) at medium levels of root colonization by AMF (40-50% colonization) there was no significant impact on the pea aphids’ ability to feed, survive, and reproduce. (ii) At high levels of root colonization by AMF (70-88% colonization), there was a positive impact on pea aphid abundance (35% increase in aphid number), growth (44% increase in aphid weight) when aphids feed on mycorrhizal plants, while pea aphid feeding significantly reduced the above-ground biomass (32% decrease in fresh shoot weight) of mycorrhizal plants. Plant gene expression analyses are ongoing which would provide a better understanding of some of the changes that make mycorrhizal plants more attractive to pea aphids.

900-068-Y Regulation of miR163 and Its Targets Under Biotic Stress in Arabidopsis Thaliana Cecilia H-T. Chow – Department of Biology, Hong Kong Baptist University Danny W-K. Ng – Department of Biology, Hong Kong Baptist University Small RNAs are important regulators for a variety of biological processes, including leaf development, flowering-time, embryogenesis and defense responses. Plant microRNAs (miRNAs) are a distinct type of small RNAs that guide the RNAinduced silencing complexes (RISC) to target transcripts based on miRNA-target complementarity. miR163 is a nonconserved miRNA and its locus has evolved recently by inverted duplication events of its target gene that belongs to the SABATH genes family of related small-molecule methyltransferases (MTs). In Arabidopsis thaliana, miR163 accumulation can be induced by alamethicin treatment, suggesting its roles in defense response pathways. To extend our knowledge regarding the physiological role of miR163 and its targets in plant-pathogen interactions, we first examined their expression in response to the bacterial pathogen, Pseudomonas syringae (Pst), in Arabidopsis thaliana. In the mir163 mutant, increase resistance against Pst was observed. In contrast, transgenic lines overexpressing miR163 showed enhanced disease sensitivity, suggesting that miR163 is a negative regulator of defense response. At the transcript level, although FAMT and PXMT1 expression was induced in both genotypes upon Pst challenge, these miR163 targets were expressed at a higher level in the mir163 mutant when compared to the wild type (Col-0). In addition, enhanced MIR163 promoter-driven β-glucuronidase (GUS) expression was detected in response to Pst challenge. These data supported that miR163and its targets act in concert to modulate plant-pathogen interactions in plant defense. Using the CAMV 35S promoter-driven expression, miR163-resistance version of FAMT and PXMT1 will be expressed in transgenic Arabidopsis. The established transgenic lines will further elucidate miR163 and its targets interactions in mediating pathogen responses. We will use data presented here to provide a foundation for understanding the role this recently evolved miRNA and its targets in plant stress responses.

900-069-Y Characterization of Nonhost Resistance in Arabidopsis Thaliana Against Clavibacter Michiganensis You Lu – University of Minnesota Carol Ishimaru – University of Minnesota, Jane Glazebrook – University of Minnesota Nonhost resistance is the most durable form of plant immunity to pathogens. It results from a variety of underlying mechanisms. Clavibacter michiganensis, a group of gram-positive phytopathogens, is non-adapted on Brassicaceae. To understand how plants in the Brassicaceae defend themselves against C. michiganensis, we screened 32 accessions of Arabidopsis thaliana and discovered the responses range from no symptom to chlorosis and necrosis. The allele(s) in A. thaliana accession Kas-1, which shows a chlorotic response following C. michiganensis inoculation, appears to be dominant over the alleles in Col-0 and Tsu-1, which confer no symptom. This variation was controlled by one locus mapped to one 53 kb region on chromosome 4. The ability to induce chlorosis in Kas-1 is conserved among all subspecies of C. michiganensis that were tested. Heat-killed cells elicited that same response as live cells. Inoculation with C. michiganensis activated MAP kinase cascades in both Kas-1 and Tsu-1. The expression of marker genes for the salicylic acid dependent and PAD4 dependent signaling pathways was strongly induced by C. michiganensis in Kas-1, Col0 and Ler-0, but not in Tsu-1. However, the titer of C. michiganensis can persist in Kas-1 and Ler-0, while it decreased in Col-0 and Tsu-1 to less than 20 percent of the inoculum over a 9-day period following inoculation. These and other results suggest the resistance of A. thaliana against C. michiganensis may be independent of hormone signaling, and the chlorosis induced by C. michiganensis may compromise the resistance. Further investigation will be required to elucidate the molecular basis of the chlorotic response induced by C. michiganensis in A. thaliana. Knowledge of nonhost resistance responses to C. michiganensis can potentially provide new avenues for breeding crops with resistance to this economically important species.

900-070-Z The Chemical Defense Responses of the Bryophyte Mnium Cuspidatum to Pythium Irregulare Philip Villani – Butler University Similar to animals and other organisms, plants are constantly being assaulted by pathogens such as bacteria, fungi and viruses. Plants have been shown to respond to pathogen challenge in two major ways including a localized hypersensitive response (HR) and/or a board plant response systematic acquired resistance (SAR). Much of our current knowledge on plant defense mechanisms comes from research on agronomic plants; however, little is know about how the more primitive plants such as mosses respond to pathogens. Physcomitrella patens is the model bryophyte species in which pathogen response research has been done; however, beyond P. patens there has been little research on other moss species. This study expands the knowledge of moss pathogen response to another species, Mnium cuspidatum. We examine biochemical responses of the moss M. cuspidatum and P. patens with respect to hydrogen peroxide production, peroxidase activity, and phenolic production in response to an elicitor, chitosan, or the plant pathogen Pythium irregulare.

900-071-Z Hyperphosphorylation of Retinoblastoma Leads to Programmed Cell Death During Effector Triggered Immunity Sophia Zebell – Duke University Xinnian Dong – Duke University, Shui Wang – Shanghai Normal University Programmed cell death (PCD) is a highly conserved phenotype of effector-triggered immunity to biotrophic pathogens. Although much is known about the upstream signaling that leads to PCD, any downstream factors that may coordinate a specific PCD gene expression program have yet to be identified. We have previously shown that during Arabidopsis thaliana incompatible interactions with the oomycete pathogen Hyaloperonospora arabidopsidis and the bacterial pathogen Pseudomonas syringae AvrRpt2, RETINOBLASTOMA-RELATED protein (RBR1) is hyperphosphorylated. This likely leads to increased concentrations of active E2F family transcription factors, which we have shown to be essential for robust effector-triggered PCD. This suggests that RB and E2F may function as direct regulators of PCD genes in plants, as they do in mammals. RBR1 is highly conserved at the amino acid level, from humans to mosses. This has allowed us to look at RBR hyperphosphorylation using the same molecular tools in many plant-pathogen interactions, testing the degree of conservation of the RBR-E2F signaling module in the regulation of effector-triggered immune PCD.

900-072-Y Towards the Understanding of Plant Immune Signaling: Filling the Gaps by Aggies (Arabidopsis Genes Governing Immune Gene Expression) Kevin Babilonia – Texas A&M University Baomin Feng, Fangjun Li, Libo Shan, Ping He Lack of adaptive immune system and specialized immune cells, plants have evolved a robust innate immune system to protect themselves against potential infections. The first layer of defense is initiated through the recognition of pathogen/microbe-associated molecular patterns (PAMPs/MAMPs) by cell surface localized pattern recognition receptors (PRRs). The activation of PRRs results in diverse defense responses including activation of MAP kinase cascade, reactive oxygen species production, stomatal closure, callose deposition, calcium influx and transcription of defense genes. Although PRR activation and regulation are well studied, the mechanisms leading to the transcriptional reprogramming of immune genes are largely unknown. We have developed a genetic screen with an EMS-mutagenized

population of Arabidopsis transgenic plants expressing a luciferase reporter gene under the control of the immune responsive marker gene FRK1 promoter. Mutants with altered luciferase activity upon MAMP treatment were identified and named as Arabidopsis genes governing immune gene expression (aggie). Aggie1 encodes Arabidopsis RNA polymerase II C-terminal domain (CTD) phosphatase-like 3 (CPL3), which negatively regulates immune gene expression and immunity via direct dephosphorylation of CTD. Aggie2 encodes poly(ADP-ribose) glycohydrolase 1 (PARG1), which is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs). The coordinative action of PARPs and PARGs plays a crucial role in a wide array of cellular responses such as DNA repair, cell division, chromatin modification and gene transcriptional regulation. Arabidopsis genome contains three PARPs and two PARGs. Importantly, Arabidopsis parp mutant displayed reduced, whereas parg mutant displayed enhanced immune gene activation and immunity to pathogen infections, suggesting that protein poly(ADP-ribosyl)ation (PARylation) post-translational modification contributes to plant immunity via regulation of plant immune gene expression. Molecular cloning and biochemical characterization of other Aggies are in progress.

900-073-Y Inhibitory Mechanism of Nodulation by Root Exposure to Light in Lotus Japonicus Aya Shimomura – Kagoshima University Ayumi Naka, Nobuyuki Miyazaki, Sayaka Moriuchi, Susumu Arima, Shusei Sato, Hideki Hirakawa, Satoshi Tabata, Makoto Hayashi, Ann Hirsch, Akihiro Suzuki Leguminous plants and rhizobia establish a symbiosis in which root nodules are formed on the host root. Nodulation is suppressed when roots are exposed to light, but the mechanism of this inhibition is unclear. Here, we report that blue light inhibits nodulation in Lotus japonicus Miyakojima MG20 roots inoculated with Mesorhizobium loti MAFF303099. Using RNA interference (RNAi), we suppressed the expression of phototropin and cryptochrome genes in hairy roots. Under blue light, plants transformed with an empty vector had no nodules, whereas plants with suppressed expression of cry1A and cry2B formed nodules. To investigate whether the inhibition of nodulation is also caused by rhizobia, we measured rhizobial proliferation. Although red light had no effect on proliferation, blue light had a strong inhibitory effect. Proliferation under blue light was partially restored in signature-tagged mutagenesis (STM) strains, in which the phototropin and cryptochrome genes are disrupted. Inoculation with the STM strains significantly increased nodulation under blue light in comparison with inoculation with M. loti. Moreover, when cry1A or cry2B RNAi plants were inoculated with the STM strains, nodulation was increased. These results suggest that inhibition of nodulation by blue light is mediated by blue light receptors of both the host plants and the symbiont. Root exposure to light is a stimulus indicating that the root is under stress and that root nodules may not function effectively. Therefore, we consider inhibition of nodulation by light as an avoidance response to preserve energy under environmental stress.

900-074-Z Macrocidins, Bioherbicidal Metabolites of the Fungus Phoma Macrostoma, Impact Carotenoid Profiles of Two Susceptible Plants Differently Michelle Hubbard – Agriculture and Agri-Food Canada Russell Hynes – Agriculture and Agri-Food Canada, Karen Bailey – Agriculture and Agri-Food Canada The fungus Phoma macrostoma is registered as a bioherbicide in Canada and the USA for control of broadleaf weeds. P. macrostoma produces macrocidins, which induce chlorosis and inhibit carotenoid biosynthesis, likely by partially inhibiting the enzyme phytoene desaturase (PDS) and acting on one or more other points in the biosynthetic pathway in dandelions and thistles. Here we explored how macrocidins impact two other susceptible plants, groundsel and chickpea, whose responses to macrocidins are unique from those of either dandelion or thistle. To fulfill this objective, we characterized changes in carotenoid and carotenoid precursor profiles over time after treatment with macrocidins as

well as chlorophyll fluorescence parameters, chlorosis severity and biomass. While groundsel was more severely impacted by macrocidins in terms of symptom severity, biomass reduction, impact on the chlorophyll fluorescent parameters Fv/Fm and OJIP curves and total carotenoid and carotenoid precursor reduction, a greater increase in percent phytoene content was measured in chickpea, implying that PDS is more completely inhibited. However, macrocidin-treatment led to a greater decrease in the beta-carotene to lutein ratio in groundsel than in chickpea. In addition, percent violaxanthin increased in macrocidin-treated groundsel, but decreased in chickpea, albeit more slowly than it increased in groundsel. The drop in beta-carotene to lutein ratio in macrocidin-treated groundsel could be due to a draw-down of beta-carotene via conversion to violaxanthin, but to other mechanism(s) in chickpea. In conclusion, macrocidins impact carotenoid biosynthesis in both groundsel and chickpeas. Variations in changes in carotenoid and their precursors suggest that different modes of action of macrocidins are more important in different susceptible plants. This could potentially reduce the likelihood of resistance to macrocidins, were it ever to develop, being transferable between species.

900-075-Z Assessing Rhizobial Dependence of Medicago Polymorpha as It Evolves in Non-native Regions Katherine Wozniak – Michigan State University Maren Friesen – Michigan State University Medicago polymorpha and its rhizobial symbiont, Ensifer medicae have evolved from thriving in the Mediterranean coast to cultivating regions as far as California and Chile. The specialized mutualism that was heavily relied upon for legume growth is now less utilized in the invasive ranges, suggesting that more generalist plant genotypes have been favored. Evolving a generalist approach in the invasive ranges would mean recruiting differing levels of bacterial diversity from the rhizosphere, instead of forming a symbiosis with only a few efficient rhizobia. This project aims to elucidate the below-ground interactions of native and invasive Medicago polymorpha genotypes using laboratory experiments with rhizobial strains and soils from both native and invasive regions. We found that there was no significant difference in plant fitness among native and invasive genotypes but the bacterial treatments were significantly different in their promotion of plant growth. Significantly higher plant fitness of invasive genotypes resulted when they were inoculated with mixed strains of invasive bacteria, and the highest plant fitness in native genotypes was obtained with one single strain of native bacteria. Native and invasive genotypes will be grown in a sterile environment that has been inoculated with soils from various regions. Both nodule and plant measurements will be analyzed to determine if there is plant benefit with either matched soil and plant genotypes, or genotypes with soils that are not from the same regions. Rhizobia that increase plant benefit will be sequenced to further assess diversity. Investigating plant-bacterial associations in invasive regions could help us to better understand how genotypes are able to colonize and flourish in the invaded range, and could have implications for prevention of parasitic plant invasions and biodiversity.

900-076-Y A Team-research Approach to Comprehensive Analysis of Arabidopsis Loci Associated with the Functions of Pseudomonas Syringae effectors Keisuke Mase – University of Minnesota Megan Siegle – University of Minnesota, Rachel Meyer – University of Minnesota, Arefat Heyder – University of Minnesota, Benjamin Spokely – University of Minnesota, Britt Swanson – University of Minnesota, Deirdre Kragh – University of Minnesota, Emily Boak-Nyberg – University of Minnesota, Heather Hanson – University of Minnesota, Jonathan Resch – University of Minnesota, Jonathan Walker – University of Minnesota, Josh Wemmer – University of Minnesota, Klothilda Lim – University of Minnesota, Melissa Rivi – University of Minnesota, Noah Strom – University of Minnesota, Samantha Dumler – University of Minnesota, Scott Miller – University of Minnesota, Shayman Lothert – University of Minnesota, Tiffany Pan – University of Minnesota, Tyler Reese – University of Minnesota, Suma

Chakravarthy – Cornell University, Hailei Wei – Cornell University, Alan Collmer – Cornell University, Fumiaki Katagiri – University of Minnesota Plant pathogens deliver effectors into plant cells to suppress the host plant immune responses to establish infection. To understand how pathogen effectors interact with plant immune signaling components, we have launched a undergraduate team-research project to systematically identify Arabidopsis genes whose allelic variation alters response to individual effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). Pto carries approximately 30 type III effector genes, and some of the effectors target the same signaling components or pathways (functional redundancy), which makes identification of the functions of individual effectors difficult. We are using combinatorial effector strains derived from Pto, which carry only limited numbers of its type III effector genes to circumvent the functional redundancy issue. To identify Arabidopsis genes that are functionally associated with the Pto effectors, we are exploring natural allelic variation in the genes that affect growth of combinatorial strains in planta. First, we established that 32 Arabidopsis accessions, which were selected for diversity in their origins and the availability of mapping populations, show substantial variation in growth of four combinatorial strains and that the patterns of variation across the accessions are substantially different among the four strains. Now we are trying to identify Arabidopsis genes underlying the phenotypic variation by QTL mapping using the Arabidopsis mapping population MAGIC (Multiparent Advanced Generation Inter-Cross) lines. For this research, we recruited and trained a team of undergraduate students every semester. Controlled plant growth environments for the experiments were provided by inexpensive homemade plant growth chambers. This experimental setup allowed us to conduct large-scale phenotyping experiments in a sustainable manner. This work was supported by an NSF grant, IOS-1121425.

900-077-Y Cotton Bacterial Blight and Xanthomonas Citri Pv. Malvacearum Diversity Anne Phillips – Washington University Jillian Burke – Danforth Plant Science Center, Anupama Vijayaraghavan – Danforth Plant Science Center, Mark Wilson – Danforth Plant Science Center, Rebecca Bart – Danforth Plant Science Center Cotton Bacterial Blignt (CBB) is a disease that negatively affects cotton yield worldwide. Since 2011, the disease has reemerged as an agronomic problem in the southern United States, the cause of which is unknown. Xanthomonas citri pv. malvacearum (Xcm), a gram negative proteobacterium, promotes the disease by injecting type three effector proteins into plant cells. We have collected isolates from infected fields as well as historical isolates to identify susceptible and resistant interactions between Xcm isolates and cotton cultivars. We will use genomics and molecular biology to understand bacterial diversity among these isolates. This information will be used to develop durable resistance strategies for new cotton varieties.

900-078-Z A Signaling Mechanism That Mediates Inhibition of Effector-triggered Immunity (ETI) Signaling by Patterntriggered Immunity (PTI) Signaling Noriyuki Hatsugai – University of Minnesota Daisuke Igarashi – University of Minnesota, Suma Chakravarthy – Cornell University, Hailei Wei – Cornell University, Alan Collmer – Cornell University, Fumiaki Katagiri – University of Minnesota Two modes of plant immunity are pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). PTI is triggered upon recognition of conserved microbe-associated molecular patterns (MAMPs) by the cognate pattern recognition receptors (PRRs). Pathogens adapted to a particular plant host deliver effectors into the plant cell that interfere with PTI signaling. ETI is triggered upon direct or indirect recognition of effectors by resistance (R) proteins. ETI

is often associated with a hypersensitive response (HR), which is characterized by rapid cell death. However, a precise molecular definition of ETI has been elusive because most experimental systems can not separate the ETI effect from the PTI and ETI interaction. Here, we report a signaling mechanism that mediates strong inhibition of ETI signaling by PTI signaling. The bacterial effector AvrRpt2 is recognized by the R protein, RPS2, resulting in HR (AvrRpt2-HR) in Arabidopsis. We have previously shown that AvrRpt2-HR induced by a bacterial strain Pseudomonas syringae pv. tomato (Pto) DC3000 avrRpt2 was abolished in an Arabidopsis quadruple mutant, dde2 ein2 pad4 sid2 (quad). We found that AvrRpt2-HR was abolished in quad even when bacterial strains that do not deliver any other effectors, such as Pto DC3000 D36E, were used to deliver AvrRpt2. However, the HR in quad was intact when AvrRpt2 was directly expressed in the plant cell using the estradiol-inducible promoter system (Ed-AvrRpt2). Unlike bacterial strains, Ed-AvrRpt2 delivers AvrRpt2 without triggering PTI. We hypothesized that PTI signaling triggered by bacterial strains inhibits an HR signaling mechanism that remained functional in quad. Interestingly, this hypothesis was supported by the results that when either of the bacterial MAMPs, flg22 or elf18, was applied to Ed-AvrRpt2plants together with estradiol, the HR was significantly decreased in quad but not in wild type. Our results provide the basis for reconstituting highly interactive signaling event.

900-079-Z The Roles of Salicylic Acid and Jasmonic Acid in Plant-pathogen Interactions in Moss Nathanael Hauck – Butler University Philip Villani Salicylic acid (SA) and jasmonic acid (JA) are two of the three phytohormones that are known to be directly involved in pathogen defense in vascular plants; however, their roles in non-vascular plants have only received limited attention. Here we report progress in determining the role of these two hormones in defense in two moss species, Amblystegium serpens and Physcomitrella patens. A. serpens was used to determine if exogenous application of either SA or JA induces resistance to Pythium irregulare infection 0, 24 or 48 hours after treatment. P. patens was used to determine if treatment with either of these hormones induces expression of putative defense genes using a qRT-PCR approach. Finally, expression of various genes that encode enzymes that function in the biosynthesis of these two hormones following inoculation of P. patens with the pathogen was also analyzed. The implications of these findings on our knowledge of the evolution of plant-pathogen interactions are discussed.

900-080-Y A Close Relationship Bewteen Spriodela Polyrhiza and the Nitrogen-fixing Bacterium Paenibacillus Sp Nathan Tivendale – University of Minnesota Changbin Chen – University of Minnesotam Jerry Cohen – University of Minnesotam Adrian Hegeman – University of Minnesotam Chanlan Chun – University of Minnesotam Todd Michael – Ibis Biosciences Nitrogen is an essential nutrient for plant growth, but plants cannot obtain nitrogen directly from the air. Instead, most plants rely on nitrogen fixation carried out by other organisms to convert N2 gas to mineral nitrogen (NO3-, NO2- or NH4+), which can then be assimilated by the plants. However, some plants, such as legumes, have developed symbiotic relationships with nitrogen-fixing bacteria to obtain nitrogen from air. Here, we report a similar phenomenon in the monocotyledonous aquatic angiosperm Spirodela polyrhiza (greater duckweed). We have observed a novel relationship between S. polyrhiza and Paenibacillus sp., a bacterial species capable of nitrogen fixation, and present four lines of evidence for this relationship: 1) the genome sequence for S. polyrhiza shows the presence of rhizobial-like genes, 2) confocal microscopy photographs clearly show the presence of rod-shaped bacteria on the surface of the duckweed roots and inside the root cap, 3) 16S rDNA sequencing of the potential symbiont revealed it to be Paenibacillus sp., a known nitrogen-fixing bacteria, and 4) when S. polyrhiza is grown on autoclaved media and supplied with 15N2 gas, the

labeled nitrogen is incorporated into the amino acids of the organism. The S. polyrhiza stock used in these experiments has been kept in culture for decades and the bacterial symbiont is not free-living in the medium. Together, these results suggest a close or symbiotic relationship between S. polyrhiza and nitrogen-fixing bacteria, which we believe was established long before the duckweed was isolated and cultured in autoclaved media.

900-081-Y The Effect of Polerovirus P0 Expression on SKP1 Cullin F-Box E3 Ubiquitin Ligase Targeting in Nicotiana Benthamiana Erick Ortiz – California State University, Fullerton Melanie Sacco – California State University, Fullerton Potato leafroll virus (PLRV) is a single-stranded RNA virus from the genus Polerovirus that induces leaf rolling and phloem necrosis in plants. Poleroviruses use the protein P0as a viral suppressor of RNA silencing to promote viral replication by degradation of the protein Argonaute (AGO1), an essential component of the RNA silencing machinery. P0 is known to interact with the SKP1 component of SKP1-Cullin1 F-box (SCF) E3 ubiquitin ligases from Arabidopsis thaliana, which target proteins with PEST motifs for degradation via the proteasome. Consistent with this interaction, P0 expression was reported to cause accumulation of proteins with K48-specific polyubiquitination, which is associated with proteasome-mediated degradation; however, P0 targets AGO1 for autophagy-mediated degradation by K63specific ubiquitination. The relationship of P0 to K48-specific ubiquitination and proteasome-mediated degradation of host proteins remains unknown. We hypothesized that P0 interferes with targeting of viral proteins for proteasomal degradation through its role as an F-box protein in the SCF E3 ligase complex. We have identified potential PEST motifs in two PLRV proteins essential for viral replication and transmission, the RNA-dependent RNA polymerase (P2) and the minor capsid read-through protein (P5). The presence of these PEST motifs in P2 and P5 suggests they are targeted by SCF E3 ligase for degradation. Co-expression assays were performed by agroinfiltration in Nicotiana benthamiana using full-length P2 as well as the read-through domain (RTD) of P5 (which contains two PEST motifs) with P0 from PLRV (P0PL). P2 accumulation was unaffected by P0PL presence, suggesting that P2 accumulation occurs independently of P0PL. The P5 RTD was not detected and may require expression of the full-length P3-P5 read-through protein for stable accumulation that can be tested in the co-expression assay. Results from P2 expression studies suggest that P0’s functions do not include diverting the SCF E3 ligase from targeting viral proteins for degradation.

900-082-Z Effect of Isoflavonoids on Bacterial Diversity Within the Soybean Rhizophere Laura White – South Dakota State University Volker Brözel – South Dakota State University, University of Pretoria, Senthil Subramanian – South Dakota State University High bacterial density and diversity near plant roots has been attributed to rhizodeposit compounds that serve as both energy sources and signal molecules. Isoflavonoids in particular are a major component of soybean rhizodeposits that regulate nod genes in rhizobia and help the plant defend against pathogens. However, it is unclear if and how these rhizodeposit compounds influence bacterial diversity. We silenced the biosynthesis of isoflavonoids using RNA interference in hairy root composite plants and examined changes in rhizosphere bacteriome diversity. Successive sonication was used to isolate soil fractions from different rhizosphere zones – distal, middle, and proximal – 3 weeks post planting. To ascertain which phylotypes were influenced by isoflavonoids, we applied pyrosequencing to the 16S rRNA gene amplicons from the proximal soil samples and resolved the resulting data via MOTHUR. The results showed that specific bacterial phylotypes within the soybean rhizosphere were influenced by isoflavonoids as well as hairy root transformation.

900-083-Z The Pseudomonas Syringae Effector Protein AvrPtoB Utilizes an Essential Subset of Host Ubiquitin-Conjugating Enzymes to Suppress Plant Immunity Bangjun Zhou – Plant Science Innovation Center and Plant Pathology Department, University of Nebraska-Lincoln; Biology Department, University of Arkansas-Little Rock, Ravi Mural – Biology Department, University of Arkansas-Little Rock, Xuanyang Chen – Biology Department, University of Arkansas-Little Rock, Matt Oates, University of Bristol, Gregory Martin – The Boyce Thompson Institute, Julian Gough – University of Bristol, Lirong Zeng – Plant Science Innovation Center and Plant Pathology Department, University of Nebraska-Lincoln; Biology Department, University of Arkansas-Little Rock The effector protein AvrPtoB of the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) encodes a modular protein where the C-terminal region encodes a ubiquitin ligase (E3). AvrPtoB can suppress both pathogen/microbeassociated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) of plants to promote the virulence of Pst, of which the latter depends on its C-terminal E3 activity. It has been suggested that AvrPtoB hijacks host ubiquitin-conjugating enzyme(s) (E2) to work with its C-terminal ubiquitin ligase in suppressing ETI. But this has not been demonstrated neither the host E2 enzyme(s) hijacked by AvrPtoB have been identified. In this study we identify from the tomato genome forty genes putatively encoding ubiquitin E2 proteins and demonstrate the majority of them encode active ubiquitin-conjugating enzymes. Phylogenetic analysis classifies the tomato E2 enzymes into thirteen subgroups. In vitro ubiquitination using AvrPtoB discovers that only members of subgroup III tomato E2s work with AvrPtoB. Knocking down the expression of subgroup III E2 genes affects the plant development and decreases the inhibition of Fen and other R protein-mediated, ETI-associated programmed cell death (PCD) by AvrPtoB and enhances the growth of Pst that triggers ETI. Silencing the subgroup III E2 genes also results in significant diminishment of reactive oxygen species (ROS) production, immunity marker genes expression, cell death suppression, and enhanced Pst growth that are associated with PTI. In vitro ubiquitination assay also reveals two plant-immunity-associated ubiquitin ligases, SPL11and CMPG1 work with subgroup III of tomato ubiquitin E2 enzymes only. Consistently, knocking down subgroup III E2 genes increases flg22-induced callose deposition likely as a result of compromising the role of SPL11 in attenuating immune signaling. These results suggest AvrPtoB has evolved to hijack an essential group of host ubiquitin E2 enzymes to promote virulence, which would be advantageous in the arm race with host plant.

900-084-Y Mapping the Upstream and Downstream Immune Components of the CALMODULIN-BINDING PROTEIN60 Family Man Zhou – University of Minnesota Jane Glazebrook – University of Minnesota Calmodulin (CaM) is a small protein which can transduce calcium signals by binding of Ca2+. After binding, CaM modulates the activities of other proteins by binding to them. Three members of the CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g, CBP60a and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT (SARD1), regulate plant immunity. CBP60g and SARD1 encode positive regulators that promote the production of salicylic acid (SA) and affect the expression of downstream defense genes in both SA dependent and independent ways, while CBP60a encodes a negative regulator. CaM binding is required for the functions of CBP60a and CBP60g, while SARD1 does not bind CaM. Upon pathogen recognition, both CBP60g and SARD1 are strongly induced, with the response of CBP60g occurring earlier than that of SARD1, while CBP60a is only modestly up-regulated. The mechanisms underlying pathogen-triggered induction of CBP60g, SARD1 and CBP60a are not known. Here, we are investigating the transcriptional control of CBP60s using cis-regulatory element analysis and yeast one hybrid screening against an Arabidopsis transcription factor library

to identify potential regulators of CBP60s. We are also using yeast two hybrid screening and Tandem affinity purification combined with mass spectrometry (TAP-MS) to identify interaction partners of CBP60s. This may help to define the functions of CBP60s and gain more insight into the Ca2+ dependent protein complexes that regulate plant immune responses. By integrating data about the transcriptional control of CBP60s and protein interactions with CBP60s, we aim to deepen our understanding of the CBP60 regulatory system.

900-085-Y Characterization of the Mechanisms That Control Cell-to-Cell Connectivity via Plasmodesmata in Response to Chitin Cecilia Cheval – John Innes Centre Christine Faulkner Recognition of chitin from fungal pathogens by pattern recognition receptors triggers a range of immune responses in plants. Among these responses is the regulation of symplastic continuity and molecular flux between cells [1]. Recent studies have demonstrated that a plasmodesmata (PD)-located, GPI anchored receptor protein LYM2 perceives chitin and triggers PD closure [2]. This contributes to resistance to a fungal pathogen. Significantly, LYM2 signalling occurs independently of other chitin-activated response pathways. LysM receptor proteins have been shown to form complexes with LysM receptor kinases and we have identified two LysM receptor kinases that are required for chitin-triggered PD closure. Thus, our hypothesis is that these receptor kinases form a complex with LYM2 to mediate this response. Additionally, our aim is to identify what signals regulate, and are regulated by, symplastic connectivity in the context of defence. For this we will examine the role of calcium, ROS and callose deposition, known components of both defence responses and PD function. Preliminary experiments suggest that ROS are essential for chitin-triggered PD closure enabling a context to dissect the hierarchy of molecular events that ultimately induce PD closure. PD closure is a newly identified plant defence response and raises a suite of questions relating to how cells communicate during pathogen attack; how do cells co-ordinate their responses? This project characterises the mechanism by which symplastic continuity is controlled during pathogen attack and thus establishes basic information that will underpin a whole new facet of research in plant defence. - Faulkner C (2013) Receptor-mediated signaling at plasmodesmata. Frontiers in plant science 4: 521 - Faulkner C, Petutschnig E, Benitez-Alfonso Y, Beck M, Robatzek S, Lipka V, Maule AJ (2013) LYM2-dependent chitin perception limits molecular flux via plasmodesmata. Proceedings of the National Academy of Sciences of the United States of America 110: 9166-9170

900-086-Z The Molecular Basis of Water-soaked Disease Lesions Enhanced by a Xanthomonas TAL Effector Allison Schwartz – University of California, Berkeley Brian Staskawicz – University of California, Berkeley Xanthomonas plant pathogens are an increasingly limiting factor of agricultural yield. Recently the major bacterial spot agent of tomatoes in the Midwestern United States has shifted from Xanthomonas euvesicatoria (Xe) to Xanthomonas gardneri (Xg), which causes significant spotting on fruits and water-soaked disease lesions on leaves. Uptake of external water plays a significant role in enhancing disease symptom development and is dependent on bacterial Type III effector delivery. Xg possesses in its Type III effector arsenal AvrHah1, a Transcription Activator Like (TAL) effector, which has

been shown to enhance water soaked symptoms in pepper, tomato, and Nicotiana benthamiana. We utilized an RNAseq approach in infected tomato leaves in order to determine the potential host susceptibility targets of AvrHah1. Two basic Helix Loop Helix (bHLH) transcription factors with predicted AvrHah1 effector binding elements were transcriptionally upregulated in wt Xg-infected leaves but not in XgΔAvrHah1-infected leaves. Both bHLH’s were confirmed as targets of AvrHah1 in transient luciferase assays. Downstream targets of the bHLH transcription factors include genes involved in cell wall modification. Because susceptible plants harbor a population of XgΔAvrHah1 comparable to wt Xg, it is possible that the host gene targets of AvrHah1 aid in lesion development in order to promote bacterial cell egression from the apoplast. An improved understanding of symptom development may inform the design of disease tolerant crops by preventing yield loss due to foliar and fruit lesions and by disrupting aspects of the pathogen life cycle.

900-087-Z Competitive Binding of WRKY Proteins to a Conserved Double W-box in the CaWRKY40 Promoter Regulates Its Synergistic Response to Pathogen and Heat Shuilin He – Fujian Agriculture and Forestry University Zhiqin Liu – Fujian Agriculture and Forestry University, Deyi Guan – Fujian Agriculture and Forestry University, Sheng Yang – Fujian Agriculture and Forestry University, Lanping Shi – Fujian Agriculture and Forestry University, Xiaoling Ma – Fujian Agriculture and Forestry University CaWRKY40 synergistically regulates pepper response to heat stress (HS) and Ralstonia solanacearum inoculation (RSI), but the mechanism is unknown. Here, 5¢ deletion analysis showed that a core region of the CaWRKY40 promoter, CaWRKY40-1889 to -1551, was primarily responsible for inducible CaWRKY40 expression. Gain- and loss-of-function assays identified a double W-box element (DWE) in this core region, which synergistically responded to HS and RSI. The WI box was crucial for the synergistic response, whereas the WII box amplified the response. Chromatin immunoprecipitation (ChIP) assays showed that DWE boundindiscriminately to transiently overexpressed CaWRKY40, CaWRKY58, and CaWRKY27 in unstressed plants, but bound only to CaWRKY40 in pepper plants subjected to HS and RSI. The WI box was crucial for preferential binding to CaWRKY40. DWE was conserved in CaWRKY40 paralogs including AtWRKY40, VvWRKY40, GmWRKY40, CplWRKY40, and other functionally associated WRKY genes in pepper and Arabidopsis. The promoter regions containing the conserved DWE phenocopied the DWECaWRKY40 in the synergistic response to HS and RSI, with slight modification by the flanking sequences. DWEAtWRKY40 also preferentially bound to AtWRKY40 in plants challenged with HS and RSI. These data suggest that the conserved synergistic response of plants to HS and pathogen infection is mediated by competitive binding of different WRKY proteins to DWE.

900-088-Y Molecular Mechanisms Regulating Cytokinin-Induced Priming Against Pathogens Dawn Hajdu – Colorado State University Evert Van Schaik – University of Fribourg, Daniel Bush – Colorado State University, Joseph Kieber – University of North Carolina at Chapel Hill, Cris Argueso – Colorado State University Priming is the indirect enhancement of the immune response of plants to pathogens. Compared to unprimed plants, the immune response from primed plants upon pathogen attack is much stronger. Recent research in Arabidopsis thaliana has shown that the plant hormone cytokinin, along with many other organic and inorganic chemicals, has a priming effect against biotrophic pathogens, a phenomenon we call cytokinin-induced priming (CIP). The molecular mechanisms behind CIP remain largely unknown. Many studies recently proposed that chromatin modifications, such as methylation/ de-methylation and acetylation/de-acetylation, may play a role in priming. We identified genes encoding transcription factors and DNA binding proteins whose expression is regulated by cytokinin. One of these genes encodes a DNA binding

protein with histone deacetylation activity (HDAC), and therefore is believed to work as a negative regulator of gene expression. Because of the similarities on how known priming agents and cytokinins potentiate defense gene expression, it is possible to hypothesize that priming in Arabidopsis by cytokinin may also involve chromatin modifications, possibly mediated by this HDAC. We are testing this hypothesis using gene expression and genetic analyses.

