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Piriformospora indica elicitation of withaferin A biosynthesis and biomass accumulation in cell suspension cultures of Withania somnifera

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Abstract

Withania somnifera, is an important medicinal plant in India, also known as Ashwagandha, that contains the bioactive compounds- withanolides and withaferin A. The endophytic fungus Piriformospora indica has been shown to be an elicitor stimulating plant growth and metabolism. Different concentrations of cell homogenate, culture filtrate and individual culture discs of P. indica were added to cell suspension and callus cultures of W. somnifera at different time intervals (10, 15, 20, 25 and 5, 10, 15, 20 days respectively) to observe the effect on cell biomass and withaferin A production. Of all the concentrations of P. indica used to study the effect on withaferin A production in cell suspension cultures, the maximum enhancement was achieved with 3 % cell homogenate (2.04 times), followed by 3 % culture filtrate (1.78 times) and culture disc (1.46 times). Quantitative PCR analysis showed an effect of P. indica elicitation on the regulatory genes of MVA, MEP and withanolides biosynthetic pathways, viz. hmgr, fpps, ss, se, cas, dxs and dxr in callus and cell suspension cultures. The highest gene expression of 11.2, 8.7 and 6.9 times was observed with hmgr among all the expressed genes in cell suspension and callus cultures with 3 % cell homogenate, 3 % culture filtrate and disc respectively, in comparison with the controls.

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Abbreviations

CAS:

Cycloaretenol synthase

DW:

Dry weight

DOXP:

Deoxy xylulose pathway

DXS:

1-deoxy-d-xylulose 5-phosphate synthase

DXR:

1-deoxy-d-xylulose 5-phosphate reductoisomerase

FPPS:

FPP synthase

HMGR:

3-Hydroxy-3-methylglutaryl coenzyme A reductase

HPLC:

High performance liquid chromatography

MS:

Murashige and Skoog

MVA:

Mevalonate pathway

MEP:

Methyl erythritol pathway

RRL:

Regional Research Laboratory

qPCR:

quantitative polymerase chain reaction

SE:

Squalene epoxidase

SS:

Squalene synthase

References

  • Ahmad M, Saleem S, Ahmad AS, Ansari MA, Yousuf S, Hoda MN, et al. (2005) Neuroprotective effects of Withania somnifera on 6-hydroxydopamine induced Parkinsonism in rats. Hum Exp Toxicol 24:137–147

    Article  PubMed  Google Scholar 

  • Archana R, Namasivayam A (1999) Antistressor effect of Withania somnifera. J Ethnopharmacol 64:91–93

    Article  CAS  PubMed  Google Scholar 

  • Atal CK, Schwarting AE (1962) Intraspecific variability in Withania somnifera. I. A preliminary survey. Llyodia 25:78–87

    Google Scholar 

  • Baldi A, Singh D, Dixit VK (2008a) Dual elicitation for improved production of withaferin a by cell suspension cultures of Withania somnifera. Appl Biochem Biotechnol 151:556–564

    Article  CAS  PubMed  Google Scholar 

  • Baldi A, Jain A, Gupta N, Srivastava AK, Bisaria VS (2008b) Co-culture of mycorrhiza-like fungi (Piriformospora indica and Sebacina vermifera) with plant cells of Linum album for enhanced production of podophyllotoxins: a first report. Biotechnol Lett 30:1671–1167

    Article  CAS  PubMed  Google Scholar 

  • Baldi A, Farkya S, Jain A, Gupta N, Mehra R, Bisaria VS, et al. (2010) Enhanced production of podophyllotoxins by co-culture of transformed linum album cells with plant growth-promoting fungi. Pure Appl Chem 82(1):227–241

    Article  CAS  Google Scholar 

  • Banerjee S, Naqvi AA, Mandal S, Ahuja PS (1994) Transformation of Withania somnifera (L.) Dunal by Agrobacterium rhizogenes: infectivity and phytochemical studies. Phytother Res 8:452–455

    Article  CAS  Google Scholar 

  • Bhattacharya SK, Muruganandam AV (2003) Adaptogenic activity of Withania somnifera an experimentalstudy using a rat model of chronic stress. Pharmacol Biochem Behav 75:547–555

    Article  CAS  PubMed  Google Scholar 

  • Bhattacharya SK, Satyan KS, Chakrabarti A (1997a) Effect of trasina, an ayurvedic herbal formulation, on pancreatic islet superoxide dismutase activity in hyperglycaemic rats. Indian J Exp Biol 35:297–299

    CAS  PubMed  Google Scholar 

  • Bruneton J (1999) Pharmacognosy, Phytochemistry, Medicinal plants. 2nd edn. Lavoisier, Paris. 464

