REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

我们研究的总体目标是从分子水平上了解控制植物无机氮同化的调控过程。 这一过程在植物生长发育的调控中起着核心作用。在拟南芥中使用分子遗传学方法，我们确定了GS，GOGAT和GDH的同工酶，控制无机-N同化成Glu/Gln，用于运输氮细胞内和细胞间的关键氨基酸。 我们的研究表明，这些基因的表达受植物的代谢状态。 例如，光对GS的转录诱导可以在没有光的情况下被蔗糖模拟。此外，蔗糖诱导的GS表达可以被氨基酸拮抗，从而导致GS活性的抑制。 这使我们假设植物有一种机制来感知氨基酸的内部水平。 这将允许植物在内部氨基酸水平高时关闭无机氮的同化。 测试这一假设，定义它的机械，并确定其组成部分，是这次更新的重点。 为了实现这一目标，我们已经开始使用分子遗传学，细胞生物学和生物化学的方法来表征拟南芥中的氨基酸传感/信号传导组件。 我们的反向遗传学研究是由氨基酸传感在进化中是原始和保守的假设驱动的。 支持这一点的是，植物中氨基酸对GS表达的抑制机制让人联想到E.杆菌 此外，我们确定了一个植物的同源物的NTR组件，PII，并显示使用PII转基因植物，PII似乎在C：N传感在叶绿体中的GS调节中发挥作用，因为它在NTR。 N同化的氨基酸产物从叶绿体输出并运输到其它细胞，我们有证据表明，Glu，主要中间体，可能作为胞外信号。 在支持这一点，我们确定了推定的传感器的细胞外谷氨酸，植物同系物的动物谷氨酸受体（iGluRs）。 我们发现植物GluRs作为配体门控离子通道发挥作用，对GLR转基因植物的研究表明它们可能在光信号转导中发挥作用，这让人想起它们在视网膜和大脑中的对应物。 这些发现表明iGluRs来源于植物和动物分化之前存在的原始氨基酸信号传导机制。 我们建议利用这种进化保守性，并测试拟南芥（或GLR突变体，我们分离）是否可以用于生物测定药物治疗GluR相关疾病的人类。 我们还将使用正向遗传学方法分离拟南芥中的氨基酸传感/信号转导突变体，这些突变体可以识别这些进化上保守的氨基酸信号转导途径或新途径的组成部分。

The overall goal of our research is to gain a molecular understanding of the regulatory processes that control the assimilation of inorganic nitrogen in plants. This process plays a central role in the regulation of plant growth and development. Using molecular-genetic approaches in Arabidopsis, we identified isoenzymes of GS, GOGAT and GDH that control the assimilation of inorganic-N into Glu/Gln, key amino acids used to transport nitrogen within and between cells. Our studies indicate that expression of these genes is regulated by the metabolic status of the plant. For example, transcriptional induction of GS by light, can be mimicked by sucrose in the absence of light. Moreover, sucrose induction of GS expression can be antagonized by amino acids, which results in repression of GS activity. This led us to hypothesize that plants have a mechanism to sense internal levels of amino acids. This would allow a plant to turn off assimilation of inorganic-N when internal levels of amino acid are high. Testing this hypothesis, defining it mechanistically, and identifying components thereof, is the focus of this renewal. Towards this goal, we have begun to characterize amino acid sensing/signaling components in Arabidopsis using molecular-genetic, cell biological, and biochemical approaches. Our reverse genetic studies are driven by the hypothesis that amino acid sensing is primitive and conserved in evolution. In support of this, the repression of GS expression by amino acids in plants is mechanistically reminiscent of the Ntr system in E. coli. Moreover, we identified a plant homologue of an Ntr component, PII, and showed using PII transgenic plants that PII appears to play a role in C:N sensing in chloroplasts an in GS regulation, as it does in Ntr. The amino acid products of N-assimilation are exported from chloroplasts and transported to other cells, and we have evidence that Glu, the prinicple intermediate, may serve as an extracellular signal . In support of this, we identified putative sensors of extracellular Glu, plant homologues of animal glutamate receptors (iGluRs). We showed plant GluRs function as ligand-gated ion channels, and studies of GLR transgenic plants indicate they may play a role in light signal transduction, reminiscent of their counterparts in the retina and brain. These findings suggest iGluRs are derived from a primitive amino acid signaling mechanism that existed before plants and animals diverged. We propose to exploit this evolutionary conservation and test whether Arabidopsis (or GLR mutants we isolate) can be used in a bioassay for drugs to treat GluR-related diseases in humans. We will also use forward genetic approaches to isolate amino acid sensing/signaling mutants in Arabidopsis which may identify components of these evolutionarily conserved amino acid signaling pathways or novel pathways.