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
This project directly addresses Subobjective 3.3 of the in-house parent project: to determine the feasibility of altering the alfalfa phenolic biosynthesis pathway to produce appropriate o-diphenol substrates for polyphenol oxidase. We previously identified a red clover gene, HCT2, crucial for biosynthesis of the polyphenol oxidase (PPO) substrate phaselic acid (caffeoyl-malate) in red clover, and showed that its expression in alfalfa results in accumulation of hydroxycinnamoyl-malate esters not normally present in that species. These esters include low levels of phaselic acid along with larger amounts of the related compounds, p-coumaroyl- and feruloyl-malate. We hypothesized that down-regulation of alfalfa’s endogenous caffeoyl-CoA O-methyltransferase (CCOMT) enzyme, which can convert caffoyl moieties to feruloyl moieties, results in enhanced production of phaselic acid. Alfalfa plants were made that both expressed the red clover transferase gene and were down-regulated for endogenous CCOMT. The resulting plants had substantially enhanced levels of phaselic acid, and surprisingly, feruloyl-malate, compared to plants expressing the red clover transferase only. Alfalfa caffeic acid O-methyltransferase (COMT; another enzyme capable of converting caffeic acid to ferulic acid) was also characterized, and we have begun to examine the role of a second red clover transferase in the differential accumulation of the various hydroxycinnamoyl-malate esters. It is anticipated one or more manuscripts detailing these results will be prepared for publication in peer-reviewed journals in 2012. These experiments are encouraging and have identified other potentially important enzymes in the biosynthetic pathway. This suggests it should be possible to make useful levels of PPO substrates in forage crops such as alfalfa. We have also begun a collaboration with an ARS scientist at the Plant Science Research Unit in Raleigh, NC to examine the role that hydroxycinnamoyl-malate esters may play in protecting plants from abiotic stresses such as ultraviolet light and ozone. Monitoring activities have included monthly meetings and frequent informal discussions of the progress of the project.