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Title:   Author  item
                                            Sullivan, Michael
  Submitted to: North American Alfalfa Improvement Conference
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 6/24/2010
                                    Publication  Date: N/A
                                    Citation: N/AInterpretive Summary:
                                Technical Abstract: In red clover, post-harvest oxidation of o-diphenol caffeic acid derivatives to o-quinones by an endogenous polyphenol oxidase (PPO) prevents breakdown of forage protein during storage (1). Agronomically important forages like alfalfa lack both PPO and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses ($100 million/yr in the U.S. alone) and release of excess nitrogen into the environment. Understanding how red clover is able to synthesize and accumulate o-diphenols will help in development of forages that take advantage of this natural system of protein protection. Our laboratory has focused on biosynthesis of phaselic acid (caffeoyl-malate), a major o-diphenol in red clover leaves that accumulates to approximately 0.5% of dry matter. Using a bioinformatics approach, we identified and cloned a novel hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase, HCT2, from red clover (2). Detailed kinetic analyses indicate the enzyme can transfer p-coumaroyl, caffeoyl-, and feruloyl moieties from their corresponding CoA derivatives to malic acid. HCT2 can carry out the reverse reaction (formation of hydroxycinnamoyl-CoA from hydroxycinnamoyl-malate) for p-coumaroyl-malate, but not for phaselic acid. An apparent lack of a 3'-hydroxylating activity capable of converting p-coumaroyl-malate to phaselic acid in red clover suggests that, in vivo, phaselic acid is formed by transfer of caffeoyl moieties to malic acid by HCT2 (3). To demonstrate the in vivo role of HCT2 in phaselic acid biosynthesis, red clover was transformed with a hairpin RNAi gene construct to silence HCT2 expression. Analysis of eleven independent transformants and their three corresponding wild type controls demonstrated a significant and substantial correlation between HCT2 mRNA levels and phaselic acid accumulation (P<0.001). In several of the HCT2-silenced plants, phaselic acid and p-coumaroyl-malate accumulated to nearly undetectable levels compared to wild type controls. These reductions resulted in easily observable phenotypes including reduced PPO-mediated browning and a reduction in blue epidermal fluorescence under UV-light. We also transformed alfalfa with the red clover HCT2 gene. Leaves of these plants have HCT2 activity approaching that of wild type red clover and accumulate p-coumaroyl- and feruloyl-malate and, to a lesser extent, phaselic acid. We are currently using these plants to better understand the phaselic acid biosynthetic pathway and as a starting point for optimization of phaselic acid accumulation in alfalfa.

1. Sullivan, M.L., and Hatfield, R.D. 2006. Crop Science 46:662.
2. Sullivan, M.L. 2009. Plant Physiology 150:1866.
3. Sullivan, M.L., and Zarnowski, R. 2010. Planta 231:319.


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