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Overexpression of squalene synthase in Withania somnifera leads to enhanced withanolide biosynthesis

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Abstract

Genetic engineering of secondary metabolic pathways is an emerging area of research for production and improvement of natural products in plant biotechnology. Here, we describe a systematic approach to manipulate a key regulatory step of isoprenoid biosynthetic pathway in Withania somnifera to study its effect on withanolide production. We generated T0 W. somnifera plants overexpressing squalene synthase (WsSQS) by Agrobacterium tumefaciens mediated transformation, which were analyzed by Gus biochemical assay and PCR of hygromycin phosphotransferase (hptII) and WsSQS. qRT-PCR analyses of various transformed tissues indicated 2–5 fold increase in WsSQS transcripts in both T0 and T1 generations. The tissue specific protein expression studies revealed 2–3 fold increase in WsSQS, which was further confirmed by enzyme activity. These observations were corroborated with the 1.5–2 fold increase in total withanolide content of the transformed tissues. However, in leaf tissue, the levels of Withaferin A and Withanolide A increased significantly up to 4–4.5 fold. These findings demonstrate genetic engineering of isoprenoid pathway in W. somnifera resulting in enhanced production of withanolides, and also provide insights into such metabolic pathways for their manipulation to improve the pharmacological content of different medicinally important plants.

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References

  • Bandyopadhyay M, Jha S, Tepfer D (2007) Changes in morphological phenotypes and withanolide composition of Ri-transformed roots of Withania somnifera. Plant Cell Rep 26(5):599–609

    Article  CAS  PubMed  Google Scholar 

  • Bhattacharya A, Ghosal S, Bhattacharya SK (2001) Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. J Ethnopharmacol 74(1):1–6

    Article  CAS  PubMed  Google Scholar 

  • D’Amato F (1977) Cytogenetics of differentiation in tissue and cell cultures. In: Applied and fundamental aspects of plant cell, tissue, and organ culture. Springer, Berlin, pp 343–357

  • Ghimire BK, Seong ES, Kim EH, Lamsal K, Yu CY, Chung IM (2010) Direct shoot organogenesis from petiole and leaf discs of Withania somnifera (L) Dunal. Afr J Biotechnol 9:7453–7461

    CAS  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Gupta SK, Dua A, Vohra BP (2003) Withania somnifera (Ashwagandha) attenuates antioxidant defense in aged spinal cord and inhibits copper induced lipid peroxidation and protein oxidative modifications. Drug Metabol Drug Interact 19(3):211–222

    Article  PubMed  Google Scholar 

  • Gupta N, Sharma P, Santosh Kumar RJ, Vishwakarma RK, Khan BM (2012) Functional characterization and differential expression studies of squalene synthase from Withania somnifera. Mol Biol Rep 39(9):8803–8812

    Article  CAS  PubMed  Google Scholar 

  • Jana CK, Hoecker J, Woods TM, Jessen HJ, Neuburger M, Gademann K (2011) Synthesis of withanolide A, biological evaluation of its neuritogenic properties, and studies on secretase inhibition. Angew Chem Int Ed Engl 50(36):8407–8411

    Article  CAS  PubMed  Google Scholar 

  • Joshi AG, Padhya MA (2010) Shoot regeneration from leaf explants of Withania somnifera (L.) Dunal. Not Sci Biol 2:63–65

    CAS  Google Scholar 

  • Kanungo S, Sahoo SL (2011) Direct organogenesis of Withania somnifera L. from apical bud. International Research. J Biotechnol 2(3):058–061

    Google Scholar 

  • Kim YS, Cho JH, Park S, Han JY, Back K, Choi YE (2011) Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum. Planta 233(2):343–355

    Article  CAS  PubMed  Google Scholar 

  • Kulkarni AA, Thengane SR, Krishnamurthy KV (2000) Direct shoot regeneration from node, internode, hypocotyl and embryo explants of Withania somnifera. Plant Cell Tissue Organ Cult 62:203–209

