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

Background: 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, bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localized PPO in red clover leaf tissue by immunogold labelling and investigated whether red clover lipid was protected in the absence of PPO-induced protein-phenol complexes and plant enzymes (lipases). Results: 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 hour) than freshly cut leaves for both palisade (61.6 and 25.6 Au label per chloroplast, respectively) and spongy mesophyll cells (94.5 and 40.6 Au label per chloroplast, 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. Conclusion: Damaging of 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 reduction of microbial lipolysis was apparent in macerated red clover tissue but not in the absence of the proteinaceous cellular matrix, suggesting that the PPO mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein-phenol complexes. (C) 2009 Society of Chemical Industr