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 PPOgene showed a nearly fivefold o-diphenol– dependent decrease in proteolysis compared to those of control alfalfa. We also demonstrate that PPO levels 10to 20-fold lower than those typically found in red clover are sufficient for proteolytic inhibition, that as little as 0.25 mmol o-diphenol mg 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. ENSILING is a popular method of preserving crops, especially in regions with humid climates. During harvest and the early stages of ensiling, disruption of plant tissues releases cellular proteases resulting in degradation of forage protein to small peptides and amino acids. As fermentation by lactic acid bacteria progresses, silage pH drops and the rate of proteolysis decreases. Unfortunately, substantial conversion of protein to NPN can take place before silage pH becomes sufficiently acidic to inhibit proteolysis. Because dairy cows and other ruminant animals poorly utilize excess NPN, loss of true protein during ensiling results in economic losses to farmers. Such losses for alfalfa alone approach $100 million annually in the United States based on an estimated loss of $70 per hectare (Rotz et al., 1993) and USDA reports of acreage devoted to alfalfa haylage production in 2004 (NASS, 2005). Further, ruminants excrete much of this NPN as urea resulting in increasing N burdens on the environment. For alfalfa, proteolytic losses during ensiling are especially high with degradation of 44 to 87% of the forage protein (Albrecht and Muck, 1991; Jones et al., 1995c; Muck, 1987; Papadopoulos and McKersie, 1983). Red clover, a legume forage with protein content similar to alfalfa, experiences up to 90% less proteolysis than alfalfa when ensiled. Jones et al. (1995a, 1995c) found that red clover tissues do not differ substantially from those of alfalfa in terms of proteolytic activities present and postharvest proteolytic inhibition in red clover is O2–dependent and involves a heat labile factor. These, along with the observations that PPO o-diphenol substrates disappear concomitantly with proteolytic inhibition (Hatfield and Muck, 1999) and that alfalfa has little if any foliar PPO or o-diphenols (Jones et al., 1995b; Sullivan et al., 2004a) suggested that the proteolytic inhibition seen in ensiled red clover is due to the action of PPO on endogenous o-diphenols. Recently, we cloned several red clover PPO genes, including the major PPO gene expressed in red clover leaves and showed that active red clover PPO enzyme could be expressed in transgenic alfalfa (Sullivan et al., 2004a). We have recently generated red clover lacking foliar PPO by expression of double-stranded PPO RNA in transgenic plants (RNAi) (Wang and Waterhouse, 2002). Using transgenic PPO-expressing alfalfa, as well as red clover plants silenced for foliar PPO expression, we have now been able to demonstrate that postharvest proteolytic inhibition in red clover is due to the action of PPO on o-diphenols and have begun to define the critical parameters of this natural system of protein protection. MATERIALS AND METHODS Plant Materials and Transformation A red clover genotype (lab designation ‘‘PPO’’) (Sullivan et al., 2004a) selected from a population of WI-2 (lot C136) germplasm (Smith andMaxwell, 1980)was used in several experiments as indicated. For experiments with transgenic red clover, two highly regenerable genotypes (designated NEWRC27 and NEWRC30) derived from a population of NEWRC germplasm (Smith and Quesenberry, 1995) were used. These red clover genotypes were transformed via Agrobacterium-mediated transformation using a modification (Sullivan and Quesenberry, 2006) of the method of Quesenberry et al. (1996). Control and red clover PPO1-expressing transgenic alfalfa, generated by Agrobacterium-mediated transformation of a highly regenerable clone of Regen-SY (Bingham, 1991), have been previously described (Sullivan et al., 2004a). All experiments utilizing transgenic plants were performed with primary M.L. Sullivan and R.D. Hatfield, U.S. Dairy Forage Research Center, ARS-USDA, 1925 Linden Drive West, Madison, WI 53705. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Received 15 June 2005. *Corresponding author ([email protected]). Published in Crop Sci. 46:662–670 (2006). Crop Physiology & Metabolism doi:10.2135/cropsci2005.06-0132 a Crop Science Society of America 677 S. Segoe Rd., Madison, WI 53711 USA Abbreviations: bp, base pair; DM, dry matter; FW, fresh weight; nkat, nanokatal; NPN, nonprotein N; PPO, polyphenol oxidase; RNAi, RNA interference; TCA, trichloroacetic acid. R e p ro d u c e d fr o m C ro p S c ie n c e . P u b lis h e d b y C ro p S c ie n c e S o c ie ty o f A m e ri c a . A ll c o p y ri g h ts re s e rv e d . 662 Published online February 1, 2006