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Innate endophytic fungus, Aspergillus terreus as biotic elicitor of withanolide A in root cell suspension cultures of Withania somnifera

  • Ramesh Kumar Kushwaha
  • Sucheta Singh
  • Shiv Shanker Pandey
  • Alok Kalra
  • Chikkarasanahalli Shivegowda Vivek BabuEmail author
Original Article
  • 31 Downloads

Abstract

In the present study, root cell suspension cultures of W. somnifera were elicited with mycelial extract (1% w/v) and culture filtrate (5% v/v) of their native endophytic fungus Aspergillus terreus 2aWF in shake flask. Culture filtrate of A. terreus 2aWF significantly elicits withanolide A at 6H (12.20 ± 0.52 µg/g FCB). However, with A. terreus 2aWF mycelial extract, withanolide A content was higher at 24H (10.29 µg/g FCB). Withanolide A content was maximum with salicylic acid (0.1 mM) treatment at 24H (8.3 ± 0.20 µg/g FCB). Further, expression analysis of withanolide pathway genes, hydrogen peroxide production, and lipid peroxidation was carried out after 48H of elicitation with 2aWF mycelial extract and culture filtrate. The expression levels of withanolides biosynthetic pathway genes, viz. HMGR, DXR, FPPS, SQS, SQE, CAS, SMT1, STE1 and CYP710A1 were quantified by real time PCR at 48H of elicitation. In all the treatments, the expression levels of key genes were significantly upregulated as compared to untreated suspension cells. Hydrogen peroxide was noticeably enhanced in SA, mycelia extract and culture filtrate, at 20% (115 ± 4.40 nM/g FCB), 42% (137.5 ± 3.62 nM/g FCB), and 27% (122.8 ± 1.25 nM/g FCB) respectively; however, lipid peroxidation was 0.288 ± 0.014, 0.305 ± 0.041 and 0.253 ± 0.007 (µM/gm FCB) respectively, higher than the control (0.201 ± 0.007 µM/gm FCB).

Keywords

Withania somnifera Withanolide A Endophyte Elicitor Suspension culture 

Notes

Acknowledgements

This work was supported by grant NWP BSC0117 (XII 5 Year Plan Network Project) from the Council of Scientific and Industrial Research (CSIR), India. Sincere thanks to Director, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, India for his encouragement. R. K. Kushwaha greatly acknowledges, ICMR for financial assistance in the form of fellowship and contingency grant for research activity. SSP acknowledges CSIR, India for financial assistance in the form of Senior Research Associateship (SRA). C. S. Vivek Babu and R. K. Kushwaha greatly acknowledges Dr. Dinesh A. Nagegowda for providing withanolides standards and primers and Dr. D. K. Venkata Rao for sharing his lab facilities.

Author contributions

RKK performed all the experiments and CSV analyzed and prepared the manuscript. All authors read and approved the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11033_2019_4641_MOESM1_ESM.docx (586 kb)
Supplementary material 1 (DOCX 585 KB)

