75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

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377 Myo-Inositol Oxygenase Is a Balance Point Between Signaling and Metabolism Shannon Alford Virginia Polytechnic Institute and State University Myo-inositol is used in plant cells as a backbone of inositol phosphate (InsP) and phosphatidylinositol phosphate (PtdInsP) signaling molecules. Myo-inositol is also a precursor for an alternative Vitamin C synthesis pathway in plants. The oxidation of myo-inositol by the enzyme Myo-Inositol OXygenase (MIOX) produces D-glucuronic acid, which is further altered to produce Vitamin C. The Arabidopsis genome contains four genes predicted to encode MIOX enzymes. Recently it was shown that plants ectopically expressing the MIOX4 gene from Arabidopsis (MIOX4 + plants) produced higher levels of Vitamin C. Since MIOX oxidizes myo-inositol, it could act as a central regulator in balancing the cell’s various needs for myo-inositol. Specifically, we are interested in whether MIOX4 + plants contain alterations in inositol signaling. We found that MIOX4 + seedlings are hyposensitive to abscisic acid ( ABA ) in a seedling germination assay. This suggests that MIOX4 + plants are altered in the ABA response pathway. Since ABA signaling has been shown to induce Ins(1,4,5)P 3 synthesis, this response may indicate that MIOX4 + plants are compromised in their ability to synthesize Ins(1,4,5)P 3 . T-DNA insertional knock-outs of the four MIOX genes have been obtained. We are examining these mutants for phenotypes and signaling alterations. Specifically, we are measuring myo-inositol levels and other metabolites by GC analysis in MIOX4 + and miox- mutant plants to determine if levels are altered. Ins(1,4,5)P 3 levels will be measured to determine if this signaling molecule is altered. Our results will help us understand how MIOX function impacts myoinositol signaling and metabolism. 378 The role of the SLEEPY1 (SLY1) F-box gene in GA regulation of seed germination in Arabidopsis Tohru Ariizumi 1 , Elizabeth Schramm 1 , Camille Steber 1, 2 1 Washington State University, 2 USDA-ARS, Wheat Genetics Unit Seed germination is a complex developmental process regulated by phytohormones. The phytohormone abscisic acid (ABA) inhibits seed germination, whereas gibberellin (GA) stimulates seed germination. In tomato and Arabidopsis, GA is clearly required for seed germination. Recent evidence suggests that GA stimulates seed germination by triggering destruction of DELLA family proteins via the SCF SLY1 E3 ubiquitin ligase and the 26S proteosome pathway. DELLA proteins are negative regulators of GA responses, and RGL2 is the main DELLA protein repressing seed germination. SLY1 appears to tranduce the GA signal by triggering DELLA destruction by ubiquitination. GA-insensitive sly1 mutants resemble GA biosynthesis mutants in that they exhibit dwarfism, late flowering, reduced fertility and increased seed dormancy. These sly1 phenotypes are not rescued by GA application and are not as severe as those seen in the ga1-3 GA biosynthesis mutant. While the ga1-3 mutant fails to germinate in the absence of GA, the seed germination rate varies greatly (3-100%) among independent seed lots of young sly1 mutants. When sly1 mutant seeds can germination, they germinate more slowly than WT and show greater sensitivity to ABA and reduced osmotic potential. The germination of dormant sly1 mutant seed lots improved following afterripening. Consistent with the notion that SLY1 regulates seed germination, a SLY1 promoter::GUS fusion shows expression in the radicle during seed germination. To better understand the sly1 mutant seed germination phenotype, we are examining the effect of these mutations on RGL2 protein accumulation. It is known that high levels of RGL2 protein in the ga1-3 mutant correlates with failure to germinate, and that mutations in RGL2 suppress the ga1-3 seed germination phenotype.
