Enzymatic functions of CmPAL1 and CmPAL2 in Chinese chestnut (Castanea mollissima BL.): Key catalysts in powdery mildew resistance via MeJA signaling.

Phenylalanine ammonia-lyase (PAL) is a key enzyme catalyzing the conversion of L-phenylalanine into trans-cinnamic acid and plays a crucial role in plant secondary metabolism and stress responses. However, the enzymatic functions of PAL proteins in Chinese chestnut remain largely unexplored. In this study, we characterized four CmPAL proteins encoded in the chestnut genome through phylogenetic analysis, chromosomal localization, protein structure, and promoter cis-element identification. Enzyme activity assays and transcript profiling revealed distinct expression patterns among the CmPAL genes, with CmPAL1 showing the highest expression in roots, whereas CmPAL2, CmPAL3, and CmPAL4 showed relatively high expression in old leaves and low expression in kernels. To validate their enzymatic functions, CmPAL1 and CmPAL2 were transiently overexpressed in Nicotiana benthamiana and heterologously expressed in Escherichia coli BL21. Both approaches confirmed their ability to catalyze the conversion of L-phenylalanine into trans-cinnamic acid. Additionally, enzyme activity and transcript levels of CmPAL genes were significantly induced under abiotic stresses (drought, cold, heat, and salt), hormonal treatments methyl jasmonate (MeJA) and salicylic acid (SA), and Blumeria farreri (Bf) infection. Notably, CmPAL activity and expression were significantly higher in Bf-infected samples treated with 10 mM MeJA than with either treatment alone, suggesting an additive effect in enhancing PAL-mediated defense responses. Overall, our findings indicate that CmPAL1 and CmPAL2 are involved in phenylpropanoid metabolism and may contribute to powdery mildew resistance through MeJA signaling.