ARS | Publication request: CLONING OF RED CLOVER AND ALFALFA POLYPHENOL OXIDASE GENES AND EXPRESSION OF ACTIVE ENZYMES IN TRANSGENIC ALFALFA

Submitted to: Molecular Breeding of Forage and Turf Conference
Publication Type: Proceedings
Publication Acceptance Date: February 14, 2003
Publication Date: February 14, 2003
Citation: Sullivan, M.L., Thoma, S., Samac, D., Hatfield, R.D. Cloning of red clover and alfalfa polyphenol oxidase genes and expression of active enzymes in transgenic alfalfa. Molecular Breeding of Forage and Turf Conference. 2003. p. 189-195

Technical 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 experiments indicate RC PPO1 is expressed predominantly in young leaf tissue, RC PPO2 is expressed most highly in flowers and petioles, and RC PPO3 may be 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. Additionally, we have recently cloned a PPO gene from alfalfa. Preliminary studies indicate alfalfa PPO expression is limited to flowers and seed pods. 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. Our transgenic alfalfa system will provide a means to further characterize the red clover and alfalfa PPO enzymes with respect to activation, substrate specificity, and role in PPO-mediated inhibition of post-harvest proteolysis.