BackgroundFurofuran lignans, the main insecticidal ingredient inPhryma leptostachya, exhibit excellent controlling efficacy against a variety of pests. During the biosynthesis of furofuran lignans, Dirigent proteins (DIRs) are thought to be dominant in the stereoselective coupling of coniferyl alcohol to form ( ±)-pinoresinol. There are DIR family members in almost every vascular plant, but members of DIRs inP. leptostachyaare unknown. To identify thePlDIRgenes and elucidate their functions in lignan biosynthesis, this study performed transcriptome-wide analysis and characterized the catalytic activity of the PlDIR1 protein.ResultsFifteen full-length uniquePlDIRgenes were identified inP. leptostachya. A phylogenetic analysis of thePlDIRsclassified them into four subfamilies (DIR-a, DIR-b/d, DIR-e, and DIR-g), and 12 conserved motifs were found among them. In tissue-specific expression analysis, except forPlDIR7, which displayed the highest transcript abundance in seeds, the otherPlDIRsshowed preferential expression in roots, leaves, and stems. Furthermore, the treatments with signaling molecules demonstrated thatPlDIRscould be significantly induced by methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (ETH), both in the roots and leaves ofP. leptostachya. In examining the tertiary structure of the protein and the critical amino acids, it was found that PlDIR1, one of the DIR-a subfamily members, might be involved in the region- and stereo-selectivity of the phenoxy radical. Accordingly, LC–MS/MS analysis demonstrated the catalytic activity of recombinant PlDIR1 protein fromEscherichia colito direct coniferyl alcohol coupling into ( +)-pinoresinol. The active sites and hydrogen bonds of the interaction between PlDIR1 andbis-quinone methide (bisQM), the intermediate in ( +)-pinoresinol formation, were analyzed by molecular docking. As a result, 18 active sites and 4 hydrogen bonds (Asp-42, Ala-113, Leu-138, Arg-143) were discovered in the PlDIR1-bisQM complex. Moreover, correlation analysis indicated that the expression profile ofPlDIR1was closely connected with lignan accumulations after SA treatment.ConclusionsThe results of this study will provide useful clues for uncoveringP. leptostachya's lignan biosynthesis pathway as well as facilitate further studies on the DIR family.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12870-023-04297-6.