900-089-Y Investigating the Roles Small RNAs Play in Plant-virus Interactions Richard Moyle – University of Queensland Lilia Carvalhais, Gayathery Subramaniam, Ekaterina Nowak, Lara-Simone Pretorius, Jessica Dalton-Morgan, Anahid AlAmery, Carys Lewington Plant viruses cause serious damage to crops all around the world. There is no effective way to treat infected plants, some of which are infected by several viruses simultaneously. Although many studies have been initiated to develop new resistance strategies against plant viral infection, little is known about the fundamental mechanisms associated with these viruses. Recent research efforts indicate endogenous and virus-derived small RNAs could play pivotal roles in plant-virus interactions. We have sequenced the genomes of local isolates of Turnip Mosaic Virus, Tomato Spotted Wilt Virus and Cucumber Mosaic Virus. Using a combination of bioinformatics, deep small RNA sequencing and transient reporter assays, we are investigating the impact viral small RNAs have on plant gene expression. Using the knowledge obtained, we are developing and testing new approaches and strategies for preventing crop losses caused by plant viruses.

900-090-Z Microbial Tradeoffs of Root Cortical Aerenchyma in Maize: Mycorrhizal Colonization and Root Pathogens in the Field Tania Galindo-Castañeda – The Pennsylvania State University Gregory Roth – The Pennsylvania State University, Kathleen Brown – The Pennsylvania State University, Jonathan Lynch – The Pennsylvania State University Root cortical aerenchyma (RCA) improves the acquisition of water and nutrients by reducing the metabolic costs of soil exploration. While the overall results so far indicate a great potential of RCA as a plant breeding target, the microbial tradeoffs of increased RCA formation need to be considered in the context of sustainable agriculture. The general objective of this research is to study the effects of RCA on the belowground microbial relations of maize. Here, analyses of the effects of RCA on the arbuscular mycorrhizal colonization (AMC) and the root rot (RR) percent of field-grown maize are presented. Three field studies with up to 40 maize lines grown under optimal fertilization regimes were used to analyze variation of RCA and to select lines with contrasting levels of RCA. Lines with the most extreme RCA values were selected to measure AMC. A similar experimental approach was used to study the effect of RCA on RR percent, although RR was measured in complete sets of lines at root sampling, regardless of their RCA levels. Natural genotypic variation in RCA in inbred and commercial hybrid lines was consistent with previous reports, with values ranging from 040%. Variation in AMC and RR was 0-70% and 0-90% respectively. No significant correlation has been found between RCA and either AMC or RR in field studies. This indicates that RCA could be used as a plant breeding target for more efficient root systems in corn, without compromising AMC and RR. Further data analysis has, however, identified other root anatomical traits as better predictors of AMC and RR in maize: cell cortical area, number of cell files, and percent cross-section area occupied by xylem vessels.

900-091-Z The Role of Ethylene in Plant-pathogen Interactions in Moss Keiffer Williams – Butler University Nathanael Hauck Ethylene is one of the three phytohormones that are known to be directly involved in pathogen defense in vascular plants; however, its role in non-vascular plants has not been examined. Here we report progress in determining its role in defense in two moss species, Amblystegium serpens and Physcomitrella patens. A. serpens was used to determine if exogenous application of either ACC, the precursor to ethylene in vascular plants, or ethephon, which is converted to ethylene in plants, induces resistance to Pythium irregulare infection 0, 24 or 48 hours after treatment. P. patens was used to determine if treatment with either of these chemicals induces expression of putative defense genes using a qRTPCR approach. Finally, expression of ACC synthase and ethylene receptor genes following inoculation of P. patens with the pathogen was also analyzed. The implications of these findings on our knowledge of the evolution of plantpathogen interactions are discussed.

900-092-Y Expression of the Pseudomonas Syringae Pv. Tomato DC3000 iaaL Gene in Arabidopsis Thaliana Inhibits Primary Root Growth Eve Mellgren – Elmhurst College Katrina De Leon Jose – Elmhurst College The plant hormone auxin is not only important for plant growth and development, but also plays a role in plantpathogen interactions. Previous studies suggest that auxin promotes disease caused by the plant pathogen Pseudomonas syringae pv. tomato DC3000 (DC3000). Interestingly, in addition to auxin synthesis genes, the DC3000 genome also contains an iaaL gene, which encodes an enzyme, indoleacetic acid-lysine synthetase, that conjugates the auxin indole-3-acetic acid (IAA) to lysine. IAA-lysine is thought to be an inactive form of auxin. To investigate the function of the DC3000 iaaL gene, a transgenic Arabidopsis thaliana line was made where the iaaL gene from DC3000 is regulated by an estradiol-inducible system. After observing induced expression of the iaaL transgene specifically in the presence of estradiol, seedlings were grown on plates containing estradiol and without estradiol. Induction of iaaL gene expression led to plants with shorter primary roots than those plants without iaaL gene expression. Thus, IaaL from DC3000 is likely conjugating IAA to lysine, inactivating it, and inhibiting primary root growth. Future studies will determine if induction of iaaL causes other phenotypes related to auxin inactivation. Furthermore, this transgenic line will also be used to manipulate IAA levels to further our understanding of how and when auxin is required for DC3000 to cause disease.

900-093-Y Parameterization of a Biological Market Model of the Legume-rhizobia Mutualism Colleen Friel – Michigan State University Teresa Clark – Michigan State University, Maren Friesen – Michigan State University, Emily Grman – Eastern Michigan University, Yair Shachar-Hill – Michigan State University Mutualisms are exceedingly common-most species in existence participate in at least one such interaction. However, mutualisms are highly context dependent; interactions that are mutually beneficial under some environmental conditions may become parasitic under different conditions. Understanding how species interactions vary under different environmental conditions is an important challenge facing ecologists. All mutualisms involve trade, whether of services such as protection or pollination, or of goods such as nutrients. Thus, it is possible to use economic principles of

game theory to mathematically model the outcomes of mutualisms. Here, we adapt and parameterize a mathematical model of the interaction between a plant and a resource-trading symbiont. The model was originally developed for plant-mycorrhizal fungi interactions, but we adapted it to the ecologically and agriculturally important legume-rhizobia symbiosis. We used growth chamber experiments with Medicago truncatula and its symbiont Sinorhizobium medicae WSM419 with a resource gradient to parameterize the model and determine the allocation to direct uptake of resources of each partner and the resource exchange ratio. The results of this model can be used to answer important questions regarding resource acquisition specialization, and community assembly across the range of resource availability in the environment. To our knowledge, this project is the first experimental parameterization of theoretical model of mutualisms and thus forms an important connection between theoretical modeling and empirical interaction studies.

900-094-Z Exploring Natural Variation of Arabidopsis Thaliana in Response to Various Bacterial Pathogen Effector Sets Jonathan Walker – University of Minnesota Rachel Meyer – University of Minnesota, Megan Siegle – University of Minnesota, Emily Boak-Nyberg – University of Minnesota, Samantha Dumler – University of Minnesota, Heather Hanson – University of Minnesota, Arefat Heyder – University of Minnesota, Deirdre Kragh – University of Minnesota, Klothilda Lim – University of Minnesota, Shayman Lothert – University of Minnesota, Tiffany Pan – University of Minnesota, Benhamin Spokely – University of Minnesota, Josh Wemmer – University of Minnesota, Suma Chakravarthy – Cornell University, Hailei Wei – Cornell University, Keisuke Mase – Cornell University, Alan Collmer – Cornell University,Fumiaki Katagiri – University of Minnesota Across the Arabidopsis thaliana accessions there could be significant variation in response to different effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). To explore if such variation exists, we performed experiments in which the leaves of 32 A. thaliana accessions were infected individually with four Pto-derived strains that carry limited sets of type III effector genes (CUCPB5115, CUCPB6032, CUCPB6080, ΔAvrPtoΔAvrPtoB). In order to collect reproducible data, the experiments were performed in homemade plant growth chambers at 22°C with a 10.5-hour light/13.5-hour dark cycle and a 75% relative humidity. Plants were then inoculated with one of the four bacterial strains at a dose of OD600=0.0002. Discs of inoculated leaves were initially collected from some plants for the bacterial colony counting immediately after inoculation to ensure this inoculation dose was constant in different accessions. After three days, the bacterial growth was measured from leaf discs from the remaining plants using the same procedure. The bacterial growth was analyzed across the 32 accessions for each bacterial strain using a single mixed-effects linear model. Our results show that there is substantial phenotypic variation among the accessions in response to Pto-derived strains with limited effector genes. Furthermore, we observed different variation patterns across the accessions in responses to the different strains. These observations strongly suggest that the A. thaliana genes that affect the response to different pathogen effectors can be mapped using natural variation. This work was supported by an NSF grant, IOS-1121425.

900-095-Z Maize Kernel Infection by Aspergillus Flavus: Is Hypoxia Tolerance a Factor? Subbaiah Chalivendra – Louisana State University Catherine DeRobertis – Louisana State University, Kenneth Damann – Louisana State University Aflatoxins, potent carcinogenic secondary metabolites of Aspergillus flavus are a serious threat to human health and agriculture due to their contamination of key crop commodities such as corn, cotton and peanuts. Previous research suggested that A. flavus isolates that colonize maize belong predominantly to the mating type MAT1-2, while there is no such mating type bias among soil isolates from the same field. Since developing maize kernels maintain a near-anoxic microenvironment, we hypothesized that this mating type-dependent virulence may be due to a differential tolerance to

hypoxia. Gene expression changes in the host tissue also suggest that hypoxia plays a key role in the pathophysiology of A. flavus. We are testing this proposal using field inoculations of developing kernels with different mating types and their combinations. Transcript levels of two key hypoxic genes, alcohol dehydrogenase (adh) and pyruvate decarboxylase (pdc) were 25% greater in MAT1-2 isolates than in MAT1-1. Aflatoxin levels were also significantly greater in MAT1-2 isolates. The biosynthesis of pseurotin A, the immunosuppressive mycotoxin, is induced by hypoxia in A. fumigatus. We are correlating the data with the expression of toxin biosynthesis genes and the tolerance of fungal isolates to oxygen deprivation in culture media. We have also observed post-transcriptional regulation of toxin biosynthesis and will be looking at the role of hypoxia in this regulatory component as well.

900-096-Y Mapping Genes of the Arabidopsis Thaliana That Affect Immunity Variation Against Pseudomonas Syringae Tiffany Pan – University of Minnesota Rachel Meyer – University of Minnesota, Megan Siegle – University of Minnesota, Emily Boak-Nyberg – University of Minnesota, Samantha Dumler – University of Minnesota, Heather Hanson – University of Minnesota, Arefat Heyder – University of Minnesota, Scott Miller – University of Minnesota, Tyler Reese – University of Minnesota, Jonathan Resch – University of Minnesota, Melissa Rivi – University of Minnesota, Britt Swanson – University of Minnesota, Noah Strom – University of Minnesota, Jonathan Walker – University of Minnesota, Keisuke Mase – University of Minnesota, Fumiaki Katagiri – University of Minnesota, Alan Collmer – Cornell University, Hailei Wei – Cornell University, Suma Chakravarthy – Cornell University Unlike animals, plants only have innate immunity and do not have immune-specialized cells. In pattern-triggered immunity (PTI), plants detect pathogen-derived molecules called microbe-associated molecular patterns using receptors on the cell surface and activate defensive responses. In order to overcome PTI, pathogens use effectors to interfere with PTI signaling by targeting particular sites of the signaling network. To better understand this process, this experiment looked to find specific plant genes whose functions may be influenced by pathogen effectors. We used the model plant, Arabidopsis thaliana to map the potential loci. Specifically, we used 512 recombinant inbred lines from the MAGIC mapping population that were derived from 19 different parent accessions. This number of plant lines (83) allowed us to use a relatively well balanced experimental design for replication. The plants were infected with one of two different strains derived from the Pseudomonas syringae pv. tomato DC3000 (Pto), CUCPB6080 and CUCPB6032. CUCPB6080 carries only two out of 28 well-expressed Pto type III effector genes while CUCPB6032 carries nine. The limited numbers of effector genes help reduce the chance of having functional redundancy among effector genes and observe the effectors’ specific effects. Immunity variations that could be observed across the MAGIC lines between these two strains were likely to be attributed to the seven effector genes different between the strains. We quantified the immune response by bacterial growth in plants. After completing this round of experiments, the QTL mapping showed no chromosomal locations that are significantly associated with the phenotypic variation with either of the strains. We are planning to increase replication to estimate more accurate bacterial growth values for each MAGIC line. Some data points in the beginning of the experiment were lost, which possibly caused the data to become unbalanced. This work was supported by an NSF grant, IOS-1121425.

900-097-Y Isolation of Possible Biocontrol Endophytic Bacteria from Solanum Tuberosum Effective Against Streptomyces Scabies Annie Flatley – Northern Michigan University Luke Ogle, Adam Noel, Erica Fraley, Alaxandra Goodman, Donna Becker Use of biological control offers a cost effective and environmentally safe method for controlling plant diseases.

Biocontrol agents that can colonize roots and live endophytically within plant tissue should allow for effective disease control. The goal of this research was to develop protocols to isolate putative Streptomyces species from potato stem and tuber tissue due to their ability to produce inhibitory compounds which could potentially reduce diseases caused by Streptomyces scabies. Endophyte isolation from Solanum tuberosum (potato) plants (stem and tuber tissues) that were grown in a biocontrol field trial were the focus of this study. Several surface sterilization processes were assessed and two different tissue-processing methods were focused on to obtain endophytes: 1) excising cross-sections of stem tissue and 2) use of a maceration technique for tuber and stem tissue. From the stem tissue, four putative Streptomyces species were isolated; one of the four isolates was inhibitory to Streptomyces scabies in agar plate bioassays. To date, two putative Streptomyces species and several other bacterial species were isolated from tuber tissue. Further characterization of these isolates are underway with the goal of discovering endophytic pathogen-inhibiting bacteria that could then be used in concert with soil-dwelling biocontrol agents to enhance disease control of the potato scab pathogen.

900-098-Z Signaling Components That Regulate Plant Immunity and Alter Actin Cytoskeletal Dynamics Yi-Ju Lu – Michigan State University Mieder Palm-Forster – Michigan State University, Brad Day – Michigan State University Actin cytoskeletal dynamics are tightly regulated by phospho-regulation of a conserved family of actin depolymerization factors (ADFs). Previous work has shown that ADFs also play an important role in modulating cell morphology, development, and resistance to pathogens through control of actin filament organization. Within this process, Arabidopsis ADF4 has been shown to regulate key processes not only associated with cytoskeletal organization, but also immune signaling processes, including effector triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI). In the current study, we employed biochemical and cell biology-based methods to elucidate the connection between actin cytoskeletal changes and plant immunity. From this, we have identified the Arabidopsis calcium-dependent protein kinase-3 (CPK3) as a key molecular switch, responsible for the phosphorylation of ADF4. As predicted, and consistent with our previous studies and working hypothesis, cpk3 knockout mutant plants showed enhanced susceptibility to Pseudomonas syringae pv. tomato DC3000 expressing the bacterial cysteine protease effector AvrPphB. As a function of PTI, stomata closure triggered by flg22, a 22 amino acids flagellin peptide, is impaired in cpk3 mutants. Lastly, using confocal microscopy, we observed an increase in bundling of actin cytoskeletal filaments, supporting a key role for CPK3 in filament organization through the activity of ADF. Our data support a model through which CPK3 functions as a key signaling node that is required for plant immunity and determines actin turnover and remodeling in Arabidopsis.

900-099-Z A Translocation Signal for Delivery of Nematode CLE Effectors to the Extracellular Space of Host Plant Cells Jianying Wang – University of Missouri Thomas Baum – Iowa State University, Eric Davis – North Carolina State University, Xiaohong Wang – Cornell University, Melissa Mitchum – University of Missouri Plant-pathogenic bacteria, fungi, oomycetes, nematodes, insect herbivores, and beneficial microbes all secrete effector proteins into plant cells to manipulate the host’s immune system or mimic plant molecules that hijack developmental and metabolic programs to promote disease, herbivory, or beneficial associations, respectively. In bacterial pathogens, a suite of effectors is translocated through the type III secretion system into plant cells which are then targeted to subcellular structures or organelles for their specific activities. However, little is known about the delivery and trafficking mechanisms of plant-parasitic nematode effectors. Here we show that CLAVATA3/ESR (CLE) peptide effectors of plantparasitic cyst nematodes have evolved a translocation signal that is recognized by host cell machinery to redirect these

peptide mimics from the cytoplasm to the apoplast of parasitized host cells where they can then be recognized by membrane-bound extracellular receptors to exert their function. We used serial deletion experiments to determine that the translocation signal, VDIT, is a 37 aa sequence within the variable domain of CLE effectors and demonstrate its conservation across cyst nematode species spanning two major genera. We show that the VDIT trafficking function is conserved across plant species, is functional across cell types, and is able to traffic other unrelated small peptides from the cytoplasm to the apoplast of host cells, including the AVR9 peptide elicitor of the fungal pathogen Cladosporium fulvum. Identification of the translocation signal for delivery of CLE effector proteins to the extracellular space of host cells provides unparalleled opportunities to determine how cyst nematodes manipulate their plant hosts to establish infection, identify points vulnerable for disruption to engineer resistance, and uncover potentially novel protein trafficking mechanisms in plants that may also serve as subcellular host targets of other pathogens effectors.

900-100-Y Cyst Nematode CLE Peptides Regulate Feeding Site Formation via the WOX4 Gene in Arabidopsis Xiaoli Guo – University of Missouri Yuki Kondo, J. Peter Etchells, Hiroo Fukuda, Xiaohong Wang, Melissa Mitchum Stem cells are important in the continuous formation of various tissues during postembryonic organogenesis. Stem cell pools in the SAM (shoot apical meristem), RAM (root apical meristem) and vascular procambium/cambium are regulated by CLE-receptor kinase-WOX signaling modules. Cyst nematode secreted CLE-like effector proteins delivered into host cells through a stylet, act as ligand mimics of plant A-type CLE peptides and are pivotal for successful parasitism. Recently, we demonstrated that CLE receptors CLAVATA1 (CLV1), the CLAVATA2 (CLV2)/CORYNE (CRN) heterodimer receptor complex and RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) from Arabidopsis and soybean, which transmit the CLV3 signal in the SAM, are required for perception of cyst nematode CLEs and proper feeding site formation. However, little is known about the downstream signaling pathways regulated by nematode CLEs for feeding site formation. Here we show that TDIF (tracheary element differentiation inhibitory factor), a B-type CLE peptide encoded by the CLE41 and CLE44 genes, is involved in beet cyst nematode Heterodera schachtii parasitism. The TDIF-TDR1 (TDIF receptor)-WOX4 pathway, which promotes procambial meristem cell proliferation, is responsive to nematode infection in developing feeding sites. Reduced nematode infection is observed in cle41 and tdr-1wox4 mutants. Syncytium size is compromised in cle41, tdr-1, wox4 and tdr-1wox4 mutants. By qRT-PCR and promoter:GUS analysis, we showed that the expression of WOX4, but not TDR, is decreased in a clv1-101clv2-101rpk2-5 mutant, suggesting WOX4 is a potential downstream target of nematode CLEs. Exogenous treatment with TDIF and nematode CLE peptide can induce WOX4 expression in wild type roots, but not in tdr1clv1clv2 mutant, which indicates the two types of CLEs synergistically regulate cell proliferation during feeding site formation. These findings highlight the role of the procambium cell proliferation pathway in cyst nematode feeding site formation.

900-101-Y Biological Diversification of Arabidopsis Triphosphate Tunnel Metalloenzymes: Involvement in and Pathogen Resistance and Leaf Senescence Wolfgang Moeder – University of Toronto Huoi Ung – University of Toronto, Keiko Yoshioka – University of Toronto The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis thaliana encodes three TTM genes. AtTTM3 displays tripolyphosphatase activity and is involved in root development. Here, we report the biochemical activity and biological function of AtTTM1 and 2 and the diversification of the biological roles between them. Biochemical analyses revealed that both display pyrophosphatase activity, making

them the only TTMs characterized so far to act on a diphosphate substrate. AtTTM2 knockout mutant plants exhibit an enhanced hypersensitive response, elevated pathogen resistance against both virulent and avirulent pathogens, and elevated accumulation of salicylic acid (SA) upon infection. On the other hand, AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects. However, promoter swap analyses revealed that they functionally can complement each other. This work provides conclusive data indicating that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work suggests a possible novel connection between pathogen resistance-related programmed cell death and senescence through phosphate metabolites.

900-102-Z Identifying Protein-protein Interactions Involved in Plant Immunity Using Promiscuous Biotin Ligase Fusions Mike Brown – Stanford University Mary Beth Mudgett – Stanford University Many plant pathogens secrete virulence factors that suppress host immunity. Determining the targets of such effectors and the host proteins these targets interact with is a useful way to elucidate immunity pathways. However, many of these interactions are too weak or transient to be detected by traditional biochemical approaches such as immunoprecipitation. To address this issue, we are employing proximity-dependent biotin identification (BioID). This approach involves fusing a protein of interest to a promiscuous biotin ligase and using affinity purification to isolate the biotinylated interacting proteins. We are implementing BioID in two ways: fusions with host proteins known to play roles in immunity and fusions with bacterial effector proteins. In the former case, the fusions can be transiently expressed in Nicotiana benthamiana or stably expressed in transgenic Arabidopsis lines. For this strategy, we are using ACIP1, an Arabidopsis protein that functions in both pattern- and effector-triggered immunity and is a target of Xanthomonas euvesicatoria (Xe) effector AvrBsT. When using effector fusions, such as the Xe proteins AvrBsT and XopD, the constructs can be expressed in a pathogen and transferred to the plant cells during normal infection. We are currently running proof-of-principle experiments and investigating ways to reduce background from biotin-containing carboxylases normally found in the hosts. BioID may prove to be an important tool for identifying the targets of bacterial effectors and the pathways involved in plant immunity.

900-103-Z Investigation of the Roles of Calmodulin-Like Proteins in Plant Immunity Jane Glazebrook – University of Minnesota Gerit Bethke, Amanda Thao, William Truman, Zhi Li We have found that a family of calmodulin-binding proteins, called CBP60s, play roles in control of plant immunity. In a study of co-expressed genes, genes encoding two calmodulin-like (CML) proteins, CML46 and CML47, were found to be co-expressed with CBP60s and with SID2, an immunity gene whose expression is affected by CBP60 proteins. We tested plants with cml46 and cml47 mutations for defects in immunity. We found that cml46 and cml47 plants were more resistant to the bacterial pathogen Pseudomonas syringae pv. maculicola Psm ES4326, than wild type plants. A double cml46 cml47 mutant was even more resistant. Many mutations affecting immunity to Pma ES4326 have alterations in salicylic acid (SA) signaling. Accordingly, expression of the SA reporter gene, PR-1, was monitored in cml46 cml47 plants. While PR-1 was expressed at similar levels in untreated wild type and cml46 cml47 plants, it was expressed at higher levels in cml46 cml47 than wild-type plants in response to stimuli including infiltration with water, infiltration with the immune-response inducing peptide flg22, or wounding. This finding suggests that CML46 and CML47 have roles in suppression of immunity.

While all eukaryotes produce calmodulin, CMLs are unique to plants. Arabidopsis has 50 CMLs in the genome, and many of these are induced by pathogen attack. We are investigating possible roles of these proteins in immunity, and working to identify proteins that interact with them.

900-104-Y Use of Two Pathogen-inhibiting Streptomyces Isolates for Biocontrol of Scab Disease of Raphanus Sativus Tyler Park – Northern Michigan University Nichole Klingler, Adam Noel, Luke Ogle, Annie Flatley, Donna Becker Streptomyces species are ubiquitous soil bacteria that are promising as biological control agents due to their prolific antibiotic production that can inhibit soil-borne plant pathogens. This includes Streptomyces scabies, which causes scab disease on underground storage organs. The goal of this research was to test two Streptomyces isolates that have known inhibitory and biocontrol abilities against S. scabies in a potato (Solanum tuberosum) agricultural system for their effectiveness in biocontrol of this pathogen in a radish (Raphanus sativus) greenhouse assay. Pathogenic S. scabies were mixed into either pasteurized or unpasteurized soil prior to planting. Radish seeds or radish roots were soaked in a spore suspension of the two biocontrol Streptomyces isolates. Controls of treatments without the pathogen and the biocontrol Streptomyces were included. Radish plants were grown to maturity. Upon harvesting, the radish taproot was rated for scab disease and tuber weight and length were determined. Disease levels were reduced in the presence of the two biocontrol Streptomyces. Taproot weight was greater in the treatments that included the biocontrol Streptomyces. In addition, evidence was sought to determine the ability of these Streptomyces isolates and other pathogen-inhibiting bacteria to enter into an endophytic relationship with the radish plant. For this, several protocols were assessed to try to optimize the isolation of endophytic bacteria from stem and taproot tissue. Surface-sterilized tissue sections (stem and taproot) or macerated stem tissue spread on agar plates have indicated the presence of several bacterial endophytes, including two putative Streptomycete isolates from the stem tissue sections. The search for additional endophytic bacteria capable of inhibiting the scab pathogen are currently underway. The results of this study are promising for the use of these Streptomyces isolates for biocontrol against scab disease in radish.

900-105-Y Identification of Key Cytokinin Pathway Components Involved in Disease Susceptibility to Plant - Parasitic Nematodes Carola De La Torre – Division of Plant Sciences and Bond Life Sciences Center, University of Missouri Demosthenis Chronis – Division of Plant Sciences and Bond Life Sciences Center, University of Missouri, Tomas Werner – Dahlem Centre of Plant Sciences, Freie Universitat Berlin, Tatsuo Kakimoto, Osaka University, Thomas Schmulling – Dahlem Centre of Plant Sciences, Freie Universitat Berlin, Melissa Mitchum – University of Missouri Cyst and root-knot nematodes are devastating root parasites and a major threat to global food security, causing billions of dollars in yield losses annually. Previous studies have demonstrated that decreasing plant cytokinin levels by constitutively overexpressing a cytokinin degradation enzyme reduced the susceptibility of Lotus japonicus to root-knot nematodes, suggesting that cytokinins play a positive role in plant–nematode interactions. However, the cytokinin pathway components that contribute to plant susceptibility to nematode infection are unknown. In Arabidopsis thaliana, the limiting step in the cytokinin biosynthesis pathway is controlled by adenine isopentenyl transferases (IPTs), while catabolism is controlled by cytokinin dehydrogenases/oxidases (CKXs). Cytokinin signals through a two component phosphorelay system that involves membrane-bound histidine kinase receptors (AHKs) which ultimately alter the phosphorylation state of Arabidopsis response regulators (ARRs). In this study, we have conducted a comparative analysis of the Arabidopsis cytokinin response to the cyst nematode, Heterodera schachtii, and the root-knot nematode, Meloidogyne incognita. We have examined cytokinin biosynthesis (IPTs), catabolism (CKXs), and signaling (AHKs, ARRs)

gene expression in nematode infection sites using qRT-PCR and reporter lines. Additionally, we have tested mutant lines of the cytokinin genes found to be upregulated in response to nematode infection. Our results unveil a distinct profile of differentially regulated cytokinin genes in response to each of these species, and sheds additional insight into their role as potential targets for engineering nematode resistance.

900-106-Z Characterization of Pediomelum Esculentum, a Native Legume, and Its Endophytes Tyrel Deutscher – South Dakota State University Volker Brozel – South Dakota State University, Senthill Subramanian – South Dakota State University, Leslie Henry – Oglala Lakota College, Neil Reese – South Dakota State University Prairie turnip (Pediomelum esculentum (Pursh) Rydb. [Fabaceae]) is a perennial legume of the Great Plains not commonly found to form nodules in the wild. The root system consists of a deep taproot and large edible tuber that stores protein and starch. The tuber is a traditional food of Native Americans, and was consumed by early European settlers. We have investigated the conditions for nodule formation in prairie turnip, and whether nitrogen is fixed in a non-nodule symbiosis. Wild plants were divided into three tissue types: herbaceous top, edible tuber, and inedible root. Bacterial cultures were isolated from these tissues on nitrogen-free media and screened for nifH. Partial 16s rRNA was sequenced, and isolates were identified as known plant-associated bacteria: Rhizobium, Burkholderia, Erwinia, Rhanella, and Xanthomonas, among others. Bright field and fluorescence imaging revealed bacteria present in the intercellular space of the tuber and sterile seedlings, indicating the seeds harbor bacteria. Imaging of nodule sections showed a unique structure with a central vasculature resembling lateral roots. The formation of root hairs is uncommon, and nodules appear to be associated with breaks in the epidermis where lateral roots emerge. This morphology is similar to the nodule-forming non-legume Parasponia,making Pediomelum esculentum an exception among legumes, and an intriguing model for further study.

900-107-Z Standing Variation in Copy Number of Nodule-Cysteine Rich Peptides Plays a Role in Rhizobial Nodulation Joseph Guhlin – University of Minnesota Kevin Silverstein – University of Minnesota, Peng Zhou – University of Minnesota, Andrew Farmer – National Center for Genome Resources, Peter Tiffin – University of Minnesota, Nevin Young – University of Minnesota Medicago truncatula is a model legume often used to study the symbiosis between legumes and nitrogen-fixing rhizobia. M. truncatula has a stable, high-quality reference genome and a well characterized collection of HapMap accessions, for which SNPs have been called through deep re-sequencing. In this study we have identified copy-number variants (CNVs) in M. truncatula HapMap accessions and performed a genome-wide association study (GWAS) on nodulation-related traits. CNVs were identified through comparison between reference read-depth and specific accession read-depths. Two nodule cysteine-rich (NCR) peptides in a 50kbp region were identified as likely contributors to total nodule number. Copy number variation in either NCR resulted in a decrease in total nodule number. A follow-up candidate gene association study with SNPs from NCRs and their promoter region identified two additional candidate NCRs at this locus, and a third found on a separate scaffold. Further analysis of the scaffold NCR suggests it belongs to the same locus. These results indicate that CNVs may play a role in phenotypic variation that has not been fully characterized by SNPonly methods, and hints that other structural variants may also play an important role in phenotype. This work was supported by NSF Plant Genome grant IOS-1237993.

900-108-Y Symbiotic and Transcriptomic Dimensions of Trifolium Diversity Maren Friesen – Michigan State University Prateek Shetty – Michigan State University, Sharon Strauss – UC Davis, Brian Anacker – UC Davis, Jean Burns – Case Western, Joseph Dunham – University of Southern California The biodiversity and ecosystem functions of natural plant communities is tied to their shared soil microbial communities. Diverse North American clover (Trifolium)-Rhizobium communities are an exemplar system in which positive and negative feedbacks can be dissected at molecular, functional, and taxonomic levels. Rhizobia are a primary bacterial symbiont of legumes such as Trifolium and infect roots to form nodules, where the bacteria fix atmospheric nitrogen into biologically available forms. We find that Trifolium plants make more large nodules in their home soils and that nodulation correlates with performance, demonstrating that symbiotic nitrogen-fixing rhizobia are an important part of the niche. Competition experiments show that plants allocate more to nodules when competing against members of their own species, suggesting that symbiotic interactions play a role in species coexistence. Nodule genotyping shows widespread sharing of rhizobium strains across Trifolium species with the majority of structuring occuring between different soils rather than species. Using 16S amplicon sequencing, we find that nodules harbor a rare, diverse community of bacteria that is largely conserved across species. However, Trifolium species structure this community to a small extent as does the source soil, but the majority of variation in community structure explained by a soil x species interaction. Root transcriptomic analysis of plants grown under differing competitive regimes shows that several genes related to symbiosis as well as playing potential roles in secondary metabolism vary in the presence of competitors. Furthermore, transcriptional responses to conspecific and congeneric competition differ. The overarching goal of this work is to bridge organismal and community scales to predict coexistence and inform variation in biological nitrogen fixation.

900-109-Y Investigating the Role of Rhg1 Encoded α-SNAPs in Rhg1-Mediated Resistance to Soybean Cyst Nematode Adam Bayless – University of Wisconsin-Madison David Cook – University of Wisconsin-Madison, Xiaoli Guo – University of Wisconsin-Madison, Andrew Bent – University of Wisconsin-Madison Heterodera glycines (Soybean cyst nematode, SCN) is a highly adapted, obligate parasite of soybean roots, and causes annual yield losses valued at over $1 billion in the U.S. alone. The Rhg1 locus (Resistance to Heterodera glycines 1) is present in most commercial SCN resistant soybean varieties and is the primary means of restricting yield loss caused by SCN. The most widely used Rhg1 locus consists of ten tandem repeats of a ~30 kb block that encodes four genes. However, the molecular mechanisms of how these genes contribute to Rhg1-mediated resistance remains unclear. Our recent work uncovered that resistance-conferring Rhg1 loci group into two classes, carrying either a high or low Rhg1 copy number and also encoding distinct alleles of an α-SNAP (alpha Soluble NSF Attachment Protein). α-SNAP is present in all eukaryotes and plays a crucial role in vesicle trafficking. α-SNAP binds to and stimulates the disassembly of cisSNARE (Soluble N-ethylmaleimide Attachment Receptors) protein complexes that form during vesicle fusion events, freeing SNARE proteins for future vesicle fusion events. Both classes of Rhg1 encode α-SNAPs with C-terminal amino acid polymorphisms at highly conserved regions. We are utilizing in vitro and in vivo approaches to examine how Rhg1encoded α-SNAPs contribute to SCN resistance and how they functionally differ from the wild type α-SNAPs in terms of impacts on vesicular trafficking.

900-110-Z Comparative Proteomic Analysis of Gossypium. Thurberi in Response to Verticillium. Dahliae Inoculation Xiaojie Yang – Economic Crop Research Institute, Henan Academy of Agriculture Sciences

Weiping Fang – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Deyi Xie – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Heqin Zhu – State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Arigculture Sciences, Wu Li – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Zhenzhen Xu – State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Arigculture Sciences, Lirong Yang – Plant Protection Research Institute, Henan Academu of Agriculture Sciences, Zhifang Li – State Key Laboratory of Cotton Biology, Institute of Cotton Research, Chinese Academy of Arigculture Sciences, Lil Sun – Texas Tech University, Lihong Nei – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Zhongjie Tang – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Shuping Lv – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Fuan Zhao – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Yao Sun – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Yuanming Zhao – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Jianan Hou – Economic Crop Research Institute, Henan Academy of Agriculture Sciences, Xiaojie Yang – Economic Crop Research Institute, Henan Academy of Agriculture Sciences Cotton production is detrimentally affected by Verticillium wilt caused by soil-borne Verticillium dahliae Kleb which infects cotton roots and colonizes and occludes the xylem vessels, resulting in leaf curl, necrosis, defoliation, vascular tissue wilt, discoloration, and most severely, plant death. In this study, the recently developed isobaric tags for relative and absolute quantification (iTRAQ) with more sensitive and accurate protein quantification was applied for the first attempt to identify differentially expressed proteins of G. thurberi inoculated with V. dahliae. The results showed that, a total of 6533 proteins were identified from the roots of G. Thurberi after inoculation with V. Dahliae, and of these identified proteins, 396 showed up- and 279 down-regulation of cotton after infected by V. Dahliae compared to the mock-infected. The further bioinformatis analysis showed that, the main functional groups of proteins that were significantly different were those involved in cell wall organization and reinforcement, disease-resistant chemicals of secondary metabolism, phytohormones signaling, pathogenesis-ralated proteins, disease-resistant proteins. Physiological and biochemical analysis showed that peroxidase activity, which promotes the biosynthesis and accumulation of lignin, was induced in hypocotyl rapidly at only one hour after inoculation with V. dahliae, and salicylic acid and jasmonic acid were also accumulated siginificantly in hypocotyl of seedlings after inoculation with V. Dahliae. These findings extend a better comprehensive elucidations on the molecular events involved in regulatory networks of response of G. Thurberi to V. Dahliae infection, which may provide a foundation for further researchs on diseaseresistant breeding in cotton.

900-111-Z Pathogen Defense Response Modulated in Aspen Root Due to Helper Bacteria in Mycorrhizal Interactions Shalaka Shinde – Argonne National Lab Sarah Zerbs, Peter Larsen, Frank Collart Plant microbial communities play an important role in terrestrial ecosystem. In forest ecosystem 80-90% of the plants are colonized by symbiotic fungi, nearly all plants are associated with mycorrhizal helper bacteria (MHB) which modulate plant response to the environment and carbon allocation. To explore the molecular mechanisms underlying these interactions we examined the plant transcriptomic impact of Aspen seedling colonization by mycorrhizal fungi and/ or helper bacteria.Aspen seedlings were inoculated with mycorrhizal fungus Laccaria bicolor and/ or helper bacteria Pseudomonas fluorescens SBW25 and incubated for five weeks in controlled conditions. Analysis of the Aspen expression profiles of uncolonized and colonized Aspen demonstrated 2482 genes were significantly differentially regulated in response to MHB colonization. Plant transporters and transcription factors were highly up-regulated during MHB colonization whereas enzyme related genes were down-regulated during the interaction. In a parallel treatment, we identified over 3000 plants genes that were significantly differentially regulated due to mycorrhizal fungal

colonization. Plant genes related to transport, stress and defense response were up-regulated while enzyme related transcripts where down regulated. In aspen seedlings colonized with both fungi and MHB, transcript profiles are more similar to the expression profile of plants colonized with mycorrhizal fungi alone versus plants colonized only with MHB. These results are consistent with a proposed hypothesis that bimolecular patterns reflect the efficient exchange and utilization of key nutrients by various members within the community. The most significant observation in root transcriptome was the down-regulation of plant defense-related genes when MHB are present in the root community. Changes in the predicted transmembrane transport capacity of root correlates with changes in expression of plant defense response highlights possible interaction mechanisms between plant root and MHB. Future studies to characterize these molecular patterns will enhance our understanding of the relationships underpinning the establishment and maintenance of forest ecosystems.

