Data_Sheet_1_Engineering Alfalfa to Produce 2-O-Caffeoyl-L-Malate (Phaselic Acid) for Preventing Post-harvest Protein Loss via Oxidation by Polyphenol Oxidase.PDF

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.

许多植物会在其组织中积累高水平的羟基肉桂酸酯与酰胺类化合物，推测其功能为抵御生物与非生物胁迫。红三叶（Trifolium pretense）叶片可积累5–15 mmol/kg鲜重（fresh weight, FW）的咖啡酸衍生物，其中包括phaselic酸（2-O-咖啡酰-L-苹果酸）。内源多酚氧化酶（polyphenol oxidase, PPO）对咖啡酰苹果酸的氧化作用已被证实，可在收获后及青贮储存过程中维持饲草蛋白含量，进而提升乳业及其他反刍动物生产系统的氮利用效率。
广泛种植的饲草作物苜蓿既缺乏PPO及其底物，收获后会出现大量蛋白流失。我们此前已在红三叶中鉴定出一种负责phaselic酸积累的羟基肉桂酰辅酶A（hydroxycinnamoyl-coenzyme A, CoA）：苹果酸羟基肉桂酰转移酶（HMT，此前被称为HCT2）。为实现在苜蓿中生成可被PPO氧化的化合物以维持饲草蛋白含量的目标，我们将红三叶的HMT基因在苜蓿中进行了异源表达。
该转基因苜蓿的叶片主要积累对香豆酰苹果酸与阿魏酰苹果酸，最高含量分别可达1.26 mmol/kg鲜重与0.25 mmol/kg鲜重。
对表达HMT的苜蓿进一步通过RNA干扰（RNA interference, RNAi）载体进行超转化，以沉默内源咖啡酰辅酶A O-甲基转移酶（caffeoyl-CoA acid O-methyltransferase, CCOMT）后，其叶片不仅积累了高水平的咖啡酰苹果酸，还同时积累了对香豆酰与阿魏酰苹果酸酯，三者最高含量分别可达2.16、2.08与3.13 mmol/kg鲜重。
苜蓿茎中咖啡酰苹果酸与对香豆酰苹果酸的积累水平更高，最高分别可达8.37 mmol/kg鲜重与3.15 mmol/kg鲜重。
体外实验表明，该水平的咖啡酰苹果酸积累足以以PPO依赖的方式抑制蛋白水解，这证实收获后蛋白保护的PPO系统可成功适配至苜蓿中。