Acta Biologica Hungarica

, Volume 64, Issue 2, pp 207–217 | Cite as

Enzymatic and Non-enzymatic Antioxidant Responses of Alfalfa Leaves and Roots Under Different Salinity Levels

  • G. DehghanEmail author
  • Leyla Amjad
  • H. Nosrati


The effect of increasing NaCl concentrations on biomass, hydrogen peroxide (H2O2), ascorbic acid (ASC), proline and total thiol, and the activity of some antioxidant enzymes in alfalfa (Medicago sativa L. cv. Gara-Yonjeh) were investigated. The dry weights of roots and shoots with increasing NaCl concentrations decreased progressively, and the strongest toxicity was detected at NaCl treatment of 200 mM. Superoxide dismutase (SOD) activity in the leaves increased gradually up to NaCl concentrations of 100, while the higher concentration of NaCl reduced SOD activity in both leaves and roots. The maximum levels of ascorbate peroxidase (APX) activity were increased at 150 mM and 100 mM NaCl in leaves and roots of Gara-Yonjeh, respectively. Peroxidase (POD) activity in roots of Gara-Yonjeh increased (82% at 200 mM) by salinity, while it decreased (43% at 200 mM) in leaves. In contrast, catalase (CAT ) activitiy increased (84% at 200 mM) in leaves, and decreased (57% at 200 mM) in the roots of Gara-Yonjeh. Electrophoresis analysis suggested that different patterns in SOD, CAT and POD isoenzymes depend on NaCl concentrations, and the staining intensities of these isoforms are supported the results obtained from the spectrophotometric determinations. In POD and CAT, activity of isoform III was detected at all concentrations, by a “low-high-low” pattern, with the maximum activity at 50 mM of NaCl. Results imply that the function of antioxidant systems in higher NaCl concentration is responsible for the salt tolerance observed in Gara-Yonjeh.


