Red clover HDT, a BAHD hydroxycinnamoyl-coenzyme A:L-3,4-dihydroxyphenylalanine(L-DOPA) hydroxycinnamoyl transferase that synthesizes clovamide and other N-hydroxycinnamoyl-aromatic amino acid amides

Authors: Sullivan, Michael; KNOLLENBERG, BENJAMIN - Pennsylvania State University

Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/13/2021
Publication Date: 11/9/2021
Citation: Sullivan, M.L., Knollenberg, B.J. 2021. Red clover HDT, a BAHD hydroxycinnamoyl-coenzyme A:L-3,4-dihydroxyphenylalanine(L-DOPA) hydroxycinnamoyl transferase that synthesizes clovamide and other N-hydroxycinnamoyl-aromatic amino acid amides. Frontiers in Plant Science. 12. Article 727461. https://doi.org/10.3389/fpls.2021.727461.
DOI: https://doi.org/10.3389/fpls.2021.727461

Interpretive Summary: Red clover accumulates high levels of two hydroxycinnamic acid compounds, phaselic acid and clovamide. These compounds likely serve important functions in protecting the plant from environmental stresses, such as exposure to ultraviolet light or ozone, or attack by insects or pathogens. Besides the likely role in protecting the plant from stress and pathogens, these compounds also work in red clover with an enzyme called polyphenol oxidase (PPO) to prevent protein breakdown during harvest and storage. Such protein losses in forage crops may cost U.S. farmers $1 to 2 billion annually and result in the release of nitrogenous waste into the environment due to poor utilization of degraded protein by dairy cattle. Because many important forages, such as alfalfa, lack both the PPO enzyme and the hydroxycinnamic acid compounds that work with PPO, understanding how plants such as red clover make these elements could help adapt the PPO-based protein protection system to other forage crops like alfalfa. Here, we identified and characterized a gene and the corresponding enzyme from red clover that is responsible for clovamide accumulation. Genetically modified alfalfa expressing the red clover gene made compounds related to clovamide, a promising first step in making the desired hydroxycinnamoyl compounds in alfalfa. Also, this work adds to the knowledge base of the genes and corresponding enzymes involved in the production of these important hydroxycinnamic acid compounds in plants. Such information will allow easier determination of the function of similar genes, and may eventually allow creation of rationally designed genes whose corresponding enzymes produce new or desired hydroxycinnamoyl compounds in plants.

Technical Abstract: Red clover leaves accumulate high levels (up to 1 to 2% of dry matter) of two caffeic acid derivatives: phaselic acid (2-O-caffeoyl-L-malate) and clovamide (N-caffeoyl-L-3,4-dihydroxyphenylalanine [L-DOPA]). These likely play roles in protecting the plant from biotic and abiotic stresses, but can also help preserve protein during harvest and storage of the forage via oxidation by an endogenous polyphenol oxidase. We previously identified and characterized HMT, a hydroxycinnamoyl-coenzyme A (CoA): malate hydroxycinnamoyl transferase from red clover. Here, we identified a hydroxycinnamoyl-CoA:L-DOPA hydroxycinnamoyl transferase (HDT) activity in unexpanded red clover leaves. Silencing of the previously cloned HMT gene reduced both HMT and HDT activities in red clover, even though the HMT enzyme lacks HDT activity. A combination of PCR with degenerate primers based on BAHD hydroxycinnamoyl-CoA transferase sequences and 5' and 3' rapid amplification of cDNA ends was used to clone two nearly identical cDNAs from red clover. When expressed in Escherichia coli, the encoded proteins were capable of transferring hydroxycinnamic acids (p-coumaric, caffeic, or ferulic) from the corresponding CoA thiolesters to the aromatic amino acids L-Phe, L-Tyr, L-DOPA, or L-Trp. Kinetic parameters for these substrates were determined. Stable expression of HDT in transgenic alfalfa resulted in foliar accumulation of p-coumaroyl- and feruloyl-L-Tyr that are not normally present in alfalfa, but not derivatives containing caffeoyl or L-DOPA moieties. Transient expression of HDT in Nicotiana benthamiana resulted in the production of caffeoyl-L-Tyr, but not clovamide. Coexpression of HDT with a tyrosine hydroxylase resulted in clovamide accumulation, indicating the host species’ pool of available amino acid (and hydroxycinnamoyl-CoA) substrates likely plays a major role in determining HDT product accumulation in planta. Finally, that HDT and HMT proteins share a high degree of identity (72%), but differ substantially in substrate specificity, is promising for further investigation of structure-function relationships of this class of enzymes, which could allow the rational design of BAHD enzymes with specific and desirable activities.