379 SPIKE1 is a guanine nucleotide exchange factor that positively regulates ROP small GTPases and controls an evolutionarily conserved pathway of actin-dependent cell morphogenesis Dipanwita Basu, Taisiya Zakharova, Eileen Mallery, Daniel Szymanski Purdue University During cell morphogenesis the actin cytoskeleton is dynamically reorganized in response to endogenous signals. The recently identified WAVE-ACTIN RELATED PROTEIN (ARP) 2/3 pathway clearly illustrates this point. Activating signals from the small GTPase ROP are thought to activate the heteromeric WAVE complex, which in turn, positively regulates the actin filament nucleation activity of ARP2/3. However, several aspects of this signaling pathway are not defined. For example, which of the 11 Arabidopsis ROPs feed into the WAVE-ARP2/3 pathway and which guanine nucleotide exchange factor(s) (GEF) promote GTP-loading of ROP upstream of the WAVE-ARP2/3 We have used a forward genetic analysis of epidermal morphogenesis to define completely, a pathway of genes from GEF to ARP2/3- dependent actin filament nucleation. These genes play important roles in polarized growth and cell-cell adhesion in a variety of tissues and organs. SPIKE1 (SPK1) is the lone plant gene product that contains a DHR2 domain, the defining feature of the DOCK family of GEFs. We will present strong genetic and biochemical data indicating that the DHR2 domain of SPK1 is critical, and that DHR2 is necessary and sufficient for activation of ROP small GTPases in vivo. Using double mutants analyses and the well known wave and arp2/3 phenotype of trichome swelling, we find that one function of SPK1 is to positively regulate this evolutionarily conserved actin filament nucleation pathway; a pathway that appears to utilize only a subset of ROPs. In Rho-signaling pathways, it is extremely difficult to link directly GEF activity to specific Rho-GTP effector targets. For example, multiple ROPs and GEFs could lie between SPK1 and the ROP-binding WAVE protein SRA1. We tested SPK1 function as a "signaling scaffold" for downstream effectors by assaying the ability of endogenous SPK1 to interact with WAVE complex proteins. We will present several lines of biochemical evidence that support the hypothesis that endogenous SPK1 is physically associated with WAVE complex proteins. The SPK1-WAVE complexes may be poised to receive and transmit ROP signals with a high degree of spatial and temporal specificity. To our knowledge this is the first example in which the in vivo function of a signaling pathway from upstream GEF to actin filament nucleation machinery has been defined in a multicellular organism. 380 The Arabidopsis Aleurone Layer Contributes to Seed Dormancy and Responds to GA, ABA and Nitric Oxide Paul Bethke 1, 2 , Natsuyo Aoyama 3 , Igor Libourel 4 , David Still 3 , Russell Jones 2 1 USDA ARS Dept of Horticulture, University of Wisconsin, Madison, USA, 2 Dept. Plant and Microbial Biology, University of California, Berkeley, USA, 3 Dept Plant Sciences, California State Polytechnic University, Pomona, USA, 4 Michigan State University, East Lansing, USA Seed dormancy is a complex phenomenon influenced by both genetic and environmental factors. ABA is central to the establishment and maintenance of seed dormancy, and gibberellins are important for germination. The volatile signaling molecule nitric oxide (NO) reduces seed dormancy in Arabidopsis. Seeds of Arabidopsis are dormant at maturity, and depending on the ecotype and environment, dormancy loss occurs within a few weeks or after several months. Arabidopsis seeds contain an embryo enveloped in an aleurone layer that is in turn surrounded by the testa. Imbibed Arabidopsis seeds remained dormant when the testa was removed, but like intact seeds germinated when treated with KCN vapors or NO gas. Removal of both the testa and the aleurone layer resulted in growth of the embryo. Aleurone cells underwent characteristic changes in ultrastructure in seeds that will germinate, and the most obvious was a reduction in the number of vacuoles per cell. The rate of this vacuolation was greatest for the cells proximal to the root tip. Vacuolation did not occur in dormant seeds and was prevented by ABA or the NO scavenger cPTIO. The effect of cPTIO was overcome by GA, and GA on its own promoted the vacuolation of aleurone cells. These data suggest that the aleurone layer is a significant determinant of Arabidopsis seed dormancy, and that this tissue is responsive to ABA, GA and NO in ways that are consistent with the physiology of dormancy and germination in this species. We also quantified mRNA abundance of key genes associated with dormancy and germination in the aleurone layer and embryo of Arabidopsis seeds. Dormant seeds of ecotype C24 were imbibed with water or with the nitric oxide (NO) scavenger cPTIO, conditions that maintain or strengthen dormancy respectively. Other seeds were treated with KCN vapors to break dormancy in an NO-dependent manner. Embryos and aleurone layers were dissected from seeds and quantitative PCR was used to measure mRNA abundance at 1, 24 and 48 h after imbibition for genes associated with GA biosynthesis (GA3ox1, GA30x2), GA responses (cysteine protease), lipid metabolism (malate synthase, isocitrate lyase), and NO synthesis (nitric oxide synthase). Our data show that genes for GA biosynthesis were strongly up-regulated in the embryos of seeds that will germinate relative to seeds that will remain dormant. GA3ox1 and GA3ox2, however, were not expressed at detectable levels in the aleurone layer, even at times when a GA-responsive cysteine protease was expressed. The nitric oxide synthase was expressed in both the embryo and the aleurone layer, and expression in the aleurone layers was strongly stimulated by the NO scavenger cPTIO.
