Cloning of Red Clover and Alfalfa Polyphenol Oxidase Genes and Expression of Active Enzymes in Transgenic Alfalfa

Abstract

Red clover contains high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. This results in a characteristic post-harvest browning reaction associated with decreased protein degradation during ensiling. To define PPO’s role in inhibition of post-harvest proteolysis, we are taking both biochemical and molecular approaches. We have cloned three unique PPO cDNAs from red clover leaves, RC PPO1-3. The cDNAs encode proteins that are predicted to be targeted to the chloroplast thylakoid lumen. RNA blotting and immunoblotting experiments indicate RC PPO1 is expressed predominantly in young leaf tissue, RC PPO2 is expressed most highly in flowers and petioles, and RC PPO3 is expressed in both leaves and flowers. We expressed the red clover cDNAs in alfalfa, which lacks both significant endogenous PPO activity and o-diphenol substrates in its leaves, to further characterize the individual proteins encoded by RC PPO1-3. The expressed proteins are active in alfalfa extracts and the individual enzymes show differences in substrate specificity, suggesting different functional roles. Additionally, we have cloned a PPO gene from alfalfa. Preliminary studies indicate alfalfa PPO expression is limited to flowers and seedpods. Expression of the cloned alfalfa PPO gene from a strong constitutive promoter in transgenic alfalfa results in low but measurable enzyme activity, suggesting a low specific activity compared to the red clover enzymes.