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Biotic elicitors enhance diosgenin production in Helicteres isora L. suspension cultures via up-regulation of CAS and HMGR genes

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Abstract

In an attempt to find an alternative and potent source of diosgenin, a steroidal saponin in great demand for its pharmaceutical importance, Helicteres isora suspension cultures were explored for diosgenin extraction. The effect of biotic elicitors on the biosynthesis of diosgenin, in suspension cultures of H. isora was studied. Bacterial as well as fungal elicitors such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Aspergillus niger were applied at varying concentrations to investigate their effects on diosgenin content. The HPLC based quantification of the treated samples proved that amongst the biotic elicitors, E. coli (1.5%) proved best with a 9.1-fold increase in diosgenin content over respective control cultures. Further, the scaling-up of the suspension culture to shake-flask and ultimately to bioreactor level were carried out for production of diosgenin. During all the scaling-up stages, diosgenin yield obtained was in the range between 7.91 and 8.64 mg l−1, where diosgenin content was increased with volume of the medium. The quantitative real-time PCR (qRT-PCR) analysis showed biotic elicitors induced the expression levels of regulatory genes in diosgenin biosynthetic pathway, the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and cycloartenol synthase (CAS), which can be positively correlated with elicited diosgenin contents in those cultures. The study holds significance as H. isora represents a cleaner and easy source of diosgenin where unlike other traditional sources, it is not admixed with other steroidal saponins, and the scaled-up levels of diosgenin achieved herein have the potential to be explored commercially.

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References

  • Ahlawat S, Saxena P, Ali A, Abdin MZ (2016) Piriformospora indica elicitation of withaferin A biosynthesis and biomass accumulation in cell suspension cultures of Withania somnifera. Symbiosis 69:37–46

    CAS  Google Scholar 

  • Ahlawat S, Saxena P, Ali A, Khan S, Abdin MZ (2017) Comparative study of withanolide production and the related transcriptional responses of biosynthetic genes in fungi elicited cell suspension culture of Withania somnifera in shake flask and bioreactor. Plant Physiol Biochem 114:19–28

    CAS  PubMed  Google Scholar 

  • Ahmed SA, Baig MM (2014) Biotic elicitor enhanced production of psoralen in suspension cultures of Psoralea corylifolia L. Saudi J Biol Sci 21(5):499–504

    CAS  PubMed  PubMed Central  Google Scholar 

  • Algar E, Gutierrez-Mañero FJ, Bonilla A, Lucas JA, Radzki W, Ramos-Solano B (2012) Pseudomonas fluorescens N21.4 metabolites enhance secondary metabolism isoflavones in soybean (Glycine max) calli cultures. J Agric Food Chem 60(44):11080–11087

    CAS  PubMed  Google Scholar 

  • Babiychuk E, Bouvier-Navé P, Compagnon V, Suzuki M, Muranaka T, Van Montagu M, Kushnir S, Schaller H (2008) Allelic mutant series reveal distinct functions for Arabidopsis cycloartenol synthase 1 in cell viability and plastid biogenesis. Proc Natl Acad Sci USA 105(8):3163–3168

    CAS  PubMed  Google Scholar 

  • Barik B, Dey AK, Das PC (1998) Helicteres isora Linn, a new source of diosgenin. Indian J Chem 20(B):938

    Google Scholar 

  • Benveniste P (2004) Biosynthesis and accumulation of sterols. Ann Rev Plant Biol 55:429–457

    CAS  Google Scholar 

  • Biswas T, Kalra A, Mathur AK, Lal RK, Singh M, Mathur A (2016) Elicitors influenced differential ginsenoside production and exudation into medium with concurrent Rg3/Rh2 panaxadiol induction in Panax quinquefolius cell suspensions. Appl Microbiol Biotechnol 100(11):4909–4922

    CAS  PubMed  Google Scholar 

  • Chattopadhyay S, Srivastava AK, Bhojwani SS, Bisaria VS (2002) Production of podophyllotoxin by plant cell cultures of Podophyllum hexandrum in bioreactor. J Biosci Bioeng 93(2):215–220

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  • Chen X, Mou Y, Ling J, Wang N, Wang X, Hu J (2015) Cyclic dipeptides produced by fungus Eupenicillium brefeldianum HMP-F96 induced extracellular alkalinization and H2O2 production in tobacco cell suspensions. World J Microbiol Biotechnol 31(1):247–253