900-112-Y Arabidopsis MED18-interacting Protein 1 Regulates Responses to Pathogens Through Acetylation of Regulatory Loci Chao-Jan Liao – Purdue University Zhibing Lai, Tesfaye Mengiste Arabidopsis MED18-interacting protein 1(MIP1) encodes a putative histone acetyltransferase (HAT) that shares biological functions with MED18 in plant defense and plant growth functions. The mip1 mutant displays impaired immune responses to infection by the fungal pathogen Botrytis cinerea. By contrast, mip1 plants show enhanced disease symptoms but wild type levels of bacterial growth when inoculated with virulent and non-pathogenic strains of Pseudomonas syringae. The mutant also shows accelerated dark induced senescence suggesting the role of MIP1 in suppressing premature senescence. These responses are accompanied with altered responses of the mutant to the plant hormones ethylene and abscisic acid. Underlying these responses, MED18 and MIP1 both regulate expression of selected immune and hormone response regulatory factors through direct association with their promoters impacting defense responses consistent with their overlapping biological functions. Interestingly, the mip1 mutation causes reduced histone H3 acetylation at chromatin of target genes further confirming the regulatory impact of MIP1 on gene expression. The impact of MIP1 on pathogen and hormone responses, its interaction with the mediator complex and other regulatory loci is presented.

900-113-Y Mechanisms of TAL Effector-Mediated Resistance and Susceptibility to Bacterial Blight of Cotton Kevin Cox – Texas A&M University, Institute for Plant Genomics and Biotechnology Fanhong Meng – Texas A&M University, Institute for Plant Genomics and Biotechnology, Katie Wilkins – Cornell University, School of Integrative Plant Science, Ping He – Texas A&M University, Institute for Plant Genomics and Biotechnology, Adam Bogdanove – Cornell University, School ofIntegrative Plant Science, Libo Shan – Texas A&M University, Institute for Plant Genomics and Biotechnology Bacterial blight of cotton (BBC) is a destructive disease caused by Xanthomonas campestris pv. malvacearum (Xcm). Xcm injects transcription activator-like (TAL) effectors, which can directly induce expression of host genes to either cause susceptibility (S) or trigger resistance (R) in different hosts. Although more than ten Xcm TAL effectorshave been identified, no corresponding S or R genes have been cloned in cotton. Xcm TAL effectorsAvrb6 and PthN play major roles in causing water-soaking in susceptible lines of cotton. In cotton lines homozygous for the r gene b6, Avrb6 triggers a defense response resulting in hypersensitive response (HR). In this study, toward understanding mechanisms of susceptibility and resistance in BBC, we aim to identify cotton targets of Avrb6 and PthN by genome-wide transcript profiling coupled with TAL effector binding site computational prediction, followed by functional characterization of

candidate genes using high-throughput virus-induced gene silencing and artificially designed TAL effectors. RNA-seq analysis revealed that GhSWEET1 is highly induced in cotton cells expressing Avrb6. Analysis of the reference genome predicted an effector binding element (EBE) site in the GhSWEET1 promoter, which we confirmed in our experimental line (Ac44) by amplicon sequencing. We further validated that GhSWEET1 was induced by Avrb6through RT-PCR in both cotton protoplasts and Xcm-cotton interaction. A transient protoplast reporter assay indicated that Avrb6 could specifically activate the GhSWEET1 promoter, and mutation of the EBE site of GhSWEET1 abolished Avrb6-mediated transactivation activity.Additional candidates and functional investigation on the role of GhSWEET1 mediating Xcm virulence will be presented.

900-114-Z Evidence for Fungal Endophyte Colonization of Healthy Leaf Tissue in Tillandsia Recurvata Ross Joseph – New College of Florida Joel Thurmond – New College of Florida, Amy Clore – New College of Florida Numerous studies have reported the presence of fungal endophytes within the asymptomatic tissues of terrestrial and aquatic plants. These findings have led to speculation that these organisms could represent a form of defensive mutualism ubiquitous in all plants but with particular diversity in tropical and subtropical regions. Despite these claims, a relatively small number of plant species have been screened for the presence of fungal endophytes to date and plant types from many environments remain highly underrepresented. Here, we present evidence of colonization by fungal endophytes in asymptomatic tissues of the bromeliad, Tillandsia recurvata, a common Florida epiphyte. Characterization of the endophytes in this study was accomplished through tissue culture methods utilizing sections taken from surface sterilized leaf tissue of T. recurvata leaves. Fungi cultured from these tissues were subcultured to achieve pure isolates. From these subcultures, DNA was extracted, amplified using ITS1F and ITS4 primers, and sequenced using the Sanger method. Sequences of the amplicons were assembled using Sequencher software and searched against the NCBI BLAST database. To date, two isolates have been tentatively identified as Exophiala capensis and Penicillium citrinum, while other isolates await sequencing. Sequenced isolate identities were further examined through morphological determination of microscopic features including conidiophores, conidiogenous cells, conidia and hyphae septation.

ABIOTIC - Zone 1000 Abiotic: General 1000-001-Y A Role for Cyanide in the Post-wildfire Development of Corms of the Geophyte Triteleia Laxa Winslow Briggs – Carnegie Institution for Science Tong-Seung Tseng – Carnegie Institution for Science Researchers in Western Australia identified several compounds in smoke that elicit seed germination in sensitive species—the karrikin family of butenolides and glyceryl nitrile. Seeds of some species respond only to karrikins, some only to glyceryl nitrile, and some to both. Many of these species germinate in large numbers following wildfire, but many other non-fire-following species including Arabidopsis thaliana also respond to one or the other of these compounds. In addition, a number of species growing from bulbs or corms respond to wildfire by flowering in large numbers. Among these are Triteleia laxa, Toxicoscordion fremontii, Chlorogalum pomeridianum, Calochortus venustus, and Calochortus alba. We tested whether either karrikins (karrikin I) or glyconitrile (a close homolog of glyceryl nitrile with the same dissociation chemistry) might induce any measurable response from Triteleia laxa corms. Sprouting, growth rate, flowering time, and percent of corms producing flowering scapes were all unaffected by treatment with 10 – 100 µM kARI (the most active of the karrikins). By contrast, 10 -100 µM glycosyl nitrile induced a dramatically more rapid emergence of roots and more rapid emergence of shoots from dormant corms than induced by water alone. Since both

glyceryl nitrile and glycosyl nitrile release very low levels of cyanide at pH values near neutrality, we suspected a cyanide effect. Corms incubated in water in a petri dish near a second petri dish containing 10 and 100 µM potassium ferrocyanide—a compound that slowly releases cyanide in the light—rooted extensively when both dishes were placed in an air-tight chamber. compared to water controls. We are currently investigating any possible flowering response to cyanide from Triteleia corms.

1000-002-Z Arabidopsis CML38, a Calcium Sensor That Localizes to Ribonucleoprotein Complexes Under Hypoxia Stress Ansul Lokdarshi Craig Conner – University of Tennessee, Carlee McClintock – University of Tennessee, Tian Li – University of Tennessee, Daniel Roberts – University of Tennessee Flooding induces wide variety of adaptions in plants that are ultimately mediated by the initial re-orientation of gene expression networks and synthesis of response proteins. A key aspect of this strategy is the formation of mRNA triage centers that function at the post-transcriptional level to regulate translation under oxygen deprivation stress. We have identified an Arabidopsis thaliana calcium sensor AtCML38, that is highly up-regulated (>300 fold) within 4-6 hrs of hypoxia treatment and colocalizes with mRNP (mRNA-ribo nucleo protein) markers tested in N.benthamiana leaves. This localization action is also supported by the co-immunoprecipitation of mRNP resident proteins from CML38pro:: CML38:YFP transgenic plants challenged with hypoxia followed by LC MS/MS analysis. Given the complexity of hypoxia regulatory strategies with respect to mRNA homeostasis for coordinated responses towards oxygen deprivation, our data suggest that CML38 may serve as a potential calcium signaling target within stress granules and P-bodies during flooding stress responses.

1000-003-Z Genetic Screen for Identification of Mutants Impaired in Reactive Oxygen Species (ROS) Sensing Esther Cattan – Bar-Ilan University Ilana Lebenthal-Loingerl1 – Bar-Ilan University, Ron Mittler – Bar-Ilan University, Gad Miller – Bar-Ilan University Reactive oxygen species (ROS) function as important regulators of many different cellular processes, including growth, development and stress responses, yet they are toxic and capable of inflicting oxidative damage to cells. It is still unknown how cells resolve this dual function conflict, but it is clear that ROS metabolism requires a tight control. In addition, there is still a huge gap in the understanding of how these signals are perceived and transduced in responding to stress. To identify mutants impaired in ROS sensing (irs mutants) and ROS signals transduction we used a genetic screen utilizing the promoter of the ROS-responsive gene Zat12, a zinc finger protein responsive to various types of abiotic stresses including oxidative stress. A population of EMS treated Zat12p::Luciferase Arabidopsis seedlings was systematically screened under moderate oxidative conditions on MS-media, for showing low expression level of the reporter. Out of ca. 50,000 initially screened mutants, only few showing reliable altered Zat12 expression were further selected for phenotypic evaluation. Three of those mutants that have been phenotypically and genotypically characterized displaying either tolerance or sensitivity to oxidative stress, were subjected to genome sequencing to identify the location of the mutation. Bioinformatics analyses revealed distinct chromosomal region enriched in SNPs, harboring ten to twenty SNP’s for each

of the three mutants. Knockout mutants for candidate genes are being tested for identification of the specific IRS gene. Here we present the progress being made in this project thus far.

1000-004-Y Control of Arabidopsis Plant Architecture by Gravity Through LAZY Gene Action Takeshi Yoshihara – University of Wisconsin-Madison Edgar Spalding –University of Wisconsin-Madison Plant architecture is a major determinant of crop performance by affecting lodging resistance, photosynthesis efficiency, and other important traits. AtLAZY1 is the best known member of a family of genes that plays a role in determining lateral shoot angle, and therefore shoot architecture, in rice, maize, and Arabidopsis. Despite considerable sequence divergence among its members within and between species, LAZY genes display five conserved regions in higher plants, three of which are also conserved in lower plants. Our research separately tested the functions of the five conserved regions in the Arabidopsis LAZY1 gene by determining if mutating them affected their ability to restore a normal inflorescence branch angle to a lazy1 knockout mutant. By this assay, LAZY1 regions I, II, and V were determined to be necessary for setting lateral branch angle. Subcellular localization studies in a transient expression system revealed that region I is required for maintaining the plasma membrane-localized pool of LAZY1. The unaffected nuclear-localized pool of the protein was apparently insufficient to rescue the branch-angle phenotype of lazy1 knockout mutants, which is due to weakened negative gravitropism. Surprisingly, mutating region II of LAZY1 produced positively gravitropic shoot branches. Mutating the LAZY2 family member impaired root gravitropism. LAZY2 was found to reside at the plasma membrane. Swapping promoters showed that LAZY2 was also able to rescue the lazy1 branch-angle phenotype with high efficiency when expressed under the AtLAZY1 promoter. Our current results indicate that i) AtLAZY1 and AtLAZY2 are functionally conserved, ii) both molecules function at the plasma membrane, and iii) different specific modifications of AtLAZY1 can cause inflorescence gravitropism to be negative, positive, or agravitropic. The possibility that LAZY1 and its relatives affect a cell polarity-setting mechanism to control the directional quality of agravitropic response is under investigation.

1000-005-Y An Integrated Approach to Model the Command Center in Plant Root Tip: Re-evaluation of Darwin’s “RootBrain” Hypothesis Sonali Sengupta – Bose Institute Sanghamitra Adak – Bose Institute, Arindam Ray – Bose Institute, Abhishek Mukherjee – Bose Institute, Arun Lahiri Majumder – Bose Institute Abiotic stress has great ability to reprogram organ growth in plants. It is debated whether such reprograming represents adaptive strategies or physiological noise. Using a phenomic study on rice root, we show that plants indeed use growth reprogramming as survival strategy. We also show that stress hurdles like salinity and soil impedance induce similar root behavior. Microarray of rice root tips challenged with salt and mechanical hurdle reveals a complex crosstalk among ~6500 transcripts. These transcripts can be mapped to three interlinked “transcription hubs”, regulating sugar and hormone signaling, redox state and calcium signaling. Our analysis suggests that root growth reprogramming is launched by a change in redox state, followed by calcium signals, leading to changes in sugar and hormonal distribution. To minimize prediction bias, we expanded the survey to 52 rice varieties and observed that all hub genes are linked to root bending irrespective of genotype. We further designed an electronic AIU (Artificial Intelligence Unit) simulating the root. Interestingly, the AIU suggests a 3-compartment processing unit, fitting the 3-hub transcriptional archetype. We propose that a command center is active for growth reprogramming in the primary root tip 3-5 days post-germination. This

center is functional even in absence of the embryonic shoot but loses capacity if the root is decapitated. Our results redefine the debated “Root-Brain” hypothesis (Darwin and Darwin, 1880) and characterize the neural network behind it.

1000-006-Z Using Genetic Approaches to Study Molecular Response to Cadaverine in Arabidopsis Thaliana Nicole Gibbs – University of Wisconsin-Madison Wolfgang Busch – Gregor Mendel Institute, Amy Jancewicz – University of Wisconsin-Madison, Patrick Masson – University of Wisconsin-Madison Polyamines are small organic bases that contribute to the regulation of growth and development through their interactions with many cellular components. Cadaverine, a diamine, modulates root architecture and has been proposed to function in heat and salt stress in plants through an unknown mechanism. To identify genes involved in cadaverine response, a forward genetic screen and genome-wide association study (GWAS) were carried out in Arabidopsis thaliana. Thirteen candidate cadaverine response mutants identified through the forward genetic screen are being characterized for specificity to cadaverine, potential contribution to cadaverine homeostasis, transport and/or response, and for their possible involvement in stress response. Causative mutations are being identified using bulked segregant analysis coupled with next-generation genome sequencing. For the genome-wide association study, 190 Arabidopsis accessions were screened to identify Single Nucleotide Polymorphisms (SNPs) associated with cadaverine response of sixteen root-growth traits. Significant associations were found for root length, root width and root angle, and the loci responsible for 11 of these associations are being investigated using a combination of expression studies and reverse genetics. A preliminary reverse genetic analysis of an association on chromosome 5 has identified a locus involved in cadaverine and stress response. Identified candidate genes will be genetically and molecularly characterized to determine function and potential involvement in stress response.

1000-007-Z The Other Abiotic Factor: Mechanical Stresses and Tissue Failure Douglas Cook – New York University Abu Dhabi Margaret Julias – New York University Abu Dhabi, Daniel Robertson – New York University Abu Dhabi Mechanical stresses act on all plants and include gravitational forces, turgor pressure, resistance of the soil to root growth, and wind loading, among others. This study examines mechanical stresses which cause failure (breakage) of septated stalks. At the level of gross physiology, maize stalks have been observed to exhibit remarkably consistent failure patterns in terms of type, location of failure initiation, and failure direction relative to the stalk physiology. The failure of maize stalks appears to be of the buckling type, and two models of failure have been proposed. In the first model, tissue failure leads to radical geometric deformation of the stalk. In the second model this causality is reversed, with geometric deformation causing tissue failure. The primary objective of this study was to resolve which of these two failure initiation models is more accurate, and thereby determine whether tissue failure or tissue deformation is the source of failure initiation. As all Poaceae plants exhibit similar structures, resolution of this question is expected to have many implications for the improvement of stalk strength. To address this question, maize stalks were subjected to three-point bending tests. A multi-modality imaging study was performed, involving high-speed imaging to visualize the failure process, digital image correlation to assess strain patterns, force measurement to assess the progression of stalk strength, scanning electron microscopy to examine failure patterns at the microscopic scale, and high resolution computed tomography scanning to assess threedimensional structural changes before and after failure. Preliminary data suggests that localized tissue failure causes a

cascade of tissue failure which then causes buckling failure of the entire stalk. This information suggests that increasing the resistance of stalk tissues to compressive stresses may be a new approach for developing stronger maize stalks.

1000-008-Y Long-Distance Signaling in Plants: Characterizing the Function of Lipid-Binding Proteins and Their Response to Abiotic Stress in the Phloem Allison Barbaglia – Michigan State University Banita Tamot – Michigan State University, Veronica Greve – Michigan State University, Olena Tetyuk – Michigan State University, Urs Benning – Michigan State University, Susanne Hoffmann-Benning – Michigan State University Unlike animals, plants cannot escape the unpredictability of their environment. To cope, they developed detection, signaling, and response mechanisms to adapt to environmental stresses. Even though many of the signals that travel through the plant are local, others require the need of phloem-mediated long-distance transport. We have identified phloem-localized lipids and their protein partners, indicating a role in long-distance signaling. These proteins could function in lipid transport/signaling by: releasing the lipid from the membrane, binding and transporting the lipid, acting as a receptor, or acting as the signal itself (Benning et al., 2012). Gaining insight on how the phloem regulates this signal transport is vital for understanding how plants respond to changes induced by their surroundings. We are focusing on three lipid-binding proteins: a putative GDSL-lipase that may release lipids into the phloem; PIG-P-like protein, a potential receptor with a predicted role in GPI-anchor synthesis; and PLAFP, a lipid-binding protein with unknown function. Previously, we have shown each protein localizes to the cell periphery, responds to stress, and bind lipids. Interestingly, PLAFP and PIG-P bind the same lipid, phosphatidic acid (PA). PLAFP is induced by ABA and PEG, a drought mimic. PA is a well-known secondary signal involved in drought/ABA response. This suggests that PLAFP-PA acts in the ABA signaling path. Given their localization within the phloem, it is possible that they could function in long-distance signaling as well. Funding: NSF-IOS grant #1144391 to SHB

1000-009-Y AtMBP-1 Affects Plant Development and ABA Responses and Is Regulated by AtSAP5 Miyoung Kang – Oklahoma State University Haggag Abdelmageed – Oklahoma State University, Randy Allen – Oklahoma State University Arabidopsis LOS2 is a bi-functional gene that encodes both enolase and the transcriptional repressor, AtMBP-1. Plants that over-express AtMBP-1 show reduced growth and delayed flowering. These developmental characteristics are correlated with increased ABA responsive gene expression. AtMBP-1 expressing plants show hypersensitivity to ABA and ectopic activation of ABA signaling pathways. The stability of AtMBP-1 is negatively regulated by AtSAP5 through the ubiquitin-dependent proteasome pathway and destabilization of AtMBP-1 is promoted by ABA treatment. Coexpression of AtMBP1 and AtSAP5 prevents AtMBP1 accumulation and reverses the dwarf phenotype associated with AtMBP-1 over-expression. AtMBP-1 down-regulates the expression of the STZ/ZAT10 and has been shown to interact with a cMyc promoter-like sequence element in the STZ/ZAT10 promoter. Binding of AtMBP1 to the STZ/ ZAT10 promoter in vivo was confirmed by chromatin immunoprecipitation assays. Thus, AtMBP-1 appears to be regulated at the post-translational level and affects stress responses through directly down-regulating the expression of target genes, including STZ/ZAT10.

1000-010-Z Identification of Novel Transcriptional Regulators of Zat12 Using Promoter-based Comparative Yeast Onehybrid Screens Gad Miller – Bar-Ilan University Bat-Hen Ben-Daniel – Chaim Wachtel, Yair Glick Reactive oxygen species (ROS) are toxic byproducts of aerobic metabolism as well as important signals involved in the regulation of many different cellular processes, including growth, development, stress responses and death. It is unknown how cells resolve the conflict between this dual function of ROS, and much of the regulatory components mediating its signaling cascades, particularly during stress, await identification. To identify ROS-responsive transcriptional regulators we targeted factors involved in Zat12. Zat12 is a C2H2 zinc finger family protein that is rapidly upregulated in response to a wide range of abiotic stresses and to ROS, both locally and systemically. For this end plants expressing luciferase under successive deletions in Zat12 promoter were used parallel with comparative yeast-one-hybrid (Y1H) screens using corresponding promoter segments as bait against control and stress prey cDNA-fusion libraries. Promoter analysis underlined ca. 200 bp fragments responding to ROS or salt stress, and the Y1H screens identified 15 known as well as novel potential Zat12-regulators. Knockout mutants for several of the identified gens showed altered expression of Zat12 and altered phenotypic response to oxidative stress, supporting a role for their involvement in it regulation. The protein of selected five potential Zat12 transcriptional regulators were further used in DNA array binding screens using a novel microfluidity technology. Obtained results verified in high confidence not only the ability of these genes’ products to specifically bind dsDNA but also retrieved their potential core target site. These results implicate several genes of unknown function in transcriptional regulation in general and more specifically in regulation of Zat12 and stress response. Our data could be valuable for future charting of the earliest signal transduction gene network responding to abiotic stress and to ROS that leads to Zat12 and other early responding genes.

1000-011-Z Fate, Toxicity and Transport of Engineered Metal- Based Nanoparticles in Crops and Their Impact on Global Food Production Om P. Dhankher – University of Massachusetts Amherst, Chaunxin Ma – University of Massachusetts Amherst, Baoshan Xing – University of Massachusetts Amherst, Jason White – Connecticut Agri. Experimental Station Widespread use of metal-based nanoparticles (NPs) such as oxides of silver (Ag), titanium (Ti), zinc (Zn), and rare earth elements (REE) including cerium (Ce) and Indium (In) in the environment has raised some serious concerns about their adverse effects on the agriculture and environmental and human health. In this study, the effects of these NPs exposure on several plants including arabidopsis, rice and soybean wereinvestigated at the physiological, biochemical, and molecular levels. Our results demonstrate that most of these NPs induce severe oxidative stress and causes significant membrane damage. The levels of malondialdehyde (MDA), an important indicator for membrane damage, and antioxidant enzymes such as SOD, CAT, APX, PPO and POD that scavenge ROS under stress conditions were also significantly higher compared the untreated plants. We also evaluated the effect of Ag and Ce NPs exposure on N fixation and nutrient displacement in soybean. To our surprise, even a very low level of Ag NPs exposure severely reduced the nodule formation in roots, N fixation, and plant growth. Exposure of plants to Ag and other metal oxides NPs also inhibited Fe and other nutrient element uptake in plants. Additionally, we explored the role of glutathione (GSH) in ameliorating the NP-induced stress in plants. Transgenic Arabidopsis and Crambe abyssinica plants

overexpessing gamma-glutamylecysteine synthase (g-ECS) produce several-fold higher levels of GSH. Transgenic lines, when exposed to Ag NPs, showed enhanced tolerance to NPs toxicity as these line grew better and had higher biomass, root length, transpiration rate compared to control plants. Our results showed that metal-based NPs could cause significant toxicity in crops and can affect crop yield. These studies will be help in understanding the fate, transport, and toxicity of NPs in the agricultural crops and to further develop strategies for mitigating the toxicity of these NPs in food crops.

1000-012-Y Comparative Transcriptome and Proteome Profiling of Arabidopsis to Reveal the Au Nanoparticle Biosynthesis Manish Tiwari – Department of Biology, WKU Sneha Krishnamurthy – Department of Biology, WKU, Devesh Shukla – Department of Biology, WKU, Nilesh Sharma – Department of Biology, WKU, Shivendra Sahi – Department of Biology, WKU The fabrication of Au nanoparticles is a subject of extensive research over past years due to wide industrial applications including photovoltaics, sensory probes, therapeutic agents, and in targeted drug delivery. The use of plants and plants based methods for such purpose seem more favorable than conventional chemical methods which produces negative environmental impacts. Despite the potential of using plants, a limited knowledge available about underlying biochemical reactions and genes involved in the process. In this study, we have investigated the effect of Au on seedling growth, root system architecture, and transcriptome, proteome and ionome shift in Arabidopsis. Au exposure alters the root system architecture and increased the primary, higher order roots and shoot area, at the same time optical and hyperspectral images clearly revealed the formation of Au nanoparticles in root and shoot. The transcriptome analysis of root shows differential expressions of several members of WRKY, MYB, BHLH gene family and many genes involved in Fe deficiency and homeostasis. Similar to transcripts expression, the members of GST, metal binding protein and redox related proteins were also induced in proteome studies showing a crucial role of GSTs. In contrast to Fe, the level of Zn, Mg, Ca, and Cu were raised in roots during the presence of Au, whereas the level all these metal reduced in shoot compared to control. Altogether, present study describes the Au crosstalk with other nutrients that shaped the root architecture, and identified some promising genes for having their role in Au nanoparticles biosynthesis.

1000-013-Y CaARP, a Pathogenesis Related-10 Protein Functions as Aldo/keto Reductase to Scavenge Cytotoxic Aldehydes Deepti Jain – Delhi University – South Campus Hitaishi Khandal – National Institue of Plant Genome Research, Jitendra Khurana – Delhi University – South Campus, Debasis Chattopadhyay – National Institue of Plant Genome Research Pathogenesis-related (PR) proteins represent a part of the multicomponent defense signalling mechanism in plants and often induced in response to a variety of stresses. PR proteins are present as multigene family and among them PR-10 family consists of low molecular weight (16-19 kDa) proteins. PR-10 proteins were first identified for their expression during pathogenesis but many PR-10 proteins have been reported for their constitutive expression and accumulation in response to various external stimuli. Although PR-10 proteins have been reported throughout the plant kingdom; however their biological function is poorly understood. Through an analysis of drought-induced changes in chickpea transcriptome, we identified a PR-10 gene (CaARP) whose transcript accumulated in response to drought, salt, ABA and oxidative stresses. Interestingly, CaARP showed the presence of conserved motifs corresponding to catalytic signature of aldo/keto reductase (AKR) of short-chain dehydrogenase/reductase (SDR) superfamily. Heterologous expression of CaARP in bacteria, yeast and plant displayed AKR activity with different aromatic and aliphatic aldehydes including

methylglyoxal (MG) and malondialdehyde (MDA) as substrates. CaARP also showed protective role against treatment with salt, oxidative stress or cytotoxic aldehydes in bacteria, yeast and plant. Arabidopsis plants expressing CaARP showed low MDA content in presence or absence of stress suggesting that the protein might work as a scavenging enzyme for cytotoxic aldehydes produced from lipid peroxidation. Additionally, CaARP was able to discriminate between reduced and oxidized form of NADP and showed structural change upon binding with NADPH. Since, PR-10 family members have been identified in a variety of angiosperms, monocots and dicots so we compared sequences of PR-10 proteins in land plants and found that most of them possess conserved motifs corresponding to catalytic signature of AKR of SDR superfamily. Our findings are thus discussed within the context of hypothesizing that PR-10 proteins act as cytotoxic aldehyde scavenger and redox-sensors.

1000-014-Z Linking Stress, Sugar Metabolism, and Nuclear Signaling in Maize: Ndpk1 (Nucleoside Diphosphate Kinase 1) Maria Angelica Sanclemente – University of Florida Kelsey Wyman – University of Florida, Wayne Avigne – University of Florida, Isabel Branstrom – University of Florida, Karen Koch – University of Florida Plant development, sugar metabolism, and tolerance to abiotic stresses depend on balanced ratios of ATP/ADP and other nucleotide phosphates (e.g. GTP/GDP, UTP/UDP). This balance is mediated by nucleoside diphosphate kinase (NDPK) which uses ATP to phosphorylate nucleoside diphosphates (e.g. GDP) producing the corresponding nucleotide triphosphate (e.g.GTP). In addition, the maize NDPK1 (ZmNDPK1) isoform has the capacity to bind non-canonical arrangements of DNA such as G4 quadruplexes. These structures are formed by stacking of guanine-base quartets in a quadruple helix. Sequences that form G4s are prevalent in the 5’ UTRs of key genes for sugar and stress responses, suggesting a potential role for NDPK in their modulation. Previous data indicate that Ndpk mRNA levels rise in seedlings growing under hypoxia. Oxygen levels can potentially alter carbohydrate metabolism, sugar signaling and energy balance. The objective of the work reported here was thus to test the capacity of ZmNdpk1 to respond to sugar levels and oxygen stress. We did this by subjecting root tips from 5-d-old seedlings to two oxygen treatments (aerobic [20% O2] or anaerobic [0.2% O2] in combination with two glucose levels (physiologically abundant [2% Gluc] and low [0.2% Gluc]). Two oxygen-responsive alcohol dehydrogenase genes (Adh1 and Adh2) were used as indicators of oxygen deficiency in the root tissues. After 24h, Ndpk1 mRNA levels were up-regulated in response to glucose abundance regardless of the oxygen treatment. Similarly, both Adh1 and Adh2 were up-regulated by elevated glucose levels in both aerobic and anaerobic conditions. However, this increase was greater under low-oxygen conditions, as expected from their role in hypoxic responses. Results are consistent with proposed roles of NDPK1 in sugar metabolism of developing tissues with high energy demands. In addition, sugar modulation of Ndpk1 and Adh1 suggest overlapping responses to sugar signals and conditions affecting metabolic balance.

1000-015-Z Different Mechanisms Modulate the Transport Activity of the MATE and ALMT-type Transporters Involved in Plant Aluminum Resistance Miguel Pineros – USDA-ARS Leon Kochian – USDA-ARS Members of the ALMT (Al-activated malate transporter) and MATE (multidrug and toxin efflux) families confer plant aluminum resistance on acid soils by mediating organic acid (OA) anion efflux, thereby immobilizing toxic aluminum (Al3+) ions at the root surface. Although similar in function, their structure and regulation differ significantly. We have integrated electrophysiological analysis with cellular imaging approaches to conduct a structural-functional analysis aimed at determining the transporters’ topology, stoichiometry, function and regulation. Electrophysiological (TEVC of X.

oocytes) analysis of various structurally altered transporters indicated that the large enhancement of ALMT transport activity by extracellular Al3+ is the result of an intrinsic regulatory mechanism that involves the interaction of the intracellular N and C-terminus regions of the ALMT protein. In contrast, MATE transporters mediate a constitutive and Al3+-insensitive OA transport, suggesting that their transport activity is modulated by additional cellular mechanisms associated with an upstream Al3+ signaling cascade. Results from electrophysiological and BiFC analysis demonstrated that calcineurin B-like (CBL5)/protein kinase (CIPK2)-mediated protein phosphorylation down regulates AtMATE1mediated citrate efflux, thereby constituting a Ca2+-regulated pathway which minimizes carbon loss (OA exudation), and regulates Al resistance both temporally and spatially as the root grows through the acid soil horizons. These studies provide a platform for better understanding the underlying transport properties critical for Al resistance, with the ultimate goal of "engineering" these transporters to enhance their ability to confer Al resistance in crop plants grown on acid soils.

1000-016-Y Ectopic Expression of Plant and Animal Peroxiredoxin Genes in Arabidopsis Plants Takashi Ueda – Florida Gulf Coast University Ryan Humphries – Florida Gulf Coast University, David Shepard – Florida Gulf Coast University, Emily Ngo – Florida Gulf Coast University, Nina Infantado – Florida Gulf Coast University, Julie Van Horn – Florida Gulf Coast University Peroxiredoxins (Prxs) are a family of ubiquitous proteins that help minimize the harmful effects of oxidative stress catalyzing the reduction of hydrogen peroxide (H2O2) and organic hydroperoxides. Full-length cDNA’s corresponding to a 1-Cys and 2-Cys Prx genes were isolated from buckwheat (Fagopyrum esculentum) and the flatback mud crab (Eurypanopeus depressus), respectively. The buckwheat 1-Cys Prx gene, designated as FePer1, is proposed to play important roles both in desiccation tolerance of embryos during seed development. The crab 2-Cys Prx gene, designated as EdPrx-1, is expressed at low level in the gill, hypodermis, and hepatopancreas tissues of the crab under non-stressed condition. However, its expression is elevated in the gills under dilute-salinity stress condition, suggesting a possible role in protection against oxidative stress caused by the increased metabolic activities associated with hyperosmoregulation. The goal of this project is to express the two Prx genes ectopically in a transgenic Arabidopsis thaliana plants to assess their effects on the plants with respect to their stress tolerance. Preliminary results will be presented.

1000-017-Y Salt Stress and Oxidative Stress in Boechera, a Comparative Study Michael Armstrong – San Diego State University Elizabeth Waters – San Diego State University In this study, supported by an ASPB SURF award, we have examined the abiotic stress tolerance of four Boechera species and have compared their stress responses to that of their relative Arabidopsis thaliana. Boechera (Brassicaceae) is a genus of over 100 species that native to North America. There are many Boechera species native to California and these species grow in a wide variety of habitats from the dry hot Mojave dessert to the much cooler and wetter Sierra Nevada Mountains. The four Boechera species studied are: B. arcuata, B. californica, B. depauperata, B. perennans. Boechera arcuata is found in coastal to inland areas. B. californica and B. perennans are both found in chaparral and desert regions. B. depauperata is a high altitude species found in the Sierra Nevada Mountains. Previously we have found significant variation among these species for tolerance to heat stress. In this study we examined both tolerance to salt and oxidative stress. Tolerance to salt stress was measured by examining the impact of salt on seed germination. Seeds were germinated with and with out the presence of salt (0, 25, 50, 100 mM NaCl). The results of these experiments indicated that B. depauperata is extremely sensitive to salt stress. The other four Boechera species and A. thaliana (Col) had similar salt stress responses. In order to examine oxidative stress we exposed 7-10 day old Boechera and A. thaliana

(Col) seedlings to Methyl Viologen (0, 25, 50 100 mM) for 24 hours. Response to oxidative stress was monitored by examining chlorophyll fluorescence and ion leakage. We have found variation among the species for tolerance to oxidative stress.

1000-018-Z A Mass Spectrometry-based Untargeted Metabolomics Study of Abiotic Stress Induced Whole Plant Metabolic Changes in Arabidopsis Thaliana Yuan Xu – University of Minnesota Dana Freund – University of Minnesota, Adrian Hegeman – University of Minnesota, Jerry Cohen – University of Minnesota Plant abiotic stress responses lead to modified gene expression and resulting in the changes of metabolism, the direct signature of biochemical activity. A mass spectrometry-based untargeted metabolomics approach was used to study whole plant metabolic changes in Arabidopsis thaliana that were induced by different abiotic stresses. Multiple abiotic stresses, including heat, cold, drought, and high light, were tested in a comparative metabolomics study such that the common and unique metabolic features of the different abiotic stress induced metabolic changes could be clearly distinguished. Arabidopsis thaliana wild type seeds were grown vertically on agar plates at 22°C under a 16-h-light/8-hdark photoperiod of 80 μmolm-2s-1 cool-white fluorescent for 11 days. 11-day-old seedlings were treated with abiotic stresses including basal heat stress (45°C for 5h), acquired heat stress (38°C for 1.5h , 22°C for 2h, 45°C for 5h), basal cold stress (3°C for 3h), acquired cold stress (3°C for 3h, -20°C for 1h), drought stress (desiccation for 2h), and high light stress (902 μmolm-2s-1 high light for 1h). Each stress group has a corresponding recovery group for plants to be recovered for 2 days after the stress treatment. Metabolic profiles of control, stress groups, and stress recovery groups were acquired using ultra performance liquid chromatography mass spectrometry (UPLC-MS). Thousands of metabolic features (m/z, retention time, intensity) were analyzed by SIEVE™ software (Thermo Scientific) for PCA and single metabolite t-test analyses. Hundreds of metabolites were significantly altered in stress groups or recovery groups compared with the control group (p-value < 0.05 from t-test). Among them, 30 metabolites of interests were confidently identified by authentic standards. These metabolites of interests include amino acids, TCA cycle intermediates, sugars, and other plant metabolites, indicating that aspects of their metabolism are modified by exposure to different abiotic stress conditions.

