The ABF4-bHLH28-COMT5 module regulates melatonin synthesis and root development for drought tolerance in citrus

Melatonin significantly influences the regulation of plant growth, development, and stress tolerance. However, the regulatory mechanisms underlying melatonin accumulation for drought tolerance in citrus are not fully understood. In this study, we first demonstrated that application of exogenous melatonin resulted in better drought tolerance by reducing water loss and maintaining redox homeostasis. Genome-wide analysis revealed presence of 96 genes involved in melatonin biosynthesis in trifoliate orange (Poncirus trifoliata L., also known as Citrus trifoliata L.). Seven caffeic acid-O-methyltransferases (COMT) genes were detected, among which PtCOMT5 was most substantially induced by drought stress and predominantly expressed in roots and leaves. Overexpression of PtCOMT5 led to enhanced drought tolerance in trifoliate orange by promoting melatonin accumulation and root development, whereas CRISPR-Cas9-mediated PtCOMT5 mutation led to opposite phenotype. Yeast one-hybrid screening and protein-DNA interaction assays confirmed that the transcription factor PtbHLH28 acts a transcriptional activator of PtCOMT5 through interacting with the gene promoter. In addition, PtbHLH28 was found to be positively regulated by PtABF4, a core member of the ABA signaling pathway. PtbHLH28 and PtABF4 were demonstrated to function in drought tolerance by regulating PtCOMT5-mediated melatonin synthesis and root development. Overall, this study elucidates the crucial role of a molecular module composed of PtABF4-PtbHLH28-PtCOMT5 in modulation of melatonin accumulation for promoting drought tolerance and root development in citrus. Our findings shed light on melatonin accumulation in plants exposed to drought stress and gain new insight into the regulatory network associated with the function of melatonin in plant development and stress response.