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WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha)

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WsWRKY1-mediated transcriptional modulation of Withania somnifera tryptophan decarboxylase gene (WsTDC) helps to regulate fruit-specific tryptamine generation for production of withanamides.

Abstract

Withania somnifera is a highly valued medicinal plant. Recent demonstration of novel indolyl metabolites called withanamides in its fruits (berries) prompted us to investigate its tryptophan decarboxylase (TDC), as tryptophan is invariably a precursor for indole moiety. TDC catalyzes conversion of tryptophan into tryptamine, and the catalytic reaction constitutes a committed metabolic step for synthesis of an array of indolyl metabolites. The TDC gene (WsTDC) was cloned from berries of the plant and expressed in E. coli. The recombinant enzyme was purified and characterized for its catalytic attributes. Catalytic and structural aspects of the enzyme indicated its regulatory/rate-limiting significance in generation of the indolyl metabolites. Novel tissue-wise and developmentally differential abundance of WsTDC transcripts reflected its preeminent role in withanamide biogenesis in the fruits. Transgenic lines overexpressing WsTDC gene showed accumulation of tryptamine at significantly higher levels, while lines silenced for WsTDC exhibited considerably depleted levels of tryptamine. Cloning and sequence analysis of promoter of WsTDC revealed the presence of W-box in it. Follow-up studies on isolation of WsWRKY1 transcription factor and its overexpression in W. somnifera revealed that WsTDC expression was substantially induced by WsWRKY1 resulting in overproduction of tryptamine. The study invokes a key role of TDC in regulating the indolyl secondary metabolites through enabling elevated flux/supply of tryptamine at multiple levels from gene expression to catalytic attributes overall coordinated by WsWRKY1. This is the first biochemical, molecular, structural, physiological and regulatory description of a fruit-functional TDC.

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Acknowledgements

RSS and NSS thank New Millenium Indian Technology Leadership Initiative (NMITLI) for NMITLI Project grant on Ashwagandha. NSS also thanks Network project BSC 0203 for the grant for research and Dr V Gupta for providing vectors. JSJ acknowledges financial support of the University Grant Commission for research fellowship.

Funding

This work was supported by a Grant-in-Aid for research on Ashwagandha from New Millenium Indian Technology Leadership Initiative (NMITLI) for NMITLI Project.

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Authors and Affiliations

  1. Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, India

    Jyoti Singh Jadaun, Amit Kumar Kushwaha, Neelam S. Sangwan, Lokesh Kumar Narnoliya, Smrati Mishra & Rajender Singh Sangwan

  2. Department of Biochemistry, Central University of Haryana, Mahendergarh, Haryana, 123031, India

    Neelam S. Sangwan

  3. Academy of Scientific and Innovative Research (AcSIR), CSIR-HRDC Campus, Sector 19, Kamla Nehru Nagar, Ghaziabad, Uttar Pradesh, 201002, India

    Rajender Singh Sangwan

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  1. Jyoti Singh Jadaun
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  2. Amit Kumar Kushwaha
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  3. Neelam S. Sangwan
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  4. Lokesh Kumar Narnoliya
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  5. Smrati Mishra
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  6. Rajender Singh Sangwan
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Contributions

Experiments were designed, directed and conceived by RSS and NSS, and performed by JSJ, AKK and LKN. Manuscript writing and data analysis was done by RSS, NSS, SM and JSJ. All the authors approve the manuscript and declare no competing financial interacts.

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Correspondence to Neelam S. Sangwan or Rajender Singh Sangwan.

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Communicated by Neal Stewart.

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Jadaun, J.S., Kushwaha, A.K., Sangwan, N.S. et al. WRKY1-mediated regulation of tryptophan decarboxylase in tryptamine generation for withanamide production in Withania somnifera (Ashwagandha). Plant Cell Rep 39, 1443–1465 (2020). https://doi.org/10.1007/s00299-020-02574-4

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