Skip to main content
SpringerLink
Log in

High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure

  • Published:
Planta Aims and scope Submit manuscript

Abstract

High-pressure freezing of chemically untreated nodules of soybean (Glycine max (L.) Merr.), in sharp contrast to chemical fixation and prefixation, appears to preserve the ultrastructure close to the native state. This is supported by the observation that the peribacteroid membrane of high-pressure-frozen samples is tightly wrapped around the bacteroids, a finding that is fully consistent with the current views on the physiology of oxygen and metabolite transport between plant cytosol and bacteroids. In soybean root nodules, the plant tissue and the enclosed bacteria are so dissimilar that conventional aldehyde-fixation procedures are unable to preserve the overall native ultrastructure. This was demonstrated by high-pressure freezing of nodules that had been pre-fixed in glutaraldehyde at various buffer molalities: no buffer strength tested preserved all ultrastructural aspects that could be seen after high-pressure freezing of chemically untreated nodules.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Appleby, C.A. (1984) Leghemoglobin and Rhizobium respiration. Annu. Rev. Plant Physiol. 35, 443–478

    Google Scholar 

  • Coetzee, J., van der Merwe, C.F. (1985) Penetration rate of glutaraldehyde in various buffers into plant tissue and gelatine gels. J. Microsc. 3, 129–136

    Google Scholar 

  • Dubochet, J., Adrian, M., Chang, J., Homo, J.-C., Lepault, J., McDowall, A.W., Schultz, P. (1988) Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, No. 2, 129–228

    Google Scholar 

  • Escaig, J. (1982) New instruments which facilitate rapid freezing at 83K. J. Microsc. 126, 221–229

    Google Scholar 

  • Hahn, M., Studer, D. (1986) Competitivness of a nif - B. japonicum mutant against the wild-type strains. FEMS Microbiol. Lett. 33, 143–148

    Google Scholar 

  • Harreveld, A. van, Crowell, J. (1964) Electron microscopy after rapid freezing on a metal surface and substitution fixation. Anat. Rec. 149, 381–385

    Google Scholar 

  • Hayat, M.A. (1989) Principles and techniques of electron microscopy: Biological applications. The Macmillan Press Ltd, London

    Google Scholar 

  • Hülser, D.F., Paschke, D., Greule, J. (1989) Gap junctions: Correlated electrophysiological recordings and ultrastructural analysis by fast freezing and freeze-fracturing. In: Electron microscopy of subcellular dynamics, pp. 33–49. Plattner, H., ed. CRC Press Inc. Boca Raton, Florida

    Google Scholar 

  • Humbel, B., Marti, T., Müller, M. (1983) Improved structural preservation by combining freeze-substitution and low temperature embedding. Beitr. Elektronenmikrosk. Direktabb. Oberfl. 16, 585–594

    Google Scholar 

  • Katinakis, P., Klein Lankhorst, R.M., Louwerse, J., van Kammen, A., van den Bos R.C. (1988) Bacteroid-encoded proteins are secreted into the peribacteroid space by Rhizobium leguminosarum. Plant. Mol. Biol. 11, 183–190

    Google Scholar 

  • Layzell, D.B., Hunt, S., Moloney, A.H.M., Fernando, S.M., Diaz del Castillo, L. (1990) Physiological, metabolic and developmental implications of oxygen regulation in legume nodules. In: Nitrogen fixation: achievements and objectives, pp. 21–32, Gresshoff, P.M., Roth, E., Stacey, G., Newton, W.E., eds. Chapman and Hall, New York

    Google Scholar 

  • Lee, R.M.K.W. (1984) A critical appraisal of the effects of fixation, dehydration and embedding on cell volume. In: The science of biological specimen preparation. pp. 61–70, Revel, J.P., Barnard T., Haggis, G.H., eds. SEM Inc., AMF O'Hare, Chicago

    Google Scholar 

  • Luft, J.H. (1961) Improvements in epoxy resing embedding methods. J. Biophys. Biochem. Cytol. 9, 409–414

    Google Scholar 

  • Mellor, R.B. (1988) Distribution of trehalase in soybean root nodule cells: implications for trehalose metabolism. J. Plant Physiol. 133, 173–177

    Google Scholar 

  • Mellor, R.B., Werner, D. (1987) Peribacteroid membrane biogenesis in mature legume root nodules. Symbiosis 3, 75–100

    Google Scholar 

  • Menco, M. (1986) A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching and freeze-substitution. J. Electr. Microsc. Technol. 4, 177–240

    Google Scholar 

  • Moor, H. (1987) Theory and practice of high pressure freezing. In: Cryotechniques in biological electron microscopy, pp. 175–191, Steinbrecht, R.A., Zierold, K. eds. Springer, Berlin

    Google Scholar 

  • Müller, M. (1988) Cryopreparation of microorganisms for electron microscopy. Methods Microbiol. 20, 1–28

    Google Scholar 

  • Müller, M., Marti, T., Kriz, S. (1980) Improved structural preservation by freeze substitution. In: Electron microscopy 1980. No. 2. pp. 720–772, Brederoo, P., de Priester, W. eds. Proc. 7th Eur. Congr. Electron Microsc. Foundation, Leiden

    Google Scholar 

  • Müller, M., Moor, H. (1984) Cryofixation of thick specimens by high pressure freezing. In: The science of biological specimen preparation, pp. 131–138. Revel, J.-P., Barnard, T., Haggis, G.H. eds. SEM, AMF O'Hare, Chicago

    Google Scholar 

  • Muller, L.L., Jack, T.J. (1975) Rapid chemical dehydration of samples for electron microscopic examinations. J. Histochem. Cytochem. 23, 107–110

