Acta Physiologiae Plantarum

, Volume 32, Issue 5, pp 925–932 | Cite as

Modulation of symbiotic efficiency and nodular antioxidant enzyme activities in two Phaseolus vulgaris genotypes under salinity

Original Paper


To analyse nodular expression of antioxidant enzymes depending on plant genotype and salinity, two Phaseolus vulgaris genotypes, tolerant BAT477 and sensitive COCOT, were inoculated with the reference strain Rhizobium tropici CIAT899 and grown under 25 and 50 mM NaCl. Plant growth, nodulation and nitrogen fixing activity measured by the acetylene reducing activity (ARA) as an indicator of nitrogenase (E.C. activity were more affected by salt concentrations in COCOT than in BAT477, particularly with 50 mM NaCl. Electrophoresis analysis of antioxidant enzymes in nodules, roots and free-living rhizobia showed that only catalase (CAT E.C. isoenzymes varied with genotype. The sensitive genotype showed lower antioxidant enzyme activities than tolerant genotype and it was more affected by salinity. In the tolerant genotype catalase and ascorbate peroxidase (APX, E.C. were inhibited by salt stress, whereas superoxide dismutase (SOD, E.C. and peroxidase (POX, E.C. were activated by salinity. Statistical analysis allowed suggesting that tolerance to salinity is associated with a differential regulation of distinct superoxide dismutase and peroxidase activities.


Antioxidant enzymes Ascorbate peroxidase Catalase Nitrogen fixing activity Nodule Peroxidase Rhizobium Salinity Superoxide dismutase 



Ascorbate peroxidase


Acetylene reducing activity

ASC-GSH cycle

Ascorbate–glutathione cycle




Ethylenediaminetetraacetic acid


3-(4,5-Dimethlthiazol-2-4)-5-5diphenyl tetrazolium bromide


Nitroblue tetrazolium


Nodule dry weight


Phenylmethylsulfonyl fluoride


Guaiacol peroxidase




Reactive oxygen species


Root dry weight


Shoot dry weight


Superoxide dismutase


N,N,N′,N′-tetramethyl ethylene diamine


Tris (hydroxymethyl) aminomethane



This work was supported by grants from the Tunisian Ministry of Scientific Research, Technology and Competency Development. We are most grateful to Dr. Inoubli T for English revision. We thank Zitoun A, for excellent technical support.