    PubMed  Google Scholar 

  • Chiu CS, Chiu YJ, Wu LY, Lu TC, Huang TH, Hsieh MT, Lu CY, Peng WH (2011) Diosgenin ameliorates cognition deficit and attenuates oxidative damage in senescent mice induced by D-galactose. Am J Chin Med 39:551–563

    CAS  PubMed  Google Scholar 

  • Chodisetti B, Rao K, Gandi S, Giri A (2013) Improved gymnemic acid production in the suspension cultures of Gymnema sylvestre through biotic elicitation. Plant Biotechnol Rep 7(4):519–525

    Google Scholar 

  • Ciura J, Szeliga M, Tyrka M (2015) Optimization of in vitro culture conditions for accumulation of diosgenin by fenugreek. J Med Plants 3(3):22–25

    Google Scholar 

  • Dangi R, Misar A, Tamhankar S, Rao S (2014) Diosgenin content in some Trigonella species. Indian J Adv Plant Res 1:47–51

    CAS  Google Scholar 

  • Deshpande HA, Bhalsing SR (2014) Isolation and characterization of diosgenin from in vitro cultured tissues of Helicteres isora L. Physiol Mol Biol Plants 20(1):89–94

    CAS  PubMed  Google Scholar 

  • Deshpande HA, Bhalsing SR (2015) Plant derived novel biomedicinal: diosgenin. Int J Pharmacog Phytochem Res 6:780–784

    Google Scholar 

  • Diarra ST, He J, Wang J, Li J (2013) Ethylene treatment improves diosgenin accumulation in in vitro cultures of Dioscorea zingiberensis via up-regulation of CAS and HMGR gene expression. Electron J Biotechnol 16(5):6. https://doi.org/10.2225/vol16-issue5-fulltext-9

    Article  CAS  Google Scholar 

  • Gas-Pascual E, Simonovik B, Schaller H, Bach TJ (2015) Inhibition of cycloartenol synthase (CAS) function in tobacco BY-2 cells. Lipids 50(8):761–772

    CAS  PubMed  Google Scholar 

  • Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48(12):909–930

    CAS  PubMed  Google Scholar 

  • Gomez P, Ortuno A, Del Río JA (2004) Ultrastructural changes and diosgenin content in cell suspensions of Trigonella foenum-graecum L. by ethylene treatment. Plant Growth Regul 44(2):93–99

    CAS  Google Scholar 

  • Gupta R, Singh A, Srivastava M, Singh V, Gupta MM, Pandey R (2017) Microbial modulation of bacoside A biosynthetic pathway and systemic defense mechanism in Bacopa monnieri under Meloidogyne incognita stress. Scientific Reports 7:41867. https://doi.org/10.1038/srep41867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hao S, Xu R, Li D, Zhu Z, Wang T, Liu K (2015) Attenuation of streptozotocin-induced lipid profile anomalies in the heart, brain, and mRNA expression of HMG-CoA reductase by diosgenin in rats. Cell Biochem Biophys 72(3):741–749

    CAS  PubMed  Google Scholar 

  • Hussain MS, Fareed S, Saba Ansari M, Rahman A, Ahmad IZ, Saeed M (2012) Current approaches toward production of secondary plant metabolites. J Pharm Bioallied Sci 4(1):10

    PubMed  PubMed Central  Google Scholar 

  • Hwang HS, Lee H, Choi YE (2015) Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis. BMC Genomics 16(1):180

    PubMed  PubMed Central  Google Scholar 

  • Ibraheim SK (2014) Effect of foliar spray with some biostimulants on growth, yield and seeds quality of pea plants grown in sandy soil. J Appl Sci Res 10(5):400–407

    CAS  Google Scholar 

  • Jaisi A, Panichayupakaranant P (2016) Increased production of plumbagin in Plumbago indica root cultures by biotic and abiotic elicitors. Biotechnol Lett 38(2):351–355. https://doi.org/10.1007/s10529-015-1969-z

    Article  CAS  PubMed  Google Scholar 

  • Jayachandran KS, Vasanthi HR, Rajamanickam GV (2009) Antilipoperoxidative and membrane stabilizing effect of diosgenin, in experimentally induced myocardial infarction. Mol Cell Biochem 327:203–210

