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Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal

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Abstract

The medicinal plant Withania somnifera is researched extensively to increase the quantity of withanolides and specifically withaferin A, which finds implications in many pharmacological activities. Due to insufficient knowledge on biosynthesis and unacceptability of transgenic approach, it is preferred to follow alternative physiological methods to increase the yield of withanolides. Prior use of elicitors like salicylic acid, methyl jasmonate, fungal extracts, and even mechanical wounding have shown to increase the withanolide biosynthesis with limited success; however, the commercial viability and logistics of application are debatable. In this investigation, we tested the simple nitrogeneous fertilizers pertaining to the enhancement of withaferin A biosynthesis. Application of ammonium sulfate improved the sterol contents required for the withanolide biosynthesis and correlated to higher expression of pathway genes like FPPS, SMT1, SMT2, SMO1, SMO2, and ODM. Increased expression of a gene homologous to allene oxide cyclase, crucial in jasmonic acid biosynthetic pathway, suggested the involvement of jasmonate signaling. High levels of WRKY gene transcripts indicated transcriptional regulation of the pathway genes. Increase in transcript level could be correlated with a corresponding increase in the protein levels for WsSMT1 and WsWRKY1. The withaferin A increase was also demonstrated in the potted plants growing in the glasshouse and in the open field. These results implicated simple physiological management of nitrogen fertilizer signal to improve the yield of secondary metabolite through probable involvement of jasmonate signal and WRKY transcription factor for the first time, in W. somnifera besides improving the foliage.

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References

  • Akhtar N, Gupta P, Sangwan NS, Sangwan RS, Trivedi PK (2012) Cloning and functional characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene from Withania somnifera: an important medicinal plant. Protoplasma 250:613–622

    Article  PubMed  Google Scholar 

  • Cai WJ, Huang JH, Zhang SQ, Wu B, Kapahi P, Zhang XM, Shen ZY (2011) Icariin and its derivative icariside II extend healthspan via insulin/IGF-1 pathway in C. elegans. Plos One 6(12):e28835. doi:10.1371/journal.pone.0028835

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chen H, Jones AD, Howe GA (2006) Constitutive activation of the jasmonate signaling pathway enhances the production of secondary metabolites in tomato. FEBS Lett 580:2540–2546

    Article  CAS  PubMed  Google Scholar 

  • Choudhary MI, Hussain S, Yousuf S, Dar A, Mudassar, Atta ur R (2005) Chlorinate and di-epoxy withanolides from Withania somnifera and their cytotoxic effects against human lung cancer cell line. Phytochem 71:2205–2209

    Article  Google Scholar 

  • Dhar N, Rana S, Razdan S, Bhat WW, Hussain A, Dhar RS, Vaishnavi S, Hamid A, Vishwakarma RA, Lattoo SK (2014) Cloning and functional characterization of three branch point oxidosqualene cyclases from Withania somnifera (L.) Dunal. J Biol Chem 289(24):17249–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dhuley JN (2000) Adaptogenic and cardioprotective action of ashwagandha in rats and frogs. J Ethnopharmacol 70:57–63

    Article  CAS  PubMed  Google Scholar 

  • Doma M, Abhaynkar G, Reddy VD, Kavi Kishor PB (2012) Carbohydrate and elicitor enhanced withanolide (withaferin A and withanolide A) accumulation in hairy root cultures of Withania somnifera (L.). Ind J Expt Biol 50:484–490

    Google Scholar 

  • Dombrecht B, Xue GP, Sprague SJ, Kirkegaard JA, Ross JJ, Reid JB, Fitt GP, Sewelam N, Schenk PM, Manners JM et al (2007) MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell 19:2225–2245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Elsakka M, Grigorescu E, Stanescu U, Stanescu U, Dorneanu V (1990) New data referring to chemistry of Withania somnifera species. Rev Med Chir Soc Med Nat Iasi 94:385–387

