Abstract
The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. 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 that display less than 5% of wild-type PPO activity. Strikingly, the PPO-silenced plants developed spontaneous necrotic lesions on their leaves in the absence of pathogen challenge (i.e. a lesion mimic phenotype). 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 plants. Silencing of PPO caused major alterations in the metabolism of phenolic compounds and their derivatives (e.g. coumaric acid and catechin) and in the expression of phenylpropanoid pathway genes. Several observed metabolic changes point to a direct role for PPO in the metabolism of tyrosine and in the biosynthesis of the hydroxycoumarin esculetin in vivo. In addition, PPO-silenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut.
Highlights
The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones
previously implicated a chloroplastlocalized phenolase (PPO) display much lower tyrosinase activities than catecholase activities, and this was true for JrPPO1 (Supplemental Fig. S2)
Vaughn et al (1988) suggested that PPOs are not involved in the metabolism of phenolic compounds in healthy, intact cells, several more recent studies have demonstrated that some PPOs do serve as biosynthetic enzymes required for the production of specialized phenolic-derived pigments and other secondary metabolites (Joy et al, 1995; Cho et al, 2003; Gandía-Herrero et al, 2005)
Summary
The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. PPOsilenced plants displayed massive (9-fold) increases in the tyrosine-derived metabolite tyramine, whose exogenous application elicits cell death in walnut and several other plant species. Overall, these results suggest that PPO plays a novel and fundamental role in secondary metabolism and acts as an indirect regulator of cell death in walnut. To better understand the functional role of the single PPO enzyme in walnut, we used RNA interference (RNAi) to generate a series of transgenic walnut lines with greatly reduced PPO activity These transgenic lines displayed a lesion mimic phenotype, spontaneously developing necrotic spots on their leaves independent of pathogen infection. Results from global transcript and metabolite profiling of the PPO-silenced lines suggest that JrPPO1 plays a fundamental role in the metabolism of Tyr in vivo and that, in the absence of PPO, the toxic metabolite tyramine accumulates to high levels in walnut leaves
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