Genome-wide analysis of the WRKY transcription factor family in Perilla frutescens and the positive role of PfWRKY38, PfWRKY59, and PfWRKY78 in regulating rosmarinic acid biosynthesis

Perilla frutescens is a plant of significant economic importance, valued for its applications in both therapeutic and cosmetic fields, in which rosmarinic acid (RA) serves as a key bioactive metabolite. Although WRKY transcription factors are known regulators of secondary metabolism, their functional roles in P. frutescens remain imperfectly characterized. To date, regulatory mechanisms for RA biosynthesis in perilla have been documented only for bHLH transcription factors. The study first comprehensive analysis of the WRKY gene family and demonstrate its involvement in RA regulation, thereby identifying a novel layer of transcriptional control. Comprehensive genome-wide analysis led to the identification of 111 PfWRKY genes. Evolutionary relationships and potential functions of these genes were investigated through phylogenetic classification, conserved motif analysis, and synteny assessment. By leveraging homology with Salvia miltiorrhiza , three candidate WRKY transcription factors ( PfWRKY38, PfWRKY59, and PfWRKY78 ) were selected, all of which showed significant transcriptional upregulation upon methyl jasmonate (MeJA) treatment. Further analyses revealed that these proteins localize to the nucleus, possess transcriptional activity, and exhibit specific binding to the W-box cis -element. Functional studies in transgenic hairy roots demonstrated that overexpression of each PfWRKY gene markedly promoted RA accumulation, increasing its levels by 2.16–2.98-fold relative to controls. This enhanced production was associated with elevated expression of key RA biosynthetic genes, including Pf4CL , PfC4H , and PfRAS . Here, we present a comprehensive genome-wide analysis of the previously unidentified WRKY gene family in P. frutescens and identifies three PfWRKY members as positive regulators of RA biosynthesis. These results provide valuable genetic tools and candidate targets for metabolic engineering and molecular breeding aimed at improving the nutraceutical and pharmaceutical value of P. frutescens . • 1.Genome-wide identification revealed 111 WRKY genes in Perilla frutescens . • 2.PfWRKY38, PfWRKY59, and PfWRKY78 act as positive regulators of RA biosynthesis. • 3.MeJA significantly induced PfWRKY38/59/78 expression in P. frutescens leaves. • 4.Overexpression lines increased rosmarinic acid content up to 2.98-fold. • 5.Transgenic hairy roots showed enhanced antioxidant activity linked to RA levels.