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

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433 The Pyrabactins: Small Molecule Agonists of the Abscisic Acid Signaling Pathway Pauline Fung, Freeman Chow, Yang Zhao, Nicholas Provart, Sean Cutler University of Toronto, Department of Botany We performed a chemical genetic screen of a 10,000 member small molecule library to identify compounds that disrupt hypocotyl cell expansion in etiolated Arabidopsis seedlings. Six compounds were identified that inhibit germination and ~750 compounds were found to reproducibly inhibit hypocotyl growth by >20%. Two of the germination inhibitors identified are structurally similar and examination of an analog series shows that a pyridine moiety is essential for the activity of these compounds that we have named the Pyrabactins (for pyridine aba activation). To delineate their mechanism of action, the effects of the Pyrabactins were examined on mutants in the GA and ABA signaling and biosynthetic pathways. These experiments demonstrate that Pyrabactins require a functional ABA signaling, but not biosynthetic, pathway to inhibit germination suggesting that Pyrabactins activate the ABA signaling pathway. To examine this hypothesis further, whole genome transcript profiling was used and these experiments reveal that ABA and Pyrabactin treatments induce strikingly similar expression profiles. Moreover, to isolate ABA-specific probe sets from germination responsive ones, transcripts that were significantly regulated by multiple germination inhibitors were identified and excluded in our microarray analysis. Collectively our genetic, physiological and transcriptional data suggest that the Pyrabactins inhibit germination by activating the ABA signaling pathway and thus define a new class of synthetic plant growth regulators. As first steps towards target identification, we have taken two approaches. A natural variation screen has identified several strains hypersensitive to Pyrabactin A and analysis of the Cold Spring Harbor Lab ecotype (CSHL1) has shown that it carries a recessive mutation in a locus designated POD1 (Pyrabactin overdoser) that segregates as a Mendelian trait and maps to the bottom arm of chromosome three; map based cloning of the POD1 locus is ongoing. Additionally an EMS screen for Pyrabactin A resistance has identified several mutations; of current interest is a dominant mutation in PYR1 (Pyrabactin resistant) that maps to the bottom arm of chromosome 4 and is currently being fine mapped. Our discovery of the Pyrabactins illustrates the successful application of forward chemical genetics in identifying new plant growth regulators. Furthermore, the Pyrabactins should be useful tools for dissection and manipulation of the ABA signaling pathway. 434 Experimental Validation of a Predicted Feedback Loop in the Circadian Gene Network of Arabidopsis Peter Gould 1 , James Locke 2 , Andrew Millar 3 , Anthony Hall 1 1 University of Liverpool, 2 Warwick University, 3 University of Edinburgh Circadian rhythms, controlled by an endogenous circadian clock, have been conserved through evolution from prokaryotes to eukaryotes. Circadian rhythms not only provide organisms with the ability to prepare for environmental changes caused by sunset and sunrise, but also allow seasonality to be determined, an advantage for flowering in plants and mating and hibernation in animals. The circadian clock generates rhythms with a period length of ~ 24 hours in a diverse range of processes from developmental to behavioural activities. Although a large percentage of genes in higher eukaryotes show circadian rhythmic expression (5-15% of genes), only a small number of these genes are known to be involved in the circadian clocks central network of interlocked feedback loops. The central network in Arabidopsis thaliana has been shown to comprise of a feedback loop in which LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), repress the expression of TIMING OF CAB EXPRESSION 1 (TOC1) which is their transcriptional activator. Although this clock mechanism mathematically is able to produce an oscillation it cannot fit with all the experimental data to date. For instance, the model would predict that the cca1; lhy double mutant would be arrhythmic but this is not the case. From experimental data the cca1; lhy double mutant infact has a short period length rhythm that persists over several days. From mathematical modelling and experimental work we present evidence for the existence of multiple feedback loops in the Arabidopsis central network. Within this network GIGANTEA (GI) has been predicted to act as a component of one of the interlocking loops that is critical for the working of the circadian clock. Through experimental work we have identified a role for GI within this model.
