Methionine Biosynthesis in Higher Plants

9728215 Leustek本项目的主题是高等植物蛋氨酸生物合成的发育调控，主要集中在关键酶胱硫氨酸合成酶(CS)，以及苏氨酸合成酶(TS)和高丝氨酸激酶(HK)。目的是研究模式维管植物拟南芥在发育过程中细胞和组织中Met的合成是如何调节的。使用报告基因方法确定CS启动子在所有细胞中的表达并不相同。在幼苗中，CS启动子在分生组织以及根和子叶的维管组织中高度活跃。在叶肉细胞中，该酶的活性显著降低。但在这种细胞类型中，它是由Thr&Amp；Lys(蛋氨酸合成抑制剂)处理的幼苗诱导的，并被Met喂养抑制。血管组织中的启动子活性不受这些处理的影响。在成熟植株中，CS启动子主要在叶片、花梗、花托和花药的维管组织中活性，而在角果和发育中的种子中不活性。用反义RNA抑制CS的表达会导致严重的生长缺陷，这种缺陷可以通过饲喂蛋氨酸部分恢复。这些植物的表型类似于A.thaliana，但DNA甲基化水平降低。该项目的下一阶段将集中于对报告基因结果的进一步探索。通过平行研究CS和TS的表达，将探索底物竞争在调节Met合成中的可能作用。两者都将通过原位mRNA和蛋白质的测量进行分析。Met及其代谢产物对细胞特异性表达的影响将通过两种方式进行研究：通过对被干扰Met合成的植物中的CS和TS的分析，以及通过对Met水平升高或S-腺苷蛋氨酸水平较低的拟南芥突变株的分析。我们将探索CS酶在植物细胞中的亚细胞定位，并对重组CS酶的动力学性质进行分析，以确定酶调节是否对Met的合成起到调节作用。最后，将克隆编码HK的cDNA，目的是研究HK酶，并研究HK的表达是如何调控的。蛋氨酸是所有生物密切参与新陈代谢的各个方面的重要氨基酸。动物不能产生蛋氨酸，它们必须从饮食中获得蛋氨酸。然而，大多数农作物都没有含有足够数量的这种关键营养物质。其中一个原因可能是植物精确地控制了它们所产生的蛋氨酸的数量。了解这种调控是如何发生的，可能会导致通过增加作物的蛋氨酸含量来提高作物的营养价值的方法。

9728215 Leustek The topic of this project is the developmental regulation of Met biosynthesis in higher plants, focusing primarily on the key enzyme cystathionine (-synthase (CS), and with some emphasis on threonine synthase (TS) and homoserine kinase (HK). The goal is to study how Met synthesis is regulated in cells and tissues during development in the model vascular plant Arabidopsis thaliana. Using a reporter gene method it was determined that expression from the CS promoter is not equal in all cells. In seedlings the CS promoter is highly active in meristems and the vascular tissues of roots and cotyledons. The activity is significantly lower in mesophyll cells. But in this cell type it is induced by treatment of seedlings with Thr & Lys (inhibitors of Met synthesis), and is repressed by Met-feeding. Promoter activity in the vascular tissues is unaffected by these treatments. In mature plants the CS promoter is active primarily in vascular tissue of leaves, the flower stalk, floral receptacle and anthers; and is inactive in siliques and developing seeds. Inhibition of CS expression with antisense-RNA causes a severe growth defect that can be partially restored by Met-feeding. The phenotype of these plants resembles that of A. thaliana with reduced levels of DNA methylation. The next phase of the project will center on further exploration of the reporter gene results. The possible role of substrate competition in regulating Met synthesis will be explored by studying CS and TS expression in parallel. Both will be analyzed by measurement of in situ mRNA and protein. The affect of Met and its metabolites on cell specific expression will be studied in two ways: through analysis of CS and TS in plants that have been treated with agents that perturb Met synthesis, and through analysis of A. thaliana mutant lines with elevated levels of Met or with lower levels of S-adenosylmethionine. The subcellular localization of the CS enzyme in plant cells will be explored and the kinetic propert ies of the recombinant CS enzyme will be analyzed in order to determine whether enzyme regulation plays any role regulating Met synthesis. Finally, the cDNA encoding HK will be cloned, the goal being to study the HK enzyme and to study how expression of HK is regulated. The amino acid methionine is of vital importance to all living organisms being intimately involved in every aspect of metabolism. Animals are unable to produce methionine and they must obtain it from their diet. Yet, most crop plants do not contain sufficient amounts of this key nutrient. One reason may be that plants control precisely the amount of methionine that they make. Understanding how this regulation takes place could lead to methods for improving the nutritional value of crops by increasing their methionine content.