Acta Biologica Hungarica

, Volume 69, Issue 4, pp 437–448 | Cite as

Sodiumnitroprusside Stimulated Production of Tropane Alkaloids and Antioxidant Enzymes Activity in Hairy Root Culture of Hyoscyamus Reticulatus L.

  • Madineh Khezerluo
  • Bahman HosseiniEmail author
  • Jafar Amiri


Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson’s or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 uM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), per-oxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 uM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.


Elicitation hairy root Hyoscyamus reticulatus L. nitric oxide tropane alkaloids 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Aberham, A., Pieri, V., Croom, E. M. J. R., Ellmerer, E., Stuppner, H. (2011) Analysis of iridoids, secoiridoids and xanthones in Centawium erythraea, Frasera carpliniensis and Gentiana lutea using LC-MS and RP-HPLC. J. Pharm. Biomed. Anal. 54, 517–525.CrossRefGoogle Scholar
  2. 2.
    Aebi, H. (1984) Catalase in vitro. Methods Enzymol. 105, 121–126.CrossRefGoogle Scholar
  3. 3.
    Amdoun, R., Khelifi, L., Khelifi-Slaoui, M., Amroune, S., Benyoussef, E. H., Vu Thi, D., Assaf-Ducrocq, C., Gontier, E. (2009) Influence of minerals and elicitation on Datlira stramonium L. tropane alkaloid production: modelization of the /ra vz/ro biochemical response. Plant Sci. 177, 81–87.CrossRefGoogle Scholar
  4. 4.
    Arasimowicz, M., Floryszak-Wieczorek, J. (2007) Nitric oxide as a bioactive signaling molecule in plant stress responses. Plant Sci. 172, 876–887.CrossRefGoogle Scholar
  5. 5.
    Bao, X., Lu, C., Frangos, J. A. (1999) Temporal gradiënt in shear but not steady shear stress induces PDGF-A and MCP-1 expression in endothelial cells: role of NO, NFKB and egr-1. Arterioscler. Thromb. Vasc. Biol. 19, 996–1003.CrossRefGoogle Scholar
  6. 6.
    Beligni, M. V., Lamattina, L. (1999) Nitric oxide protects against cellular damage produced by methylviologen herbicides in potato plants. J. Biol. Chem. 3, 199–208.Google Scholar
  7. 7.
    Bertani, G. (1951) Studies onlysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J. Bacteriol. 62, 293–300.PubMedGoogle Scholar
  8. 8.
    Chandra, S., Chandra, R. (2011) Engineering secondary metabolite production in hairy roots. Phytochem. Rev. 10, 371–395.CrossRefGoogle Scholar
  9. 9.
    Del Rio, L. A., Corpas, F. J., Barroso, J. B. (2004) Nitric oxide synthase activity in plants. Phytochem. 65, 783–792.CrossRefGoogle Scholar
  10. 10.
    Delledonne, M., Xia, Y., Dixon, R. A., Lamb, C. (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394, 585–588.CrossRefGoogle Scholar
  11. 11.
    Delledonne, M., Zeier, J., Marocco, A., Lamb, C. (2001) Signal interactions between nitric oxide and reactive oxygen intermediates in the plant hypersensitive disease resistance response. Proc. Acad Nat. Sci. Phila. 98, 13454–13459.CrossRefGoogle Scholar
  12. 12.
    Dixon, R. A. (2001) Natural products and plant disease resistance. Nature 411, 843–847.CrossRefGoogle Scholar
  13. 13.
    Duan, X., Su, X., You, Y., Qu, H., Li, Y., Jiang, Y. (2007) Effect of nitric oxide on pericarp browing of harvested logan fruit in relation to phenolic metabolism. Food Chem. 104, 571–576.CrossRefGoogle Scholar
  14. 14.
    Durner, J., Wendehenne, D., Klessig, D. F. (1998) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc. Acad. Nat. Sci. Phila. 95, 10328–10333.CrossRefGoogle Scholar
  15. 15.
    Esfandiari, E., Mahboob, S. A., Shekari, F. (2008) Destructive effect of active oxygen species, plant defence mechanisms and its necessary 10*. Agro Plant Breed Cong Iran. 1–22.Google Scholar
  16. 16.
    Fadzillah, N. M., Yusuf N., Mahmood, M. (2006) Paraquat (Methyl viologen) toxicity in centella asiatica callus cultures. Pertanika J. Trop. Agric. Sci. 29, 57–66.Google Scholar
  17. 17.
    Fu, J., Huang, B., Zhang, G. (2000) Physiological and biochemical change during seed filling in relation to leaf senescence in soybean. Bio. Plantarum 4, 545–548.CrossRefGoogle Scholar
  18. 18.
    Gould, K. S., Klinguer, A., Pugin, A., Wendehenne, D. (2003) Nitric oxide production in tobacco leaf cells: a generalized stress response? Plant Cell Environ. 26, 1851–1862.CrossRefGoogle Scholar
