REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

我们正在使用拟南芥作为模型来识别结构 和控制氮同化为谷氨酰胺和 利用分子、生物化学和遗传方法研究谷氨酸。氮气 同化为这些氨基酸会影响植物的生长，并最终 种子的数量和质量。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 合成酶(GS)、谷氨酸合成酶(FD-GOGAT或NADH-GOGAT)，以及 谷氨酸脱氢酶(GDH)。每种酶的基因家族 拟南芥包含独立调节的成员，编码不同的 同工酶。尽管在许多国家进行了数十年的体外研究 物种，植物中GS、GOGAT和GDH同工酶的活体作用可以 只是猜想而已。我们建议进行第一次系统隔离 在GS、Fd-GOGAT、GOGAT、 NADH-GOGAT或GDH，在单一物种中。我们已经证明了这是可能的 分离氮素同化基因缺陷的拟南芥突变体 使用同工酶筛选，对生长表型无偏见。我们提出了一个 突变后代的详细特征，以量化影响 在初级氮等过程中单一同工酶的损失 结实过程中的同化、光呼吸和氮素动员。 这项分析将确定关键和限速酶 植物氮素利用的效率。此外，我们还可能揭开 调控基因中的突变。我们已经开始研究这种机制。 控制氮同化基因的调控。基于基因 在调节研究中，我们开发了一个“代谢控制”模型， 提出了这些氮同化基因是受调控以响应 植物中碳氮代谢产物的比率。我们建议 基因筛查以发现这种机器的组件并在 递给两个假定的调控基因。我们的具体目标是：1)隔离 通过以下方式在叶绿体GS2中增加拟南芥GDH突变体和突变体 同工酶筛选，2)现有拟南芥突变体的特性 Fd-GOGAT(GLS1)两个基因中的一个缺陷和分离突变株 第二个基因，3)在细胞质GS1或NADH-GOGAT中产生突变 在转基因植物中表达显性-负性亚基，4)检测 代谢控制模型和定义代谢物感觉，5)定义 使用基因筛选的调控途径的组件并定义 候选基因在体内的功能。我们对生物多样性的基础研究 调控拟南芥氮素同化的机制可能有 对改善作物氮素利用的影响不受此影响 分子遗传学研究。此外，由于代谢信号也 在动物身上发生，对这一过程的洞察可能更容易获得 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.