MINISYMPOSIUM ABSTRACTS

Minisymposium 1: Abiotic: Water MS-1-0 Overview of Area by Chair, Carla Antonio, Instituto De Tecnologia Quimica E Biologica-Plant Metabolomics Laboratory (ITQB-UNL)

MS-1-1 Regulation of Primary Metabolism in Response to Flooding Stress as Revealed by 13C-stable Isotope Redistribution Carla Antonio – ITQB-UNL 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 – RWTH Aachen University Track Name: Biotic + Abiotic 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.

MS-1-2 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 Track Name: Biotic + Abiotic

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 oxygen-sensor 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). Currently we are investigating N-end rule pathway regulation of response to other abiotic stresses in barley.

MS-1-3 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 Track Name: Biotic + Abiotic 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.

MS-1-4 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 Track Name: Biotic + Abiotic 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.

MS-1-5 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 Track Name: Biotic + Abiotic 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.

Minisymposium 2: Bioinformatics MS-2-0 Overview of Area by Chair, Shahid M. Mukhtar, University of Alabama at Birmingham

MS-2-1 Getting to the Edge: Network Biology Identifies Essential Hubs in Arabidopsis Interactions Networks Shahid Mukhtar – UAB Hadia Ahmed – UAB Track Name: Genomics Diverse natural systems ranging between the extremes of the biological spectrum - the microscale of the cell and the macroscale of socioecosystems - have been explored to understand their hierarchical structures. These biological networks possess ubiquitous properties such as scale-freeness and small-world nature. Systems-level network analyses and computational modeling can provide global understanding of molecular mechanisms governing biological processes under diverse cellular states. The research in my laboratory focuses at the interface of plant-pathogen interactions and bioinformatics. We employ systems-level approaches, in particular network-based analyses, to understand how macromolecular networks are organized in the cells and how pathogen proteins perturb such networks to promote diseases. Previously, we generated proteome-scale interactome maps including Arabidopsis interactions network (Science 2011), Arabidopsis-pathogens interactions networks (AI-1; Science 2011, Cell Host & Microbe 2014). Currently, we are employing network decomposition analyses on AI-1 and MIND1 (Membrane-based Interactome Database; Science 2014) to understand the structure and organization of these networks. Integration of large-scale Arabidopsis co-expression and phenotypic data into various layers of Arabidopsis networks will provide novel insights in understanding the properties of essential/non-essential proteins. These data also evidenced that pathogen effectors target host non-essential hub proteins to modulate immune responses. Our large-scale integrative network-based analysis is expected to (a) provide a comprehensive understanding of the relationships between phytopathogens and plants, (b) infer and assess biological functions, (c) understand biological processes and molecular pathways, and (d) predict and prioritize candidates for further investigations.

MS-2-2 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 Track Name: Genomics 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 DE-SC0000997

MS-2-3 Gene by Environment Interaction in the Maize Ionome Alexandra Asaro – Washington University in St. Louis/Donald Danforth Plant Science Center

Greg Ziegler – United States Department of Agriculture/Agricultural Research Service/Plant Genetics Research Unit/Donald Danforth Plant Science Center, Cathrine Ziyomo – Donald Danforth Plant Science Center, Owen Hoekenga – Genomics Consultant, Brian Dilkes – Department of Horticulture and Landscape Architecture, Purdue University, Ivan Baxter – United States Department of Agriculture/Agricultural Research Service/Plant Genetics Research Unit/Donald Danforth Plant Science Center Track Name: Genomics Plant elemental profiles are determined by interactions between a plant’s genetic background and its growth environment. In order to investigate genotype by environment interactions, we analyzed the maize (Zea mays L.) kernel ionomes of intermated B73 x Mo17 (IBM) recombinant inbreds grown in multiple locations. We measured the levels of 21 mineral nutrients in seeds from eight experiments in four locations over five different years using ICP-MS. Quantitative trait locus (QTL) mapping of these element composition data and a set of 4,217 biallelic markers was implemented with the R packages R/QTL and QTLRel. We evaluated several different methods identifiying QTL by environment interactions (QEIs). Overall, we were able to identify a large number of QTL and QEIs, indicating that elemental profiles are highly heritable and responsive to the environment. We also performed a principal components analysis (PCA) on the elemental composition data and then used these principle components in place of the elemental phenotypes for QTL mapping. Analysis of the PCs identified additional loci affecting the ionome that were not detected in single element scans, suggesting pleiotropic alleles with multi-element effects. We were also able to correlate weather data and other location-specific variables with the elemental traits to identify several potential environmental drivers of ionomic variation.

MS-2-4 How to Scale Data, Science, and People Using Biological Cyberinfrastructure Jason Williams – iPlant/CSHL

The iPlant – iPlant Collaborative Track Name: Genomics At all levels of scientific investigation, valuable opportunities are realized by having the ability to scale questions; single-gene 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.

MS-2-5 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 Track Name: Genomics 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 cross-compatible 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.

Minisymposium 3: Calcium Signaling MS-3-0 Overview of Area by Chair, Tina Romeis, FU Berlin

MS-3-1 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 Track Name: Diverse-Talk 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 post-translational 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.

MS-3-2 Visualization of Real-time Cytosolic Ca2+ Elevation Using a Novel Optogenetic Ca2+ Sensor Yi Ma – University of Connecticut Gerald Berkowitz – University of Connecticut Track Name: Diverse-Talk Ca2+ is a ubiquitous secondary messenger involved in numerous plant signal 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.

MS-3-3 Purine Nucleotide-regulated Calcium Signalling in Stress and Development Julia Davies – University of Cambridge Track Name: Diverse-Talk 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.

MS-3-4 Coordination of Pollen Tube Growth by Ca2+: Channels and Downstream Mechanims José Feijó – University of Maryland Track Name: Diverse-Talk Pollen transcriptomics revealed the expression of of about 7.000 genes in pollen, but theoretical modelling suggests that the cooperation of all of these into the processes of wall surface and cytoplasmic volume production is a minimal condition to explain most of the morphogenic events that characterize these cells. Spatial and temporal integration of

extended biochemical and biophysical processes is mandatory, and in the past we have proposed that ion dynamics can be a common regulator of fundamental growth processes. Modifications of Hodgkin-Huxley equation to space rather than time, allows the prediction of precise physical conditions in the growing tip consistent with an ion based coordination of cell growth and morphogenesis. Validation of these assumptions implies integration of various levels of organization, and we’re presently focusing on the genetic tools necessary to test some of the predictions. I will report on advances on the biology of Glutamate- Receptor Like (GLR) Ca2+-channels. These channels are hypothesized to participate on the generation of the Ca2+ focused gradient characteristic of functional pollen tubes, and eventually regulate Ca2+ fluxes into other compartments as well. I will present data suggesting an evolutionary conservation of these channels related to a male reproductive function. Further, detailed genetic analysis of GLR family genes, coupled with theoretical analysis of the Ca2+ cytosolic concentration vs. extracellular fluxes, revealed that rather than being strictly plasma membrane channels, they may be differentially distributed throughout the endomembrane system. On the absence of specific peptide tags, we propose that these localizations may be regulated by an underlying mechanism based on specialized sorting chaperone proteins. If this hypothesis is confirmed, the integrated study of of GLRs may start the foundation of a completely new view of Ca2+ signalling and homeostasis in plant cells.

