Submitted to: Plant Physiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/16/2004
Publication Date: 10/1/2004
Citation: Sullivan, M.L., Hatfield, R.D., Thoma, S., Samac, D.A. 2004. Cloning of red clover polyphenol oxidase cdnas and expression of active protein in escherichia coli and transgenic alfalfa. Plant Physiology. 136:3234-3244. Interpretive Summary: Red clover leaves contain high levels of the enzyme polyphenol oxidase (PPO) and compounds called o-diphenols. The action of PPO upon o-diphenols results in a familiar post-harvest browning reaction. Although the browning reaction is often associated with a negative impact on food quality (for example, browning of fruits and vegetables), in red clover preserved by ensiling, the browning reaction is associated with maintaining protein of this forage crop during storage. To understand the role of PPO and o-diphenols in preventing post-harvest protein loss in red clover, we isolated genes from red clover that correspond to PPO. We transferred the red clover genes to alfalfa, a forage crop lacking significant amounts of PPO in its leaves. The genetically modified alfalfa produced functional red clover PPO enzyme. This research is an important first step in defining the role of PPO and o-diphenols in preventing post-harvest protein loss in ensiled forage crops. This research will provide background information needed to develop an effective treatment for preventing protein loss during ensiling. Adapting red clover's natural system of protein preservation for other forage crops, such as alfalfa, would have direct economic benefits to farmers and a positive impact on the environment.
Technical Abstract: Red clover (Trifolium pratense) leaves contain high levels of polyphenol oxidase (PPO) activity and o-diphenol substrates. Wounding of leaves during harvest and ensiling results in browning of leaf tissues from activity of PPO on the o-diphenols. In association with browning, leaf proteins remain undegraded during ensiling, presumably due to PPO-generated o-quinone inhibition of leaf proteases. We cloned three red clover PPO cDNAs, PPO1, PPO2, and PPO3, from a leaf cDNA library. Sequence comparisons among the three red clover PPO clones indicated they are 87% to 90% identical at the nucleotide level (80%'83% amino acid identity). All three encode proteins predicted to localize to the chloroplast thylakoid lumen. RNA-blotting and immunoblotting experiments indicated PPO1 is expressed primarily in young leaves, PPO2 in flowers and petioles, and PPO3 in leaves and possibly flowers. We expressed mature PPO1 in Escherichia coli. A portion of the expressed protein was soluble and functional in an assay for PPO activity. We also expressed the red clover PPO cDNAs under the control of a constitutive promoter in alfalfa (Medicago sativa). The expressed red clover PPO proteins were active in alfalfa extracts as evidenced by o-diphenol-dependant extract browning and quantitative assays of PPO activity. Proteolysis in leaf extracts of alfalfa expressing red clover PPO1 was dramatically reduced in the presence of an o-diphenol compared to controls. Transgenic alfalfa expressing red clover PPO should prove an excellent model system to further characterize the red clover PPO enzymes and PPO-mediated inhibition of postharvest proteolysis in forage plants.