    Google Scholar 

  • Newcomb, W., Jackson, S., Racette, S., Torrey, J.G. (1990) Ultrastructure of infected cells in the actinorhizal root nodules of Gymnostoma papuanum (Casuarinaceae) prepared by high pressure freezing and chemical fixation. Protoplasma 157, 172–181

    Google Scholar 

  • Powell, R., Gannon, F. (1988) The Leghaemoglobins. BioEssays 9, 117–121

    Google Scholar 

  • Ramseier, T.M., Kaluza, B., Studer, D., Gloudemans, T., Bisseling, T., Jordan, P.M., Jones, M.R., Zuber, M., Hennecke, H. (1989) Cloning of a DNA region from B. japonicum encoding pleiotropic functions in heme metabolism and respiration. Arch. Microbiol. 151, 203–212

    Google Scholar 

  • Ramseier, T.M., Winteler, H.V., Hennecke, H. (1991) Discovery and sequence analysis of bacterial genes involved in the biogenesis of c-type cytochromes. J. Biol. Chem. 266, 7793–7803

    Google Scholar 

  • Regensburger, B., Meyer, L., Filser, M., Weber, J., Studer, D., Lamb, J.W., Fischer, H.-M., Hahn, M., Hennecke, H. (1986) Bradyrhizobium japonicum mutants defective in root nodule bacteroid development and nitrogen fixation. Arch. Microbiol. 144, 355–366

    Google Scholar 

  • Reynolds, E.W. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212

    Google Scholar 

  • Robertson, J.G., Lyttleton, P., Tapper, B.A. (1984a) The role of peribacteroid membrane in legume root nodules. In: Advances in nitrogen fixation research, pp. 475–481, Veeger, C., Newton, W.E., eds. Nijhoff/Junk, The Hague

    Google Scholar 

  • Robertson, G.R., Wells, B., Bisseling, T., Farnden, K.J.F., Johnston, A.W.B. (1984b) Immuno-gold localization of leghaemoglobin in cytoplasm in nitrogen-fixing root nodules of pea. Nature 311, 254–256

    Google Scholar 

  • Roland, J.C., Vian, B. (1991) General preparation and staining of thin sections. In: Electron microscopy of plant cells, pp. 1–66, Hall, J.L., Hawes, C., eds. Academic Press, London

    Google Scholar 

  • Steinbrecht, R.A., Müller, M. (1987) Freeze-substitution and freeze-drying. In: Cryotechniques in biological electron microscopy, pp. 149–172, Steinbrecht, R.A., Zierold, K. eds. SpringerVerlag, Berlin Heidelberg

    Google Scholar 

  • Studer, D., Gloudemans, T., Franssen, H.J., Fischer, H.-M., Bisseling, T., Hennecke, H. (1987) Involvement of the bacterial nitrogen fixation regulatory gene (nifA) in control of nodule-specific host-plant gene expression. Eur. J. Cell Biol. 45, 177–184

    Google Scholar 

  • Studer, D., Michel, M., Müller, M. (1989) High pressure freezing comes of age. Scanning Microscopy, Suppl. 3, 253–269

    Google Scholar 

  • Todd, W.J. (1986) Effects of specimen preparation on the apparent ultrastructure of microorganisms. In: Ultrastructure techniques for microorganisms, pp. 87–99, Aldrich, H.C., Todd, W.J., eds. Plenum Press, New York

    Google Scholar 

  • Udvardi, M.K., Price, G.D., Gresshoff, P.M., Day, D.A. (1988) A dicarboxylate transporter on the peribacteroid membrane of soybean nodules. FEBS Lett. 231, 36–40

    Google Scholar 

  • Vandenbosch, K.A., Newcomb, E.H. (1988) The occurrence of leghemoglobin protein in the uninfected interstitial cells of soybean root nodules. Planta 175, 442–451

    Google Scholar 

  • Vincent, J.M. (1970) A manual for the practical study of the root nodule bacteria. Blackwell, Oxford

    Google Scholar 

  • Werner, D., Mörschel, E. (1978) Differentiation of nodules of Glycine max. with an ineffective strain of Rhizobium japonicum. Planta 141, 169–177

    Google Scholar 

  • White, D.L., Andrews, S.B., Faller, J.W., Barrnett, R.J. (1976) The chemical nature of osmium tetroxide fixation and staining of membranes by X-ray photoelectron spectroscopy. Biochim. Biophys. Acta 436, 577–592

    Google Scholar 

Download references

Author information

Author notes

  1. Daniel Studer

    Present address: MIB, Universität Bern, Murtenstrasse 35, CH-3010, Bern, Switzerland

Authors and Affiliations

  1. Laboratorium für Elektronenmikroskopie I, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092, Zürich, Switzerland

    Daniel Studer & Martin Müller

  2. Institut für Mikrobiologie, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092, Zürich, Switzerland

    Daniel Studer & Hauke Hennecke

Authors
  1. Daniel Studer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hauke Hennecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Martin Müller
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

We thank E. Martinoia (Pflanzenwissenschaften, ETH, Zürich, Switzerland) and H. Hohenberg (Heinrich-Pette-Institut, Hamburg, FRG) for critical and constructive discussion and D. Duff (Anorganische Chemie, ETH, Zürich) and M.L. Yaffee (Laboratorium für Elektronenmikroskopie I, ETH, Zürich) for help with the manuscript. This study was supported by a research grant from the ETH.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Studer, D., Hennecke, H. & Müller, M. High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure. Planta 188, 155–163 (1992). https://doi.org/10.1007/BF00216809

Download citation

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00216809

Key words

Search

Navigation