MS-3-5 Genetic Regulators of Arabidopsis Circadian Oscillations of Cytosolic-free Calcium Timothy Hearn – University of Cambridge Alex Webb – University of Cambridge Track Name: Diverse-Talk 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.

Minisymposium 4: Fruit Development MS-4-0 Overview of Area by Chairs, Zhongchi Liu, Univ. of Maryland, and Robert G. Franks, Ph.D., North Carolina State University

MS-4-1 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 Track Name: Development 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. In addition, receptacle is a unique tissue in strawberry and we have identified receptacle-specific genes and miRNAs. One such miRNA is highly and specifically expressed in the receptacle and initiates phased siRNAs that target a large family of F-Box genes. Another receptacle-specific gene encodes ahomolog of FLOWERING LOCUS T (FT). Promoter::GUS construct showed that this gene is transcribed in the vasculature connecting the seeds and the fleshy receptacle fruit. Functional studies are being carried out to determine the fruit-specific miRNA andFT in strawberry fruit development. Our studies are beginning to reveal the molecular underpinnings of fruit set and early stage fruit development.

MS-4-2 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 Track Name: Development 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.

MS-4-3 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 Track Name: Development 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.

MS-4-4 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 Track Name: Development 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.

MS-4-5 Domain-Specific Transcriptional Profiles from the Arabidopsis Gynoecium Reveal Robust Developmental Mechanisms Required for Ovule Development Robert Franks – NCSU Gonzalo Villarino – NCSU, Bhupinder Sehra – NCSU, Miguel Flores – NCSU, Eva Sundberg – Swedish University of Agricultural Sciences Track Name: Development 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.

Minisymposium 5: Membrane Dynamics MS-5-0 Overview of Area by Chair, Karin Schumacher, Heidelberg University

MS-5-1 Protein Trafficking – the Only Constant Thing Is Change Karin Schumacher Heidelberg University Stefan Scholl – COS, Upendo Lupanga – COS

Track Name: Diverse-Talk 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.

MS-5-2 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 Track Name: Diverse-Talk 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.

MS-5-3 Post Golgi Vesicle Trafficking During Plant Cell Elongation and Cell Division Natasha Worden – University of California Davis Destiny Davis – University of California Davis, Georgia Drakakaki – University of California Davis Track Name: Diverse-Talk The plant endomembrane system controls many aspects of plant signaling and development including deposition of the cell wall during both cell division and elongation. Using a combination of chemical genomics and proteomics we studied

components of the endomembrane system that are present in the trans-Golgi Network (TGN), a site of polysaccharide trafficking and recycling of endosomal components. We have identified novel pharmacological inhibitors, which target cell wall deposition during cell elongation and cell division.

The small molecule, CESTRIN, is a novel inhibitor of cellulose synthase localization. It reduces the cellulose content in plants, and interferes with the interplay of cellulose synthases with microtubules. This behavior affords the identification of new players involved in the organization of cellulose synthase complexes at the plasma membrane. Cellulose synthase proteins are present in Syntaxin of Plants 61 (SYP61) vesicles and this localization is increased under CESTRIN application. In a complementary proteomic approach, we separated TGN vesicles identified by the syntaxin SYP61, and analyzed their proteome. We are currently characterizing several components of the SYP61 vesicle proteome of with no assigned function and investigating their involvement in cell wall deposition.

Endosidin 7 (ES7) is an additional small molecule that targets callose synthase activity during late cytokinesis and arrests cell plate maturation. The temporal interplay between cell-plate-specific post-Golgi vesicle populations and callose accumulation reveals the unique contributions of secretory and endosomal vesicles, and provides a detailed framework for the understanding of cell plate formation. Our data detail the essential role of callose during the late stages of cell plate maturation and establish the temporal relationship between vesicles and regulatory proteins at the cell plate assembly matrix during polysaccharide deposition.

MS-5-4 Endocytosis of Plant Receptor Kinases Eugenia Russinova – VIB-UGent Track Name: Diverse-Talk 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 suffice, 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.

MS-5-5 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 Track Name: Diverse-Talk 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).

Minisymposium 6: Abiotic: Salt & Minerals MS-6-0 Overview of Area by Chair Miguel Piñeros, Agricultural Research Service - US Department of Agriculture

MS-6-1 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 Track Name: Biotic + Abiotic 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.

MS-6-2 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 Track Name: Biotic + Abiotic 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.

MS-6-3 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 Track Name: Biotic + Abiotic 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 G-protein 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.

MS-6-4 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 Track Name: Biotic + Abiotic 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 co-expressing 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.

MS-6-5 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) Track Name: Biotic + Abiotic 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 Fe-deficient 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.

Minisymposium 7: Biochem of Bioenergy MS-7-0 Overview of Area by Chair, Harvey Millar, the University of Western Australia

MS-7-1 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 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 Track Name: Biochem 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.

MS-7-2 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 Track Name: Biochem 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 phosphorylation for the plant monolignol biosynthetic pathway. Here, we present the discovery of phosphorylation mediated on/off regulation of enzyme activity for 5-hydroxyconiferaldehyde O-methyltransferase 2 (PtrAldOMT2), an enzyme central to monolignol biosynthesis for lignification in stem differentiating xylem (SDX) of Populus trichocarpa. Phosphorylation turned off the PtrAldOMT2 activity, as demonstrated in vitro using purified phosphorylated and unphosphorylated recombinant PtrAldOMT2. Protein extracts of P. trichocarpa SDX, which contains endogenous kinases, also phosphorylated recombinant PtrAldOMT2 and turned off the recombinant protein activity. Similarly, ATP/Mn2+–activated phosphorylation of SDX protein extracts reduced the endogenous SDX PtrAldOMT2 activity by approximately 60%, and dephosphorylation fully restored the activity. Global shotgun proteomic analysis of phosphopeptide enriched P. trichocarpa SDX protein fractions identified PtrAldOMT2 mono-phosphorylation at Ser 123 or Ser 125 in vivo. Phosphorylation-site mutagenesis verified the PtrAldOMT2 phosphorylation at Ser 123 or Ser 125, and confirmed the functional importance of these phosphorylation sites for O-methyltransferase activity. The PtrAldOMT2 Ser 123 phosphorylation site is conserved across 93% of AldOMTs from 46 diverse plant species, and 98% of the AldOMTs have either Ser 123 or Ser 125. PtrAldOMT2 is a homodimeric cytosolic enzyme expressed more abundantly in syringyl lignin-rich fiber cells than in guaiacyl lignin-rich vessel cells. The reversible phosphorylation of PtrAldOMT2 is likely to have an important role in regulating syringyl monolignol biosynthesis of P. trichocarpa.

