Annals of Forest Science

, Volume 66, Issue 2, pp 211–211

Effect of salt on ROS homeostasis, lipid peroxidation and antioxidant mechanisms in Pinus pinaster suspension cells

  • Herlânder Azevedo
  • Vítor Amorim-Silva
  • Rui M. Tavares
Original Article


  • • In the Pinus genus, information on the effectiveness of oxidative defence mechanisms during exposure to salt is lacking. The effect of salt stress imposition on ROS homeostasis was investigated using maritime pine (Pinus pinaster Ait.) suspension cells as a model system.

  • • Cells were maintained in MS-based medium, exposed to salt (50, 100 and 150 mM NaCl) and analysed for biomass production, evidencing a decreasing growth capacity. Use of 100 mM NaCl imposed severe salt stress without affecting cell viability, being chosen for subsequent studies on the ROS homeostasis of salt shock-treated suspension cells.

  • • Increased total ROS levels were evident on the second day of salt exposure, but a superoxide ion transient burst was immediately noticeable. Additionally, lipid peroxide formation seemed to correlate with superoxide ion breakdown. In-gel superoxide dismutase activity evidenced a FeSOD homodimer with strongly increasing activity between hours 12–48 of salt stress imposition. Subsequently, P. pinaster Fe-Sod1 and csApx1 genes were isolated from a cDNA library and expression was shown to increase within 12–24 h.

  • • Results show that severe salt treatment generates oxidative stress in P. pinaster cells despite the induction of antioxidant systems, and suggest a putative involvement of ROS in salt stress signalling.


H2O2 maritime pine NaCl superoxide 

Effet du stress salin sur l’homéostasie des formes réactives d’oxygène, la peroxydation des lipides et les mécanismes antioxydants dans des suspensions cellulaires de Pinus pinaster


  • • Les informations sur les mécanismes de défense oxydative du pin en réponse à un stress salin sont rares. L’effet d’une exposition au sel sur l’homéostasie des formes réactives d’oxygène (FRO) a été étudié en utilisant une suspension cellulaire de pin maritime (Pinus pinaster Ait.) comme modèle.

  • • Les cellules cultivées dans un milieu MS modifié ont été exposées au sel (50, 100 et 150 mM NaCl) et l’analyse de la production de biomasse a révélé une réduction de leur croissance. Une concentration de 100 mM NaCl, stress sévère qui n’affecte cependant pas la viabilité cellulaire, a été choisie pour les études suivantes.

  • • L’augmentation des teneurs en FRO est évidente le jour suivant l’enrichissement du milieu en sels mais une production transitoire d’ions superoxyde est immédiatement constatée. De plus, l’apparition de produits issus de la peroxydation des lipides semble concomitante à la disparition des ions superoxyde. La mesure par tests in-gel de l’activité de la superoxyde dismutase supporte l’implication d’un homodimère de FeSOD dont l’activité augmente fortement au bout de 12 et jusqu’à 48 h d’exposition au sel. Les gènes Fe-Sod1 et csApx1, isolés d’une banque d’ADNc de P. pinaster, voient leur expression augmenter au bout de 12 h et jusqu’à 24 h de traitement.

  • • Les résultats montrent que de fortes concentrations de sels provoquent un stress oxydatif dans les cellules de P. pinaster malgré l’induction de réponses antioxydantes et suggèrent l’implication des ERO dans les voies de transduction du stress salin.


