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Title:   Author  item
                                            LEE, MICHAEL - Aberystwyth Universityitem
                                            TWEED, JOHN - Aberystwyth Universityitem
                                            Sullivan, Michael
  Submitted to: Grass and Forage Science
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 3/21/2012
                                    Publication  Date: 1/1/2013
                                    Citation: Lee, M.R., Tweed, J.K., Sullivan, M.L. 2013. Oxidation of ortho-diphenols in red clover with and without polyphenol oxidase (PPO) activity and their role in PPO activation and inactivation. Grass and Forage Science. 68(1):83-92.Interpretive Summary: Polyphenol oxidase (PPO), an enzyme present in the tissues of many plants, carries out oxidation of compounds called o-diphenols. Secondary reactions of these oxidized o-diphenols with proteins and other cellular constituents are often associated with browning seen in fresh produce. Leaves of red clover have particularly high levels of PPO and o-diphenol PPO substrates. Previous studies have demonstrated that in red clover, these high PPO and substrate levels have beneficial effects when the forage is used as feed for ruminant animals. First, PPO prevents protein losses during storage. Second, the protein is less degraded by rumen microorganisms and more efficiently utilized by the animal. These have positive economic (farmers can spend less on protein supplements) and environmental (more efficiently utilized protein means less nitrogen waste is released into the environment via animal waste) impacts. Finally, PPO present in red clover prevents breakdown of fatty acids during storage of the forage and by microbes in the animal’s rumen. It also prevents conversion of polyunsaturated fats to saturated fats by microbes in the animal's rumen. Together, these lead to increased levels of beneficial polyunsaturated fatty acids in the resulting animal products (milk and meat), although exactly how PPO prevents fatty acid breakdown and conversion from polyunsaturated to saturated fatty acids by rumen microbes is not clear. The experiments presented here address how extent of post-harvest processing affects PPO activity and oxidation of o-diphenols. Tissue damage resulted in short-term increases in PPO activity that eventually declined to nearly undetectable levels. These activity changes were the result of oxidized o-diphnols whose initial reaction with the PPO protein stimulates enzyme activity. However, subsequent chemical reactions of these PPO-bound o-diphenols result in inactivation of the enzyme. Experiments with red clover plants lacking PPO activity found that even without PPO, o-diphenols become oxidized and react with cellular proteins. Greater tissue damage results in more rapid oxidation of the o-diphenols. These results provide insights into the post-harvest activation of PPO and the oxidation reactions of o-diphenols with and without PPO activity, and should help develop strategies to take best advantage of this natural system of protein and lipid protection.
                                Technical Abstract: Oxidation of phenol to quinone with its subsequent binding and complexing with protein in red clover (Trifolium pretense L.) to be fed to ruminant livestock has been shown to improve nitrogen use efficiency and improve the deposition of polyunsaturated fats into animal products. This oxidation has almost exclusively been attributed to the activity of the enzyme group, polyphenol oxidases (PPO), during conservation. However, during conservation PPO has been shown to be inactivated, and oxidation of phenolic substrate could 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 lightly damaged or frozen at -20 degrees C as the heavily damaged. Material was left at room temperature and sampled at regular intervals for 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 catalyzed during the first 2 h by extent of damage that increased 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-induced oxidation in PBP formation in wilted red clover was reported, showing the importance of o-diphenolic substrate concentration and not just PPO activity in PBP formation during prolonged wilting.


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