    CAS  PubMed  Google Scholar 

  • Khanahmadi M, Paek KY (2017) Bioreactor technology for sustainable production of valuable plant metabolites: challenges and advances. In: Abdullah S, Chai-Ling H, Wagstaff C (eds) Crop improvement. Springer, Berlin, pp 169–189. https://doi.org/10.1007/978-3-319-65079-1_8

    Chapter  Google Scholar 

  • Kumar V, Desai D, Shriram V (2014) Hairy root induction in Helicteres isora L. and production of diosgenin in hairy roots. Nat Prod Bioprospect 4:107–112. https://doi.org/10.1007/s13659-014-0011-9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kumar P, Chaturvedi R, Sundar D, Bisaria VS (2016) Piriformospora indica enhances the production of pentacyclic triterpenoids in Lantana camara L. suspension cultures. Plant Cell Tissue Organ Cult 125(1):23–29

    CAS  Google Scholar 

  • Kusakari K, Yokoyama M, Inomata S, Gozu Y, Katagiri C, Sugimoto Y (2012) Large-scale production of saikosaponins through root culturing of Bupleurum falcatum L. using modified airlift reactors. J Biosci Bioeng 113(1):99–105

    CAS  PubMed  Google Scholar 

  • Lattanzio V, Lattanzio VMT, Cardinali A (2006) Role of phenolics in the resistance mechanisms of plants against fungal pathogens and insects. In: Imperato F (ed) Phytochemistry: advances in research. Research Signpost, Kerala, pp 23–67

    Google Scholar 

  • Le KC, Im WT, Paek KY, Park SY (2018) Biotic elicitation of ginsenoside metabolism of mutant adventitious root culture in Panax ginseng. Appl Microbiol Biotechnol 102(4):1687–1697

    CAS  PubMed  Google Scholar 

  • Leivar P, Antolín-Llovera M, Ferrero S, Closa M, Arró M, Ferrer A, Boronat A, Campos N (2011) Multilevel control of Arabidopsis 3-hydroxy-3-methylglutaryl coenzyme A reductase by protein phosphatase 2A. Plant Cell 23:1494–1511

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li P, Mao Z, Lou J, Li Y, Mou Y, Lu S, Peng Y, Zhou L (2011a) Enhancement of diosgenin production in Dioscorea zingiberensis cell cultures by oligosaccharides from its endophytic fungus Fusarium oxysporum Dzf17. Molecules 16(12):10631–10644

    PubMed  PubMed Central  Google Scholar 

  • Li P, Mou Y, Shan T, Xu J, Li Y, Lu S, Zhou L (2011b) Effects of polysaccharide elicitors from endophytic Fusarium oxysporium Dzf17 on growth and diosgenin production in cell suspension culture of Dioscorea zingiberensis. Molecules 16(11):9003–9016

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li P, Mou Y, Lu S, Sun W, Lou J, Yin C, Zhou L (2012) Quantitative determination of diosgenin in Dioscorea zingiberensis cell cultures by microplate-spectrophotometry and high-performance liquid chromatography. Afr J Pharm Pharmacol 6:1186–1193

    CAS  Google Scholar 

  • Liagre B, Vergne-Salle P, Corbiere C, Charissoux JL, Beneytout JL (2004) Diosgenin, a plant steroid, induces apoptosis in human rheumatoid arthritis synoviocytes with cyclooxygenase-2 overexpression. Arthritis Res Ther 6:R373

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lu M, Wong H, Teng W (2001) Effects of elicitation on the production of saponin in cell culture of Panax ginseng. Plant Cell Rep 20(7):674–677

    CAS  Google Scholar 

  • Manero FJ, Algar E, Martín Gómez MS, Saco Sierra MD, Solano BR (2012) Elicitation of secondary metabolism in Hypericum perforatum by rhizosphere bacteria and derived elicitors in seedlings and shoot cultures. Pharmac Biol 50(10):1201–1209

    CAS  Google Scholar 

  • Mirunalini S, Shahira R (2011) Novel effect of diosgenin—a plant derived steroid: a review. Pharmacologyonline 1:726–736

