The transcription factor CitPH4 regulates plant defense-related metabolite biosynthesis in citrus

Wild citrus (Citrus L.) exhibits high disease resistance accompanied by high-acidity fruit, whereas cultivated citrus produces tastier fruit but is more susceptible to disease. This is a common phenomenon, but the underlying molecular mechanisms remain unknown. Citrus PH4 (CitPH4) is a key transcription factor promoting citric acid accumulation in fruits. Accordingly, CitPH4 expression decreased during citrus domestication, along with a reduction in citric acid levels. Here, we demonstrate that a CitPH4-knockout mutant exhibits an acidless phenotype and displays substantially lower resistance to citrus diseases. Metabolome and transcriptome analyses of CitPH4-overexpressing citrus callus, Arabidopsis, and CitPH4-knockout citrus fruits revealed that quercetin, pipecolic acid (Pip), and N-hydroxypipecolic acid (NHP) are pivotal defense-related metabolites. Application of quercetin and Pip inhibited the growth of Xcc and Penicillium italicum, while NHP inhibited the growth of P. italicum and Huanglongbing. Biochemical experiments demonstrated that CitPH4 enhances the expression of quercetin and NHP biosynthesis genes by binding to their promoters. Moreover, Pip and quercetin contents were positively associated with citric acid content in the pulp of fruits from natural citrus populations. Finally, the heterologous expression of CitPH4 in Arabidopsis promoted the expression of stress response genes and enhanced its resistance to the fungal pathogen Botrytis cinerea. The overexpression of CitPH4 in tobacco (Nicotiana tabacum) enhanced disease resistance. This study reveals the mechanism by which CitPH4 regulates disease resistance and fruit acidity, providing a conceptual strategy to control fruit acidity and resistance to devastating diseases.