A cascade of <scp>BoABF2</scp> ‐ <scp>BoMYB96</scp> ‐ <scp>BoMIEL1</scp> 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.