ELUCIDATING THE ROLES OF HYDROXYCINNAMOYL TRANSFERASES AND P-COUMAROYL 3-HYDROXYLASES IN O-DIPHENOL BIOSYNTHESIS IN RED CLOVER
Project Number: 3655-21000-046-08
Start Date: Dec 01, 2008
End Date: Nov 30, 2011
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.
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.