Plant mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities

Polyphenol oxidase (PPO) is an enzyme involved in the browning reaction of red clover leaves, when cut or crushed and exposed to air. PPO starts the browning process by oxidizing endogenous phenols to quinones, which contain electrophilic sites. These sites react with nucleophilic sites of other compounds such as proteins. The leaf tissue of two lines of red clover (cv. Milvus, a genotypic mutant with reduced PPO activity (LowPPO) and the wild-type, NormalPPO) were extracted in phosphate-citrate buffer, and a third treatment was prepared by extracting the LowPPO leaves in phosphate-citrate buffer plus 50 mM ascorbate to inhibit PPO activity (AscPPO). These extracts were compared over a 12 h time course in terms of proteolytic and lipolytic activity. Characterization of the tissues showed PPO activities of 9.11, 1.85 and 0 optical density g-1 fresh weight min-1, which were reflected in the extent of phenol (derived from quinones) binding to protein after 12 h incubation 102.3, 83.2 and 45.8 mg bound phenol g-1 protein (p <0.001) for NormalPPO, LowPPO and AscPPO, respectively. Proteolysis measured as free amino acids released into the incubation was significantly reduced (p <0.001) with increasing PPO activity, with values after the 12 h incubation of 0.03, 0.08 and 0.14 g g-1 protein for NormalPPO, LowPPO and AscPPO, respectively. Lipolysis, measured as the proportional decline in the membrane lipid polar fraction, was likewise reduced (p <0.001) with increasing PPO activity, with values after the 12 h incubation of 0.12, 0.20 and 0.22 for NormalPPO, LowPPO and AscPPO, respectively. Changes that occurred in the lipid fractions (polar fraction, diacylglycerol, triacylglycerol and free fatty acids) during the incubations are also reported and discussed. These results support the selection of forages high in PPO activity to reduce protein and lipid loses in silo and potentially in the rumen.