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Acorus tatarinowii (A. tatarinowii) is a medicinal plant of the Araceae family. Currently, pharmacology focuses on the study of volatile oils, but there are few reports of another important secondary metabolite, lignan. Dirigent protein is thought to play an important role in plant secondary metabolism and responds to a variety of biotic and abiotic stresses. However, the DIR gene family of A. tatarinowii has not been systematically analyzed, and it is unknown whether it affects lignan synthesis. In this study, a total of 27 AtsDIRs were identified by comprehensive analysis of the genome of the medicinal plant A. tatarinowii , and the candidate gene AtsDIR2 3 that may be involved in lignan synthesis was screened through bioinformatics and transcriptome analysis. It is worth noting that AtsDIR23 is significantly expressed in rhizomes and is a member of the DIR-a subfamily. Subsequently, subcellular localization revealed that AtsDIR23 was localized in chloroplasts. The functional verification of AtsDIR2 3 b y the transient transformation of A. tatarinowii and the stable transformation of Arabidopsis thaliana showed that the content of lignans in overexpressed plants increased. Co-expression analysis screening revealed the MYB transcription factor (AtsMYB91) that is highly correlated with AtsDIR23 expression, while yeast one-hybrid assays and double luciferase experiments showed that AtsMYB91 negatively regulated the expression of AtsDIR2 3 b y binding to the AtsDIR23 promoter. In conclusion, AtsDIR23 can promote the accumulation of lignans, which provides a reference for further research on the regulation of lignans by DIR genes. [ABSTRACT FROM AUTHOR]