Biologia Plantarum

, Volume 54, Issue 3, pp 461–470 | Cite as

Effect of abiotic stresses on the activity of antioxidative enzymes and contents of phytohormones in wild type and AtCKX2 transgenic tobacco plants

  • Z. Mýtinová
  • V. Motyka
  • D. Haisel
  • A. Gaudinová
  • Z. Lubovská
  • N. Wilhelmová
Original Papers


The responses of antioxidant enzymes (AOE) ascorbate peroxidase (APX), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT) in soluble protein extracts from leaves and roots of tobacco (Nicotiana tabacum L. cv. Samsun NN) plants to the drought stress, salinity and enhanced zinc concentration were investigated. The studied tobacco included wild-type (WT) and transgenic plants (AtCKX2) harbouring the cytokinin oxidase/dehydrogenase gene under control of 35S promoter from Arabidopsis thaliana (AtCKX2). The transgenic plants exhibited highly enhanced CKX activity and decreased contents of cytokinins and abscisic acid in both leaves and roots, altered phenotype, retarded growth, and postponed senescence onset. Under control conditions, the AtCKX2 plants exhibited noticeably higher activity of GR in leaves and APX and SOD in roots. CAT activity in leaves always decreased upon stresses in WT while increased in AtCKX2 plants. On the contrary, the SOD activity was enhanced in WT but declined in AtCKX2 leaves. In roots, the APX activity prevailingly increased in WT while mainly decreased in AtCKX2 in response to the stresses. Both WT and AtCKX2 leaves as well as roots exhibited elevated abscisic acid content and increased CKX activity under all stresses while endogenous CKs and IAA contents were not much affected by stress treatments in either WT or transgenic plants.

Additional key words

abscisic acid cytokinin cytokinin oxidase/dehydrogenase drought salinity zinc 



abscisic acid


transgenic tobacco harbouring cytokinin oxidase/dehydrogenase gene from Arabidopsis thaliana


antioxidative enzymes


ascorbate peroxidase










cytokinin oxidase/dehydrogenase


deepoxidation state


glutathione reductase


superoxide dismutase


wild type


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This work was financially supported by the Grant Agency of the Czech Republic, projects No.522/03/0312 and 206/06/1306. The authors wish to thank Ing. P. Dobrev, Ing. J. Malbeck and Ing. A. Trávníčková (Institute of Experimental Botany AS CR, Prague, Czech Republic) for HPLC separation and MS analyses of phytohormones and M. Korecká for excellent technical assistance. Z.M. and V.M. contributed equally to this work.


