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Induction of root colonization by Piriformospora indica leads to enhanced asiaticoside production in Centella asiatica

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Abstract

Centella asiatica (Indian pennywort) has wide application in Indian and Chinese traditional medicines with documented evidence for wound healing and neuroprotective and anti-aging potential. Asiaticoside, a trisaccharide triterpene, is the most medicinally active compound in the plant. β-Amyrin synthase and squalene synthase have been identified as the two key genes in the triterpenoid pathway which regulate the production of asiaticoside in C. asiatica. The paper reports salient findings of our study utilizing the growth-promoting endophytic fungus Piriformospora indica to successfully colonize roots of C. asiatica in vitro cultures for investigating the effect of the mutualistic association on asiaticoside production. Co-cultivation of P. indica resulted in the rapid enhancement of root and shoot biomass of host plant, which was visible after 7 days of culture and continued up to 45 days. P. indica co-cultivation also favored the synthesis of asiaticosides, as evidenced by HPLC analysis which indicated about twofold increase (0.53% (w/w) in leaves and 0.23% (w/w) in whole plant) over control (0.33% (w/w) in leaves and 0.14% (w/w) in whole plant). Real-time PCR results confirmed the strong upregulation of squalene synthase and β-amyrin synthase transcripts in P. indica-challenged plants compared with the control. Our data demonstrate the potential use of P. indica as a means to enhance plant secondary metabolite production in planta with scope for further field evaluation.

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References

  • Aziz ZA, Davey MR, Power JB, Anthony P, Smith RM, Lowe KC (2007) Production of asiaticoside and madecassoside in Centella asiatica in vitro and in vivo. Biol Plant 51:34–42

    Article  CAS  Google Scholar 

  • Baldi A, Srivastava AK, Bisaria VS (2009) Fungal elicitors for enhanced production of secondary metabolites in plant cell suspension cultures. Soil Biol 18:373–380

    Article  CAS  Google Scholar 

  • Barazani O, Benderoth M, Groten K, Kuhlemeier N, Baldwin IT (2005) Piriformospora indica and Sebacina vermifera increase growth performance at the expense of herbivore resistance in Nicotiana attenuata. Oecologia 146:234–243

    Article  PubMed  Google Scholar 

  • Bolton MD (2009) Primary metabolism and plant defense—fuel for the fire. Mol Plant Microbe Interact 22:487–497

    Article  PubMed  CAS  Google Scholar 

  • Bradwejn J, Zhou Y (2000) A double-blind, placebo-controlled study on the effects of Gotu Kola (Centella asiatica) on acoustic startle response in healthy subjects. J Clin Psychopharmacol 20:680–684

    Article  PubMed  CAS  Google Scholar 

  • Brinkhause B, Lindner M (2000) Chemical, pharmacological and clinical profile of the East Asian medical plant Centella asiatica. Phytomedicine 7:427–448

    Article  Google Scholar 

  • Buetehorn B, Rhody D, Franken P (2000) Isolation and characterization of Pitef1 encoding the translation elongation factor EF-1-alpha of the root endophyte Piriformospora indica. Plant Biol 2:687–692

    Article  Google Scholar 

  • Devarenne TP, Ghosh A, Chappell J (2002) Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco. Plant Physiol 129:1095–1106

    Article  PubMed  CAS  Google Scholar 

  • Gao FK, Dai CC, Liu XZ (2010) Mechanisms of fungal endophytes in plant protection against pathogens. Afr J Microbiol Res 4:1346–1351

    Google Scholar 

  • Haralampidis K, Trojanowska M, Osbourn AE (2002) Biosynthesis of triterpenoid saponins in plants. Adv Biochem Eng Biotechnol 75:31–49

    PubMed  CAS  Google Scholar 

  • Hausen BM (1993) Centella asiatica (Indian pennywort), an effective therapeutic but a weak sensitizer. Contact Dermatitis 29:175–179

    Article  PubMed  CAS  Google Scholar 

  • Hayashi H, Huang PY, Inoue K (2003) Up-regulation of soyasaponin biosynthesis by methyl jasmonate in cultured cells of Glycyrrhiza glabra. Plant Cell Physiol 44:404–411

    Article  PubMed  CAS  Google Scholar 

  • Hernandez-Vazquez L, Bonfill M, Moyano E, Cusido RM, Navarro-Ocaña A, Palazon J (2010) Conversion of α-amyrin into centellosides by plant cell cultures of Centella asiatica. Biotechnol Lett 32:315–319