MS-7-3 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 Track Name: Biochem 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.

MS-7-4 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 Track Name: Biochem 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 XS-P1 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.

MS-7-5 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

Track Name: Biochem

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 DE-SC0000997.

Minisymposium 8: Development MS-8-0 Overview of Area by Chair, Laurie Smith, University of California San Diego

MS-8-1 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 Track Name: Diverse-Talk 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 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., (2002). Development 129: 1261-1272. 2. Burch-Smith, T.M., et al. (2011). PNAS Plus 108: E1451-;E1460.3. Stonebloom, S., (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.

MS-8-2 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 Track Name: Diverse-Talk 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 map-based 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.

MS-8-3 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 Track Name: Diverse-Talk 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 (FT) in Arabidopsis, advances determinate growth while its closely-related antagonist, SELF-PRUNING (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.

MS-8-4 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 Track Name: Diverse-Talk 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.

MS-8-5 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 Track Name: Diverse-Talk

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.

Minisymposium 9: Plant Architecture MS-9-0 Overview of Area by Chair, Dave Jackson, PhD, Cold Spring Harbor Lab

MS-9-1 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 Track Name: Development 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.

MS-9-2 Control of Arabidopsis Plant Architecture by Gravity Through LAZY Gene Action Takeshi Yoshihara – University of Wisconsin-Madison Edgar Spalding –University of Wisconsin-Madison Track Name: Development 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.

MS-9-3 How Can We Understand Plants as Integrated Systems? Christopher Topp – Danforth Center Track Name: Development 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.

MS-9-4 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 Track Name: Development A major question in plant biology remains what genetic factors determine grass leaf architecture e.g., leaf length, width, angle (blade deflection from the culm). Collectively, such morphological traits directly influence canopy structure and light penetration, photoassimilate production, and, important to crops, 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 Mo17. We cloned the underlying gene, drl1, and identified its paralogous enhancer, drl2, using positional cloning and generated a second drl1 allele by Ds 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: 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 regulate proper development of important agronomic traits in leaf and floral organs.

MS-9-5 Root Hydropatterning: Local Water Availability Acts as a Signal for Lateral Root Initiation Neil Robbins – Standford University 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

Track Name: Development 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.

Minisymposium 10: Biotic Interactions - 1 MS-10-0 Overview of Area by Chair, Antje Heese, University of Missouri-Columbia

MS-10-1 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 Track Name: Biotic + Abiotic Carina Collins, Lauren Bond, John Smith, Daniel Salamango, Scott Peck and Antje Heese Div. of Biochemistry, IPG, Univ. of Missouri, Columbia, MO, 65211, USA, 221 Schweitzer Hall, 3 Div. of Plant Sciences, 271H Bond Life Sciences Center 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 Col-0 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.

MS-10-2 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 Track Name: Biotic + Abiotic 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 stress. 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.

MS-10-3 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

Track Name: Biotic + Abiotic 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 effector-triggered 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.

MS-10-4 Recognition of Insect Eggs by Arabidopsis Thaliana Andre Schmiesing – University of Lausanne Philippe Reymond Track Name: Biotic + Abiotic 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 pathogen-responsive 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 pathogen-associated 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.

MS-10-5 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 Track Name: Biotic + Abiotic Dissertation Body 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.

Minisymposium 11: Non-polar Metabolites: Signals and Regulation MS-11-0 Overview of Area by Chair, Dean DellaPenna, Michigan State University

MS-11-1 New Roles for Carotenoids and Carotenoid-derived Signals in Regulating Cold Tolerance and Plant Development Barry Pogson – Australian National University Track Name: Biochem 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 (Van Norman et al. PNAS 2014, Avendaño-Vázquez et al 2014, The Plant Cell) and new roles for carotenoids and their derivatives in cold tolerance of cyanobacteria and Arabidopsis.

MS-11-2 The ORANGE Proteins Are the Major Posttranscriptional Regulators of Phytoene Synthase in Controlling Carotenoid Biosynthesis Li Li – USDA-ARS Xiangjun Zhou – Cornell University, Ralf Welsch – University of Freiburg Track Name: Biochem 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.

MS-11-3 Volatile Cues Provide Honest Information for Partner Choice in a Horizontally Transmitted Mutualism María del Rosario Razo-Belmán – Cinvestav Irapuato Martin Heil – Cinvestav Irapuato Track Name: Biochem 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 trade-offs 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.

MS-11-4 “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 Track Name: Biochem

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.

MS-11-5 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 Track Name: Biochem 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.

Minisymposium 12: Biotic Interactions – 2 MS-12-0 Overview of Area by Chair, Rebecca Bart, DDPSC

MS-12-1 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 Track Name: Biotic + Abiotic 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.

MS-12-2 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 Track Name: Biotic + Abiotic 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, test and confirm the minimal set of molecular determinants that define pathovar specificity within the Xanthomonas genus. We are combining traditional gene knockout strategies with a novel comparative genomics approach. Finally, several exciting new phenotyping techniques will be discussed.

MS-12-3 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 – UW-Madison, Patrick McMinn – UW-Madison, Alice Teillet – UW-Madison Track Name: Biotic + Abiotic 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) mechanistic impacts of the non-canonical alpha-SNAP residues present in the resistance-conferring version of that Rhg1 protein, 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.

MS-12-4 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 Track Name: Biotic + Abiotic 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.

MS-12-5 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 Track Name: Biotic + Abiotic

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 REGION (CLE) genes, MtCLE12 and MtCLE13, are induced in the nodule meristem of Medicago truncatula plants undergoing nodule initiation 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 or signal stability. 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 phenotypes in these mutants. 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/HPAT enzymes and their involvement in AON. This work is supported by NSF IOS#1146014 and the Clemson Creative Inquiry Program.

Minisymposium 13: Pollen MS-13-0 Overview of Area by Chair, Emily Indriolo, New Mexico State University

MS-13-1 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

Track Name: Development 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 species-specific 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.

MS-13-2 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 Track Name: Development 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.

MS-13-3 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 Track Name: Development Flowering plants have many methods to prevent self-fertilization, one method is the ability to reject self-pollen by self-incompatibility. 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 withSRK 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.

MS-13-4 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 Track Name: Development Abstract Summary 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 stigma-specific 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 self-incompatible 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.

MS-13-5 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 Track Name: Development 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.

Minisymposium 14: Genomic Diversity: Food Crops

MS-14-0 Overview of Area by Chair, C Robin Buell, Michigan State University

MS-14-1 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 Track Name: Genomics 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.

MS-14-2 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 Track Name: Genomics 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 population-scale re-sequencing of a diverse panel of 260 wheat accessions. A panel was selected to capture the genetic diversity of

the major 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.

