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

Abstract

Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. The biosynthesis of TIAs is tissue specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. Here, we describe a C. roseus WRKY transcription factor, CrWRKY1, that is preferentially expressed in roots and induced by the phytohormones jasmonate, gibberellic acid, and ethylene. The overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially Tryptophan Decarboxylase (TDC), as well as the transcriptional repressors ZCT1 (for zinc-finger C. roseus transcription factor 1), ZCT2, and ZCT3. However, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3, and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX repressor domain to CrWRKY1, resulted in the downregulation of TDC and ZCTs but the up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid, and C. roseus protoplast assays. Up-regulation of TDC increased TDC activity, tryptamine concentration, and resistance to 4-methyl tryptophan inhibition of CrWRKY1 hairy roots. Compared with control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors including ORCA3, CrMYC2, and ZCTs may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants.

Unraveling the light-specific metabolic and regulatory signatures of rice through combined in silico modeling and multi-omics analysis

“…The high enrichment of ABRE-like motifs associated with bZIP and ABI3 TFs under B prompts us to speculate a critical role of ABA in the expression of light-harvesting chlorophyll a/b-binding (LHCB) proteins in addition to the inhibition of hypocotyl elongation by down-regulating ethylene biosynthesis as discussed earlier. Similarly, the identification of B-responsive ZnF and WRKY TFs in conjunction with significantly higher terpenoid contents in B than in other colors allowed us to hypothesize that these TFs could play a pivotal role in B-mediated secondary metabolite synthesis (Suttipanta et al, 2011). However, the high enrichment of putative Ethylene Responsive Element cis-elements among the upregulated genes under R (Table I) and up-regulation of corresponding ERF (Os06g0592500: ethyleneresponsive transcriptional coactivator) TF suggests that the R-specific ethylene signaling is possibly important for the elongation of hypocotyl.…”

Regulation of Specialized Metabolism by WRKY Transcription Factors

“…are jasmonate response factors and could potentially regulate TIA biosynthesis (Schluttenhofer et al, 2014). CrWRKY1 from C. roseus directly regulates the expression of TRYPTOPHAN DECARBOXYLASE, the enzyme synthesizing the indolic tryptamine precursor (Suttipanta et al, 2011). Additionally, metabolites from two branches of the TIA pathway, catharanthine and serpentine, accumulate differently in CrWRKY1 RNA interference lines of hairy root cultures, indicating that CrWRKY1 regulates genes governing metabolite flux within the pathway.…”

“…In contrast, WRKY TFs have not commonly been considered key regulators of specialized metabolism because of (1) their known association with stress tolerance, (2) the emphasis on other characterized master regulators of specialized metabolism, such as basic helix-loop-helix (bHLH) and ethylene response factors, (3) the difficulty of identifying metabolic mutant phenotypes, (4) the potential redundancy of multiple WRKY TF's regulating the same biosynthetic genes, and (5) a general lack of mechanistic understanding of the transcriptional regulation of specialized metabolism. Nevertheless, accumulating evidence suggests that certain WRKYs regulate the production of valuable natural products by regulating metabolite biosynthetic genes Ma et al, 2009;Suttipanta et al, 2011). WRKYs regulating products from phenylpropanoids, alkaloids, and terpenes, the three major classes of plant metabolites, have been identified (Table I).…”

“…Analysis of Catharanthus spp. terpene indole alkaloid (TIA) biosynthetic gene promoters revealed that W-boxes are a commonly present ciselement, suggesting that WRKY TFs are important in regulating alkaloid production (Suttipanta et al, 2011). At least 25% of WRKY TFs in Catharanthus spp.…”

Methyl jasmonate treatment increases podophyllotoxin production in Podophyllum hexandrum roots under glasshouse conditions

“…Elicitation of medicinal plant species by jasmonates activates transcription factors followed by upregulation of the production of structurally divergent secondary metabolites, such as nicotine and artemisinin (De Geyter et al 2012). Most elicitation studies have been performed on cell suspension and hairy root cultures (van der Fits 2000; Häkkinen et al 2004;Baldi and Dixit 2008;Todd et al 2010;Shoji et al 2010;Suttipanta et al 2011). Also the podophyllotoxin production in P. hexandrum suspension cultures was increased by seven to eight-fold after stimulation with methyl jasmonate (Bhattacharyya et al 2012).…”