H2O2 pin maritime NaCl superoxide 


  1. Able A.J., Guest D.I., and Sutherland M.W., 1998. Use of a new tetrazolium-based assay to study the production of superoxide radicals by tobacco cell cultures challenged with avirulent zoospores of Phytophthora parasitica var nicotianae. Plant. Physiol. 117: 491–499.PubMedCrossRefGoogle Scholar
  2. Allan A.C., Lapidot M., Culver J.N., and Fluhr R., 2001. An early tobacco mosaic virus-induced oxidative burst in tobacco indicates extracellular perception of the virus coat protein. Plant Physiol. 126: 97–108.PubMedCrossRefGoogle Scholar
  3. Asada K., 1994. Production and action of active oxygen species in photosynthetic tissues. In: Foyer C.H. and Mullineaux P.M. (Eds.), Production and action of active oxygen species in photosynthetic tissues, CRC Press, Boca Raton, pp. 77–104.Google Scholar
  4. Azevedo H., Lino-Neto T., and Tavares R.M., 2003. An improved method for high-quality RNA isolation from needles of adult maritime pine trees. Plant Mol. Biol. Rep. 21: 333–338.CrossRefGoogle Scholar
  5. Azevedo H., Dias A.C.P., and Tavares R.M., 2008a. Establishment and characterization of Pinus pinaster suspension cell cultures. Plant Cell Tiss. Organ Cult. 93: 115–121.CrossRefGoogle Scholar
  6. Azevedo H., Lino-Neto T., and Tavares R.M. 2008b. The necrotroph Botrytis cinerea induces a non-host Type II resistance mechanism in Pinus pinaster suspension-cultured cells. Plant Cell Physiol. 49: 386–395.PubMedCrossRefGoogle Scholar
  7. Beauchamp C. and Fridovich I., 1971. Superoxide dismutase: improved assay and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276–286.PubMedCrossRefGoogle Scholar
  8. Bor M., Ozdemir F., and Turkan I., 2003. 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.CrossRefGoogle Scholar
  9. Boveris A. and Chance B., 1973. The mitochondrial generation of hydrogen peroxide. Biochem. J. 134: 707–716.PubMedGoogle Scholar
  10. Bowler C., Slooten L., Vandenbraden S., Rycke R.D., Botterman J., Sybesma C., Montagu M.V., and Inze D., 1991. Manganese superoxide dismutase can reduce cellular damage mediated by oxygen radicals in transgenic plants. EMBO J. 10: 1723–1732.PubMedGoogle Scholar
  11. Corpas F.J., Gomez M., Hernandez J.A., and Del Rio L.A., 1993. Metabolism of activated oxygen in peroxisomes from two Pisum sativum L. cultivars with different sensitivity to sodium chloride. J. Plant Physiol. 141: 160–165.Google Scholar
  12. Couée I., Sulmon C., Gouesbet G., and El Amrani A., 2006. Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J. Exp. Bot. 57: 449–459.PubMedCrossRefGoogle Scholar
  13. Elkahouia S., Hernández J.A., Abdellyc C., Ghrira R., and Limama F., 2005. Effects of salt on lipid peroxidation and antioxidant enzyme activities of Catharanthus roseus suspension cells. Plant Sci. 168: 607–613.CrossRefGoogle Scholar
  14. Fridovich I., 1985. Biological effects of the superoxide radical. Arch. Biochem. Biophys. 247: 1–11.CrossRefGoogle Scholar
  15. Hernandez J.A., Corpas F.J., Gomez M., del Rio L.A., and Sevilla F., 1993. Salt-induced oxidative stress mediated by activated oxygen species in pea leaf mitochondria. Physiol. Plant. 89: 103–110.CrossRefGoogle Scholar
  16. Kaminaka H., Morita S., Tokumoto M., Masumura T., and Tanaka K., 1999. Differential gene expressions of rice superoxide dismutase isoforms to oxidative and environmental stresses. Free Rad. Res. 31: 219–225.CrossRefGoogle Scholar
  17. Koca H., Bor M., Ozdemir F., and Turkan I., 2007. The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars. Environ. Exp. Bot. 60: 344–351.CrossRefGoogle Scholar
  18. Kurepa J., Hérouart D., Van Montagu M., and Inzé D., 1997. Differential expression of CuZn- and Fe-superoxide dismutase genes of tobacco during development, oxidative stress, and hormonal treatments. Plant Cell Physiol. 38: 463–470.PubMedGoogle Scholar
  19. Lino-Neto T., 2001. Role of oxidative stress enzymes during Zantedeschia aethiopica spathe whitening and regreening, Minho University, Braga, 284 p.Google Scholar
