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Methods for the study of nitrogen assimilation and transport in grain legumes

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Summary

The study of nitrogen assimilation and transport in grain legumes is a necessary preliminary to selecting plant cultivars for breeding programs aimed at increasing the contribution of nitrogen fixation to plant production and seed yield. Methods used in such studies are spread widely throughout the literature and have not always been sufficiently tested. This paper presents a comprehensive set of reliable techniques particularly adapted for use in laboratories in developing countries which lack sophisticated and expensive instruments. A continuous flow system for the measurement of N2 fixation (acetylene reduction) using simple and inexpensive components is described, as are techniques for the estimation of nodule respiration, hydrogen evolution and plant nitrate reductase activity. A number of colorimetric techniques have been adapted for the analysis of nitrate-, amino-, amide-, ureide- and total-N in small volumes of xylem (bleeding) sap.

Résumé

L'étude de l'assimilation et du transport de l'azote dans les légumineuses à graines est une nécessité préliminaire pour la sélection des cultivars destinés aux croisements visant à accroître la fixation de l'azote pour la croissance des plantes et le rendement en graines. Les méthodes employées pour ces études sont éparpillées dans la littérature et n'ont pas été suffisamment testées. Dans cet article, il est présenté une étude d'ensemble de méthodes fiables, particulièrement adaptées aux laboratoires des pays en voie de développement qui ne di sposent pas d'instrumentation sophistiquée et coûteuse. Il est notamment décrit un système simple et peu onéreux de flux continu pour mesurer la fixation de l'azote par réduction de l'acétylène, ainsi que des techniques pour déterminer la respiration des nodules, le dégagement d'hydrogéne et l'activité nitrate-réductase des plantes. Plusieurs techniques colorimétriques ont été adaptées à l'analyse de l'azote nitrique, aminé, uréidique et total, dans de faibles volumes de suc de xylème prélevé par saignée.

Resumen

El estudio de la asimilación y del transporte de nitrógeno en leguminosas de grano es un preámbulo necesario para la selección de cultivares obtenidos en programas de mejora que tengan como objetivo el incremento de la contribución de la fijación de nitrógeno a la producción de plantas y de semillas. Los métodos utilizados en dichos estudios estan dispersos en la literatura y no siempre se han comprobado lo suficiente. Este trabajo presenta un amplio conjunto de técnicas seguras, que se adaptan particularmente bien para su uso en laboratorios de países en desarrollo que carecen de instrumental caro y sofisticado. Se describe un sistema de flujo continuo para la medida de la fijación de N2 (reducción de acetileno) que utiliza componentes sencillos y baratos, tembién se describen técnicas para estimar la respiración de los nódulos, la evolución del hidrógeno y la actividad nitratò-reductasa de la planta. Se han adaptado varios métodos colorimétricos para el análisis del Nitrógeno total y del Nitrógeno combinado (en forma de aminas, amidas y derivados de la urea) contenidos en pequeños volumenes de savia bruta (xilema).

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References

  • Atkins, C. A. 1982 Ureide metabolism and the significance of ureides in legumes. InAdvances in Agricultural Microbiology, ed. Subba Rao, N.S. pp. 53–58. New Delhi: Oxford and IBH Publishing.

    Google Scholar 

  • von Bohley, P. 1967 Reihenbestimmugen von Stickstoff im Ultramikromolstab. Kjeldajlveraschung und Phenol-Hypochorit-Reaktion.Hoppe-Seyler's Zeitschrift fur physiologische Chemie 348, 100–110.

    Google Scholar 

  • Bremner, J. M. 1965a Total nitrogen. InMethods of Soil Analysis Monograph No. 9, ed. Black, C. A. pp. 1149–1178. Madison, USA: American Society of Agronomy.

    Google Scholar 

  • Bremner, J. M. 1965b Inorganic forms of nitrogen. InMethods of Soil Analysis. Monograph No. 9, ed. Black, C.A. pp. 1179–1237. Madison, USA: American Society of Agronomy.

