Body of Abstract: The medicinal plant, Catharanthus roseus, produces the valuable chemotherapeutic terpenoid indole alkaloids (TIAs), vincristine and vinblastine. The biosynthesis of TIAs is induced by jasmonates (JAs) through the action of transcription factors (TFs), including those from the basic helix-loop-helix (bHLH) family such as MYC2a. However, in Arabidopsis thaliana, JA also induces the bHLH subgroup JAMs, which attenuate JA-mediated responses by competing with activators for promoter binding. Here we elucidate the role of JAM-type genes as repressors of TIA biosynthesis in C. roseus.
We identified three JAM-type genes, CrJAM2, CrJAM3, and CrRMT1 in C. roseus. In a transient expression screen, the overexpression of CrJAM2 and CrJAM3 repressed several promoters of enzymes and TFs involved in TIA biosynthesis. Similarly, the stable overexpression of CrJAM2 and CrJAM3 in hairy root cultures repressed the basal expression of most enzymes and transcriptional activators involved in TIA biosynthesis. To increase TIA biosynthesis, we used CRISPR/Cas9 to introduce mutations in the native JAMs in C. roseus hairy root cultures. CRISPR/Cas9 mediated gene editing was extremely efficient in C. roseus hairy roots, resulting in 100% mutations in the native CrJAMs at every guide location. These mutant lines exhibited increased basal expression of most TIA biosynthetic genes. This is the first time CRISPR/Cas9 is reported and applied in C. roseus. These results highlight the role of CrJAMs as repressors of TIA biosynthesis in C. roseus and of CRISPR/Cas9 as a strategy for unjamming the tight regulation of TIA biosynthesis and potentially of other plant specialized metabolic pathways.