    Google Scholar 

  • Morlacchi P, Wilson WK, Xiong Q, Bhaduri A, Sttivend D, Kolesnikova MD, Matsuda SP (2009) Product profile of PEN3: the last unexamined oxidosqualene cyclase in Arabidopsis thaliana. Org Lett 11(12):2627–2630

    CAS  PubMed  Google Scholar 

  • Mulabagal V, Tsay HS (2004) Plant cell cultures-an alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng 2(1):29–48

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497

    CAS  Google Scholar 

  • Narayani M, Srivastava S (2017) Elicitation: a stimulation of stress in in vitro plant cell/tissue cultures for enhancement of secondary metabolite production. Phytochem Rev 16(6):1227–1252

    CAS  Google Scholar 

  • Nes WD (2011) Biosynthesis of cholesterol and other sterols. Chem Rev 111(10):6423–6451

    CAS  PubMed  PubMed Central  Google Scholar 

  • Nogues I, Brilli F, Loreto F (2006) Dimethylallyl diphosphate and geranyl diphosphate pools of plant species characterized by different isoprenoid emissions. Plant Physiol 141(2):721–730

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pal T, Malhotra N, Chanumolu SK, Chauhan RS (2015) Next-generation sequencing (NGS) transcriptomes reveal association of multiple genes and pathways contributing to secondary metabolites accumulation in tuberous roots of Aconitum heterophyllum Wall. Planta 242(1):239–258

    CAS  PubMed  Google Scholar 

  • Patel K, Gadewar M, Tahilyani V, Patel DK (2012) A review on pharmacological and analytical aspects of diosgenin: a concise report. Nat Prod Bioprospect 2:46–52

    CAS  PubMed Central  Google Scholar 

  • Patra N, Srivastava AK (2016) Artemisinin production by plant hairy root cultures in gas-and liquid-phase bioreactors. Plant Cell Rep 35(1):143–153

    CAS  PubMed  Google Scholar 

  • Phillips DR, Rasbery JM, Bartel B, Matsuda SP (2006) Biosynthetic diversity in plant triterpene cyclization. Curr Opin Plant Biol 9(3):305–314

    CAS  PubMed  Google Scholar 

  • Rao SR, Ravishankar GA (2002) Plant cell cultures: chemical factories of secondary metabolites. Biotechnol Adv 20(2):101–153

    CAS  PubMed  Google Scholar 

  • Rokem JS, Tal B, Goldberg I (1985) Methods for increasing diosgenin production by Dioscorea cells in suspension cultures. J Nat Prod 48(2):210–222

    CAS  Google Scholar 

  • Selim S, Al Jaouni S (2015) Anticancer and apoptotic effects on cell proliferation of diosgenin isolated from Costus speciosus (Koen.) Sm. BMC Compl Altern Med 15:301

    Google Scholar 

  • Sethi G, Shanmugam M, Warrier S, Merarchi M, Arfuso F, Kumar A, Bishayee A (2018) Pro-apoptotic and anti-cancer properties of diosgenin: a comprehensive and critical review. Nutrients 10:645

    PubMed Central  Google Scholar 

  • Shaikh S, Shriram V, Khare T, Kumar V (2018) Establishment of callus and cell suspension cultures of Helicteres isora L. Res Plant Biol 8:1–4. https://doi.org/10.25081/ripb.2018.v8.3366

    Article  CAS  Google Scholar 

  • Sharma S, Malhotra N, Sood H (2016) Expression analysis of steroid pathway genes revealed positive correlation with diosgenin biosynthesis in Trillium govanianum. Acta Physiol Plant 38(11):272

    Google Scholar 

  • Simic SG, Tusevski O, Maury S, Hano C, Delaunay A, Chabbert B, Lamblin F, Lainé E, Joseph C, Hagège D (2015) Fungal elicitor-mediated enhancement in phenylpropanoid and naphtodianthrone contents of Hypericum perforatum L. cell cultures. Plant Cell Tissue Organ Cult 122(1):213–226

    Google Scholar 

  • Singh RS, Gara RK, Bhardwaj PK, Kaachra A, Malik S, Kumar R, Sharma M, Ahuja PS, Kumar S (2010) Expression of 3-hydroxy-3-methylglutaryl-CoA reductase, p-hydroxybenzoate-m-geranyltransferase and genes of phenylpropanoid pathway exhibits positive correlation with shikonins content in arnebia [Arnebia euchroma (Royle) Johnston]. BMC Mol Biol 11(1):88