1000-019-Z Phenotyping (screening) for Heat (and Drought) Tolerance in Bean (Phaseolus Spp.) Using New and Conventional Fluorescence and Gas Exchange Parameters Wayne Loescher – Michigan State University Jesse Traub – Michigan State University, Muhammad Naeem – Michigan State University, James Kelly – Michigan State University, Greg Austic – Michigan State University, David Kramer – Michigan State University Drought and heat tolerance are both increasingly important considerations in agriculture and plant breeding. As the global climate warms, more agricultural areas around the world will experience not only more incidences of heat stress but of drought stress as well. To develop new varieties tolerant to these stresses, plant breeders will need to conduct extensive screens for these traits in released cultivars, breeding lines, and older landrace varieties. While developed varieties ultimately will need to be evaluated in the field, the variability of weather patterns from year to year makes it difficult to replicate or even induce heat or drought stress in the field. Thus, screening and phenotyping studies in more controlled greenhouse and growth chamber settings can serve to supplement studies in the field. Heat stress especially can be easily and reproducibly imposed in controlled environments and can thus function as a general stress test for screening purposes. This is particularly important for our model system, the common bean (Phaseolus vulgaris L.), which

is not only the most widely human consumed legume, but also an important protein source in the developing world where heat and drought stresses are common. Accordingly, we have been testing both new and advanced technologies to assess their applicability to identifying the effects of heat and drought on diverse bean genotypes. To date, our results show how new instrumentation and certain photosynthetic parameters1 (efficiency, photoprotection, and photoinhibition) along with gas exchange measurements (CO2 uptake and stomatal conductance) can quickly, effectively, and efficiently demonstrate real differences in heat and drought tolerance. 1Baker, N., Harbinson, J., & Kramer, D. M. (2007) Plant, Cell & Environment 30, 1107-1125.

1000-020-Y Inositol Phosphate and Energy Sensing in Plants Glenda Gillaspy – Virginia Tech Sarah Williams – Virginia Tech, Olusegun Adepoju – Virginia Tech, Eric Land – North Carolina State University, Janet Donahue – Virginia Tech, Imara Perera – North Carolina State University Myo-inositol phosphates (InsPs) are critical signaling molecules used by eukaryotes, with roles in nutrient, energy, and abiotic stress signaling. A molecule called Ins(1,4,5)P3 has been extensively studied with respect to it’s involvement in ABA signaling in plants. One controversy within the InsP field has centered around whether InsP6 is the bonafide signaling molecule (i.e. second messenger) in the pathway, with Ins(1,4,5)P3 acting only as a precursor. Our work examines yet another potential second messenger from this pathway, the inositol pyrophosphates. Inositol pyrophosphates contain diphospho- or triphospho- (PPx) moieties at one or more positions on the inositol ring, resulting in seven or even eight phosphates (InsP7 and InsP8). We have recently shown that plants accumulate InsP7 and InsP8 in several different tissues. In addition, we have identified two highly similar genes, AtVIP1 and AtVIP2, that allow for InsP7 accumulation in yeast mutants incapable of synthesizing InsP7. Mutants lacking expression of both AtVIP1 and AtVIP2 genes have been identified and are viable. Preliminary data indicate these mutants have defects in responses to energy conditions. Work is on-going to determine whether these mutants have altered InsP and PPx-InsP levels. Two mutants have been isolated that have elevated InsP7 and InsP8 levels, and these mutants are also being tested for changes in energy sensing.

1000-021-Y Characterization of Drought- and Heat-responsive MicroRNAs in Switchgrass Chandra Obul Reddy Puli Vandana Hivrale – Oklahoma State University, Yun Zheng – Faculty of Life Science and Technology Kunming University of Science and Technology, Guru Jagadeeswaran – Oklahoma State University, Gopal Kakani – Oklahoma State University, Abdelali Barakat – Univeristy of South Dakota, Ramanjulu Sunkar – Oklahoma State University Switchgrass has emerged as one of the important biofuel crops due to its ability to produce biomass on marginal lands and withstand hot and dry summers. Although switchgrass is relatively drought- or heat-tolerant but the molecular basis of such tolerance is poorly understood. Recent investigations revealed that microRNAs (miRNAs) play a crucial role in plant acclimation to stress conditions. To identify miRNAs that might be important for tolerating drought or heat small RNAs from leaves of heat or drought-exposed switchgrass were sequenced using Illumina platform. Sequence analysis enabled the identification of 29 conserved miRNA families and 62 novel miRNA families. Notably, the abundances of several conserved and novel miRNAs were dramatically altered following drought or heat. Using at least one fold (log2) cut off, we observed that 13 conserved miRNA families were differentially regulated by both stress treatments. Additionally, five and four conserved miRNA families were specifically regulated by drought and heat, respectively. Similarly, using a more stringent cut off of two fold (log2) regulation, we found 5 and 16 novel families were upregulated

but 6 and 7 novel miRNA families were downregulated under drought and heat, respectively. Both conserved and novel miRNA regulation during drought or heat is overlapping as well as distinct. The stress-altered expression of a subset of miRNAs and their targets was validated using semi quantitative RT-PCR. Thus, the switchgrass plants exposed to drought or heat revealed similarities as well as differences with respect to miRNA regulation, which could be important for enduring stress conditions.

1000-022-Z Isolation and Characterization of Endophytic Bacteria from a Naturally Occurring Uranium-tolerant Species, Prosopis Juliflora and Their Potential Use for Phytoremediation of Uranium Sravani Kunduru – Yogi Vemana University Chandra Obul Reddy Puli – Yogi Vemana University, Jayanna Naik B – Yogi Vemana University, Krishna Kumar G – Yogi Vemana University, Suresh Raju K – Yogi Vemana University, Chandra Sekhar Akila – Yogi Vemana University, Ramachandra Reddy Arjula – University of Hyderabad, Ramanjulu Sunkar – Oklahoma State University Plant associated endophytic bacteria plays a pivotal role in improving the efficiency of phytoremediation process. Although the remediation of uranium contaminated soils using microorganisms was well studied, the role of endophyte symbiotic bacteria in phytoremediation of uranium was poorly understood. In this study, seasonal surveys carried out to identify the flora at the Tummalapalle uranium mine tailings of India, Honey Mesquite (Prosopis juliflora (SW.DC), a hardy plant, was identified as candidate species for phytoremediation of uranium contaminated soils. Further, to explore the role of endophytic bacteria in uranium accumulation, three endophytic bacterial strains were isolated and characterized from the root tissues of Prosopis grown on uranium mine tailings. The three isolates were identified as Bacillus amyloliquefaciens (PJ-4); Stenotrophomonas maltophilia (PJ-6) and Pseudomonas sp. (PJ-12) based on the 16S rDNA gene sequence analysis. The subsequent studies revealed that they all exhibited tolerance to uranium metal up to 150 mg L-1,resistance to various antibiotics and could produce siderophores. Atomic force microscopy studies displayed that bacterial cell surface was not affected by the uranium toxicity. Further, XRD and FTIR analysis revealed the involvement of carboxyl, amide and phosphate groups of bacterial cell wall in metal uptake process. The SEM-EDX analysis suggested that the metal might have sequestered into the cell cytoplasm by displacing cellular potassium through ion exchange. The TEM image analysis further revealed, uranium metal complex was initially deposited in the periplasmic-membrane-cell wall and subsequently sequestered into the intracellular components. Further, plant inoculation studies of these isolates both in pure and consortium to the non-host plant Brassica juncea, significantly enhanced the accumulation of uranium with improved growth. The results suggests that, endophytes from a natural uranium tolerant plants are a most promising resource and may be the potential candidates of bio-inoculants for enhancing the phytoremediation efficiency of uranium.

1000-023-Z The Arabidopsis Selenoprotein O-Like Is a Novel Chlorplast Kinase Involved in Oxidative Stress Response Aviah Zilberstein Yosef Fichman – Tel Aviv University, Zsuzsanna Koncz – Max-Planck Institute For Plant Breeding Research, Noam Reznik – Tel Aviv University, Csaba Koncz – Max-Planck Institute For Plant breeding Research, Hillel Fromm – Tel Aviv University A forward genetic screen for mutations affecting stress-induced silencing of PROLINE DEHYDROGENASE 1 (PRODH1) in Arabidopsis was applied to identify novel genes involved in abiotic stress responses. A T-DNA knockout mutant of the SELENOPROTEIN O-LIKE (SELO) gene showed enhanced drought tolerance and reduced levels of hydrogen peroxide. AtSELO- GFP was localized to the chloroplasts and its overexpression conferred increased sensitivity to water shortage.

AtSELO shares conserved SEC-like motif (SCSS) with orthologous SELOs from diverse organisms, some of them contain selenocysteine (more nucleophilic than cysteine) in the SCSS motif, and are associated with redox activities. Genetic complementation assays performed in yeast selo mutant with yeast ScSELO ortholog (FMP40) correlated changes in ROS levels with the presence of SEC-like domain, indicating involvement of this motif in monitoring cellular ROS levels. Another set of motifs identified in human HsSELO corresponds to an atypical kinase domain (Dudkiewicz et al., 2012). ATP-binding and kinase assays performed with a biotin labelled cross-linkable ATP analogue and 32P-γ-ATP, respectively, demonstrated that AtSELO can bind ATP and undergo autophosphorylation in vitro. Both full length and SCSS-deleted forms of AtSELO showed autophosphorylation, indicating that SCSS is not essential for the kinase activity. Currently, the AtSELO kinase motifs are being studied by site-directed mutagenesis, and protein interactions are examined to better understand how this novel chloroplast kinase is involved in modulating oxidative/redox responses. Dudkiewicz et al. (2012) PLoS ONE 7: e32138.

1000-024-Y Revealing a Topsy-turvy Relationship Between Ribosome Abundance and Plant Growth Using Arabidopsis Thaliana Natural Variation Hirofumi Ishihara – Max Planck Institute of Molecular Plant Physiology Eva-Theresa Pyl – Max-Planck-Institute of Molecular Plant Physiology, Waltraud Schulze – University of Hohenheim, Toshihiro Obata – Max-Planck-Institute of Molecular Plant Physiology, Andre Scheffel – Max-Planck-Institute of Molecular Plant Physiology, Alisdair Fernie – Max-Planck-Institute of Molecular Plant Physiology, Ronan Sulpice – National University of ireland Galway, Mark Stitt – Max-Planck-Institute of Molecular Plant Physiology, Ribosomes are the machinery in which proteins are translated. It is known that when some animals and bacteria are grown in optimal conditions, ribosome abundance is positively correlated with growth rate. At the same time production of ribosomes as well as increased protein synthesis and degradation require a huge investment of energy and resources. In plants, all energy and materials that are needed for the production and utilization of ribosomes come from photosynthesis, and are therefore limited especially in the night. We have hypothesized that if plants could regulate their ribosome pool efficiently they would grow faster. To find this out, we investigated ribosome abundance in 20 Arabidopsis thaliana accessions which differ in their growth rates. The total abundance of ribosomes and the amounts loaded in polysomes are negatively correlated with growth specifically at the end of the night. A new method was developed to measure the global rates of protein synthesis and degradation in soil-grown Arabidopsis plants. The results indicated that higher rates of protein synthesis in the small accessions allow maintenance of higher protein degradation but at the cost of a lower energetic efficiency of growth. These results point to a basic trade-off between protein synthesis/degradation and maximization of growth rate that these slower-growing accessions show more robustness and flexibility in responses to environmental conditions.

1000-025-Y Dual Expression of Colocasia Esculenta Metallothionein and AtPCS1 Increased Metal Tolerance Yeonok Kim – Chonnam University Hunseung Kang – Chonnam University Metallothioneins (MTs) and phytochelatin (PC) are typical metal-binding proteins that regulate the homeostasis and detoxification of metals in plant. Experimental evidences about correlational roles between MT and PC has been limited thus far. To investigate the biological role of Colocasia esculenta MT (CeMT2b) and Arabidopsis phytochelatin synthase (AtPCS1), we generated each transgenic plant and double gene overexpressors (CeMT-AtPCS). Under Cd stress, CeMT2b transgenic arabidopsis displayed much better seedling growth than the wild type. Furthermore, CeMT-AtPCS showed

better seedling growth than CeMT2b. Under Cu stress, much better seedling growth in CeMT2b and CeMT-AtPCS was investigated comparing to wild type. But the Cu tolerance of CeMT-AtPCS was similar with CeMT2b. Cd accumulation and total nonprotein thiol content in CeMT-AtPCS was higher than CeMT2b and wild type.A ccordingly, current results indicates that dual expression of MT and PCS is good strategy for phytoremediation and CeMT-AtPCS would be ideal candidate for phytoremediation.

1000-026-Z Variation in Yield Loss to Ozone of Diverse Inbred and Hybrid Maize Lines Andrew Leakey Gorka Erice, Tiago Tomaz, Lorena Rios-Acosta, Christopher Montes, Anna Molineaux, Ines Resano, Crystal Sorgini, Craig Yendrek, Alison Morse, Linda Young, Patrick Brown, Lauren McIntyre, Elizabeth Ainsworth Tropospheric ozone is an air pollutant that costs ~$14-26 billion in global crop losses and is projected to worsen in the future. Fifty-two inbred lines, including the nested association mapping (NAM) population founder lines, and 26 hybrids made from crossing B73 to the NAM founders and Mo17 were tested for ozone sensitivity under ambient (40 ppb) versus elevatedozone concentrations (100 ppb) at the Free Air Concentration Enrichment (FACE) site in Illinois in 2014. Across all inbred genotypes, total ear mass was 7% lower at elevated ozone. However, there was significant genetic variation in response with yield loss ranging from 0% in tolerant genotypes to 76 % in the most sensitive genotype. Likewise, yield across hybrid genotypes averaged 9 %, but varied from 0 – 26 %. Notably, maize reference line B73 was insensitive to growth at elevated ozone whereas Mo17 showed significantly lower ear mass, delayed silking and lower ear height. In addition, yield loss of 17 % was observed in hybrid B73 x Mo17. These results suggest that the extensive germplasm resources available for quantitative genetic analysis of phenotypic variation in B73 and Mo17 will facilitate investigation of oxidative stress tolerance in maize. Ultimately, the variation in yield loss to elevated ozone among genotypes has the potential to be exploited to improve the stress tolerance of maize. Future work includes completing the analysis of yield components, the assessment of key vegetative and reproductive traits and to determine the transcriptional responses and genetic loci associated with ozone sensitivity.

1000-027-Z Different Mechanisms Modulate the Transport Activity of the MATE and ALMT-type Transporters Involved in Plant Aluminum Miguel Piñeros – USDA-ARS Members of the ALMT (Al-activated malate transporter) and MATE (multidrug and toxin efflux) families confer plant aluminum resistance on acid soils by mediating organic acid (OA) anion efflux, thereby immobilizing toxic aluminum (Al3+) ions at the root surface. Although similar in function, their structure and regulation differ significantly. We have integrated electrophysiological analysis with cellular imaging approaches to conduct a structural-functional analysis aimed at determining the transporters’ topology, stoichiometry, function and regulation. Electrophysiological (TEVC of X. oocytes) analysis of various structurally altered transporters indicated that the large enhancement of ALMT transport activity by extracellular Al3+ is the result of an intrinsic regulatory mechanism that involves the interaction of the intracellular N and C-terminus regions of the ALMT protein. In contrast, MATE transporters mediate a constitutive and Al3+-insensitive OA transport, suggesting that their transport activity is modulated by additional cellular mechanisms associated with an upstream Al3+signaling cascade. Results from electrophysiological and BiFC analysis demonstrated that calcineurin B-like (CBL5)/protein kinase (CIPK2)-mediated protein phosphorylation down regulates AtMATE1mediated citrate efflux, thereby constituting a Ca2+-regulated pathway which minimizes carbon loss (OA exudation), and regulates Al resistance both temporally and spatially as the root grows through the acid soil horizons. These studies provide a platform for better understanding the underlying transport properties critical for Al resistance, with the

ultimate goal of "engineering" these transporters to enhance their ability to confer Al resistance in crop plants grown on acid soils.

Abiotic: Light 1000-028-Y HEMERA Is a Transcriptional Coactivator That Couples the Proteolysis and Transcriptional Activity of Phytochrome-Interacting Factors Meng Chen – Duke University Yongjian Qiu – Duke University, Meina Li – Duke University, Elise Pasoreck – Duke University, Lingyun Long – Duke University, Yiting Shi – Duke University, Rafaelo Galvão – Duke University, Conrad Chou – Duke University, He Wang – Duke University, Amanda Sun – Duke Univeristy Phytochromes are red and far-red photoreceptors that control plant development and growth by regulating the expression of hundreds of light responsive genes. A central mechanism by which phytochromes regulate transcription is by triggering the proteolysis of a family of growth-promoting basic helix-loop-helix transcription factors, the PHYTOCHROME-INTERACTING FACTORs (PIFs). We have previously shown that the degradation of PIF1 and PIF3 is dependent on a phytochrome signaling component named HEMERA (HMR). However, the mechanism of the HMRmediated PIF degradation remains unknown. Here, we provide genetic evidence that knocking out four PIFs, PIF1,3,4,5, rescues the long hypocotyl phenotype of hmr-5, indicating that HMR regulates hypocotyl growth by promoting PIF degradation. Surprisingly, although PIF1 and PIF3 accumulate in the hmr mutant, they fail to activate a subset of growthrelated PIF target genes, suggesting that HMR is also required for the transcriptional activity of PIFs. We show that HMR interacts directly with all PIFs. The HMR-PIF interaction is mediated mainly by HMR’s N-terminal half and PIFs’ conserved active-phytochrome B-binding motif. Moreover, HMR is a transcriptional coactivator with an acidic nine-amino-acid transcriptional activation domain (9aaTAD). Furthermore, A missense mutation in HMR’s 9aaTAD, which impairs the expression of PIF targets, also attenuates the destruction of PIF1 and PIF3. These in vivo results support a novel phytochrome signaling mechanism, in which HMR mediates downstream photomorphogenetic responses by binding directly to PIFs and coupling the degradation of PIF1 and PIF3 with the transactivation of PIF target genes via the 9aaTAD. We propose that this HMR-dependent, transactivation-coupled mechanism of PIF degradation allows hypocotyl growth to be quantitatively controlled by phytochromes in the light.

1000-029-Y Creation and Characterization of LRB (Light-Response BTB) /PIF (Phytochrome-Interacting Factor) Mutant Lines in Arabidopsis Thaliana Luke Helminiak – University of Wisconsin-Eau Claire Kari Carothers – University of Wisconsin-Eau Claire, Derek Gingerich – University of Wisconsin-Eau Claire Light-Response BTB 1 and 2 (LRB1 and LRB2) are negative regulators of phytochrome (phy) action in the red light signaling pathway in the plant Arabidopsis thaliana. These genes encode BTB (Bric-a-Brac, Tramtrack, Broad Complex) domain-containing proteins that act as target adapters in BTB/Cullin3 E3 ubiquitin-ligase complexes. Plants with disruptions of the LRB genes have reduced light-dependent degradation of phytochromes and exhibit hypersensitivity to red light. Phytochrome-Interacting Factor (PIF) genes encode transcriptional regulators that also act as negative regulators in the red response pathway. They directly interact with phytochromes and are degraded in a light and phytochrome-dependent fashion. Recently published evidence shows that LRB1 and 2 can bind to a PIF3-phyB complex and induce ubiquitylation and degradation of both PIF3 and phyB, demonstrating a close link between LRB and PIF action. To better understand how these key components of the phytochrome pathway interact and how they work

collectively to regulate light responses we are taking a genetic approach, creating Arabidopsis lines with mutations in both LRB and PIF genes. Currently, we are attempting to generate lrb1 lrb2 pif3, lrb1 lrb2 pif4, lrb1 lrb2 pif7, lrb1 lrb2 pif3 pif4, lrb1 lrb2 pif3 pif7, and lrb1 lrb2 pif4 pif7 lines from populations segregating mutations in these genes. Data on our progress creating and analyzing the phenotypes of these lines will be presented.

1000-030-Z CUL4-COP1-SPA E3 Ubiquitin Ligase Is Necessary for the Rapid Light-induced Degradation of PIF1 Enamul Huq – University of Texas at Austin Ling Zhu – University of Texas at Austin, Qingyun Bu – University of Texas at Austin, Xiaosa Xu – University of Texas at Austin, Inyup Paik – University of Texas at Austin, Xi Huang – Peking University, Ute Hoecker – University of Cologne, Xing Wang Deng – Peking University Plants undergo contrasting developmental programs in dark and light. Photomorphogenesis, a light adapted program is repressed in the dark by the CUL4COP1-SPA E3 ubiquitin ligase and the basic helix-loop-helix transcription factors called Phytochrome Interacting Factors (PIFs). These repressors synergistically degrade positively acting transcription factor in the dark to prevent photomorphogenesis. In response to light, the phytochrome family of sensory photoreceptors translocates into nucleus and inhibits these negative regulators to promote photomorphogenesis. Here we show that the CUL4COP1-SPA E3 ubiquitin ligase functions positively by degrading its cofactor PIF1 in response to light. The lightinduced ubiquitylation and subsequent degradation of PIF1 is reduced in the cop1, spaQ and cul4 backgrounds. PIF1 forms complexes with COP1, SPA1 and CUL4 in response to light. cop1 and spaQ seeds display strong hyposensitive response to far-red light for germination. Thus, the CUL4COP1-SPA E3 ubiquitin ligase displays dual opposing roles in fine tuning photomorphogenesis in the dark and light. These data show a novel signaling mechanism where an E3 ubiquitin ligase attenuates its activity by degrading its cofactor in response to light.

1000-031-Z Uncovering the Mechanism Underpinning Photoswitching Between the Inactive and Active Conformers of Phytochromes E. Sethe Burgie – University of Wisconsin-Madison Junrui Zhang – Stanford University, Richard D. Vierstra – University of Wisconsin-Madison Phytochromes influence nearly all aspects of plant morphogenesis. Central to signaling is their ability to reversibly photointerconvert between a dark-adapted, red light-absorbing Pr conformer that is biologically-inactive and a far-red light-absorbing Pfr conformer that is generated only upon photoactivation and is biologically active. Over the last decade substantial progress has been made toward understanding phytochromes at the atomic level, and the rudiments of its photoconversion mechanism. However, the molecular basis for photoswitching remains unclear, especially with regard to the photostate-dependent positions of the bilin chromophore within its GAF-domain binding pocket, and how these movements are linked allosterically with the appended domain(s) responsible for signal output. Using the photosensory module of a model bacterial phytochrome, we show for the first time the structure of the bilin chromophore as Pfr. When compared to the structure of Pr for the same phytochrome, a ‘toggle’ mechanism for photoconversion emerges, which provides unanticipated insights toward the role(s) of key amino acids during photocycling and for the stability of the Pfr conformer. The importance of these residues was then confirmed by the photochemical analysis of site-directed mutants predicted to perturb photocycling. We have also analyzed the phytochrome as a whole for photostate dependent conformational changes by analyzing differences in proteolytic sensitivity between Pr and Pfr. Together, these studies provide clarity for the structural transitions that underpin phytochrome photoconversion, which should enhance our ability to manipulate the activit(ies) of plant phytochromes in attempts to design variants with beneficial agronomic properties.

1000-032-Y Characterization of Arabidopsis Thaliana Light Mutants Identified in an lrb1 lrb2 Genetic Suppressor Screen Kevin Mayer – University of Wisconsin-Eau Claire, Weston Orendorff – University of Wisconsin-Eau Claire, Derek Gingerich – University of Wisconsin-Eau Claire The ability to respond to the amount and quality of light is vital to plant growth and development. Red and far-red light are detected by the phytochrome (phy) photoreceptors, which initiate responses to these wavelengths. We had previously identified two genes in Arabidopsis thaliana, LRB1 and LRB2 (Light-Response BTB1 and 2) as potent negative regulators of phy-mediated red light responses. LRB1 and LRB2 encode functionally redundant E3 ubiquitin-ligase target adapters that recent evidence has shown mediate ubiquitylation and degradation of phys. To further investigate the function of the LRB genes and to potentially isolate other components of the red pathway, we conducted genetic screens in Arabidopsis to identify mutations which suppress the red light hypersensitive phenotype produced by disruption of the LRB1 and 2 genes. More than 100 putative suppressor mutants were identified in an initial screen of >30,000 M2 individuals. We have been working since to confirm and characterize these lines. Three of the lines have mutations in the phyB gene, based on results from mapping and/or complementation analyses. In one the phyB mutation disrupts proper splicing of the first exon of the gene and subsequent production of the phyB protein. We are currently sequencing the phyB gene in the other two lines. These mutations may provide insight into phyB structure, function, or interactions. These results and our progress characterizing other suppressor mutant lines will be presented.

1000-033-Y Kinematic Analysis of Cryptochrome 1 and Phytochrome B Growth Phenotypes in Arabidopsis Hypocotyls Responding to Light Edgar Spalding – University of Wisconsin Aashish Thite – University of Wisconsin, Guosheng Wu – University of Wisconsin, Nathan Miller – University of Wisconsin Genetic screens for seedlings with abnormally long hypocotyls identified mutations in genes encoding the cryptochrome 1 (cry1) and phytochrome B (phyB) photoreceptors. The former grows tall in blue light, the latter in red light. Monitoring the development of cry1 and phyB phenotypes with time-lapse image analysis showed that in both cases an initial phase of hypocotyl growth inhibition is followed by an escape phase that does not occur in the wild type. That difference produces the obvious endpoint length phenotype. A new image analysis tool was developed to add an extra dimension to the description, namely a spatial map of elemental growth rate that shows where along the hypocotyl axis mutant growth occurs to create the phenotype. The method relies on tracking patches of texture along the hypocotyl axis from frame to frame to create an axial velocity profile that is differentiated to produce the elemental growth rate profile. When mapped back to the hypocotyl, different growth patterns were apparent in cry1 and phyB mutants. Fast growth driving the escape from inhibition in the case of cry1 seedlings irradiated with blue light developed within the apicalmost 1 mm of the hypocotyl, above the region that was growing fastest (more than 20 % h-1) at the onset of light. In the case of phyB seedlings irradiated with red light, fast growth driving the escape occurs in the same region of the hypocotyl that experienced the initial inhibition, 3-5 mm below the apex. Thus, the long hypocotyl phenotypes of these two different photoreceptor mutants arise from different spatial patterns of growth control. These results will help target cellular-level investigations of photoreceptor function to the portions of the hypocotyl from where the phenotype, and by extension the photoreceptor function, is known to emerge.

1000-034-Z Dissecting Arabidopsis Photomorphogenesis in a Post-transcriptional Perspective Meng-Chun Lin – Institute of Plant and Microbial Biology, Academia Sinica, Huang-Lung Tsai – Institute of Planr and Microbial Biology, Academia Sinica, Shu-Hsing Wu – Institute of Plant and Microbial Biology, Academia Sinica Post-transcriptional control of gene expression plays pivotal roles on plant growth and development. However, the posttranscriptional regulation of photomorphogenesis remains largely unexplored. We recently reported that HUAENHANCER 1(HEN1), a methyltransferase responsible for small RNA (sRNA) stability, is a negative regulator of phoromorphogenesis. Also, we know that HEN1 regulates photomorphogenesis through the actions of microRNAs (miRs) in repressing the expression of positive regulator (ELONGATED HYPOCOTYL 5, HY5) and negative regulators (TEOSINTE BRANCHED 1, CYCLOIDEA, AND PCF FAMILY, TCPs). The discovery has shed light on the small RNA-mediated control of photomorphogenesis. By integrating small RNA sequencing, mRNA transcriptome and degradome analyses, we are able to identify additional novel sRNA-target pairs that participate in regulating the phoromorphogenic process. We have found that 17% (57 out of 338) of Arabidopsis miRs are light regulated in de-etiolating seedlings. In addition, we have verified that miR163 acts as a negative regulator of photomorphogenesis, while one of its targets, At5g38100, is also a negative regulator of hypocotyl elongation. Moreover, light irradiation results in a transiently increased accumulation of miR396s, which repress the levels of target genes, including the GROWTH REGULATORY FACTORS (GRFs), to control photomorphogenesis. In summary, our survey suggests that light regulates expression of many MIRs and multiple miRNAs impose post-transcriptional regulation on their target genes. These together contribute to an optimal photomorphogenic development when Arabidopsis seedlings experience initial light exposure.

1000-035-Z AtYAK1 Promotes Arabidopsis Growth Vigor Under Sub-optimal Light Environment Wen-Yu Huang – Institute of Plant and Microbial Biology, Academia Sinica Yi-Chen Wu – Institute of Plant and Microbial Biology, Academia Sinica, Hsin-Yi Pu – Institute of Plant and Microbial Biology, Academia Sinica, Shu-Hsing Wu – Institute of Plant and Microbial Biology, Academia Sinica Arabidopsis YAK1 (AtYAK1, homolog of yeast YAK1 protein) belongs to the dual-specificity tyrosine-phosphorylation regulated kinases (DYRKs) superfamily. DYRKs regulate multiple developmental processes in diverse eukaryotes. However, their function in plants remains largely unknown. In this study, we found that plants demand AtYAK1 especially under low light condition for various developmental processes. atyak1 mutant has short siliques due to reduced male fertility when grown under low intensity of light or under 12-h light/12-h dark conditions. Interestingly, the decreased fertility of atyak1 can be improved by increased light intensity or day length, meaning that AtYAK1 optimizes plants’ fertility when light is limited in the environment. AtYAK1 not only involves in light-mediated pollen development but also regulates light responses in young seedlings. Seedlings lacking AtYAK1 is less sensitive to light in aspects of light-induced hypocotyl shortening, anthocyanin accumulation and the expression of light-inducible genes when compared to wild-type seedlings. The circadian period length shortening in response to increasing light intensity was also defective in atyak1 mutant under low light condition. In sum, our results reveal that AtYAK1 helps achieve growth and developmental vigor in plants under sub-optimal light conditions.

1000-036-Y The Plant Defense Hormone Salicylic Acid Mediates Growth Response of Shade Avoidance Syndrome in Arabidopsis Kazunari Nozue – University of California Davis Upendra Kumar Devisetty – University of California Davis, Saradadevi Lekkala – University of California Davis, Patricia Mueller-Moule – University of California Davis, Julin Maloof – University of California Davis

Both plant growth and defense are energy demanding processes; therefore plants need to prioritize growth or defense to optimize use of limited resources. Recently key transcription factors and co-repressors were found to be involved in growth/defense trade-off, however this trade-off was primarily studied by genetic manipulation of plants. The only natural situation where growth/defense trade-offs are known to occur is when plants are grown at high density. In this situation plants prioritize growth to compete with the neighbors for light and exhibit a suite of developmental and physiological responses known as the shade avoidance syndrome (SAS). Current knowledge of how plants undergoing SAS prioritize growth over defense is limited. To tease out the complex responses and learn about their temporal progression we analyzed time-course transcriptomes of ten Arabidopsis shade avoidance mutants that showed reduced petiole growth response to shade (phyB, hy5, yucQ, jar1, kat1, mida9, pif3, pif4/5, spt, PAR1-RNAi). Surprisingly we found that misexpressed genes in nine of these mutants were enriched for members of a salicylic acid related co-expressed module, leading to the hypothesis that salicylic acid pathway genes are involved in shade-induced growth responses in petioles. Indeed, reduction of SA levels (NahG transgenic plant) abolished shade-induced growth responses in the petiole, strongly supporting our hypothesis. Thus our data indicates that SA plays a role in growth-defense trade-off in SAS. Detailed analysis of timecourse transcriptome analysis in these shade avoidance response mutants will be presented.

1000-037-Y DAUGHTER-OF-HEMERA Is a Novel Component of Early Phytochrome Signaling That Initiates Arabidopsis Photomorphogenesis Emily Yang – Duke University He Wang – Duke University, Jun Cao – Max Planck Institute for Developmental Biology, Detlef Weigel – Max Planck Institute for Developmental Biology, Meng Chen – Duke University Phytochromes are red and far-red photoreceptors that initiate photomorphogenesis during seedling development by massively reprogramming the Arabidopsis transcriptome. The earliest light response at the cellular level is the translocation of phytochromes from the cytoplasm to discrete photosensory subnuclear domains named photobodies. Photobody-localization of phytochrome B is tightly correlated with the degradation of a group of growth-promoting transcription factors, the PHYTOCHROME INTERACTING FACTORs (PIFs) and consequently the inhibition of hypocotyl growth. However, the early light signaling mechanisms of photobody assembly and its roles in PIF degradation are still not fully understood. We have recently identified HEMERA (HMR), a transcriptional coactivator required for both photobody assembly and the degradation of PIF1 and PIF3. Surprisingly, studies of HMR by our laboratory and others revealed unexpectedly that HMR is required for not only phytochrome signaling but also chloroplast development. The hmr mutant represents the founding member of a new class of mutants that are tall, albino and seedling lethal. Because tall-and-albino mutants had been ignored from previous genetic screens for light signaling mutants, we hypothesize that this new mutation class defines missing components of early phytochrome signaling. To test this hypothesis, we carried out a forward genetic screen for hmr-like mutants. This screen has identified DAUGHTER-OF-HEMERA (DOH). Similar to HMR, DOH is required for both phytochrome signaling and chloroplast development. DOH participates in both red and far-red signaling and acts genetically downstream of phytochrome A and B. We show that DOH works in concert with HMR in regulating photobody assembly and the expression of PIF-dependent light responsive genes. Moreover, DOH is also required for HMR-dependent transcriptional regulation in the chloroplasts. Together, this study identifies a novel component in early phytochrome signaling, and it further demonstrates a mechanistic link between phytochrome signaling and chloroplast development during the initiation of photomorphogenesis.

1000-038-Z Role of AtRAD4 and Predicted RAD4 Interacting Proteins in Arabidopsis UV Tolerance Dana Schroeder – University of Manitoba Triparna Lahari – University of Manitoba Plants are exposed to a range of solar radiation, including damaging ultraviolet (UV) light. All organisms have pathways which identify and repair UV-damaged DNA. The light independent DNA repair pathway nucleotide excision repair (NER) is largely conserved in plants, mammals and yeast. Deficiencies in NER can result in serious disorders, for example, mutation of the human nucleotide excision repair gene XPC can result in the disease Xeroderma pigmentosum. The yeast (Saccharomyces cereviciae) homologue of human XPC is RAD4 (Radiation sensitive 4) and the Arabidopsis homologue is AtRAD4. Based on homology, the Arabidopsis Interaction Viewer (AIV) predicts interactions between AtRAD4 and Arabidopsis homologues of other known NER components such as RAD23. In addition, the AIV predicts interaction between AtRAD4 and novel RNI (Ribonuclease Inhibitor)-like superfamily proteins we refer to as RNI1 and RNI2. Another uncharacterized RNI-like superfamily protein, RNI3, is a close homologue of RNI1. In our current research, we are studying the role of RAD4 and the predicted RAD4 interacting proteins RNI1-3 in UV tolerance using loss of function and overexpression lines, as well as YFP tags to examine cellular localization. Our progress will be reported.

1000-039-Z Investigating the Role of Vitamin B6 in Light Tolerance of Natural Plant Communities Elizabeth Rueschhoff – Indiana University Southeast Jared Scott – Indiana University Southeast Vitamin B6 is a required coenzyme for a variety of cellular processes. The biosynthetic pathway of vitamin B6 has been well characterized in model organisms such as Arabidopsis thaliana. Roles of vitamin B6 have been well documented and include amino acid metabolism, carbohydrate metabolism, chlorophyll biosynthesis and auxin and ethylene metabolism. In addition, vitamin B6 has been shown to be a potent antioxidant, able to quench singlet oxygen at rates comparable to that of vitamin C and vitamin E. Vitamin B6 has been shown to also quench superoxide. While vitamin B6 metabolism is well characterized in model organisms, little research has been conducted to study the effects of vitamin B6 in natural environments. Because plants produce higher levels of singlet oxygen during photosynthesis, and vitamin B6 has been shown to be a quencher of singlet oxygen, we hypothesize that vitamin B6 may play a role in plant tolerance to high light environments. We collected five different species of plants (25 samples of each species) from a high light environment and five different species of plants (25 samples of each species) from a low light environment. We extracted the vitamin B6 from these plants and are currently performing yeast auxotrophic assays to quantify vitamin B6 levels in these samples. Our goals are to determine if plants that are adapted to grow in high light environments contain more vitamin B6 than those that are adapted to low light environments and if there are differences in vitamin B6 content in a variety of plant species.

