Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa.
نویسندگان
چکیده
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.
منابع مشابه
Cloning of an alfalfa polyphenol oxidase gene and evaluation of its potential in preventing postharvest protein degradation
BACKGROUND: Ensiling forages often leads to degradation of protein to non-protein 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 a...
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Many forages experience significant proteolytic losses when preserved by ensiling. Such losses in alfalfa (Medicago sativa L.) are especially high, with degradation of 44 to 87% of the forage protein to nonprotein N (NPN). 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-dip...
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ورودعنوان ژورنال:
- Plant physiology
دوره 136 2 شماره
صفحات -
تاریخ انتشار 2004