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

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337 Interaction-dependent localization of flavonoid enzymes in Arabidopsis Melissa Ramirez, Brenda S. Winkel Virginia Polytechnic Institute and State University As early as the 1940’s, the idea of multienzyme complexes began surfacing as a way the cell might organize enzymes to enhance the efficiency of metabolism. It has now become evident that macromolecular interaction is a fundamental aspect of cellular biochemistry. The Winkel laboratory uses the flavonoid pathway of Arabidopsis as a model to understand the assembly and regulation of enzyme complexes. This pathway is a well-characterized specialized plant metabolic system, the products of which comprise a diverse set of compounds that are critical for plant survival and reproduction. While the central steps of flavonoid biosynthesis are well detailed, a full understanding of the biochemistry of the pathway is complicated by the fact that many of the enzymes can utilize multiple substrates and may also physically interact with each other. Until recently, it was believed that flavonoid biosynthesis occurred exclusively in the cytoplasm and that the products were then transported to various sites of action within the cell. However, new evidence indicates the presence of at least two flavonoid enzymes in the nucleus, suggesting that the synthesis of nuclear flavonoids may occur in situ. Additional studies are needed to explore the possibility that subcellular localization of flavonoid enzymes is affected by or dependent upon specific protein-protein interactions and that this localization determines the types and cellular locations of end products that are produced in response to diverse biotic and environmental cues. The study presented here begins to dissect the molecular basis of the observed dual localization of chalcone synthase (CHS) and chalcone isomerase (CHI), the first two enzymes of the flavonoid pathway. Epi-fluorescence and confocal laser scanning microscopy are being used to examine the localization of these enzymes expressed as fusions to green fluorescent protein (GFP) in protoplasts and stably-transformed plants. Analyses are being performed in both wild type cells and mutants that are depleted in various flavonoid enzymes in order to explore the possible effects of specific protein-protein interactions on this localization. Site-directed mutagenesis is being used in parallel experiments to test the functionality of a putative nuclear localization signal in CHS, which overlaps with the predicted CHI interaction interface. This work represents an essential step in elucidating the mechanisms that organize related metabolic enzymes within the crowded environment of the cell interior. 338 FAC1-Directed Herbicide Toxicity Correlates With Expanded Adenine Nucleotide Pools Richard Sabina Medical College of Wisconsin EMBRYONIC FACTOR 1 (FAC1) is an early expressed plant gene and knockout lines reveal that it is essential for the zygote to embryo transition in Arabidopsis thaliana (Plant J 42:743,2005). FAC1 encodes an AMP deaminase (AMPD), which is also the intracellular target for a class of modified nucleosides produced by fungal pathogens that are converted in plant cells to transitionstate inhibitors (monophosphates) of this enzyme (Plant Physiol 114:119,1997;Bioorg Med Chem Lett 9:1985,1999). Exposure to these herbicides results in a rapid 2-3 fold increase in ATP levels, but reportedly not ADP or AMP, and eventual cessation of seedling growth with paling and necrosis at the apical meristem. The x-ray crystal structure of Arabidopsis FAC1 bound to a transition-state analog has provided a glimpse of the complete active site (J Biol Chem 281:In Press,2006). However, the mechanistic basis for lethality associated with a genetic or herbicide-induced limitation in FAC1 catalytic activity is unknown. OBJECTIVE: Explore the underlying mechanisms for FAC1-directed toxicity by considering the immediate metabolic consequences of an inability to deaminate AMP in plant cells. APPROACH: Monitor relative growth and quantify intracellular adenine nucleotides (AXP) and IMP in treated and untreated control Arabidopsis seedlings following systemic exposure to sublethal and lethal doses of deaminoformycin (DF) and attempt rescue with purine nucleosides and bases. METHODS: 5 to 7 d.o. seedlings were placed on M&S salt + 1% sucrose agar in 24-well plates with and without DF (300 nM or 22 uM) and/or a purine nucleoside or base (0.5-1 mM). Plants were grown under long-day conditions (16 h light/8 h dark) for 7-9 days, roots were excised and the remaining tissue was weighed and ground to a powder under liquid nitrogen. Ice-cold 10% (w/v) TCA was added and the frozen tissue was homogenized on ice for 2 min with a motorized pestel. The homogenate was centrifuged at 14,000Xg for 2 min at 4C and the supernatant was neutralized with an equal volume of 0.5 M tri-n-octylamine in freon. All samples were frozen immediately in dry ice and stored at -80C. Metabolites were separated by anion-exchange HPLC. RESULTS: DF inhibits plant growth and wet weight is inversely correlated with the levels of ALL adenine nucleotides and the AMP/IMP ratio, an in vivo index of AMPD activity. Downstream catabolites do not rescue seedlings from a lethal dose of DF. The correlations between decreased AMPD activity, increased AXP pools, and reduced seedling growth point to mechanisms related to upstream effects of FAC1 inhibition. This may involve hormonal imbalance due to increased cytokinin synthesis or disruption of 14-3-3 protein function by AMP. This work was supported by a grant from the Research Affairs Committee (RAC) at the Medical College of Wisconsin and through a cooperative agreement with Bayer CropScience GmbH, Frankfurt am Main, Germany.
