Unexpected Suppression of Tomato Fruit Cuticle Formation by Overexpression of a Key Cutin Biosynthetic Enzyme

Fruit cuticles regulate the diffusion of water, gases and solutes, functioning as essential protective interfaces against environmental and biotic stresses. Engineering cuticle structure and mechanics therefore represents a promising strategy for improving fruit quality and resilience. To enhance biosynthesis of cutin, the major structural component of the cuticle, we generated transgenic tomato lines expressing the Arabidopsis thaliana GLYCEROL-3-PHOSPHATE sn-2-ACYLTRANSFERASE 4 (AtGPAT4) gene specifically in exocarp tissues. AtGPAT4 is a key enzyme providing acylglycerol intermediates for cutin polymer assembly. We performed comprehensive phenotypic, microscopic, spectroscopic, transcriptional and surface nanomechanical analyses to monitor the structural consequences of AtGPAT4 overexpression and evaluated functional traits including water loss and susceptibility to the Botrytis cinerea fungus. Contrary to expectations, exocarp-targeted expression of AtGPAT4 generated fruits with cuticles that had diminished levels of cutin and epicuticular waxes, together with decreased cuticular stiffness and resistance to deformation, and whole fruit firmness. Evidently, these structural and chemical alterations did not affect thermal stability or water retention, but they increased susceptibility to fungal infection by B. cinerea. Although the engineering outcomes were unintended, they offered mechanistic insights into how manipulation of cutin biosynthesis pathways reshapes cuticle architecture, polymer composition and nanomechanical performance. This work underscores the complexity of engineering fruit surface traits and offers new perspectives for future biotechnological strategies aimed at improving cuticle robustness and pathogen resistance.