A differentially regulated AP 2/ ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus

Catharanthus roseus produces bioactive terpenoid indole alkaloids ( TIA s), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid ( JA )‐responsive AP 2/ ERF transcription factor ( TF ), ORCA 3, and its regulator, Cr MYC 2, play key roles in TIA biosynthesis. ORCA 3 forms a physical cluster with two uncharacterized AP 2/ ERF s, ORCA 4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA 4, while overlapping functionally with ORCA 3, modulates an additional set of TIA genes. Unlike ORCA 3, ORCA 4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, Cr MYC 2 is capable of activating ORCA 3 and co‐regulating TIA pathway genes concomitantly with ORCA 3. The ORCA gene cluster and Cr MYC 2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, Cr MAPKK 1. Overexpression of Cr MAPKK 1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post‐translational regulatory mechanisms that govern TIA biosynthesis in C. roseus .