ARS | Publication request: Characteristics of Polyphenol Oxidases from Red Clover (Trifolium Pratense)

Submitted to: Plant Biology Annual Meeting
Publication Type: Abstract
Publication Acceptance Date: March 30, 2007
Publication Date: July 8, 2007
Citation: Schmitz, G.E., Hatfield, R.D., Sullivan, M.L. 2007. Characteristics of polyphenol oxidases from red clover (Trifolium pratense)[abstract]. In: Program and Abstract Book of the Plant Biology and Botany 2007 Joint Congress, July 8-11, 2007, Chicago, Illinois. p. 23.

Technical Abstract: Polyphenol oxidase (PPO, EC 1.14.18.1 or EC 1.10.3.1) catalyzes the oxidation of o-diphenols to o-quinones which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense). Production of o-quinones in red clover inhibits post-harvest proteolysis during the ensiling process. Three red clover cDNAs encoding PPOs were expressed individually in alfalfa (Medicago sativa L.) that lacks foliar PPO activity and o-diphenols. Physical and biochemical characteristics of the red clover PPOs in alfalfa extracts were determined. In transgenic alfalfa extracts, red clover PPOs exist in a latent state and are activated (~10- to 40-fold) by long incubations (>2 days) at ambient temperature or short incubations (<10 minutes) at 65°C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively. Latent PPO1 is activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to ~7, and activity plateaus at higher pH. Red clover PPOs were analyzed for their ability to oxidize various o-diphenols. Activation of the PPOs resulted in only minor changes to substrate specificity when compared to the latent forms of the enzymes. Activation of the PPOs increases the Vmax of the enzymes, but has little effect on their affinity for caffeic acid. Knowledge of red clover PPOs may improve our understanding of their in vivo function(s) and how their roles in post-harvest physiology may be exploited to improve the ensiling of forages.