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United States Department of Agriculture

Agricultural Research Service

Title: Cloning of Red Clover Polyphenol Oxidase Cdnas and Characterization of Active Protein Expressed in Transgenic Alfalfa

item Sullivan, Michael
item Thoma, Sharon - EDGEWOOD COLLEGE
item Samac, Deborah - PRU ST PAUL, MN
item Hatfield, Ronald

Submitted to: Plant Physiology
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2002
Publication Date: August 4, 2002

Technical Abstract: Red clover contains high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates resulting in a characteristic post-harvest browning reaction associated with decreased protein degradation during ensiling. To define PPO's role in inhibiting post-harvest proteolysis, we are using both biochemical and molecular approaches. We have cloned three unique PPO cDNAs from red clover leaves. The cDNAs encode proteins that are >76% identical to each other and <60% identical with those of other cloned plant PPO genes. The protein sequences suggest red clover PPO is targeted to the chloroplast thylakoid lumen and processed from a 70 kilodalton protein to a 60 kilodalton protein. 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 is expressed in both leaves and flowers. We expressed the red clover cDNAs in alfalfa, which lacks significant endogenous PPO activity, to further characterize the individual proteins encoded by RC PPO1-3. In alfalfa, the red clover PPO cDNAs produce proteins similar in size to PPO derived from red clover extracts as determined by SDS-PAGE and immunoblotting. Further, the expressed protein is active in alfalfa extracts, and this activity increases over time in vitro and may be associated with a proteolytic cleavage of the PPO enzyme. Analyses of the activity derived from transgenic alfalfa indicate the individual red clover enzymes have different substrate specificites. Our transgenic alfalfa system will provide a means to further characterize the red clover PPO enzymes with respect to activation, substrate specificity, and role in PPO-mediated inhibition of post-harvest proteolysis.

Last Modified: 4/19/2015
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