2010 Annual Report 1a.Objectives (from AD-416) The overall objective of the proposed reseach is to understand the enzymes and pathways responsible for red clover's ability to accumulate relatively high levels of o-diphenols, a crucial component of a natural system of protein protection for ensiled forages. This study will focus on what we believe are key enzymes in the pathway, hydroxycinnamoyl transferases (HCTs) and p-coumaroyl 3-hydroxylase (C3H) with three researchable objectives:. 1)Identify, isolate, and characterize red clover gene sequences encoding HCTs;. 2)Characterize red clover HCTs and a red clover C3H (CYP98A44) with respect to substrate specificity and reaction characteristics; and. 3)Establish the relevance of specific HCTs to biosynthesis and accumulation of specific o-diphenols in vivo. Insights gained from the proposed research will help achieve the longer range goal of recreating o-diphenol biosynthetic pathways in alfalfa and other forage crops. Expected deliverables include peer-reviewed publications, as well as enzymes and antibodies that will be useful tools for this research and to scientists studying secondary metabolism in a wide variety of plant species. 1b.Approach (from AD-416) The proposed objectives will be accomplished using several complementary approaches including genomics, molecular biology, biochemistry, and reverse genetics. HCT genes will be cloned using standard PCR-based approaches. Enzymatic properties of the various enzymes will be assessed by expression in E. coli and/or yeast. In vivo functions of the enzymes will be elucidated by overexpression in alfalfa and gene silencing in red clover. 3.Progress Report We conducted a detailed kinetic analysis of red clover HCT2, a novel hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase with various substrates. Additionally, several transgenic red clover lines down-regulated for HCT2 were generated and analyzed for content of phaselic acid (caffeoyl-malate), the major o-diphenol present in red clover. The down-regulated plants show greatly reduced or undetectable levels of phaselic acid. These results, along with the HCT2 kinetic data, establish a crucial role for HCT2 in phaselic acid biosynthesis and define a biochemical pathway not previously described for synthesis of phaselic acid and other hydroxycinnamoyl-malate esters. A manuscript detailing these findings is currently in preparation, to be submitted to a peer-reviewed journal by the end of fiscal year 2010. To assess whether HCT2 is sufficient for phaselic acid biosynthesis, transgenic alfalfa (which does not normally make phaselic acid or other hydroxycinnamoyl-malate esters) was made, expressing the red clover HCT2 gene. These plants accumulate p-coumaroyl-, feruloyl-, and caffeoyl-malate (phaselic acid). Experiments are in progress to understand why these alfalfa plants, in contrast to red clover, accumulate mostly p-coumaroyl- and feruloyl-malate instead of phaselic acid. Monitoring activities have included monthly meetings and frequent informal discussions of the progress of the project.