MS-14-3 Neofunctionalization of homoeologs explains multiple mechanisms of chromosome pairing control associated with the Ph1 Gene of polyploid wheat Kulvinder Gill – Washington State University Ramanjot Kaur – Washington State University, Kanwardeep Singh – Washington State University, Ragupathi Nagarajan – Washington State University, Amita Mohan – Washington State University Track Name: Genomics

Most of the higher plants, including some of the most important crop plants, are polyploids or ancient polyploids. The Ph1 gene regulating chromosome pairing in wheat by differentiating homologous from homoeologous/orthologous pairing was discovered in 1958 but was so far not been cloned. Multiple studies have shown that the Ph1 gene effect is mainly manifested by the 5B copy of the gene although the 5D copy was also shown to have a weak effect. This was evident from the higher order pairing observed in the absence of 5B that was not prevalent in the absence of 5D. Moreover, increased dosage of 5A or 5D does not compensate for the loss of 5B. Several conflicting hypotheses have also been proposed for the Ph1 gene function. We have identified a gene (C-Ph1) silencing of which resulted in a phenotype similar to that of the Ph1 gene mutations. The gene has three structural copies that have both different structure and expression pattern suggesting multiple functions explaining the observations and hypotheses for the Ph1 gene action. The 5B copy of the gene has a novel 60bp insertion resulting in two alternate splice variants that are very different from the 5D or 5A copies. The 5A has a large 366bp deletion relative to 5D copy possibly rendering the corresponding protein ineffective. Specifically, the expression of the 5B copy increased 39-fold between late prophaseI and metaphaseI coinciding with the expected stage for the Ph1 gene function. The gene also has orthologs in other diploid species that resemble more with the 5D copy suggesting it to be the conserved and ancestral version of the gene. Novel function of the 5B in regulating homoeologs chromosome pairing evolved due to polyploid-specific insertions, alternate splicing, and/or highly specific expression during MI stage.

MS-14-4 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

Track Name: Genomics 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.

MS-14-5 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 Track Name: Genomics 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.

Minisymposium 15: Gene Regulation SPONSORED BY MOLECULAR PLANT

MS-15-0 Overview of Area by Chair Ann Loraine, University of North Carolina at Chapel Hill

MS-15-1 Role of SR45a in Alternative Splicing and Stress Response April Estrada – UNC Charlotte Nowlan Freese – UNC Charlotte, Ann Loraine – UNC Charlotte Track Name: Diverse-Talk 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.

MS-15-2 Identification of Proteins Required for Modification of Organelle Transcripts by RNA Editing Xiaowen Shi – Cornell University Maureen Hanson – Cornell University, Stephane Bentolila – Cornell University Track Name: Diverse-Talk 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.

MS-15-3 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 Track Name: Diverse-Talk 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/AvrBs2- and 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.

MS-15-4 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 Track Name: Diverse-Talk

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 SBP-domain 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.

MS-15-5 Application of Short Tandem Target Mimics (STTMs) for Deciphering Functions and Regulatory Networks of MicroRNAs in Crop Plants Sachin Teotia – Michigan Technological University Lina Shi – Michigan Technological University, Haiping Liu – Michigan Technological University, Guiliang Tang – Michigan Technological University Track Name: Diverse-Talk microRNAs are small non-coding RNAs that suppress the expression of their complementary target genes at the post-transcriptional 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 are studied.

Minisymposium 16: Genetics & Evolution MS-16-0 Overview of Area by Chair, Thomas Brutnell, Donald Danforth Plant Science Center

MS-16-1 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 Track Name: Genomics 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 norgDNAs 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 norgDNAs in complete reference assemblies of eleven species, O. sativa ssp. japonica, O. sativa ssp., 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 norgDNAs 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 norgDNAs 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.

MS-16-2 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 Track Name: Genomics 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.

MS-16-3 Identification and Functional Characterization of a New Protein Kinase Gene Family SRF Involved in Plant Stress Resistance Ning Yuan – Clemson University, Hong Luo – Clemson University

Track Name: Genomics Environmental stress is an important factor that significantly impacts plant development. Broad understanding of molecular mechanisms underlying plant stress response allows development of novel molecular strategies in genetically engineering crop species for enhanced performance under adverse conditions. We have identified a new Arabidopsis protein kinase gene family SRF (actor) comprising of four members (SRF1-4) whose expressions are all strongly regulated by salt and drought stresses. These four genes are highly conserved and clustered in the same chromosome region, suggesting that they may have evolved from the same ancestor via gene duplication. Subcellular localization using SRF-GFP (rotein) fusion protein reporter system revealed that they are all localized on plasma membrane, indicating that they may function similarly in plant stress response signaling. Gene expression analysis using RT-PCR and promoter-GUS reporter system revealed different expression patterns of the four SRF genes, suggesting their similar, but temporally and spatially differential functions in plants. Simultaneous knockout of SRF1 and 2 using RNA interference significantly enhanced plant abiotic stress tolerance, while overexpression of SRF1 increased plant pathogen resistance. These results indicate that the SRF gene family is involved in both abiotic and biotic stress triggered signaling pathways. To further understand the function of SRF, we are individually overexpressing the four genes in Arabidopsis, and these overexpression lines together with wild type control, RNAi knockout and T-DNA insertion mutant lines will be assessed for response to different stresses. Bimolecular fluorescence complementation assay will be conducted to investigate whether the four proteins work as dimers or not. Yeast two hybrid assay will be performed to identify ligands or co-regulators of SRF proteins.

MS-16-4 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 Track Name: Genomics 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.

MS-16-5 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 Track Name: Genomics 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.

Minisymposium 17: Hormone Signaling MS-17-0 Overview of Area by Chair, Joseph Kieber, University of North Carolina

MS-17-1 Cytokinin: Beyond Two Component Signaling Joseph Kieber – UNC Tracy Raines – UNC, Carly Sacks – UNC, Christian Burr – UNC Track Name: Diverse-Talk 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 two-component 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 type-B 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 two-component 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.

