The CsMYB44-csi-miR0008-CsCER1 module regulates cuticular wax biosynthesis and drought tolerance in citrus

Cuticular wax covering aboveground organs serves as the first line of defense shielding plants from nonstomatal water loss and diverse environmental stresses. While there have been several wax-related genes identified, the molecular mechanisms responsible for the control of wax biosynthesis remain poorly understood in citrus, particularly at the posttranscriptional level. Here, we demonstrated that the CsMYB44-csi-miR0008-CsCER1 module is responsible for regulating drought tolerance in citrus through its control of cuticular wax biosynthesis. In this study, microRNA (miRNA) sequencing analyses of 'Newhall' navel oranges and the wax-deficient 'Ganqi 3' mutant variety led to the identification of a novel cuticular wax biosynthesis-related miRNA, csi-miR0008. csi-miR0008 suppresses the expression of CsCER1, an aldehyde decarbonylase-encoding gene associated with n-alkane biosynthesis. The leaves of csi-miR0008-silencing and CsCER1-overexpressing plants exhibited increases in total wax levels, with particularly pronounced increases in n-alkane levels, contributing to enhanced drought tolerance. csi-miR0008-overexpressing and CsCER1-silencing plants exhibited the opposite phenotypes. CsMYB44 was confirmed to promote wax accumulation by directly inhibiting the expression of csi-miR0008. Taken together, our study offers new insight into the mechanisms responsible for the posttranscriptional control of citrus cuticular wax biosynthesis, while also providing a foundation for the breeding of novel citrus varieties exhibiting enhanced drought tolerance.