Furofuran-type lignans, mainly sesamin and sesamolin, are the most representative functional active ingredients in sesame (Sesamum indicum L.). Their exceptional antioxidant properties, medicinal benefits, and health-promoting functions have garnered significant attention. Dirigent (DIR) proteins, found in vascular plants, are crucial for the biosynthesis of secondary metabolites, like lignans, and essential for responding to abiotic and biotic stresses. Despite their importance, they have yet to be systematically analyzed, especially those involved in lignan synthesis in sesame. This study unveiled 44 DIR genes in sesame. Phylogenetic analysis categorized these SiDIRs into five subgroups (DIR-a, DIR-b/d, DIR-e, DIR-f, and DIR-g), aligning with conserved motifs and gene structures analyses. Expression analysis unveiled distinct tissue-specific and hormone-responsive expression patterns among the SiDIR gene family members. Particularly, SiDIR21, a member of the DIR-a subgroup, exhibited robust expression in lignan-accumulating tissues and consistently high expression levels in germplasm during seed development with high sesamin content. Furthermore, SiDIR21 overexpression in hairy roots significantly increased sesamin and sesamolin contents, confirming its role in lignan synthesis. Overall, our study offers a valuable resource for exploring SiDIRs’ functions and the lignan biosynthesis pathway in sesame.
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