MS-17-2 Study the Function of EIN2 C-terminus in the Nucleus in Ethylene Response Hong Qiao – UT Austin Bin Qi – UT Austin, Fan Zhang – UT Austin

Track Name: Diverse-Talk Ethylene is one of the most important plant hormones. It plays pleotropic roles in many aspects of plant life such as seeds germination, hypocotyl elongation, cell expansion, stem cell maintenance and adaption to stress conditions. Molecular genetic dissection has revealed that ethylene signaling pathway is function in a liner manner from the perception by the receptors on the ER membrane to EIN3 dependent transcriptional regulation in the nucleus. A model for hormone signaling has emerged in which the perception of ethylene by the receptors alters the activity of CTR1, which in turn, by an unknown mechanism, functions to relieve repression of EIN2, resulting in activation of EIN3/EIL1-dependent transcription and the activation of an ethylene response. EIN2, an ER membrane resident protein, is the key regulation of ethylene signaling, lose of its function cause completely ethylene insensitive phenotype. A few years ago, the study demonstrated that the F-box proteins ETP1 and ETP2 via 26S proteasome negatively regulate the levels of EIN2. Recently study showed that EIN2 triggers its endoplasmic reticulum (ER)– to–nucleus translocation. ER-tethered EIN2 shows CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) kinase–dependent phosphorylation. Ethylene triggers dephosphorylation at several sites and proteolytic cleavage at one of these sites, resulting in nuclear translocation of a carboxyl-terminal EIN2 fragment. These findings uncover a mechanism of subcellular communication whereby ethylene stimulates phosphorylation-dependent cleavage and nuclear movement of the EIN2-C′ peptide, linking hormone perception and signaling components in the ER with nuclear-localized transcriptional regulators. The discovery of the functional mechanism of EIN2 opens a new avenue for fully understanding ethylene regulations. In this project, we focus on the function of EIN2 in the nucleus and its regulation in ethylene response.

MS-17-3 Identification and Characterization of the Polarly-localized TRANSPORTER of IBA1 Lucia Strader – Washington University in St Louis Marta Michniewicz – Washington University in St Louis

Track Name: Diverse-Talk 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.

MS-17-4 Electrophysiological Studies of ABCB4 and PIN2 Ion Channel Activities Stephen Des Lauriers – University of Wisconsin-Madison Edgar Spalding – University of Wisconsin-Madison Track Name: Diverse-Talk 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.

MS-17-5 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 Track Name: Diverse-Talk 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.

Minisymposium 18: Biotic Interactions - 3 MS-18-0 Overview of Area by Chair, Heidi M. Appel, PhD, University of Missouri

MS-18-1 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 Track Name: Biotic + Abiotic 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.

MS-18-2 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 Track Name: Biotic + Abiotic 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 non-acetylated 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.

MS-18-3 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 Track Name: Biotic + Abiotic 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.

MS-18-4 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 Track Name: Biotic + Abiotic 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.

MS-18-5 Partner Manipulation Stabilizes a Horizontally Transmitted Mutualism Martin Heil – CINVESTAV Irapuato Track Name: Biotic + Abiotic 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 [Heil, 2005 #793" href="#_ENREF_1"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.

Minisymposium 19: Reproductive Barriers: Molecular and Evolutionary MS-19-0 Overview of Area by Chair, Bruce McClure

MS-19-1 Does Parent-offspring Conflict Drive the Evolution of Hybrid Seed Lethality in Mimulus? John Willis – Duke University Elen Oneal – Duke University Track Name: Development

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.

MS-19-2 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 Tovar-Mendez – University of Missouri Columbia, Bruce McClure – University of Missouri Columbia Track Name: Development 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.

MS-19-3 The Genetics and Evolution of Reinforcement Robin Hopkins – Harvard University Track Name: Development 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.

MS-19-4 Pollen Tube Attractants as Potential Sexual Isolating Barriers in Wild Tomato Species (Solanum Sect. Lycopersicon) Leonie Moyle – Indiana University James Pease – Indiana University, David Haak – Indiana University, Matthew Hahn – Indiana University Track Name: Development 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.

MS-19-5 Molecular Dialogues Between Pollen and Pistil Mark Johnson – Brown University Alexander Leydon Track Name: Development 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.

Minisymposium 20: Specialized Cells and Their Metabolites MS-20-0 Overview of Area by Chair, M. David Marks, University of Minnesota

MS-20-1 Patterning Mechanisms of Cytoskeletal and Cell Wall Systems During Cell Morphogenesis Dan Szymanski – Purdue University Makoto Yanagisawa – Purdue University, Anastasia Desyatova – University of Nebraska Lincoln, Samual Belteton – Purdue University, Eileen Mallery – Purdue University, Joseph Turner – University of Nebraska Lincoln

Track Name: Biochem 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 cell shape change 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.

MS-20-2 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 Track Name: Biochem 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.

MS-20-3 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 Track Name: Biochem 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.

MS-20-4 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 Roberts Noble Foundation Track Name: Biochem 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 flavonoid- and 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 compound7,4-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.

MS-20-5 Biochemical and Phenotypic Characterization of Gibberellin Production in Rhizobia Ryan Nett – Iowa State University Xuan Lu – Iowa State University, Reuben Peters – Iowa State University Track Name: Biochem Gibberellins (GAs) are crucial growth and development hormones in plants, but can also be produced by some plant-associated 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.

Minisymposium 21: Abiotic: Stress MS-21-0 Overview of Area by Co-chairs Gustavo MacIntosh, Iowa State University and Adán Colón-Carmona, University of Massachusetts Boston

MS-21-1 H+-PPases from Salt Tolerance to Sucrose Transport Roberto Gaxiola – Arizona State University Track Name: Biotic + Abiotic Plant H+-PPases have been traditionally considered bona fide vacuolar markers despite several reports also showing their plasma membrane localization in phloem cells. The vacuolar role of this enzyme has been thoroughly documented. However, its role at the plasma membrane of phloem cells remains an unresolved question. Initial studies found that over-expression of AVP1 in Arabidopsis resulted in enhanced salt tolerance and drought resistance. The salt-tolerant phenotype was explained by an increased capacity of Na+ uptake into vacuoles and the drought-resistance phenotype was attributed to an enhanced vacuolar osmoregulatory capacity. Moreover, a study on the variation of salinity tolerance in Arabidopsis ecotypes reported a positive relationship between salt-tolerance and AVP1 expression. In addition to salt and drought tolerance, increased shoot and root biomass and yield, enhanced root acidification and improved nutrient use efficiencies have been also reported. These phenotypes are not well explained by the traditional model of type I H+-PPases operating solely on the vacuole to energize the tonoplast. In this work, we provide a body of genetic, physiological, and histochemical evidence consistent with the hypothesis that this plasma membrane localized H+-PPase works in reverse as a synthase to maintain PPi homeostasis required for sucrose uptake in phloem cells. This novel role in sucrose loading explains the increase in biomass reported in a plethora of plant species as a result of H+-PPase up-regulation. The potential of regulating the allocation of sugar represents a means to improve plant productivity.

MS-21-2 Anthocyanins and Stress Erich Grotewold – The Ohio State University Track Name: Biotic + Abiotic

Anthocyanins are induced in response to a number of biotic and abiotic stress conditions, including drought, high salinity, excessive light, and low temperatures. While the specific function of these pigments in providing tolerance to these various stress conditions is unknown, hypotheses include that they function as ROS scavengers, photoprotectants, and stress signals. We have recently determined that Arabidopsis (Arabidopsis thaliana) responds to different abiotic stresses by accumulating distinct anthocyanin profiles, suggesting that not all anthocyanins have the same function. Here, we discuss these findings in the context of other studies, and describe results trying to reconcile the localization (tissue and subcellular) in the context of their proposed functions.

