Defining the roles of polyphenol oxidase in dopamine and esculetin synthesis in plants

Although the enzyme polyphenol oxidase (PPO) is well studied for its role in postharvest browning of commercial crops, its specific physiological function in plants is poorly understood. Previous research on the physiological role of PPO in walnut (Juglans regia) suggested that PPO is involved in secondary metabolism, namely in dopamine and esculetin biosynthesis. Dopamine is a precursor to plant-synthesized benzylisoquinoline alkaloids (BIAs) such as codeine and morphine, and esculetin is an antioxidant with promising anticancer properties. This study was performed to further define the role of PPO in the synthesis of esculetin and dopamine-derived metabolites in plants, using walnut, California poppy (Eschscholzia californica), and red clover (Trifolium pratense) as experimental systems. PPO enzyme kinetics and substrate specificity were characterized in each plant system, and PPO function was studied in plants displaying suppressed PPO gene expression (walnut, red clover) or inhibited PPO enzyme activity (poppy). Substrate specificity assays with red clover PPO revealed a lack of activity with substrates relevant to dopamine and esculetin biosynthesis. GC-MS analysis revealed the absence of both dopamine and esculetin in wild-type lines. Collectively, these results suggest that PPO is not involved in dopamine or esculetin biosynthesis in red clover. In walnut, in vitro enzyme kinetic assays suggest that tyramine and caffeic acid are the preferred substrates in PPO-mediated dopamine and esculetin synthesis, respectively. Poppy cell cultures in which PPO enzyme activity was chemically inhibited using the inhibitor kojic acid had significantly reduced BIA content in the culture supernatant compared to control samples. Overall, these results suggest that the previously characterized role of walnut PPO in dopamine and esculetin biosynthesis is not shared in all plant species. Instead, this PPO function may be taxonomically limited, much like previously characterized roles for specific PPO enzymes in betalain synthesis in beets (order Caryophyllales) and aurone synthesis in snapdragons (Antirrhinum majus).