Organization of Plant Metabolic Pathways and Subcellular Trafficking of Phytochemicals

植物的代谢途径是如何在细胞内组织的，以及植物的代谢物是如何在细胞内或细胞间运输的，我们所知甚少。这些都是对植物至关重要的问题，因为许多代谢物是有毒的，或者是在远离它们聚集的地方合成的。类黄酮色素及其代谢物的生物合成为研究大型多酶复合物在植物化学物质生物合成中的作用提供了一个有用的系统，并揭示了植物代谢物在细胞内运输的基本机制。本项目的两个主要目标是：1)研究玉米类黄酮生物合成两个分支对应的酶的亚细胞定位和关联；2)利用诱导型自荧光化合物探索植物代谢物的不同运输途径。为了完成第一个任务，gfp标记的与玉米类黄酮生物合成的两个主要分支相对应的酶的亚细胞定位将在该途径中存在和不存在其他酶的情况下进行研究。该通路的每个分支都可以使用特定的转录因子独立诱导。类黄酮生物合成酶物理相互作用的可能性也将被研究，使用互补的遗传和生化方法。总之，这些研究将确定与代谢途径的不同分支相对应的多酶复合物是否形成，以及这些复合物是否定位于细胞中的不同位置。对于第二个目标，先前鉴定的绿色和黄色自动荧光化合物将被分离并进行化学表征。绿色自荧光化合物积累在绿色荧光体（GFBs）结构中，这些结构以前在植物中不被识别，并与质膜融合。这些GFBs将被分离，并与它们相关的蛋白质将被表征。这些蛋白质将为研究这些小体的生物发生、货物运输和融合到特定膜结构域提供有用的标记。

Very little is known on how plant metabolic pathways are physically organized in the cell, or how plant metabolites are transported within or between cells. These are problems of central importance for plants, because many metabolites are toxic or are synthesized distantly from where they accumulate. The biosynthesis of flavonoid pigments and related metabolites provides a useful system to investigate the significance of the formation of large multi-enzyme complexes in the biosynthesis of phytochemicals, and to dissect the basic mechanisms by which plant metabolites are transported within cells. The two main goals of this project are: 1) to investigate the subcellular localization and association of enzymes corresponding to two branches of maize flavonoid biosynthesis, and 2) to explore the distinct trafficking pathways of plant metabolites using inducible auto-fluorescent compounds. To accomplish the first task, the subcellular localization of GFP-tagged enzymes corresponding to the two main branches of maize flavonoid biosynthesis will be examined in the presence and absence of the other enzymes in the pathway. Each branch of the pathway can be independently induced using specific transcription factors. The possibility that flavonoid biosynthetic enzymes physically interact will also be investigated, using complementary genetic and biochemical approaches. Together, these studies will determine whether multi-enzyme complexes corresponding to different branches of a metabolic pathway form, and whether these complexes localize to different places in the cell. For the second goal, previously identified green and yellow auto-fluorescent compounds will be isolated and chemically characterized. The green auto-fluorescent compounds accumulate in structures, the green fluorescent bodies (GFBs), previously not recognized in plants, and which fuse with the plasma membrane. These GFBs will be isolated and the proteins associated with them will be characterized. These proteins will provide useful markers to investigate the biogenesis, cargo transport and fusion of these bodies to particular membrane domains.