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75 Integrating Membrane Transport w
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79 PREP: Partnership for Research a
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83 Characterization of Orthologous
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87 High-density Arabidopsis Protein
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91 Approaches to Study Homologous R
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95 Predicting the Redundome: A Geno
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99 ReIN: an interactive tool to cre
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103 Proteomic services at the Europ
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107 Ubiquitin Lys 63 Chain Forming
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111 Characterization of the Anti-mi
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115 Molecular Genetic Analysis of P
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119 CLB19, a Novel Pentatricopeptid
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123 The Arabidopsis CDC48 Adapter P
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127 AtCKT1, a Novel Transporter for
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131 Sub-Cellular Localization of DR
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135 Discovering the function of WAV
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139 WRINKLED1, an AP2/EREBP Class T
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143 The Ubiquitin-Specific Protease
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147 Systemic signal transduction of
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151 Dissection of Flowering Pathway
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155 Dissecting genetic pathways und
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159 HANABA TARANU (HAN) Organizes A
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163 What are SCAMPS doing in plants
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167 Analysis of DNA methylation and
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171 Identification of TIA as a Myb-
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175 DEMETER DNA Glycosylase Regulat
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179 Investigation Of The Connection
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183 Genetic Analysis of Vascular Pa
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187 Arabidopsis BRANCHED genes are
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191 Regulation Of BDL Gene Expressi
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195 ARROW1 (ARO1), a Novel Regulato
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199 The Trichome Pock Phenotype of
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203 Control of Leaf Vascular Patter
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207 FAMA Controls The Switch Betwee
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211 Subtilisin-like serine protease
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215 TRIDENT and VARICOSE encode com
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219 Analysis of a Mutant, #1-63, wh
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223 Quantitative Trait Analysis of
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227 LEAFY and the Evolution of Rose
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231 Overexpression of Arabidopsis S
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235 Ethylene Signaling Components A
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239 Accumulation of Reactive Oxygen
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243 Intracellular Production Of Rea
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247 Large-Scale Screen and Isolatio
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251 Ontogeny of the Arabidopsis Cir
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255 Cell type-specific expression a
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259 Characterization of Flowering T
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263 Involvement of Cytokinins and T
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267 Identification of new actors of
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271 Scarecrow-like Protein SCL14 In
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Page 103 and 104: 279 Mechanisms controlling coordina
Page 105 and 106: 283 GLZ1, a member of family 8 glyc
Page 107 and 108: 287 Arabidopsis as a Model System t
Page 109 and 110: 291 The NIMIN-NPR1 Connection: A Mo
Page 111 and 112: 295 A Search for Transcriptional Re
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Page 115 and 116: 303 Regulation of AGAMOUS Expressio
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Page 119 and 120: 311 Centromeric Retrotransposons: P
Page 121 and 122: 315 Finding Intron Sequences That E
Page 123 and 124: 319 Biochemical Characterization Of
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Page 127 and 128: 327 The Role of Tryptophan Metaboli
Page 129 and 130: 331 A Putative Bifunctional Wax Est
Page 131 and 132: 335 Analysis of the Complex BIO3 /
Page 133 and 134: 339 Exploring of Arabidopsis non-ho
Page 135 and 136: 343 A Yeast-2-Hybrid Assay Reveals
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Page 139 and 140: 351 Potent Induction of flowering b
Page 141 and 142: 355 Phylogenetic Analysis of Arabid
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Page 145 and 146: 363 Progress Towards the Cloning of
Page 147 and 148: 367 Natural Variation in Infloresce
Page 149 and 150: 371 Natural Variation for Seed Dorm
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Page 155 and 156: 383 The RAD23 Family of Ubiquitin-L
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Page 159 and 160: 391 Characterization of the Sugar-I
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Page 167 and 168: 407 Functional Requirements for PIF
Page 169 and 170: 411 Using High-throughput Chemical
Page 171 and 172: 415 The F-box Protein PPS Functions
Page 173 and 174: 419 Round The Clock - The Molecular
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Page 177 and 178: 427 Integration of light and abscis
Page 179 and 180: 431 Functional analysis of ROP10 sm
Page 181 and 182: 435 The Importance Of RecQ Like Hel
Page 183: 439 A Comparison of Full Versus Par
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75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

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