References

  1. 1.
    Raghavan A, Shah ZA (2015) Withania somnifera: a pre-clinical study on neuroregenerative therapy for stroke. Neural Regen Res 10(2):183–185Google Scholar
  2. 2.
    Sachdeva H, Sehgal R, Kaur S (2013) Studies on the protective and immunomodulatory efficacy of Withania somnifera along with cisplatin against experimental visceral leishmaniasis. Parasitol Res 112(6):2269–2280Google Scholar
  3. 3.
    Antony ML, Lee J, Hahm ER, Kim SH, Marcus AI, Kumari V, Ji X, Yang Z, Vowell CL, Wipf P, Uechi GT, Yates NA, Romero G, Sarkar SN, Singh SV (2013) Growth arrest by the antitumor steroidal lactone withaferin A in human breast cancer cells is associated with down-regulation and covalent binding at cysteine 303 of beta-tubulin. J Biol Chem 289(3):1852–1865Google Scholar
  4. 4.
    Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazon J (2009) Steroidal lactones from Withania somnifera, an ancient plant for novel medicine. Molecules 14(7):2373–2393Google Scholar
  5. 5.
    Li AX, Sun M, Li X (2017) Withaferin-A induces apoptosis in osteosarcoma U2OS cell line via generation of ROS and disruption of mitochondrial membrane potential. Eur Rev Med Pharmacol Sci 21(6):1368–1374Google Scholar
  6. 6.
    Yu Y, Katiyar SP, Sundar D, Kaul Z, Miyako E, Zhang Z, Kaul SC, Reddel RR, Wadhwa R (2017) Withaferin-A kills cancer cells with and without telomerase: chemical, computational and experimental evidences. Cell Death Dis 8(4):e2755Google Scholar
  7. 7.
    Zhang QZ, Guo YD, Li HM, Wang RZ, Guo SG, Du YF (2017) Protection against cerebral infarction by Withaferin A involves inhibition of neuronal apoptosis, activation of PI3K/Akt signaling pathway, and reduced intimal hyperplasia via inhibition of VSMC migration and matrix metalloproteinases. Advances in Medical Sciences 62(1):186–192Google Scholar
  8. 8.
    Singh P, Guleri R, Angurala A, Kaur K, Kaul SC, Wadhwa R, Pati PK (2017) Addressing challenges to enhance the bioactives of Withania somnifera through organ, tissue, and cell culture based approaches. Biomed Res Int 2017:3278494Google Scholar
  9. 9.
    Ciddi V (2006) Withaferin A from cell cultures of Withania somnifera. Indian J Pharm Sci 68(4):490–492Google Scholar
  10. 10.
    Sharada M, Ahuja A, Suri KA, Vij SP, Khajuria RK, Verma V, Kumar A (2006) Withanolide production by in vitro cultures of Withania somnifera and its association with differentiation. Biol Plant 51(1):161–164Google Scholar
  11. 11.
    Sabir F, Sangwan NS, Chaurasiya ND, Misra LN, Sangwan RS (2008) In vitro withanolide production by Withania somnifera L. cultures. Z Naturforsch C 63(5–6):409–413Google Scholar
  12. 12.
    Baldi A, Singh D, Dixit VK (2008) Dual elicitation for improved production of withaferin A by cell suspension cultures of Withania somnifera. Appl Biochem Biotechnol 151(2–3):556–564Google Scholar
  13. 13.
    Nagella P, Murthy HN (2010) Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresour Technol 101(17):6735–6739Google Scholar
  14. 14.
    Nagella P, Murthy HN (2011) Effects of macro elements and nitrogen source on biomass accumulation and withanolide—a production from cell suspension cultures of Withania somnifera (L.). Plant Cell Tissue Organ Cult 104(1):119–124Google Scholar
  15. 15.
    Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathi A (2012) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.). Ind Crops Prod 37(1):124–129Google Scholar
  16. 16.
    Sivanandhan G, Dev GK, Jeyaraj M, Rajesh M, Arjunan A, Muthuselvam M, Manickavasagam M, Selvaraj N, Ganapathi A (2013) Increased production of withanolide A, withanone, and withaferinA in hairy root cultures of Withania somnifera (L.) Dunal elicited with methyl jasmonate and salicylic acid. Plant Cell Tissue Organ Cult 114(1):121–129Google Scholar
  17. 17.
    Sivanandhan G, Dev GK, Jeyaraj M, Rajesh M, Muthuselvam M, Selvaraj N, Manickavasagam M, Ganapathi A (2013) A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma 250(4):885–898Google Scholar
  18. 18.
    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:e104005Google Scholar
  19. 19.
    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. Enzyme Microb Technol 21(2):97–101Google Scholar
  20. 20.
    Namdeo A, Patil S, Fulzele DP (2002) Influence of fungal elicitors on production of ajmalicine by cell cultures of Catharanthus roseus. Biotechnol Prog 18(1):159–162Google Scholar
  21. 21.
    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(1):37–46Google Scholar
  22. 22.
    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 114:19–28Google Scholar
  23. 23.