    Google Scholar 

  • Bremner, J. M. &Mulvaney, C. S. 1982 Nitrogen-total. InMethods of Soil Analysis. Part 2. Chemical and Microbiological Properties, ed. Page, A. L., Miller, R. H. & Keeney, D.R. pp. 595–624. Madison, USA: American Society of Agronomy.

    Google Scholar 

  • Cataldo, D. A., Haroon, M., Schrader, L. E. &Youngs, V. L. 1975 Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid.Communications in Soil Science and Plant Analysis 6, 71–80.

    Google Scholar 

  • Connell, G. E., Dixon, G. H. &Hanes, C. S. 1955 Quantitative chromatographic methods for the study of enzyme transpeptidation reactions.Canadian Journal of Biochemistry and Physiology 33, 416–427.

    Google Scholar 

  • Dart, P. J., Day, J. M. &Harris, D. 1972 Assay of nitrogenase activity by acetylene reduction. InUse of Isotopes for Study of Fertiliser Utilisation by Legume Crops. FAO-IAEA Technical Report No. 149. pp. 85–100. Vienna: International Atomic Energy Agency.

    Google Scholar 

  • Felker, P. 1977 Micro-determination of nitrogen in seed protein extracts with the salicylate-dichlorocyanurate color reaction.Analytical Chemistry 49, 1080.

    Google Scholar 

  • Fishbeck, K., Evans, H. J. &Boersma, L. L. 1973 Measurement of nitrogenase activity of intact legume symbiontsin situ using the acetylene reduction assay.Agronomy Journal 65, 429–433.

    Google Scholar 

  • Goh, K. M. 1972 Comparison and evaluation of method for including nitrate in the total nitrogen determination of soils.Journal of the Science of Food and Agriculture 23, 275–284.

    Google Scholar 

  • Habte, M. 1983 Apparatus for the nitrogenase (C2H2−C2H4) assay of intact whole plant-soil systems.Soil Biology and Biochemistry 15, 719–720.

    Google Scholar 

  • Hardy, R. W. F. &Havelka, U. D. 1975 Nitrogen fixation research, key to world food.Science, N.Y. 188, 633–643.

    Google Scholar 

  • Herridge, D. F. 1982 Use of the ureide technique to describe the nitrogen economy of field-grown soybeans.Plant Physiology 70, 7–11.

    Google Scholar 

  • Jaworski, E. G. 1971 Nitrate reductase assay in intact plant tissues.Biochemical and Biophysical Research Communications 43, 1274–1279.

    Google Scholar 

  • Keeney, D. R. &Nelson, D. W. 1982 Nitrogen-inorganic forms. InMethods of Soil Analysis. Part 2. Chemical and Microbiological Properties, eds. Page, A. L., Miller, R. H. & Keeney, D. R. pp. 643–698. Madison, USA: American Society of Agronomy.

    Google Scholar 

  • Layzell, B., Rainbird, R. M., Atkins, C. A. &Pate, J. S. 1979 Economy of photosynthate use in nitrogen-fixing legume nodules.Plant Physiology 64, 888–891.

    Google Scholar 

  • Liao, C. F. H. 1981 Devarda's alloy method for total nitrogen determination.Soil Science Society of America Journal 45, 852–855.

    Google Scholar 

  • Mague, T. H. &Burris, R. H. 1972 Reduction of acetylene and nitrogen by field-grown soybeans.New Phytologist 72, 275–286.

    Google Scholar 

  • Mahon, J. D. 1979 Environmental and genotypic effects on the respiration associated with symbiotic nitrogen fixation in peas.Plant Physiology 63, 892–897.

    Google Scholar 

  • Matheson, A. T., Tigane, E. &Hanes, C. S. 1961 Quantitative chromatographic methods Part 5. An improved ninhydrin-hydrindantin reagent.Canadian Journal of Biochemistry and Physiology 39, 417–425.

    Google Scholar 

  • Matsumoto, T., Yamamoto, Y. &Yatazawa, M. 1976 Role of root nodules in the nitrogen nutrition of soybeans. 2. Fluctuations in allantoin concentration of the bleeding sap.Journal of the Science of Soil and Manure 46, 463–469.