1000-040-Y Effect of Light Quality on the Expression of Glycoalkaloid Biosynthetic Genes Contributing to Potato Glycoalkaloid Accumulation Hwang Bae Sohn – Highland Agriculture Research Institute, National Institute of Crop Science, RDA Manjulatha Mekapogu – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Hwang-Bae Sohn – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Yu-Young Lee – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Hyang-Mi Park – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Yong-Ik Jin – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Su-Young Hong – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Jong-Tak Suh – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Kibum Kewon –

Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Jin-Cheol Jeong – Highland Agriculture Research Institute, National Institute of Crop Science, RDA, Yul-Ho Kim – Highland Agriculture Research Institute, National Institute of Crop Science, RDA Light quality and light intensity have severe impact on potato glycoalkaloid (PGA) biosynthesis, causing the quality degradation of potato by greening and accelerating PGA induced toxicity. In this study, tubers from low and high PGA accumulating cultivars, Atlantic and Haryoung responded differently when exposed to seven light sources with different wavelengths viz., purple, red, blue, green, yellow, UV and fluorescent lights. Atlantic remained insensitive to light, but Haryoung tuber showed varied PGA contents in different light qualities with yellow light exhibiting a 44% reduction in the PGA accumulation compared to fluorescent light. Further screening of selected light sources including red, green, yellow and fluorescent light by investigating the expression pattern of key genes and PGA contents, showed lower transcript levels of key genes in yellow light which in-turn reflected in the least amount of PGA accumulation with a 22% reduction of total PGA in yellow light compared to fluorescent light in Haryoung tuber peel, clearly implicating a suppression in the PGA accumulation in the yellow light compared to other light sources. This suggest that yellow light can be used as an alternative light source instead of fluorescent light which would help in minimizing the quality degradation and PGA induced toxicity.

Abiotic: Temperature 1000-041-Y Ecological Variations and Role of Heat Shock Protein in Artimisia Judiaca in Response to Temperature Regimes of Tabuk, Saudi Arabia Zahid Abbas – University of Tabuk Shalini Saggu – University of Tabuk, Hasibur Rehman – University of Tabuk, Abid A. Ansari – University of Tabuk Artemisia judaica L. (Compositae) are shrubby herbs growing wildly in Tabuk region and distributed in the desert regions. This region is characterized by extremely variable environmental conditions where the temperature varies from extreme low to extreme high. These temperature regimes have a profound effect on morphology, growth physiology and biochemistry of the plants. The plant samples were collected from Tabuk-Jordan road (760 meter above sea level) in the month of January, April, July and October 2013 to evaluate the effect of temperature dynamics on Artimisia judiaca in four different seasons. Physiological, biochemical alterations and heat shock proteins (HSPs) were studied during these seasons in order to evaluate the environmental adaptation and stress tolerance in response to temperature variations. Plant growth parameters showed a significant increase in height, fresh and dry matter accumulation, total chlorophyll, N,P,K, artimisinin and leaf relative water contents investigated in the month of April and October. Plant growth was suppressed and an active role of CAT, POD and SOD was observed to cope with the extreme low temperature in January and extreme high temperature in July 2013. However, the plants collected in October and April did not show a statistical difference. An induction in the expression of HSP90 were recorded in all the plants collected during April and October 2013 with no statistical significant difference. Therefore, based on the results it is recommended that during April and October the environmental conditions are best suitable for growth, development and medicinal use of Artimisia. .

1000-042-Z Temperature Stress Protection of Pollen Formation by the Unfolded Protein Response Renu Srivastava – Iowa State University Yan Deng – Iowa State University, Teagen Quilichini – University of British Columbia, Haili Dong – Iowa State University, Harry Horner – Iowa State University, Stephen Howell – Iowa State University

The unfolded protein response (UPR) is elicited by ER stress during vegetative growth in plants. The UPR is constitutively active in flowers, particularly in anthers, where there are high demands for secretion during pollen development. Pollen development and pollination are the most sensitive stages to temperature stress in most plants and we have found that the UPR protects male gametophyte development from temperature stress. Double mutants ire1a ire1b and ire1b bzip28 affecting the UPR signaling pathway in Arabidopsis are conditionally male sterile at modestly elevated temperatures. The temperature-sensitive defects in the double mutants are sporophytic and disrupt the tapetum in its support of male gametophyte development. The mutant defects at elevated temperatures disrupt the deposition of the pollen coat, which leads to the clumping and collapse of many pollen grains. In comparison to wild type, the double mutants showed a strong shift in intrinsic fluorescence of developing pollen indicating compositional changes in the pollen wall. Pollen in the ire1a ireb mutant phenocopied quartet (qrt) mutants despite the fact that there was no suppression of QRT gene expression in this mutant. The impact of the double mutants on the stress transcriptome in vegetative tissues was quite different from that in flowers where it affects the expression of genes encoding components of the vesicle transport system and pollen coat formation. These findings are consistent with the concept that the UPR protects pollen formation from temperature stress by promoting the expression of genes encoding components of the pollen coat and of genes involved in the orderly transport of coat substrates to the pollen wall.

1000-043-Z Heat Stress During Development Alters Post-harvest Sugar Contents and Chip Processing Quality of Potato Tubers Paul Bethke – USDA ARS and University of Wisconsin-Madison Amy Wiberley-Bradford – University of Wisconsin-Madison, James Busse – USDA ARS - Madison WI Environmental stresses that result in altered carbohydrate metabolism and increased contents of the reducing sugars glucose and fructose decrease the value of potatoes grown for chip production because such tubers produce darkcolored, defective chips that are unacceptable to chip processors and consumers. Stem-end chip defect (SECD), which causes highly localized regions of dark color along the vasculature of potato chips at a position corresponding to the basal end of the tuber, is an economically important tuber quality defect that occurs erratically across locations and production years. Controlled-environment studies were conducted over 3 years to test the hypothesis that exposure of potato plants to short periods of high temperature stress causes SECD. Multiple potato cultivars were grown under wellwatered control conditions with 25˚C day and 18˚C night temperatures. A high temperature stress, with temperatures of 35˚C during the day and 29˚C during the night, was imposed for 3 to 14 days. Plants were returned to control conditions until vines had senesced naturally, typically 45-60 days later, when tubers were harvested. Little SECD was observed in chips prepared at harvest from control or heat-stressed tubers. SECD was abundant in chips from heat-stressed, but not control, tubers after 30 or more days in storage at 13˚C. Heat stress increased tuber glucose and fructose contents, and invertase activity increased as SECD severity increased. Different genotypes varied widely in susceptibility to heat stressinduced reducing sugar accumulation. In a separate experiment, lines of potato in which vacuolar acid invertase expression and activity were strongly suppressed using RNA-interference had reduced SECD severity in field-grown tubers, suggesting that vacuolar invertase activity produced highly localized accumulations of glucose and fructose that are the precursors of SECD formation.

1000-044-Y Regulation of Sugar Metabolism in Chipping Potatoes During and After Cooling for Low-temperature Storage Amy Wiberley-Bradford James Busse – USDA-ARS-VCRU, Paul Bethke – University of Wisconsin-Madison Regulation of sugar metabolism in cold-stored potato tubers has significant ramifications for potato chip and fry

producers and consumers. Low-temperature storage reduces losses due to sprouting and disease, but it can also induce accumulation of glucose and fructose. These reducing sugars react with free amino acids during frying to produce darkcolored and bitter-tasting products that have elevated levels of acrylamide, a neurotoxin and possible carcinogen. Glucose and fructose result from the action of vacuolar acid invertase (VInv) on sucrose produced during starch degradation. In this study, wild-type tubers and tubers in which RNA interference decreased VInv expression were used to study the regulation of sugar metabolism during and after the cooling of tubers for low temperature storage. Suppression of VInv expression was observed to decrease reducing sugar accumulation and improve chip color, though the differences in sugar contents did not affect the expression of other genes involved in starch metabolism. In addition, transcriptional control of genes central to carbohydrate metabolism was found to occur in three overlapping phases during cooling. Taken together, the data indicate that reducing sugar accumulation is partially controlled by temperature-regulated changes in sucrose accumulation, resulting from increased starch degradation by β-amylase and decreased starch resynthesis by ADP-glucose pyrophosphorylase and granule-bound starch synthase, as well as by differences in VInv that persist throughout storage.

1000-045-Y CDPK Function in Cold Priming in Arabidopsis Thaliana Sieke Schaepe – Free University Berlin Tina Romeis – Free University Berlin CDPKs are calcium-binding protein kinases which were shown to play important roles in early signal transduction pathways in abiotic and biotic stress responses. Cold stress leads to diverse changes in metabolism and morphology of the whole plant and a transcriptional reprogramming of around 14% of all genes in Arabidopsis thaliana. We established a priming protocol where plants are primed by mild chilling stress (4°C, 3 d) to a later cold stress of subzero degree (triggering stimulus) with a memory phase of 3 days in-between. Candidate AtCPK isoforms have been identified which show differential expression during cold stress. A mutant of these candidates displays an enhanced priming capacity and memory, as well as altered sugar, amino acid and lipid marker metabolites, while the corresponding OE lines show reduced priming effects. Current work addresses the molecular function of this CPK to elucidate the biochemical mechanism of cold priming and memory.

1000-046-Z The Negative Effect of High Temperature Stress on Reproduction in Arabidopsis Thaliana Vanessa Lundsgaard-Nielsen – University of Toronto Dinesh Christendat – University of Toronto, Tammy L Sage – University of Toronto The adverse effect of high temperature (HT) on pollen development is a primary reason for reduced seed set in natural and arable ecosystems. We have identified a gene, HTT - High Temperature Tolerance - in Arabidopsis that encodes a plastid-targeted protein functioning in the removal of reactive carbonyl species (RCS). RCS are formed downstream of reactive oxygen species and damage cells by oxidatively modifying proteins, rendering them nonfunctional. In the HT tolerant Arabidopsis Ler, the gene is induced in the anther, pollen remains viable and HT has little effect on seed set. Cvi pollen is not viable, has low seed set, and the gene is not induced by HT. The loss of pollen viability in Cvi and htt1-1 at HT is associated with abnormal plastid development, lipid accumulation, and autophaged mitochondria. We assessed the degree of protein carbonylation and used mass spectrometry (MS) to identify carbonylated proteins to test the hypothesis that HTT functions to maintain plastid homeostasis and pollen viability during HT stress. Pollen plastids in Arabidopsis are essential for pollen maturation due to their critical role in fatty acid and carbohydrate metabolism. Consistent with our hypothesis, carbonylated protein levels were higher in Cvi and htt1-1 than Ler at HT. MS revealed carbonylated proteins present in Cvi and htt1-1 at HT that would normally function in lipid biosynthesis, carbohydrate

metabolism, protein folding, amino acid metabolism, as well as detoxification of ROS and other compounds. Many of the proteins that were carbonylated at HT were degraded, and several proteins involved in the protein degradation process were also found to be carbonylated. Our results provide a novel role in HTT in ensuring the removal of RCS to maintain pollen viability.

1000-047-Z Identifying Targets of the SUMO E3 Ligase SIZ1 Using a Pathway Null Mutant and Mass Spectrometric Analysis in Arabidopsis Thaliana Therese Rytz – University of Wisconsin – Madison Mark Scalf – University of Wisconsin – Madison, Joseph Walker – University of Wisconsin – Madison, David Gemperline – University of Wisconsin – Madison, Lloyd Smith – University of Wisconsin – Madison, Richard Vierstra – University of Wisconsin – Madison The post-translational modification of proteins by Small Ubiquitin-like Modifier (SUMO) is an essential process. Genetic studies indicate that SUMOylation plays a critical role in protecting plants against numerous environmental challenges. Using proteomic methods in Arabidopsis, we identified hundreds of SUMO targets involved in a wide array of nuclear events, including transcription, chromatin remodeling, and RNA biology. Upon exposure to heat shock and other abiotic stresses, the SUMOylation level of these proteins rises dramatically and reversibly, suggesting that SUMOylation serves as a rapid mechanism for regulating multiple nuclear activities during stress. To further study SUMO conjugation, we are attempting to assign ligases to individual targets using novel mass spectrometric approaches. Of particular interest is the ligase SIZ1, which directs much of the heat shock-induced SUMOylation and is required for thermotolerance. To identify the proteins modified by SIZ1, we compared the wild-type SUMOylome of the 6His-SUMO line (sum1-1 sum2-1 6HisSUMO1 H89R) to that of the same background harboring the siz1-2 null mutation. After purification of SUMO conjugates facilitated by the 6His-tagged SUMO variant, mass spectrometric quantification of the preparations using a label-free approach enabled the identification of SIZ1 targets whose SUMOylation is absent or altered in the mutant. Besides increasing the catalog of known SUMO conjugates to over 900 proteins, we identified a collection of targets with statistically reduced SUMOylation in the siz1-2 background. Our data indicates that not only is SIZ1 involved in the SUMOylation of major developmental regulators, such as chromatin modifiers and co-repressors, but that the ligase also modifies multiple subunits and co-factors of specific regulatory complexes. Additionally, we found multiple stress response transcription factors that require SIZ1 for SUMOylation, highlighting the role that SIZ1 plays in the response to heat-shock. Studies of these individual targets will lead to a better understanding of SUMO during stress.

1000-048-Y FCA Mediates Thermal Adaptation of Stem Growth by Attenuating PIF4 Action in Arabidopsis Chung-Mo Park – Seoul National University Hyo-Jun Lee – Seoul National University, Jun-Ho Ha – Seoul National University, Jae-Yong Ryu – Seoul National University Climate modeling studies anticipate that global warming would profoundly influence worldwide biodiversity and distribution of plants and crop yield in near future. High ambient temperature influences diverse aspects of plant growth and development, among which, plant architectural adaptations, such as hypocotyl growth, leaf stem elongation, and increase of leaf elevation (hyponasty), are most prominent. These adaptive responses contribute to optimal growth by minimizing the risk of potential heat damage caused by the heat from the soil surface and enhancing evaporative leaf cooling. The Arabidopsis basic helix-loop-helix (bHLH) transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) regulates high temperature-induced adaptive responses by modulating auxin biosynthesis. At high temperature, PIF4 directly activates YUCCA8 (YUC8) gene encoding an auxin biosynthetic enzyme, resulting in auxin accumulation that leads to elongation of hypocotyl and leaf stem. Here, we demonstrate that the RNA-binding protein FCA attenuates the

PIF4 action by suppressing its binding to YUC8 promoter via chromatin remodeling at high ambient temperature. At 28oC, auxin content was elevated in FCA-deficient mutants that exhibit elongated stems but reduced in FCAoverexpressing transgenic plants that exhibit reduced stem growth compared with those in wild-type plants. We propose that the FCA-mediated remodeling of YUC8 chromatin tunes down the PIF4-induced architectural changes to achieve thermal adaptation of stem growth at high ambient temperature.

1000-049-Y Pectin Methylesterase 34, PME34, Contributing to Regulation of Stomatal Movement, Is Required for Heat Stress Response in Arabidopsis Tsung-Luo Jinn – National Taiwan University Pectin, a major component of the primary cell wall, is synthesized in the Golgi apparatus and exported to the cell wall in a highly methylesterified form, then de-methylesterified by pectin methylesterase (PME, E.C. 3.1.1.11). The effect of PME on the pectin methylesterification status plays a key role in plant development and plant–pathogen interactions, but its role under heat stress (HS) has been poorly studied. At least 66 potential PME genes are annotated in Arabidopsis (Arabidopsis thaliana). Thermotolerance assay of 53 PME homologous-T-DNA insertion lines revealed 2 null-mutant alleles of PME34 (At3g49220) both consistently showed reduced thermotolerance; nevertheless their impairment was independently associated with the expression of HS-related genes. PME34 transcript induction depended on abscisic acid and was high expression in guard cells. We showed PME34 mutation has a defect in the control of stomatal movement with irregular action of PME and polygalacturonase (PG, EC 3.2.1.15) resulted in a heat-sensitive phenotype. Hence, PME34 has a role in the regulation of transpiration by controlling the degree of stomatal aperture that was achieved by enzymatic actives during HS response. PME34 is required for regulating guard cell-wall flexibility to mediate HS tolerance in Arabidopsis.

1000-050-Z Different Classes of Chaperones Coordinate Protein Refolding and Degradation Fionn McLoughlin – University of Massachusetts Amherst Eman Basha – University of Arizona, Minsoo Kim – University of Massachusetts Amherst, Mary Fowler – University of Massachusetts Amherst, Elizabeth Vierling – University of Massachusetts Amherst During heat stress, proteins that are vital to cell survival become unfolded and can form aggregates that are toxic to the cell. Both molecular chaperones and ATP-dependent proteases are required to protect cells from accumulation of protein aggregates. One category of molecular chaperones, of which several members are induced by heat stress, is the small Heat Shock Proteins (sHSPs). These chaperones are able to prevent irreversible protein aggregation by binding to exposed hydrophobic patches of misfolded proteins. Knock-down lines of two conserved cytosolic sHSP classes (CI and CII) in Arabidopsis thaliana were both more sensitive to heat stress. To further investigate which proteins are targeted and to identify sHSP co-factors, affinity purification was conducted which led to the identification of multiple sHSP substrates, including translation elongation factors (eEF1Balpha, beta and gamma) and fructose-bisphosphate-aldolase. In both CI- and CII-sHSP knock-down lines these proteins remained in the insoluble cell fraction longer than in wild type plants. The chaperone HSP101 is an important cytosolic protein disaggregase, which is crucial for effectively refolding aggregated substrates. The same proteins found associated with sHSPs, were also retained longer in the insoluble cell fraction in an Hsp101 null mutant, providing further evidence for interaction between sHSPs and Hsp101 in resolubilizing heat-aggregated proteins. Localization studies revealed that HSP101-GFP accumulated in cytosolic foci and co-localized with CI-sHSPs, suggesting that these events occur in specialized cellular structures. To determine how aggregated proteins are further processed, affinity purification on HSP101 was conducted and several subunits of the 26Sproteasome lid-complex were found associated with HSP101. Poly-ubiquitinated proteins accumulated to higher levels

in the hsp101 mutant after heat stress showing that in addition to protein refolding, HSP101 also play a role in protein degradation. Interacting with the 26S-proteasome could facilitate protein degradation and play a role in determining the fate of substrate proteins.

1000-051-Z The Phenotypic and Transcriptomic Responses to Heat Stress for Arabidopsis Thaliana from Contrasting Climates Nana Zhang Stephen Tonsor Abiotic stress responses are key to wild species survival and to high yields in agriculture. Plants respond to stress through various morphological and physiological adaptations, each of which has different costs and benefits. Thus plants growing in contrasting climates are expected to have evolved contrasting mechanisms of adaptation to stress. Meanwhile, these phenotypic responses are modified through altering the expression level of thousands of stressresponsive genes. Thus understanding the response patterns of both the phenotype and transcriptome will uncover both the natural variation of various phenotypic responses and the underlying mechanisms for these adaptive stress responses. Using the natural populations of a model species, Arabidopsis thaliana, my research fills in the gap between the adaptive mechanisms and their underlying genomic and environmental causes. Plants adopt two main phenotypic strategies: avoidance and tolerance. Avoidance involves adjustments of internal states, reducing exposure to stress. Tolerance repairs or prevents the resulting biochemical damage from stress. Prior lower-level stress conditions often improve subsequent severe stress response (called acclimation). In order to explore the natural variation of avoidance and tolerance under heat stress, eight natural populations of Arabidopsis thaliana from two ends of an elevation gradient in northern Spain, were grown in the growth chambers. Three temperature treatments were conducted at flowering stage: normal, moderately hot (acclimation temperature), and high heat. Variation in avoidance and tolerance were quantified by performing various morphological and physiological measures. These data showed a novel contrasting relationship in that populations from hotter, dryer climates displayed relatively less avoidance but more tolerance than populations from colder, wetter climates. Next, messenger RNA has been extracted and is currently being sequenced with Illumina Hiseq 2500. After alignment and analysis, differentially expressed genes will be annotated for functional classification based on the Gene Ontology online database. These transcriptome data will then be correlated with the phenotypic variation, thus providing possible explanations on mechanisms. Gene expression data will also be compared with detailed micro-climate characteristics, thus providing important clues on the constraints in response to specific climate variable(s). This study is one of the first to explore natural variations in full transcriptome with high throughput sequencing technologies in lines from a large number of natural plant populations. This work will provide important information for implementing effective agriculture strategies.

1000-052-Y Upregulation of CONSTANS and FLOWERING LOCUS T Under Cool Night-time Temperatures Hannah Kinmonth-Schultz – University of Washington Soo-Hyung Kim – University of Washington, Takato Imaizumi – University of Washington The timing of flowering is regulated by day length and ambient temperature. In nature, diurnal temperature fluctuations occur in conjunction with light/dark cycles, and temperature cycles influence plant development. To understand flowering mechanisms in more natural conditions, we explored the effect of daily light and temperature changes on

Arabidopsis thaliana. We exposed seedlings to different day-length and day/night temperature treatments and assessed expression profiles of genes involved in flowering regulation. Warm-day, cool-night temperature cycles increased CONSTANS (CO) mRNA and protein levels at night. CO mRNA induction was diminished in flowering bhlh (fbh)-quadruple mutants. Day/night temperature changes altered the day-length-dependent patterns of FLOWERING LOCUS T (FT) expression. FT levels were suppressed at dusk, but higher at the end of cooler nights. Dusk suppression occurred in younger seedlings and was alleviated in short vegetative phase (svp)mutants. The increase at the end of the night was maintained over two weeks in long days. FT levels correlated strongly with flowering time across treatments. Here we show that day/night temperature changes mediate photoperiodic flowering by altering FT expression patterns. FBHdependent induction of CO is enhanced by cooler nighttime temperatures, and CO protein is stabilized. In addition, cooler temperatures suppress FT at dusk, through activity of SVP, when plants are young, perhaps to suppress precocious flowering. Our results suggest a mechanism through which day length anddiurnal temperature changes combine to modulate flowering time.

1000-053-Y NUCLEAR FACTOR-Y, Subunit B2 Is Involved in Freezing Tolerance in Arabidopsis Yajun Wu – South Dakota State University Mingxiang Liang, Rokebul Anower, David Hole, Jeffery Harper, Dong Chen, Jixiang Wu, Yajun Wu Nuclear factor Ys (NF-Ys), also known as heme-activated proteins (HAPs), are transcription factors and have multiple roles in plant growth and development, such as embryogenesis, flowering time control, and stress tolerance. In a previous study, we reported that NF-YB2 (i.e. HAP3b) promotes flowering time through the long day photoperiod pathway. We report in this study that NF-YB2 is involved in controlling plant cold stress response. Transcript profiling and gene expressionanalysis indicated that NF-YB2 repressed expression of CBF3 and several CBF3-regulated genes under room temperature growth conditions. Freezing tests showed that NF-YB2-overexpression plants were more susceptible to freezing stress while nf-yb2 (i.e. hap3b), a null allele mutant line, were more tolerant to the stress compared to wild-type plants based on survival rate and leaf electrolyte leakage of plants. Our data support a model that NF-YB2 may act as a negative regulator of CBF3 in cold stress tolerance.

1000-054-Z Expression of CBF-like Genes in Alfalfa (Medicago Sativa L.) Praveena Kanchupati – South Dakota State University Rokebul Anower, Yajun Wu As an effort to improve freezing tolerance in alfalfa, we have recently discovered a germplasm that is naturally adapted to the Grand River National Grassland environment in South Dakota and showed greater freezing tolerance compared to some of the known freezing tolerant genotypes. The objective of this study is to address the molecular basis of the freezing tolerance in this novel germplasm. C-repeat binding factor (CBFs) is a key transcription factor in Arabidopsis along with many other plants and plays an important role in response to low temperature and freezing stress. A survey of Medicago truncatula (a close relative to alfalfa) genome resulted in the identification of 19 CBF-like genes. Phylogenetic analysis grouped them into 5 distinct clusters based on their homology to Arabidopsis and soybean CBFs. Expression profiling of the gene family under cold stress revealed a diverse pattern of induction. Five genes, which showed different patterns of induction under cold stress, were selected for further analysis. The transcript levels of these genes differ in diurnal and developmental regulation. We are currently conducting additional analysis to understand how these genes are regulated differently.

1000-055-Z Unraveling the Genetic Basis of Frost Tolerance in Switchgrass (Panicum Virgatum L.) Christian Schwoyer – University of Georgia Katrien Devos – University of Georgia, Jeff Bennetzen – University of Georgia, Peng Qi – University of Georgia, Bochra Bahri – University of Georgia, Orville Lindstrom – University of Georgia, Guillaume Daverdin – University of Georgia, Debkanta Chakraborty – University of Georgia, Srini Chaluvadi – University of Georgia With the demand for an environmentally responsible fuel source ever-present, Panicum virgatum L. (switchgrass) has become a promising candidate lignocellulosic fuel crop. Switchgrass is a perennial, C4 grass native to North America and a promising biofuel crop thanks to its ability to thrive on marginal soils unsuitable for other crops. Switchgrass can be classified under two main ecotypes: upland and lowland, which are native to the Great Plains and Southern US respectively. In their native ranges, the lowland ecotype produces roughly 30% more biomass compared to its upland counterpart. However, this increased production of the lowland ecotype is lost when it’s grown in northern latitudes. The main cause of this decrease in yield is due to high levels of winterkill. Upland ecotypes have developed frost tolerance mechanisms that allow them to withstand the effects of winterkill; these mechanisms are currently unknown. To dissect this issue, we developed a mapping population derived from crossing two F1 sibs that were the result of a cross between an upland and lowland parent, thereby being heterozygous for all loci for both ecotypes. We will create a de novo genetic map of this population using Genotyping-by-Sequencing (GBS). This map will act as the framework for later analyses. Our mapping population is currently undergoing cold-acclimatization and freezing tests in controlled, greenhouse environments, as well as exposing the population (~400 individuals) to an ongoing field trial. In the future, we will combine Quantitative Trait Loci (QTL) analysis with expression QTL (eQTL) analysis, focusing on genes that are differentially expressed between the two ecotypes under cold acclimatization to identify candidate genes. Understanding the genetic factors responsible for frost tolerance in switchgrass is critical for development of switchgrass varieties that combine the high biomass yield of the lowland ecotype with the frost tolerance of the upland ecotype.

1000-056-Y State Transitions in Winter Stressed Conifers as a Function of Growth Light Environment Jacob Jerrard – University of Saint Thomas, Amy Verhoeven – University of Saint Thomas During winter conifer needles are exposed to subfreezing temperatures and daily light fluctuations. Plants use state transitions, the reversible phosphorylation of light harvesting proteins (LHCII), to cope with fluctuating light. This process involves increases in LHCII phosphorylation in low light conditions and decreases in dark and high light conditions and is thought to optimize energy partitioning between photosystems. State transitions have been well characterized in summer conditions but have not been investigated in overwintering plants. We asked the following: (1) Is light-dependent phosphorylation of photosynthetic proteins different in winter versus summer conifers? (2) Are the phosphorylation states of these proteins dependent on growth light environment? To address this, winter and summer samples from three species [Pinus strobus (white pine), Abies balsamea (balsam fir) and Picea glauca (white spruce)] were gathered in dark, low, and high light conditions from both south and north (self shaded) tree sides. Environmental parameters and chlorophyll fluorescence were measured at the time of sampling. Thylakoids were isolated and western blots probed with anti-phosphothreonine to determine LHCII phosphorylation status. Chlorophyll fluorescence data showed expected differences in photosynthetic efficiency in response to both light and season, with dramatic Fv/Fm decreases observed during winter relative to summer. North facing needles showed lower levels of Chl a/b relative to south facing needles in pine and spruce, consistent with acclimation to lower light conditions. The relative phosphorylation of LHCII followed expected patterns in summer samples, with maximal phosphorylation occurring in low light conditions. In winter LHII remained phosphorylated in all conditions (dark, low light and high light), and overall this relative phosphorylation was more pronounced in the north facing needles relative to those facing south. The

sustained phosphorylation of LHCII in winter needles, even in high light conditions, suggests a role for phosphorylated LHCII in maintenance of winter-induced photosystem downregulation.

1000-057-Y The Role of Cold Cues on Life History Timing in Mimulus Guttatus Matthew Rubin – Syracuse University Jannice Friedman Cold treatment of seeds (stratification) and cold treatments of leaves (vernalization) are important for timing of germination and flowering in plants. Genetic variation for response to these cues may be a result of environment and past selective regimes. Thus, stratification and vernalization regulate development in some angiosperms. Previous work has shown that stratification breaks seed dormancy and accelerates germination timing, and can produce polymorphisms in seed germination. Vernalization is important for reproduction in plants because it silences a repressor of flowering in eudicots and induces an activator of flowering in monocots. To investigate the effects of stratification and vernalization on germination and flowering proportion and timing, we grew seed from annual and perennial populations of Mimulus gutattus, that span abroad geographic range that experience very different temperatures and seasonality. The experiment included 4 stratification treatments (0, 5, 10, 15 days of cold) and two vernalization treatments (short days, followed by long days; and short days followed by 4 weeks of 4oC, followed by long days). Perennials have higher germination (approx. 80%) than annuals (approx. 60%) and stratification accelerates germination timing in annuals. We also found that stratification overrides the vernalization requirement for flowering in inland perennials, and cold treatment at either the seed or leaf stage synchronizes flowering time; indicating vernalization is not required for plants to flower, but accelerates the flowering process. These findings support a mechanism in which cold treatment at either the seed or leaf stage ensures synchronous flowering of all plants for inland perennial populations. This suggests that there is a threshold at which flowering is accelerated irrespective of whether the plant was vernalized.

1000-058-Z Genetic Mapping and Analysis of Cold-tolerance Genes in Rice Yao Shi – Marquette University Michael Schlappi – Marquette University, Aaron Jackson – Dale Bumpers National Rice Research Center, USDA Natural variation in plants determines their tolerance to biotic and abiotic stresses. The tolerance to one of the most important abiotic stresses, cold, is greater in plants that originated from temperate zones compared to those from tropical zones due to their distinct abilities to deal with the osmotic and oxidative damage caused by cold stress. Theoretically, cold-tolerance genes are mostly correlated with detoxification, osmoprotection, chaperone, and ion/water transport functions. Due to the complex regulatory network of cold tolerance in plants, the overall coldtolerance capability probably results from a combination of multiple genes that function synergistically. In agreement with this, using the global USDA mini-core collection of rice and genome-wide association study (GWAS), we identified 37 cold-tolerance quantitative trait loci (QTL) over all 12 rice chromosomes. Of the 37 QTL, 7 were novel and not yet reported in previous studies, while the other 30 overlapped with or were near previously published QTL. We show here that LOC_Os04g24110, a glucosyltransferase gene, is a promising cold-tolerance candidate gene associated with LTSS4-1, a novel large effect QTL identified by GWAS mapping. To validate and fine map GWAS based QTL, we generated a bi-parental mapping population derived from a cross between a cold-tolerant accession from Russia and a cold-sensitive accession from Chad. This approach validated the LTSS4-1 QTL and uncovered a large-effect QTL on chromosome 8, qPGC8, which contains eight promising candidate genes associated with it. Potential mechanisms for chilling tolerance associated with LTSS4-1 and qPGC8 will be discussed.

Abiotic: Water 1000-059-Z Analysis of Inbreeding-stress Interactions in a RIL Population of Brassica Rapa Carmela Rosaria Guadagno – University of Wyoming Brent Ewers – University of Wyoming, Cynthia Weinig – University of Wyoming, Stanley DeVore – University of Wyoming In plant biology, recombinant inbred lines (RILs) are currently one important tool to primary map any trait that differs between the parental strains used to generate the population. Thanks to segregating variation at many loci, the homozygous genotypes show a wide array of phenotypic variation, which often exceeds the one between the two parental genotypes. This pronounced range of phenotypes makes RILs populations valuable materials for genetic studies, such as the detection of quantitative trait loci (QTL). Also, the same mapping population can be maintained for long periods of time and it can be repeatedly reproduced to map all kinds of different traits. However, the analysis of RILs can be biased by inbreeding depression, namely the presence of deleterious recessive alleles in the offspring of related individuals. In natural populations, a long history of intensive study has shown the presence of inbreeding–stress interactions. Although low vigor due to inbreeding depression is commonly accepted for some RILs of breeding populations, there is a gap in knowledge about its interaction with stressors. If these two sources of reduced fitness interact in a non-additive manner, thresholds for RILs population lifetime may be reduced. Inbreeding-stress interactions may also bias the predictions of models that utilize RILs populations assigning independent effects to these two fonts of disturbance. Here we propose to identify categories of stressors that are more or less likely to induce inbreeding-stress interactions. Specifically, we analyze RILs derived from a rapid cycling (IMB211) x Sarson (R500) cross of Brassica rapa. We utilize leaf chlorophyll a fluorescence as a good indicator of plant tolerance to environmental fluctuations, in order to tune the stress level across genotypes. Moreover, the redox state changes over time are monitored to clarify additional factors, which may be important in explaining the relevance of inbreeding-stress interactions in RILs populations.

1000-060-Y Chemical Genetics Mutants Impaired in Biotic-to-abiotic Interference Signaling in Arabidopsis Thaliana Jiyoung Park – University of California Tae-Houn Kim – Duksung Women’s University, Aaron Stephan – University of California, Rebecca Schwab – Max Planck Institute for Developmental Biology, Detlef Weigel – Max Planck Institute for Devel Drought and pathogen infection are two major factors limiting crop yield. Plants in the field are often exposed to more than one stress simultaneously. The plant hormone abscisic acid (ABA) is crucial for resistance to abiotic stresses including drought. Although knowledge of how plants defend against either of these stress forms has advanced, the interplay of the two tolerance mechanisms remains to be elucidated. A recent study using chemical genetics provides evidence for cross-interference, which occurs from biotic stress signaling to antagonize ABA signal transduction (1). A small molecule, DFPM, rapidly inhibits ABA-dependent gene expression and ABA-induced stomatal closure by activating pathogen effector-triggered immunity (ETI). Further studies revealed DFPM requires early central transducers of ETI, EDS1 and PAD4, and disrupts ABA signaling at the level of Ca2+signaling. To dissect this interference signaling, Arabidopsis thaliana harboring an ABA-inducible marker pRAB18:GFP have been EMS mutagenized and screened for altered responses to ABA and DFPM. Four rda (resistant to DFPM inhibition of ABA signaling) mutants were isolated for hyposensitivity to DFPM-mediated inhibition of ABA reporter expression. To map causative mutations, rda mutants were backcrossed into the parental line, and the F2 mapping population was used for whole genome re-sequencing (WGS). WGS results revealed an insertion site of the ABA reporter pRAB18:GFP in chromosome1. Furthermore, custom-made ShoreMap-based interval mapping identified one region clearly enriched with mutant-specific SNPs. In the region, no

SNP-carrying gene is known to have a role in ABA/Ca2+ signaling or pathogen resistance. Fine mapping is ongoing to identify the causative mutation for the rda phenotype. Our study shows chemical genetics screening combined with next generation sequencing-based mapping is identifying novel components involved in cross-talk mechanisms between pathogen resistance signaling and ABA signaling in plants. 1. T.H. Kim et al., Current Biology 21:990-997 (2011)

1000-061-Y Regulation of Primary Metabolism in Response to Flooding Stress as Revealed by 13C-stable Isotope Redistribution Carola Paepke – Max Planck Institute Molecular Plant Physiology Marcio Rocha – Universidade Federal do Amazonas, Houssein Diab – University of Angers, Anis Limami – University of Angers, Toshihiro Obata – Max Planck Institute Molecular Plant Physiology, Alisdair Fernie – Max Planck Institute Molecular Plant Physiology, Joost van Dongen – Aachen University Due to their sessile nature, plants cannot escape from regularly changing environmental and seasonal conditions that adversely affect their growth and development. Their survival depends largely on the initiation of highly complex adaptive responses involving stress sensing, signal transduction, and the activation of a number of stress-related genes and metabolites. Central metabolism including carbon, nitrogen and energy metabolism, is essential for plant life, and flexibility to reconfigure these primary metabolic pathways to sustain cellular homeostasis is crucial for plants to develop strategies that allow them to survive. Advances in understanding the global changes occurring in plant metabolism under specific abiotic stress conditions are fundamental to enhance plant fitness and increase stress tolerance. Current challenges in the analysis of the complex plant primary metabolome will be presented, focusing on a study of the metabolic adaptations of wild-type roots of the crop legume soybean (Glycine max) to hypoxia with GC-TOF-MS metabolite profiling. To address the redistribution of carbon through the metabolic pathways, stable isotope-labelling experiments were used to better understand the control and regulation of primary metabolic networks under hypoxia. 13C-pyruvate labelling was performed to compare flux through the TCA cycle, fermentation, alanine metabolism, and the γ-amino butyric acid (GABA)-shunt, whereas 13C-glutamate was performed to address the flux via glutamate to succinate. Our combined labelling data reveal the inhibition of the TCA-cycle enzyme succinate-dehydrogenase, also known as Complex II of the mitochondrial electron transport chain, explaining the bifurcation of the cycle and the down regulation of the rate of respiration measured during hypoxic stress. Moreover, an alternative carbon flux that would explain the accumulation of alanine (Ala), GABA, and succinate upon hypoxia via pathways mediated by an Ala- and GABA-shunt is suggested.