  19. 19.
    Hashimoto, T., Yamad, Y. (1987) Purification and characterization of hyoscyamine 6P-hydroxylase from root culture of Hyoscyamus niger L. Eur. J. Biochem. 194, 277–285.CrossRefGoogle Scholar
  20. 20.
    Hayat, S., Mori, M., Pichtel, J., Ahmad, A. (2010) Nitric oxide in plant physiology. Wiley-Blackwell.Google Scholar
  21. 21.
    Hu, X., Neill, S., Cai, W. (2003) Nitric oxide mediates elicitor-induced saponin synthesis in cell cultures of Panax ginseng. Funct. Plant Biol. 30, 901–907.CrossRefGoogle Scholar
  22. 22.
    Kamada, H., Okamura, N., Satake, M., Harada, H., Shimomura, K. (1986) Alkaloid production by hairy root cultures in Atropa belladonna. Plant Cell Rep. 5, 239–242.CrossRefGoogle Scholar
  23. 23.
    Kopyra, M., Gwozdz, E. A. (2003) Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus. Plant Physiol. Bioch. 41, 1011–1017.CrossRefGoogle Scholar
  24. 24.
    Macadam, J. W., Nelson, C. J., Sharp, R. E. (1992) Peroxidaes activity in the leaf elongation zone of tall fescue. Plant Physiol. 99, 872–878.CrossRefGoogle Scholar
  25. 25.
    Mittler, R. (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7, 405–110.CrossRefGoogle Scholar
  26. 26.
    Murashige, T., Skoog, F. (1962) Arevised medium for rapid growth and bioassays with tobacco tissue cultures, Physiol. Plantarum 15, 473–176.CrossRefGoogle Scholar
  27. 27.
    Nakano, Y., Asada, K. (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880.Google Scholar
  28. 28.
    Nasibi, F., Kalantari, K. M. (2009) Influence of nitric oxide in protection of tomato seedling against oxidative stress induced by osmotic stress, Acta Physiol. Plant 31, 1037–1044.CrossRefGoogle Scholar
  29. 29.
    Nasibi, F., Manochehri Kalantari, K., Khodashenas, M. (2010) Effect of sodium nitroprusside (SNP) on some biochemical characteristics of tomato seedlings (Lycopersicum esculentum) under drought stress. J. Agri. Sci. Nature Res. 16, 2–16.Google Scholar
  30. 30.
    Navarre, D. A., Wendehenne, D., Durner, J., Noad, R., Klessig, D. F. (2000) Nitric oxide modulates the activity of tobacco aconitase. Plant Physiol. 122, 573–582.CrossRefGoogle Scholar
  31. 31.
    Neill, J., Radhika, D., Hancock, J. (2003) Nitric oxide signaling in plant. New Phytol. 159, 11–35.CrossRefGoogle Scholar
  32. 32.
    Oksman-Caldentey, K. M., Inze, D. (2004) Plant cell factories in the post-genome era: new ways to produce designer secondary metabolites. Trends Plant Sci. 9, 440–143.CrossRefGoogle Scholar
  33. 33.
    Palazon, J., Navarro-Ocana, A., Hernandez-Vazquez, L., Mirjalili, M. H. (2008) Application of metabolic engineering to the production of scopolamine. Molecules 13, 1722–1742.CrossRefGoogle Scholar
  34. 34.
    Parsa, M., Garoosi, G. A., Haddad, R. (2013) Cloning and study the bioinfomatic trait of tropinone reductase-II (TR II) gene from Hyoscyamus niger. J. Cell Tissu 3, 307–318.Google Scholar
  35. 35.
    Schmidt, H. W., Walter, U. (1994) NO at work. Cell. 78, 919–925.CrossRefGoogle Scholar
  36. 36.
    Seidel, V., Windhovel. J., Eaton, G., Alfermann, A. W., Arroo, R. R. J., Medarde, M., Petersen, M., Wolley, J. G. (2002) Biosynthesis of podophyllotoxin in Linum album cell cultures. Planta 215, 1013–1039.CrossRefGoogle Scholar
  37. 37.
    Tripathi, L., Tripathi, J. N. (2003) Role of biotechnology in medicinal plants. Tropical J. Pharm. Res. 2, 243–253.Google Scholar
  38. 38.
    Vanleberghe, G. C., Mclntosh, L. (1996) Signals regulating the expression of the nuclear gene encoding alternative oxidase of plant mitochondria. Plant Physiol. 111, 589–595.CrossRefGoogle Scholar
  39. 39.
    Wang, J. W., Zheng, L. R., Wu, J. Y., Tan., R. X. (2006) Involvement of nitric oxide in oxidative burst, phenylalanine ammonia-lyase activation and Taxol production induced by low-energy ultrasound in Taxusyunnanensis cell suspension cultures. ’Nitric Oxide-Biol. Ch. 15, 351–358.CrossRefGoogle Scholar

Copyright information

© Akadémiai Kiadó Zrt. 2018

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors and Affiliations

  • Madineh Khezerluo
    • 1
  • Bahman Hosseini
    • 1
    Email author
  • Jafar Amiri
    • 1
  1. 1.Department of Horticultural ScienceUrmia UniversityUrmiaIran

Personalised recommendations