  • Chaturvedi HC, Jain M, Kidwai NR (2007) Cloning of medicinal plants through tissue culture. J Exp Biol 45:937–948

    CAS  Google Scholar 

  • Chaudhary G, Sharma U, Jagannathan NR, Gupta YK (2003) Evaluation of Withania somnifera in a middle cerebral artery occlusion model of stroke in rats. Clin Exp Pharmacol Physiol 30:399–404

    Article  CAS  PubMed  Google Scholar 

  • Chaurasiya ND, Sangwan NS, Sabir F, Misra LN, Sangwan RS (2012) Withanolide biosynthesis recruits both mevalonate and DOXP pathways of isoprenogenesis in Ashwagandha Withania somnifera L. (Dunal). Plant Cell Rep. doi:10.1007/s00299-012-1302-4

    PubMed  Google Scholar 

  • Ciddi V (2006) Withaferin A from cell cultures of Withania somnifera. Indian J Pharm Sci 68:490–492

    Article  CAS  Google Scholar 

  • Devi PU, Sharada AC, Solomon FE, Kamath MS (1992) In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor Sarcoma 180. Indian J Exp Biol 30:169–172

    CAS  PubMed  Google Scholar 

  • Dhar RS, Verma V, Suri KA, Sangwan RS, Satti NK, Qazi GN, et al. (2006) Phytochemical and genetic analysis in selected chemotypes of Withania somnifera. Phytochem 67:2269–2276

    Article  CAS  Google Scholar 

  • Dhuley JN (1998) Effect of ashwagandha on lipid peroxidation in stress-induced animals. J Ethnopharmacol 60:173–178

    Article  CAS  PubMed  Google Scholar 

  • Dhuley JN (2001) Nootropic-like effect of ashwagandha (Withania somnifera L) in mice. Phytother Res 15:524–528

    Article  CAS  PubMed  Google Scholar 

  • Eilert U (1987) Elicitation: methodology and aspects of application. In: Constabel F, Vasil I (eds) Cell culture and somatic cell genetics of plants. Academic Press, San Diego, pp. 153–196

    Google Scholar 

  • Franken P (2012) The plant strengthening root endophyte Piriformospora indica: potential application and the biology behind. Appl Microbiol Biotechnol 96:1455–1464

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Furmanowa M, Gajdzis-Kuls D, Ruszkowska J, Czarnocki Z, Obidoska G, Upadhyay SN, et al. (2001) In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity. Planta Med 67:146–149

    Article  CAS  PubMed  Google Scholar 

  • Ganzera M, Choudhary MI, Khan IA (2003) Quantitative HPLC analysis of withanolides in Withania somnifera. Fitoterapia 74:68–76

    Article  CAS  PubMed  Google Scholar 

  • Jain S, Shukla SD, Sharma K, et al. (2001) Neuroprotective effects of Withania somnifera Dunn in hippocampal sub-regions of female albino rat. Phytother Res 15:544–548

    Article  CAS  PubMed  Google Scholar 

  • Kaefer E (1977) Meiotic and mitotic recombination in Aspergillus and its chromosomal aberrations. Adv Genet 19:33–131

    Article  Google Scholar 

  • Ketchum REB, Gibson DM, Croteau RB, Shuler ML (1999) The kinetics of taxoid accumulation in cell suspension culture of taxus following elicitation with methyl jasmonate. Biotechnol Bioeng 62:97–105

    Article  CAS  PubMed  Google Scholar 

  • Kirson I, Glotter E, Lavie D, Abraham A (1971) Constituents of Withania somnifera Dun part XII the withanolides of an Indian chemotype. J Chem Soc C:Org 2032–2044

  • Kumar A, Kaul MK, Bhan MK, Khanna P, Suri KA (2007) Morphological and chemical variation in 25 collections of the Indian medicinal plant, Withania somnifera (L.) Dunal (Solanaceae). Genet Resour Crop Evol 54:655–660

    Article  CAS  Google Scholar 

  • Kumar V, Rajauria G, Sahai V, Bisaria VS (2012) Culture filtrate of root endophytic fungus Piriformospora indica promotes the growth and lignan production of Linum album hairy root cultures. Process Biochem 47:901–907

    Article  CAS  Google Scholar 

  • Malla R, Singh A, Zeyaullah MD, Yadav V, Verma A, Varma A, Rai M (2002) Piriformospora indica and plant growth promoting rhizobacteria: an appraisal. In: Rao GP, Manoharchari C, Bhat DJ, Rajak RC, Lakhanpal TN (eds) Frontiers of fungal diversity in India. International Book Distributing Co, Lucknow, pp. 401–419

