3β-hydroxysteroid-dehydrogenase/decarboxylase 2 ( HSD2 ) is essential for fruit cuticle formation in tomato

Abstract

The plant cuticle is a multilayered structure composed of cutin, cutin-linked polysaccharides and diverse waxes. It covers aerial plant tissues to prevent water loss and pathogen invasion, and modulates key crop quality traits. Despite substantial progress in research on cuticle formation, its molecular regulatory mechanisms remain largely elusive. In this study, we functionally characterized a tomato mutant exhibiting severe fruit suberization, named CM (cuticle mutation). Genetic analysis revealed that the mutant phenotype is governed by a single recessive gene. Using bulked segregant analysis (BSA), we mapped the causal locus to SNP45564049 in the fourth exon of SlHSD2, a G-to-A substitution producing a glycine-to-serine change. CRISPR/Cas9-mediated knockout of SlHSD2 successfully reproduced the CM mutant phenotype and altered the profile of pericarp cutin monomers. Integrated transcriptomic and widely targeted metabolomic analyses showed that SlHSD2 mutation reshapes the expression of cutin, suberin and wax biosynthetic genes, perturbs fatty acid metabolism and the accumulation of core cutin monomers, and alters numerous metabolites including alkaloids, flavonoids, lipids and phenolic acids, ultimately impairing cuticle development. We further found that the SBP-box transcription factor SlCNR acts as a negative regulator of fruit cutin deposition, directly binding the GTAC motifs in the SlHSD2 promoter to represses its expression. In summary, SlHSD2 is a newly identified downstream target of SlCNR that is essential for tomato fruit cuticle development via the regulation of lipid metabolism. This study expands the regulatory network of fruit cuticle formation and provides valuable gene resources for crop genetic improvement and stress-resistant breeding.