Biochemistry of Signal-Responsive Transcription in Plants

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9728789 Arias The overall objective of this project is to understand how specific plant TGA factors contribute to as-1 dependent gene expression in plants. Towards this end, the investigator's laboratory developed transient and stable transfection systems with tobacco protoplasts and cells to evaluate the trans-activation of target reporter genes by endogenous or transiently-expressed factors in response to defined stimuli. With one of these model systems they found that two distinct as-1 binding proteins, termed TGA1a and VBP1, increase transcription in response to auxins and xenobiotic stress through a mechanism that does not involve a change in DNA-binding, dimerization, or nuclear localization functions of their bZIP domains. The first goal is to identify by in vivo gain-of-function assays the specific domains and residues that determine basal and auxin-responsive activation by TGA1a. The second goal is to directly test by in vivo labeling and phosphopeptide mapping, whether auxin induces a change in the phosphorylation state of transiently-expressed TGA1a. The importance of the phosphoacceptor site to trans-activation and other functions will be evaluated with mutant forms of these factors in transient-expression assays. The third goal is to use protein-binding assays to characterize a 120 kDA tobacco protein that selectively and stably associates with TGA1a. The binding site for this protein will be mapped with transiently-expressed, mutant forms of TGA1a in a co-immunoprecipitation assay. The question of whether the association of this protein with TGA1a is auxin-responsive will also be investigated. Completion of these objectives will provide important new information on how plant hormone and chemical-stress cues stimulate the activity of a plant DNA-binding protein that regulates gene expression. The proposed studies are likely to make significant contributions towards current efforts at understanding how gene expression in eukaryotes, and especially in plants, is regulated. Such knowledge may lead to the development of novel regulatory systems for controlling the expression of economically-important genes in plants.