BnaA8.RAP2.1 negatively regulates cuticular wax accumulation and drought tolerance in Brassica napus.

Cuticular wax is an important component of the plant surface barrier that contributes to water retention and drought adaptation. However, the transcriptional mechanisms that negatively regulate wax accumulation remain poorly understood. Here, we identify BnaA8.RAP2.1, an AP2/ERF transcriptional repressor, as a negative regulator of cuticular wax accumulation and drought tolerance in Brassica napus. Loss- and gain-of-function analyses revealed reciprocal effects of BnaA8.RAP2.1 on wax deposition, cuticle permeability, water loss, and drought tolerance. BnaA8.RAP2.1-overexpressing plants exhibited reduced wax accumulation, increased cuticle permeability and water loss, and greater drought sensitivity, whereas knockout plants showed the opposite phenotypes. Stomatal aperture measurements further revealed genotype-dependent responses under drought, suggesting that altered stomatal responses may also contributes to BnaA8.RAP2.1-mediated drought phenotypes. Mechanistically, BnaA8.RAP2.1 directly bound to and repressed the promoters of BnCER1-2 and BnGCN5, as demonstrated by ChIP-qPCR, EMSA, and dual-luciferase assays. Genetic rescue analysis showed that BnCER1-2 overexpression partially restored wax accumulation, water retention, and drought tolerance in RAP2.1-overexpression plants, supporting BnCER1-2 as a functional downstream target. Moreover, constitutive expression of BnaA8.RAP2.1 in Arabidopsis rap2.1 mutant backgrounds produced wax- and drought-related phenotypes consistent with the negative regulatory activity of BnaA8.RAP2.1 in a heterologous system. Together, our findings establish BnaA8.RAP2.1 as a transcriptional repressor of cuticular wax accumulation and reveal that its effects on drought tolerance involve both cuticle-associated water retention and stomatal responses, providing a potential target for improving drought resilience in rapeseed.