    Article  Google Scholar 

  • Kumar OA, Jyothirmayee G, Tata SS (2011) Multiple shoot regeneration from nodal explants of Ashwagandha (Withania somnifera) (L.) Dunal. Asian J Exp Biol Sci 2(4):636-640

  • Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45(8):976–984

    Article  CAS  PubMed  Google Scholar 

  • Lockley WJS, Rees HH, Goodwin TW (1976) Biosynthesis of steroidal withanolides in Withania somnifera. Phytochemistry 15:937–939

    Article  CAS  Google Scholar 

  • Logesh P, Settu A, Thangavel K, Ganapathi A (2010) Direct in vitro regeneration of Withania somnifera (L.) Dunal through leaf disc culture. Int J Biol Technol 1:1–4

    CAS  Google Scholar 

  • Lu H-Y, Liu J-M, Zhang H-C, Yin T, Gao S-L (2008) Ri-mediated Transformation of Glycyrrhiza uralensis with a Squalene Synthase Gene (GuSQS1) for Production of Glycyrrhizin. Plant Mol Biol Rep 26:1–11

    Article  CAS  Google Scholar 

  • Manickam VS, Mathavan RE, Antonisamy R (2000) Regeneration of Indian ginseng plantlets from stem callus. Plant Cell Tissue Organ Cult 62:181–185

    Article  Google Scholar 

  • Manwar JV, Mahadik KR, Paradkar AR, Takle SP, Sathiyanarayanan L, Patil SV (2012) Determination of withanolides from the roots and herbal formulation of Withania somnifera by HPLC using DAD and ELSD detector. Der Pharmacia Sinica 3(1):41–46

    CAS  Google Scholar 

  • 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):2373–2393

    Article  CAS  PubMed  Google Scholar 

  • Mirjalili MH, Moyano E, Bonfill M, Cusido RM, Palazon J (2011) Overexpression of the Arabidopsis thaliana squalene synthase gene in Withania coagulans hairy root cultures. Biol Plantarum 55(2):357–360

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, Hahn EJ, Paek KY (2008) Establishment of Withania somnifera hairy root cultures for the production of withanolide A. J Integr Plant Biol 50(8):975–981

    Article  CAS  PubMed  Google Scholar 

  • Oksman-Caldentey KM, Inze D (2004) Plant cell factories in the post-genomic era: new ways to produce designer secondary metabolites. Trends Plant Sci 9(9):433–440

    Article  CAS  PubMed  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(2):133–141

    Article  CAS  PubMed  Google Scholar 

  • Pandit J, Danley DE, Schulte GK, Mazzalupo S, Pauly TA, Hayward CM, Hamanaka ES, Thompson JF, Harwood HJ Jr (2000) Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis. J Biol Chem 275(39):30610–30617

    Article  CAS  PubMed  Google Scholar 

  • Patel N, Patel P, Kumari U, Kendurkar SV, Khan BM (2014) Microprojectile bombardment assisted agroinfection increases transformation efficiency of Withania somnifera (L.). Res Biotechnol 5(4):13–24

    Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucl Acids Res 29(9):e45

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Rani G, Virk GS, Nagpal A (2003) Callus induction and plantlet regeneration in Withania somnifera (L.) Dunal. In Vitro Cell Dev Biol Plant 39:468–474

    Article  Google Scholar 

  • Ray S, Jha S (1999) Withanolide synthesis in cultures of Withania somnifera transformed with Agrobacterium tumefaciens. Plant Sci 146:1–7

    Article  CAS  Google Scholar 

  • Ray S, Jha S (2001) Production of withaferin A in shoot cultures of Withania somnifera. Planta Med 67:432–436

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Seo JW, Jeong JH, Shin CG, Lo SC, Han SS, Yu KW, Harada E, Han JY, Choi YE (2005) Overexpression of squalene synthase in Eleutherococcus senticosus increases phytosterol and triterpene accumulation. Phytochemistry 66(8):869–877. doi:10.1016/j.phytochem.2005.02.016