1000-062-Z Manipulating Stomatal Density Enhances Drought Tolerance Without Deleterious Effect on Nutrient Uptake Christopher Hepworth – The University of Sheffield Jon Hughes – The University of Sheffield, Timothy Doheny-Adams – The University of York, Lee Hunt – The University of Sheffield, Duncan Cameron – The University of Sheffield, Julie Gray – The University of Sheffield Under climate change it is predicted that the availability of water for agriculture will decrease across many parts of the world, making drought tolerant crops increasingly important. An obvious route to achieving drought tolerance would be to reduce transpirational water loss through stomata. However, recent high profile work has suggested a reduction in stomatal conductance and thus transpiration may impact on nutrient uptake; restricting the accumulation of nutrients

essential for both plant growth and human nutrition. Clearly, solving drought tolerance at the expense of nutrient content is a non-sustainable strategy, especially in light of predicted depletion in global phosphate reserves over the next half century Arabidopsis thaliana mutants specifically manipulated to have differing levels of stomatal development were used to determine whether plants with increased or decreased rates of transpiration would have altered drought tolerance and/or altered mass flow to and nutrient uptake by, the roots. Using natural abundance stable isotope (13C) profiling and 15N and 33P isotope tracers, we show the impact of manipulating plant water loss on the relationship between WUE, drought tolerance and nutrient uptake. Our results indicate alterations to root architecture, rate of stomatal closure and soil water content may mitigate the effect of a reduction in water towards the rhizosphere, suggesting drought tolerance may be possible without deleterious effects on plant nutrient content. This work has now been translated into barley and wheat and we show for the first time the functional effect of Epidermal Patterning Factor manipulation on stomatal development and drought tolerance in monocots. We discuss the feasibility of this method of drought tolerance for future crop breeding.

1000-063-Z Mapping the Tomato Root Gene Expression in Drought and Flooding Kaisa Kajala – UC Davis Siobhan Brady – UC Davis, Donnelly West – UC Davis Extreme precipitation patterns – droughts and flooding – are increasing with climate change. Plant development is a plastic process affected by environmental cues, and different plants respond to these water stresses differently. For maintaining agricultural yields it is critical to understand how crop species regulate their developmental patterns to adapt to the unfavorable environments. Our work aims to elucidate how major constituents of a delicious bowl of chili (tomatoes, legumes and rice) develop in response to drought and flooding, and how these environmental cues are integrated into plant development. We have developed a toolbox with a dozen of cell- and tissue-type specific promoters for roots of domesticated tomato Solanum lycopersicum cultivar M82 and wild drought-tolerant tomato S. pennellii. These promoters will allow us to profile gene expression changes on the level of specific cell-types. Furthermore, to investigate the regulatory level of the stress responses, we have optimized two molecular methods for isolating different regulatory compartments in tomato: nuclei by Isolation of Nuclei Tagged in specific Cell-Types (INTACT) and ribosomes by Translating Ribosome Affinity Purification (TRAP). This will allow us to map the stress-responsive changes to the epigenetic marks, nuclear transcriptome and the ribosomal transcriptome (“translatome”) and to understand how the cellular and morphological changes of flooded and droughted roots are regulated on a genetic level.

1000-064-Y Root to Shoot Signaling During Drought Stress: Closing the Gap Between Stomatal Control and Long-distance Drought Signals Annika Kreye – Cornell University Miguel Pineros – USDA-ARS, Peter Melcher – Ithaca College, Taryn Bauerle – Cornell University Stomatal conductance (gs) is mainly determined by the plant water status. Despite a thorough understanding of the role of short-distance signals in guard cell regulation, the relationship between long-distance signals and stomatal regulation

is, by contrast, extremely nascent. Likewise, the significance of embolism events for the onset of stomatal closure has yet to be determined. In this study, we simultaneously and continuously monitored changes in hydraulic and electrical long-distance signals noninvasively using acoustic emission measurements and surface electrodes, throughout intact sunflowers prior to and upon imposing two different levels of drought stress. We hypothesized that 1) Long-distance signals leading to stomatal closure originate in plant organs (petioles and roots), most prone to cavitation events; and 2) The interaction between hydraulic and electrical long-distance signals in plants leads to changes in stomatal aperture during drought stress. Results show that stomatal closure onset is highly correlated with the onset of hydraulic and electrical long-distance signals. Stomatal conductance, as well as net photosynthesis (A), remained stable over the course of the experiment until drought was induced via PEG8000 or natural dry-down treatments. The addition of -3.0 MPa PEG solution increased acoustic emission events (AEE) first in the petiole (98%) and then in the stem (92%). AEE of the petiole peaked at the same time as a decrease in gs was recorded. Likewise, changes in surface electrical potential (SEP) were found in both drought treatments, with the onset being correlated with the petiole AEE peaks. Additionally, the initial onset of cavitation events led to increases in gs, suggesting that the release of water from embolism serves as a temporary hydraulic capacitance system that is linked to stomatal responses. The observed change in gs to cavitation and electrical signaling provides insight to the intricate connections that exist across plant organ systems in response to drought.

1000-065-Y Changes in Water Availability Induce Folding of Intrinsically Disordered Stress Proteins from Plants César L Cuevas-Velazquez – Instituto de Biotecnologia-Universidad Nacional Autonoma de Mexico Gloria Saab-Rincón – Instituto de Biotecnologia-Universidad Nacional Autonoma de Mexico, Carlos Amero – Centro de Investigaciones Químicas, Universidad Autónoma de Mexico, Maria Martinez – The Scripps Research Institute, Gerard Kroon – The Scripps Research Institute, Jane Dyson – The Scripps Research Institute, Alejandra Covarrubias – Instituto de Biotecnologia-Universidad Nacional Autonoma de Mexico Late Embryogenesis Abundant (LEA) proteins are a broadly distributed group of proteins involved in the plant response to water deficit. Most of them are hydrophilins because of their high hydrophilicity and content of small amino acids, which are predicted to be intrinsically disordered proteins. By partial dehydration and freeze-thaw in vitro assays, it was shown that some LEA proteins protect other proteins from the effects of water-limitation and it was suggested that this might occur through protein-protein interactions. We have hypothesized that their putative flexible nature and their conserved motifs play critical roles in the interaction with their partners, allowing them to recognize diverse molecular targets. To get insight into their structure and its relation to their function, we have characterized the structural properties of two Arabidopsis group 4 LEA proteins (AtLEA4-2 and AtLEA4-5) using circular dichroism and nuclear magnetic resonance. We showed that both are intrinsically unstructured in aqueous solution; however, their intrinsic potential to form secondary structure was exhibited by treatment with a structure inducer, which promoted a significant a-helix formation (up to 88%). Importantly, a decrease in water availability and molecular crowding also induced folding in both LEA proteins. Moreover, we demonstrated that the N-terminal conserved region of AtLEA4-5 protein is necessary and sufficient to allow the conformational change to a-helix and to prevent the inactivation of reporter enzymes after several in vitro freeze-thaw cycles, at low molar ratios, supporting the hypothesis of direct protein-protein interaction as a protection mechanism. The structural and functional characterization described in this study demonstrates that particular regions in group 4 LEA proteins and their structural flexibility are required to protect molecular partners from the deleterious effects of water deficit. We are grateful to CONACyT (132258) and DGAPA-UNAM (IN208212). C.L. Cuevas was supported by a CONACyT PhD fellowship.

1000-066-Z Comparison of Drought Stress-induced Responses of Photosynthesis and Grain Yield Between a Normal and a Waxy Corn Hybrids (Zea Mays L.) During Tassel Emergence Seonghyu Shin – NICS Sang Gon Kim – NICS, Jin-Seok Lee – NICS, Beom-Young Son – NICS, Jiyoung Shon – NICS, Shingu Kang – NICS, Kang Bo Shim – NICS, Woonho Yang – NICS Drought stress during flowering stage brings about severe impact on corn yield. This study was to determine how much the drought stress during tassel emergence causes loss of grain yield in two distinct Korean elite F1 hybrid corns of ‘Gwangpyeongok’, a normal dent corn and ‘Ilmichal’, a waxy corn (wx/wx). Drought treatment was imposed by withholding water right before tassel emergence and then was relieved by re-watering at 0, 5, 10, 15, and 20 days after visible wilting, respectively. The relative leaf water content of drought-treated plants for 10 days of visible wilting was reduced by about 44% and fully recovered at 10 days after re-watering in the both hybrids, compared to the wellwatered. The photosynthesis and the leaf conductance rates of drought-treated plants for 10 days of visible wilting decreased by about 90% and recovered to levels of the well-watered at 10 days after re-watering in Ilmichal unlike Gwangpyeongok, equivalent to 77% of the well-watered, suggesting Ilmichal can make more rapid recovery from drought stress. For the both hybrids, response of grain yield to consecutive days of water-withholding during tassel emergence was best fitted with sigmoid function. Probit analysis revealed that the consecutive days of visible wilting to cause 50% reduction of grain yield was 4.9 days for Gwangpyeongok and 6.7 ones for Ilmichal, suggesting that every single day of water-withholding since visible wilting during tassel emergence can cause 7.0% and 9.4% reduction of grain yield in Ilmichal and Gwangpyeongok , respectively. These results indicate that Ilmichal (wx/wx) is less vulnerable to drought stress at tassel emergence than Gwangpyeongok.

1000-067-Z Enhanced Waterlogging Tolerance in Barley by Manipulation of Expression of the N-end Rule Pathway E3 Ligase PROTEOLYSIS6 Guillermina Mendiondo – The University of Nottingham Daniel Gibbs – University of Birmingham, Mirian Szurman-Zubrzycka – University of Silesia, Michael Holdsworth – The University of Nottingham Tolerance of crops to low oxygen (hypoxia) during flooding is a key target for food security. In Arabidopsis thaliana (L.) Heynh. the N-end rule pathway of targeted proteolysis controls plant responses to hypoxia by regulating the stability of group VII Ethylene Response Factor (ERFVII) transcription factors, controlled by the oxidation status of amino terminal (Nt)-Cysteine (Cys). Here we show that the barley (Hordeum vulgare L.) ERFVII BERF1 is a substrate of the N-end rule pathway in vitro. Furthermore we show that Nt-Cys acts as a sensor for hypoxia in vivo, as the stability of the oxygensensor reporter protein MCGGAIL-GUS increased in waterlogged transgenic plants. Transgenic RNAi barley plants, with reduced expression of the N-end rule pathway N-recognin E3 ligase PROTEOLYSIS6 (HvPRT6), showed increased expression of hypoxia-associated genes and altered seed germination phenotypes. In addition, in response to waterlogging transgenic plants showed sustained biomass, enhanced yield, retention of chlorophyll and enhanced induction of hypoxia-related genes. HvPRT6 RNAi plants also showed reduced chlorophyll degradation in response to continued darkness, often associated with waterlogged conditions. Barley Targeting Induced Local Lesions In Genomes (TILLING) lines, containing mutant alleles of HvPRT6, also showed increased expression of hypoxia-related genes and phenotypes similar to RNAi lines. We conclude that the N-end rule pathway represents an important target for plant breeding to enhance tolerance to waterlogging in barley and other cereals (Mendiondo et al. 2015). Nowadays, we are studying how the N-end rule pathway controls another abiotic stress such drought and salinity in Barley.

1000-068-Y Scaffold Protein RACK1A Inhibitor Compounds Provide Abscisic Acid Hypersensitivity to Allow Crop Plants Withstand Drought and Salt Stresses Hemayet Ullah – Howard University Mercy Sabila – Howard University, Ahsan Rahman – Howard University, Albandari Albehishi – Howard University, Rachel Darko – Howard University, Kyaira Ware – Howard University, Sivanesan Dakshanamurthy – Georgetown University RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in single cell eukaryote yeast to human and plays regulatoryroles in diverse signal transduction and stress response pathways. Loss of function mutant in Arabidopsis indicates that RACK1A regulates diverse environmental stress resistance and developmental pathways through a negative regulation of stress hormone abscisic acid ABA) signaling pathway . It is hypothesized that chemical knock-out, as opposed to genetic knock-out, of RACK1A will provide a functional advantage in protecting plants from diverse stress through the effect of hypersensitivity to the ABA. Site directed mutagenesis studies indicated that the key post-translational modifications like sumoylation at K273 and tyrosine phosphorylation of Y248 residues dictate the RACK1A’s potential to interact with other proteins. In order to facilitate the identification of small compounds binding to the functional pocket, the crystal structure of RACK1A protein is deduced at 2.4 A resolution. Deduced crystal structure of RACK1A is used to identify dozens of small compounds that could potentially bind to the Y248 pocket. The compounds could potentially inhibit Y248 prosphorylation and bind to purified recombinant RACK1 protein with a kD value in the micro-molar ranges. The compounds can effectively elicit ABA signaling pathways through activating the ABI5 gene expression as evaluated in the ABA induced seed germination, growth inhibition assays. The effectiveness of the compounds in regulating diverse environmental stress responses are evaluated in different crop plants. Here we present, evidence that the compounds are effective in regulating salt and drought stress responses in wide variety of crop plants including in tomato, beans, and pepper. To allow ease of application to crop plants, the compounds are being formulated as fertilizer additives.

1000-069-Y Starch Degradation Induced by Abscisic Acid Confers Resistance to Osmotic Stress in Arabidopsis Thaliana Matthias Thalmann – University of Zürich Diana Pazmino – University of Zürich, Daniel Horrer – University of Zürich, Arianna Nigro – University of Zürich, Diana Santelia – University of Zürich Water stress is a major factor limiting the productive potential of crop plants. One of the most prominent responses of plants to water stress is the accumulation within the cell of large amounts of compatible solutes (e.g. sugars), which retain water and protect proteins and membranes against damage and denaturation caused by dehydration. There is increasing evidence that starch degradation plays a major role in this process. In our study, we showed that in Arabidopsis thaliana leaves starch is rapidly degraded upon osmotic stress, as indicated by the reduced starch content and the accumulation of the starch catabolite maltose. Two specialized starch degrading enzymes α-amylase 3 (AMY3) and β-amylase 1 (BAM1), which are not required for night time leaf starch degradation, work synergistically to degrade starch during stress. After being released from starch, maltose is further metabolised to other sugars, mainly sucrose. Sucrose is subsequently exported to the roots, where it promotes primary root elongation and lateral root formation to counteract the stress. Notably, amy3bam1 mutants are more sensitive to osmotic stress, as they fail to degrade starch in response to stress and have reduced root growth compared to the wild type. We also showed that BAM1 and AMY3 transcripts accumulate upon osmotic stress in an ABA dependent manner. Moreover, mutants impaired in ABA synthesis or signalling cannot degrade starch during osmotic stress, whereas application of exogenous ABA triggers starch degradation even in the absence of stress. These results suggest that ABA is most likely the trigger of the observed daytime-stress mediated starch degradation.

1000-070-Z High-throughput Phenotyping of Natural Variation in Brachypodium Distachyon Under Combinations of Progressive Drought and Heat Stress Malia Gehan – Donald Danforth Plant Science Center Noah Fahlgren – Donald Danforth Plant Science Center, Tracy Hummel – Donald Danforth Plant Science Center, Stephanie Turnipseed – Donald Danforth Plant Science Center, Todd Mockler – Donald Danforth Plant Science Center The world population relies on grasses, such as rice, and wheat, as principal sources of calories, but these crops are not expected to meet global demand by 2050. The geographical distribution of food and bioenergy crops is limited by several factors including temperature, soil salinity, and water availability. To tackle the daunting challenge of producing more food and fuel with fewer inputs a variety of strategies to improve and sustain crop yields will be explored. These strategies may include: mining natural variation of wild crop relatives to breed crops that require less water; increasing crop temperature tolerance to expand the geographical range in which they grow; and altering the architecture of crops so they can maintain productivity while being grown more densely. These research objectives can be achieved with a variety of methodologies, but they will require both high-throughput DNA sequencing and phenotyping technologies. A current bottleneck in agricultural science is the ability to efficiently quantify plant traits (phenotypes) through time. The development of high-throughput phenotyping technologies and open-source, flexible, and translatable analysis tools that extract agronomically important traits is the main focus of this research. B. distachyon is a C3 model grass that is closely related to wheat, and rice, which has similar architectural features. An accession panel of 143 B. distachyon accessions was screened under, control, drought, heat, and drought and heat conditions and the resulting imaging data was examined for traits that approximate water-use-efficiency, biomass accumulation, tiller count, and plant water content (near-infrared imaging) using new open-source phenomics software tools. Importantly, this digital physiology data was grounded with manually measured traits.

1000-071-Z The Growth Zone of Maize Leaves Is an Ideal Model System for Systems Biology Approaches to Investigate the Effects of Drought Stress on Organ Growth Regulation Viktoriya Avramova – University of Antwerp Hamada AbdElgawad – University of Antwerp, Yves Guisez – University of Antwerp, Han Asard – University of Antwerp, Gerrit Beemster – University of Antwerp Drought is the most important yield-limiting factor under natural and agricultural conditions and therefore detailed knowledge of its impact on plant growth regulation is crucial. The maize leaf represents an attractive system for growth studies, because of its spatial gradient, allowing sampling of dividing, expanding and mature cells at the same time, and its big size, providing enough material for molecular analyses: a big advantage over the model plant Arabidopsis thaliana. By means of a kinematic analysis we showed that drought inhibits leaf growth by reducing cell division rates in the meristem and cell expansion rates in the elongation zone. A transcriptome analysis provided a molecular basis for the observed inhibition of cell division rates, but also pointed at distinct effects on the development of the photosynthetic machinery, and antioxidant and redox systems. The effects on these pathways were subsequently confirmed by detailed biochemical analysis of the activity of key enzymes and quantification of metabolites. We demonstrated the functional significance of the identified transcriptional and physiological changes, showing that 1. Increasing the antioxidant capacity in the growth zone, by overexpression of iron superoxide dismutase, increases leaf

growth under control and drought conditions. 2. Increased expression of photosynthesis genes under stress facilitates faster growth upon re-watering compared to unstressed controls.

1000-072-Y The Interactome of Soybean GmWRKY53 Using Yeast 2-Hybrid Library Screening to Saturation Prateek Tripathi – University of Southern California Roel Rabara, Mani Choudhary, Marissa Miller, Ying-Sheng Huang, Stéphanie Blachon, Qingxi Shen, Paul Rushton Soybean GmWRKY53 functions in both biotic and abiotic stress signalling. Using GmWRKY53 as a bait yeast two-hybrid library screening to saturation isolated multiple independent fragments for many interacting proteins, enabling delineation of minimal interacting domains and computation of a confidence score. Multiple independent clones coding for the LATE ELONGATED HYPOCOTYL clock protein GmLCL2 (MYB114) were isolated and the binding site for GmWRKY53 was mapped to ninety amino acids separate from the MYB domain. This suggests a direct input from the clock on GmWRKY53 activity. The GmWRKY53-interacting proteins also included three water stress-inducible AP2/ERF transcription factors. One of these (Glyma03g26310) is one of the most strongly water stress induced genes in soybean roots, suggesting that GmWRKY53/ERF complexes regulate water stress responses.

1000-073-Y The Resurrection Plant Tripogon Loliiformis Manipulates Trehalose Metabolism to Induce Autophagy and Tolerate Desiccation Brett Williams – Queensland University of Technology Isaac Njaci – Queensland University of Technology, Lalehvash Moghaddam – Queensland University of Technology, Hao Long – Queensland University of Technology, Martin Dickman – Texas A & M University, Xiuren Zhang – Texas A & M University, Sagadevan Mundree – Queensland University of Technology Over the next fifty years there will be a massive challenge to sustain an ever-increasing global population. With climate forecasts predicting increasing erratic weather globally and reduced crop yields, how do we fulfil this ensuing supplydemand gap? A small group of angiosperms termed resurrection plants have the ability to tolerate desiccation and return from a dormant state upon availability of water may serve as a unique genetic resource for the generation of resilient crops that can survive and yield during unfavourable conditions. Here, we use one such plant, Tripogon loliiformis, to answer two intriguing questions on resurrection plant resilience; Do pre-existing tissues “resurrect” or are they “awakened” from a dormant state upon the addition of water?, and What mechanisms are implemented to protect cells during desiccation? We show that T. loliiformis leaf tissues are alive in desiccated plants and that pre-existing tissues resurrect upon the addition of water. Notably we describe a unique regulatory role for the non-reducing sugar trehalose in the activation of autophagy pathways in T.loliiformis. Transcriptome, Gas Chromatography Mass Spectrometry and confocal microscopy analyses correlated trehalose accumulation with the onset of autophagy in dehydrating T. loliiformis shoot tissues. These results were supported in vitro with the observation of autophagy in hydrated T. loliiformis leaves following treatment with a 25 mM trehalose solution. Autophagy can promote survival of cells by removal of damaged organelles and misfolded proteins to suppress PCD and nutrient recycling to delay the onset of senescence. These findings illustrate how resurrection plants manipulate sugar metabolism to survive desiccation and may provide potential targets for the development of stress tolerant crops.

1000-074-Z Leaf Age and Water Availability Effects on Leaf Anatomy and Mesophyll Conductance of Wheat (Triticum Aestivum L.)

Eisrat Jahan Understanding the effects of different light levels and leaf age on photosynthetic capacity is important as these are major factors for growth and development, in terms of net carbon gain. Mesophyll conductance (gm) influences photosynthetic rate and leaf intrinsic water-use efficiency (A/gsw), and has been found to respond to environmental conditions and leaf anatomical traits. To understand the combined effect of leaf age, water limitation and irradiance on gm in wheat (Triticum aestivum L.), we used a leaf gas exchange system coupled to a tunable-diode laser absorption spectrometer to measure carbon isotope discrimination and allow estimation of gm, together with cross sectional microscopic analysis to determine the surface area of chloroplasts exposed to the intercellular air space (Sc). Both Sc and gm decreased as leaves aged. Photosynthetic rate (A) and gm increased with increasing photon flux density (PFD) from 300 to 1300 µmol m-2 s-1 but decreased with increasing leaf age from young to old. However, gm did not increase with increasing PFD for old leaves. Stomatal conductance (gsc) and gm were significantly higher for irrigated compared with drought leaves, and gm increased with increasing PFD for both irrigated and drought plants, but only young leaves. gm and A were more strongly correlated than gm and gsc for both irrigated and drought conditions. These results underscore a high level of variability in wheat gm in response to environmental and physiological conditions, and will allow estimation of the relative limitations to photosynthesis as leaves age.

1000-075-Z Carbohydrate Metabolism and the Trehalose Biosynthetic Pathway in Maize Kernels Under Sucrose Starvation Mark Lagrimini – University of Nebraska-Lincoln Samuel Bledsoe, Clemence Henry, Regina Feil, John Lunn, Mark Stitt High temperatures and inadequate rainfall at certain stages in crop development can have disastrous consequences to yield. In maize, drought occurring near or during the flowering stage often causes significant kernel abortion that greatly impacts potential yield. The trehalose biosynthetic pathway has recently been found to be important in plant metabolism in response to stress in higher order plants. Trehalose is currently known throughout the plant and animal kingdoms as an osmoprotectant, high energy fuel source, structural component, and involved in pathogen response. New insight on the role of the trehalose pathway focuses on the sugar phosphate intermediate trehalose-6-phosphate (Tre6P) and its regulatory role on the Sucrose non-fermenting Related Kinase 1 (SnRK1). SnRK1 has been shown to be a central regulator of numerous catabolic and anabolic events critical to plant metabolism. Slight changes in trace Tre6P levels within the plant often cause dramatic phenotypes suggesting that Tre6P is acting as a metabolic switch in response to carbon availability. Understanding the role of the trehalose and SnRK1 pathways in higher plant species such as maize will provide a better understanding of carbon partitioning in plants especially as it pertains to kernel abortion and potentially increasing yields. This study first explores the practicality of in vitro kernel culture as a means to evaluate sink strength in the context of regulation by Tre6P and the SnRK1 pathway on maize inbred B73, a Nested Association Mapping population (NAM) parent line and the model organisms for this study. Sink strength is then characterized for 14 additional NAM inbred parent lines as well as a more detailed analysis of Tre6P and SnRK1 involvement for selected inbred lines Oh43 and M37W. The findings of this study greatly support the involvement of the SnRK1 pathway in response to sucrose starvation stress and its regulation by Tre6P.

1000-076-Y Biochemical Analyses of Sorghum Varieties Reveal Differential Mechanisms of Tolerating Drought Chukwuma Ogbaga – University of Illinois Piotr Stepien, Beth Dyson, Giles Johnson

We have examined the biochemical responses of two sorghum cultivars of differing drought tolerance, Samsorg 17 (more drought tolerant) and Samsorg 40 (less drought tolerant), to sustained drought. Plants were exposed to different degrees of drought and then maintained at that level for five days. Responses were examined in terms of metabolic changes and the expression of drought induced proteins - Heat Shock Proteins (HSPs) and dehydrins (DHNs). Generalised phenotypic changes were studied using Fourier transform infrared (FT-IR) Spectroscopy and non-targeted Gas Chromatography Mass Spectrometry (GC-MS) was employed to detect changes in metabolites, while changes in protein expression were examined using Western blot analysis. Different response profiles of metabolites, HSPs and DHNs were observed in the two cultivars. Metabolic changes involved variation in amino acids, polysaccharides and their derivatives. A total of 188 compounds, with 142 known metabolites and 46 unknown small molecules, were detected in the two sorghum varieties. Under water deficit conditions, Samsorg 17 accumulated sugars and sugar alcohols, while in Samsorg 40 amino acids increased in concentration. This study suggest that the two Sorghum varieties adopt distinct approaches in response to drought, with Samsorg 17 being better able to maintain leaf function under severe drought conditions.

1000-077-Y Genome-Wide Association Study of Drought Resistance in Cultivated Sunflower Seedlings Rishi Masalia – University of Georgia John Burke – University of Georgia Of all abiotic stresses that negatively impact crop productivity, drought is considered to be one of the most devastating. Cultivated sunflower (Helianthus annuus) is grown in predominantly rainfed (as opposed to irrigated) regions and drought at the seedling stage critically curtails stand establishment and potential yield. Functionally, resistance to water deficits have been well characterized through osmotic adjustment, early flowering, and improved water uptake strategies, however the underlying genetic mechanisms governing these strategies are still poorly understood. In the last decade, an appreciable genomic and transcriptomic effort has taken place to elucidate the mechanisms underlying drought resistance in both crop and model plant species, but little of this work has translated to improved breeding efforts. One contributing factor is that drought resistance is comprised of many genes of small effect, which are troublesome to detect. To address this concern our project utilizes an established sunflower association mapping (SAM) population with a preliminary dataset of roughly 4M SNP markers (MAF ≥10%) across 288 inbred lines to identify candidate genes involved in drought resistance. With increased recombination events and marker density this population allows for the detection of genomic regions and genes of small effect associated with this complex phenotype. To assess phenotypic variation across treatments (drought, control) and inbred lines, polyethylene glycol molecular weight 6000 (PEG-6000) was used to impose a controlled chemical drought in five-day-old seedlings for a tenday duration. Though this project is still ongoing, we have identified associations across the sunflower genome for 18 quantitative seedling vigor traits, most of which focus on root architecture and growth. This poster focuses primarily on preliminary results, methodological advances in the implementation of drought stress, and strategies for highthroughput phenotyping.

1000-078-Z OsbZIP23 and OsbZIP45, Members of the Rice Basic Leucine Zipper Transcription Factor Family, Are Involved in Drought Tolerance Su-Hyun Park – Crop Biotechnology Institute, GreenBio Science and Technology, Seoul National University Jin Seo Jeong – Seoul National University, Kang Hyun Lee – Seoul National University, Youn Shic Kim – Seoul National University, Yang Do Choi – Seoul National University, Ju-Kon Kim – Seoul National University

Many stress-inducible genes including the transcription factor basic leucine zipper (bZIP) are involved in the response of plants to environmental stresses. bZIPs are composed of two domains, a basic region for DNA binding and a leucine zipper region for dimerization. In this study, two drought-induced bZIP genes OsbZIP23 and OsbZIP45 were identifiedin rice. The transcription factors are orthologs of Arabidopsis bZIPs belonging to groups A and G, respectively, and are known to be involved in drought tolerance. To investigate the regulation of OsbZIP23 and OsbZIP45 expression in rice, quantitative RT-PCR was performed using RNAs from plants grown at drought stress conditions and different developmental stages. Expression of OsbZIP23 and OsbZIP45 showed positive correlation with drought tolerance. To further understand the functions of OsbZIP23 and OsbZIP45, we overexpressed OsbZIP23 and OsbZIP45 in rice by using PGD1 promoter. Results of phenotypic and chlorophyll fluorescence analysis on PGD1:OsbZIP23 and PGD1:OsbZIP45 plants showed enhanced tolerance to drought stress. These results suggest that OsbZIP23 and OsbZIP45 are involved in drought stress response in rice and have a great potential for engineering drought tolerant crops.

1000-079-Z Nitrogen Reallocation and Remobilization During Desiccation of Sporobolus Stapfianus Abou Yobi – USDA-ARS Douglas Allen – USDA-ARS, Melvin Oliver – USDA-ARS Perennial grasses have evolved mechanisms to respond to soil nitrogen availability that involve nitrogen remobilization within the plant. Movement occurs from mature to developing leaves during active growth or through redirection to roots for storage during dry seasons or at the onset of winter. In resurrection grasses, however, drying induced leaf senescence is limited to mature leaves. Our results have shown that during dehydration, many amino acids accumulate significantly in immature leaves, but not in roots. We hypothesized that the nitrogen in the mature leaves is remobilized into immature leaves for storage and utilization when the plant rehydrates. Our transcriptomic analysis, however, has revealed an increase in the abundance of transcripts encoding enzymes in amino acids biosynthetic pathways during the dehydration of immature leaves, which suggests a role of biosynthesis in the accumulation of amino acids as well. To better understand nitrogen movement and potential carriers; we used a hydroponic system to conduct a pulse experiment with 15N-labeled nitrogen source. Our results demonstrated that several 15N-labeled amino acids increased in abundance during the dehydration of immature leaves coincident with a decrease in their abundance in mature leaves. Consistent with our previous findings, asparagine and alanine decreased in all the organs investigated, which suggests that they are catabolized during dehydration, perhaps redirecting the nitrogen into other amino acids. Lastly, with few exceptions, the 15N fraction of most amino acids increased or remained unchanged in the dehydrated roots, demonstrating that roots were not the source of the labeled nitrogen in the immature leaves. Overall, we conclude that the observed accumulation of amino acids in the leaves of desiccating immature leaves of S. stapfianus occurs as the result of both enhanced biosynthesis using stored nitrogen and a remobilization of nitrogen from mature desiccation sensitive leaves as they senesce.

1000-080-Y Is miR399 Regulated by a Target Mimic in the Maize Primary Root Growth Zone Under Water Deficit Stress? Candace Seeve – USDA-ARS Ramanjulu Sunkar – Oklahoma State University, Robert Sharp – University of Missouri, Melvin Oliver – USDA-ARS MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate the expression of protein-coding genes by binding to complementary regions within the target gene transcript, thereby guiding cleavage of the transcript by the nuclease Argonaute, or repressing its translation. The expression of miRNAs is regulated both transcriptionally and posttranscriptionally. Post-transcriptional regulation occurs during processing of the precursor miRNA transcript to generate the mature miRNA, or by interaction with a class of long, non-coding RNAs called miRNA target mimics that bind to the

miRNA via a near-complementary region without being cleaved. Using an established vermiculite-based seedling culture system to impose defined low water potential conditions (-0.3 MPa and -1.6 MPa, for 26 h), we identified more than 40 miRNAs by RNA-Seq that significantly responded to water deficits in the growth zone (apical 10 mm) of the maize primary root. Among these, members of the zma-miR399 family accumulated to significant levels (above the wellwatered control). However, we were able to identify only two target transcripts regulated by the zma-miR399 members during exposure to water deficits using either degradome sequencing, or by quantitative PCR (qPCR) directed at published in silico predicted zma-miR399 targets. Blastn searches of the NCBI maize EST libraries and contig assemblies revealed a non-protein coding RNA containing a sequence that is nearly complementary to the zma-miR399 sequence. qPCR assays revealed that the mimic transcript abundance increased under water deficit conditions. We hypothesize that this non-coding RNA is a target mimic that sequesters miR399 and disrupts its regulation of other target genes. miR399 target mimics have been identified in several species, however, to our knowledge this is the first report of a miR399 target mimic in maize. It is also the first report that miR399, a well-characterized regulator of phosphate homeostasis, and its target mimic are concurrently induced under water deficit stresses.

1000-081-Y Molecular Regulation of Remobilization of Carbon Reserve in Straws of Super Rice Guanqun Wang – The Chinese University of Hong Kong Jianhua Zhang – The Chinese University of Hong Kong Hybrid rice cultivars have large panicles with huge yield potential but realization of the yield potential is limited by low grain filling rate and incomplete use of pre-stored carbon reserve in the straws. In contrast to most conventional rice cultivars, carbon reserve in straws may increase, rather than decrease, at the middle to late grain filling stage in some super rice cultivars. Kernels of late-flowering are poorly filled as a consequence. There is in most cases food leftover, unused in their straws at maturity. In our early research we found that proper soil drying at the middle to late grain filling stage can promote the remobilization of this carbon reserve. Grain filling can be enhanced with better developed kernels. The molecular mechanisms, however, are not clear for the regulation of food remobilization in the temporary storage organs, i.e. the sheath and stem, under the mild soil drying. Our hypothesis is that carbon remobilization is regulated by biochemical steps inside the straws (the stem and sheath). Since super rice shows unique variations in grain filling rate and carbon remobilization, we believe there must exist some key regulatory factors controlling the sugar and starch metabolism in the stem and sheath that determine how quickly the carbon flow can be and where the carbon flow should go in the later grain filling stage.

1000-082-Z Tissular Localization of Transcripts and Proteins Induced by Water Deficit in Arabidopsis Thaliana: Group 4 LEA Proteins Coral Martinez – Instituto de Buotecnologia UNAM Cesar Luis Cuevas – Instituto de Biotecnologia UNAM, David Rendon – Instituto de Biotecnologia UNAM, Alejandra Covarrubias – Instituto de Biotecnologia UNAM Late Embryogenesis Abundant (LEA) proteins accumulate during the last stages of seed development and in vegetative tissues in plants under water deficit. These proteins play relevant roles in dehydration tolerance in plants; however, little is known about their function and localization in plant organs and tissues. Previous work uncovered the relevance of a group 4 LEA protein in plant tolerance to water deficit. This group is formed by three members (AtLEA4-1, AtLEA4-2 y AtLEA4-5) and it was shown that mutant plants deficient in any of these members are more susceptible to water deficit. Although LEA proteins may perform similar functions, the fact that the deficiency of these proteins exhibit detectable phenotype that is not compensated by the others members of the group could be explained by specific spatio-temporal

distribution of each of these proteins during water deficit. In this work we focused on the localization of AtLEA4-5 transcript and protein, the gene from group 4 that shows the highest response to water deficit conditions. By using transgenic Arabidopsis plants containing transcriptional fusions of the AtLEA4-5 promoter with a reporter gene (GUS), we were able to detect expression of the reporter protein in primary and lateral roots of seedlings subjected to water deficit; whereas in the aerial part of these stressed seedlings, expression was specifically detected in leaf primordia. In mature embryo we detect expression, predominantly in radicle. At protein level, it was localized by translational fusion of the AtLEA4-5 protein with fluorescent reporter protein EGFP. Protein was localized in primary and lateral roots, more abundantly in vascular tissues of seedlings subjected to water deficit. In embryo we detect expression, predominantly in radicle as well. Interestingly, at the cellular level, this protein was found widely distributed in the cytoplasm and nucleus of epidermal cells of radicle embryos.

1000-083-Z OsHIGD2 Is Involved in Hypoxia-induced Stem Growth in Deepwater Rice Soong-Taek Hwang – Kunsan National University Dongsu Choi – Kunsan National University Recent evidence supports ethylene as a critical factor in modulating plant responses to flooding stress. We found that hypoxia also promotes stem elongation in deepwater rice even in the absence of ethylene. Five HIGD (Hypoxia-Induced Gene Domain) protein genes were identified from deepwater rice in a series of gene-chip microarray analyses for profiling genes that are specifically regulated by submergence. Among genes with differential expression patterns, OsHIGD2 showed rapid induction by hypoxia as well as submergence. Coexpression database search confirmed that OsHIGD2 shows closely-related expression pattern with OsADH1 (Alcohol DeHydrogenase1). We also looked for OsHIGD2-interacting partners by yeast two hybrid assay using OsHIGD2 as bait. As a result, we found and confirmed 6 proteins as OsHIGD2-interacting ones, most of which are related to regulation of redox potential or fermentation. In this report, we present evidences that HIGD2 plays a role in a mitochondrial mechanism required to tolerate low oxygen stress.