    Article  PubMed  CAS  Google Scholar 

  • Jakobsen L, Abbott K, Robson AD (1992) External hyphae of vesicular–arbuscular mycorrhizal fungi associated with Trifolium subterraneum. I. Spread of hyphae and phosphorus inflow into roots. New Phytol 120:371–380

    Article  CAS  Google Scholar 

  • Jenner L, Romby P, Rees B, Schulze-Briese C, Springer M, Ehresmann C, Ehresmann B, Moras D, Yusupova G, Yusupov M (2005) Translational operator of mRNA on the ribosome. How repressor proteins exclude ribosome binding. Science 308:120–123

    Article  PubMed  CAS  Google Scholar 

  • Khan SA, Hamayun M, Yoon HJ (2008) Plant growth promotion and Penicillium citrinum. BMC Microbiol 8:231

    Article  PubMed  Google Scholar 

  • Kim OT, Kim MY, Hong MH, Ahn JC, Hwang B (2004) Stimulation of asiaticoside production from Centella asiatica whole plant cultures by elicitors. Plant Cell Rep 23:339–344

    Article  PubMed  CAS  Google Scholar 

  • Kim OT, Kim MY, Huh SM, Bai DG, Ahn JC, Hwang B (2005) Cloning of a cDNA probably encoding oxidosqualene cyclase associated with asiaticoside biosynthesis from Centella asiatica (L.) Urban. Plant Cell Rep 24:304–311

    Article  PubMed  CAS  Google Scholar 

  • Kim OT, Bang KH, Shin YS, Lee MJ, Jung SJ, Hyun DY, Kim YC, Seong NS, Cha SW, Hwang B (2007) Enhanced production of asiaticoside from hairy root cultures of Centella asiatica (L.) Urban elicited by methyl jasmonate. Plant Cell Rep 26:1941–1949

    Article  PubMed  CAS  Google Scholar 

  • Mangas S, Bonfill M, Osuna L, Moyano T, Tortoriello J, Guido RM, Piñol MT, Palazón J (2006) The effect of methyl jasmonate on triterpene and sterol metabolism of Centella asiatica, Ruscus aculeatus and Galphimia glauca cultured plants. Phytochemistry 67:2041–2049

    Article  PubMed  CAS  Google Scholar 

  • Mangas S, Moyano E, Osuna L, Cusido RM, Bonfill M, Palazón J (2008) Triterpenoid saponin content and the expression level of some related genes in calli of Centella asiatica. Biotechnol Lett 30:1853–1859

    Article  PubMed  CAS  Google Scholar 

  • Maquart FX, Chastang F, Simeon A, Birembaut P, Gillery P, Wegrowski Y (1999) Triterpenes from Centella asiatica stimulate extracellular matrix accumulation in rat experimental wounds. Eur J Dermatol 9:289–296

    PubMed  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Peskan-Berghöfer T, Shahollari B, Giong PH, Hehl S, Markert C, Blanke V, Kost G, Varma A, Oelmüller R (2004) Association of Piriformospora indica with Arabidopsis thaliana roots represents a novel system to study beneficial plant–microbe interactions and involves early plant protein modifications in the endoplasmic reticulum and at the plasma membrane. Physiol Plant 122:465–477

    Article  Google Scholar 

  • Pham GH, Singh KR, Sachdev M, Prasad R, Kaldorf M, Buscot F, Oelmuller R, Peskan T, Weiss M, Hampp R, Varma A (2004) Axenic cultures of Piriformosora indica. In: Varma A, Abbott L, Werner D, Hampp R (eds) Plant surface microbiology. Springer, Berlin, pp 593–616

    Google Scholar 

  • Phillip JM, Hayman DS (1970) Improved procedures for clearing roots and staining parasitic and VAM fungi for rapid assessment of infection. Trans Br Mycol Soc 55:158–161

    Article  Google Scholar 

  • Rai MK, Varma A (2005) Arbuscular mycorrhizae—like biotechnological potential of Piriformospora indica, which promotes the growth of Adhatoda vasica. Electron J Biotechnol 8:107–112

    Article  Google Scholar 

  • Rogers SO, Bendich AJ (1994) Extraction of total cellular DNA from plants, algae and fungi. In: Gelvin SB, Schilperoort RA (eds) Plant molecular biology manual, 2nd edn. Kluwer, Dordrecht, pp 1–8

    Google Scholar 

  • Serfling A, Wirsel SGR, Lind V, Deising HB (2007) Performance of the biocontrol fungus Piriformospora indica on wheat under green house and field conditions. Phytopathology 97:523–531

