|Evans, Merici - EDGEWOOD COLLEGE-WI|
|Thoma, Sharon - EDGEWOOD COLLEGE-WI|
Submitted to: Meeting Abstract
Publication Type: Proceedings
Publication Acceptance Date: March 22, 2003
Publication Date: March 22, 2003
Citation: SULLIVAN,M.L., EVANS,M., THOMA,S., HATFIELD,R.D., ALFALFA POLYPHENOL OXIDASE IS EXPRESSED IN FLOWERS AND DEVELOPING SEEDS, MEETING ABSTRACT, 2003. 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 no activity. This apparent lack of PPO activity could be due to the lack of a functional gene or the lack of PPO expression in the tissues we have examined. Alternatively, the PPO enzyme in alfalfa could have substrate specificities that are significantly different from those of previously characterized PPOs and cannot be detected in our standard activity assays. To distinguish among these possibilities, we cloned an alfalfa PPO gene by screening an alfalfa genomic library with an ~190 bp PCR fragment corresponding to one of the conserved copper binding regions present in PPO enzymes. A single PPO gene was isolated and sequenced. Southern blot analysis showed that there are two to three related DNA sequences in the genome of the alfalfa clone examined. In RNA blotting experiments, we were able to detect PPO mRNA in alfalfa flowers and seed pods, but not in leaves, stems or apical shoots. Preliminary experiments utilizing a promoter-GUS fusion in transgenic alfalfa show an expression pattern consistent with the RNA blotting experiments. Active PPO protein was expressed in leaf tissue of transgenic alfalfa using a strong constitutive promoter, and will be a source of enzyme for several further studies of the enzyme.