Gene expression patterns, localisation and substrates of polyphenol oxidase in red clover (Trifolium pratense L.)

Polyphenol oxidase (PPO) genes and their corresponding enzyme activity occur in many plants; natural PPO substrates and enzyme/substrate localisation are less well characterised. 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 M. sativa leaves. PPO enzyme presence and activity were co-localised in leaves and nodules by cytochemistry. Substrates and PPO activity were localised in developing squashed cell layer of nodules, suggesting PPO may have a developmental role in nodules.