The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate-responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus

Jasmonates are plant signalling molecules that play key roles in defence against insects and certain pathogens, among others by controlling the biosynthesis of protective secondary metabolites. In Catharanthus roseus, the AP2/ERF-domain transcription factor ORCA3 controls the jasmonate-responsive expression of several genes encoding enzymes involved in terpenoid indole alkaloid biosynthesis. ORCA3 gene expression is itself induced by jasmonate. The ORCA3 promoter contains an autonomous jasmonate-responsive element (JRE) composed of a quantitative sequence responsible for the high level of expression and a qualitative sequence that acts as an on/off switch in response to methyl-jasmonate (MeJA). Here, we identify the basic helix-loop-helix (bHLH) transcription factor CrMYC2 as the major activator of MeJA-responsive ORCA3 gene expression. The CrMYC2 gene is an immediate-early jasmonate-responsive gene. CrMYC2 binds to the qualitative sequence in the ORCA3 JRE in vitro, and transactivates reporter gene expression via this sequence in transient assays. Knock-down of the CrMYC2 expression level via RNA interference caused a strong reduction in the level of MeJA-responsive ORCA3 mRNA accumulation. In addition, MeJA-responsive expression of the related transcription factor gene ORCA2 was significantly reduced. Our results show that MeJA-responsive expression of alkaloid biosynthesis genes in C. roseus is controlled by a transcription factor cascade consisting of the bHLH protein CrMYC2 regulating ORCA gene expression, and the AP2/ERF-domain transcription factors ORCA2 and ORCA3, which in turn regulate a subset of alkaloid biosynthesis genes.