435 The Importance Of RecQ Like Helicases From Plants In DNA Repair And Recombination Frank Hartung, Stefanie Suer, Holger Puchta University of Karlsruhe, Faculty of botany II RecQ helicases are conserved throughout all kingdoms of life regarding both their overall structure and function. The originally described RecQ protein from E. coli is a DNA helicase and functions as a suppressor of homologous recombination. Eukaryotic RecQ like proteins are also 3´ to 5´ DNA helicases resolving different recombinogenic DNA structures during replication and DNA repair processes. Recently, even strand annealing activity has been reported for different mammalian RecQ like proteins. The RecQ helicases are key factors in a number of DNA repair and recombination pathways involved in the maintenance of genome integrity. In bacteria only one RecQ protein exists but in eukaryotes the number and structure of RecQ like proteins vary strongly between different organisms. Knockouts of several RecQ like genes cause severe diseases (i.e. WRN, BLM and RTS) in men or harmful cellular phenotypes in yeast. These diseases are often accompanied by genomic instability and cancer predisposition. Despite the fact that plants in general do not develop cancer, the largest number of RecQ like genes per organism has been found in them until now. Arabidopsis and rice possess seven different RecQ like genes each and also the moss Physcomitrella patens seem to have a comparable number. Therefore we can anticipate essential and also different roles of the RecQ like proteins in plant DNA metabolism. A major part of our analyses is focussed on T-DNA knockout mutants of several RecQ like genes in Arabidopsis thaliana, investigating their behavior in DNA repair and recombination pathways. We found miscellaneous phenotypes of single and double mutants regarding the homologous recombination (HR) frequency using ß-glucuronidase containing HR reporter lines. Furthermore, we also analysed the genes of different RecQ interacting proteins in respect to their role in HR. 436 GATEWAY vectors for plant genome analysis Mansour Karimi, Bjorn De Meyer, Rudy Vanderhaegen, Christiane Genetello, Dirk Inze, Pierre Hilson Department of Plant System Biology (PSB), Vlaams Interuniversitair Instituut voor Biotechnologie, Universiteit Gent, Technologiepark 927, B-9052 Gent, Belgium The Gateway TM technology (Invitrogen) has been developed to facilitate the transfer of DNA segments into and between plasmids by recombinational cloning. We have constructed a large collection of Gateway-compatible destination vectors for a wide range of gene function analyses in transgenic plant cells. Taking advantage of the novel Multisite recombination Gateway cassettes, plant binary destination vectors have also been created in which two or three segments can be transferred from source entry clones and pasted contiguously in a single LR clonase in vitro reaction. We are currently creating a collection of universal pEntry clones in diverse Multisite pDonor vectors to expand the range of plant expression constructs that can be produced straightforwardly using the Multisite Gateway system. Furthermore, vectors for the analysis of monocotyledons genome have been created. All our destination vectors carry one of three plant selectable markers coding for resistance to kanamycin (nptII), hygromycin (hpt) or glufosinate ammonium (bar). All recombinational cloning cassettes are also available in high copy number plasmids. This collection of vectors is a useful resource for the plant research community and can be obtained on line (http://www.psb.ugent.be/gateway). The web site also includes other accessions and provides recombinational cloning instructions, as well as experimentally verified sequences, maps and Vector NTI files for each plasmid. References: Karimi et al. (2002) GATEWAY(TM) vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci. 7:193-195. Joubes et al. (2004) Conditional, recombinase-mediated expression of genes in plant cell cultures. Plant J. 37:889-896. Karimi et al. (2005) Modular cloning in plant cells. Trends Plant Sci. 10:103-105.
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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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275 Protein Prenylation Is Required
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279 Mechanisms controlling coordina
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283 GLZ1, a member of family 8 glyc
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287 Arabidopsis as a Model System t
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291 The NIMIN-NPR1 Connection: A Mo
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295 A Search for Transcriptional Re
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299 Identification of genes contrib
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303 Regulation of AGAMOUS Expressio
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307 Genetic Control of the Interplo
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311 Centromeric Retrotransposons: P
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315 Finding Intron Sequences That E
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319 Biochemical Characterization Of
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323 Fluorescent amino acid sensors
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327 The Role of Tryptophan Metaboli
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