JrPPO1/2 play distinct roles in regulating walnut fruit browning by different spatiotemporal expression and enzymatic characteristics.

Polyphenol oxidase (PPO) activity drives walnut fruit browning, but the roles of its only two-family genes, JrPPO1 and JrPPO2, remain unclear. This study explores the spatiotemporal expression and enzymatic characteristics of JrPPO1 and JrPPO2 in walnut. Treatment with the PPO activator CuSO and HO accelerated fruit browning and up-regulated JrPPO1/2 expression, whereas treatment with the PPO inhibitor ascorbic acid delayed browning, down-regulating JrPPO1 and up-regulating JrPPO2 expression. Compared to mJrPPO1, mJrPPO2 can exhibited better enzyme activity at higher temperatures (47 °C) and in more acidic environments (pH 4.25). mJrPPO2 exhibited a higher substrate specificity over mJrPPO1, and the preferred substrates are catechol, chlorogenic acid, and epicatechin. Additionally, mJrPPO2 adapted better to low concentration of oxygen (as low as 1.0% O) and slightly elevated CO levels compared to mJrPPO1. Subcellular localization and spatiotemporal expression patterns showed that JrPPO1 is only expressed in green tissues and located in chloroplasts, while JrPPO2 is also located in chloroplasts, partly associated with membranes, and is expressed in both green and non-green tissues. Silencing JrPPO1/2 with virus-induced gene silencing (VIGS) reduced fruit browning, maintained higher total phenols, and decreased MDA production. Notably, silencing JrPPO1 had a greater impact on browning than JrPPO2, indicating JrPPO1's greater contribution to PPO activity and fruit browning in walnut fruits. Consequently, JrPPO1 can be effectively regulated both at the molecular level and by manipulating environmental conditions, to achieve the objective of controlling fruit browning.