Functional characterization of LeWRKY24 targeting LeSAT2 to promote acylshikonin biosynthesis

(R)-shikonin/(S)-alkannin and their derivatives (collectively denoted as shikonins) are abundantly distributed in Boraginaceae roots. Acetylshikonin, as a crucial derivative of (R)-shikonin, possesses substantial research significance. Previous investigations have shown that LeSAT2, specifically catalyzes the acylation of (R)-shikonin to yield various acylated (R)-shikonin derivatives, including acetylshikonin. Additionally, electrophoretic mobility shift assays (EMSA) indicated that LeWRKY24 transcription factors may be involved in the transcriptional regulation of LeSAT2. For this study, the interaction between LeWRKY24 and LeSAT2 was further verified using dual-luciferase reporter (DLR) and yeast one-hybrid (Y1H) assays. The LeWRKY24 overexpression vector was constructed and introduced into Echium plantagineum to induce transgenic hairy roots. High-performance liquid chromatography (HPLC) analysis showed that in LeWRKY24-overexpressing hairy root lines, a significant increase was observed in the total pigment content, and in particular, the content of acetylshikonin was dramatically upregulated by as much as 200-fold. Transcriptome data revealed that genes associated with shikonin biosynthesis in the overexpression lines were significantly upregulated, activating the metabolic pathway, which explains the molecular mechanism underlying this regulatory effect. Our results also confirm that LeWRKY24 possesses cross-species regulatory universality in acylshikonin biosynthesis, laying a foundation for its widespread application in the metabolic engineering of Boraginaceae plants.