Biochemistry of Signal-Responsive Transcription in Plants

9728789 Arias这个项目的总体目标是了解特定的植物TGA因子如何影响植物中依赖AS-1的基因表达。为此，研究人员的实验室利用烟草原生质体和细胞开发了瞬时和稳定的转基因系统，以评估内源性或瞬时表达的因子对特定刺激的反式激活目标报告基因。通过其中一个模型系统，他们发现两种不同的AS-1结合蛋白，称为TGA1a和VBP1，通过一种不涉及其bZIP结构域的DNA结合、二聚化或核定位功能改变的机制来增加转录，以响应生长素和外源压力。第一个目标是通过体内功能增益分析确定决定TGA1a基础和生长素反应激活的特定结构域和残基。第二个目标是通过体内标记和磷酸肽定位直接测试生长素是否诱导瞬时表达的TGA1a的磷酸化状态的变化。在瞬时表达分析中，将用这些因子的突变形式来评估磷受体位置对反式激活和其他功能的重要性。第三个目标是使用蛋白质结合试验来表征选择性和稳定地与TGA1a结合的120kda烟草蛋白。在免疫共沉淀试验中，该蛋白的结合位点将与瞬时表达的突变形式的TGA1a进行定位。这种蛋白与TGA1a的关联是否对生长素有反应的问题也将被调查。这些目标的完成将为植物激素和化学胁迫信号如何刺激调节基因表达的植物DNA结合蛋白的活性提供重要的新信息。拟议的研究可能会对目前理解真核生物，特别是植物中的基因表达是如何调控的努力做出重大贡献。这些知识可能会导致开发新的调控系统来控制植物中重要的经济基因的表达。

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.