Advertisement

Plant and Soil

, Volume 158, Issue 2, pp 151–162 | Cite as

Measurement of nitrogenase activity in legume root nodules: In defense of the acetylene reduction assay

  • J. Kevin Vessey
Review

Abstract

The closed acetylene reduction assay has been used as a measure of nitrogenase activity and an indicator of N2 fixation in Rhizobium/legume symbioses for 25 years. However, starting 10 years ago this assay has come under harsh criticism as being inaccurate. Currently, confusion exists regarding the conditions under which the acetylene reduction assay can be used accurately, or whether it can be used at all as a measure of nitrogenase activity. This article reviews the circumstance that has lead to this confusion. The author argues that under the proper assay conditions and with the appropriate checks, the closed acetylene reduction assay is still a valuable tool in assessing relative differences in nitrogenase activity in Rhizobium/legume symbioses.

Key words

acetylene reduction gas exchange hydrogen evolution nitrogen fixation oxygen diffusion rhizobia 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Appleby, C A 1984 Leghmoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35, 443–478.Google Scholar
  2. Atkins, C A, Fernando, M, Hunt, S and Layzell, D B 1992 A metabolic connection between nitrogenase activity and the synthesis of ureides in nodulated soybean. Physiol. Plant. 84, 441–447.Google Scholar
  3. Balandreau, J and Dommergues, Y 1971 Measure in situ de l'activité nitrogénasique. C.R. Acad. Sc. Paris 273, 2020–2023.Google Scholar
  4. Bedmar, E J and Phillips, D A 1984 Pisum sativum cultivar effects on hydrogen metabolism in Rhizobium. Can. J. Bot. 62, 1682–1686.Google Scholar
  5. Begersen, F J 1962 Oxygenation of leghaemoglobin in soybean root-nodules in relation to the external oxygen tension. Nature 194, 1059–1061.Google Scholar
  6. Bergersen, F J 1970 The quantitative relationship between nitrogen fixation and the acetylene reduction assay. Aust. J. Biol. Sci. 23, 1015–1025.Google Scholar
  7. Boddey, R M and Urquiaga, S 1992 Calculations and assumptions involved in the use of the A-value and N-15 isotope dilution techniques for the estimation of the contribution of plant-associated biological N2 fixation. Plant and Soil 145, 151–155.Google Scholar
  8. Bremer, E and Van Kessel, C 1990 Appraisal of the nitrogen-15 natural abundance method for quantifying dinitrogen fixation. Soil Sci. Soc. Am. J. 54, 404–411.Google Scholar
  9. Burris, R H 1991 Nitrogenases. J. Biol. Chem. 266, 9339–9342.Google Scholar
  10. Denison, R F, Weisz, P R and Sinclair, T R 1983 Analysis of acetylene reduction rates of soybean nodules at low acetylene concentrations. Plant Physiol. 73, 648–651.Google Scholar
  11. Dilworth, M 1966 Acetylene reduction by nitrogen-fixing preparations from Clostridium pasteurianum. Biochem. Biophys. Acta 127, 285–294.Google Scholar
  12. Ding, M M, Yi, W M and Liao, L Y 1986 A survey of the N2ase activity of nodules of tree legumes, including Tamarindus indica. Nitro. Fixing Tree Res. Rep. 4, 9–10.Google Scholar
  13. Döbereiner, J 1980 Forage grasses and grain legumes. In Methods for Evaluating Biological Nitrogen Fixation. Ed. F J Bergersen. pp 535–556. John Wiley, New York.Google Scholar
  14. Drevon, J J, Kalia, V C, Heckmann, M O and Pedelahore, P 1988 In situ open-flow assay of acetylene reduction activity by soybean root nodules: influence of acetylene and oxygen. Plant Physiol. Biochem 26, 73–78.Google Scholar
  15. Durand, J L, Sheehy, J E and Minchin, F R 1987 Nitrogenase activity, photosynthesis and nodule water potential in soyabean plants experiencing water deprivation. J. Exp. Bot. 187, 311–321.Google Scholar