    Verma P, Khan SA, Mathur AK, Shanker K, Kalra A (2014) Fungal endophytes enhanced the growth and production kinetics of Vinca minor hairy roots and cell suspensions grown in bioreactor. Plant Cell Tissue Organ Cult 118(2):257–268Google Scholar
  24. 24.
    Verma P, Khan SA, Mathur AK, Ghosh S, Shanker K, Kalra A (2014) Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor. Protoplasma 251(6):1359–1371Google Scholar
  25. 25.
    Senthil K, Jayakodi M, Thirugnanasambantham P, Lee SC, Duraisamy P, Purushotham PM, Rajasekaran K, Nancy Charles S, Mariam Roy I, Nagappan AK, Kim GS, Lee YS, Natesan S, Min TS, Yang TJ (2015) Transcriptome analysis reveals in vitro cultured Withania somnifera leaf and root tissues as a promising source for targeted withanolide biosynthesis. BMC Genom 16:14Google Scholar
  26. 26.
    Singh S, Pal S, Shanker K, Chanotiya CS, Gupta MM, Dwivedi UN, Shasany AK (2014) Sterol partitioning by HMGR and DXR for routing intermediates toward withanolide biosynthesis. Physiol Plant 152(4):617–633Google Scholar
  27. 27.
    Jadaun JS, Sangwan NS, Narnoliya LK, Singh N, Bansal S, Mishra B, Sangwan RS (2016) Over-expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose-scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis. Physiol Plant 159(4):381–400.  https://doi.org/10.1111/ppl.12507 Google Scholar
  28. 28.
    Mishra S, Bansal S, Mishra B, Sangwan RS, Asha, Jadaun JS, Sangwan NS (2016) RNAi and homologous over-expression based functional approaches reveal triterpenoid synthase gene-cycloartenol synthase is involved in downstream withanolide biosynthesis in Withania somnifera. PLoS One 11:e0149691Google Scholar
  29. 29.
    Grover A, Samuel G, Bisaria VS, Sundar D (2013) Enhanced withanolide production by overexpression of squalene synthase in Withania somnifera. J Biosci Bioeng 115(6):680–685.  https://doi.org/10.1016/j.jbiosc.2012.12.011 Google Scholar
  30. 30.
    Patel N, Patel P, Kendurkar SV, Thulasiram HV, Khan BM (2015) Overexpression of squalene synthase in Withania somnifera leads to enhanced withanolide biosynthesis. Plant Cell Tissue Organ Cult 122(9):409–420Google Scholar
  31. 31.
    Singh AK, Dwivedi V, Rai A, Pal S, Reddy SG, Rao DK, 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 13(9):1287–1299.  https://doi.org/10.1111/pbi.12347 Google Scholar
  32. 32.
    Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497Google Scholar
  33. 33.
    Rani G, Grover IS (1999) In vitro callus induction and regeneration studies in Withania somnifera. Plant Cell Tissue Organ Cult 57(1):23–27Google Scholar
  34. 34.
    Rana S, Bhat WW, Dhar N, Pandith SA, Razdan S, Vishwakarma R, Lattoo SK (2014) Molecular characterization of two A-type P450s, WsCYP98A and WsCYP76A from Withania somnifera (L.) Dunal: expression analysis and withanolide accumulation in response to exogenous elicitations. BMC Biotechnol 14:89Google Scholar
  35. 35.
    Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101–1108Google Scholar
  36. 36.
    Junglee S, Urban L, Sallanon H, Lopez-Lauri F (2014) Optimized assay for hydrogen peroxide determination in plant tissue using potassium iodide. Am J Analyt Chem 5(11):730–736.  https://doi.org/10.4236/ajac.2014.511081 Google Scholar
  37. 37.
    Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125(1):189–198Google Scholar
  38. 38.
    Fernández-Bautista N, Domínguez-Núñez JA, Moreno MC, Berrocal-Lobo M (2016) Plant tissue trypan blue staining during phytopathogen infection. Bio-protocol 6(24): e2078Google Scholar
  39. 39.
    Tamagnone L, Merida A, Stacey N, Plaskitt K, Parr A, Chang CF, Lynn D, Dow JM, Roberts K, Martin C (1998) Inhibition of phenolic acid metabolism results in precocious cell death and altered cell morphology in leaves of transgenic tobacco plants. Plant Cell 10(11):1801–1816Google Scholar
  40. 40.
    Steward N, Martin R, Engasser JM, Goergen JL (1999) A new methodology for plant cell viability assessment using intracellular esterase activity. Plant Cell Report 19(16):171–176Google Scholar
  41. 41.
    Forni C, Frattarelli A, Damiano C (1999) Different size, shape and growth behaviour of cells in suspension cultures of strawberry (Fragaria x ananassa Duch.). Plant Biosyst 133(2):205–212Google Scholar
  42. 42.
    Ouyang J, Wang XD, Zhao B, Wang YC (2005) Enhanced production of phenylethanoid glycosides by precursor feeding to cell culture of Cistanche deserticola. Process Biochem 40(11):3480–3484Google Scholar
  43. 43.
    Sivanandhan G, Selvaraj N, Ganapathi A, Manickavasagam M (2014) Improved production of withanolides in shoot suspension culture of Withania somnifera (L.) Dunal by seaweed extracts. Plant Cell Tissue Organ Cult 119(1):221–225Google Scholar
  44. 44.
