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Title:   Author  item
                                            Sullivan, Michaelitem
                                            Hatfield, Ronald
  Submitted to: Crop Science
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 9/22/2005
                                    Publication  Date: 2/1/2006
                                    Citation: Sullivan, M.L., Hatfield, R.D. 2006. Polyphenol oxidase and o-diphenols inhibit post-harvest proteolysis in red clover and alfalfa. Crop Science. 46:662-670.Interpretive Summary: Ensiling is a popular method of preserving forage, particularly in the cool humid regions of the U.S. 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 cows. 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. Removing PPO or o-diphenols from red clover extracts by genetic and biochemical means, respectively, results in increased protein degradation. Likewise, 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 provided appropriate o-diphenols are added to it. Using laboratory scale silos, alfalfa treated with PPO and o-diphenols had 25% less protein degradation than untreated alfalfa. Assuming the process could be scaled up, this would result in at least a $25 million annual savings to dairy framers, not including the potential economic benefit from reduced nitrogen in the environment. The PPO process has the potential to treat silages of all types to prevent excess nitrogen loss, ultimately increasing profitability of operations for farmers and significantly lessening nitrogen pollution to the environment.
                                Technical Abstract: Many forages experience significant proteolytic losses when preserved by ensiling. Such losses in alfalfa (Medicago sativa L.) are especially high, with degradation of 44-87% of the forage protein to non-protein nitrogen. In contrast, red clover (Trifolium pratense L.) has up to 90% less proteolysis during ensiling. Here we demonstrate that the combination of polyphenol oxidase (PPO) and o-diphenol PPO substrates, both abundantly present in red clover, is responsible for post-harvest proteolytic inhibition in this forage crop. Proteolysis in red clover leaf extracts increased nearly five-fold when endogenous o-diphenols were removed by gel filtration, but returned to starting levels by adding back an exogenous o-diphenol. Proteolysis in leaf extracts of red clover plants silenced for PPO expression was dramatically increased compared to control plants. Leaf extracts of transgenic alfalfa expressing a red clover PPO gene showed a nearly five-fold o-diphenol-dependent decrease in proteolysis compared to those of control alfalfa. We also demonstrate that PPO levels ten- to twenty-fold lower than those typically found in red clover are sufficient for proteolytic inhibition, that as little as 0.25 mmol o-diphenol mg-1 protein has a substantial impact on proteolysis, that a wide variety of o-diphenols are functional substrates in proteolytic inhibition, and that proteolysis is reduced for PPO-expressing alfalfa in small scale ensiling experiments. Together, these results indicate that PPO and o-diphenols can be an effective treatment to prevent protein loss in ensiled forage crops.


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