Medicago sativa L. Gara-Yonje antioxidant system salt stress isoenzyme pattern 


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  1. 1.
    Badawi, G. H., Yamauchi, Y., Shimada, E., Sasaki, R., Kawano, N., Tanaka, K., Tanaka, K. (2004) Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum) chloroplasts. Plant Sci. 166, 919–928.CrossRefGoogle Scholar
  2. 2.
    Bartels, D., Sunkar, R. (2005) Drought and salt tolerance in plants. Crit. Rev. Plant Sci. 24, 23–58.CrossRefGoogle Scholar
  3. 3.
    Bates, L. S., Waldren, R. P., Teare, I. D. (1973) Rapid determination of free proline for water stress studies. Plant Soil 39, 205–207.Google Scholar
  4. 4.
    Beauchamp, C., Fridovich, I. (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287.CrossRefGoogle Scholar
  5. 5.
    Boominathan, R., Doran, P. M. (2002) Ni-induced oxidative stress in roots of the Ni hyperaccumulator, Alyssum bertolonii. New Phytol. 156, 205–215.CrossRefGoogle Scholar
  6. 6.
    Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Annu. Rev. Biochem. 72, 248–254.CrossRefGoogle Scholar
  7. 7.
    Cobbett, C. S. (2000) Phytochelatins biosynthesis and function in heavy-metal detoxification. Curr. Opin. Plant Biol. 3, 211–216.CrossRefGoogle Scholar
  8. 8.
    Ellman, G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70–77.CrossRefGoogle Scholar
  9. 9.
    Ghamsari, L., Keyhani, E., Golkhoo, S. (2007) Kinetics properties of guaiacol peroxidase activity in Crocus sativus L. corm during rooting. Iran Biomed. J. 11, 137–146.PubMedGoogle Scholar
  10. 10.
    Hajiboland, R., Amjad, L. (2007) Does antioxidant capacity of leaves play a role in growth response to selenium at different sulfur nutritional status? Plant Soil Environ. 53, 207–215.CrossRefGoogle Scholar
  11. 11.
    Howieson, J., Ballard, R. (2004) Optimising the legume symbiosis in stressful and competitive environments within southern Australia: some contemporary thoughts. Soil Biol. Biochem. 36, 1261–1273.CrossRefGoogle Scholar
  12. 12.
    Hu, M. L. (1994) Measurement of protein thiol groups and glutathione in plasma. Methods Enzymol. 233, 380–385.CrossRefGoogle Scholar
  13. 13.
    Jebara, S., Jebara, M., Limam, F., Aouani, M. E. (2005) Changes in ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salt stress. J. Plant. Physiol. 162, 929–936.CrossRefGoogle Scholar
  14. 14.
    Kim, S. Y., Lim, J. H., Park, M. R., Kim, Y. J., Park, T. I. I., Seo, Y. W. (2005) Enhanced antioxidant enzymes are associated with reduced hydrogen peroxide in barley roots under salt stress. J. Biochem. Mol. Biol. 38, 218–224.PubMedGoogle Scholar
  15. 15.
    Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.CrossRefGoogle Scholar
  16. 16.
    Lee, D. H., Kim, Y. S., Lee, C. B. (2001) The inductive responses of the antioxidant enzymes by salt stress in the rice (Oryza sativa L.). J. Plant Physiol. 158, 737–745.CrossRefGoogle Scholar
  17. 17.
    Lin, C. C., Kao, C. H. (1996) Proline accumulation is associated with inhibition of rice seedling root growth caused by NaCl. Plant Sci. 114, 121–128.CrossRefGoogle Scholar
  18. 18.
    Marschner, H. (1995) Mineral Nutrition of Higher Plants. London, Academic Press.Google Scholar
  19. 19.
    Martía, M. C., Camejoa, D., Fernández-García, N., Rellán-Álvarezb, R., Marquesc, S., Sevilla, F., Jiménez, A. (2009) Effect of oil refinery sludges on the growth and antioxidant system of alfalfa plants. J. Hazard. Mater. 171, 879–885.CrossRefGoogle Scholar
  20. 20.
    Moran, J. F., Becana, M., Iturbe-Ormaetxe, I., Frechilla, S., Klucas, R. V., Aparicio-Tejo, P. (1994) Drought induces oxidative stress in pea plants. Planta 194, 346–325.Google Scholar
  21. 21.
    Naya, L., Ladrera, R., Ramos, J., Gonzalez, E. M., Arrese-Igor, C., Minchin, F. R., Becana, M. (2007) The response of carbon metabolism and antioxidant defenses of alfalfa nodules to drought stress and to the subsequent recovery of plants. Plant Physiol. 144, 1104–1114.CrossRefGoogle Scholar
  22. 22.
    Noctor, G., Foyer, C. H. (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 249–279.CrossRefGoogle Scholar
  23. 23.
    Obinger, C., Maj, M., Nicholls, P., Loewen, P. (1997) Activity, peroxide compound formation, and heme d synthesis in Escherichia coli HPII catalase. Arch. Biochem. Biophys. 342, 58–67.CrossRefGoogle Scholar
  24. 24.
    Okeri, H. A., Alonge, P. O. (2006) Determination of the ascorbic acid content of two medicinal plants in Nigeria. Pak. J. Pharm. Sci. 19, 39–44.Google Scholar
  25. 25.
    Platiša, J., Veljovic-Jovanovic, S., Kukavica, B., Vinterhalter, B., Smigockic, A., Ninkovic, S. (2008) Induction of peroxidases and superoxide dismutases in transformed embryogenic calli of alfalfa (Medicago sativa L.). J. Plant Physiol. 165, 895–900.CrossRefGoogle Scholar
  26. 26.
    Poustini, K., Siosemardeh, E. A., Ranjbar, E. M. (2007) Proline accumulation as a response to salt stress in 30 wheat (Triticum aestivum L.) cultivars differing in salt tolerance. Genet. Resour. Crop Evol. 54, 925–934.CrossRefGoogle Scholar
  27. 27.
    Tambussi, E. A., Bartoli, C. G., Belrano, J., Guiamet, J. J., Arans, J. L. (2000) Oxidative damage to thylakoid protein in water stressed leaves of wheat (Triticum aestivum). Physiol. Plant 108, 398–404.CrossRefGoogle Scholar
  28. 28.
    Tejera Garcí, N. A., Iribarne, C., Palma, F., Lluch, C. (2007) Inhibition of the catalase activity from Phaseolus vulgaris and Medicago sativa by sodium chloride. Plant Physiol. Biochem. 45, 535–541.CrossRefGoogle Scholar
  29. 29.
    Tennant, D. (1975) A test of modified line intersect method of estimating root length. J. Ecol. 63, 995–1001.CrossRefGoogle Scholar
  30. 30.
    Vaidyanathan, H., Sivakumar, P., Chakrabarty, R., Thomas, G. (2003) Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)-differential response in salt-tolerant and sensitive varieties. Plant Sci. 165, 1411–1418.CrossRefGoogle Scholar
  31. 31.
    Velikova, V., Yordanov, I., Edreva, A. (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants. Protective role of exogenous polyamines. Plant Sci. 151, 59–66.CrossRefGoogle Scholar
  32. 32.
    Wang, W. B., Kim, Y. H., Lee, H. S., Kim, K. Y., Deng, X. P., Kwak, S. S. (2009) Analysis of antioxidant enzyme activity during germination of alfalfa under salt and drought stresses. Plant Physiol. Biochem. 47, 570–577.CrossRefGoogle Scholar
  33. 33.
    Winterbourn, C. C., McGrath, B. W., Carrell, R. W. (1976) Reactions involving superoxide and normal and unstable haemoglobins. Biochem. J. 155, 493–502.CrossRefGoogle Scholar
  34. 34.
    Woodbury, W., Spencer, A. K., Stahman, M. A. (1971) An improved procedure using ferricyanide for detecting catalase isozymes. Anal. Biochem. 44, 301–305.CrossRefGoogle Scholar
  35. 35.
    Zhou, Z. S., Huang, S. Q., Guo, K., Kant Mehta, S., Zhang, P. C., Yang, Z. M. (2007) Metabolical adaptations to mercury-induced oxidative stress in roots of Medicago sativa L. J. Inorg. Biochem. 101, 1–9.CrossRefGoogle Scholar
  36. 36.
    Zhu, J. K. (2002) Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53, 247–273.CrossRefGoogle Scholar

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© Akadémiai Kiadó, Budapest 2013

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Authors and Affiliations

  1. 1.Department of Plant Biology, Faculty of Natural ScienceUniversity of TabrizTabrizIran

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