MS-21-3 Linking Stress, Sugar Metabolism, and Nuclear Signaling in Maize: Ndpk1 (Nucleoside Diphosphate Kinase 1) Maria Sanclemente – University of Florida Track Name: Biotic + Abiotic

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.

MS-21-4 Small Compounds Targeting Tyrosine Phosphorylation of RACK1A Protein Confer ABA Hypersensitivity Hemayet Ullah – Howard University Track Name: Biotic + Abiotic 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. MS-21-5 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 Track Name: Biotic + Abiotic

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 AtMATE1-mediated 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.

Minisymposium 22: Metabolic Engineering MS-22-0 Overview of Area by Chair, Toni M. Kutchan, Donald Danforth Plant Science Center

MS-22-1 Plants as Synthetic Biology Platforms Toni M Kutchan – Donald Danforth Plant Science Center Track Name: Biochem 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.

MS-22-2 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, Ed Cahoon – University of Nebraska – Lincoln, Mike Pollard – Michigan State University, John Ohlrogge – Michigan State University

Track Name: Biochem 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-acetyl-1,2-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 acetyl-TAGs. Future field growth of these lines will produce large quantities of MCFA acetyl-TAGs for product testing.

MS-22-3 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 Track Name: Biochem 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 benzenoid-phenylpropanoid 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, IL11-4-1) 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 feedback-insensitive 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 flavor-associated 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.

MS-22-4 Overexpression of Proton-pumping Pyrophosphatases Stimulates Phloem Loading and Long-distance Transport in Arabidopsis Umesh Yadav – University of North Texas Aswad Khadilkar – University of North Texas, Carolina Salazar – University of North Texas, Vladimir Shulaev – University of North Texas, Julio Paez-Valencia – Arizona State University, Roberto Gaxiola – Arizona State University, Brian Ayre – University of North Texas Track Name: Biochem 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.

MS-22-5 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 Track Name: Biochem 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 evidence-based genome-scale metabolic reconstruction of maize, with significant improvements in the quality of the gene-reaction 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.

Minisymposium 23: Epigenetics MS-23-0 Overview of Area by Chair, Hong Gu Kang, Texas State University

MS-23-1 Genetic and Epigenetic Variation in Crop Legume Species Scott Jackson – University of Geogia/CAGT Kyung Do Kim – University of Georgia, Moaine El Baidouri – University of Georgia, Carolina Chavarro – University of Georgia, Marc Libault – University of Oklahoma

Track Name: Genomics Legumes are important sources of protein for human consumption, forage for animals and through association with soil-borne 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.

MS-23-2 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 Track Name: Genomics 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 nuclear-localized. 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.

MS-23-3 AtMBP-1 Affects Plant Development and ABA Responses and Is Regulated by AtSAP5 Miyoung Kang – Oklahoma State University Haggag Abdelmageed – Oklahoma State University Track Name: Genomics 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. Co-expression 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.

MS-23-4 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, Chi-Wah Tseung – University of Florida, Matthew Evans – Carnegie Institution for Science - Stanford, A. Mark Settles – University of Florida Track Name: Genomics Genomic imprinting in plants is an epigenetic phenomenon by which a subset of genes is expressed in a parent-of-origin–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 maternal-effect 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.

MS-23-5 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 Track Name: Genomics 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. Phloem-purified 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 mobility-shift 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.

Minisymposium 24: Cell Growth MS-24-0 Overview of Area by Chair, Magdalena Bezanilla, University of Massachusetts Amherst

MS-24-1 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, Steve Briggs – University of California, San Diego Track Name: Development 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.

MS-24-2 Myosin VIII Links Actin to Microtubules During Cell Division and Polarized Growth Magdalena Bezanilla – University of Massachusetts Amherst Track Name: Development 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.

MS-24-3 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

Track Name: Development Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) initiates both rapid and long-term signaling events in plant and animal cells and is necessary to fend off microbial invaders. Within minutes, innate immune signaling results in changes to cytosolic calcium levels, protein phosphorylation, 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 we have quantified the actin remodeling that occurs during pattern-triggered immunity (PTI) in Arabidopsis epidermal cells. We find that epidermal cells respond to a diverse array of bacterial and fungal MAMPs by transiently increasing actin dynamics and significantly elevating the density of filament arrays. Genetic analyses demonstrate that actin remodeling requires perception of MAMPs via known components of Arabidopsis PRRs. To elucidate which actin-binding proteins are involved, we screened a collection of homozygous mutants; 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 several MAMPs and are more susceptible than wild-type plants to the biotrophic bacterial pathogen, Pseudomonas syringae, as well as the necrotrophic fungus, Alternaria brassicola. Because phospholipase D (PLD) activity and phosphatidic acid (PA) fluxes are implicated in PTI, and CP is PA-binding protein, we tested 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. As a next step, we aim to identify which of the 12 PLD isoforms are responsible for these cytoskeletal rearrangements.

MS-24-4 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

Track Name: Development Small peptide signals, including CEPs (terminally encoded eptides), 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 up-regulated by N-limitations or -starvation and elevated CO2 levels 1. 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 levels 1. 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 nodulation 1. 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 modifications 2. 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).

MS-24-5 The HOPS Subunit AtVPS41 Is Involved in Homotypic Vacuole Fusion Jiameng Zheng – North Carolina State University Marcela Rojas-Pierce – North Carolina State University Track Name: Development 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, because 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.

Minisymposium 25: Ecophysiology MS-25-0 Overview of Area by Chair, Frank Dohleman, Monsanto Company

MS-25-1 Canopy Position Has a Profound Effect on Soybean Seed Composition Ivan Baxter – Missouri State University Kunzhi Li – Kunming University of Science and Technology, Randall Nelson – USDA-ARS, Alexander Ulanov – University of Illinois at Urbana-Champaign, Steve Huber – USDA-ARS Track Name: Diverse-Talk 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.

MS-25-2 Long-term Biological Consequences of the Chernobyl Accident in Chronic 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

Track Name: Diverse-Talk 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 rates. 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.

MS-25-3 Determining the Efficiency of Photorespiratory Recycling and the Impact to Net CO2 Assimilation Using Gas Exchange Berkley Walker – USDA/ARS, University of Illinois Paul South – USDA/ARS, University of Illinois, Donald Ort – USDA/ARS, University of Illinois Track Name: Diverse-Talk 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.

MS-25-4 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 Track Name: Diverse-Talk 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 weed-regulated 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.

