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 was directly related to Objective 3 of the parent project: Determine the biochemical/chemical/genetic basis for biological systems needed to inhibit degradation of forage proteins during harvest, storage and utilization to minimize nitrogen waste from dairy production systems (e.g., polyphenol oxidase, PPO/o-diphenols systems, other polyphenol systems including tannins). Enzymatic characterization of HCT2, a novel hydroxycinnamoyl-CoA:malate transferase from red clover, defined a new pathway whereby hydroxycinnamoyl-malate esters are produced in plants. This included both in vitro analyses (kinetics) and in vivo analysis (reverse genetics via gene silencing). Characterization of activities of a second enzyme, C3H, helped define the enzyme’s place in the biosynthetic pathway of phaselic acid, the major hydroxycinnamoyl-malate ester in red clover. It was shown that expression of the red clover HCT2 gene in alfalfa leads to accumulation of hydroxycinnamoyl-malate esters not usually present in this species, including small amounts of phaselic acid. Unlike red clover, the predominant hydroxycinnamoyl-malate esters that accumulate in alfalfa are p-coumaroyl-malate and feruloyl-malate. As part of a functional polyphenol oxidase (PPO) system in plants, o-diphenol substrates must also be produced. In red clover, the two main substrates are phaselic acid and clovamide. These substrates are formed by special enzymes (transferases) that combine caffeic acid with malate (phaselic acid) or L-dopa (clovamide). The transferase HCT2 (phaselic acid) was studied in detail to understand how it functions within red clover. Insertion of the HCT2 gene into alfalfa produced small amounts of phaselic acid, but produced larger amounts of p-coumarate and ferulate conjugates with malate. To optimize phaselic acid accumulation, we examined the roles of likely up- and down-stream genes in this biosynthetic pathway. Alfalfa plants expressing red clover HCT2 were also altered to down-regulate endogenous caffeoyl-CoA O-methyltransferase (CCOMT), an enzyme partially responsible for making feruloyl moieties in vivo. These plants showed increased accumulation of phaselic acid. HCT2-expressing alfalfa plants were also supertransformed with HCT1, an enzyme involved in making caffeoyl moieties in vivo. These plants had a slight, but significant, increase in phaselic acid accumulation. We are continuing this work and are in the process of assessing whether two red clover transferase genes related to HCT2 (HCT3 and HCT4) have roles in biosynthesis of phaselic acid or other caffeoyl derivatives in forage legumes by gene silencing in red clover and over-expression in alfalfa. These findings will serve as the foundation for recreating similar pathways in alfalfa and other forage crops that do not produce phaselic acid or other similar compounds that are part of a natural system of post-harvest protein protection in red clover. Adaptation of this system from red clover to alfalfa could save U.S. farmers 100 million dollars annually and reduce nitrogen emissions from animal production operations. This work may also have implications for plant resistance to abiotic stress, since hydroxycinnamoyl-malate esters such as phaselic acid may act as ultraviolet protectants in vivo and may afford some protection against ozone.