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ICAR 2011 University of Wisconsin–Madison 272 The Role of the Conserved Protein, CACTIN, in Arabidopsis. An Exciting, Essential, and Unique Eukaryotic Gene. Katherine Baldwin, Patrick Masson University of Wisconsin, Madison, WI, USA CACTIN is a conserved gene found across eukaryotes and encodes a protein without recognizable functional domains. Mutations in the CACTIN gene result in lethality in Drosophila, C. elegans, Toxoplasma, and Arabidopsis. In flies, CACTIN plays a role in the NFkB pathway. There is no recognized NF-kB pathway in plants or C.elegans. Our studies using Arabidopsis suggest that CACTIN plays a critical role in embryogenesis and possibly a function in root gravitropism. Like in animal systems, Arabidopsis fluorescently tagged CACTIN localizes to nuclear speckles. By yeast-two-hybrid, we found that CACTIN binds to a putative component of the spliceosome. We propose that CACTIN maybe a peripheral part of the eukaryotic spliceosome. We present further experiments in Arabidopsis to test this hypothesis as well as examining its functional conservation between plants and animals. 273 AGD1, a Class 1 ARF-GAP that Localizes to Punctate Bodies of the Endomembrane System, Regulates Multiple Components of Root Hair Growth in Arabidopsis Cheolmin Yoo 1 , Satoshi Naramoto 2 , J. Alan Sparks 1 , Hiroo Fukuda 2 , Elison Blancaflor 1 1 Samuel Roberts Noble Foundation, Ardmore, Oklahoma, USA, 2 University of Tokyo, Tokyo, Japan Mutations to the Arabidopsis thaliana AGD1 gene, which encodes a class 1 Adenosine Diphosphate (ADP)-Ribosylation Factor (ARF)-Guanosine Trinucleotide (GTPase) Activating Protein (GAP), triggers the formation of wavy root hairs. To better understand the position of AGD1 within the intricate signaling pathways that govern root hair polarity, we studied the localization of a functional AGD1-green fluorescent protein (GFP) fusion in living root hairs, evaluated various components of root hair tip growth in agd1 mutants, and analyzed double mutants of agd1 and other loci involved in root hair development. AGD1- GFP decorated punctate bodies in the root hair that were reminiscent of fluorescently labeled endomembrane compartments. However, AGD1-GFP foci were distinct from several endomembrane markers and localization of AGD1 punctate structures persisted despite exposure to Brefeldin A or Wortmannin. Targeting of RabA4b-GTPase, ROP2-GTPase and phosphatidylinositol-4-phosphate, which are essential components of tip growth maintenance, were altered in agd1 root hairs. Furthermore, dampened tip cytosolic calcium oscillations and disrupted tonoplast dynamics were observed in root hairs of agd1. Double mutant analysis indicated that RHD4, which encodes a phosphatidylinositol-4-phosphate phosphatase, is epistatic to AGD1. On the other hand, double mutants to AGD1 and ACT2, a root hair-expressed vegetative actin isoform, displayed additive root hair defects. Taken together, our results support a model that positions AGD1 downstream of phosphoinositide metabolism in controlling cytoskeletal, cytosolic calcium , ROP2 and RabA4b-mediated root hair polarity. 274 Dissecting The Requirement For Plant RanGAP1 Subcellular Targeting And GAP Activity For Its Cellular And Developmental Functions Joanna Boruc, Thushani Rodrigo-Peiris, Iris Meier The Ohio State University, Columbus (OH), USA RanGAP is an accessory protein of Ran signaling, which is involved in nucleocytoplasmic transport, spindle organization and post-mitotic nuclear assembly. These functions of RanGAP are conserved across higher eukaryotic organisms. However, we have recently discovered a novel function for higher plant RanGAP in cytokinesis, possibly reflecting the phragmoplast-dependent division of plant cells. We have shown that Arabidopsis RanGAP1 is a continuous marker of the cell division plane and it is required for proper cell division and plant development, likely independent of its role in nucleocytoplasmic trafficking. We have created Arabidopsis mutant lines with decreasing RanGAP levels and increasing phenotypic severity. These mutants are a valuable tool to dissect the requirement for the GAP activity and/or mitotic subcellular addresses of RanGAP for its role in plant cell division and development. We have identified a point mutation in the targeting domain of RanGAP that blocks all subcellular positioning during mitosis, but which still complements a temperature-sensitive yeast RanGAP mutant strain. Moreover, we have constructed point mutations that block the GAP activity, but do not interfere with the subcellular positioning. Through the complementation of the mutant plants with these mutant versions of the protein, we are currently testing which features of Arabidopsis RanGAP are required for its roles in cell division and plant development. These data will further evaluate our hypothesis of a separate evolution of RanGAP targeting mechanisms and its subfunctionalization in different kingdoms. 275 GT-2 Family Transcription Factors Regulate Cell Growth in Arabidopsis Christian Breuer, Ayako Kawamura, Keiko Sugimoto RIKEN Plant Science Center Plant organ growth is regulated by two distinct processes, first, the cell number which is controlled by cell proliferation, and second, the size of individual cells, which is regulated by cell expansion and cell growth. In Arabidopsis, various studies have shown a positive correlation between cell size and ploidy, but the molecular mechanisms underlying this observation are still unclear. We have previously identified GT-2-LIKE1 (GTL1) as a repressor of ploidy-dependent cell growth in trichomes. GTL1 is a nuclear protein, and further expression analysis revealed that GTL1 is expressed in various cell types, except meristematic cells, throughout plant development, suggesting that GTL1 might function as a general transcriptional regulator of plant cell growth. Ubiquitous expression of GTL1 arrests early seedling growth, and thus, we have used strong cell- and tissue-specific promoters to allow an assessment of the potential role of GTL1 in regulating growth, ploidy and size of plant cells. Detailed examination of these ectopic lines confirms that GTL1 Poster: Cell Biology
