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

9514177种Cramer植物产生大量的异戊二烯类化合物(10,000种)，这些化合物在生长发育、电子传递、光合作用、抗氧化保护、繁殖和抗病方面发挥作用。从共同的中央途径中间体中适当地在空间和时间上产生特定的异戊二烯终产物构成了一个重大的调控挑战。这项拟议的研究将解决植物类异戊二烯途径是否被组织在不同的代谢单位或“代谢物”中，作为将途径流量引导到特定最终产物的机制。在茄科植物中，3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)和角鲨烯合成酶(角鲨烯合成酶)的HMG1亚型与甾醇的生物合成是协调调节的。来自各种实验方法的数据表明，除了在基因水平上的差异调控外，蛋白质-蛋白质相互作用和/或特定HMGRs的亚细胞定位在将中间体分配到特定异戊二烯类化合物的生产中可能是重要的。在这个项目中，将检验番茄HMG1和HMG2分别在生理上不同的途径或代谢产物中发挥作用，从而产生植物甾醇或植物保卫素的假设。HMGR同工酶水平将通过引入受结构性启动子或防御诱导启动子调控的hmg1或hmg2序列来改变。HMG1和HMG2转基因项目将被区别标记，以便于免疫定位研究和蛋白质稳定性评估。许多植物特有的异戊二烯类化合物在农业、工业和制药应用(如天然杀虫剂、橡胶和紫杉醇)方面具有重要价值。拟议中的实验将提供重要的新信息，对于理解类异戊二烯生物合成所涉及的复杂生化和细胞机制至关重要。

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.