A cascade of BoABF2-BoMYB96-BoMIEL1 regulates wax accumulation in kale

Cuticular wax plays an important role in drought resistance. However, the underlying regulatory mechanisms for wax accumulation remain unclear. Moreover, the regulatory networks of drought-induced wax accumulation remain largely unknown. Here, BoMYB96 was identified as a candidate gene for wax content variation in kale (Brassica oleracea var. acephala) through whole-genome quantitative trait locus resequencing analysis. Two BoMYB96 alleles, BoMYB96^S0836 and BoMYB96^6BZ, were identified based on single-nucleotide polymorphisms in the coding region. Overexpression and RNAi-mediated gene silencing analyses showed that BoMYB96 positively regulates wax accumulation and drought tolerance. BoMYB96 directly promoted the expression of three wax biosynthetic genes, BoCER3, BoKCS1 and BoKCR1. BoMYB96^S0836 exhibited higher transcriptional activation of the three genes than BoMYB96^6BZ. Drought-inducible BoABF2 activated the expression of BoMYB96^S0836 by directly binding to its promoter, and the positive role of BoABF2 in wax biosynthesis was validated using CRISPR/Cas9. The E3 ubiquitin ligase BoMIEL1 interacted with BoMYB96 and mediated its ubiquitination and degradation. Western blot analysis showed that the protein levels of BoMYB96^S0836 and BoMIEL1 were significantly increased and decreased under drought, respectively. Overexpression of BoMIEL1 significantly suppressed wax accumulation. This study reveals the BoABF2-BoMYB96-BoMIEL1 regulatory cascade that affects wax accumulation and provides a new genetic resource for kale breeding to improve drought resistance.