MS-25-5 Environmental Benefits of Crop Improvement Frank Dohleman – Monsanto Company Track Name: Diverse-Talk Over the past 50 years, global production of select grain and oilseed crops in have increased by 330%, while land-area used to produce these crops has increased by only 46%. Agricultural innovation has resulted in continued crop production efficiency via an increase in crop yield of 244% across that time. Frequent assertions are made that these yield increases are achievable only through agricultural intensification, and come at the expense the regional and global environment. The interactions of cropping systems with the environment is an important one, and the impacts of the environment on crop yield, and our ability to produce more are well established. Here I will address the other side of the interaction, our current understanding of the impact of agricultural innovation on environmental parameters including climate change, nutrient cycling and crop water use. An assessment of the environmental impacts of historical

agricultural innovation will allow us to better understand and communicate the societal benefits of technology as we continue our focus to conserve more.

Minisymposium 26: Abiotic: Light & Temperature

MS-26-0 Overview of Area by Chair, Meng Chen, Duke University

MS-26-1 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 Track Name: Biotic + Abiotic 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.

MS-26-2 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 Track Name: Biotic + Abiotic 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 light-induced 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.

MS-26-3 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 Track Name: Biotic + Abiotic 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 HMR-mediated 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 growth-related 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 HMRs 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 HMRs 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.

MS-26-4 The Negative Effect of High Temperature Stress on Reproduction in Arabidopsis Thaliana Vanessa Lundsgaard-Nielsen – University of Toronto, Department of Ecology and Evolutionary Biology Dinesh Christendat – University of Toronto, Department of Cell and Systems Biology, Tammy L Sage – University of Toronto, Department of Ecology and Evolutionary Biology Track Name: Biotic + Abiotic

The adverse effect of high temperature (HT) stress on pollen development is a major cause of reduced seed set in arable ecosystems. We have identified a gene, HTT (High Temperature Tolerance) in Arabidopsis that encodes a plastid-targeted protein functioning in removal of reactive carbonyl species (RCS). RCS are formed following lipid peroxidation and damage cells by oxidizing proteins. In the HT tolerant Arabidopsis Ler, gene expression is induced by HT in the anther, pollen remains viable and plants produce seed. In contrast, Cvi pollen is not viable at HT, has no 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 and lipid accumulation. We assessed protein carbonylation and used mass spectrometry 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. Proteomic analysis indicated that Cvi and htt1-1 accumulated carbonylated proteins that function in carbohydrate metabolism, fatty acid transport and lipid biosynthesis. Notably, 78% of carbonylated proteins operate within other cellular compartments emphasizing the importance of plastid proteome homeostasis for pollen viability at HT. Other carbonylated proteins produced at HT in Cvi and htt1-1 included those involved in protein folding, amino acid metabolism and detoxification of ROS and other compounds. Proteins known to operate in the heat-induced signal transduction pathway and the plant ubiquitin-proteosome degradation pathway were also carbonylated. Our results provide a novel role for HTT in ensuring the removal of RCS to maintain pollen viability.

MS-26-5 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 Track Name: Biotic + Abiotic 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 dark-colored, 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 well-watered control conditions with 25ËšC day and 18ËšC night temperatures. A high temperature stress, with temperatures of 35ËšC during the day and 29ËšC 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 13ËšC. Heat stress increased tuber glucose and fructose contents, and invertase activity increased as SECD severity increased. Different genotypes varied widely in susceptibility to heat stress-induced 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.

Minisymposium 27: Education MS-27-0 Overview of Area by Co-chairs, Kathleen Archer, Trinity College, Hartford Connecticut and Erin Friedman, Lynchburg College

MS-27-1 The Sounds of Science Collaboration Between Plant Biologists and Music Composition Students Berkley Walker – USDA/ARS, University of Illinois Kyle Shaw – University of Illinois, M.O. Abbott – University of Illinois, Sam Gingher – University of Illinois, Aleel Grennan – University of Illinois, Kc Grennan – Xen Design Inc.Deepak Jaiswal – University of Illinois, Johannes Kromdijk – University of Illinois, Justin McGrath – University of Illinois, Barry Morse – University of Illinois, Mary Pietrowicz – University of Illinois, Charles Pignon – University of Illinois, Ursula Ruiz-Vera – University of Illinois, Jeffrey Shroyer – Jeffry Shroyer Studios, Lucas Smith – University of Illinois, Benjamin D. Whiting – University of Illinois, Craig Yendrek – University of Illinois Track Name: Diverse-Talk

We live in an exciting time for both plant science and new music. Never in human history have we been able to learn so much and express ourselves so freely as right now. Yet despite this ability both scientists and musicians struggle to find audience for their work. The Sounds of Science was designed to expose new audiences to the plant science research that is fundamentally shifting our understanding of how plants work and how we can sustainably use them to meet growing demands for food, fiber and fuel. We developed this collaboration by pairing plant scientists with music composition students. The scientists provided primary research data for the composition students to produce original musical works. These pieces were then showcased in a performance also featuring a short talk from the scientists about why the data were important. This performance was video recorded and distributed via YouTube and public television. The Sounds of Science Collaboration proved to be a successful and scalable way to expose new audiences to cutting edge plant science research.

MS-27-2 Sharing the Wealth: Mentoring Other Colleagues and the Next Generation of Undergraduate Teachers in Evidence-Based (scientific) Teaching Sue Wick – University of Minnesota Robin Wright – University of Minnesota Track Name: Diverse-Talk There have been numerous calls to reform undergraduate biology education, and various programs established to promote aspects of teaching known to be effective: evidence-based, student-centered, and employing active learning. Often neglected in these programs are the teachers of the future, the graduate students and postdocs. Colleagues, including those in other disciplines, also can be eager to learn how to put principles of effective teaching into practice. Members of the College of Biological Sciences at UMN have designed a few options for sharing our practice of scientific teaching. One is an evening workshop series for grad students and postdocs to expand upon what they learned in Preparing Future Faculty classes. Participants clustered in disciplinary groups to identify a topic from an undergraduate class that could be the basis of an active learning module. For some this was a concept that they knew is difficult for

undergraduates. Using backward design, each group articulated learning objectives, assessments, and classroom activities to address the topic, and presented the materials to the rest of the workshop participants for feedback. By coordinating with faculty, some of these modules were field tested in classrooms, allowing for further refinement. Another option we developed was the “Crash Course in Scientific Teaching,” a one-day intensive workshop for faculty and postdocs from an assortment of departments and colleges. After presenters laid out the principles of active learning, discipline-specific groups practiced using backward design to modify one or more elements of a course. Some of the participants in both programs opted for ongoing mentoring in scientific teaching. Our experience indicates that increasing the critical mass of instructors who have experienced scientific teaching, and forming a community of practice that can continue to offer encouragement and feedback, greatly increases the likelihood that scientific teaching will actually be implemented in courses.

