Novel Roles for the Polyphenol Oxidase Enzyme in Secondary Metabolism and the Regulation of Cell Death in Walnut    

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, 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.

Walnut: past and future of genetic improvement

“…Polyphenol oxidase (PPO) is responsible for catalyzing oxidation of phenolic compounds including tannins into highly reactive quinones. Originally only a single PPO gene, JrPPO1 was observed in walnut (Escobar 2013;Araji et al 2014). Using the genome sequence information, a second gene JrPPO2 has now been reported and is preferentially expressed in callus tissue .…”

Aurone synthase is a catechol oxidase with hydroxylase activity and provides insights into the mechanism of plant polyphenol oxidases

“…However, in most cases, their individual physiological role and especially their natural substrates remain unknown. Beside aurone formation, their specific involvement in biosynthetic processes was shown only in a few cases (1,(7)(8)(9)(10)(11). Recently, Sullivan (7) wondered whether there exist more PPOs that are involved in the plant's secondary metabolism and whether the few so far known specialized PPOs represent only "the tip of the iceberg.…”

Crystallization and preliminary X-ray crystallographic analysis of polyphenol oxidase fromJuglans regia(jrPPO1)

“…The study of tyrosinases in plants has focused on their role in the process of post-harvest browning, whereby cut or damaged plant tissues turn brown owing to the polymerization of tyrosinase-derived quinones (Queiroz et al, 2008). Walnut presents an interesting model to further explore the function of tyrosinase in plants based on the high content of various phenolic compounds in walnut tissues (Colaric et al, 2005;Solar et al, 2006;Araji et al, 2014).…”

“…Recently, a tyrosinase from walnut leaves (Juglans regia; jrPPO1; UniProt COLU17) was isolated and characterized as having a monophenolase activity (Escobar et al, 2008;Araji et al, 2014;Zekiri et al, 2014), thus making jrPPO1 an attractive enzyme for crystallographic studies of a plant tyrosinase. Although it exhibits monophenolase activity, jrPPO1 has the highest sequence identity to catechol oxidase from V. vinifera (sequence identity 62%; UniProt P4331; Virador et al, 2010) and has only weak identity (<20%) to bacterial and fungal tyrosinases (Matoba et al, 2006;Ismaya et al, 2011;Sendovski et al, 2011;Mauracher, Molitor, Michael et al, 2014).…”