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Submitted to: Phytochemical Society of North America Meeting and Newsletter
Publication Type: Abstract Only Publication Acceptance Date: 7/23/2015 Publication Date: 8/8/2015 Citation: Sullivan, M.L. 2015. Engineering alfalfa to accumulate useful caffeic acid derivatives and characterization of hydroxycinnamoyl-CoA transferases from legumes [abstract]. Phytochemical Society of North America. Paper No. S4-8. Interpretive Summary: Technical Abstract: Some forages crops, such as red clover, accumulate high levels of caffeic acid derivatives. Oxidation of these o-diphenols to quinones by endogenous polyphenol oxidases (PPOs) and the subsequent reactions of these quinones (probably with endogenous plant proteases) result in a significant reduction in post-harvest protein degradation. Reduced protein loss in forage crops should lead to more efficient nitrogen utilization in ruminant animal production systems, providing both economic and environmental benefits. One of the most commonly grown legume forage crops, alfalfa, does not accumulate PPO-utilizable phenolics. For this reason, we have been working to engineer biosynthetic pathways for accumulation of caffeic acid derivatives in this crop. Having identified a hydroxycinnamoyl-CoA:malate transferase (HMT) in red clover responsible for phaselic acid (caffeoyl-malate) accumulation, we transferred this gene to alfalfa. Leaves of the resulting transgenic alfalfa accumulated mostly p-coumaroyl and feruloyl-malate rather than the desired caffeoyl derivative. Simultaneous down-regulation of endogenous caffeoyl-CoA O-methyl transferase (CCOMT) resulted in increased accumulation of phaselic acid in alfalfa leaves and even higher levels in stems, possibly due to the relatively higher levels of HCT and C3H in this tissue that would normally be involved in the production of monolignols. The levels of phaselic acid produced in these plants should be sufficient to significantly impact post-harvest proteolysis. We are currently testing this via in vitro assays and small-scale ensiling experiments. We have also identified a gene from red clover encoding a hydroxycinnamoyl-CoA transferase responsible for clovamide (caffeoyl-L-DOPA amide) production, a gene from bean encoding a hydroxycinnamoyl-CoA transferase capable of transferring hydroxycinnamoyl moieties to tetrahydroxylated adipic acid isomers, and hydroxycinnamoyl-CoA transferase activities from bean and perennial peanut capable of transferring hydroxycinnamoyl moieties to malic and tartaric acid, respectively. Characterization of these enzymes should provide insights into the structure/function relationships of this class of BAHD acyltransferases. |
