Submitted to: Plant Physiology
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2002
Publication Date: August 3, 2002
Technical Abstract: Ensiling is a popular method for preserving forages for animal production systems in the humid regions of the U.S. and Europe. A major problem in silage production is the extensive protein degradation that occurs during the ensiling process, resulting in economic losses of $110 million per year (U.S.) and negative environmental impacts due to increased animal excretion of nitrogen. Red clover silage retains over 80% of its protein as true protein resulting in improved animal performance. Reduced proteolysis in red cover is correlated with the presence of a soluble polyphenol oxidase (PPO) and soluble polyphenols that produce the typical browning reaction upon exposure to oxygen. We have partially purified red clover PPO and determined some of its characteristics. Red clover PPO has an apparent molecular weight of 65 kD and has optimal activity at near neutral pH. Caffeic acid is a preferred substrate compared to a wide range of other O-diphenols. O-diphenols with a propenyl side chain appear to be required for effective binding to and reaction with the PPO. The PPO enzyme effectively utilizes caffeic acid conjugates such as chlorogenic acid, clovamide, and phasic acid. Browning reactions occur immediately upon rupture of red clover leaf cells and crude extracts can rapidly lose PPO activity if the enzyme is not separated from endogenous phenolics. Red clover extracts containing PPO and o-diphenol substrates inhibit protein degradation in model proteases systems. Determining the molecular mechanism of this inhibition is part of our on going research program.