Cloning and Characterization of Red Clover Polyphenol Oxidase cDNAs and Expression of Active Protein in Escherichia coli and Transgenic Alfalfa

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. RNAblotting 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.Ensiling crops is a popular method of preserving forage for animal feed, especially in the humid northern regions of the United States. During harvesting and early stages of ensiling, plant membranes are ruptured, releasing proteolytic enzymes that rapidly degrade available substrates. Once fermentation by lactobacteria has progressed sufficiently to lower silage pH to less than about 5, proteolytic activity slows significantly. The wider the window between harvest and reaching a low pH, the more extensive is the proteolytic degradation. Unfortunately, excessive proteolysis in ensiled forages results in economic losses to farmers (Rotz et al., 1993) amounting to approximately $100 million annually. This economic loss is due to purchasing additional protein to supplement diets because of poor utilization of the nonprotein nitrogen products of proteolysis (ammonia, amino acids, and small peptides) by ruminant animals. Ruminants excrete much of this nonprotein nitrogen as urea, resulting in increasing nitrogen burdens upon the environment.For some ensiled forages, such as alfalfa (Medicago sativa), proteolytic losses are especially high with degradation of 44% to 87% of the forage protein (Papadopoulos and McKersie, 1983;Muck, 1987). By contrast, red clover (Trifolium pratense), a forage with protein content similar to alfalfa, has up to 90% less proteolysis than alfalfa during ens...

Red clover polyphenol oxidase and lipid metabolism

“…eoplasts, chloroplasts and leucoplasts). In red clover at least three genes are encoding three different PPO enzymes, namely PPO1, PPO2 and PPO3 (Sullivan et al, 2004). They were found to be 87% to 90% identical on the nucleotide level.…”

The effect of high polyphenol oxidase grass silage on metabolism of polyunsaturated fatty acids and nitrogen across the rumen of beef steers1

“…The potential to exploit PPO has led to transgenic approaches to introduce PPO into agriculturally important crops such as alfalfa (Medicago sativa; Sullivan et al, 2004). Lee et al (2006b) screened 6 grass species for PPO activity and noted activity in cocksfoot (Dactylis glomerata) comparable to red clover.…”

Modern methods for genetic improvement of Trifolium pratense.

“…A PPO gene family in red clover has been reported as a cluster of at least six genes . The three paralogous genes PPO1, PPO2 and PPO3, having high homology and thus suggesting a recent evolutionary event, were identified by Sullivan et al (2004). Multiple PPO1 copies have been characterized.…”

“…DNA sequences show high homology within species. Red clover PPO genes are differentially expressed in aerial and root tissues (Sullivan et al 2004). PPO1/5 and PPO4 have proved to be useful genetic markers for red clover breeding purposes.…”