    Google Scholar 

  • Matsumoto, T., Yatazawa, M. &Yamamoto, Y. 1977 Effects of exogenous nitrogen-compounds on the concentrations of allantoin and various constituents in several organs of soybean plants.Plant and Cell Physiology 18, 613–614.

    Google Scholar 

  • McClure, P. R. &Israel, D. W. 1979 Transport of nitrogen in the xylem of soybean plants.Plant Physiology 64, 411–416.

    Google Scholar 

  • McClure, P. R., Israel, D. W. &Volk, R. J. 1980 Evaluation of the relative ureide content of xylem sap as an indicator of N2 fixation in soybeans—Greenhouse studies.Plant Physiology 66, 720–725.

    Google Scholar 

  • McKnight, T. 1949 Efficiency of isolates ofRhizobium in the cowpea group with proposed additions to this group.Queensland Journal of Agricultural Science 6, 61–76.

    Google Scholar 

  • Minchin, F. R. &Pate, J. S. 1973 The carbon balance of a legume and the functional economy of its root nodules.Journal of Experimental Botany 24, 259–271.

    Google Scholar 

  • Minchin, F. R., Summerfield, R. J., Hadley, P., Roberts, E. H. &Rawsthorne, S. 1981 Carbon and nitrogen nutrition of nodulated roots of grain legumes.Plant, Cell Environment 4, 5–26.

    Google Scholar 

  • Minchin, F. R., Witty, J. F., Sheehy, J. E. &Muller, M. 1983 A major error in the acetylene reduction assay: Decreases in nodular nitrogenase activity under assay conditions.Journal of Experimental Botany 34, 641–649.

    Google Scholar 

  • Mitchell, H. L. 1972 Microdetermination of nitrogen in plant tissues.Journal of the Association of Official Agricultural Chemists 55, 1–3.

    Google Scholar 

  • Montanheiro, M. N. S., Saito, S. M. T., Reichardt, K. & Libardi, P. L. 1979 Controle de tensões de água do solo em vasos. InCongresso Brasileiro de Ciência do Solo. p. 68. Manaus: Sociedade Brasileira de Ciência do Solo, resumo no. 189.

  • Neves, M. C. P., Minchin, F. R. &Summerfield, R. J. 1981 Carbon metabolism, nitrogen assimilation and seed yield of cowpea (Vigna unguiculata) plants dependent on nitrate-nitrogen for on one of two strains ofRhizobium.Tropical Agriculture 58, 115–132.

    Google Scholar 

  • Pace, G. M., MacKown, C. T. &Volk, R. J. 1982 Minimizing nitrate reduction during Kjeldahl digestion of plant tissue extracts and stem exudates. Applications to15N studies.Plant Physiology 69, 32–36.

    Google Scholar 

  • Pate, J. S. 1977 Functional biology of dinitrogen fixation by legumes. InA Treatise on Dinitrogen Fixation. Section III, ed. Hardy, R. W. F. & Silver, W. S. pp. 473–517. New York, USA: John Wiley and Sons.

    Google Scholar 

  • Pulver, E. L., Brockman, F., Nangju, D. &Wien, H. 1978 IITA's programme on N fixation. InIsotopes in Biological Dinitrogen Fixation. pp. 269–284. Vienna: International Atomic Energy Agency.

    Google Scholar 

  • Rainbird, R. M., Atkins, C. A., Pate, J. S. &Sanford, P. 1983 Significance of hydrogen evolution in the carbon and nitrogen economy of nodulated cowpea.Plant Physiology 71, 122–127.

    Google Scholar 

  • Schubert, K. R. &Evans, H. J. 1976 Hydrogen evolution: A major factor affecting the efficiency of nitrogen fixation in nodulated symbionts.Proceedings of the National Academy of Sciences of the United States of America 73, 1207–1211.

