REGULATION OF AMINO ACID BIOSYNTHESIS GENES IN PLANTS

我们研究的总体目标是对控制植物吸收无机氮的调控过程有一个分子上的了解。这一过程在植物生长发育的调控中起着核心作用。利用分子遗传学方法，我们在拟南芥中鉴定了GS、GOGAT和GDH同工酶，这些同工酶控制无机氮到Glu/Gln的同化，Glu/Gln是细胞内和细胞间运输氮的关键氨基酸。我们的研究表明，这些基因的表达受到植物代谢状态的调节。例如，光对GS的转录诱导，可以在没有光的情况下被蔗糖模仿。此外，蔗糖诱导GS的表达可被氨基酸拮抗，从而抑制GS的活性。这导致我们假设，植物有一种机制来感知氨基酸的内部水平。这将允许植物在体内氨基酸水平较高时关闭对无机氮的同化。检验这一假设，机械地定义它，并确定它的组成部分，是这次更新的重点。为了实现这一目标，我们已经开始利用分子遗传学、细胞生物学和生物化学的方法对拟南芥中的氨基酸感应/信号成分进行研究。我们的反向遗传学研究是由氨基酸感知在进化中是原始的和保守的假设驱动的。为了支持这一点，氨基酸在植物中对GS表达的抑制在机械上使人想起大肠杆菌中的NTR系统。此外，我们鉴定了NTR组分PII的植物同源物，并使用PII转基因植株表明PII似乎在叶绿体的C：N感知和GS调节中发挥作用，就像它在NTR中所做的那样。N-同化的氨基酸产物从叶绿体输出并运输到其他细胞，我们有证据表明，主要的中间产物Glu可能作为细胞外信号。为了支持这一点，我们确定了可能的细胞外Glu传感器，即动物谷氨酸受体的植物同源物(IGluRs)。我们展示了植物GluRs作为配体门控离子通道的功能，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同行。这些发现表明，iGluRs起源于一种原始的氨基酸信号机制，这种机制在植物和动物分化之前就存在了。我们建议利用这一进化保守，并测试拟南芥(或我们分离的GLR突变体)是否可以用于治疗人类GluR相关疾病的药物的生物测试。我们还将使用正向遗传方法来分离拟南芥中的氨基酸感知/信号突变体，这可能会识别这些进化上保守的氨基酸信号通路或新途径的成分。

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.