  1. Abdelghani, M.O., Suty, L., Chen, J.N., Renaudin, J.-P., Teyssendier de la Serve, B.: Cytokinins modulate the steady-state levels of light-dependent and light-independent proteins and mRNAs in tobacco cell suspensions. — Plant Sci. 77: 29–40, 1991.CrossRefGoogle Scholar
  2. Alscher, R.G., Donahue, J.L., Cramer, C.L.: Reactive oxygen species and antioxidants, relationships in green cells. — Physiol. Plant. 100: 224–233, 1997.CrossRefGoogle Scholar
  3. Arbona, V., Flors, V., Jacas, J., García-Augustín, P., Gómez-Cadenas, A.: Enzymatic and non-enzymatic antioxidant responses of Carrizo citrage, a salt-sensitive citrus rootstock, to different levels of salinity. — Plant Cell Physiol. 44: 388–394, 2003.Google Scholar
  4. Bonnet, M., Camares, O., Veisseire, P.: Effects of zinc and influence of Acremoniom lolii on growth parameters, chlorophyll a fluorescence and antioxidant enzyme activities of ryegrass (Lolium perenne L. cv Apollo). — J. exp. Bot. 51: 945–953, 2000.CrossRefPubMedGoogle Scholar
  5. Bor, M., Özdemir, F., Türkan, I.: The effect of salt stress on lipid peroxidation and antioxidants in leaves of sugar beet Beta vulgaris L. and wild beet Beta maritima L. — Plant Sci. 164: 77–84, 2003.CrossRefGoogle Scholar
  6. Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. — Anal Biochem. 72: 248–254, 1976.CrossRefPubMedGoogle Scholar
  7. Brugière, N., Jiao, S., Hantke, S., Zinselmeier, C., Roessler, J.A., Niu, X., Jones, R.J., Habben, J.E.: Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress. — Plant Physiol. 132: 1228–1240, 2003.CrossRefPubMedGoogle Scholar
  8. Cakmak, I: Possible roles of zinc on protecting plant calls from damage by reactive oxygen species. — New Phytol. 146: 185–205, 2000.CrossRefGoogle Scholar
  9. Christmann, A., Grill, E., Meinhard, M.: Abscisic acid signalling. — In: Hirt H., Shinozaki K. (ed.): Plant Responses to Abiotic Stress. Pp. 39–71. Springer-Verlag, Berlin — Heidelberg 2003.Google Scholar
  10. Clijsters, H., Cuypers, A., Vangronsfeld, J.: Physiological responses to heavy metals in higher plants. Defence against oxidative stress. — Z. Naturforsch. Biosci. 54: 730–734, 1999.Google Scholar
  11. Contour-Ansel, D., Torres-Franklin, M.L., Cruz de Carvalho, M.H., D’Arcy-Lameta, A., Zuily-Fodil, Y.: Glutathione reductase in leaves of cowpea, cloning of two cDNAs, expression and enzymatic activity under progressive drought stress, desiccation and abscisic acid treatment. — Ann. Bot. 98: 1279–1287, 2006.CrossRefPubMedGoogle Scholar
  12. Cowan, A.K., Cairns, A.L.P., Bartels-Rahm, B.: Regulation of abscisic acid metabolism: towards a metabolic basis for abscisic acid-cytokinin antagonism — J. exp Bot. 50: 595–603, 1999.CrossRefGoogle Scholar
  13. Cuypers, A., Vangronsveld, J., Clijsters, H.: The redox status of plant cells (AsA and GSH) is sensitive to zinc imposed oxidative stress in roots and primary leaves of Phaseolus vulgaris. — Plant Physiol. Biochem. 39: 657–664, 2001.CrossRefGoogle Scholar
  14. Cuypers, A., Vangronsveld, J., Clijsters, H.: Peroxidases in roots and primary leaves of Phaseolus vulgaris copper and zinc phytotoxicity, a comparison. — J. Plant Physiol. 159: 869–876, 2002.CrossRefGoogle Scholar
  15. Dionisio-Sese, M., Tobita, S.: Antioxidant responses of rice seedlings to salinity stress. — Plant Sci. 135: 1–9, 1998.CrossRefGoogle Scholar
  16. Dixit, V., Pandey, V., Shyam, R.: Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). — J. exp. Bot 52: 1101–1109, 2001.CrossRefPubMedGoogle Scholar
  17. Dobrev, P., Kamínek, M.: Fast and efficient separation of cytokinins from auxin and abscisic acid and their purification using mixed-mode solid-phase extraction. — J. Chromatogr. A 950: 21–29, 2002.CrossRefPubMedGoogle Scholar
  18. Dobrev, P.I., Havlíček, L., Vágner, M., Malbeck, J., Kamínek, M.: Purification and determination of plant hormones auxin and abscisic acid using solid phase extraction and two-dimensional high performance liquid chromatography. — J. Chromatogr. A 1075: 159–166, 2005.CrossRefPubMedGoogle Scholar
