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

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345 Physiological function of HMA2 and HMA4 in Arabidopsis thaliana Edwin Wong, Renee Jarvis, Christopher Cobbett The University of Melbourne Type-1 B P-type ATPases utilize ATP-hydrolysis to transport heavy metals across various biological membranes. Current study in our lab is focusing on the characterization of two of the eight P-1 B transporters in Arabidopsis thaliana, HMA2 and HMA4. Genetic analysis has shown HMA2 and HMA4 are essential for zinc homeostasis: a hma2;hma4 double mutant is severely zinc deficient (1). Symptoms include chlorosis, stunting and failure to develop pollen, and these phenotypes are reversible by the application of exogenous zinc. Promoter-GUS reporter constructs have shown HMA2 and HMA4 have parallel expression patterns in vascular tissues and in developing anthers. Expression of the HMA proteins in yeast has effects on cadmium resistance and zinc dependence. As divalent cation transporters, HMA2 and HMA4 may function in Cd translocation and detoxification in addition to maintaining Zn homeostasis. In plants, phytochelatins are a major determinant of Cd detoxification. Upon exposure to heavy metals, these heavy metal-binding peptides are synthesized enzymatically from glutathione by CAD1-encoded phytochelatin synthase. cad1 mutants are phytochelatin deficient and hypersensitive to Cd (2). We have created multiple cad1;hma mutant lines. These are being analysed for altered Cd-sensitivity and Cd uptake and translocation. Both HMA2 and HMA4 have potential metalbinding motifs. For example, in the N-terminal region, HMA2 has GICC instead of the more common metal binding motif GMxCxxC and, in an extended C-terminal domain there are multiple di-Cysteines and a poly-His region. We are expressing various mutant derivatives of HMA2 in the hma2;hma4 double mutant in an attempt to understand the in planta physiological significance of these motifs. (1) Hussain et al. 2004 Plant Cell 16: 1327-1339. (2) Ha et al. 1999 Plant Physiol: 107: 1059-1066. 346 Identification and Characterization of Pyruvate Decarboxylase (pdc) Gene Family Members in Arabidopsis Songqing Ye, Jerry Cohen Graduate Program in Plant Biology, Department of Horticultural Science, and Microbial and Plant Genomics Institute, University of Minnesota, Saint Paul, MN 55108 USA Pyruvate decarboxylase (PDC, EC 4.1.1.1) converts, by decarboxylation, pruvate into acetaldehyde and then alcohol dehydrogenase (ADH, EC 1.1.1.1) reduces acetaldehyde into ethanol. This set of reactions shunts the main glycolytic pathway into ethanol fermentation, instead of entry into the tricarboxylic acid cycle, so it is very important for plants as they respond to anaerobic stress. By searching the Arabidopsis database, we found that there are four genes having the high sequence similarity to the pdc genes reported from bacteria. The 4 putative ipdc/pdc genes are: pdc4 (At5g01320), pdc3 (At5g01330), pdc2 (At5g54960), and pdc1 (At4g33070). A phylogenetic tree, based on the full amino acid sequences of known pdc genes was constructed using a neighbor-joining method. All plant putative pdc genes were shown to be conserved and clustered together. Pdc1 and pdc2 share high sequence similarity (82% identity). Both pdc3 and pdc4 are located on chromosome 5, separated by 1.6kb, and show 92% identity. We have cloned these genes from cDNA (ABRC) or a cDNA library and expressed and purified the recombinant proteins by the His-tag technique. We found that AtPDC2 is functional PDC according to its measured biochemical activity. The optimal pH for the recombinant protein was 6.2 and the Km was 3.5 mM. Typically, PDCs require the cofactors Mg 2+ and thiamin pyrophosphate (TPP). We found 0.5 mM TPP and 5 mM Mg 2+ resulted in the highest activity, however, the plant enzyme still showed around 40% of its activity when supplied with only a single cofactor or without any cofactor, compared with an optimal combination of both cofactors. In bacteria and fungi, PDCs have bi-functional activities and always show some IPDC (indole pyruvate decarboxlase) activity as well. However, we did not find any IPDC activity for AtPDC2 suggesting that uniquely this plant enzyme is mono-functional. Also, AtPDC1 and AtPDC3 have not measurable PDC activity. This work was supported, in part, by a grant from the U.S. Department of Energy.
