RNA-seq and Metabolite Profiling Reveal Novel Functions for the Polyphenol Oxidase Enzyme in Walnut (Juglans regia)

The enzyme polyphenol oxidase (PPO; EC 1.14.18.1) catalyzes the oxidation of phenolic compounds into highly reactive quinones. PPO-derived quinones cause the postharvest browning of cut/bruised fruit, but the native physiological functions of PPO in plants remain unclear.  Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines which display < 5% of wild-type PPO activity. Surprisingly, these PPO-silenced plants develop spontaneous necrotic lesions on their leaves, and these lesions do not appear to be associated with pathogen infection or disease. To gain a clearer perspective on the potential functions of PPO and its possible connection to cell death, we compared the leaf transcriptomes and metabolomes of wild-type and PPO-silenced walnuts. Major alterations were observed in the abundance of phenolic metabolites (e.g. dopamine, catechin) and in the expression of genes involved in central pathways of phenolic metabolism (e.g. 4-coumarate:coenzyme A ligase and cinnamate-4-hydroxylase) in PPO-silenced plants. Many of these alterations could be traced to the ability of PPO to utilize coumaric acid, tyrosine, and tyramine as substrates, activities which were verified via in vitro enzyme activity assays. Notably, PPO-silenced plants displayed a massive (8-fold) increase in serotonin, a derivative of tryptophan that has been shown to trigger apoptosis in several plant species. Overall, these results suggest that PPO plays a novel and fundamental role in the metabolism of aromatic amino acids and their derivatives (flavonoids, phenylpropanoids) in plants.