Isoprenoid Pathway Partitioning: HMGR Isoforms and Sterol vs. Phytoalexin Synthesis

植物产生大量的类异戊二烯化合物（1万种），这些化合物在生长发育、电子传递、光合作用、抗氧化保护、繁殖和抗病等方面发挥作用。从常见的中枢通路中间体中适当的空间和时间生产特定的类异戊二烯终产物提出了重大的监管挑战。拟议的研究将解决植物类异戊二烯途径是否组织在不同的代谢单位或“代谢物”中，作为指导途径通量进入特定最终产物的机制。在茄属植物中，3-羟基-3甲基戊二酰辅酶a还原酶（HMGR，产生甲羟戊二酸的限速酶）和角鲨烯合成酶（使法尼基中间体合成甾醇的关键分支酶）的HMG1异构体在甾醇生物合成过程中受到协调调节。来自多种实验方法的数据表明，除了基因水平上的差异调控外，特异性hmgr的蛋白-蛋白相互作用和/或亚细胞定位可能对将中间体分配到特定类异戊二烯的生产中很重要。在这个项目中，将测试番茄HMG1和HMG2在生理上不同的途径或代谢中分别导致植物甾醇或植物抗毒素产生的假设。通过引入hmg1或hmg2序列来改变HMGR同工酶的水平，这些序列由组成型启动子或防御诱导型启动子调控。HMG1和HMG2转基因项目将进行差异标记，以促进免疫定位研究和蛋白质稳定性评估。许多植物特有的类异戊二烯具有重要的农业、工业和制药应用价值（例如，天然农药、橡胶和紫杉醇）。所提出的实验将为理解类异戊二烯生物合成中涉及的复杂生化和细胞机制提供重要的新信息。

9514177 Cramer Plants produce a vast array of isoprenoid compounds (10,000) that function in growth and development, electron transport, photosynthesis, anti-oxidant protection, reproduction, and disease resistance. The appropriate spatial and temporal production of specific isoprenoid endproducts from common central-pathway intermediates poses a significant regulatory challenge. The proposed reserch will address whether the plant isoprenoid pathway is organized in distinct metabolic units or "metabolons" as a mechanism to direct pathway flux into specific endproducts. In Solanaceous plants, the HMG1 isoform of 3-hydroxy-3methylglutaryl CoA reductase (HMGR, the rate-limiting enzyme producing mevalonic acid) and squalene synthetase (the key branch enzyme committing farnesyl intermediates to sterol synthesis) are coordinately regulated in association with sterol biosynthesis. Data from a variety of experimental approaches suggest that, in addition to differential regulation at the gene level, protein-protein interaction and/or subcellular localization of specific HMGRs may be important in partitioning intermediates into production of specific isoprenoids. In this project, the hypothesis that tomato HMG1 and HMG2 function in physiologically distinct pathways or metabolons leading to production of phytosterols or phytoalexins, respectively, will be tested. HMGR isozyme levels will be altered by introducing hmg1 or hmg2 sequences regulated by either constitutive or defense-inducible promoters. The HMG1 and HMG2 transgene projects will be differentially tagged to facilitate immunolocalization studies and assessment of protein stability. Many plantspecific isoprenoids have significant value for agricultural, industrial, and pharmaceutical applications (e.g., natural pesticides, rubber, and taxol). The proposed experiments will provide significant new information critical for understanding the complex biochemical and cellular mechanisms involved in isoprenoid biosynthesis.