339 Exploring of Arabidopsis non-host resistance via high resolution profiling of plant secondary metabolites Dierk Scheel, Christoph Boettcher, Edda von Roepenack-Lahaye, Stephan Clemens Leibniz Institute of Plant Biochemistry, Halle, Germany We developed a platform for the highly sensitive profiling of mostly secondary metabolites, employing capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (CapLC-ESI-QTOF- MS). This approach achieves a very good coverage of Arabidopsis secondary metabolism. A recent compilation listed six biosynthetic classes: nitrogen-containing compounds, phenylpropanoids, benzenoids, polyketides such as flavonoids, terpenes and fatty acid derivatives. Metabolites of five of these classes can clearly be detected by CapLC-ESI-QTOF- MS. Furthermore, in-source fragmentation and targeted tandem MS analysis allow to obtain structural information on unknown compounds. This is of paramount importance given, for instance, the conservatively estimated 5000 metabolites in Arabidopsis thaliana of which maybe only 500 are annotated today. Databases for LC-MS spectra and for known and “theoretically” occurring compounds in the Brassicaceae help in structural elucidation and in cataloguing the Arabidopsis metabolome. A systematic evaluation of matrix effects has shown that the good separation achieved allows reproducible quantification. The platform and its applicability as a general method for biochemical phenotyping of Arabidopsis mutants will be presented. In particular, we show here profiling data of Arabidopsis mutants with altered non-host resistance against fungal and oomycete pathogens. 340 Arabidopsis Sucrose Transporter AtSUC9: High Affinity Sucrose Transport, Intragenic Control of Expression and Comparative Substrate Specificity Alicia Sivitz, Anke Reinders, Meghan Johnson University of Minnesota Transport of sucrose across membranes is controlled by sucrose transporter proteins (SUTs or SUCs). Plants have small gene families of SUTs; the Arabidopsis genome contains seven SUT genes. Here, the Arabidopsis thaliana sucrose transporter AtSUC9 (At5g06170) was expressed in Xenopus oocytes and revealed to have an ultra-high affinity for sucrose (K0.5 = 0.066 +/- 0.025 mM) compared to other plant sucrose transporters. AtSUC9 also showed low specificity and transported a wide range of glucosides including helicin, salicin, arbutin, maltose, fraxin, esculin, turanose, and alphamethyl D glucose. AtSUC9 substrate specificity was found to be similar to AtSUC2 (At1g22710). The ability of AtSUC9, AtSUC2, HvSUT1 (from barley) and ShSUT1 (from sugarcane) to transport a variety of substrates was compared, and the results indicate that Type I and Type II sucrose transporters have different substrate specificities. AtSUC9 expression was found in sink tissues throughout the shoots and in flowers. AtSUC9 expression was dependent on intragenic sequence, which was also true for AtSUC1 (At1g71880), but not AtSUC2. In summary, the novel transporter AtSUC9 has a much higher affinity for sucrose than any other plant sucrose transporters, indicating that AtSUC9 is uniquely suited to function in maintaining very low sucrose concentrations in the wall space around shoot sink cells.
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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
Page 81 and 82: 235 Ethylene Signaling Components A
Page 83 and 84: 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
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
Page 113 and 114: 299 Identification of genes contrib
Page 115 and 116: 303 Regulation of AGAMOUS Expressio
Page 117 and 118: 307 Genetic Control of the Interplo
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
Page 125 and 126: 323 Fluorescent amino acid sensors
Page 127 and 128: 327 The Role of Tryptophan Metaboli
Page 129 and 130: 331 A Putative Bifunctional Wax Est
Page 131: 335 Analysis of the Complex BIO3 /
Page 135 and 136: 343 A Yeast-2-Hybrid Assay Reveals
Page 137 and 138: 347 Genetic Mechanisms of Glucosino
Page 139 and 140: 351 Potent Induction of flowering b
Page 141 and 142: 355 Phylogenetic Analysis of Arabid
Page 143 and 144: 359 eQTL-mapping reveals AMP2A, a c
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
Page 151 and 152: 375 Natural genetic and epigenetic
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Page 155 and 156: 383 The RAD23 Family of Ubiquitin-L
Page 157 and 158: 387 Molecular Functions of ARL2 and
Page 159 and 160: 391 Characterization of the Sugar-I
Page 161 and 162: 395 Functional analysis of phosphat
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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
Page 179 and 180: 431 Functional analysis of ROP10 sm
Page 181 and 182: 435 The Importance Of RecQ Like Hel
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439 A Comparison of Full Versus Par
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