Identification of WRKY transcription factors involved in regulating the biosynthesis of the anti-cancer drug camptothecin in .

Camptothecin is a chemotherapeutic drug widely used to treat various cancers. is an ideal plant model for the study of camptothecin production, with various advantages for studying camptothecin biosynthesis and regulation. The DNA-binding WRKY transcription factors have a key regulatory role in secondary metabolite biosynthesis in plants. However, little is currently known about their involvement in camptothecin biosynthesis in . We identified 46 genes unevenly distributed on the 11 chromosomes of . Phylogenetic and multiple sequence alignment analyses divided the OpWRKY proteins into three subfamilies. Based on spatial expression and co-expression, we targeted the candidate gene . Overexpression of significantly reduced the accumulation of camptothecin compared with the control. Conversely, camptothecin accumulation increased in knockout lines. Further biochemical assays showed that negatively regulates camptothecin biosynthesis from both the iridoid and shikimate pathways by directly downregulating the gene expression of , , , and . Our data provide direct evidence for the involvement of WRKYs in the regulation of camptothecin biosynthesis and offer valuable information for enriching the production of camptothecin in plant systems.