Functional diversification of WRINKLED3 integrates fatty acid metabolism with insecticidal acylsugar production in Solanaceae species

Acylsugars are defensive glycolipids in Solanaceae glandular trichomes, and their biosynthesis offers an exemplary system for understanding the evolutionary mechanisms of plant chemical defense, yet their transcriptional regulation is poorly understood. Here, we identified and characterized WRINKLED3 (WRI3), which is critical for acylsugar biosynthesis in tomato. SlWRI3 is specifically expressed in trichome tip cells, and its knockout reduces acylsugar accumulation. Using transcriptomics, DNA-protein interaction assays, and metabolomics, we demonstrate that SlWRI3 acts via a dual regulatory mechanism: directly activating the acyltransferase gene SlASAT1 for the initial acylation of sucrose core and upregulating multiple acetyl-CoA carboxylase (ACCase) subunits to provide acyl chain precursors. Silencing these ACCase genes similarly decreased acylsugar levels. Phylogenetic analysis indicates that the function of WRI3 in acylsugar biosynthesis is evolutionarily conserved in Solanaceae. These findings elucidate how a primary metabolism-associated regulator was repurposed to coordinate precursor supply and specialized metabolite production, deepening our understanding of plant metabolic evolution, providing a target for engineering pest-resistance in Solanaceae crops.