    PubMed  PubMed Central  Google Scholar 

  • Sirotkin AV, Alexa R, Alwasel S, Harrath AH (2019) The phytoestrogen, diosgenin, directly stimulates ovarian cell functions in two farm animal species. Domest Animal Endocrinol 69:35–41

    CAS  Google Scholar 

  • Son SH, Choi SM, Lee YH, Choi KB, Yun SR, Kim JK, Park HJ, Kwon OW, Noh EW, Seon JH, Park YG (2000) Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor. Plant Cell Rep 19(6):628–633

    CAS  PubMed  Google Scholar 

  • Suzuki M, Kamide Y, Nagata N, Seki H, Ohyama K, Kato H, Masuda K, Sato S, Kato T, Tabata S, Yoshida S (2004) Loss of function of 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) in Arabidopsis leads to dwarfing, early senescence and male sterility, and reduced sterol levels. Plant J 37(5):750–761

    CAS  PubMed  Google Scholar 

  • Tashackori H, Sharifi M, Chashmi NA, Safaie N, Behmanesh M (2016) Induced-differential changes on lignan and phenolic acid compounds in Linum album hairy roots by fungal extract of Piriformospora indica. Plant Cell Tissue Organ Cult 127(1):187–194

    CAS  Google Scholar 

  • Wang YJ, Pan KL, Hsieh TC, Chang TY, Lin WH, Hsu JT (2011) Diosgenin, a plant-derived sapogenin, exhibits antiviral activity in vitro against hepatitis C virus. J Nat Prod 74(4):580–584

    CAS  PubMed  Google Scholar 

  • Werner S, Maschke RW, Eibl D, Eibl R (2017) Bioreactor technology for sustainable production of plant cell-derived products. In: Pavlov A, Bley T (eds) Bioprocessing of plant in vitro systems. Springer, Berlin, pp 1–20. https://doi.org/10.1007/978-3-319-32004-5_6-1

    Chapter  Google Scholar 

  • Xue Z, Duan L, Liu D, Guo J, Ge S, Dicks J, ÓMáille P, Osbourn A, Qi X (2012) Divergent evolution of oxidosqualene cyclases in plants. New Phytol 193(4):1022–1038

    CAS  PubMed  Google Scholar 

  • Yang D, Fang Y, Xia P, Zhang X, Liang Z (2018) Diverse responses of tanshinone biosynthesis to biotic and abiotic elicitors in hairy root cultures of Salvia miltiorrhiza and Salvia castanea Diels f. tomentosa. Gene 643:61–67

    CAS  PubMed  Google Scholar 

  • Zhang Y, Tang L, An X, Fu E, Ma C (2009) Modification of cellulase and its application to extraction of diosgenin from Dioscorea zingiberensis CH Wright. Biochem Eng J 47(1–3):80–86

    Google Scholar 

  • Zhou L, Yang G, Sun H, Tang J, Yang J, Wang Y, Garran TA, Guo L (2017) Effects of different doses of cadmium on secondary metabolites and gene expression in Artemisia annua L. Front Med 11(1):137–146

    PubMed  Google Scholar 

  • Zhu YL, Huang W, Ni JR, Liu W, Li H (2010) Production of diosgenin from Dioscorea zingiberensis tubers through enzymatic saccharification and microbial transformation. Appl Microbiol Biotechnol 85(5):1409–1416

    CAS  PubMed  Google Scholar 

  • Zhu JH, Li HL, Guo D, Wang Y, Dai HF, Mei WL, Peng SQ (2018) Identification, characterization and expression analysis of genes involved in steroidal saponin biosynthesis in Dracaena cambodiana. J Plant Res 131:555–562

    CAS  PubMed  Google Scholar 

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Acknowledgements

The financial support for conducting this from Savitribai Phule Pune University, Pune in the form of Research Grant (No. OSD/BCUD/392/132) is acknowledged.

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Shaikh, S., Shriram, V., Khare, T. et al. Biotic elicitors enhance diosgenin production in Helicteres isora L. suspension cultures via up-regulation of CAS and HMGR genes. Physiol Mol Biol Plants 26, 593–604 (2020). https://doi.org/10.1007/s12298-020-00774-6

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