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

Abstract

SUMMARYJasmonates 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.

The Transcription Factor CrWRKY1 Positively Regulates the Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus

“…The repression of ORF by CrWRKY1-SRDX derepresses the ORCA3 promoter, leading to the up-regulation of ORCA3. Alternatively, CrWRKY1 regulates ORCA3 by repressing CrMYC2, which controls the JA-responsive expression of ORCA3 (Zhang et al, 2011). Our results are in agreement with recent observations showing that JA induction increases TIA accumulation in C. roseus hairy roots, yet, overexpression of ORCA3 does not enhance TIA production (Peebles et al, 2009).…”

“…TIA biosynthesis responds strongly to a group of chemical or fungal elicitors that are known to regulate gene transcription, providing an explanation for why the manipulation of TFs often results in substantial effects to the TIA pathway (El-Sayed and Verpoorte, 2007). Promoter analyses of several structural and TF genes related to the C. roseus TIA pathway have revealed the presence of chemical-and elicitorresponsive cis-elements, leading to the cloning of jasmonate-responsive TFs including the AP2-like ORCA2 and ORCA3 (Menke et al, 1999;Ouwerkerk and Memelink, 1999;, the G box-binding factors CrGBF1 and CrGBF2 (Sibéril et al, 2001), the P box-binding factor CrBPF1 (van der , the zinc-finger repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3 (Pauw et al, 2004), and the basic helix-loop-helix TF CrMYC2 (Zhang et al, 2011).…”

The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus

“…This complexity undoubtedly requires an array of TFs (1), many of which remain to be discovered. The expression of several of these TFs may be JA inducible as part of the JA signaling cascade, as previously shown for ORCA3 and MYC2 (13,15). This feature has turned out to be a powerful tool in the past to find pathway-regulating TFs in different plant species (1,4,35) and has been also successfully exploited here to identify the TF BIS1 as a regulator of the iridoid pathway.…”

Regulation of Specialized Metabolism by WRKY Transcription Factors

“…The phytohormone jasmonate is a key elicitor of specialized metabolism (Aerts et al, 1994;Singh et al, 1998;Qi et al, 2011;Lenka et al, 2012), primarily through the induction of key TFs, such as MYC2 and ethylene response factors, that regulate the biosynthetic genes (van der Fits and Memelink, 2000;Shoji et al, 2010;Shoji and Hashimoto, 2011;Zhang et al, 2011;Patra et al, 2013;Schweizer et al, 2013;Sears et al, 2014). Study of the grape (Vitis vinifera) WRKY family found that 80% of WRKY genes are jasmonate responsive .…”