The polyphenol oxidase gene JrPPO2 is a candidate integrator for mycorrhizal symbiosis and stress adaptation in walnut

Polyphenol oxidase (PPO) is a key class of copper-containing enzymes involved in plant development, stress adaption, and enzymatic browning, while the specific PPO genes responsible for these processes in walnut ( Juglans regia L.) remain poorly characterized. This study provides a comprehensive characterization of their bioinformatic features, expression patterns, and functional divergence, with a novel focus on their responses to arbuscular mycorrhizal fungi (AMF). In this study, two PPO genes, designated as JrPPO1 and JrPPO2 , were identified in the walnut genome. They encode acidic, hydrophilic chloroplast proteins (610–615 amino acids) and cluster within phylogenetic Subgroup IV, showing closest affinity to pecan PPOs . The two proteins shared conserved motifs, domain architecture, and structural features dominated by random coils and alphahelices. Both JrPPO genes are located on chromosome 3 and possess diverse cis-regulatory elements in their promoters. They exhibit distinct tissue-specific expression, with JrPPO1 predominantly expressed in leaves and JrPPO2 in roots. Under various treatments, JrPPO1 was specifically upregulated by indoleacetic acid (IAA) but downregulated by other cues. In contrast, JrPPO2 displayed a broad-response profile, being upregulated by IAA, high temperature, manganese stress, and most notably by inoculation with four out of five tested AMF species ( Funneliformis mosseae, Acaulospora scrobiculata, Diversispora spurca , and Paraglomus occultum ), a finding not previously reported. Root colonization assessment revealed that colonization rates varied significantly among AMF species and were significantly negatively correlated with JrPPO1 expression and positively with JrPPO2 expression. Consistent with JrPPO2 expression, total PPO activity in roots was significantly enhanced under these same inducing treatments (IAA, manganese stress, and the four AMF species). The distinct, AMF species-dependent regulation of JrPPO2 , coupled with its expression-activity correlation, suggests that JrPPO2 is a strong candidate gene potentially involved in mediating walnut-AMF interactions and integrating symbiotic signals with abiotic stress responses. These findings reveal that despite structural conservation, JrPPO1 and JrPPO2 have likely evolved specialized functions, as evidenced by their divergent expression patterns and differential responses to environmental and hormonal cues and mycorrhizal inoculation. This functional divergence highlights JrPPO2 as a potential target for strategies aimed at enhancing stress resilience in walnut.