Oxidation of ortho-diphenols in red clover with and without polyphenol oxidase (PPO) activity and their role in PPO activation and inactivation

Oxidation of phenol to quinone with its subsequent binding and complexing with protein in red clover (Trifolium pratense L.) when fed to ruminant livestock has been shown to improve nitrogen-use efficiency and the deposition of polyunsaturated fats into animal products. This oxidation has, almost exclusively, been attributed to the activity of the enzyme group polyphenol oxidases (PPOs) and, specifically, catecholase during conservation. However, during conservation, temporal inactivation of PPO occurs and oxidation of phenolic substrate may occur through other mechanisms not associated with PPO. T1 progeny of a cross between a red clover clone having normal levels of foliar PPO activity and a transgenic PPO-silenced red clover plant with undetectable levels of PPO activity were divided into two populations based on the presence of the silencing transgene and lack of measurable PPO activity (PPO−) or the absence of the silencing transgene and normal levels of PPO activity (PPO+). This material was subsequently used to determine the relevant extent of phenolic oxidation in the presence and absence of PPO activity under two damage regimes (heavy and light). PPO+ and PPO− material was passed through a garden shredder as the light-damaged regime, or frozen at −20°C as the heavy damaged regime. Material was left at room temperature and sampled at regular intervals for the determination of PPO enzyme activity (active and total), PPO activation (conversion from latent to active forms) and formation of protein-bound phenol (PBP) as a measure of oxidation. Experiments with red clover leaf extracts were carried out to determine the mechanism for the temporal activation and inactivation (loss of enzymatic activity) of PPO during wilting. PBP formation was evident in both PPO− and PPO+ treatments with the response further catalysed during the first 2 h by extent of damage that increased the activation of PPO, but which also resulted in a more rapid temporal inactivation of PPO. PBP formation, PPO activation and PPO inactivation were all shown to be mediated predominantly by quinone binding. A role of non-PPO oxidation in PBP formation in wilted red clover is reported, showing the importance of o-diphenolic substrate concentration and not just PPO activity to increase nitrogen use efficiency and the deposition of polyunsaturated fats into animal products