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 down-regulation 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.
Highlights
Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs
We aimed to isolate WRKY transcription factors (TFs) that are induced by methyl jasmonate (MJ) and have corresponding expression profiles as key TIA pathway genes
Sequence alignment (Fig. 1A) revealed that CrWRKY1 shares high amino acid sequence identity with Nicotiana tabacum NtWRKY3 (42%), Glycine max GmWRKY58 (45%), and Arabidopsis AtWRKY70 (40%), all of which are classified as group III WRKY TFs (Zhang and Wang, 2005)
Summary
Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semisynthetic anticancer drugs. 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. 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. When Medicago truncatula is exposed to fungal elicitors or methyl jasmonate (MJ), a large number of WRKY genes are up-regulated, some of which are involved in the production of defense compounds such as flavonoids and terpenoids (Naoumkina et al, 2008). Despite three reported sequences of putative C. roseus WRKY genes, obtained from EST sequencing (Murata et al, 2006), no WRKY TF from C. roseus has been functionally characterized to date
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