    Google Scholar 

  • Marero LM, Jin JH, Shin JH, Lee HJ, Chung IS, Lee HJ (1997) Effect of fungal elicitation on indirubin production from a suspension culture of Polygonum tinctorium. Enzym Microb Technol 21:97–101

    Article  CAS  Google Scholar 

  • MaujiRam KMA, Jha P, Khan S, Kiran U, Abdin MZ, et al. (2010) HMG-CoA reductase limits artemisinin biosynthesis and accumulation in Artemisia annua L. plants. Acta Physiol Plant 32:859–866

    Article  Google Scholar 

  • Mir BA, Koul S, Kumar A, Kaul MK, Soodan AS, Raina SN (2010) Intraspecific variation in the internal transcribed spacer (ITS) regions of rDNA in Withania somnifera (L.) Dunal. Indian J Biotechnol 9:325–328

    CAS  Google Scholar 

  • Mirjalili MH, Fakhr-Tabatabaei SM, Alizadeh H, Ghassempour A, Mirzajani F (2009) Genetic and withaferin A analysis of Iranian natural populations of withania somnifera and W. coagulans by RAPD and HPTLC. Nat Prod Commun 4:337–346

    CAS  PubMed  Google Scholar 

  • Mishra LC, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (ashwagandha) a review. Altern Med Rev 5:334–346

    CAS  PubMed  Google Scholar 

  • Mohan R, Hammers HJ, Bargagna-Mohan P, Zhan XH, Herbstritt CJ, Ruiz A, et al. (2004) Withaferin a is a potent inhibitor of angiogenesis. Angiogenesis 7:115–122

    Article  CAS  PubMed  Google Scholar 

  • Mohanty I, Arya DS, Dinda A, Talwar KK, Joshi S, Gupta SK (2004) Mechanisms of cardioprotective effect of Withania somnifera in experimentally induced myocardial infarction. Basic Clin Pharmacol Toxicol 94:184–190

    Article  CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497

    Article  CAS  Google Scholar 

  • Murphy JG, Rafferty SM, Cassells AC (2000) Stimulation of wild strawberry (Fragaria vesca) arbuscular mycorrhizas by addition of shellfish waste to the growth substrate: interaction between mycorrhization, substrate amendment and susceptibility to red core (Phytophthora fragariae). Appl Soil Ecol 15:153–158

    Article  Google Scholar 

  • Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Paek KY, et al. (2008) Establishment of Withania somnifera hairy root cultures for the production of withanolide a. J Int Plant Biol 50:975–981

    Article  CAS  Google Scholar 

  • Nagella P, Murthy HN (2010) Establishment of cell suspension cultures of Withania somnifera for the production of withanolide a. Bioresour Technol 101:6735–6739

    Article  CAS  PubMed  Google Scholar 

  • Nagella P, Murthy HN (2011) Effects of macroelements and nitrogen source on biomass accumulation and withanolide-a production from cell suspension cultures of Withania somnifera (L.) Dunal. Plant Cell Tissue Organ Cult 104:119–124

    Article  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • Namdeo A, Patil S, Fulzele DP (2002) Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus. Biotechnol Prog 18:159–162

    Article  CAS  PubMed  Google Scholar 

  • Negi MS, Singh A, Lakshmikumaran M (2000) Genetic variation and relationship among and within Withania species as revealed by AFLP markers. Genome 43:975–980

    Article  CAS  PubMed  Google Scholar 

  • Negi MS, Sabharwal V, Wilson N, Lakshmikumaran MS (2006) Comparative analysis of the efficiency of SAMPL and AFLP in assessing genetic relationships among Withania somnifera genotypes. Curr Sci 91:464–471

    CAS  Google Scholar 

  • Panda S, Kar A (1997) Evidence for free radical scavenging activity of Ashwagandha root powder in mice. Indian J Physiol Pharmacol 41:424–426

    CAS  PubMed  Google Scholar 

  • Panda S, Kar A (1998) Changes in thyroid hormone concentrations after administration of ashwagandha root extract to adult male mice. J Pharm Pharmacol 50:1065–1068

    Article  CAS  PubMed  Google Scholar 

  • Pozo M, Cordier C, Dumas-Gautod E, Gianinazzi SJ, Barea JM, Azcon-Aguilar C (2002) Localized versus systemic effect of arbuscular mycorrhizal fungi on defence responses to Phytophthora infection in tomato plants. J Exp Bot 53:525–534

    Article  CAS  PubMed  Google Scholar 

  • Rai M, Acharya D, Singh A, Varma A (2001) Positive growth responses of the medicinal plants Spilanthes calva and Withania somnifera to inoculation by Piriformospora indica in a field trial. Mycorrhiza 11:123–128