MS-27-3 Introducing Basic Biotechnology Teaching Techniques in High Schools in Sub-Saharan Africa – Ghana Marian Quain – Crops Research Institute Marceline Egnin – Tuskegee University, James Asibuo – CSIR - CRI Ghana, Eric Acquah – CSIR-CRI, Ruth Prempeh – CSIR-CRI, Victor Amankwaah – CSIR-CRI Track Name: Diverse-Talk Modern Biotechnology, is the science where genes that are responsible for particular traits are manipulated in the laboratory under controlled environment to develop products or impart specific capabilities of organisms. Several fields of science come into play in its application and interpretation however, in the developing countries such as Ghana, this approach is lacking and this greatly affects the understanding and relevance of molecular and related sciences in the field of Biotechnology. Considering our current curricula for High School, which pipelines students into higher education, there is an obvious gap between the emerging state-of-the-art science, its understanding, application, and awareness students and teachers in sub-Saharan Africa have of modern biotechnology. Currently fermentation is the only mention of biotechnology in biology and related high school science textbooks in Ghana. This project therefore sought to introduce modern biotechnology in an applicable way to educate teachers (tutors) and students. Specific gaps in the teaching of Biotechnology and Genomics in Ghanaian high schools were identified; teaching Model for high school teachers to implement Modern agriculture biotechnology were communicated through hands-on workshops to train high school teachers in basic concept of biotechnology and genomics. Fourteen Biology, Agricultural and related sciences teachers from Junior to Senior high school classrooms benefited from a 4-day intensive hands-on training utilizing paper models and dry labs to enhance their understanding and delivery of plant biology with emphasis in biotechnology. This was followed by visits to schools of participating teachers to monitor adoption and curriculum implementation of modules. A website was established to link PIs with teachers and the general public. Following ten months of implementation of the project, 87 teachers, about 1327 students from more than 10 senior high schools as well as some 75 persons of other groups has benefited from the introduction to Modern Biotechnology.

MS-27-4 Introducing CourseSource: a Peer-Reviewed, Open-Access Journal Developed in Partnership with Scientific Societies That Publishes Evidence-Based Resources for Teaching Undergraduate Biology Jessamina Blum Sue Wick – University of Minnesota, Robin Wright – University of Minnesota Track Name: Diverse-Talk

Over the past 20 years, many national reports have called for transformation of undergraduate science education. For example, in 2012, the President’s Council of Advisors on Science and Technology advocated evidence-based pedagogy as a means to produce an additional 1,000,000 U.S. STEM graduates over the next decade. In response to such calls to action, colleges and universities nationwide are approaching their undergraduate STEM courses in new ways, but this change has been slow and fragmented. One of the greatest challenges to STEM education transformation is the time, energy, and vision that instructors need to create evidence-based lessons, courses, and curricula. The Vision and Change in Undergraduate Biology Education Report highlighted this need and recommended creation of a repository of “student-centered, outcomes-oriented” resources. With support from the Howard Hughes Medical Institute and in collaboration with professional societies, including the ASPB and BSA, we have created a peer-reviewed, open-access journal called CourseSource that meets this need (see coursesource.org). CourseSource publishes field-tested lessons that implement best practices in scientific teaching and that address learning goals determined by the associated scientific society. This poster presentation will describe CourseSource article formats, the submission process, and review criteria, as well as provide advice about potential submissions related to plant biology and other disciplines.

MS-27-5 Leaves of Green: A Project-driven Workshop for Question-based Exploration of Plant Biology in the High School Curriculum Mark Lubkowitz – Saint Michael’s College Track Name: Diverse-Talk To foster inquiry-based learning of plant biology in high schools, we have developed a four day workshop that is contributing to the curriculum in seventeen high schools in Vermont, New Hampshire, and New York. During the workshop, teams of one teacher and two students learn cost effective methods for experimentally addressing questions about photosynthesis and carbohydrate partitioning, and how carbohydrate partitioning affects crop yields, biofuel production, and atmospheric CO2 levels. Each team completes an independent project, which is shared with all participants, such that every teacher leaves with a list of questions and experimental approaches to adopt in their classroom. Furthermore, by having teachers and students participate, teachers are able to develop and test their respective modules with students. Our exit surveys and follow-up communications demonstrate that the workshop is having a large curricular impact at the participating schools, and elements have been adopted in an impressive number of courses. Furthermore, our model can be adapted to the college curriculum. It is now a core component of the Plant Biology course at Saint Michael’s College, where students work with local farms to experimentally answer plant growth- and yield-related questions using the methods developed in the workshop. This “principle to practice” approach is proving quite successful as an authentic way to learn plant biology for high school and college students and is a service to the local farming community.

Minisymposium 28: Space Biology MS-28-0 Overview of Area by Chair, Anna-Lisa Paul

MS-28-1 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

Track Name: Diverse-Talk 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.

MS-28-2 Arabidopsis in Space: From Microgravity to Mechanical Signaling and Back Simon Gilroy – University of Wisconsin-Madison Won-Gyu Choi – University of Wisconsin-Madison, Sarah Swanson – University of Wisconsin-Madison , Richard Barker – University of Wisconsin-Madison, Su-Hwa Kim – University of Wisconsin-Madison Track Name: Diverse-Talk 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.

MS-28-3 Identification of a Novel Protein Involved in Actin-mediated Directional Root Skewing from Ground- and Space-based Plant Experiments Elison Blancaflor – Samuel Roberts Noble Foundation J. Alan Sparks – The Samuel Roberts Noble Foundation, Taegun Kwon – The Samuel Roberts Noble Foundation Track Name: Diverse-Talk 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).

MS-28-4 Plant Gravitropic Signal Transduction: A Network Analysis Leads to Gene Discovery Sarah Wyatt – Ohio University Colin Kruse – Ohio University Track Name: Diverse-Talk 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.

MS-28-5 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 Track Name: Diverse-Talk 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.

Minisymposium 29: Transcriptional Networks in Development MS-29-0 Overview of Area by Chair, Andrea L. Eveland, Donald Danforth Plant Science Center

MS-29-1 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

Track Name: Development 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.

MS-29-2 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

Track Name: Development 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 two-hybrid 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.

MS-29-3 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 Track Name: Development 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 over-expression 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.

MS-29-4 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, Michael Pautler – Cold Spring Harbor Laboratory, Hui Jiang – Donald Danforth Plant Science Center, Thomas Brutnell – Donald Danforth Plant Science Center, David Jackson – Cold Spring Harbor Laboratory, Elizabeth Kellogg – Donald Danforth Plant Science Center

Track Name: Development 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.

MS-29-5 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 Track Name: Development 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 Opaque-2 (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 O2-binding 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.

Minisymposium 30: Biotechnology MS-30-0 Overview of Area by Chair Maureen Hanson, Cornell University

MS-30-1 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

Track Name: Biochem 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 C-terminal 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

MS-30-2 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 Track Name: Biochem 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.

MS-30-3 Homo-dimerization and Ligand Binding by the Leucine-rich Repeat Domain at RHG1/RFS2 Underlying Partial Resistance to Two Soybean Pathogens David Lightfoot – SIUC Track Name: Biochem 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 131-485, 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.

MS-30-4 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

Track Name: Biochem 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.

MS-30-5 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 Track Name: Biochem 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.