    Article  CAS  PubMed  Google Scholar 

  • Sharada M, Ahuja A, Suri KA, Vij SP, Khajuria RK, Verma V, Kumar A (2007) Withanolide production by in vitro cultures of Withania somnifera and its association with differentiation. Biol Plantarum 51(1):161–164

    Article  CAS  Google Scholar 

  • Sharma V, Gupta AP, Bhandari P, Gupta RC, Singh B (2007) A validated and densitometric HPTLC method for the quantification of Withaferin-A and Withanolide-A in different plant parts of two morphotypes of Withania somnifera. Chromatographia 66:801–804

    Article  CAS  Google Scholar 

  • Sharma MM, Ali DJ, Batra A (2010) Plant regeneration through in vitro somatic embryogenesis in ashwagandha (Withania somnifera L. Dunal). Researcher 2:1–6

    Google Scholar 

  • Sivanandhan G, Mariashibu TS, Arun M, Rajesh M, Kasthurirengan S, Selvaraj N, Ganapathi A (2011) The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants. Acta Physiol Plant 33:2279–2288

    Article  CAS  Google Scholar 

  • Sivanesan I (2007) Direct regeneration from apical bud explants of Withania somnifera Dunal. Indian J Biotechnol 6:125–127

    CAS  Google Scholar 

  • Sivanesan I, Murugesan K (2008) An efficient regeneration from nodal explants of Withania somnifera Dunal. Asian J Plant Sci 7:551–556

    Article  CAS  Google Scholar 

  • Tozawa R, Ishibashi S, Osuga J, Yagyu H, Oka T, Chen Z, Ohashi K, Perrey S, Shionoiri F, Yahagi N, Harada K, Gotoda T, Yazaki Y, Yamada N (1999) Embryonic lethality and defective neural tube closure in mice lacking squalene synthase. J Biol Chem 274(43):30843–30848

    Article  CAS  PubMed  Google Scholar 

  • Udayakumar R, Kasthurirengan S, Mariashibu TS, Rayan JJS, Ganapathi A, Kim SC, Kim JJ, Choi CW (2014) Agrobacterium-mediated genetic transformation of Withania somnifera using nodal explants. Acta Physiol Plant 36:1969–1980

    Article  CAS  Google Scholar 

  • Vogeli U, Chappell J (1988) Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor. Plant Physiol 88(4):1291–1296

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wentzinger LF, Bach TJ, Hartmann MA (2002) Inhibition of squalene synthase and squalene epoxidase in tobacco cells triggers an up-regulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase. Plant Physiol 130(1):334–346

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Winters M (2006) Ancient medicine, modern use: Withania somnifera and its potential role in integrative oncology. Altern Med Rev 11:269–277

    PubMed  Google Scholar 

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Acknowledgments

The authors are thankful to S. Haldar and A. Shukla for their help in LC–MS. The authors thank Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial support and University Grants Commission (UGC), New Delhi, India for providing fellowship.

Conflict of interest

The authors declare that they have no conflict of interest and the publication of the work has been approved by all co-authors.

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Authors and Affiliations

  1. Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India

    Neha Patel, Parth Patel, Shuchishweta V. Kendurkar & Bashir M. Khan

  2. Organic Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, Maharashtra, India

    Hirekodathakallu V. Thulasiram

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  1. Neha Patel
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  2. Parth Patel
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Correspondence to Bashir M. Khan.

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Patel, N., Patel, P., Kendurkar, S.V. et al. Overexpression of squalene synthase in Withania somnifera leads to enhanced withanolide biosynthesis. Plant Cell Tiss Organ Cult 122, 409–420 (2015). https://doi.org/10.1007/s11240-015-0778-3

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  • DOI: https://doi.org/10.1007/s11240-015-0778-3

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