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.
Among the accomplishments this year were the following: We conducted preliminary kinetic analysis of two hydroxycinnamoyl transferase (HCT) enzymes (HCT1 and HCT2) with various substrates that suggest HCT2 is involved in biosynthesis of the major red clover o-diphenol, phaselic acid. These data, along with gene sequences encoding the enzymes, have been published in a peer-reviewed journal (in press at the time of this report). Complete gene sequences for two additional HCTs have been cloned and sequenced, and the corresponding proteins have been produced in E. coli for characterization of the enzymatic activities. Several red clover lines down-regulated for HCT2 have been generated, and characterization of the plants has been initiated. Red clover comarate 3’-hydroxylase (C3H, CYP98A44) has been expressed in yeast, and its enzymatic activity has been characterized, providing insights into its role and position in the o-diphenol biosynthetic pathway. A manuscript detailing this work on C3H is being prepared and is expected to be submitted to a peer-reviewed journal by the end of fiscal year 2009. We have also characterized antiserum raised against red clover C3H. This antiserum recognizes related enzymes from other plant species and will be useful to scientists working on phenylpropanoid biosynthesis. Monitoring activities have included monthly meetings and frequent informal discussions of project progress.