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

Can Wang; Xiaolong Hao; Yao Wang; Itay Maoz; Wei Zhou; Zhigang Zhou; Guoyin Kai

doi:10.1093/hr/uhac099

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

Hortic Res. 2022 Apr 22:9:uhac099. doi: 10.1093/hr/uhac099. eCollection 2022.

Authors

Can Wang¹, Xiaolong Hao¹, Yao Wang¹, Itay Maoz², Wei Zhou¹, Zhigang Zhou³, Guoyin Kai¹

Affiliations

¹ Laboratory for Core Technology of TCM Quality Improvement and Transformation, The Third Affiliated Hospital, School of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, Zhejiang, 310053, China.
² Department of Postharvest Science, ARO, The Volcani Center, HaMaccabim Rd 68, POB 15159, Rishon LeZion, 7528809, Israel.
³ Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Conferred by Ministry of Education, Shanghai Ocean University, Shanghai, 201306, China.

Abstract

Camptothecin is a chemotherapeutic drug widely used to treat various cancers. Ophiorrhiza pumila 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 O. pumila. We identified 46 OpWRKY genes unevenly distributed on the 11 chromosomes of O. pumila. 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 OpWRKY6. Overexpression of OpWRKY6 significantly reduced the accumulation of camptothecin compared with the control. Conversely, camptothecin accumulation increased in OpWRKY6 knockout lines. Further biochemical assays showed that OpWRKY6 negatively regulates camptothecin biosynthesis from both the iridoid and shikimate pathways by directly downregulating the gene expression of OpGES, Op10HGO, Op7DLH, and OpTDC. 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.