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

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437 High-throughput platform for expression in Arabidopsis protoplasts and cultured cells Le Son Long Nguyen, Rudy Vanderhaeghen, Mansour Karimi, Alain Goossens, Pierre Hilson Department of Plant Systems Biology (PSB), Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Ghent, Belgium Sequence information and genome annotation are improving at an impressive pace but functional ontology is still inexistent or rudimentary for most genes. Even though they are not suited for complex organismal studies, assays in plant protoplasts and cultured cells have proven very useful to dissect a broad range of molecular mechanisms including signaling and metabolic pathways as well as transcriptional regulatory networks. We are building the framework enabling large-scale systematic screens in such cells. (1) Protocols for transfection of tobacco and Arabidopsis protoplasts have been adapted for automated handling in multi-well plates. Our current experimental set-up allows processing of up to 200 samples per day. (2) Vectors were constructed compatible with efficient Gateway recombinational cloning (www. psb.ugent.be/gateway) and designed for gene overexpression, promoter studies or silencing via RNA interference. They include reporter plasmids for standardized assays based on the dual firefly/Renilla luciferase enzymatic system (3) We are currently developing the hardware and software necessary to record and analyze cell growth, in vivo reporter gene expression and other cellular features at the microscopic level. References: De Sutter, V., Vanderhaeghen, R., Tilleman, S., Lammertyn, F., Vanhoutte, I., Karimi M., Inzé, D., Goossens, A. and Hilson, P. (2005) Exploration of jasmonate signalling via automated and standardized transient expression assays in tobacco cells. Plant J. 44, 1065-1076. Hilson, P. (2006) Cloned sequence repertoires for small- and large-scale biology. Trends Plant Sci. 11, 133-141. Karimi, M., De Meyer, B. and Hilson, P. (2005) Modular cloning in plant cells. Trends Plant Sci. 10, 103-105. Underwood, B.A., Vanderhaeghen, R., Whitford, R., Town, C.D. and Hilson, P. (2006) Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations. Plant Biotechnol. J. 4, 317-324. 438 HY5 Interacting Proteins Isolated by Genome Wide Screen of Protein-Protein Interaction of Plant Transcription Factors Hye Jin Kim, Ju Hwoan Kang, Young Hun Song, Su Young Shin, Na Young Song, Geon Hui Son, Jong Hong Department of Biochemistry and Environmental Biotechnology Research Center, Division of Applied Life Science, Gyeongsang National University, Jinju, 660-701, Korea The comprehensive analysis of transcription factor interaction can provide fundamentally important information for understanding the complex regulation of gene expression in plant growth and development. The construction of transcription factor (TF) ORFeome library in a yeast two hybrid vectors can provide a valuable tool in isolating novel interacting TFs to the protein of interest. We have constructed Arabdisopsis TF ORFeome library which contains 1400 TFs in a correct reading frame to the prey vector (pDEST32 Gal4AD) using gateway recombination cloning system (Invitrogen). The Arabidopsis transcription factor HY5 is one of the best characterized bZIP transcription factor that act as positive regulator of photomorphogenesis. The HY5 is also involved in other developmental process such as root development, hormone responses, and flowering. This indicates that HY5 regulate many genes by interacting with multiple regulatory factors. Using the full length HY5 protein as a bait we have isolated 49 different transcription factors which include 8 B-box zinc finger factor, 9 bZIP factors, 6 C2H2, 5 AP2/EREBP, 2 MYB , 4 TCP proteins, and others. Thirty TFs also interacted with HYH, a HY5 homolog, an indication that the interaction identified by high throughput analysis is specific. In addition, more than 20 HY5 interacting proteins are shown to interact with COP1, a negative regulator that specifically targets HY5 for degradation via 26S proteosome in the dark through direct physical interaction. This study shows that the Arabidopsis TF ORFeom library is a powerful tool to identify many novel interacting factors of biological interest (Supported by BK21 program and CFGC of 21C Frontier Research Program, KOSEF)
