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CROP PHYSIOLOGY & METABOLISM

Polyphenol Oxidase and o-Diphenols Inhibit Postharvest Proteolysis in Red Clover and Alfalfa

U.S. Dairy Forage Research Center, ARS-USDA, 1925 Linden Drive West, Madison, WI 53705

^* Corresponding author (mlsulliv{at}wisc.edu)

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 o-diphenols 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 µmol 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.

Abbreviations: bp, base pair • DM, dry matter • FW, fresh weight • nkat, nanokatal • NPN, nonprotein N • PPO, polyphenol oxidase • RNAi, RNA interference • TCA, trichloroacetic acid

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