REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

本提案的长期目标是了解全球 控制植物中的基因表达。 我们特别 研究协调基因表达的机制 属于整合的氨基酸生物合成途径。 见解 在我们最近的研究中， 谷氨酰胺合成酶（GS）基因家族的实验室， 天冬酰胺合成酶（AS）。 这些关键词的综合表达 酶用于调节氮的同化和运输， 植物 我们的研究表明，GS的个别成员 和AS基因家族编码不同的产物， 在特定细胞类型中差异表达， 时间的发展，并响应环境信号。 光诱导叶绿体GS2核基因的表达 并伴随抑制AS 1基因的表达。 的 从光照下的GS2表达转换为黑暗下的AS1表达 具有显著的生物学意义， 植物 我们打算阐明光的新机制- AS1的抑制表达，特别注意其 作为诱导型启动子系统的潜力。 几个模型将在 测试以确定GS2的光激活和光激活是否与GS2的光激活相关。 AS1的阻遏由"阻遏物/激活物"蛋白介导，或 不同的因素。 GS和AS基因的表达也是协调的 当氮必须从富氮源中动员出来， 代谢下沉。 因此，胞质GS3A和AS 1的基因是 在发芽过程中和固氮根中协同诱导 结节 我们建议确定参与韧皮部的因素- 这些基因的特异性和发育调控表达。 初步结果表明，一些因素相互作用， GS和AS基因的启动子，并表明， 这些基因的整合调控是复杂的， 多方面的 拟议的研究协调的因素， GS和AS基因在发育过程中的调节将提供一些 第一次深入了解植物基因表达的全局控制。 该提案的具体目标是：1）定义光 参与GS2表达激活的调节元件（LRE） 和/或使用报告基因通过光抑制（nLRE）AS1表达 转基因植物的基因系统。 我们将阐释这部小说 光抑制基因表达机制及开发AS 1的应用 作为植物中的"暗"诱导型启动子系统。 2)确定 "浅色"和"深色"提取物中独特结合活性的性质 用GS 2和AS 1启动子的顺式元件检测。 3)识别 DNA序列参与韧皮部特异性和发育 GS3A和AS 1在子叶中的表达受到调控， 固定结节。 4)检测韧皮部特异性DNA结合活性 利用新的纯化方法。 5)表征cDNA 克隆编码DNA结合蛋白因子，并确定性质 因子：因子相互作用。 6)DNA结合因子的检测能力 在体外激活或抑制转录。 7)确定细胞- 克隆因子特异性和体内调节。 8)试验模型 整合GS和AS调节因子异位表达， 携带GS-GUS和AS-GUS转基因的转基因植物。 九、 确定因子的异位表达是否调节内源性 GS和AS基因表达和/或影响氮同化。

The long-term objectives of this proposal ar to understand global control of gene expression in plants. In particular we are investigating the mechanisms which coordinate the expression of genes belonging to integrated amino acid biosynthetic pathways. Insights into this process are beginning to emerge from recent work in our laboratory on the gene families for glutamine synthetase (GS) and asparagine synthetase (AS). The integrated expression of these key enzymes serves to regulate nitrogen assimilation and transport in plants. Our studies have revealed that individual members of the GS and AS gene families encode distinct products which are differentially expressed in particular cell-types, at particular times in development, and in response to environmental signals. Light induces the expression of the nuclear gene for chloroplast GS2 and concomitantly represses the expression of the AS1 gene. The switch from GS2 expression in the light to AS1 expression in the dark is dramatic and has biological significance to nitrogen economy in plants. We propose to elucidate the novel mechanism of light- repressed expression of AS1 with particular attention to its potential as an inducible promoter system. Several models will be tested to determine whether light-activation of GS2 and light- repression of AS1 are mediated by a "repressor/activator" protein or by distinct factors. GS and AS gene expression are also coordinated when nitrogen must be mobilized from nitrogen-rich sources to metabolic sinks. Accordingly, genes for cytosolic GS3A and AS1 are coordinately induced during germination and in nitrogen-fixing root nodules. We propose to identify factors involved in the phloem- specific and developmentally-regulated expression of these genes. Preliminary results suggest that a number of factors interact with the promoters of the GS and AS genes and suggest that the mechanisms underlying the integrated regulation of these genes are complex and multifaceted. The proposed studies on factors which coordinate the regulation of GS and AS genes during development will provide some of the first insights into global control of gene expression in plants. The specific aims of this proposal are to: 1) Define Light Regulatory Elements (LREs) involved in activation of GS2 expression and/or repression (nLRE) of AS1 expression by light using a reporter gene system in transgenic plants. We will elucidate the novel mechanism of light-repressed gene expression and develop AS1 for use as a "dark" inducible promoter system in plants. 2) Determine the nature of unique binding activities in "light" and "dark" extracts detected with cis-elements of the GS2 and AS1 promoters. 3) Identify DNA sequences involved in phloem-specific and developmentally regulated expression of GS3A and AS1 in cotyledons and nitrogen- fixing nodules. 4) Detect phloem-specific DNA binding activities utilizing a novel purification procedure. 5) Characterize cDNA clones encoding DNA binding protein factors and determine the nature of factor:factor interactions. 6) Test ability of DNA-binding factor to activate or repress transcription in vitro. 7) Determine cell- specific and in vivo regulation of cloned factor. 8) Test models for integrated GS and AS regulation by ectopic expression of factor(s) in transgenic plants harboring GS-GUS and AS-GUS transgenes. 9) Determine whether ectopic expression of factor modulates endogenous GS and AS gene expression and/or affects nitrogen assimilation.