Transcription factor CitMYB16 positively regulates cutin and wax biosynthesis in citrus by directly activating CitDCR and CitKCS2

The cuticle forms a protective barrier on fruit surfaces, playing crucial roles in limiting water loss, reducing fruit cracking, and resisting pathogen invasion. However, the molecular mechanisms underlying cuticle accumulation in citrus fruits remain unclear due to the challenges associated with isolating and analyzing cuticle components. In this study, ZK (Citrus trifoliata) and HJ (Citrus reticulata) with extremely different cuticle contents were identified from nine citrus varieties, providing ideal materials for studying cuticle accumulation in citrus. Gene co-expression network analysis and correlation analysis identified an R2R3-MYB transcription factor, CitMYB16, as a potential key regulator of cuticle formation. Transgenic experiments involving overexpression (in tomato and citrus fruit) and VIGS (in citrus fruit) revealed that CitMYB16 promotes the accumulation of both cutin and wax. Further analysis demonstrated that CitMYB16 directly binds to the promoters of DEFECTIVE IN CUTICULAR RIDGES (CitDCR, a key gene in cutin monomer formation) and CitKCS2 (involved in wax biosynthesis) and activates their expression. Moreover, functional analysis through stable overexpression in tomato and transient transformation in citrus fruit confirmed that CitDCR contributes to the accumulation of the cutin monomer 9(10),16-dihydroxy hexadecanoic acid. Collectively, these results indicate that CitMYB16 positively regulates the accumulation of cutin and wax, thereby promoting cuticle development in citrus fruit. Our findings provide new insights into the transcriptional regulation of cutin and wax metabolism during fruit ripening.