Characterization of polyphenol oxidase in walnut

The polyphenol oxidase enzyme (PPO) is almost ubiquitous in Kingdom Plantae. PPO catalyzes the oxidation of phenolic compounds to quinones, which polymerize to form brown-colored phytomelanins. Although the physiological function of PPO in plants is unclear, recent studies have suggested that PPO has potential roles in seed coat coloration, pathogen resistance, and insect resistance. Walnut (Juglans regia) generates an exceptionally diverse group of phenolic compounds in leaf and hull tissues, but little is known about PPO in walnut. Recently, we cloned a single PPO-encoding gene from walnut and designated the gene jrPPOJ. In order to investigate the physiological function(s) of this walnut PPO, we constructed transgene vectors designed to either overexpress or silence jrPPOJ. Eight jrPPOJ-silenced transgenic walnut lines with PPO-activities :=::: 1% of wild type were identified, but none of the plants transformed with the jrPPOJ overexpression construct displayed elevated PPO activity. Using protein extracts from the leaves of wild-type and jrPPOJ-silenced walnuts, we identified the likely phenolic substrates for JrPPOl in planta and performed basic biochemical characterization of the enzyme. Preliminary studies suggest that PPO may play a role in defense against the causal agent of walnut blight, Xanthomonas campestris pv. juglandis, since this pathogen appears to replicate more rapidly in the leaves of jrPPOJ-silenced plants than in wild-type plants. PPO may also have an as-yet uncharacterized role in the control of cell death, since jrP PO ]-silenced lines exhibit a lesionmimic phenotype (necrotic leaf spots) whose severity increases over the course of the growing season. Keywords: Polyphenol Oxidase (PPO); Phenolic Compounds; Walnut (Juglans regia); Xanthomonas campestris; Enzyme