347 Genetic Mechanisms of Glucosinolates Hydrolysis in Crucifers Zhiyong Zhang, James Ober, Daniel Kliebenstein University of California-Davis Glucosinolates are sulfur-rich plant secondary metabolites present in all cruciferous plants that require hydrolysis for activation. Their breakdown products have a wide range of biological activities in plant-herbivore and plant-pathogen interactions and anti-carcinogenic properties that are determined by the resulting chemical structure. In Arabidopsis and Brassica, hydrolysis by the enzyme, myrosinase, produces the bioactive nitriles, epithionitriles or isothiocyanates depending upon the plants genotype and the glucosinolates structure. Two QTL, ESP and ESM1, function to determine formation of epithionitriles and nitriles. We have cloned both QTL and they have opposing functions. ESP directs nitrile formation while ESM1 inhibits nitrile formation. Both proteins have been shown to interact with myrosinase in other species suggesting that the myrosinase protein complex functions to control the structural outcome of glucosinolate hydrolysis. We have extended this work to show that ESP and ESM1 control the in planta formation of Indole-3- acetonitrile, a potential auxin precursor. In addition to the biochemical function, we are studying the downstream impact on Arabidopsis/Insect interactions. The glucosinolate hydrolysis profile change towards isothiocyanate formation controlled by ESM1 is associated with resistance to Trichoplusia ni. We have recently identified associated variation within Brassica vegetables that could allow for potential improvement of human nutrition. Work on associating this phenotypic variation with genetic variation within ESP and ESM1 will be presented. We have recently identified associated variation within vegetables that could allow for potential improvement of human nutrition. The myrosinase protein complex is comprised of a large number of proteins. A number of these proteins are ESP or ESM1 homologues. Interestingly, the ESP and ESM1 homologues are interspersed in the same two gene clusters on Chromosome 1, and another is on Chromosome 3. Thus, suggesting that they are undergoing parallel evolution. These homologues show high similarity with ESM1 or ESP in both gene structure and protein features. We are currently using T-DNA Knockouts, 35S-overexpression and biochemistry to test if these homologues also control structural specificity during glucosinolate hydrolysis. Preliminary genomic analysis will be presented. Department of Plant Sciences, University of California, Davis, CA 95616. To whom correspondence should be addressed: kliebenstein@ucdavis.edu 348 Isolation for mutants of higher accumulated elements and pigments from rice FOX lines Youichi Kondou 1 , Mika Kawashima 1 , Takanari Ichikawa 1 , Akie Ishikawa 1 , Hirohiko Hirochika 2 , Minami Matsui 1 1 PSC, RIKEN, 2 National Institute of Agrobiological Sciences FOX hunting system is novel gain-of-function technique. In this system, random over-expression of a normalized plant's full-length cDNA library cause dominant gain-of-function mutations and it is expected that plant acquire useful phenotype. We are establishing rice FOX lines using 13,000 non-redundant rice full-length cDNAs to introduce them into Arabidopsis plants. At present, we are screening these lines with various trait for exploration of useful rice genes. In this study, we will introduce screening strategy for isolation of mutants of higher accumulated elements and pigments (Elements and Pigments) in rosette leaves as examples of useful phenotype. We already isolated some mutants by prescreening in T1 generation from about 3,000 lines. This research is supported by the Special coordination funds for Promoting Science and Technology entitled Rapid identification of useful traits using rice full-length cDNAs.
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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
Page 85 and 86: 243 Intracellular Production Of Rea
Page 87 and 88: 247 Large-Scale Screen and Isolatio
Page 89 and 90: 251 Ontogeny of the Arabidopsis Cir
Page 91 and 92: 255 Cell type-specific expression a
Page 93 and 94: 259 Characterization of Flowering T
Page 95 and 96: 263 Involvement of Cytokinins and T
Page 97 and 98: 267 Identification of new actors of
Page 99 and 100: 271 Scarecrow-like Protein SCL14 In
Page 101 and 102: 275 Protein Prenylation Is Required
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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 127 and 128: 327 The Role of Tryptophan Metaboli
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Page 131 and 132: 335 Analysis of the Complex BIO3 /
Page 133 and 134: 339 Exploring of Arabidopsis non-ho
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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
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Page 149 and 150: 371 Natural Variation for Seed Dorm
Page 151 and 152: 375 Natural genetic and epigenetic
Page 153 and 154: 379 SPIKE1 is a guanine nucleotide
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
Page 175 and 176: 423 Protein Prenylation Process Neg
Page 177 and 178: 427 Integration of light and abscis
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Page 181 and 182: 435 The Importance Of RecQ Like Hel
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75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

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