  1. Aebi H (1984) Catalase in vitro. Methods Enzymol 105:121–126CrossRefPubMedGoogle Scholar
  2. Amako K, Chen GX, Asada K (1994) Separate assays specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol 35:497–504Google Scholar
  3. Anderson MD, Prasad TK, Stewart CR (1995) Changes in isozymes profiles of catalase, peroxidase, and gluthatione reductase during acclimation to chilling in mesocotyls of maize seedling. Plant Physiol 109:1247–1257PubMedGoogle Scholar
  4. Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44:276–287CrossRefPubMedGoogle Scholar
  5. Becana M, Dalton DA, Moran JF, Iturbe-Ormaetxe I, Matamoros MA, Rubio MC (2000) Reactive oxygen species and antioxidants in legume nodules. Physiol Plant 109:372–381CrossRefGoogle Scholar
  6. Blokhina O, Virolaonen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress. Anal Bot 91:179–194CrossRefGoogle Scholar
  7. Bradford M (1976) A rapid and sensitive method for the quantification of microgram quantities of proteins utilising the principal of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  8. Comba ME, Benavides MP, Tomaro ML (1998) Effect of salinity on antioxidant defence system in soybean root nodules. Aust J Plant Physiol 25:665–671CrossRefGoogle Scholar
  9. Cruz de Carvalho MH, Arcy-Lameta A, Roy-Macauley H, Gareil M, El Maarouf H, Pham-Thi, Zuily-Fodil Y (2001) Aspartic protease in leaves of common bean (Phaseolus vulgaris L.) and cowpea (Vigna unguiculata L. Walp): enzymatic activity, gene expression and relation to drought susceptibility. FEBS Lett 492:242–246Google Scholar
  10. Delgado MJ, Garrido JM, Ligero F, Lluch C (1993) Nitrogen fixation and carbon metabolism by nodules and bacteroide of pea plants under sodium chloride stress. Physiol Plant 89:824–829CrossRefGoogle Scholar
  11. Drevon JJ, Abdelly C, Amarger N, Aouani ME, Aurag J, Gherbi H, Jebara M et al (2001) An interdisciplinary research strategy to improve symbiotic nitrogen fixation and yield of common bean (Phaseolus vulgaris) in salinised areas of the Mediterranean basin. J Biotechnol 91:257–268CrossRefPubMedGoogle Scholar
  12. Gogorcena Y, Gordon AJ, Escuredo PR, Minchin FR, Witty JF, Moran JF, Becana M (1997) N2 fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants. Plant Physiol 113:1193–1201PubMedGoogle Scholar
  13. Gonzalez EM, Galvez L, Royuria M, Aparicio-Tejo PM, Arresse-igor C (2001) Insights into regulation of nitrogen fixation in pea nodules: lessons from drought, abscisic acid and increased photoassimilate availability. Agronomy 21:607–613CrossRefGoogle Scholar
  14. Hardy RWF, Holston RD, Jakson EK, Burns RC (1968) The acetylene-ethylene assay for nitrogen fixation: laboratory and field evaluation. Plant Physiol 43:1185–1208CrossRefPubMedGoogle Scholar
  15. Jamet A, Sigaud S, Van de Sype G, Puppo A, Hérouart D (2003) Expression of the bacterial catalase genes during Sinorhizobium meliloti- Medicago sativa symbiosis and their crucial role during the infection process. Mol Plant Microbe Int 16:217–225CrossRefGoogle Scholar
  16. Jebara M, Drevon JJ (2001) Genotypic variation in nodule conductance to the oxygen diffusion in common bean (Phaseolus vulgaris L.). Agronomy 21:667–674CrossRefGoogle Scholar
  17. Jebara M, Drevon JJ, Aouani ME (2001) Effects of hydroponic culture system and NaCl on interactions between common bean lines and native rhizobia from Tunisian soils. Agronomy 21:601–605CrossRefGoogle Scholar
  18. Jebara S, Jebara M, Limam F, Aouani ME (2005) Changes of ascorbate peroxidase, catalase, guaiacol peroxidase and superoxide dismutase activities in common bean (Phaseolus vulgaris) nodules under salinity. J Plant Physiol 162:929–936CrossRefPubMedGoogle Scholar
  19. Johnson SM, Doherty SJ, Croy RRD (2003) Biphasic superoxide generation in potato tubers. A self amplifying response to stress. Plant Physiol 13:1440–1449CrossRefGoogle Scholar
  20. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685CrossRefGoogle Scholar
  21. Matamoros MA, Dalton DA, Ramos J, Clemente MR, Rubio MC, Becana M (2003) Biochemistry and molecular biology of antioxidants in the rhizobia–legume symbiosis. Plant Physiol 133:499–509CrossRefPubMedGoogle Scholar
