Immunogold Labelling to Localize Polyphenol Oxidase (PPO) During Wilting of Red Clover Leaf Tissue and the Effect of Removing Cellular Matrices on PPO Protection of Glycerol-Based Lipid in the Rumen

Authors: LEE, MICHAEL - Aberystwyth University; TWEED, JOHN - Aberystwyth University; COOKSON, ALAN - Aberystwyth University; Sullivan, Michael

Submitted to: Journal of the Science of Food and Agriculture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/30/2009
Publication Date: 12/11/2009
Citation: Lee, M.R., Tweed, J.K., Cookson, A., Sullivan, M.L. 2010. Immunogold Labelling to Localize Polyphenol Oxidase (PPO) During Wilting of Red Clover Leaf Tissue and the Effect of Removing Cellular Matrices on PPO Protection of Glycerol-Based Lipid in the Rumen. Journal of the Science of Food and Agriculture. 90(3):503-510.

Interpretive Summary: Polyphenol oxidase (PPO), an enzyme present in the tissues of many plants, carries out oxidation reactions that are often associated with browning seen in fresh produce. Leaves of red clover have particularly high levels of PPO. Previous studies have demonstrated that, in red clover, these high PPO 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 animals 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 exactly where in red clover leaf tissues the PPO enzyme is found and at what levels, and also how PPO is able to protect lipids in the rumen. PPO was present in mesophyll cells of clover leaves and increased as harvested leaves were allowed to wilt. This increase may reflect conversion of PPO from an inactive to active form. Protection of lipids in the rumen was found to require red clover protein, suggesting complexation of lipids with protein due to the presence of PPO results in the protective effect. These results provide insights into the post-harvest activation of PPO and how PPO can exert its effects in the rumen and should help develop strategies to take best advantage of this natural system of protein and lipid protection.

Technical Abstract: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones which can react with nucleophilic cellular constituents (e.g. proteins), forming protein-phenol complexes that may reduce protein solubility and bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localised PPO in red clover leaf tissue by immunogold labeling and investigated whether red clover lipid was protected in the absence of PPO-induced protein-phenol complexes and plant enzymes (lipases). PPO protein was detected to a greater extent (P<0.001) within the chloroplasts of mesophyll cells in stressed (cut/crushed and wilted for 1 h) than freshly cut leaves for both palisade (61.6 and 25.6 Au-label chloroplast-1, respectively) and spongy mesophyll cells (94.5 and 40.6 Au-label chloroplast-1, respectively). Hydrolysis of lipid and C18 polyunsaturated fatty acid biohydrogenation during in vitro batch culture was lower (P<0.05) for wild type red clover than for red clover with PPO expression reduced to undetectable levels, but only when cellular matrices containing protein-phenol complexes were present. Damage to the leaves resulted in over a doubling of PPO detected within mesophyll cells, potentially as a consequence of conversion of the enzyme from latent to active form. PPO’s reduction of microbial lipolysis was apparent in macerated red clover tissue, but not in the absence of the proteinaceous cellular matrix, suggesting PPO’s mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein-phenol complexes.