  19. Gaudinová, A., Dobrev, P., Malbeck, J., Špak, J., Trávníčková, A., Vaňková, R., Motyka, V.: [The effect of biotic and abiotic stress on phytohormone levels and metabolism in radish.] — In: Hnilička, F. (ed.): Vliv Abiotických a Biotických Stresorů na Vlastnosti Rostlin. Pp. 81–84. Česká Zemědělská Univerzita, Praha 2004. [In Czech.]Google Scholar
  20. Gaudinová, A., Dobrev, P.I., Šolcová, B., Novák, O., Strnad, M., Friedecký, D., Motyka, V.: The involvement of cytokinin oxidase/dehydrogenase and zeatin reductase in regulation of cytokinin levels in pea (Pisum sativum L.) leaves. — J. Plant Growth Regul. 24: 188–200, 2005.CrossRefGoogle Scholar
  21. Gidrol, X., Lin, W.S., Degousee, N., Yip, S.F., Kush, A.: Accumulation of reactive oxygen species and oxidation of cytokinin in germinating soybean seeds. — Eur. J. Biochem. 224: 221–28, 1994.Google Scholar
  22. Goldberg, D.M., Spooner, R.J.: Glutathione reductase. — In: Bergmayer, H.U. (ed.): Methods in Enzymatic Analysis. Vol. 3. Pp. 259–265. Verlag-Chemie, Weinheim 1983.Google Scholar
  23. Hacisalihoglu, G., Hart, J.J., Wang, Y.H., Cakmak, I., Kochian, L.V.: Zinc efficiency is correlated with enhanced expression and activity of zinc-requiring enzymes in wheat. — Plant Physiol. 131: 595–602, 2003.CrossRefPubMedGoogle Scholar
  24. Hare, P.D., Cress, W.A., Van Staden, J.: The involvement of cytokinins in plant responses to environmental stress. — Plant Growth Regul. 23: 79–103, 1997.CrossRefGoogle Scholar
  25. Hernandéz, J.A., Campillo, A., Jiménez, A., Alarcón, J., Sevilla, F.: Response of antioxidant systems and leaf water relations to NaCl stress in pea plants. — New Phytol. 141: 241–251, 1999.CrossRefGoogle Scholar
  26. Liu, X., Huang, B.: Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass. — Crop. Sci. 40: 503–510, 2000.CrossRefGoogle Scholar
  27. Mittler, R.: Oxidative stress, antioxidants and stress tolerance. — Trends Plant Sci. 7: 405–410, 2002.CrossRefPubMedGoogle Scholar
  28. Moran, J.F., Becana, M., Iturbe-Ormaetxe, I., Frwchilla, S., Klucas, V.: Drought induces oxidative stress in pea plants. — Planta 194: 346–352, 1994.CrossRefGoogle Scholar
  29. Motyka, V., Vaňková, R., Čapková, V., Petrášek, J., Kamínek, M., Schmülling, T.: Cytokinin-induced upregulation of cytokinin oxidase activity in tobacco includes changes in enzyme glycosylation and secretion. — Physiol. Plant. 111: 11–21, 2003.CrossRefGoogle Scholar
  30. Mýtinová, Z., Haisel, D., Wilhelmová, N.: Photosynthesis and protective mechanisms in transgenic tobacco leaves with overexpressed cytokinin oxidase/dehydrogenase and thus lowered cytokinin content during ageing. — Photosynthetica 44: 599–605, 2006.Google Scholar
  31. Mýtinová, Z., Wilhelmová, N., Gaudinová, A., Motyka, V.: [Comparison of abiotic stresses on antioxidative enzymatic system in plants differing in their cytokinin metabolism.] — In: Bláha, L. (ed.): Vliv Abiotických a Biotických Stresorů na Vlastnosti Rostlin. Pp. 211–217. Výzkumný Ústav Rostlinné Výroby, Praha 2005. [In Czech.]Google Scholar
  32. Nakano, Z, Asada, K.: Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplast. — Plant Cell Physiol. 22: 867–880, 1981.Google Scholar
  33. Procházková, D., Wilhelmová, N.: Antioxidant protection during ageing and senescence in transgenic tobacco with enhanced activity of cytokinin oxidase/dehydrogenase. — Biol. Plant. 53: 691–696, 2009.CrossRefGoogle Scholar
  34. Rios-Gondalez, K., Erdei, L., Lips, S.H.: The activity of antioxidant enzymes in maize and sunflower seedlings as affected by salinity and different nitrogen sources. — Plant Sci. 162: 923–930, 2002.CrossRefGoogle Scholar
  35. Savouré, A., Thorin, D., Davey, M., Xue-Juh, H., Mauro, S., Van Montagu, M., Inzé, D., Verbruggen, N.: NaCl and CuSO4 treatment trigger distinct oxidative defence mechanisms in Nicotiana plumbaginifolia L. — Plant Cell Environ. 22: 387–396, 1999.CrossRefGoogle Scholar
  36. Singh, S.S., Letham, D.S., Jameson, P.E., Zang, R., Parker, C.W., Bandenoch-Jones, J. Noodén, L.D.: Cytokinin biochemistry in relation to leaf senescence. — Plant Physiol. 88: 788–794, 1988.Google Scholar
