CsERF003, CsMYB7 and CsMYB102 promote cuticular wax accumulation by upregulating CsKCS2 at fruit ripening in Citrus sinensis

Cuticular wax is a hydrophobic protective layer covering the surface of citrus fruit, which plays an important role in preventing fruit water loss and improving fruit appearance quality. Our previous study has demonstrated that CsKCS2 is highly expressed at fruit ripening and involved in fruit cuticular wax synthesis in Citrus sinensis. However, the upstream regulators of CsKCS2 and the transcription factors regulating fruit cuticular wax synthesis at ripening remain unclear. Here, we performed a gene co-expression analysis with the published transcriptomic data of five developmental stages of Citrus sinensis fruit. The results showed that the gene expression trend of the blue module was synchronized with the accumulation pattern of fruit cuticular wax during fruit development and ripening. Further dual luciferase assay suggested that the promoter of CsKCS2 could be activated by CsERF003, CsMYB7 and CsMYB102 in the blue module. Moreover, ectopic expression in Arabidopsis revealed that CsERF003, CsMYB7 and CsMYB102 could increase cuticular wax content in leaves by upregulating the expression of wax-related genes (such as KCS, CER1, CER3 and CER4). Our results provide new insights into the transcriptional regulation of fruit cuticular wax synthesis, and important information for the mining of regulatory factors involved in cuticular wax synthesis in Citrus sinensis fruit.