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United States Department of Agriculture

Agricultural Research Service

Research Project: ELUCIDATING THE ROLES OF HYDROXYCINNAMOYL TRANSFERASES AND P-COUMAROYL 3-HYDROXYLASES IN O-DIPHENOL BIOSYNTHESIS IN RED CLOVER Project Number: 3655-21000-046-08
Project Type: Reimbursable

Start Date: Dec 01, 2008
End Date: Nov 30, 2011

Objective:
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.

Approach:
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.

Last Modified: 9/2/2014
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