    Yi GE, Robin AH, Yang K, Park JI, Hwang BH, Nou IS (2016) Exogenous methyl jasmonate and salicylic acid induce subspecies-specific patterns of glucosinolate accumulation and gene expression in Brassica oleracea L. Molecules 21:1417Google Scholar
  45. 45.
    Zang YX, Ge JL, Huang LH, Gao F, Lv XS, Zheng WW, Hong SB, Zhu ZJ (2015) Leaf and root glucosinolate profiles of Chinese cabbage (Brassica rapa ssp. pekinensis) as a systemic response to methyl jasmonate and salicylic acid elicitation. J Zhejiang Univ Sci B 16(8):696–708Google Scholar
  46. 46.
    Shahzad R, Waqas M, Khan AL, Hamayun M, Kang SM, Lee IJ (2015) Foliar application of methyl jasmonate induced physio-hormonal changes in Pisum sativum under diverse temperature regimes. Plant Physiol Biochem 96:406–416Google Scholar
  47. 47.
    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(6):901–907Google Scholar
  48. 48.
    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):23–29Google Scholar
  49. 49.
    Saxena P, Ahlawat A, Ali A, Abdin MZ (2016) Gene expression analysis of the withanolide biosynthetic pathway in hairy root cultures of Withania somnifera elicited with methyl jasmonate and the fungus Piriformospora indica. Symbiosis 71(2):143–154Google Scholar
  50. 50.
    Sanz MK, Hernandez XE, Tonn CE, Guerreiro E (2000) Enhancement of tessaric acid production in Tessaria absinthioides cell suspension cultures. Plant Cell Rep 19(8):821–824Google Scholar
  51. 51.
    Kushwaha RK, Singh S, Pandey SS, Kalra A, Vivek Babu CS (2019) Fungal endophytes attune withanolide biosynthesis in Withania somnifera, prime to enhanced withanolide A content in leaves and roots. World J Microbiol Biotechnol 35(2):20.  https://doi.org/10.1007/s11274-019-2593-1 Google Scholar
  52. 52.
    Singh G, Tiwari M, Singh SP, Singh S, Trivedi PK, Misra P (2016) Silencing of sterol glycosyltransferases modulates the withanolide biosynthesis and leads to compromised basal immunity of Withania somnifera. Sci Rep 6:25562.  https://doi.org/10.1038/srep25562 Google Scholar
  53. 53.
    Srivastava S, Sanchita, Singh R, Srivastava G, Sharma A (2018) Comparative study of withanolide biosynthesis-related miRNAs in root and leaf tissues of Withania somnifera. Appl Biochem Biotechnol 185(4):1145–1159Google Scholar
  54. 54.
    Sivanandhan G, Arunachalam C, Selvaraj N, Sulaiman AA, Lim YP, Ganapathi A (2015) Expression of important pathway genes involved in withanolides biosynthesis in hairy root culture of Withania somnifera upon treatment with Gracilaria edulis and Sargassum wightii. Plant Physiol Biochem 91:61–64Google Scholar
  55. 55.
    Gupta P, Agarwal AV, Akhtar N, Sangwan RS, Singh SP, Trivedi PK (2012) Cloning and characterization of 2-C-methyl-d-erythritol-4-phosphate pathway genes for isoprenoid biosynthesis from Indian ginseng, Withania somnifera. Protoplasma 250(1):285–295Google Scholar
  56. 56.
    Rao MV, Paliyath G, Ormrod DP, Murr DP, Watkins CB (1997) Influence of salicylic acid on H2O2 production, oxidative stress, and H2O2-metabolizing enzymes. Salicylic acid-mediated oxidative damage requires H2O2. Plant Physiol 115(1):137–149Google Scholar
  57. 57.
    Leon J, Lawton MA, Raskin I (1995) Hydrogen peroxide stimulates salicylic acid biosynthesis in tobacco. Plant Physiol 108(4):1673–1678Google Scholar
  58. 58.
    Hao W, Guo H, Zhang J, Hu G, Yao Y, Dong J (2014) Hydrogen peroxide is involved in salicylic acid-elicited rosmarinic acid production in Salvia miltiorrhiza cell cultures. Sci World J 2014:843764Google Scholar
  59. 59.
    Kawano T, Muto S (2000) Mechanism of peroxidase actions for salicylic acid-induced generation of active oxygen species and an increase in cytosolic calcium in tobacco cell suspension culture. J Exp Bot 51(135):685–693Google Scholar
  60. 60.
    Garg N, Manchanda G (2009) ROS generation in plants: Boon or bane? Plant Biosyst 143(1):81–96Google Scholar
  61. 61.
    Rath I, Barz W (2000) The role of lipid peroxidation in aluminium toxicity in soybean cell suspension cultures. ‎Z Naturforsch B 55(11–12):957–964Google Scholar
  62. 62.
    Helaly MN, El-Hosieny HAR, El-Sarkassy NM, Fuller MP (2017) Growth, lipid peroxidation, organic solutes, and anti-oxidative enzyme content in drought-stressed date palm embryogenic callus suspension induced by polyethylene glycol. In Vitro Cell Dev Biol Plant 53(2):133–141Google Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Microbial Technology LaboratoryCSIR-Central Institute of Medicinal and Aromatic Plants, Research CentreBangaloreIndia
  2. 2.Microbial Technology DivisionCSIR-Central Institute of Medicinal and Aromatic PlantsLucknowIndia
  3. 3.Academy of Scientific and Innovative Research, (AcSIR)GhaziabadIndia

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