Targeting the tomato fruit cuticle by gene overexpression and editing with the fruit epidermis-preferential nsLTP promoter

The thick cuticle covering and embedding epidermal cells of tomato (Solanum lycopersicum) fruit, a model for cuticle studies, plays important roles in fruit protection and quality. To further our understanding of the influence of cuticle components on cuticle architecture and properties, the next step is to engineer cuticle composition. Ideally, to avoid indirect effects at whole-plant level, a fruit-and epidermis-specific promoter should be used. Here, to overexpress and edit target genes in tomato, we selected a non-specific Lipid Transfer Protein 2 promoter (pronsLTP) with preferential activity in fruit epidermis, as shown by NLS-GFP fluorescence analysis. Overexpression of SlMYB75 driven by pronsLTP induced anthocyanin accumulation specifically in the growing fruit epidermis, consistent with the cuticle deposition pattern. In keeping with changes in anthocyanins, the cuticle composition in flavonoids and phenolic acids was altered, as shown by Raman microspectroscopy, as were several cuticle properties. We then edited the carotenoid PSY1 gene with a pronsLTP-driven CRISPR/Cas9 system. Different PSY1 knock-out alleles were detected predominantly in fruit exocarp and mesocarp of color-impaired pronsLTP::Cas9-Psy1 mutants, and very little in the leaf. A mutant carrying a heritable knock-out mutation in both fruit and leaf was also detected, indicating that mutants obtained with pronsLTP may require screening before being studied. Altogether, our results indicate that pronsLTP2 can efficiently result in gene overexpression and editing in fruit epidermis. Implications of these findings are important for the functional analysis and genetic engineering of cuticle-related genes in tomato.