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Title:   Author  item
                                            Hatfield, Ronalditem
                                            Sullivan, Michaelitem
                                            Muck, Richard
  Submitted to: International Silage Conference
                                    Publication Type: Proceedings
                                    Publication Acceptance Date: 2/11/2005
                                    Publication  Date: 7/3/2005
                                    Citation: Hatfield, R.D., Sullivan, M.L. Muck, R.E. 2005. Using the red clover polyphenol oxidase gene to inhibit proteolytic activity in alfalfa. In: Park, R.S., Stronge, M.D., editors. Silage Production and Utilization. XIVth International Silage Conference, a Satellite Workshop of the XXth International Grassland Congress, July 2005, Belfast, Northern Ireland. p. 212.Interpretive Summary:
                                Technical Abstract: Preserving high quality forage in cool humid regions of agricultural production remains a challenge due to potentially high levels of protein degradation during ensiling. Red clover is an exception maintaining its high protein levels during ensiling. Decreased proteolytic activity in red clover is due to polyphenol oxidase (PPO) activity and appropriate o-diphenol substrates.This work highlights potential strategies for utilizing PPO as a means of decreasing proteolytic degradation during the ensiling of alfalfa and other forages. Since alfalfa does not produce o-diphenols, PPO-transformed alfalfa (PPO-Alf) is an excellent system for defining factors critical for PPO-mediated proteolytic inhibition. The amount of PPO activity measured in transformed alfalfa (PPO-Alf) was at levels similar to that found in red clover using the o-diphenol caffeic acid as the primary PPO substrate. Addition of caffeic acid to PPO-Alf extracts resulted in approximately 80% inhibition of proteolytic activity. Caffeic acid and chlorogenic acid o-diphenols were the most rapidly utilized substrates with other o-diphenols showing considerably less activity. However, a wide range of o-diphenols were effective in decreasing proteolysis over incubation times of 4 hours or longer. Addition of PPO-Alf extracts to oat leaf extracts along with caffeic acid resulted in a 50% reduction in proteolysis. Preliminary experiments ensiling PPO-Alf in mini-silos with the addition of o-diphenols resulted in a 25% decrease in proteolysis over control silos. These results indicate that strategies can be developed to utilize the PPO system to decrease protein losses during ensiling of forges.


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