    Google Scholar 

  • Schubert, K. R., Engelke, J. A., Russel, S. A. &Evans, H. J. 1977 Hydrogen reactions of nodulated leguminous plants. I. Effect of rhizobial strain and plant age.Plant Physiology 60, 651–654.

    Google Scholar 

  • Schubert, K. R. &Ryle, J. A. 1980 The energy requirements for N fixation in nodulated legumes. InAdvances in Legume Science, ed. Summerfield, R. J. & Bunting, A. H. pp. 85–96. Kew: Royal Botanic Gardens.

    Google Scholar 

  • Sinha, S. K., Khanna-Chopra, R., Chatterjee, S. R. &Abrol, Y. P. 1978 Composition of bleeding sap inVigna radiata.Physiologia Plantarum 42, 45–58.

    Google Scholar 

  • Sprent, J. I. 1980 Root nodule anatomy, type of export product and evolutionary origin in some leguminosae.Plant and Cell Environmental 3, 35–43.

    Google Scholar 

  • Streeter, J. G. 1972 Nitrogen nutrition of field-grown soybean plants. 1. Seasonal variations in soil nitrogen and nitrogen composition of stem exudate.Agronomy Journal 64, 311–314.

    Google Scholar 

  • Thomas, R. J., Feller, U. &Erismann, K. H. 1979 The effect of different inorganic nitrogen sources and plant age on the composition of bleeding sap ofPhaseolus vulgaris.New Phytologist 82, 657–669.

    Google Scholar 

  • Thomas, R. J. &Schrader, L. T. 1981 Ureide metabolism in higher plants.Phytochemistry 20, 361–371.

    Google Scholar 

  • Vincent, J. M. 1970A Manual for the Practical Study of the Root-Nodule Bacteria. International Biological Programme Handbook No. 15. Oxford: Blackwell Scientific Publications.

    Google Scholar 

  • Vogels, G. D. &Van Der Drift, C. 1970 Differential analyses of glyoxylate derivatives.Analytical Biochemistry 33, 143–157.

    Google Scholar 

  • von Bohley, P. 1967 Reihenbestimmungen von Stickstoff im Ultramikromolstab. Kjeldajlveraschung und Phenol-Hypochorit-Reaktion.Hoppe-Seyler's Zeitschrift fur Physiologische Chemie 348, 100–110.

    Google Scholar 

  • Witty, J. F., Minchin, F. R. &Sheehy, J. E. 1983 Carbon costs of nitrogenase activity in legume root nodules determined using acetylene and oxygen.Journal of Experimental Botany 34, 951–963.

    Google Scholar 

  • Wych, R. D. &Rains, R. W. 1978 Simultaneous measurement of nitrogen fixation estimated by acetylene-ethylene assay and nitrate absorption by soybeans.Plant Physiology 62, 443–448.

    Google Scholar 

  • Young, E. G. &Conway, C. F. 1942 On the estimation of allantoin by the Rimini-Schryver reaction.Journal of Biological Chemistry 142, 839–853.

    Google Scholar 

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Author notes

  1. R. J. Thomas (Consultant bilateral convenium CNPq-British Council)

    Present address: Hill Farming Research Organisation, EH26 0PY, Penicuik, Midlothian, Scotland, UK

Authors and Affiliations

  1. EMBRAPA-Programa Nacional de Pesquisa em Biologia do Solo, Km 47, Seropedica, 23851, Rio de Janeiro, Brazil

    R. M. Boddey (Consultant IICA/EMBRAPA World Bank Project), J. A. R. Pereira, M. Hungria, R. J. Thomas (Consultant bilateral convenium CNPq-British Council) & M. C. P. Neves

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  1. R. M. Boddey
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  2. J. A. R. Pereira
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  3. M. Hungria
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  4. R. J. Thomas
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  5. M. C. P. Neves
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Boddey, R.M., Pereira, J.A.R., Hungria, M. et al. Methods for the study of nitrogen assimilation and transport in grain legumes. Mircen Journal 3, 3–22 (1987). https://doi.org/10.1007/BF01090491

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  • DOI: https://doi.org/10.1007/BF01090491

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