Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2013
Publication Date: 6/24/2013
Publication URL: http://handle.nal.usda.gov/10113/60096
Citation: Webb, K.J., Cookson, A., Allison, G., Sullivan, M.L., Winters, A.L. 2013. Gene expression patterns, localization, and substrates of polyphenol oxidase in red clover (Trifolium pratense L.). Journal of Agricultural and Food Chemistry. 61(31):7421-7430. Interpretive Summary: Polyphenol oxidases (PPO) are enzymes present in nearly all plants. PPOs oxidize o-diphenolic compounds present in plants to o-quinones. These quinones are highly reactive and undergo a number of secondary chemical reactions. These secondary reactions are most widely associated with post-harvest browning in fruits, vegetables, and other agricultural crops. Quinone reactions resulting from the action of PPO have also been shown by ARS scientists to protect forage protein during storage. Despite their presence in a wide variety of plant species, relatively little is known about what roles PPO enzymes play in plant growth and development. In this study, several red clover PPO enzymes were characterized in order to gain insights into their possible functions in plants. This characterization included examining in which tissues the PPO enzymes were produced, where within the cells of those tissues the PPO enzymes were present, levels and location of naturally occurring o-diphenols used by PPOs, and how well different PPO enzymes use various naturally occurring o-diphenols. PPO and o-diphenol levels were highest in leaves, and red clover PPOs effectively utilized the most abundant potential o-diphenol substrates, caffeic acid derivatives, consistent with a potential role in defense against pathogens. Interestingly, PPO and PPO substrates were also present in certain cells of root nodules, the specialized structures that allow legumes like red clover to fix nitrogen. This finding suggests that PPO plays a role in nitrogen fixation. These findings will be useful to other research scientists interested in plant pathogen interactions, symbiotic nitrogen fixation, and PPO enzymes in general.
Technical Abstract: Polyphenol oxidase (PPO) genes and their corresponding enzyme activity occur in many plants; natural PPO substrates and enzyme/substrate localization are less well characterized. Leaf and root PPO activity in Arabidopsis and five legumes were compared with high-PPO red clover (Trifolium pratense L.). Red clover PPO enzyme activity decreased leaves > stem > nodules > peduncle = petiole > embryo; PPO1 and PPO4 genes were expressed early in leaf emergence, while PPO4 and PPO5 predominated in mature leaves. PPO1 was expressed in embryos and nodules. PPO substrates, phaselic acid, and clovamide were detected in leaves, and clovamide in nodules. Phaselic acid and clovamide, along with caffeic and chlorogenic acids, were suitable substrates for PPO1, PPO4, and PPO5 genes expressed in Medigo sativa leaves. PPO enzyme presence and activity were co-localized in leaves and nodules by cytochemistry. Substrates and PPO activity were localized in developing squashed cell layer of nodules, suggesting PPO may have a developmental role in nodules.