  37. Slovik, S., Daeter, W., Hartung, W.: Compartmental redistribution and long-distance transport of abscisic acid (ABA) in plants as influenced by environmental changes in the rhizosphere — a biomathematic model. — J. exp. Bot. 46: 881–894, 1995.CrossRefGoogle Scholar
  38. Sumithra, K., Jutur, P.P., Carmel, B.D., Reddy, A.R.: Salinity induced changes in two cultivars of Vigna radiata: responses of antioxidative and proline metabolism. — Plant Growth Regul. 50: 11–22, 2006.CrossRefGoogle Scholar
  39. Thomas, D.J., Avenson, T.J., Thomas, J.B., Herbert, S.K.: A cyanobacterium lacking iron superoxide dismutase is sensitised to oxidative stress induced with methyl viologen but is not sensitised to oxidative stress induced with norflurazon. — Plant Physiol. 116: 1593–1602, 1998.CrossRefPubMedGoogle Scholar
  40. Tripathi, B.N., Gaur, J.P.: Relationship between copper- and zinc-induced oxidative stress and proline accumulation in Scenedesmus sp. — Planta 219: 397–404, 2004.CrossRefPubMedGoogle Scholar
  41. Ukeda, H., Maeda, S., Ishii, T., Sawamura, M.: Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3′-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase. — Anal. Biochem. 251: 206–209, 1997.CrossRefPubMedGoogle Scholar
  42. Ünyayar, S., Keleş, Y., Çekiç, F.Ö.: The antioxidative response of two species with different drought tolerances as a result of drought and cadmium stress combination. — Plant Soil Environ. 51: 57–64, 2005.Google Scholar
  43. Vaillant, N., Monnet, F., Hitmi, A., Sallanon, H., Coudret, A.: Comparative study of responses in four Datura species to a zinc stress. — Chemosphere 59: 1005–1013, 2005.CrossRefPubMedGoogle Scholar
  44. Wang, H., Liu, R.L, Jin, J.Y.: Effects of zinc and soil moisture on photosynthetic rate and chlorophyll fluorescence parameters of maize. — Biol. Plant. 53: 191–194, 2009.CrossRefGoogle Scholar
  45. Wang, W., Vinocur, B., Altman, A.: Plant response to drought, salinity and extreme temperature, towards genetic engineering for stress tolerance. — Planta 218: 1–14, 2003.CrossRefPubMedGoogle Scholar
  46. Wang, W.X., Vinocur, B., Shoseyov, O., Altman, A.: Biotechnology of plant osmotic stress tolerance, physiological and molecular consideration. — Acta Hort. 560: 285–292, 2001.Google Scholar
  47. Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H., Schmülling, T.: Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. — Plant Cell 15: 2532–2550, 2003.CrossRefPubMedGoogle Scholar
  48. Werner, T., Motyka, V., Strand, M., Schmülling, T.: Regulation of plant growth by cytokinin. — Proc. nat. Acad. Sci. USA 98:10487–10492, 2001.CrossRefPubMedGoogle Scholar
  49. Wilkinson, S., Davies, W.J.: Xylem sap pH increase: a drought signal received at the apoplastic face of the guard cell that involves the suppression of saturable abscisic acid uptake by the epidermal symplast. — Plant Physiol. 113: 559–573, 1997.PubMedGoogle Scholar
  50. Zeevaart, J.A.D., Creelman, R.A.: Metabolism and physiology of abscisic acid. — Annu. Rev. Plant Physiol. Plant mol. Biol. 39: 439–473, 1988.CrossRefGoogle Scholar
  51. Zhang, J., Kirkham, M.B.: Antioxidant responses to drought in sunflower and sorghum seedlings. — New Phytol. 132: 361–373, 1996.CrossRefGoogle Scholar
  52. Zhu, Z., Wei, G., Li, J., Qian, Q., Yu, J.: Silicon alleviates salt stress and increases antioxidant enzymes activity in leaves of salt-stressed cucumber (Cucumis sativus L.). — Plant Sci. 167: 527–533, 2004.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Z. Mýtinová
    • 1
    • 2
  • V. Motyka
    • 3
  • D. Haisel
    • 1
    • 2
  • A. Gaudinová
    • 3
  • Z. Lubovská
    • 1
    • 2
  • N. Wilhelmová
    • 2
  1. 1.Faculty of ScienceCharles UniversityPraha 2Czech Republic
  2. 2.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPragueCzech Republic
  3. 3.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicPragueCzech Republic

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