1000-084-Y Making an Aquaporin Water-tight: The Structural Basis of Selectivity of Plant Nodulin 26-Intrinsic Proteins Daniel Roberts – The University of Tennessee Tian Li – The University of Tennessee, Zachary Beamer – The University of Tennessee The evolution of land plants led to an amplification and diversification of the aquaporin superfamily of membrane channels. Among the subfamilies of plant-specific aquaporin-like channels are the nodulin-26 intrinsic proteins (NIPs) which are multifunctional transporters of water, ammonia, glycerol and metalloid nutrients that participate in a number of osmoregulatory and metabolic functions. Structurally, NIPs share the canonical hourglass fold and overall pore architecture of the aquaporin superfamily, but possess substitutions within the aromatic arginine (ar/R) selectivity filter that lead to distinct transport functions. The ar/R is formed by the confluence of four conserved amino acids within the pore and control transport selectivity based on pore diameter, hydrophobicity and hydrogen bonding. The nine proteins of the Arabidopsis NIP subfamily are subdivided into two ar/R subgroups: NIP subgroup I, which are permeated by glycerol, water and ammonia, and NIP subgroup II, which serve as plasma membrane boric acid channels which are essential for boron homeostasis under limiting conditions. NIP II proteins also lack aquaporin activity and are essentially "water tight". NIP I and II proteins differ principally by the substitution of a conserved alanine (NIP subgroup II) for a conserved tryptophan (NIP subgroup I) in the helix 2 position of the ar/R filter. Based on transport analyses and molecular dynamics simulation, a model is proposed through which the alanine substitution results in both the selectivity for critical metalloid nutrients such as boric acid while simultaneously restricting water flow through the ar/R

selectivity filter. A mechanism involving two different rotameric states of the conserved arginine residue in the ar/R region is proposed to be responsible for the water-tight character of the pore. This mechanism is proposed to facilitate the selective uptake of metalloid nutrients while preventing water flux, perhaps as a turgor maintenance function of the pore. (Supported in part by NSF IOS-1121465).

1000-085-Y Sensory Potentiation of Rapid Hyperosmotic-induced Ca2+ Responses in Arabidposis Thaliana Aaron Stephan – University of California San Diego Hans-Henning Kunz, Julian Schroeder Plants experience hyperosmotic stress when faced with drought or saline soils, but it is currently unclear how plants perceive this stress in a dynamic environment. Hyperosmotic stress induces a rapid rise in intracellular Ca2+ in plants. Here we show that these Ca2+ response profiles in the reference plant Arabidopsis thalianaexhibit a compensatory mechanism that enhances response magnitudes to smaller relative shifts in stimulus intensity — a phenomenon we term “sensory potentiation”. The sensing, signaling, and potentiation occur in roots and can be modulated. We present evidence that organellar transporters play a role in elicitation of rapid hyperosmotic-induced Ca2+ responses, but do not significantly affect potentiation of the response. Together, these detailed Ca2+ response profiles indicate that plants perceive both relative shifts and absolute intensities of stress.

1000-086-Z A Tale of Two Tomatoes: Cell Fate Plasticity in the Shoot Apical Meristem During Water Stress Donnelly West – UC Davis Kaisa Kajala – UC Davis, Siobhan Brady – UC Davis, Neelima Sinha – UC Davis All plants have to respond quickly to environmental conditions or perish. However, domesticated plant species lack some of the response vigor seen in their close wild relatives. In order to investigate the responses to water stress in tolerant and susceptible species of tomato (Solanum lycopersicum and Solanum pennellii), we couple the comparison of classic anatomy and morphology with comparative genetics. To parse out the genetics of specific cell populations in the shoot apical meristem, differentiating leaf tissue, and leaf vascular tissue, we use cell-type specific isolation techniques for nuclei (Isolation of Nuclei TAgged in specific Cell Types - INTACT) and ribosomes (Translating Ribosome Affinity Purification - TRAP). The INTACT and TRAP methods allow isolation of transcriptional, translational, and chromosomal regulation information in morphologically indistinguishable, yet genetically distinct cell populations. Responses in developmental genetics to waterlogging or insufficient-watering conditions result in significant morphological differences. Precisely identifying these gene networks can help illuminate cellular fate, tissue-type commitment, and water-stress response.

1000-087-Z Polyamines-mediated Regulation of Antioxidative Genes in Soybean Seedlings Under Excess Moisture Stress Gagandip Sidhu – University of Manitoba Belay T. Ayele – University of Manitoba Excessive soil moisture affects crop growth and developmental processes and in turn yields, partly through inducing the production of reactive oxygen species (ROS), which lead to oxidative cellular damage. Early developmental stages are especially sensitive to such damage. Polyamines, naturally occurring plant growth substances, have been shown to promote the activity of antioxidative enzymes and thereby reduce ROS production and enhance crop performance under stress conditions. To assess the role of polyamines, spermidine and spermine, in enhancing the enzymatic ROS

scavenging capacity of plants under stress condition, this study examined the expression patterns of genes encoding ascorbate peroxidase 1(APX1), catalase 1(CAT1) and superoxide dismutase 1(SOD1) in soybean seedlings grown from seeds treated with spermidine or spermine. Our data shows that treatments of seeds with spermine led to increased expression of all the three antioxidative genes both in the cotyledon and embryonic axis tissues of seedlings grown under excess soil moisture. Relative to the other genes, the effect of spermine on the expression of CAT1 was much higher in both cotyledon (5-fold) and embryo axis (6-fold) tissues. No substantial changes in the expression of the target genes from both cotyledon and embryo axis tissues were apparent in response to treatment with spermidine.

1000-088-Y Sub1 Rice: Reprogramming of Transcription and Metabolism upon Desubmergence Is Linked to Photosynthetic Recovery Anna Locke – University of California, Riverside Gregory Barding – California Polytechnic State University, Pomona, Cynthia Larive – University of California, Riverside, Julia Bailey-Serres – University of California, Riverside Rice has two major strategies for surviving complete submergence due to flooding: rapid elongation and growth quiescence. The quiescence trait is conferred by an ethylene-responsive transcription factor, SUB1A, and it has been introduced via marker-assisted breeding into high-yielding, farmer-preferred rice varieties grown in flood-prone regions of South and Southeast Asia. During submergence, SUB1A upregulation and protein accumulation restricts carbohydrate consumption and gibberellic acid (GA)-induced elongation by promoting accumulation of the GA signaling repressors SLR1 and SLRL1. SUB1A’s growth-restriction mechanism during submergence is accompanied with enhancement of post-submergence recovery. To better understand SUB1A function in gene, metabolism, and growth regulation, we performed transcriptomic and metabolomic analyses over a 24 hour post-submergence recovery period in near-isogenic M202 and M202(Sub1) rice varieties. Following a 3-day submergence event, 41 metabolites were measured at five time points with GC-MS, NMR, and RPIP-UPLC-MS; transcript accumulation was monitored for the same time points with RNA-seq. These analyses demonstrated that SUB1A continues to alter metabolism following desubmergence, particularly at the levels of starch/sucrose catabolism and photosynthesis. Although the beneficial effects of SUB1A during submergence have primarily been associated with restricted carbohydrate breakdown, GO term analysis showed that genes associated with photosynthesis are consistently more highly expressed in submergencetolerant Sub1 plants than in intolerant plants. This up-regulation of photosynthesis genes at the transcriptional level may result in better maintenance of the photosynthetic apparatus in Sub1 plants during submergence, allowing plants to more rapidly resume physiological function upon reoxygenation. Consistent with this, gas exchange measurements confirm the recovery of photosynthesis only a few hours after desubmergence in Sub1 plants, while it is still depressed in submergence-intolerant plants. Thus, the enhanced survival and recovery from submergence conferred by SUB1A, shown previously to involve multiple pathways, includes regulation of the photosynthetic apparatus, which may also impact other abiotic stress responses.

1000-089-Y Aging in Moss: Lipid Peroxidation by ROS Does Not Limit Longevity in Dry Storage Tyler Sternhagen – University of South Dakota Karen Koster – University of South Dakota Desiccation tolerance generally enables the long-term survival of seeds and other organisms; however, longevity is limited by damage that accumulates in dry tissues, even under optimal conditions. We have begun studying the types of damage incurred by dry tissues using the moss Physcomitrella patens, in which desiccation tolerance can be induced by treatment with exogenous abscisic acid (ABA). We determined how the longevity of dried, desiccation-tolerant

Physcomitrella was affected by relative humidity (RH) at 24°C. We observed that only 50% of the moss survived when stored in darkness at 75% RH for one week, however, moss survival did not decrease at 32% RH until after four weeks. Reactive oxygen species (ROS) are reported to cause significant damage to dry tissues and thereby limit storage longevity. ROS do this, in part, by oxidizing lipids to form hydroperoxides, which compromise membrane structure. To assess this possibility, we measured lipid hydroperoxide contents in Physcomitrella and determined that levels increased approximately 10-fold during dehydration, but did not increase further during four weeks of dried storage at either 32% or 75% RH in the dark. Hydroperoxide content did not differ between moss samples stored at 32% and 75% RH. Membrane damage was evident in moss that did not survive storage; protoplasts failed to re-expand during rehydration, indicating that their plasma membranes were no longer intact. Collectively, these data suggest that lipid hydroperoxides resulting from ROS attack do not accumulate in dried Physcomitrella during storage and are not the primary cause of the loss of viability during storage of the dried moss.

1000-090-Z Small Compounds Targeting Tyrosine Phosphorylation of RACK1A Protein Confer ABA Hypersensitivity Hemayet Ullah – Howard University RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in single cell eukaryote yeast to human and plays regulatoryroles in diverse signal transduction and stress response pathways. Loss of function mutant in Arabidopsis indicates that RACK1A regulates diverse environmental stress resistance and developmental pathways through a negative regulation of stress hormone abscisic acid ABA) signaling pathway . It is hypothesized that chemical knock-out, as opposed to genetic knock-out, of RACK1A will provide a functional advantage in protecting plants from diverse stress through the effect of hypersensitivity to the ABA. Site directed mutagenesis studies indicated that the key post-translational modifications like sumoylation at K273 and tyrosine phosphorylation of Y248 residues dictate the RACK1A’s potential to interact with other proteins. In order to facilitate the identification of small compounds binding to the functional pocket, the crystal structure of RACK1A protein is deduced at 2.4 A resolution. Deduced crystal structure of RACK1A is used to identify dozens of small compounds that could potentially bind to the Y248 pocket. The compounds could potentially inhibit Y248 prosphorylation and bind to purified recombinant RACK1 protein with a kD value in the micro-molar ranges. The compounds can effectively elicit ABA signaling pathways through activating the ABI5 gene expression as evaluated in the ABA induced seed germination, growth inhibition assays. The effectiveness of the compounds in regulating diverse environmental stress responses are evaluated in different crop plants. Here we present, evidence that the compounds are effective in regulating salt and drought stress responses in wide variety of crop plants including in tomato, beans, and pepper. To allow ease of application to crop plants, the compounds are being formulated as fertilizer additives.

Abiotic: Salt and Minerals 1000-091-Z A Putative Transcription Factor Is a Novel Regulator of Copper Homeostasis That Acts Together with SQUAMOSA Promoter Binding Protein–like7 to Contribute Flower Development in Arabidopsis Thaliana Jiapei Yan – Cornell University Hail Jung – Cornell University, Michael Rutzke – Robert W. Holley Center for Agriculture and Health, USDA-ARS, Leon V. Kochian Kochian – Robert W. Holley Center for Agriculture and Health, USDA-ARS, Chen Jiao – Boyce Thompson Institute, Zhangjun Fei – Boyce Thompson Institue, Olena Vatamaniuk – Cornell University The transition metal copper (Cu) is among the most important mineral nutrients and is essential for plant growth and development. However, Cu is toxic if it is present in cells in excess. To maintain Cu homeostasis, plants have evolved

sophisticated regulatory mechanisms. SPL7 has been shown to play a central role in this regulatory network and is the only transcription factor with a documented role in Cu homeostasis. We have found recently that a member of the basic helix-loop-helix family of TFs that we designated CCIT1, is transcriptionally regulated by Cu availability and localizes to the nucleus in A. thaliana. The ccit1-1 knockout allele is hypersensitive to Cu deficiency. Importantly, the genomic fragment consisting of the CCIT1 promoter and genomic sequence complements ccit1-1 sensitivity to Cu deficiency. Previous transcriptome analyses have identified CCIT1 among the downstream targets of SPL7. However, our genetic studies using the spl7-1 and ccit1-1 single mutants and the ccit1spl7 double mutant suggest that CCIT1 does not act immediately downstream of SPL7. First, we found that whereas spl7-1 and ccit1-1 single mutants develop to the adult stage and are fertile, the double ccit1-1spl7-1 mutant is not viable even when grown under Cu replete conditions. A higher but not toxic concentration of Cu allows the double mutant to develop to the reproductive stage; however, plants remain infertile due to pollen sterility and altered flower morphology. Second, we found that transcriptional response of CCIT1 to Cu deficiency in roots and flowers is, in part, independent of SPL7. Together, these data suggest that CCIT1 and SPL7 act in a parallel interacting Cu regulatory pathway and that this pathway is also important for flower development. Components of this pathway as revealed by Y1H and RNAseq data and the contribution to flower development will be discussed.

1000-092-Y Characterization of the Vascular-mediated Long-distance Signaling System Involved in Plant Phosphate Homeostasis Zhaoliang Zhang – University of California Davis Byung-Kook Ham – University of California Davis, Akiko Yoshida – University of Tokyo, Yi Zheng – Cornell University, Zhangjun Fei – Cornell University, Leon Kochian – Cornell University, William Lucas – University of California Davis Phosphate (Pi) is an essential plant macronutrient, but low availability of Pi in most soils often imposes serious limitations on crop production. Pi fertilizer application is not a sustainable strategy, as global Pi reserves are rapidly being depleted. Thus, for global food security, it will be critical to develop a comprehensive understanding of the mechanisms used by plants to acquire and efficiently utilize Pi. It is known that root-to-shoot and shoot-to-root signaling systems are essential for nutrient acquisition and adaption to fluctuating nutrient conditions; however, the signaling agents in these pathways remain largely unknown. In our project, we use cucumber as a model plant to detect systemic signal(s) that regulate Pi homeostasis in cucumber. A sand-culture system was used to establish the temporal-spatial Pi deficiency response throughout the plant. Based on these findings, RNA was extracted from the lamina of the third leaf, phloem sap collected from the stem just beneath the third node, roots and shoot apex and processed by RNA-Seq to establish transcriptome profiles of the early responses to Pi deficiency. These studies identified the presence of highly tissue-specific responses to Pi deficiency. Interestingly, we detected more than 100 mRNAs and 28 miRNAs that were upregulated in phloem sap within 12 h of imposing a Pi deficiency condition to the roots. More importantly, more than 1000 long-distance mobile mRNAs responsive to Pi deficiency were further identified by using a combination of heterografting between cucumber and watermelon and RNA sequencing. Using this system, we were able to identify mobile mRNAs being delivered from source leaves to the shoot apex, developing leaves and to roots. We are currently determining the target cells/tissues in the roots and shoot apex. Our goal is to develop a gene regulatory network plants use to adapt to the ever changing Pi availability in soil.

1000-093-Y The Activity of Ammonium Transporter Is Regulated by Signaling Regulator CBL-CIPK Network Cheng-Hsun Ho – Department of Plant Biology, Carnegie institute for Science Wolf Frommer – Department of Plant Biology, Carnegie institute for Science Nitrogen is quantitatively the most important mineral nutrient. Ammonium is one of the three major forms of nitrogen acquired by plants. Assimilation of ammonium into amino acids is less energy consuming relative to nitrate. However, ammonium, when given alone, leads to toxicity in bacteria, fungi, animals and plants. Therefore, ammonium transport (AMT) is highly regulated. Our lab identified the first ammonium transporters from any organism, and uncovered an allosteric regulatory mechanism that can be used to rapidly shut down cellular import of ammonium. Besides of it’s transport activity, AMT have While we could show that phosphorylation of the C-terminus is important for allosteric inactivation of AMT complexes, the protein kinases required for the phosphorylation of the C-terminus AMTs and the components participated in the signaling pathway are still unknown. In this study, CBLs-CIPKs (Calcineurin B-like and CBL-interacting protein kinase, respectively), primary signaling regulators involved in all major abiotic and biotic pathways, were co-expressed with AMT1;1 in the heterologous Xenopus laevis oocyte system using Two Electrode Voltage Clamp. Results shown that multi CBLs and CIPKs significantly affect the transport activity of AMT1;1 while coexpressing in oocytes. These results demonstrate CBL-CIPK network novelly participates in the regulation of AMTs’ activity and candidates that may control allosteric inhibition of AMT complexes through interaction and phosphorylation. Other members of AMT family will be further tested with same CIPKs in oocytes to determine whether other members of the AMT family using the same phosphorylation mechanism. The in vivo phosphorylation status of AMT1;1 and ammonium signal responses in CIPKs mutants and the ammonium toxicity growth assays will also be further determined in planta to understand the mechanism of regulation as well as the biology of ammonium toxicity. Our research here promises to make important contributions towards the development of more nutrient efficient plats for sustainable agriculture.

1000-094-Z A Set of LRR-RLK Genes Quantitatively Regulates Root Growth Under Iron-limited Conditions in Arabidopsis Santosh Satbhai – Gregor Mendel Institute of Molecular Plant Biology (GMI) Youssef Belkhadir – Gregor Mendel Institute of Molecular Plant Biology (GMI), Wolfgang Busch – Gregor Mendel Institute of Molecular Plant Biology (GMI) Iron (Fe) is an important mineral micronutrient for plants and animals. Low availability of Fe limits plant growth because Fe forms insoluble ferric oxides in soil. Root architecture is a key trait for Fe acquisition and the survival of plants on Fedeficient soils. Consequently, plants have evolved adaptive mechanisms that coordinate root growth and development with Fe availability. To unravel Fe dependent development and the underlying mechanisms, we have studied the early root development using Arabidopsis thaliana (Arabidopsis) natural accessions under Fe-deficient condition. We used a diverse set of 450 natural accessions of Arabidopsis to identify genes that quantitatively regulate root growth responses to Fe deficiency using genome wide association mapping. Strikingly, these plants showed high variation in their primary root elongation when grown on Fe-deficient medium. We identified more than 20 statistically significant genomic loci that are associated with changes in root growth rate upon Fe deficiency. Among genes in proximity of these associations, a cluster of 3 Leucine-rich repeat receptor-like protein kinases (LRR-RLK) genes and a kinase gene showed strong signatures of epistatic interactions. Each of the single mutant lines of these signaling genes displayed a significant root growth rate reduction on Fe deficient media but not on full media, showing that this gene cluster is involved in growth regulation under Fe limited conditions. Due to their tissue specific expression pattern, we hypothesize that these four genes are coordinating growth responses in different tissues of the root. We are currently testing this hypothesis and exploring the epistatic interactions of these genes and their alleles. The identification of previously unknown genes

and their epistatic interaction has important implications for the generation of crops with improved nutritional quality and increased growth in Fe-deficient soils.

1000-095-Z The Role of High-affinity Phosphate Transporters 1;8 and 1;9 in Arabidopsis Thaliana Irina Boico – North Carolina State University Marcela Rojas-Pierce – North Carolina State University Phosphorus (P) is an essential macronutrient critical for plant growth and development. Its uptake, in the inorganic phosphate (H2PO4-, Pi) form, occurs via phosphate transporters. Pi is scarce in many soils worldwide, thus limiting plant productivity. In Arabidopsis thaliana one of the adaptive mechanisms to Pi shortage is the up-regulation of high-affinity phosphate transporters of the PHOSPHATE TRANSPORTER 1 Family (PHT1). PHT1;8 and PHT1;9 share 78% nucleotide identity and encode for proteins that are 79% identical, thus might have similar functions. Recently, PHT1;8 and PHT1;9 have been implicated in the Pi translocation from root to shoot, however, the exact role of PHT1;8 and PHT1;9 in acclimation to Pi stress and the cellular mechanisms to regulate accumulation of these proteins remain unclear. A reverse genetic approach was used to characterize the function of PHT1;8 and PHT1;9. Loss of PHT1;8, PHT1;9 or both resulted in reduced basal phosphate levels and longer root hairs. Upon Pi stress, pht1;8 and pht1;8pht1;9 double have reduced tolerance to low Pi, increased rhizosphere acidification and increased anthocyanin accumulation in shoots. All mutants also show compensatory effects with increased transcript levels of other PHT1 family genes. In addition, fluorescent-protein translational fusions to PHT1;8 and PHT1;9 were used to identify their subcellular localization. Transient expression studies in Nicotiana benthamiana show that both proteins localize to endoplasmic reticulum (ER) and co-localize with an ER marker. However, in stably-transformed Arabidopsis thaliana PHT1;8 and PHT1;9 localize predominantly to ER and to the plasma membrane (PM). The PM pool of PHT1;8 and PHT1;9 is sensitive to Brefeldin A (BFA), suggesting their active recycling at the PM. Altogether, our data suggests that PHT1;8 and 1;9 have a dual localization (ER and PM) and both are required for proper adaptation to low levels of Pi.

1000-096-Y The Heterotrimeric G-protein β Subunit, AGB1, Plays Multiple Roles in the Arabidopsis Salinity Response Yunqing Yu – Penn State University Sarah M. Assmann – Penn State University Salinity is one of the major problems affecting plant growth and crop yield. Salinity stress includes both osmotic and ionic toxicity. Sodium homeostasis is influenced by Na+ uptake and extrusion, vacuolar Na+ compartmentation, and root to shoot Na+ translocation via transpiration. In Arabidopsis thaliana, heterotrimeric G proteins consist of one canonical Gα (GPA1), one Gβ (AGB1) and three Gγ (AGG1, AGG2 and AGG3) subunits. G proteins play multiple roles in plant development and in responses to environmental stresses. The knockout mutant of the Arabidopsis heterotrimeric Gprotein Gβ subunit, agb1, is hypersensitive to salt, exhibiting a leaf bleaching phenotype. We show that AGB1 is mainly involved in the ionic toxicity component of salinity stress and plays roles in multiple processes of Na+ homeostasis. agb1 mutants accumulate more Na+ and less K+ in both shoots and roots of hydroponically grown plants, as measured by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES). agb1 plants have higher root to shoot translocation rates of radiolabelled 24Na+ under transpiring conditions, as a result of larger stomatal apertures and increased stomatal conductance under both control and salt treated conditions. 24Na+ tracer experiments also show that 24Na+ uptake rates by excised roots of agb1 and wild-type are initially equal, but that agb1 has higher net Na+ uptake at 90 min, implicating possible involvement of AGB1 in regulation of Na+ efflux. Supplemental external Ca2+ alleviates the salt hypersensitivity of agb1 by reducing Na+ accumulation to below the toxicity threshold. Our results

provide new insights into the regulatory pathways underlying plant responses to salinity stress, an important agricultural problem.

1000-097-Y Boron, a Hidden Toxin in Flue Gas Desulfurization Water Lawrence Davis – K-State U Joni Mengarelli – K-State U, Madhubhashini Gulkaduwa – K-State U, Ganga Hettiarachchi – K-State U Coal fired power plants generate large quantities of heavily contaminated water while removing volatile or aerosolized sulfur from the flue gases by addition of calcium, usually a lime slurry. The overall process yields solid gypsum (sometimes marketed as gypsoil) plus a byproduct called flue gas desulfurization water (FGD). The composition of FGD is poorly defined because it depends on the source of coal, conditions of combustion, and the lime source material used to precipitate flue gas sulfur. The final FGD solution is typically high in magnesium, chloride, sodium, and supersaturated with gypsum. Boron concentrations in FGD waters (10-500 mg/L) exceed state irrigation water limits (~1-10 mg/L B) many-fold. Contaminating mercury can be removed to attain very low levels, while selenium and arsenic precipitate under reducing conditions as selenide and arsenide in vegetated treatment wetlands. Simple anions and cations are removable by ion exchange techniques if necessary. However, boron (as borate) has very low affinity for ion exchangers. It is poorly bound by soil organic matter. It can be sorbed by vicinal hydroxyl groups of sugars, in a very expensive treatment process. Tests were done with a local source of FGD water to determine its toxicity to sunflowers grown hydroponically. Simulated FGD water, with added Na, Ca, Mg, Cl, and sulfate was compared. Essential nutrients were added to all solutions to approximate ½ strength Hoagland’s solution. While added salts simulating FGD water inhibited sunflower dry weight gain 10-20%, actual FGD water was a much stronger inhibitor. Added boron caused further growth inhibition, whether supplied in actual or simulated FGD water. A 20 mg/L B level gave up to 40% inhibition of growth over two weeks. These results have major implications for FGD water disposal. It must be diluted well below 5 mg/L before use with plants, including treatment wetlands.

1000-098-Z An Initial Survey on the Role of Auxin at Salinity Condition by the Moss (Physcomitrella Patens) Haniyeh Koochak – Institute of Botany, Technische Universität Dresden Nowadays, moss Physcomitrella patens has become a novel plant model for the state-of-the-art physiological, molecular and genetic analyses. Since compared to other plants like Arabidopsis thaliana, P. patens exhibits a high degree of abiotic stress tolerance, this organism is used for the investigation of various types of stress and their influence on the plants. The gametophytic tissue of this plant consists of one native auxin indole-3-acetic acid (IAA). The stable transgenic P. patens (GH3::GUS line) is including auxin-inducible reporter gene system. Actually, the expression of β-glucuronidase (GUS) gene drives by the auxin-inducible promoters GH3. GUS reporter gene will be used to determine auxin levels in P. patens. In this research, during 50 days, GH3::GUS line on the medium supplemented with different concentrations of NaCl, was used in order to assay the GUS activity. Along with this step total protein content in the plants was measured for these all samples, as well.

The consequence of this part prepared us with preliminary data to be followed by more experiments leading to find the influence of salt as abiotic stress-factor on cellular processes and molecular pathways.

1000-099-Z Mycorrhizal Lycopersicon Esculentum Membrane’s Gene Expression and Physiological Responses to Silver Nanoparticles Azam Noori – State University of New York-ESF Jason White – Connecticut Agricultural Experiment Stationm Lee Newman – State University of New York-ESF Progress in Nanotechnology during last decades caused vast usage of nanomaterials. Among different types of nanomaterials, silver nanoparticles (Ag-NPs) have vast application in different area such as biolabeling, filters and antimicrobials. Antimicrobial properties of Silver NPs led to the use of these nanometals in different fields of medicine, cosmetics, health and agriculture. Diverse application of these products increases their release in to the environment and the risk of taken up by plants. In this study we attempt to determine the impact of Ag-NPs on plants physiological and molecular responses in mycorrhizal and non-mycorrhizal plants. Rhizophagus intraradices spores were used to colonize Lycopersicon esculentum (tomato) roots. Grown mycorrhizal and non-mycorrhizal L. esculentum for 60 days were exposed to 25 and 75 ppm of 2nm silver nanoparticles for 20 days. Roots, stems and leaves length and weight as well as mycorrhizal colonization were measured in each treatment. Plants (roots, stems and leaves) and soil were analyzed for Ag-NPs content using Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). The highest amount of silver was detected in roots and the lowest in stems. Ag-NPs content increased significantly in Mycorrhizal and non-mycorrhizal plants by increasing NPs exposure. Gene expression of aquaporines; TIP and PIP, proton transporter (LAH1) and potassium channel (KC1) was measured using Q-PCR. Beta tubulin was used as reference gene. The gene expression of two aquaporins genes (PIP and TIP) increased 3 and 1.45 times respectively in plants exposed to 75 ppm Ag-NPs. The gene expression of LAH1 and KC1 increased 1.54 and 5.78 times respectively in exposed plants to 75 ppm Ag-NPs compare to control. Chlorophyll and anthocyanin content were measured in leaves. Anthocyanin content increased significantly in exposed plants to 75 ppm Ag-NPs both in mycorrhizal and non-mycorrhizal plants.

1000-100-Y Natural Genetic Variation in Aluminum Sensitive 1 May Confer Differential Stress Tolerance in Tomato Susan Bush – Macalester College Keo Corak – Macalester College Wild relatives of crops such as tomato carry natural genetic diversity that can provide differing levels of tolerance to environmental stresses; for example, the wild tomato Solanum pennellii grows in the deserts of Chile and Peru. In many plants, toxicity due to aluminum exposure is common in soils with low pH, up to 30% of arable land. A previous study identified conserved codon changes in the Aluminum Sensitive 1 (ALS1) gene between S. pennellii and domesticated S. lycopersicum, suggesting adaptive aluminum tolerance in the wild species. ALS1 is a half-type ABC transporter protein found at the vacuolar membrane that may be involved in chelating aluminum for tolerance. We have examined the morphological and gene expression changes in domesticated S. lycopersicum, compared to tomatoes with an introgression of a region of S. pennellii chromosome 3, carrying the wild allele of ALS1. Preliminary work suggests differences in adventitious root development and expression of stress-responsive genes. Application of this work may permit development of domesticated tomatoes suitable for production in suboptimal soils.

1000-101-Y Overexpressing AtNHX1 and SOS1 Simultaneously in Arabidopsis Additively Improve Salt Stresses Tolerance Li Sun – Texas Tech University Necla Pehlivan – Recep Tayyip Erdogan University, Jarrett Philip – Texas Tech University, Guoxin Sheng – Zhejiang Academy of Agricultural Sciences, Xiaojie Yang – Henan Academy of Agricultural Sciences, Hong Zhang – Texas Tech University Abiotic stresses are major factors limiting agricultural production, which are becoming more serious due to climate changes. Revolutionary biomaterials such as transgenic crops are needed to make agriculture sustainable in foreseeable future. Among different stresses, high salinity adversely impacted over 800 million hectares of arable land in the world nowadays. After many efforts have been made, AtNHX1 and SOS1 were separately identified that might be used to improve salt stress tolerance in transgenic plants. We stacked both genes and transformed them into Arabidopsis. Plants overexpressing both genes perform much better in the presence of higher concentrations of sodium chloride, which shows an attainable way of making better transgenic crops with desirable traits coming from multiple genes.

1000-102-Z The Only DELLA Protein in Rice, SLR1, Is Regulated by Salinity Stress and Post-translational Modifications Nuno Gonçalves – ITQB-UNL/iBET André Cordeiro – ITQB-UNL/iBET, Margarida Rosa – ITQB-UNL, Mafalda Rodrigues – ITQB-UNL/iBET, Margarida Oliveira – ITQB-UNL/iBET, Isabel Abreu – iBET DELLA proteins are fundamental modulators of gibberellin hormonal response. Besides having been described as regulators of plant growth and development, their accumulation was shown to be involved in plant response to abiotic stress. Their regulation is mainly done at the post-translational level. In Arabidopsis, there are 5 DELLA proteins, while only one exists in most cereals. Our aim is to characterize SLR1, the only DELLA in rice, and its role in response to abiotic stress and regulation by post-translational modifications (PTMs). Rice is the most sensitive crop to high-salinity conditions. Therefore we started by analyzing how SLR1 knockout lines perceive salt stress differently from wild-type plants, which show tissue-specific and stress-induced SLR1 accumulation, at seedling stage. At the booting stage, we have evaluated commercial rice varieties for correlation between SLR1 protein levels and salt-stress tolerance, resulting in yield maintenance. Recently, a new layer of DELLA regulation was added, when the SUMOylation of DELLA proteins in Arabidopsis was shown to enhance their stability. Still, there is no information how that affects DELLA stress response. We already established that SLR1 is SUMOylated in in-vitro rice-SUMOylation assays. Now we are exploring the impact of SLR1 SUMOylation in the response of rice to salt and drought stresses. These are the first steps in unveiling SLR1 function in abiotic stress and the fine-tuning of its regulation by PTMs. The knowledge gathered will help understanding how DELLA stress tolerance mechanisms can be established as tools to design better adapted crops, able to grow in increasingly challenging conditions.

1000-103-Z Engineering OsCyp2 as a Potential Candidate for Amelioration of Multiple Environmental Stress in Rice Suchismita Roy – Jawaharlal Nehru University Snehlata Singla Pareek – ICGEB, India, Ashwani Pareek – Jawaharlal Nehru University Agricultural productivity is greatly affected by unfavourable environmental factors to which crops are exposed. Current agricultural productivity is unable to meet the food demand of a continuously increasing population. The need of the

hour is to adapt agriculture to increased uncertainty. Identifying suitable genes may serve as repositories for future individual gene-based functional genomics for providing better agronomic traits. Global transcript profiling in rice has shown that salinity stress regulates a large set of genes. One such gene, which showed up-regulation under salinity stress is cyclophilin. Cyclophilins are a set of PPIases considered to be omnipresent at subcellular compartments, involved in a wide variety of cellular processes. Cyclophilin 2 (OsCyp2) is a cytosolic member of the immunophilin family from Oryza and is a 519 bp gene encoding 172 amino acids. qRT-PCR data suggests that transcript abundance of OsCyp2 is regulated under different stress conditions in a developmental and organ specific manner. Localization studies confirmed that OsCyp2 is localized in both cytosol and nucleus, indicating its possible interaction with several other proteins. Stable overexpressing transgenic lines of this gene in Oryza sativa imparted tolerance towards multiple abiotic stresses as evidenced by higher germination efficiency, higher root length, more number of lateral root formation, higher shoot length, chlorophyll content, and K+/Na+ ratio as well observed in the transgenic plants, under stress conditions. Transgenic plants also showed reduced MDA content, electrolyte leakage, and catalase and ascorbate peroxidase enzyme activity content under stress conditions suggesting better ion homeostasis than WT plants. RNAi lines of OsCyp2 gene validated this result with an observed phenotypic change in the seed morphology, seed weight and yield quality. Thus, overexpression of this gene is apposite for genetic engineering for better grain quality, yield and tolerance towards unfavourable conditions.

1000-104-Y The Function of Intracellular Vesicle Trafficking Under Salt Stress: A Role of AtECA4 Salt Stress Response Hanh Nguyen - POSTECH Inhwan Hwang In eukaryotic cells, the cellular space and activity are compartmentalized into membrane-surrounded organelles. Thus eukaryotic cells must have mechanisms to transport molecules between these organelles. The processes called exocytosis and endocytosis are involved in trafficking of molecules between these organelles. These trafficking pathways are involved in transporting molecules for house-keeping functions as well as to the processes involved in adaption to the environmental changes. The latter process is critical for plants, in particular, because plants are sessile organisms and are constantly exposed to ever-changing environment. Earlier studies have shown that the cell can regulate stress response by adjusting the rate of trafficking to and from the plasma membrane (PM). In addition, recent biochemical and genetic studies provide evidence that ROSs generate by PM-localized NADPH oxidases are signaling molecules that mediate responses to abiotic and biotic stresses. In this study, we examined whether an A/ENTH domain-containing protein AtECA4 is involved in abiotic stress response by linking between vesicle trafficking and salt stress. AtECA4 is an adaptor protein which localizes to the cell plate in dividing cells, and to the PM and punctate spot patterns in nondividing cell. Salt stress induces its protein expression and induces its PM localization. By studying the phenotype of a mutant that has a T-DNA insertion into AtECA4, we found that this mutant plants are hypersensitive to salt stress, showing a lower germination rate, shorter root length, and less chlorophyll content, compared to WT plant, under the salt stress conditions. Besides, the ateca4 plants do not generate ROS burst in response to salt stress. These results suggest that AtECA4 plays a role in salt stress response. Additionally, we are studying the trafficking of cargos to examine how AtECA4 regulates vesicle trafficking during salt stress.

1000-105-Y The Role of Glutathione in Iron Deficiency Response and Tolerance in Arabidopsis Yi-Wen Wang – Academia Sinica Varanavasiappan Shanmugam – Tamil Nadu Agricultural University, Munkhtsetseg Tsednee – Academia Sinica, Krithika Karunakaran – Academia Sinica, Kuo-Chen Yeh – Academia Sinica

Iron (Fe) deficiency is a common problem in agriculture and it affects both plant’s productivity and nutritional quality. Identifying the key factors involved in the tolerance of Fe deficiency is therefore imperative. In the present study, the glutathione deficient mutant, zir1 was found to be more sensitive to Fe deficiency and it grows poorly under alkaline soil. Other glutathione deficient mutants also showed various degrees of sensitivity under Fe limited conditions. In wild type, the glutathione level was induced under Fe deficiency and blocking glutathione biosynthesis led to increased sensitivity to Fe deficiency. Alternatively, overexpressing glutathione enhanced the tolerance to Fe deficiency. Under Fe limited conditions, glutathione deficient mutants accumulated low Fe content only in shoot tissues, whereas zir1 had defect in both shoot and root Fe levels. The key genes involved in root Fe uptake are expressed lower in zir1 when compared to wild type. In addition, our split root experiment suggests that in zir1 the systemic signals that govern the expression of root Fe uptake genes are still active. Furthermore, zir1 contains lower accumulation of nitric oxide (NO) and NO reservoir s-nitrosoglutathione (GSNO). NO is a signaling molecule involved in the up-regulation of root Fe uptake related genes. Interestingly, the NO mediated inductions of Fe uptake genes are dependent of glutathione supply in zir1. These results suggest that glutathione plays a role in Fe deficiency tolerance and NO-mediated Fe deficiency signaling in Arabidopsis.