  16. Fishbeck, K, Evans, H J and Boersma, L L 1973 Measurement of nitrogenase activity of intact legume symbionts in situ using the acetylene reduction assay. Agron. J. 65, 429–433.Google Scholar
  17. Guerin, V, Trinchant, J C and Rigaud, J 1990 Nitrogen fixation (C2H2 reduction) by broad bean (Vicia faba L.) nodules and bacteroids under water-restricted conditions. Plant Physiol. 92, 595–601.Google Scholar
  18. Handley, L L and Raven, J A 1992 The use of natural abundance of nitrogen isotopes in plant physiology and ecology. Plant Cell Environ. 15, 965–985.Google Scholar
  19. Hardy, R W F, Holsten, R D, Jackson, E K and Burns, R C 1968 The acetylene — ethylene assay for N2 fixation: Laboratory and field evaluation. Plant Physiol. 43, 1185–1207.Google Scholar
  20. Hunt, S, King, B J, Canvin, D T and Layzell, D B 1987 Steady and non-steady gas exchange characteristics of soybean nodules in relation to the oxygen diffusion barrier. Plant Physiol. 84, 164–172.Google Scholar
  21. Hunt, S, Gaito, S and Layzell, D B 1988 Model of gas exchange and diffusion in legume nodules. II: Characterization of the diffusion barrier and estimation of the infected cell concentration of CO2, H2 and N2. Planta 173, 128–141.Google Scholar
  22. Hunt, S and Layzell, D B 1993. Gas exchange of legume nodules and the regulation of nitrogenase activity. Ann. rev. Plant Physiol. Mol. Biol. 44, 483–511.Google Scholar
  23. Imsande, J 1991 Regulation of nodule efficiency by the undisturbed soybean plant. J. Exp. Bot. 42, 687–691.Google Scholar
  24. Imsande, J and Ralston, E J 1981 Hydroponic growth and the non-destructive assay for dinitrogen fixation. Plant Physiol. 68, 1380–1384.Google Scholar
  25. James, E K, Sprent, J I, Minchin, F R and Brewin, N J 1991 Intercellular location of glycoprotein in soybean nodules: Effect of altered rhizosphere oxygen concentration. Plant Cell Environ. 14, 467–476.Google Scholar
  26. Johnsen, K H and Apsley, D K 1991 A simple method for measuring acetylene reduction of intact, nodulated locust seedlings. Tree Physiol. 9, 501–506.Google Scholar
  27. Knowles, R 1980 Nitrogen fixation in natural plant communities and soils. In Methods for Evaluating Biological Nitrogen Fixation. Ed. F J Bergersen. pp 557–582. John Wiley, New York.Google Scholar
  28. Kuzma, M M, Hunt, S and Layzell, D B 1993 Role of oxygen in the limitation and inhibition of nitrogenase activity and respiration rate in individual soybean nodules. Plant Physiol. 101, 161–169.Google Scholar
  29. Layzell, D B 1990 N2 fixation, NO3 - reduction and NH4 + assimilation. In Plant Physiology, Biochemistry and Molecular Biology. Eds. D T Dennis and D HTurpin. pp 389–406. Longman, Essex, UK.Google Scholar
  30. Layzell, D B, Weagle, G E and Canvin, D T 1984 A highly sensitive flow through H2 gas analyzer for use in N2 fixation studies. Plant Physiol. 75, 582–585.Google Scholar
  31. Mahon, J D 1977 Root and nodule respiration in relation to acetylene reduction in intact nodulated peas. Plant Physiol. 60, 812–816.Google Scholar
  32. Merderski, H J and Streeter, J G 1977 Continuous, automated acetylene reduction assays using intact plants. Plant Physiol. 59, 1076–1081.Google Scholar
  33. Minchin, F R, Becana, M and Sprent, J I 1989. Short-term inhibition of legume N2 fixation by nitrate. 2. Nitrate effects on nodule oxyen diffusion. Planta 180, 46–52.Google Scholar
  34. Minchin, F R, Sheehy, J E, Witty, J F and Muller, M 1983 A major error in the acetylene reduction assay: Decreases in nodular nitrogenase activity under assay conditions. J. Exp. Bot. 34, 641–649.Google Scholar
