CrJAMs are transcriptional repressors of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

Catharanthus roseus produces monoterpenoid indole alkaloids (MIAs) in response to the defense hormone jasmonate (JA). In Arabidopsis, JA regulates defense processes through MYC2 and JA-Associated MYC2a-like transcription factors (JAMs). Here we characterize two previously identified C. roseus JAMs, CrJAM2 and CrJAM3, as regulators of MIA biosynthesis. Functional screens using viral-induced gene silencing and transient promoter activity assays showed that CrJAMs repress the MIA pathway; silencing both CrJAM2 and CrJAM3 increased expression of MIA pathway genes and MIA levels while their individual overexpression repressed promoter activity. To assess their activity broadly across MIA regulatory and biosynthetic genes, we generated C. roseus hairy root cultures either constitutively overexpressing individual CrJAMs or CRISPR-Cas9 creating mutations in multiple CrJAMs, and profiled their expression by RNA-Seq. CrJAM2 overexpression decreased expression of most MIA biosynthetic genes relative to the GUS control, while double mutant lines showed increased basal expression of these genes relative to non-targeting controls. Notably, methyl JA treatment perturbs this equilibrium, reversing these trends at 24 hrs in both CrJAM overexpression and mutant lines relative to basal conditions. In summary, CrJAMs broadly repress MIA regulatory and biosynthetic gene expression and together with CrJAZ and CrMYC2a regulates MIA biosynthesis.