Organization of Plant Metabolic Pathways and Subcellular Trafficking of Phytochemicals

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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.