    Article  PubMed  CAS  Google Scholar 

  • Sun C, Johnson JM, Cai D, Sherameti I, Oelmuller R, Lou B (2010) Piriformospora indica confers drought tolerance in Chinese cabbage leaves by stimulating antioxidant enzymes, the expression of drought-related genes and the plastid-localized CAS protein. J Plant Physiol 167:1009–1017

    Article  PubMed  CAS  Google Scholar 

  • Varma A, Verma S, Sudha S, Sahay N, Bütehorn B, Franken P (1999) Piriformospora indica, a cultivable plant-growth-promoting root endophyte. Appl Environ Microbiol 65:2741–2744

    PubMed  CAS  Google Scholar 

  • Verma S, Varma A, Rexer KH, Hassel A, Kost G, Sarabhoy A, Bisen P, Bütenhorn B, Franken P (1998) Piriformospora indica, gen. et sp. nov., a root colonizing fungus. Mycologia 90:896–903

    Article  CAS  Google Scholar 

  • Visweswari G, Siva Prasad K, Lokanatha V, Rajendra W (2010) The antiepileptic effect of Centella asiatica on the activities of Na+/K+, Mg2+ and Ca2+-ATPases in rat brain during pentylenetetrazol-induced epilepsy. Indian J Pharmacol 42:82–86

    Google Scholar 

  • Waller F, Achatz FB, Baltruschat H, Fodor J, Becker K, Fischer M, Heier T, Huckelhoven R, Neumann C, von Wettstein D, Franken P, Kogel KH (2005) The endophytic fungus Piriformospora indica reprograms barley to salt-stress tolerance, disease resistance, and higher yield. Proc Natl Acad Sci 102:13386–13391

    Article  PubMed  CAS  Google Scholar 

  • Weiss M, Selosse MA, Rexer KH, Urban A, Oberwinkler F (2004) Sebacinales: a hitherto overlooked cosm of heterobasidiomycetes with a broad mycorrhizal potential. Mycol Res 108:1003–1010

    Article  PubMed  Google Scholar 

  • Widgerow AD, Chait LA (2000) New innovations in scar management. Aesthet Plast Surg 24:227–234

    Article  CAS  Google Scholar 

  • Wright CS (1984) Structural comparison of the two distinct sugar binding sites in wheat germ agglutinin isolectin II. J Mol Biol 178:91–104

    Article  PubMed  CAS  Google Scholar 

  • Yendo ACA, Costa FD, Gosmann G, Fett-Neto AG (2010) Production of plant bioactive triterpenoid saponins: elicitation strategies and target genes to improve yields. Mol Biotechnol 46:94–104

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The authors thank Natural Remedies Pvt. Ltd, Bangalore, Karnataka, India, for HPLC analysis and the National Institute for Interdisciplinary Science and Technology (Council of Scientific and Industrial Research, Government of India), Thiruvananthapuram, Kerala, India, for SEM analysis. We also thank the Indian Council of Medical Research (ICMR, grant no. No.3/1/JRF/74/MPD-2007) for providing the Senior Research Fellowship.

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Authors and Affiliations

  1. Division of Plant Molecular Biology, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, 695 014, Kerala, India

    Jisha Satheesan & Manjula Sakunthala

  2. Division of Microbiology, Kerala Agricultural University, Thiruvananthapuram, 695 522, Kerala, India

    Anith K Narayanan

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  1. Jisha Satheesan
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  2. Anith K Narayanan
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  3. Manjula Sakunthala
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Correspondence to Manjula Sakunthala.

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ESM. 1

Analysis of P. indica colonization by Trypan blue staining. Spores of P. indica inside the root of C. asiatica (JPEG 9 kb)

High resolution (TIFF 3513 kb)

ESM. 2

SEM images of the control non-colonized C. asiatica (a) and P. indica co-cultured roots after 10 h (b). Arrow indicates fungal penetration into the root (JPEG 25 kb)

High resolution (TIFF 985 kb)

ESM. 3

PCR analysis of PITEF in root (a) and leaf tissue of P. indica-colonized plant (b). Lane 1 Tissues from the non-colonized plants; lane 2 tissues from P. indica-colonized plant; lane 3 P. indica as the positive control; lane 4 negative control (JPEG 9 kb)

High resolution (TIFF 939 kb)

ESM. 4

Pathway showing the biosynthesis of asiaticoside (JPEG 1 kb)

High resolution (TIFF 490 kb)

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Satheesan, J., Narayanan, A.K. & Sakunthala, M. Induction of root colonization by Piriformospora indica leads to enhanced asiaticoside production in Centella asiatica . Mycorrhiza 22, 195–202 (2012). https://doi.org/10.1007/s00572-011-0394-y

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