  35. Minchin, F R, Sheehy, J E and Witty, J F 1986 Further errors in the acetylene reduction assay: Effect of plant disturbance. J. Exp. Bot. 37, 1581–1591.Google Scholar
  36. Olsson, J E and Gresshoff, P M 1984 Analysis of the acetylene reducing ability of root nodule protoplasts isolated from Glycine max. Z. Pflanzenphysiol. 113, 471–476.Google Scholar
  37. Parsons, R, Raven, J A and Sprent, J I 1992 A simple open flow system used to measure acetylene reduction activity of Sesbania rostrata stem and root nodules. J. Exp. Bot. 43, 595–604.Google Scholar
  38. Schubert, K R and Evans, H J 1976 Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts. Proc. Natl. Acad. Sci. USA 73, 1207–1211.Google Scholar
  39. Sheehy, J E 1991 Theory of a crop enclosure system for measuring nitrogen fixation, photosynthesis, respiration and biological processes in the soil. Ann. Bot. 67, 123–130.Google Scholar
  40. Sheehy, J E, Bergersen, F J, Minchin, F R and Witty, J 1987 A simulation study of gaseous diffusion resistance, nodule pressure gradients and biological nitrogen fixation in soyabean nodules. Ann. Bot. 60, 345–351.Google Scholar
  41. Sheehy, J E, Woodward, F I and Gosse, G 1991 Measurement of N2 fixation (C2H2), photosynthesis and respiration using an open system in the natural environment. Ann. Bot. 67, 131–136.Google Scholar
  42. Sung, L, Moloney, A H, Hunt, S and Layzell, D B 1991 The effect of excision on O2 diffusion and metabolism in soybean nodules. Physiol. Plant. 83, 67–74.Google Scholar
  43. Tjepkema, J D and Yocum, C 1974 Measurement of O2 partial pressure within soybean nodules by oxygen microelectrode. Planta 119, 351–360.Google Scholar
  44. Turner, G L and Gibson, A H 1980 Measurement of nitrogen fixation by indirect means. In Methods for Evaluating Biological Nitrogen Fixation. Ed. F J Bergersen. pp 111–138. John Wiley, New York.Google Scholar
  45. Vessey, J K 1992 Cultivar differences in assimilate partitioning and capacity to maintain N2 fixation rate in pea during pod-filling. Plant and Soil 139, 185–194.Google Scholar
  46. Vessey, J K, Walsh, K B and Layzell, D B 1988a Oxygen limitation of N2 fixation in stem-girdled and nitrate-treated soybean. Physiol. Plant. 73, 113–121.Google Scholar
  47. Vessey, J K, Walsh, K B and Layzell, D B 1988b Can a limitation in phloem supply to nodules account for the inhibitory effect of nitrate on nitrogenase activity in soybean? Physiol. Plant. 74, 137–146.Google Scholar
  48. Vikman, P A and Vessey, J K 1992 The decline in N2 fixation rate in common bean with the onset of pod-filling: Fact or artifact. Plant and Soil 147, 95–105.Google Scholar
  49. Walsh, K W, Vessey, J K and Layzell, D B 1987 Carbohydrate supply and N2 fixation in soybean: The effect of varied daylength and stem girdling. Plant Physiol. 85, 137–144.Google Scholar
  50. Witty, J F and Minchin, F R 1988 Measurement of nitrogen fixation by the acetylene reduction assay; Myths and mysteries. In Nitrogen Fixation by Legumes in Mediterranean Agriculture. Eds. D P Beck and L AMateron. pp 331–334. Martinus Nijhoff Publishers, Dordrecht/Boston/London.Google Scholar
  51. Witty, J F, Minchin, F R, Sheehy, J S and Minquez, M I 1984 Acetylene-induced changes in the oxygen diffusion resistance and nitrogenase activity of legume root nodules. Ann. Bot. 53, 13–20.Google Scholar
  52. Wolle, D, Kim, C H, Dean, D and Howard, J B 1992 Ionic interactions in the nitrogenase complex: properties of Fe-protein containing substitutions for Arg-100. J. Biol. Chem. 267, 3667–3673.Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • J. Kevin Vessey
    • 1
  1. 1.Department of Plant ScienceUniversity of ManitobaWinnipegCanada

Personalised recommendations