THREE POLYPHENOL OXIDASES FROM RED CLOVER (TRIFOLIUM PRATENSE) DIFFER IN ENZYMATIC ACTIVITIES AND ACTIVATION PROPERTIES

Authors: Schmitz, George; Sullivan, Michael; Hatfield, Ronald

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/26/2007
Publication Date: 12/11/2007
Citation: Schmitz, G.E., Sullivan, M.L., Hatfield, R.D. 2008. Three polyphenol oxidases from red clover (Trifolium pratense) differ in enzymatic activities and activation properties. Journal of Agricultural and Food Chemistry. 56(1):272-280.

Interpretive Summary: Ensiling is a popular method of preserving forage. A shortcoming of this method is that for many ensiled forages, there is a significant amount of protein degradation during the process. This unfortunately results in poor utilization of the resulting protein fragments by ruminants such as dairy or beef cattle. Large amounts of the original plant nitrogen are simply excreted from the animal rather than being converted to useful products such as milk and meat. It is estimated that this nitrogen loss costs dairy farmers alone $100 million per year and contributes to additional nitrogen waste in the environment. An exception with respect to these large protein losses is red clover, which, compared to alfalfa, has up to 90% less nitrogen loss from protein degradation during ensiling. Red clover contains the enzyme polyphenol oxidase (PPO) and o-diphenol compounds that together act to inhibit protein degradation. Expressing a red clover PPO gene in alfalfa, which normally has little or no PPO activity of its own, results in decreased protein degradation in extracts of the modified plants if appropriate o-diphenols are supplied. A PPO-based process could be applied to silages of all types to prevent excess nitrogen loss, ultimately increasing profitability of operations for farmers and significantly lessening nitrogen pollution to the environment. To take full advantage of this natural process of protein preservation, we have begun characterizing the red clover PPO enzymes. The red clover PPOs expressed in alfalfa are highly stable and increase in activity within a few days of harvesting, which indicates these enzymes would be durable in an ensiling process for alfalfa. The three red clover PPOs show substantial activity towards several different o-diphenols, which vary in their cost and commercial availability and will affect the cost-effectiveness of this process. Knowledge of the properties of the red clover PPOs expressed in alfalfa will help scientists develop and refine a PPO system for ensiling of alfalfa or other forages.

Technical Abstract: Polyphenol oxidases (PPOs) oxidize o-diphenols to o-quinones, which cause browning reactions in many wounded fruits, vegetables, and plants including the forage crop red clover (Trifolium pratense L.). Production of o-quinones in red clover inhibits postharvest proteolysis during the ensiling process. The cDNAs encoding three red clover PPOs were expressed individually in alfalfa (Medicago sativa L.), which lacks detectable endogenous foliar PPO activity and o-diphenols. Several 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 (10-40-fold increase in activity) by long incubations (>2 days) at ambient temperature or short incubations (<10 min) at 65 °C. PPO1 appears to be more stable at high temperatures than PPO2 or PPO3. During incubation at ambient temperature, the molecular masses of the PPO enzymes were reduced by approximately 20 kDa. The apparent pH optima of latent PPO1, PPO2, and PPO3 are 5.5, 6.9, and 5.1, respectively, and latent PPO1 is slightly activated (~5-fold) by low pH. Activation of the PPOs shifts the pH optima to ~7, and the activated PPOs retain substantial levels of activity as the pH increases above their optima. The latent and activated PPOs were surveyed for ability to oxidize various o-diphenols, and activation of the PPOs had little effect on substrate specificity. Activation increases the Vmax but not the affinity of the PPO enzymes for caffeic acid. Results indicate red clover PPOs undergo structural and kinetic changes during activation and provide new insights to their effects in postharvest physiology.