1000-106-Z Measuring the Effect of Copper Deprivation on Lipid Droplet Formation in Chlamydomonas Reinhardtii Eric Ahrens – University of Nebraska-Kearney Brandon Karlin – University of Nebraska-Kearney, Ashleigh Teten – University of Nebraska-Kearney, Paul Twigg – University of Nebraska-Kearney Chlamydomonas reinhardtii is a model alga for the study of many processes. With the increasing emphasis on biofuels, Chlamydomonas and other algal genera have become more closely examined as possible fuel sources. Recently, it has been demonstrated that Chlamydomonas will produce large amounts of lipid droplets under conditions of nitrogen deprivation. The goal of this study was to examine how lipid accumulation was affected by copper deprivation. Copper is a required element for Chlamydomonas growth and is a cofactor of superoxide dismutase. Oxidative stress has also been shown to cause lipid accumulation and zinc was chosen as a target for this reason. We will present our lipid accumulation time course separately showing the effects of zinc, nitrogen, and a combination of both zinc and nitrogen deprivation. This project was funded by the NSF-EPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094).

1000-107-Z Brachypodium Distachyon as a Model System for Studies of Copper Transport in Cereal Crops Jiapei Yan – Cornell University Hail Jung, Sheena Gayomba, Olena Vatamaniuk – Cornell University Copper (Cu) is an essential micronutrient but is also toxic when is accumulated in cells in excess. Copper bioavailability, and thus crop productivity on agricultural soils depends on the soil type and agricultural practices. For example, Cu deficiency develops on alkaline soils, which occupy approximately 30% of the world’s arable land, and on organic soils. While Cu deficiency can be remedied by the application of Cu-based fertilizers, the repeated use of fertilizers as well as Cu-containing pesticides, has led to the build-up of toxic levels of Cu in soils. In this regard, organic farming has emerged as a preferred production system that relies on natural fertilizers; however, natural fertilizers increase soil organic matter and thus, further reduce Cu bioavailability. Sensitivity to Cu bioavailability in soils varies among crop species with wheat being most sensitive to Cu deficiency. However, the genetic, molecular, and physiological basis underlying the increased sensitivity to Cu availability are unknown. Given the complexity of the wheat genome, we used Brachypodium distachyon (brachypodium) as a wheat proxy to study the molecular mechanisms of Cu homeostasis. We focused on

members of the CTR/COPT family of Cu transporters because their homologs in A. thaliana are transcriptionally upregulated in Cu-limited conditions and are involved either in Cu uptake from soils into the root, or long-distance transport and distribution of Cu in photosynthetic tissues. We will present data suggesting that increased sensitivity to Cu deficiency in some grass species may arise from lower efficiency of Cu uptake and tissue partitioning systems. The presented work also reinforces the importance of using brachypodium as a model for the comprehensive analyses of Cu homeostasis in cereal crops.

1000-108-Y CsNIP2;1 Is a Plasma Membrane Transporter That Is Involved in Urea Uptake in Cucumis Sativus L Jiapei Yan – Cornell University Lu Zhang – China Agricultural University, Olena Vatamaniuk – Cornell University, Xiangge Du – China Agricultural University Urea is an important source of nitrogen (N) for the growth and development of plants. Urea occurs naturally in soils from the degradation of nitrogenous compounds and is the major N source in agricultural fertilizers. Therefore, the identification and characterization of urea transporters in higher plants is important for fundamental understanding of urea-based N-nutrition in plants and for designing novel strategies for improving the use-efficiency of urea-based Nfertilizers. Progress in this area, however, is hampered due to scarce knowledge of plant urea transporters. From what is known, urea uptake from the soil into plant roots is mediated by two types of transporters: the major intrinsic proteins (MIPs) and the DUR3 orthologues, mediating low- and high-affinity urea transport, respectively. We have recently characterized a MIP family member from Cucumis sativus L, CsNIP2;1, with regard to its contribution to urea transport. We found that 1) CsNIP2;1 resides at the plasma membrane and functionally complements growth defect of the ureauptake deficient yeast mutant; 2) Expression of CsNIP2;1 is upregulated by urea in roots but not in shoots; 3) Expression of CsNIP2;1 is the highest in cotyledon and hypocotyl immediately after germination. 4) Ectopic expression of CsNIP2;1 improves growth of the dur3-3 A. thaliana on urea as a single N source. Together, our data suggest CsNIP2;1 is urea uptake transporter that contributes to N nutrition and might be particularly important at early developmental stages of Cucumis sativus L.

1000-109-Y Physiological and Molecular Characterization of Nitrate Uptake in Sugarcane (Saccharum Spp.) Antonio Figueira – Universidade de Sao Paulo Luis Henrique Serezino – Universidade de Sao Paulo, Joni Lima – Universidade de Sao Paulo, Marielle Vitti – Universidade de Sao Paulo, Renato Vicentini – Universidade Estadual de Campinas The expansion of sugarcane (Saccharum spp.) cultivated area to marginal lands and the need to maintain high yields have led to increasing application of nitrogen fertilizers. However, this practice represents high economic and environmental costs. Sugarcane exhibits low nitrogen use efficiency (NUE) in comparison to other C4 grass crops. To better understand the low efficiency in nitrate uptake by sugarcane, this study carried out the physiological characterization of nitrate uptake by kinetic analysis using 15N, together with the expression profiling of genes coding for the major nitrate transporters (NRTs) related to uptake, namely ScNRT1.1, ScNRT2.1, and ScNAR2.1. To understand the reasons for the reduced ability to acquire nitrate by sugarcane roots, plants of cultivar ’SP80-3280’ were exposed to various N supplement conditions to investigate the regulation of the uptake process. The lower efficiency in nitrate acquisition compared to ammonium was corroborated and demonstrated for low N condition. Short-term 15N-labeled influx studies showed a correlation with nitrate transport in sugarcane roots under sufficient or at low N external concentrations and/or low N status. Ammonium negatively regulates nitrate uptake by modulating the expression of genes involved in this process. Plants under N deficiency exhibited a late regulation of high affinity uptake systems

(HATS) responsible for nitrate uptake. The lack of correlation between 15N influx and transcript accumulation of nitrate transporter genes suggests the existence of a post-transcriptional regulation of HATS in roots subjected to nitrate resupply. This regulation may be one of the mechanisms responsible for the low nitrate uptake by sugarcane roots, when compared to other C4 species.

1000-110-Z Finding Transcription Factors That Control Plant Responses to Nitrate--- from DNA Cis-elements to Nitrate Regulatory Network Peizhu Guan Nigel Crawford Nitrate is both nutrient and signal. It plays a key role in plant metabolism and development. The proper and precise responses to nitrate are dependent on a whole spectrum of spatial and temporal gene expression. However, the regulators of transcription and the regulatory mechanisms underlying the patterns of gene expression remain largely unknown. By using nitrate enhancer DNA to screen a comprehensive library of Arabidopsis transcription factors in the yeast one-hybrid system, the transcription factor gene TEOSINTE BRANCHED1/CYCLOIDEA/ PROLIFERATING CELL FACTOR1-20 (TCP20) was first identified. TCP20 belongs to an ancient, plant-specific TCP gene family whose origins predate the emergence of land plants. TCP genes regulate shoot, flower and embryo development. Our work demonstrated that TCP20 functions as an essential part of the systemic signaling pathway that directs nitrate foraging by Arabidopsis roots. Root foraging is a prominent example of phenotypic plasticity where plants proliferate lateral roots preferentially in nutrient-rich zones to compete for nutrients in diverse soil microenvironments. Our systematic approach holds promise for identifying key transcription factors and regulatory mechanisms in nitrate signaling and transcriptional programming. An understanding of these molecular mechanisms will assist in efforts to improve nitrogen use efficiency (NUE) in agriculture and thereby reduce greenhouse gas emissions and pollution of ecosystems.

1000-111-Z Correlation of Leaf Sheath Transcriptome Profiles with Physiological Traits of Bread Wheat Cultivars Under Salinity Stress Fuminori Takahashi – RIKEN CSRS Joanne Tilbrook – ACPFG, Christine Trittermann – ACPFG, Bettina Berger – The Plant Accelerator, Stuart Roy – ACPFG, Motoaki Seki – RIKEN CSRS, Kazuo Shinozaki – RIKEN CSRS, Mark Tester – KAUST Salinity stress has significant negative effects on plant biomass production and crop yield. Salinity tolerance is controlled by complex systems of gene expression and ion transport. The relationship between specific features of mild salinity stress adaptation and gene expression was analyzed using four commercial varieties of bread wheat (Triticum aestivum) that have different levels of salinity tolerance. The high-throughput phenotyping system in The Plant Accelerator at the Australian Plant Phenomics Facility revealed variation in shoot relative growth rate and salinity tolerance among the four cultivars. Comparative analysis of gene expression in the leaf sheaths identified genes whose functions are potentially linked to shoot biomass development and salinity tolerance. Early responses to mild salinity stress through changes in gene expression have an influence on the acquisition of stress tolerance and improvement in biomass accumulation during the early “osmotic” phase of salinity stress. In addition, results revealed transcript profiles for the wheat cultivars that were different from those of usual stress-inducible genes, but were related to those of plant growth. These findings suggest that, in the process of breeding, selection of specific traits with various salinity stress-inducible genes in commercial bread wheat has led to adaptation to mild salinity conditions.

1000-112-Y Salt-regulated Genes or Proteins in Imperata Cylindrica Revealed by Transcriptomic and 2D-DIGE Proteomic Analyses Ing-Feng Chang Min-Jey Tsai, Ting-Ying Wu, Ping Kao, Ren-Jiu Xu Cogon grass (Imperata cylindrica L. Beauv. var. major), one of the top-ten weeds and a C4 plant, is widespread and distributed. In particular, Chuwei ecotype from mangrove in Taiwan was found to be salt tolerant. However, the salt tolerance mechanism is unknown. The present study was designed to identify salt-responsive genes and proteins in Chuwei ecotype which can be involved in salt tolerance mechanism. Total RNAs from shoot and root of Chuwei ecotype were isolated and subjected to next generation sequencing for transcriptome profiling. These transcript sequences were used to design probes for microarray analyses. A microarray analysis was performed to identify salt stress responsive genes up-regulated in response to 150 mM salt stress. Real-time PCR was introduced to confirm gene expression. Moreover, a proteomic analysis using 2D-difference in gel electrophoresis (DIGE) was carried out to identify salt stress responsive proteins up-regulated in response to 150 mM salt stress. The up-regulated proteins include photosystem proteins and antioxidant proteins. Taken together, results from microarray and proteomic study indicates that photosynthesis genes are up-regulated in the salt stress response in the salt-tolerant ecotype of Imperata cylindrica. Upregulation of photosynthesis genes may serve one of the salt tolerant mechanisms in Chuwei ecotype of Imperata cylindrica.

1000-113-Y Overexpression of OsNUC1 in Arabidopsis Enhances Root Growth and Photosynthesis via Transcriptome Modification Supachitra Chadchawan Thanikarn Udomchalothorn, Kitiporn Plaimas, Luca Comai, Teerapong Buaboocha OsNUC1 gene encodes rice nucleolin and it has been shown to involve in salt stress responses. Overexpression of the full-length OsNUC1 gene in Arabidopsis resulted in the significant root growth enhancement under salt stress condition. The transgenic Arabidopsis seeds with OsNUC1 over-expression also showed the hypersensitive response to ABA during germination. Under salt stress, the net photosynthesis rate of the transgenic lines was increased, while the net photosynthesis rate of wild type was decreased. Based on transcriptome analysis of the transgenic lines, in comparison with wild type, it was revealed that the gene expression of more than 1,900 gene were significantly changed due to the over-expression of OsNUC1 gene in normal grown plants, while under salt stress condition the gene expression of 999 genes was found to be significantly different between the transgenic and wild type plants. Gene enrichment analysis showed that the over-expression of OsNUC1 gene decreased the expression of genes involving in photosynthesis, while in salt stress condition, the expression of the genes involving in light-harvesting complex was increased in the transgenic lines. These results suggested the role of OsNUC1 in the modification of the transcriptome, especially the gene transcripts responsible for photosynthesis.

1000-114-Z Phytoremediation Strategy Using Transgenic Salt Tolerant Sorghum to Dispose Salt Waste Water from Oil Fields Hanady Adam – Western Kentucky University-Owensboro Ginger Gilbert – Western Kentucky University-Owensboro, Chandrakanth Emani – Western Kentucky UniversityOwensboro The present investigationaims at effectively employing the process of phytoremediation (‘phyto’-plants; ‘remedium’-

restoring balance) using transgenic salt-tolerant sorghum plants genetically engineered for increased salt stress tolerance to remove oil field saltwater contamination, a significant environmental problem in many oil-production areas. The approach is an environmental friendly approach that utilizes the natural process of transpiration and the inherent ability of a salt tolerant species to dispose salt water. The salt tolerant sorghum plants in the present study will be genetically engineered with a DREB1A (dehydration response element binding) to enhance their general abiotic stress response that increases their salt tolerant ability. Additionally the sorghum plants have a natural ability to grow in petroleum-contaminated soil with a capacity to degrade total petroleum hydrocarbon contaminants. This provides the oil operators and oil industry with a feasible vegetation-based remediation approach for surface soils near oil wells contaminated with petroleum hydrocarbons and for effectively disposing salt wastewater instead of relying on expensive options such as hauling and ground injection in dedicated sites.

1000-115-Z Dissecting Salt Tolerance in Soybean by Profiling Differential Physiological Responses Jade Newsome As salinization of soils grows due to intense cultivation and natural deposition of salts over time, the need to develop crops with higher tolerance to saline conditions becomes critical. Though differences in ion uptake among soybean genotypes are well documented, the key mechanisms to cope with salt stress employed by tolerant cultivars on the whole-plant level are still largely unknown. Objectives of the current research focus on characterization of the differential physiological responses to salt stress in foliar tissues between sensitive and tolerant cultivars. Reciprocal grafts were performed between sensitive and tolerant genotypes. Following daily flooding with 100 mM NaCl or H2O, photosynthetic rates were measured and tissue analyzed for ion content. To further assess tolerance mechanisms, nongrafted plants were subjected to the same treatment as above and malondialdehyde (MDA) accumulation in leaves was measured as a marker of oxidative stress and lipid peroxidation. Due to the high levels of ions accumulated in foliar tissues of sensitive plants, higher levels of oxidative damage and thus higher MDA levels are expected in sensitive plants relative to tolerant plants. Foliar tissues from these plants were also analyzed for phytohormone content to evaluate potential stress signaling mechanisms. Results suggest that salicylic acid levels may be reduced in sensitive plants under salt stress while abscisic acid levels appear to be induced in both sensitive and tolerant plants under salt stress. Results indicate that tolerant cultivars utilize a combination of mechanisms to cope with salt stress including heightened exclusion of ions as well as enhanced ability to maintain cellular homeostasis in foliar tissues relative to sensitive cultivars. Further experimentation is necessary to validate these findings, and will be complemented with evaluation of expression of key genes involved in ion uptake, redox balancing, and phytohormone synthesis.

1000-116-Y Virus Induced Gene Silencing of Candidate Genes in the Iron-efficiency Response of Soybean Adrienne Moran Lauter – USDA-ARS Lindsay Rutter – Iowa State University, Steven Whitham – Iowa State University, Dianne Cook – Iowa State University, Michelle Graham – USDA-ARS, Iowa State University Iron Deficiency Chlorosis (IDC) is an important problem for soybeans grown on calcareous soil, leading to yield loss. We have conducted transcriptomic studies of leaf and root tissue from the iron efficient cultivar, Clark, at 30, 60 and 120 minutes after reduction of iron from hydroponic media. In leaves, we see an increase in the number of differentially expressed genes (DEGs) over time. However in roots, there is a decrease in the number of DEGs over time. Both leaves and roots regulate the expression of DEGs involved in hormone signaling, regulation of DNA replication and iron uptake utilization, which are key aspects of the soybean iron-efficiency response. Despite the similarity in pathways identified

in leaves and roots, there is very little overlap in individual DEGs between the tissues and time points. This data suggests the involvement of many transcription factors in eliciting rapid changes in gene expression. To further characterize iron deficiency responsive genes, we are using Virus Induced Gene Silencing (VIGS) to elucidate the function of 50 candidate genes. Over half of the silencing constructs produced phenotypes with altered growth, development or in leaf chlorophyll content, in soil grown plants. VIGS constructs resulting in interesting phenotypes in soil grown plants are now being tested on plants grown hydroponically with differing iron concentrations. In addition, these constructs are being tested for their role in other abiotic stress responses.

WHOLE-PLANT BIOLOGY - Zone 1100 Whole-Plant: Environmental and Ecophysiological 1100-001-Y Physicochemical and Biochemical Comparison of Nigerian and Sudanese Acacia Gum Exudates Ibrahim Babale Gashua – University of Wolverhampton, UK P.A. Williams – University of Glyndwr, UK Gum Arabic is a plant gum exudate harvested from Acacia trees (A. senegal and A.seyal) native to the semi-arid region of the sub-Saharan Africa. These exudates have been widely studied with particular emphasis on the gum obtained from Sudan. The aim of the current study was to investigate the physiochemical characteristics and associative behaviour of two samples of gum Arabic harvested from trees of Acacia senegal at two different geographical locations in Nigeria (which have not been studied previously) and too compare their properties with previously studied samples of gum Arabic harvested from trees of Acacia senegal and Acacia seyal originating from Sudan. The physicochemical characterisation was carried out using GPC/MALLS coupled to a variety of detectors, while dynamic light scattering techniques were used to investigate the association behaviour of the samples in aqueous solution and in 0.1M NaNo3. The results of which, support the hypothesis that gum Arabic forms multilayers at the oil-water interface due to protein glucuronic acid electrostatic interaction [1]. The GPC elution profiles obtained using light scattering (LS), refractive index (RI) and UV detection at 280nm for the Sudanese and Nigerian Acacia senegal gum samples are similar, but are significantly lower than the values obtained for the gum obtained from Acacia seyal and are consistent with the literature. References 1. Padala, SR, Williams, PA., Phillips, GO. Adsorption of Gum Arabic, Egg White Protein, and Their Mixtures at the OilWater Interface in Limonene Oil-in-Water Emulsions. Journal of Agriculture and Food Chemistry, 2009, 57(11), 4964 4973.

1100-002-Z Differential Epidermal Cell Expansion in Sun and Shade Unifies Changes to Vein and Stomatal Density in Herbs and Woody Plants Madeline Carins Murphy – University of Tasmania Gregory Jordan – University of Tasmania, Timothy Brodribb – University of Tasmania Modification of leaf area (or epidermal cell area) has been proposed as a mechanism to balance leaf water supply (driven by minor veins) with demand (generated by stomata) during acclimation to sun and shade. This has been observed in woody species but not in herbs. We compared the acclimation of minor veins and stomata in woody and herbaceous plants using four phylogenetic pairs of species (one of each habit). Leaf lignin concentration was also quantified to assess whether construction costs of herbaceous leaf veins differ from woody species and how much leaf

weight was occupied by minor veins. Coordinated changes to minor veins and stomata in all species were mediated by a common relationship between epidermal cell area and vein and stomatal density. This coordination is likely driven by the significant cost of minor veins (13.07 % of the dry leaf weight in sun leaves and 21.64 % in shade leaves). Relative costs of venation increased in the shade, intensifying selective pressure towards economising the investment in minor vein density. Modulation of epidermal cell area appears to be a general mechanism to maximise investment efficiency in leaf anatomical traits that control leaf water fluxes independent of leaf habit or ecology.

1100-003-Z Long-term Biological Consequences of the Chernobyl Accident in Chronically Irradiated Plant Populations Polina Volkova – Russian Institute of Radiology and Agroecology Stanislav Geras’kin – Russian Institute of Radiology and Agroecology, Elizaveta Kazakova – Russian Institute of Radiology and Agroecology The Chernobyl accident was one of the most severe radiation disasters in the world. Long-term consequences for biota have been studying for more than 25 years after that accident. It is known that chronic stress exposure can change an amount or structure of the intra-population variability. Therefore, it is necessary to analyze the relationships between genetic polymorphism and chronic radiation exposure. These studies are useful for development of principles and standards of the radiation protection of biota and also can be used in biomonitoring after accident situations. For investigation we have chosen 6 areas in the Bryansk region, the most contaminated after the Chernobyl accident region of Russia. Our study conducted in several districts of Bryansk region, which are characterized the most dose rate. Experimental sites similar to climate characteristics, stand of trees is homogeneous, pine trees take up a significant part of phytocenosis. Six populations of Pinus sylvestris L. growing under the different levels of radiation exposure were chosen as objects in our study. Experiment has been carrying on since 2003 and within its framework were investigated cytogenetic characteristics, genetic structure, reproductive ability, radioadaptation, morphological characteristics and biochemistry changing in the experimental populations. We identified changes in a genetic structure of studied populations. Frequency of the rare alleles, indices of genotypic diversity and proportions of the rare alleles increased along the level of radiation exposure. A frequency of mutational events in isozyme loci was enhanced. Nevertheless, no significant influence of radiation exposure in studied dose rates range (7-130 mGy/year) on enzymes activities was found. During all experimental years, frequency of aberrant cells in the most exposed populations has been significantly higher than on the reference sites. This confirms that even relatively low doses of chronic radiation exposure can effect on genetic structure and microevolution of chronic irradiated plant populations.

1100-004-Y Temperature Has a Stronger Effect Than Photoperiod in Regulating Seasonal Photosynthetic Performance in White Spruce (Picea Glauca) Joseph Stinziano – The University of Western Ontario Danielle Way – The University of Western Ontario Elevated temperatures associated with global climate change are expected to extend the growing season in high latitude forests, potentially increasing seasonal carbon uptake and helping offset anthropogenic CO2 emissions. However, trees use not only late season declines in temperature to sense when to prepare for winter dormancy, but also declines in day length. Because photoperiod is not affected by climate change, day length signals might constrain the ability of forests to extend their growing season in a warmer climate. We hypothesized that photoperiod is more important than

temperature in regulating seasonal photosynthetic capacity in a dominant boreal conifer, white spruce (Picea glauca), as changes in photoperiod are a more reliable cue of seasonality than changes in temperature. White spruce were grown under combinations of either naturally declining or constant temperature and photoperiod treatments, to simulate autumn environmental cues. Two different warming treatments both delayed autumn photosynthetic capacity declines, while photoperiod treatments had little effect on patterns of seasonal carbon uptake capacity. Weekly variation in leaf nitrogen (reflecting leaf protein concentration) was closely coupled to changes in photosynthetic pigments, although photosynthetic nitrogen use efficiency increased under warming. In contrast, water use efficiency remained constant across all treatments. These data suggest that temperature is more important than photoperiod in regulating seasonal changes in photosynthetic parameters in white spruce, implying that as the climate warms, the growing season length for boreal conifers may be extended, enhancing carbon sequestration in forest ecosystems.

1100-005-Y Climatic and Competitive Constraints Drive Clinal Variation in Populus Tremuloides Leaf Morphology Alec Baird – University of Washington Janneke Hille Ris Lambers – University of Washington, Elizabeth Van Volkenburgh – University of Washington A clear understanding of the relationship between leaf morphology and climate is essential to predict plant responses to anthropogenic climate change. To explore this topic, I collected leaves from 90 Populus tremuloides (quaking aspen) saplings in summer of 2014 across the species’ elevational range (2600 – 3200m), which covers significant precipitation and temperature gradients, in the La Plata Mountains, Colorado. For each sapling, I measured the specific leaf area (cm2/g), and growth rate (cm/year) with age calculated from annual ring analysis. Competition for sunlight was quantified as continuous leaf area index (LAI) analyses via fisheye photos. There is a strong positive relationship between SLA and leaf area index, with individuals under open canopies producing leaves with less surface area but greater thickness, while individuals under more closed canopies produce leaves with greater surface area but less thickness. There was a slight significant difference in SLA across elevations, with low elevation individuals producing leaves of lower SLA while individuals of middle and higher elevations produced leaves of greater SLA values. We found a positive interaction between elevation and LAI, such that shaded individuals at high elevations produced higher SLA leaves than shaded individuals at low elevation. These results suggest that light availability, as opposed to climate, drives major changes in SLA. Annual growth showed no significant relationships as a function of SLA, LAI, and the interaction between the two and elevation. These results indicate that light availability is the strongest driver of intra-specific variation in leaf morphology, and surprisingly, that juvenile aspen growth is not strongly constrained by either light availability or geographic variation in drought stress. Perhaps aspen understories are more microclimatically homogenous than macroclimatic gradients might suggest, or that clonality or variation in traits other than leaf morphology sustain growth rates even under water and light limitation.

1100-006-Z An Analysis and Evaluation of the Sensitivity of an Agroecosystem Model to Variation in Key Photosynthetic Parameters Andy VanLoocke – Iowa State University Selina Teh – Iowa State University, Rajsanjyot Kantipudi – Iowa State University, Robert Koester – University of Illinois, Elisabeth Ainsworth – USDA ARS One major challenge in assessing the accuracy of agroecosystem models that are based on the Farquhar et al. (1980) photosynthesis model is obtaining data on crops or genotypes that have a broad range of photosynthetic capacity measured under consistent environmental conditions. The objective of this study is to assess the ability of an agroecosystem model, Agro-IBIS, to accurately simulate the sensitivity of crop yield to variation in ecosphysiological

parameters using data collected on 24 field grown soybean genotypes. Our approach has two components 1) conduct multiple model simulations with combinations over a range of parameter values and determine the response of model outputs, and 2) compare the predicted model response to observed variations of the input parameters and the resulting variations in yield for soybeans grown under field conditions. The first component uses model simulations to create response surfaces of soybean yield for thousands of combinations of key physiological parameters including the maximum rate of electron transport (Jmax), the maximum velocity of carboxylation by Rubisco (Vc,max), and specific leaf area. The second component uses physiology, gas exchange and yield data collected from a common garden experiment in which soybean cultivars with release dates spanning from the 1920’s to 2000’s were grown. The model analyses were compared to the observed response surfaces and regions of model improvement were identified. Preliminary results suggest that the modeled predictions of yield are similar to observed yield but are more sensitive to changes in Vc,max and Jmax than measured yield. The next step in our analysis will be to determine the source of the model error leading to this increased sensitivity. We will test the hypothesis that inaccurate model responses are due to co-dependencies amongst model parameters which are not currently included in the analysis.

1100-007-Z Regulation of PIF3-like and Other Genes Associated with Weed Stress in Soybean David Horvath – USDA-ARS Stephanie Hansen – South Dakota State University, Sharon Clay – South Dakota State University, Janet Miller – South Dakota State University, Ronald Pierik – Universiteit Utrecht, Netherlands, David Clay – South Dakota State University, Brian Scheffler – USDA-ARS, Changhui Yan – North Dakota State University Weeds reduce yield in soybeans through incompletely defined mechanisms even when not directly competing for resources. Thus it should be possible to increase productivity under weedy conditions by making crops blind to weeds. This requires an understanding of how crops perceive and respond to weeds. The effects of weeds on soybean transcription were evaluated in field conditions over four years. RNASeq data collected from 6 biological samples of soybeans growing with or without weeds identified two PIF3-like genes and 67 other genes as differentially expressed in response to weed pressure. The relationship of these weed-induced PIF3 genes to genes involved in shade avoidance responses in arabidopsis suggest that these genes may be important in soybean weed responses. Given the likely role of these genes in the yield-reducing shade avoidance syndrome, we suggest these PIF3 genes could be targets for manipulating weed tolerance in soybean. We further investigated the regulation of weed-induced PIF3s and these other genes in greenhouse and growth chamber conditions. The weed-induced soybean PIF3 genes are induced when soybeans are grown in the presence of weeds even when the weed is prevented from directly competing with the soybeans for light, nutrients, or water. However, in growth chambers with high red:far ratios, expression of few weedregulated genes were altered even when weeds were grown in direct root-to-root contact with the weeds. Additionally, only a subset of weed-regulated genes observed under field conditions were differentially regulated in greenhouse conditions where red:far red light ratios were very large in the morning and evening due to artificial lighting. We further investigated the role of light quality on induction of the weed-induced PIF3 genes by growing soybeans in pots surrounded by tufts of far red reflecting plastic. We conclude that the red:far red light ratio induces these weed-induced PIF3 genes.

1100-008-Y Volatile Organic Compounds Act as Antimicrobial Agents and as Inducers of Resistance to Anthracnose in Phaseolus Vulgaris Elizabeth Quintana – Postdoctoral Cinvestav Irapuato Martin Heil – Research Professor Cinvestav Irapuato

Plants have evolved multiple strategies to resist both abiotic and biotic stress. The release of volatile organic compounds (VOCs) is mainly discussed in the context of indirect defense via tritrophic interactions but in fact, VOCs are multifunctional and play important roles as direct resistance agents , as signals that induce resistance-related responses in the emitting plant itself and in neighboring plants, and as damage-associated molecular patterns (DAMPs). Here, we report that VOCs released from resistant cultivars of bean (Phaseolus vulgaris) in response to inoculation with the fungal pathogen, Colletotrichum lindemuthianum (the causal agent of Anthracnose), cause airborne resistance in eighboring, susceptible plants. Experiments aimed at understanding the underlying mechanisms revealed the priming of marker genes of plant resistance to pathogens (pathogenesis-related [PR] 1, 2 and 4). One VOC, limonene, was sufficient to induce resistance to the fungal pathogen in bean plants, as it had been described earlier for nonanal-induced resistance to the bacterial pathogen, Pseudomonas syringae. Independently of these responses in the receiver plant, VOCs such as limonene and b-linalool directly and irreversibly inhibited the germination of fungal spores in vitro and in vivo and the formation of spores by life mycelia in vitro. Plant-derived VOCs can induce transcriptional responses related to pathogen resistance in receiver plants and cause associational resistance via their direct, antimicrobial effects.

1100-009-Y Determining the Efficiency of Photorespiratory Recycling and the Impact to Net CO2 Assimilation Using Gas Exchange Berkley Walker РUSDA/ARS, University of Illinois Doug Orr Orr РRothamsted Research, Andr̩ Alc̢ntara РRothamsted Research, Elizabete Carmo-Silva РRothamsted Research, Carl Bernacchi РUSDA/ARS, University of Illinois, Martin Parry РRothamsted Research, Paul South РUSDAARS, Donald Ort РUSDA-ARS Photorespiration comprises the second largest flux of CO2 in an illuminated C3 leaf. Photorespired CO2 significantly decreases the efficiency of net CO2 assimilation and subsequent plant yield. This CO2 loss occurs in the mitochondria, but traditional models of photosynthetic gas exchange implicitly assume it occurs in the chloroplast. This could underestimate the amount of CO2 lost from photorespiration by ignoring possible loss of CO2 from the mitochondria directly to the intercellular space. The stoichiometry of this CO2 loss per Rubisco oxygenation may also increase in photorespiratory mutants, but it is unclear if this loss of efficiency explains lowered net photosynthetic rates. We hypothesized that plants have evolved to minimize photorespiratory loss of CO2 through anatomical configuration and possibly redundancy in pathways for photorespiratory flux. We tested this hypothesis using CO2 gas exchange and electron microscopy on a diverse group of agronomically important species. Thus far we have found little evidence for significant CO2 release from the mitochondria to the intercellular airspace using a novel gas exchange analysis. We also present gas exchange and biochemical data in Arabidopsis mutants lacking the glycerate/glycolate antiporter PLGG1 and the genes required for peroxisomal reduction of hydroxypyruvate (hprpmdh1pmdh2). Despite disruption to photorespiration, these mutants are able to fix CO2 as efficiency as wild type plants even under conditions of high photorespiration at low light. At high light, they have reduced photosynthetic rates that are explained by Rubisco deactivation and reduced electron transport and not decreases to the efficiency of photorespiratory recycling of carbon. These findings support the hypothesis that photorespiration has evolved to be efficient and likely possesses alternative pathways. The work with plgg1 and hprpmdh1pmdh2 further suggests that photorespiration is able to deactivate Rubisco and the light reactions of photosynthesis.

1100-010-Z Evolution of Freezing Tolerance in the American Oaks Jeannine Cavender-Bares – University of Minnesota Frank Hoerner – University of Minnesota/Franklinville Experimental Station, Jose Meireles – University of Minnesota, Matthew Kaproth – University of Minnesota, Andrew Hipp – Morton Arboretum

A long-standing ecological hypothesis is that species are limited to the north by minimum temperature and to the south by competition, resulting in an evolutionary trade-off between freezing tolerance and growth rate. We investigated the extent to which the climatic origins of populations of 33 oak species (genus Quercus) that spanned a tropical-temperate gradient and span the major phylogenetic lineages with the genus were associated with freezing tolerance and growth. Acorns from multiple populations for each species were grown under common tropical and temperate conditions. Growth rate, seed mass, and stem freezing vulnerability using the electrolyte leakage method were compared with source mean minimum temperatures. Data were analyzed using phylogenetic generalized least squares regression of traits on climatic variables as well as an adaptation modeling approach that jointly estimates the effects of adaptation and phylogenetic inertia in the evolution of traits toward shifting environmental predictors. This approach combines the strengths of phylogenetic comparative analysis and optimality testing in understanding the role of natural selection in trait evolution. We find that climatic distributions predict freezing tolerance, cold temperature acclimation potential and growth rates. Our analyses support the hypothesis that the evolution of freezing tolerance and growth among species and of tradeoffs in functional traits contribute to species distribution limits across major climatic gradients and species turnover across habitats.

1100-011-Z Patterns in Sap Flow and Water Use in Sympatric Oak Species Along a Hydrologic Gradient Jennifer Teshera-Levye – University of Minnesota Brianna Miles – Colorado State University, Valery Terwilliger – University of Kansas, Jeannine Cavender-Bares – University of Minnesota Co-occurrence of closely-related sympatric species may be promoted by partitioning variation in resources and stress factors along environmental gradients. Some species may become specialized to particular niches within the larger ecosystem; there is a trade-off, however, between increased specialization and the ability to respond plastically to changing environmental conditions. Understanding the degree of variability in physiological responses to different environmental conditions will be particularly important as climate shifts and weather patterns become more extreme. We investigate this problem in three co-occurring species of oak (Quercus alba, Quercus palustrus, and Quercus falcata) at the Smithsonian Environmental Research Center in Maryland. Over the course of two summers, we measured sap flow, whole plant hydraulic conductivity, water use efficiency in 13 individuals of each species. The species co-occur along an elevation gradient ranging from floodplain to a drier upland habitat; though their distributions overlap, the peak distribution of Q. palustrus occurs in the wetter lowland relative to the other two species, whose peak distributions occur farther upland. In addition to sampling across this habitat gradient, sapflow was monitored during both a wet summer and a drought summer, enabling us to look at both inter-specific variation and intra-specific variation in water use along the hydrologic gradient and between years. Teasing apart these differences helps to clarify how much trait variation exists within a population after specializing to a particular part of its range, potentially enabling us to make more accurate predictions of tree responses to changing future climates.

1100-012-Y Mechanisms Underlying Elevation Stratification in Southwestern US Oak Species Are Not Revealed by Leaf Wilting Point and Stem Freezing Injury Tests Beth Fallon – University of Minnesota, Plant Biological Sciences Dept. Jeannine Cavender-Bares – University of Minnesota, Dept. of Ecology, Evolution & Behavior The vegetation zones in the mountains of the southwestern U.S. have long been correlated with temperature and precipitation gradients. Resurveys of historical transects have demonstrated upward shifts in elevation means that may

be due to climate warming. Determining the role of physiological tolerances in demarcating elevation range limits is important in deciphering mechanisms of stratification and predicting ecosystem vulnerability. We examined six sympatric oak species (Quercus L.), which partition space along the climatic gradients of the Chiricahua Mountains in southeastern Arizona, to evaluate the influence of physiological tolerances to local climate on elevation ranges. We surveyed randomly-stratified sites spanning all elevations to examine associations of species with local climate. The oak species occupy overlapping elevation ranges, yet differ in mean elevation and mean climate variables. We established monitoring sites (sites=12, N=138) and examined pre-dawn water potential during both summer drought and after seasonal rains. Species differed in water access during both precipitation regimes (p