  20. Loreto F. and Velikova V., 2001. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products and reduces lipid peroxidation of cellular membranes. Plant Physiol. 127: 1781–1787.PubMedCrossRefGoogle Scholar
  21. Masood A., Shah N.A., Zeeshan M., and Abraham G., 2006. Differential response of antioxidant enzymes to salinity stress in two varieties of Azolla (Azolla pinnata and Azolla filiculoides). Environ. Exp. Bot. 58: 216–222.CrossRefGoogle Scholar
  22. Mittler R., 2002. Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci. 7: 405–410.PubMedCrossRefGoogle Scholar
  23. Mittler R., Vanderauwera S., Gollery M., and Van Breusegem F., 2004. Reactive oxygen gene network of plants. Trends Plant Sci. 9: 490–498.PubMedCrossRefGoogle Scholar
  24. Murashige T. and Skoog F., 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.CrossRefGoogle Scholar
  25. Noctor G. and Foyer C.H., 1998. Ascorbate and glutathione: keeping active oxygen under control. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49: 249–279.PubMedCrossRefGoogle Scholar
  26. Osmond C.B. and Grace S.C., 1995. Perspectives on photoinhibition and photorespiration in the field: quintessential inefficiencies of the light and dark reactions of photosynthesis. J. Exp. Bot. 46: 1351–1362.Google Scholar
  27. Perl A., Perl-treves R., Galili G., Aviv D., Shalgi E., Malkin S., and Galun E., 1993. Enhanced oxidative-stress defence in transgenic potato plants expressing tomato Cu, Zn superoxide dismutase. Theor. Appl. Genet. 85: 568–576.CrossRefGoogle Scholar
  28. Petersen D.R., Reichard J., Kolaja K.L., and Hartley D.P., 1999. 4-Hydroxynonenal and malondialdehyde heatic protein adducts in rats treated with carbon tetrachloride: immuno-chemical dection and lobular localization. Toxicol. Appl. Pharm. 161: 23–33.CrossRefGoogle Scholar
  29. Radic S., Radic-Stojkovic M., and Pevalek-Kozlina B., 2006. Influence of NaCl and mannitol on peroxidase activity and lipid peroxidation in Centaurea ragusina L. roots and shoots. J. Plant Physiol. 163: 1284–1292.PubMedCrossRefGoogle Scholar
  30. Ruiz J.M., Blasco B., Rivero R.M., and Romero L., 2005. Nicotine-free and salt-tolerant tobacco plants obtained by grafting to salinity-resistant rootstocks of tomato. Physiol. Plant. 124: 465–475.CrossRefGoogle Scholar
  31. Sedmak J.J. and Grossberg S.E., 1977. A rapid, sensitive, and versatile assay for protein using Coomassie Brilliant Blue G250. Anal. Biochem. 79: 544–552.PubMedCrossRefGoogle Scholar
  32. Shimon-Kerner N., Mills D., and Merchuk J.C., 2000. Sugar utilization and invertase activity in hairy-root and cell-suspension cultures of Symphytum officinale. Plant Cell Tissue Organ Cult. 62: 89–94.CrossRefGoogle Scholar
  33. Van Breusegem F., Bailey-Serres J., and Mittler R., 2008. Unraveling the tapestry of networks involving reactive oxygen species in plants. Plant Physiol. 147: 978–984.PubMedCrossRefGoogle Scholar
  34. Verniquet F., Gaillard J., Neuberger M., and Douce R., 1991. Rapid inactivation of plant aconitase by hydrogen peroxide. Biochem. J. 276: 643–648.PubMedGoogle Scholar
  35. Volokita M., 1991. The carboxy-terminal end of glycolate oxidase directs a foreign protein into tobacco leaf peroxisomes. Plant J. 1: 361–366.PubMedCrossRefGoogle Scholar
  36. Wang J., Zhang H., and Allen R.D., 1999. Overexpression of an Arabidopsis peroxisomal ascorbate peroxidase gene in tobacco increases protection against oxidative stress. Plant Cell Physiol. 40: 725–732.PubMedGoogle Scholar
  37. Zhu J.-K., 2001. Plant salt tolerance. Trends Plant Sci. 6: 66–71.PubMedCrossRefGoogle Scholar

Copyright information

© Springer S+B Media B.V. 2009

Authors and Affiliations

  • Herlânder Azevedo
    • 1
  • Vítor Amorim-Silva
    • 1
  • Rui M. Tavares
    • 1
  1. 1.Departamento de BiologiaUniversidade do MinhoBragaPortugal

Personalised recommendations