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, previously called HCT2) 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.
Highlights
Hydroxycinnamoyl esters and amides can serve a variety of roles in plants
In arabidopsis and some other Brassicaceae, hydroxycinnamoyl-malate esters are synthesized via the action of a hydroxycinnamoyl-glucose hydroxycinnamoyl transferase (Lehfeldt et al, 2000), in common bean (Phaseolus vulgaris L.) (Sullivan, 2017) and red clover (Sullivan, 2009), we identified BAHD family (D’Auria, 2006) hydroxycinnamoylCoA:malate hydroxycinnamoyl transferases capable of transferring hydroxycinnamic acids from a coenzyme A (CoA) thiolester to malic acid (Figure 1)
Because polyphenol oxidase (PPO)-oxidizable compounds like caffeoyl-malate are an important part of a natural system of post-harvest protein protection in some legume forage crops (Sullivan and Hatfield, 2006; Sullivan and Zeller, 2012; Sullivan and Foster, 2013), we sought to determine whether alfalfa, an important forage crop lacking both foliar expression of PPO and accumulation of PPO substrates, could be engineered to accumulate caffeoyl-malate
Summary
Hydroxycinnamoyl esters and amides can serve a variety of roles in plants. One well characterized example is p-coumaroyl-shikimate, which is essentially a biosynthetic intermediate between p-coumaroyl-Coenzyme A (CoA) and caffeoyl-CoA by being the substrate for coumaroyl 3 hydroxylase (C3 H) (Schoch et al, 2006). Upon tissue breakdown and release of PPO, oxidation of these caffeic acid derivatives to the corresponding quinones, and the subsequent secondary reactions of the quinones can lead to the familiar browning reaction common in injured or post-harvest plant materials (Sullivan et al, 2004; Sullivan and Hatfield, 2006; Sullivan and Foster, 2013) These reactions appear to have a role in protecting plants against herbivory and pathogens (Thipyapong et al, 2004). We hypothesized that hydroxycinnamoyl-malate ester accumulation, caffeoyl-malate accumulation, might be higher in stems than in leaves for alfalfa expressing HMT and/or CCOMT RNAi. For representative plants analyzed above, stem hydroxycinnamoylmalate content was analyzed and compared to that of leaves, and differences were evaluated using the Wilcoxon sign-rank test.
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