Functional diversification of CsCAD14 orchestrates synergistic phenylpropanoid biosynthesis to defend gray blight disease in tea plants

Phenylpropanoid metabolism is a crucial secondary metabolic pathway in plants. Numerous neofunctionalized and specialized enzymes emerged during plant terrestrialization, yielding metabolites contributing to environmental adaptation. Here, a cinnamyl alcohol dehydrogenase from tea plant ( Camellia sinensis ), CsCAD14, exhibited dual activity catalyzing monolignol and hydroxycinnamic acid biosynthesis. Antifungal assays showed that monolignols and hydroxycinnamic acids synergistically inhibited Pestalotiopsis growth. Integrated functional characterizations revealed that CsCAD14 enhanced gray blight disease resistance by increasing monolignol and hydroxycinnamic acid levels compared to CsCAD11 featured with single-enzymatic activity. Notably, genetic analysis of F ₁ progeny from crossing susceptible and resistant tea cultivars identified a nonsynonymous mutation in the CsCAD14 coding region, which abolished dual-enzymatic activity and was associated with pathogen susceptibility. Furthermore, evolutionary analysis revealed a positively selected amino acid substitution (L192V) driving CAD functional diversification from single-enzymatic activity in algae to dual-enzymatic activity in land plants. These findings uncover neofunctionalization of dual-activity CADs evolved for phenylpropanoid defense to enhance fungal disease resistance during plant adaptation to terrestrial environments.