Cloning of an alfalfa polyphenol oxidase gene and evaluation of its potential in preventing postharvest protein degradation

BACKGROUND: Ensiling forages often leads to degradation of protein to non-protein nitrogen (NPN), which is poorly utilized by ruminants. Postharvest protein degradation is especially high in alfalfa (Medicago sativa L.). In contrast, red clover (Trifolium pratense L.) has up to 90% less protein loss during ensiling due to polyphenol oxidase (PPO) forming o-quinones from endogenous o-diphenols and subsequent binding of o-quinones to cytoplasmic proteins. Here we determined whether an endogenous PPO might be exploited for postharvest protein protection in alfalfa. RESULTS: We isolated an alfalfa PPO gene (MsPPO1) that shares limited sequence identity (70-72%) with red clover PPO genes. MsPPO1 is expressed primarily in flowers and developing seed pods, but not in leaves or stems. Expression of MsPPO1 from a strong constitutive promoter in transgenic alfalfa results in accumulation of PPO transcripts in leaves, but little enzyme activity is detected using a variety of o-diphenol substrates unless assayed in the presence of sodium dodecyl sulfate (SDS). Under this SDS-activated condition, preference of MsPPO1 for tested substrates is catechol ≥ (-)-epicatechin > caffeic acid. PPO activity in unactivated MsPPO1-alfalfa extracts is sufficient to inhibit proteolysis in the presence of catechol, but not caffeic acid or (-)-epicatechin. Inhibition is less than in extracts of alfalfa expressing the red clover PPO1 gene. CONCLUSION: Endogenous alfalfa PPO, even if expressed in appropriate target tissues, would be less effective at preventing proteolytic losses in ensiled forages than red clover PPO.