    Article  CAS  PubMed  Google Scholar 

  • Rai D, Bhatia G, Sen T, Palit G (2003) Anti-stress effects of Ginkgo biloba and Panax ginseng a comparative study. J Pharmacol Sci 93:458–464

    Article  CAS  PubMed  Google Scholar 

  • Ramachandra Rao S, Ravishankar GA (2002) Plant cell cultures:chemical factories of secondary metabolites. Biotechnol Adv 20:10–153

    Article  Google Scholar 

  • Ray S, Jha S (2001) Production of withaferin a in shoot cultures of Withania somnifera Dunal. Planta Med 67:432–437

    Article  CAS  PubMed  Google Scholar 

  • Ray S, Ghosh B, Se S (1996) Withanolide production by root cultures of Withania somnifera transformed with Agrobacterium rhizogens. Planta Med 62:571–573

    Article  CAS  PubMed  Google Scholar 

  • Rijhwani S, Shanks JV (1998) Effect of elicitor dosage and exposure time on biosynthesis of indole alkaloids by Catharanthus roseus hairy root cultures. Biotechnol Prog 14:442–449

    Article  CAS  PubMed  Google Scholar 

  • Roja G, Heble MR (1991) Tissue cultures of Withania somnifera: morphogenesis and withanolide synthesis. Phytother Res 5:185–187

    Article  CAS  Google Scholar 

  • Sabir F, Sangwan RS, Singh J, Misra LN, Pathak N, Sangwan NS (2011) Biotransformation of withanolides by cell suspension cultures of Withania somnifera (Dunal). Plant Biotech Rep 5:127–134

    Article  Google Scholar 

  • Sangwan RS, Chaurasiya NS, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide a biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera Dunal), a main medicinal plant in Ayurveda. Chem Pharm Bull 55:1371–1375

    Article  CAS  PubMed  Google Scholar 

  • Schaller H, Grausem B, Benveniste P, Chye ML, Tan CT, et al. (1995) Expression of the Hevea brasiliensis (H.B.K.) müll. Arg. 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 in tobacco results in sterol overproduction. Plant Physiol 109:761–770

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sharma G, Agrawal V (2013) Marked enhancement in the artemisinin content and biomass productivity in Artemisia annua L. shoots co-cultivated with Piriformospora indica. World J Microbiol Biotechnol 29:1133–1138

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, et al. (2012a) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Ind Crop Prod 37:124–129

    Article  CAS  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, et al. (2012b) 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 168:681–696

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Muthuselvam M, et al. (2013a) A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma 250:885–898

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Manickavasagam M, et al. (2013b) 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 9:104005

    Article  Google Scholar 

  • Umadevi M, Rajeswari R, Sharmila Rahale C, Selvavenkadesh S, Pushpa R, Bhowmik D (2012) et al Traditional and medicinal uses of Withania somnifera. The Pharma Innovation 1:102–110

  • Varma A, Sudha S, Franken P (1999) Piriformospora indica- a cultivable plant growth promoting root endophyte with similarities to arbuscular mycorrhizal fungi. Appl Environ Microbiol 65:2741–2744

    CAS  PubMed  PubMed Central  Google Scholar 

  • Varma A, Bakshi M, Lou B, Hartmann A, Oelmueller R (2012) Piriformospora indica: a novel plant growth promoting mycorrhizal fungus. Agric Res 1:117–131

    Article  Google Scholar 

  • Verma S, Varma A, Rexer KH, Kost G, Sarbhoy A, Bisen P, Butehorn B, Franken P (1998) Piriformospora indica, gen. nov. sp. nov., a new root-colonizing fungus. Mycologia 95:896–903

    Article  Google Scholar 

  • Verpoorte R, Contin A, Memelink J (2002) Biotechnology for the production of plant secondary metabolites. Phytochem Rev 1:13–25

    Article  CAS  Google Scholar 

  • Vitali G, Conte L, Nicoletti M (1996) Withanolide composition and in vitro culture of Italian Withania somnifera. Planta Med 62:287–288

    Article  CAS  PubMed  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 

  • Ziauddin M, Phansalkar N, Patki P, Diwanay S, Patwardhan B (1996) Studies on the immunomodulatory effects of Ashwagandha. J Ethnopharmacol 50:69–76

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Seema Ahlawat is thankful to Department of Science and Technology, Government of India, for providing WoS-A Fellowship.

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Ahlawat, S., Saxena, P., Ali, A. et al. Piriformospora indica elicitation of withaferin A biosynthesis and biomass accumulation in cell suspension cultures of Withania somnifera . Symbiosis 69, 37–46 (2016). https://doi.org/10.1007/s13199-015-0364-9

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