Engineering alfalfa to produce 2-O-caffeoyl-L-malate (phaselic acid) for preventing post-harvest protein loss via oxidation by polyphenol oxidase

Authors: Sullivan, Michael; GREEN, HEATHER - University Of Wisconsin; VERDONK, JULIAN - Wageningen University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/9/2020
Publication Date: 1/13/2021
Citation: Sullivan, M.L., Green, H.A., Verdonk, J.C. 2021. Engineering alfalfa to produce 2-O-caffeoyl-L-malate (phaselic acid) for preventing post-harvest protein loss via oxidation by polyphenol oxidase. Frontiers in Plant Science. 11. Article 610399. https://doi.org/10.3389/fpls.2020.610399.
DOI: https://doi.org/10.3389/fpls.2020.610399

Interpretive Summary: Degradation of protein to peptides and amino acids in ensiled forages such as alfalfa is a problem because excess nonprotein nitrogen is poorly utilized by ruminant animals. Reducing protein losses just 10% could save U.S. farmers $ 200 to 400 million annually and reduce release of excess nitrogen into the environment. We previously identified a system of protein protection in red clover consisting of the enzyme polyphenol oxidase (PPO) and PPO-oxidizable o-diphenols. Since the widely grown forage crop alfalfa lacks both PPO and o-diphenols, adapting the red clover system to alfalfa requires providing both components, either by physically adding them or by genetically modifying the forages to make them. Here, we show that transgenic expression in alfalfa of a red clover enzyme (HMT [hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase]) involved in making one of its major o-diphenolic compounds, phaselic acid, along with downregulation of an alfalfa gene (CCOMT [caffeoyl-CoA O-methyltransferase]) results in accumulation of phaselic acid to levels comparable to those found in red clover. In reconstitution experiments, this level of phaselic acid could reduce protein degradation in the presence of the PPO enzyme. These results show the red clover system of protein protection can be adapted to alfalfa as well as demonstrating the high degree to which alfalfa specialized metabolism can be redirected to make useful compounds.

Technical Abstract: Many plants accumulate high levels of hydroxycinnamoyl esters and amides in their tissues, presumably to protect against biotic and abiotic stress. Red clover (Trifolium pretense) leaves accumulate high levels (5-15 mmol/kg fresh weight [FW]) of caffeic acid derivatives, including phaselic acid (2-O-caffeoyl-L-malate). Oxidation of caffeoyl-malate by an endogenous polyphenol oxidase (PPO) has been shown to help preserve forage protein after harvest and during storage as silage, which should improve N use efficiency in dairy and other ruminant production systems. The widely grown forage alfalfa lacks both PPO and PPO substrates and experiences substantial loss of protein following harvest. We previously identified a hydroxycinnamoyl-coenzyme A (CoA):malate hydroxycinnamoyl transferase (HMT) responsible for phaselic accumulation in red clover. With the goal of producing PPO-oxidizable compounds in alfalfa to help preserve forage protein, we expressed red clover HMT in alfalfa. Leaves of these alfalfa accumulated mainly p-coumaroyl- and feruloyl-malate (up to 1.26 and 0.25 mmol/kg FW, respectively). Leaves of HMT-expressing alfalfa supertransformed with an RNA interference (RNAi) construct to silence endogenous caffeoyl-CoA acid O-methyltransferase (CCOMT) accumulated high levels of caffeoyl-malate, as well as the p-coumaroyl and feruloyl esters (up to 2.16, 2.08, and 3.13 mmol/kg FW, respectively). Even higher levels of caffeoyl- and p-coumaroyl-malate were seen in stems (up to 8.37 and 3.15 mmol/kg FW, respectively). This level of caffeoyl-malate accumulation was sufficient to inhibit proteolysis in a PPO-dependent manner in in vitro experiments, indicating that the PPO system of post-harvest protein protection can be successfully adapted to alfalfa.