  22. Mhadhbi H, Jebara M, Limam F, Aouani ME (2004) Rhizobial strain involvement in plant growth, nodule protein composition and antioxidants enzymes expression of chickpea–rhizobia symbioses: modulation by salinity. Plant Physiol Biochem 42:717–722CrossRefPubMedGoogle Scholar
  23. Mittler R, Zilinskas BA (1993) Detection of ascorbate peroxidase activity in native gels by inhibition of ascorbate-dependent reduction of nitroblue tetrazolium. Anal Biochem 212:540–546CrossRefPubMedGoogle Scholar
  24. Moran JF, James EK, Rubio MC, Sarath G, Klucas RV, Becana M (2003) Functional characterization and expression of a cytosolic iron-superoxide dismutase from Cowpea root nodules. Plant Physiol 133:773–782CrossRefPubMedGoogle Scholar
  25. Reddy RA, Chaitanya VK, Vivekanandan M (2004) Drought-induced responses of photosynthesis and antioxidant metabolism in higher plants. J Plant Physiol 161:1189–1202CrossRefGoogle Scholar
  26. Ross EJH, Kramer SB, Dalton DA (1999) Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems. Phytochem 52:1203–1210CrossRefGoogle Scholar
  27. Rubio MC, Gonzalez EM, Minchin FR, Webb KJ, Arrese-Igor C, Ramos J, Becana M (2002) Effects of water stress on antioxidant enzymes of leaves and nodules of transgenic alfalfa overexpressing superoxide dismutases. Physiol Plant 115:531–540CrossRefPubMedGoogle Scholar
  28. Santos R, Hérouart D, Puppo A, Touati D (2000) Critical protective role of bacterial superoxide dismutase in Rhizobium–legume symbiosis. Mol Microbiol 38:750–759CrossRefPubMedGoogle Scholar
  29. Sassi Aydi S, Aydi S, Gonzalez E, Abdelly C (2008) Osmotic stress affects water relations, growth, and nitrogen fixation in Phaseolus vulgaris plants. Acta Physiol Plant 30:441–449CrossRefGoogle Scholar
  30. Sassi S, Gonzalez EM, Aydi S, Arrese-Igor C, Abdelly C (2008) Tolerance of common bean to long-term osmotic stress is related to nodule carbon flux and antioxidant defences: evidence from two cultivars with contrasting tolerance. Plant Soil 312:39–48CrossRefGoogle Scholar
  31. Scandalios JG, Guan L, Polidords AN (1997) Catalases in plants: gene structure, proprieties, and expression. In: Scandalios JG (ed) Oxidative stress and the molecular biology of antioxidant defences. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 343–406Google Scholar
  32. Sheokand S, Dhandi S, Swaraj K (1995) Studies on nodule functioning and hydrogen peroxide scavenging enzymes under salt stress in chickpea nodule. Plant Physiol Biochem 33:561–566Google Scholar
  33. Swaraj K, Laura JS, Bishnoi NR (1993) Nitrate induced nodule senescence and changes in activities of enzymes scavenging H2O2 in clusterbean (Cyamopsis tetragonaloba Taub.). J Plant Physiol 41:202–205Google Scholar
  34. Tejera NA, Campos R, Sanjuan J, Lluch C (2004) Nitrogenase and antioxidant enzyme activities in Phaseolus vulgaris nodules formed by Rhizobium tropici isogenic strains with varying tolerance to salt stress. J Plant Physiol 3:329–338CrossRefGoogle Scholar
  35. Vadez V, Drevon JJ (2001) Genotypic variability in phosphorus use efficiency for symbiotic N2 fixation in common bean (Phaseolus vulgaris). Agronomy 21:691–699CrossRefGoogle Scholar
  36. Vadez V, Rodier F, Payre H, Drevon JJ (1996) Nodule conductance to O2 and nitrogenase-linked respiration in bean genotypes varying in the tolerance of N2 fixation to P deficiency. Plant Physiol Biochem 34:871–878Google Scholar
  37. Vallejos CE (1983) Enzyme activity staining. In: Tanksley SD, Orton TJ (eds) Isozymes in plant genetics and breeding, Part A. Elseviers, Amsterdam, p 469–516Google Scholar
  38. Velagaleti RR, Marsh S, Kramer D (1990) Genotypic differences in growth and nitrogen fixation among soybean (Glycin Max (L.) Merr.) cultivars grown under salt stress. Trop Agric 67:169–177Google Scholar
  39. Yu Q, Rengel Z (1999) Micronutrient deficiency influences plant growth and activities of superoxide dismutase in narrow-neafed lupins. Ann Bot 83:175–182CrossRefGoogle Scholar
  40. Zhu JK (2001) Plant salt tolerance. Plant Sci 6:66–71Google Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2010

Authors and Affiliations

  • Salwa Jebara
    • 1
  • Jean Jacque Drevon
    • 2
  • Moez Jebara
    • 1
  1. 1.Centre of Biotechnology Borj Cedria (CBBC)Hammam LifTunisia
  2. 2.UMR1222 Rhizosphère & Symbiose INRA-AGRO.MMontpellier Cedex 01France

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