REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

我们正在使用拟南芥作为模型，以确定结构 和控制氮同化成谷氨酰胺的调节基因， 利用分子、生物化学和遗传学方法研究谷氨酸。 氮 同化成这些氨基酸影响植物生长， 种子数量和质量。 因此，我们对基因的基础研究， 在植物中控制这一过程与人类和动物间接相关 营养 正在研究的结构基因是：谷氨酰胺 谷氨酸合成酶（Fd-GOGAT或NADH-GOGAT），和 谷氨酸脱氢酶（GDH）。 中每种酶的基因家族 拟南芥含有独立调控的成员编码不同的 同工酶 尽管数十年的体外研究在许多国家进行， 物种，GS，GOGAT和GDH同工酶在植物中的体内作用可以 只会被折磨。 我们建议进行第一次系统隔离 在GS，Fd-GOGAT， NADH-GOGAT或GDH，在单个物种中。 我们已经证明， 分离氮同化基因缺陷的拟南芥突变体 使用对生长表型无偏倚的同工酶筛选。 我们提出了一个 突变后代的详细表征，以量化影响 一个同工酶的损失过程中，如初级氮 同化，光呼吸和氮动员过程中的种子。 该分析将确定控制蛋白质合成的关键酶和限速酶。 植物利用氮的效率。 此外，我们还可能发现 调节基因的突变体。 我们已经开始研究 控制氮同化基因的调控。 基于基因 在调节研究中，我们开发了一种“代谢控制”模型， 提出这些氮同化基因的调节是响应于 植物中碳与氮代谢物的比例。 我们提出 基因筛选，以揭示这一机制的组成部分， 两个假定的调控基因。 我们的具体目标是：1）隔离 另外的拟南芥gdh突变体和叶绿体GS 2中的突变体， 同工酶筛选，2）表征现有拟南芥突变体 Fd-GOGAT（gls 1）的两个基因之一有缺陷， 第二个基因，3）通过以下方法在胞质GS1或NADH-GOGAT中产生突变体： 在转基因植物中表达显性负性亚基，4）测试 代谢控制模型，并定义代谢物的意义，5）定义 使用基因筛选的调控途径的组成部分，并确定 候选基因的体内功能。 我们的基础研究 调节拟南芥氮同化作用的机制可能 提高作物氮肥利用率的意义 分子遗传学研究 此外，作为代谢信号， 发生在动物身上，对这一过程的了解可能更容易获得 在拟南芥中使用分子遗传策略。

We are using Arabidopsis thaliana as a model to identify the structural and regulatory genes controlling nitrogen assimilation into glutamine and glutamate using molecular, biochemical and genetic approaches. Nitrogen assimilation into these amino acids affects plant growth and ultimately seeds quantity and quality. Thus, our basic studies on the genes that control this process in plants relate indirectly to human and animal nutrition. The structural genes under investigation are: glutamine synthetase (GS), glutamate synthase (Fd-GOGAT or NADH-GOGAT), and glutamate dehydrogenase (GDH). The gene families for each enzyme in Arabidopsis contain independently regulated members encoding distinct isoenzymes. Despite decades of in vitro studies conducted in many species, the in vivo roles of GS, GOGAT and GDH isoenzymes in plants can only be conjectured. We propose to conduct the first systematic isolation of plant mutants specifically defective in each isoenzyme of GS, Fd-GOGAT, NADH-GOGAT or GDH, in a single species. We have shown that it is possible to isolate Arabidopsis mutants defective in nitrogen assimilatory genes using isoenzyme screens unbiased for growth phenotype. We propose a detailed characterization of the mutant progeny, to quantify the effects of the loss of a single isoenzyme on processes such as primary nitrogen assimilation, photorespiration, and nitrogen mobilization during seed set. This analysis will define the key and rate-limiting enzymes that control the efficiency of nitrogen use in plants. In addition we may also uncover mutants in regulatory genes. We have begun to investigate the mechanisms controlling the regulation of nitrogen assimilatory genes. Based on gene regulation studies, we have developed a "metabolic control" model that proposes these nitrogen assimilatory genes are regulated in response to the ratio of carbon to nitrogen metabolites in a plant. We propose genetic screens to uncover the components of this machinery and have in hand two putative regulatory genes. Our specific aims are: 1) Isolate additional Arabidopsis gdh mutants and mutants in chloroplastic GS2 by isoenzyme screening, 2) Characterize existing Arabidopsis mutants defective in one of two genes for Fd-GOGAT (gls1) and isolate mutants in the second gene, 3) Create mutants in cytosolic GS1 or NADH-GOGAT by expressing dominant-negative subunits in transgenic plants, 4) Test the metabolic control model and define the metabolites senses, 5) Define components of the regulatory pathway using genetic screens and define the in vivo function of candidate genes in hand. Our basic studies on the mechanisms that regulate nitrogen assimilation in Arabidopsis may have implications for improving nitrogen use in crops not amenable to such molecular-genetic studies. Furthermore, as metabolic signaling also occurs in animals, insights into this process may be more readily obtained using a molecular-genetic strategy in Arabidopsis.