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Title:   Author  item
                                            Sullivan, Michaelitem
                                            Hatfield, Ronalditem
                                            Samac, Deborah

                                                         - *Debby*
  Submitted to: Journal of the Science of Food and Agriculture
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/17/2008
                                    Publication  Date: 4/16/2008
                                    Citation: Sullivan, M.L., Hatfield, R.D., Samac, D.A. 2008. Cloning of an alfalfa polyphenol oxidase gene and evaluation of its potential in preventing postharvest protein degradation. Journal of the Science of Food and Agriculture. 88(8):1406-1414.Interpretive Summary: Protein breakdown in alfalfa preserved by ensiling is a major problem because dairy cows poorly utilize the resulting nonprotein nitrogen breakdown products. Because of poor utilization of this nonprotein nitrogen, cattle must be fed protein supplements which increases feed costs and the amount of nitrogen in manure that is lost to the environment. In red clover, breakdown of protein in the ensiled forage is prevented by the oxidation of o-diphenol compounds by the enzyme polyphenol oxidase (PPO). Although alfalfa normally lacks these components, post-harvest protein breakdown was similarly prevented in genetically modified alfalfa expressing the red clover PPO enzyme when o-diphenol PPO substrates were supplied. As a first step in determining whether a similar PPO naturally present in alfalfa might be exploited for post harvest protein protection in that forage, we isolated and characterized a gene encoding the PPO enzyme in alfalfa. The alfalfa PPO gene is active in flowers and seed pods, but not leaves or stems, the tissues most relevant to silage production. When the alfalfa PPO gene was expressed to high levels in leaves of genetically modified alfalfa, little PPO enzyme activity was detected. The alfalfa PPO enzyme was able slightly decrease the level of post harvest protein degradation in an alfalfa extract, but to a much lesser extent than is seen for red clover PPO. These results suggest that even if the alfalfa PPO gene is expressed in tissues relevant to silage production (leaves and stems), it lacks useful enzyme activity for preserving forage protein. Consequently, exploitation of the PPO/o-diphenol system to preserve protein in ensiled alfalfa (which could save U.S. farmers up to $100 million annually) will likely require the development of management practices (co-ensiling of different forages, for example). Alternatively, suitable genetically modified alfalfa expressing a more active PPO enzyme such as that in red clover could be used for silage production. The data of this study will also be useful to other researchers interested in PPO enzymes.
                                Technical Abstract: BACKGROUND: Ensiling forages often leads to degradation of protein to nonprotein nitrogen (NPN), which is poorly utilized by ruminants. Postharvest protein degradation is especially high in alfalfa (Medicago sativa L.). In contrast, red clover (Trifolium pratense L.) has up to 90% less protein loss during ensiling due to polyphenol oxidase (PPO) forming o-quinones from endogenous o-diphenols and subsequent binding of o-quinones to cytoplasmic proteins. Here we determined whether an endogenous PPO might be exploited for postharvest protein protection in alfalfa. RESULTS: We isolated an alfalfa PPO gene (MsPPO1) that shares limited sequence identity (70-72%) with red clover PPO genes. MsPPO1 is expressed primarily in flowers and developing seedpods, but not in leaves or stems. Expression of MsPPO1 from a strong constitutive promoter in transgenic alfalfa results in accumulation of PPO transcripts in leaves, but little enzyme activity is detected using a variety of o-diphenol substrates unless assayed in the presence of sodium dodecyl sulfate (SDS). Under this SDS-activated condition, preference of MsPPO1 for tested substrates is catechol  (-)-epicatechin > caffeic acid. PPO activity in unactivated MsPPO1-alfalfa extracts is sufficient to inhibit proteolysis in the presence of catechol, but not caffeic acid or (-)-epicatechin. Inhibition is less than in extracts of alfalfa expressing the red clover PPO1 gene. CONCLUSION: Endogenous alfalfa PPO, even if expressed in appropriate target tissues would be less effective at preventing proteolytic losses in ensiled forages than red clover PPO.


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