Genome-wide identification of the ECERIFERUM (CER) gene family in cabbage and critical role of BoCER4.1 in wax biosynthesis.

The ECERIFERUM (CER) gene family is essential for the biosynthesis of plant cuticular wax. In this study, 32 BoCER genes were identified in cabbage through genome-wide analysis. We found that the BoCER genes are highly conserved with their homologous counterparts in Arabidopsis thaliana. However, there was a significant divergence in the expression pattern among the BoCER paralogs, which suggests the occurrence of functional specialization during evolution. The expression analysis also showed that most of the BoCER genes are expressed in the aboveground part. Cis-regulatory element analysis suggested that BoCER genes could potentially be regulated through coordinated light and hormonal signaling. Furthermore, the abscisic acid and drought treatments markedly upregulated multiple BoCER genes, highlighting their involvement in abiotic stress responses. The functional analysis using CRISPR/Cas9-mediated knockout showed that BoCER4.1 governs the biosynthesis of alcohol. In situ hybridization localized the expression of BoCER4.1 to the tapetum, microspores, stem epidermis, and vascular bundles, while subcellular localization assays indicated its location in the endoplasmic reticulum, which aligns it with the biosynthetic machinery for wax. A phenotypic analysis revealed that the cuticles of the BoCER4.1 mutants were more permeable, and this was characterized by accelerated water loss and chlorophyll leaching. Correspondingly, the drought resistance of cababge with BoCER4.1 knockout was significantly reduced, accompanied by increased malondialdehyde content and decreased proline content under drought condition. This study provides new insights into the role of BoCERs in wax biosynthesis of cabbage, and also provides scientific basis for genetic improvement of drought resistance in cabbage.