439 A Comparison of Full Versus Partial 15 N Incorporation for Metabolic Labeling in Arabidopsis Thaliana Edward Huttlin 2, 1 , Adrian Hegeman 2, 1 , Amy Harms 1 , Michael Sussman 2, 1 1 University of Wisconsin Biotechnology Center, 2 University of Wisconsin Biochemistry Department Over the past several years a variety of isotope-assisted quantitative proteomics techniques have been developed, allowing use of tandem mass spectrometry for the simultaneous comparison of the identities and abundances of hundreds or thousands of proteins within pairs of biological samples. These approaches include in vitro labeling techniques such as ICAT and 18 O labeling which introduce an isotopically-labeled tag onto each sample during sample preparation, as well as in vivo metabolic labeling techniques in which an isotopic label is incorporated into the organism from its media during normal growth and development. For all of these approaches, after incorporation of either a light or a heavy isotopic tag, the control and experimental samples are combined and processed together. Because the tagged peptides are essentially chemically identical, they provide an excellent internal control for all subsequent steps in sample preparation and analysis. Yet using mass spectrometry we can differentiate among peptides from each sample due to the mass difference between the heavy and light isotopic tags. Because metabolic labeling allows combination of control and experimental samples through all steps of sample processing including tissue homogenization and protein extraction, it provides perhaps the ideal internal control. While metabolic labeling provides an elegant control for all steps in sample preparation, presently some technical challenges limit its widespread application, especially in intact organisms such as plants. First, achieving full incorporation of an isotopic label such as 15 N into plants and other higher organisms is challenging and may require unnatural growth conditions that limit the biological questions to which it may be applied. Additionally, when applied in a high throughput manner these approaches require informatics tools for automated data analysis. We have compared traditional ubiquitous 15 N metabolic labeling in Arabidopsis with a new approach using partial incorporation of 15 N (based on Whitelegge et al. Phytochemistry 65 (2004) 1507-1515) to produce changes in the isotopic envelopes of each peptide that can be used for quantitative comparison. While both approaches require significant informatics tools for analysis, the latter is perhaps more amenable for labeling under a wider variety of conditions. We will present a comparison of traditional metabolic labeling with partial metabolic labeling in Arabidopsis whole tissue with respect to numbers of peptide and protein identifications as well as quantitative accuracy and dynamic range. We will also discuss our current strategies for automated data analysis using both approaches. 440 Understanding the TGA transcriptional network using ChIP-chip and expression arrays Francoise Thibaud-Nissen 1 , Christopher Johnson 2 , Hank Wu 1 , Julia Redman 1 , Yuelin Zhang 3 , Xin Li 3 , Jonathan Arias 2 , Christopher Town 1 1 The Institute for Genomic Research, Rockville MD, USA., 2 University of Maryland, Baltimore County, MD, USA., 3 University of British Columbia, Vancouver BC, Canada In order to understand the transcriptional network of TGA factors in Arabidopsis and its role in systemic acquired resistance, we are conducting ChIP-chip studies using TGA antibodies and analysis of SA-induced changes in expression in wild-type and tga mutants. The TGA transcription factors TGA2, TGA5 and TGA6 have been found to play a redundant and essential role in the onset of systemic acquired resistance. In a preliminary analysis we have identified over 50 putative binding sites for TGA2 through a ChIP-chip approach using Arabidopsis whole-genome arrays, 55% of which contain a single palindromic octamer TGACGTCA. Additional ChIP-chip data using TGA5 show substantial overlap in the binding sites of TGA2 and TGA5. In parallel, the effect of the SA treatment on gene expression in wild-type plants and tga2tga5, tga6 and tga2tga5tga6 mutants was determined using Affymetrix ATH1 arrays. We observed substantial and very similar changes in expression upon SA application in the wild-type, the single and the double mutant. In particular, genes coding for kinases and proteins involved in the response to biotic stimuli are overrepresented among the genes differentially regulated by SA. However, SA induced very few changes in gene expression in the triple mutant. Hierarchical clustering of experimental samples based on gene expression revealed that the 18-hour mock-treated samples of the triple mutant cluster with SA-treated samples of the wild-type, single and double mutants, indicative of high basal levels of genes usually induced by SA in the untreated tga2tga5tga6 plants. Research supported by the NSF 2010 Program
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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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331 A Putative Bifunctional Wax Est
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