9319733 VanEtten The objective of this proposal is to further the understanding of the biosynthesis of (+) pisatin, the pterocarpanoid phytoalexin produced by pea. Pterocarpans exist as either of two stereoisomers in nature although the (-) isomer is more common. Recent results suggest that the biosynthesis of (+) pisatin and (-) pterocarpans share an intermediate that has a (-) chirality. Synthesis of (+) pisatin is also unusual in that the oxygen on one of its asymmetric carbons derives from H2O, whereas the oxygen on the analogous carbon of (-) pterocarpans is derived from O2. These observations imply that fundamentally different mechanisms establish the chirality and incorporation of oxygen in (+) pisatin compared to the (-) pterocarpans. Genes that encode the enzymes which produce the (-) chirality intermediate and the putative terminal step in pisatin biosynthesis have been cloned. These genes, and the enzymes they encode, will be used to resolve the origin of chirality in (+) pisatin by combining traditional tracer studies and biochemical analyses with the production of transgenic pea tissue lacking each of these enzymes. Such experiments should establish conclusively whether the enzymes are involved in pisatin biosynthesis and, if so, will subsequently be used to identify the novel steps in this pathway. Furthermore, this work will define an experimental system in which the biological function of this phytoalexin can be delineated. %%% Many plants synthesize antibiotics, called phytoalexins, in response to microbial infection. Production of these antimicrobial compounds is believed to be one mechanism used by plants to defend against disease. The objective of this grant proposal is to identify the biochemical steps involved in the production of the phytoalexin pisatin made by garden pea. We will use standard biochemical procedures in addition to making specific mutations in suspected steps to characterize the pathway. The information gained f rom this research may eventually be used to clone genes involved in the biosynthesis of phytoalexins. The cloned genes could be used to engineer new biosynthetic pathways in plants with the goal of augmenting the plant's disease resistance mechanisms. This research program is funded jointly by the Metabolic Biochemistry Program, Division of Molecular & Cellular Biosciences, and the Integrative Plant Biology Program, Division of Integrative Biology and Neuroscience. ***