ICAR 2011 University of Wisconsin–Madison can negatively regulate cell growth and ploidy of any cell type tested in Arabidopsis. In addition, we are able to show the essential role of endoreduplication for early differentiation processes in plant cells. GTL1 is a member of the GT2 trihelix transcription factor family has two closely related homologs in Arabidopsis, DF1 and GT2. Loss-of-function alleles for DF1 and GT2 do not exhibit any obvious phenotypes. Furthermore GTL1, DF1 and GT2 show similar expression patterns and ectopic expression studies of DF1 and GT2 also cause a dramatic decrease in cellular growth. These results suggest a conserved function among the three homologs, and confirming our initial assumption of functional redundancy within the gene family. Investigations to reveal direct down-stream targets of these transcription factors are currently ongoing and are considered as a subject for discussion. 276 Identification of Nuclear Genes Encoding Chloroplast-Localized Proteins Required for Embryo Development in Arabidopsis Nicole Bryant 1 , Johnny Lloyd 1 , Colleen Sweeney 1 , Fumiyoshi Myouga 2 , David Meinke 1 1 Oklahoma State University, Stillwater, OK, USA, 2 RIKEN Plant Science Center, Yokohama, Japan We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis. Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with an embryo-defective (119 genes), plant-pigment (121 genes), gametophyte (3 genes) or alternate (138 genes) phenotype. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat (PPR) proteins well-represented among EMB datasets. The essential chloroplast accD locus, which is necessary for fatty acid biosynthesis, appears to explain why chloroplast translation is required for embryo development in Arabidopsis. Plant species that can tolerate the loss of chloroplast translation, including Brassica and maize, have evolved a compensation mechanism that allows an alternate form of the enzyme produced in the cytosol to carry out fatty acid biosynthesis in the chloroplast. Research supported by the NSF Arabidopsis 2010 Program. 277 Abstract Withdrawn 278 SWEET Sugar Transporters For Cellular Efflux Highjacked For Nutrition Of Pathogens Li-Qing Chen 3 , Bi-Huei Hou 3 , Sylvie Lalonde 3 , Mara Hartung 3 , Xiao-Qing Qu 3 , Jung-Gun Kim 4 , William Underwood 1 , Ginny Antony 2 , Frank White 2 , Shauna Somerville 1 , Mary Beth Mudgett 4 , Wolf Frommer 3 1 Energy Bioscience Institute, Berkeley, CA, USA, 2 Kansas State University, Manhattan, KS, USA, 3 Carnegie Institution for Science, Stanford, CA, USA, 4 Stanford University, Stanford, CA, USA Sugar efflux is essential for processes requiring cellular exchange of carbon and energy in multicellular organisms. Examples in plants include nectar secretion, pollen nutrition, release of sugars from mesophyll cells as a first step for phloem loading, seed nutrition, and carbon sequestration in storage tissues. Despite the widespread importance, sugar effluxers have remained elusive. A novel class of sugar transporters, the SWEETs, was identified using FRET glucose nanosensors, which dynamically monitor subcellular glucose levels in live cells (Chen et al., 2010). The SWEETs form a broadly conserved gene family with 17 members in Arabidopsis, 7 in Chaenorhabditis elegans and 1 in human. AtSWEET1 and AtSWEET8/RPG1 function as uniporters. AtSWEET8/RPG1 is expressed in the tapetum and is essential for pollen viability (Guan et al, 2008). The rice homologs OsSWEET11/Xa13/Os8N3 and OsSWEET14/Os11N3 are highjacked by bacterial pathogens by means of direct binding of a bacterial effector to the promoter (Chen et al., 2010 and Antony et al., 2010). OsSWEET11 promoter mutations that abrogate this binding are responsible for resistance to Xanothomonas oryzae in a wide spectrum of rice lines. Both OsSWEET11 and OsSWEET14 mediate glucose transport when expressed in HEK293T cells or Xenopus oocytes, suggestive of a role in local sugar efflux induced by the pathogens. This mechanism of pathogen nutrition may be general because several pathogens induce Arabidopsis SWEETs during infection, though each pathogen tested induces a different set of SWEETs. 279 The Localization of APYRASE1/2 and Their Roles in Regulating of Growth and Development Tsan Yu Chiu, Stanley Roux UT, Austin, TX, USA Apyrases (EC 3.6.1.5) are known as enzymes that hydrolyze both di- and triphosphate nucleotides and are widely identified in eukaryotic cells. They are categorized as E-type ATPases, which have the characteristics of broad divalent cation (Mg 2+ , Ca 2+ ) requirement for activation, and insensitivity to inhibitors of F-type, P-type, and V-type ATPases. Apyrases are well characterized in animals and yeast. In animals it plays a crucial role in terminating signal transduction initiated by extracellular nucleotides extracellular ATP (eATP). In yeast, GDA1 is involved in regulating protein glycosylation, sugar level control and membrane integrity. Poster: Cell Biology
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1 Suppression of plant immunity by
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Kenobi, Kim Kim.Kenobi nottingham.a
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