    CAS  PubMed  Google Scholar 

  • Fits L, Memelink J (2000) ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 289:295–297

    Article  PubMed  Google Scholar 

  • Gupta P, Agarwal AV, Akhtar N, Sangwan RS, Singh SP, Trivedi PK (2013a) Cloning and characterization of 2-C-methyl-D-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera. Protoplasma 250:285–295

    Article  CAS  PubMed  Google Scholar 

  • Gupta P, Akhtar N, Tewari SK, Sangwan RS, Trivedi PK (2011) Differential expression of farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in response to elicitors. Plant Growth Regul 65:93–100

    Article  CAS  Google Scholar 

  • Gupta P, Goel R, Pathak S, Srivastava A, Singh SP, Sangwan RS, Asif MH, Trivedi PK (2013b) De novo assembly, functional annotation and comparative analysis of Withania somnifera leaf and root transcriptomes to identify putative genes involved in the withanolides biosynthesis. PLoS ONE 8:doi:10.1371/journal.pone.0062714.

  • Hause B, Hause G, Kutter C, Miersch O, Wasternack C (2003) Enzymes of jasmonate biosynthesis occur in tomato sieve elements. Plant Cell Physiol 44:643–648

    Article  CAS  PubMed  Google Scholar 

  • Hause B, Stenzel I, Miersch O, Maucher H, Kramell R, Ziegler J, Wasternack C (2000) Tissue-specific oxylipin signature of tomato flowers—allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles. Plant J 24:113–126

    Article  CAS  PubMed  Google Scholar 

  • Jayaprakasam B, Zhang Y, Seeram NP, Nair MG (2003) Growth inhibition of human tumor cell lines by withanolides from Withania somnifera leaves. Life Sci 74:125–132

    Article  CAS  PubMed  Google Scholar 

  • Kaileh M, Berghe WV, Heyerick A, Horion J, Piette J, Libert C, De Keukeleire D, Essawi T, Haegeman G (2007) Withaferin A strongly elicits IKK hyperphosphorylation concomitant with potent inhibition of its kinase activity. J Biol Chem 282:4253–4264

    Article  CAS  PubMed  Google Scholar 

  • Kato N, Dubouzet E, Kokabu Y, Yoshida S, Taniguchi Y, Dubouzet JG, Yazaki K, Sato F (2007) Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica. Plant Cell Physiol 48(1):8–18

    Article  CAS  PubMed  Google Scholar 

  • Kaul MK, Kumar A, Ahuja A, Mir BA, Suri KA, Qazi GN (2009) Production dynamics of withaferin A in Withania somnifera (L.) Dunal complex. Nat Prod Res 23:1304–11

    Article  CAS  PubMed  Google Scholar 

  • Kraiser T, Gras DE, Gutierrez AG, Gonzalez B, Gutierrez RA (2011) A holistic view of nitrogen acquisition in plants. J Exp Bot 62:1455–1466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Koeduka T, Fridman E, Gang DR, Vassao DG, Jackson BL, Kish CM et al (2006) Eugenol and isoeugenol, characteristic aromatic constituents of spices, are biosynthesized via reduction of a coniferyl alcohol ester. Proc Natl Acad Sci U S A 103:10128–10133

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li S, Zhang P, Zhang M, Fu C, Yu L (2013) Functional analysis of a WRKY transcription factor involved in transcriptional activation of the DBAT gene in Taxus chinensis. Plant Biol 15(1):19–26

    Article  PubMed  Google Scholar 

  • Ma D, Pu G, Lei C, Ma L, Wang H, Guo Y, Chen J, Du Z, Wang H, Li G, Ye H, Liu B (2009) Isolation and characterization of AaWRKY1, an Artemisia annua transcription factor that regulates the amorpha-4,11-diene synthase gene, a key gene of artemisinin biosynthesis. Plant Cell Physiol 50(12):2146–2161

