ARS | Publication request: ISOLATION AND CHARACTERIZATION OF POLYPHENOL OXIDASE GENES FROM ALFALFA

Submitted to: Plant Physiology
Publication Type: Abstract Only
Publication Acceptance Date: February 28, 2002
Publication Date: August 3, 2002

Technical Abstract: Polyphenol oxidase (PPO) catalyzes the oxidation of o-diphenols to o-quinones. We are interested in how PPO enhances post-harvest quality of forage crops. Whereas red clover contains high levels of PPO activity, alfalfa contains little or none. This apparent lack of PPO activity in alfalfa could be due to lack of a functional gene or lack of PPO expression in the aerial tissues we have examined. Alternatively, the PPO enzyme in alfalfa could have substrate specificities that are significantly different from those of previously characterized PPOs making it non-detectible in standard activity assays. To distinguish among these possibilities, we are cloning alfalfa PPO genes. Primers flanking a conserved CuA binding site present in other plant PPOs were used in PCR reactions. These primers amplified an approximately 190-bp fragment from alfalfa genomic DNA. The fragment had 68 % nucleotide identity with a red clover PPO gene and the encoded product would have 60 % amino acid identity with the red clover enzyme. In RNA blotting experiments, we have been unable to detect PPO mRNA in alfalfa leaves, stems, flowers, apical shoots, or seed pods using the PCR-derived gene fragment as a probe. We are currently using the PCR fragment to screen an alfalfa genomic library for full-length PPO clones. Any clones obtained will be used for further expression studies and protein production in E. coli and transgenic plants.