Polyphenol oxidase and o-diphenols inhibit postharvest proteolysis in red clover and alfalfa

Many forages experience significant proteolytic losses when preserved by ensiling. Such losses in alfalfa (Medicago sativa L.) are especially high, with degradation of 44 to 87% of the forage protein to nonprotein N (NPN). In contrast, red clover (Trifolium pratense L.) has up to 90% less proteolysis during ensiling. Here we demonstrate that the combination of polyphenol oxidase (PPO) and o-diphenol PPO substrates, both abundantly present in red clover, is responsible for postharvest proteolytic inhibition in this forage crop. Proteolysis in red clover leaf extracts increased nearly fivefold when endogenous odiphenols were removed by gel filtration but returned to starting levels by adding back an exogenous o-diphenol. Proteolysis in leaf extracts of red clover plants silenced for PPO expression was dramatically increased compared to control plants. Leaf extracts of transgenic alfalfa expressing a red clover PPO gene showed a nearly fivefold o-diphenol-dependent decrease in proteolysis compared to those of control alfalfa. We also demonstrate that PPO levels 10- to 20-fold lower than those typically found in red clover are sufficient for proteolytic inhibition, that as little as 0.25 mu mol o-diphenol mg(-1) protein has a substantial impact on proteolysis, that a wide variety of o-diphenols are functional substrates in proteolytic inhibition, and that proteolysis is reduced for PPO-expressing alfalfa in small-scale ensiling experiments. Together, these results indicate that PPO and o-diphenols can be an effective treatment to prevent protein loss in ensiled forage crops.