Submitted to: Gordon Research Conference Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 3/8/2005
Publication Date: 7/11/2005
Citation: Sullivan, M.L. 2005. Cloning and characterization of genes involved in o-diphenol biosynthesis in red clover. Gordon Research Conference on Plant Metabolic Engineering, July 10-15, 2005, Tilton School, Tilton, NH.
Technical Abstract: In red clover leaves, the oxidation of abundant o-diphenol caffeic acid derivatives by the enzyme polyphenol oxidase (PPO) prevents post-harvest protein loss, enhancing the quality of this forage when it is preserved by ensiling. Unfortunately, many important forage crops, such as alfalfa, lack significant levels of both PPO and o-diphenols in their leaves and stems and consequently experience significant proteolytic losses when ensiled. We are trying to develop strategies to minimize post-harvest protein losses in a variety of forage crops based on the PPO/o-diphenol system. In plants, caffeic acid and its derivatives are intermediates in the synthesis of monolignols, although in some plants like red clover these caffeic acid derivatives accumulate to relatively high levels in leaves. Understanding how red clover leaves synthesize such high levels of o-diphenols could greatly facilitate engineering similar pathways in crops like alfalfa. Work in Arabidopsis, basil, and other plant species is beginning to identify key enzymes involved in the biosynthesis of o-diphenols and related phenylpropanoid compounds. We have isolated cDNA clones from a red clover leaf cDNA library corresponding to some of these enzymes including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), p-coumaroyl CoA ligase (4CL), hydroxycinnomoyl transferase (HCT), and p-coumaric acid 3-hydroxylase (C3H). There is likely overlap of enzymes involved in the biosynthesis of o-diphenol PPO substrates and other important phenols (e.g. monolignols, phytoalexins), so we are currently characterizing the expression patterns of these genes in red clover as they relate to the accumulation of caffeic acid derivatives. The functional roles of the encoded enzymes in o-diphenol biosynthesis will be determined utilizing gene silencing and overexpression experiments in transgenic red clover and alfalfa, respectively.