Regulatory Mechanisms of Metabolic Repression in Higher Plants

本研究的目的是了解高等植物光合基因代谢抑制的调控机制。该实验室最近的研究表明，糖和醋酸盐特异性地协同抑制7个玉米光合基因启动子。这种抑制在这些代谢物的生理浓度下是有效的，并且超越了光、细胞类型和发育阶段的调节。转录的代谢抑制似乎是玉米的一个主要调控回路，并且易于使用本实验室建立的原生质体瞬时表达方法进行生化和分子遗传学分析。该项目可分为两部分：第一部分是继续研究直接参与葡萄糖和醋酸盐介导的光合基因抑制的转录机制。利用一个已被充分表征的玉米光合基因启动子（cabZm5启动子）与一个可测定的报告基因融合，研究者将开始定义介导代谢抑制的顺式调控DNA元件。研究者还将描述随后和/或介导抑制的dna -蛋白质相互作用的变化。第二部分旨在了解葡萄糖浓度是如何被感知和转导的。各种葡萄糖类似物、化学抑制剂和玉米己糖激酶突变体将被用来确定葡萄糖磷酸化或代谢是否需要抑制。由于初步结果表明cAMP可能是玉米系统（如酵母和脊椎动物）中相关的第二信使，因此将研究影响cAMP或cAMP依赖性蛋白激酶的药物的影响。最后，为了验证原生质体瞬时表达实验中产生的调控概念，研究者将开始创建转基因玉米系统并分析转基因玉米植株。该项目的结果将为植物生长发育所必需的代谢物介导的信号转导和基因调控提供急需的见解。

The goal of this research project is to understand the regulatory mechanisms mediating metabolic repression of photosynthetic genes in higher plants. Recent studies in this laboratory have shown that sugars and acetate specifically and coordinately repress seven maize photosynthetic gene promoters. The repression is effective at physiological concentrations of these metabolites, and overrides regulation by light, cell type, and developmental stage. Metabolic repression of transcription appears to be a major regulatory circuit in maize and is susceptible to biochemical and molecular genetic analyses using protoplast transient expression methods established in this laboratory. The project can be divided into two parts: the first part is to continue the study of transcriptional mechanisms directly involved in glucose- and acetate-mediated photosynthetic gene repression. Using a well characterized maize photosynthetic gene promoter (the cabZm5 promoter) fused to an assayable reporter gene, the investigator will begin to define the cis-acting regulatory DNA elements mediating metabolic repression. The investigator will also characterize the changes in DNA-protein interactions which follow and/or mediate repression. The second part aims to understand how the glucose concentration is sensed and transduced. Various glucose analogues, chemical inhibitors, and maize hexokinase mutants will be used to determine whether glucose phosphorylation or metabolism is required for repression. Because preliminary results suggest cAMP can be a relevant second messenger in the maize system (as in yeast and vertebrates), the effects of agents impacting cAMP or cAMP-dependent protein kinases will be investigated. Finally, to test regulatory concepts generated by work using protoplast transient expression assays, the investigator will begin to create a transgenic maize system and analyze transgenic maize plants. The results from this project will provide much needed insight into metabolite-mediated signal transduction and gene regulation essential to plant growth and development.