    Article  CAS  PubMed  Google Scholar 

  • Mannan A, Liu C, Arsenault PR, Towler MJ, Vail DR, Lorence A, Weathers PJ (2010) DMSO triggers the generation of ROS leading to an increase in artemisinin and dihydroartemisinic acid in Artemisia annua shoot cultures. Plant Cell Rep 29(2):143–152. doi:10.1007/s00299-009-0807-y

    Article  CAS  PubMed  Google Scholar 

  • Misra A, Chanotiya CS, Gupta MM, Dwivedi UN, Shasany AK (2012) Characterization of cytochrome P450 monooxygenases isolated from trichome enriched fraction of Artemisia annua L. leaf. Gene 510:193–201

    Article  CAS  PubMed  Google Scholar 

  • Naidu PS, Singh A, Kulkarni SK (2003) Effect of Withania somnifera root extract on haloperidol induced orofacial dyskinesia: possible mechanism of action. J Med Food 6:107–114

    Article  PubMed  Google Scholar 

  • Pal S, Singh S, Shukla AK, Gupta MM, Khanuja SPS, Shasany AK (2011) Comparative withanolide profiles, gene isolation, and differential gene expression in the leaves and roots of Withania somnifera. J Hort Sci Biotechnol 86:391–397

    CAS  Google Scholar 

  • Pandey V, Misra P, Chaturvedi P, Mishra MK, Trivedi PK, Tuli R (2010) Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant. Plant Cell Rep 29:133–141

    Article  CAS  PubMed  Google Scholar 

  • Pandey SP, Somssich IE (2009) The role of WRKY transcription factors in plant immunity. Plant Physiol 150:1648–1655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Patterson K, Cakmak T, Cooper A, Lager I, Rasmusson AG, Escobar MA (2010) Distinct signalling pathways and transcriptome response signatures differentiate ammonium- and nitrate-supplied plants. Plant Cell Environ 33:1486–501

    CAS  PubMed  PubMed Central  Google Scholar 

  • Razdan S, Bhat WW, Rana S, Dhar N, Lattoo SK, Dhar RS, Vishwakarma RA (2013) Molecular characterization and promoter analysis of squalene epoxidase gene from Withania somnifera (L.) Dunal. Mol Bio Rep 40(2):905–916

    Article  CAS  Google Scholar 

  • Sangwan RS, Agarwal K, Luthra R, Thakur RS, Sangwan NS (1993) Biotransformation of arteannuic acid into arteannuin-B and artemisinin in Artemisia annua. Phytochem 34:1301–1302

    Article  CAS  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Lal P, Misra LN, Tuli R, Sangwan NS (2008) Withanolide A is inherently de novo biosynthesized in roots of the medicinal plant ashwagandha (Withania somnifera). Physiol Plant 133:278–287

    Article  CAS  PubMed  Google Scholar 

  • Schluttenhofer C, Pattanaik S, Patra B, Yuan L (2014) Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling. BMC Genomics 15:502. doi:10.1186/1471-2164-15-502

    Article  PubMed  PubMed Central  Google Scholar 

  • Singh AK, Dwivedi V, Rai A, Pal S, Reddy SGE, Rao DKV, Shasany AK, Nagegowda DA (2015) Virus induced gene silencing of Withania somnifera squalene synthase negatively regulates sterol and defence related genes resulting in reduced withanolides and biotic stress tolerance. Plant Biotechnol J. doi:10.1111/pbi.12347

    Google Scholar 

  • Singh S, Pal S, Shanker K, Chanotiya CS, Gupta MM, Dwivedi UN, Shasany AK (2014) Sterol partitioning by HMGR and DXR for routing intermediates towards withanolide biosynthesis. Physiol Plant. doi:10.1111/ppl.12213

    Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Dev GK, Manickavasagam M, Selvaraj N, Ganapathi A (2012) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168(3):681–96

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Kapil DG, Jeyaraj M, Rajesh M, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Increased production of withanolide A, withanone and withaferin A in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114:121–129

    Article  CAS  Google Scholar 

  • Sivanandhan G, Selvaraj N, Ganapathi A, Manickavasagam M (2014) Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor. PLoS ONE 9(8):e104005. doi:10.1371/journal.pone.0104005

    Article  PubMed  PubMed Central  Google Scholar 

  • Stenzel I, Hause B, Miersch O, Kurz T, Maucher H, Weichert H, Ziegler J, Feussner I, Wasternack C (2003) Jasmonate biosynthesis and the allene oxide cyclase family of Arabidopsis thaliana. Plant Mol Biol 51:895–911

    Article  CAS  PubMed  Google Scholar 

  • Suttipanta N, Pattanaik S, Kulshrestha M, Patra B, Singh SK, Yuan L (2011) The transcription factor CrWRKY1 positively regulates the terpenoid indole alkaloid biosynthesis in Catharanthus roseus. Plant Physiol 157(4):2081–2093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants: H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11:1187–1194

    Article  CAS  Google Scholar 

  • Xu Y-H, Wang J-W, Wang S, Wang J-Y, Chen X-Y (2004) Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-δ-cadinene synthase-A. Plant Physiol 135:507–515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang H, Forde BG (1998) An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture. Science 279:407–409

    Article  CAS  PubMed  Google Scholar 

  • Zhang Y, Ye H, Li G (2003) Effect of horseradish peroxidase on the biosynthesis of artemisinin in Artemisia annua in vitro. China J Appl Environ Biol 9:616–618

    CAS  Google Scholar 

  • Zhao J, Davis LC, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333

    Article  CAS  PubMed  Google Scholar 

  • Ziegler J, Stenzel I, Hause B, Maucher H, Miersch O, Hamberg M, Grimm M, Ganal M, Claus Wasternack C (2000) Molecular cloning of allene oxide cyclase: the enzyme establishing the stereochemistry of octadecanoids and jasmonates. J Biol Chem 275:19132–19138

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors express their sincere gratitude to the Director, CIMAP for his keen interest and providing facilities for the experiments. The seed and plant material was provided by the National Gene Bank for Medicinal and Aromatic Plants. This work was supported by CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow-226015 from the Twelfth Five Year Plan project of CSIR (BSC0203). S. P. (AcSIR) was supported by CSIR-SRF.

Author contributions

S. P. and A. K. Y. have helped in the experimentation and analysis; M. M. G. has helped in withanolide and sterol experiments and analysis; R. K. V has helped in the soil and data analysis. A. K.S. and D.A.N. have helped in the analysis of the transcription factors; A.P. and S. R. have helped in raising the polyclonal antibodies for western blot experiments. A. K. S. has helped in the planning, experimentation, analysis, interpretation, and manuscript writing.

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Authors and Affiliations

  1. Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, Uttar Pradesh, India

    Shaifali Pal, Anup Kumar Singh, Shubhra Rastogi, Dinesh A. Nagegowda, Anirban Pal & Ajit Kumar Shasany

  2. Academy of Scientific and Innovative Research, New Delhi, India

    Shaifali Pal & Ajit Kumar Shasany

  3. Analytical Chemistry Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, Uttar Pradesh, India

    Akhilesh Kumar Yadav & Madan Mohan Gupta

  4. Soil Science Division, CSIR-Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP, Lucknow, 226015, Uttar Pradesh, India

    Rajesh Kumar Verma

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  1. Shaifali Pal
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Correspondence to Ajit Kumar Shasany.

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Pal, S., Yadav, A.K., Singh, A.K. et al. Nitrogen treatment enhances sterols and withaferin A through transcriptional activation of jasmonate pathway, WRKY transcription factors, and biosynthesis genes in Withania somnifera (L.) Dunal. Protoplasma 254, 389–399 (2017). https://doi.org/10.1007/s00709-016-0959-x

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