Abstract
Transmission electron microscopy and immunocytological labeling were used to study the distribution and ontological occurrence of dinitrogenase reductase (Fe-protein) of nitrogenase in cyanobacterial symbionts within young leaves of the water-ferns Azolla filiculoides Lamarck, A. caroliniana Willdenow, and A. pinnata R. Brown. Rabbit anti-dinitrogenase reductase antisera and goat anti-rabbit-immunoglobulin G antibody conjugated to colloidal gold were used as probes. Western blot analyses showed that a polypeptide of approx. 36 kDa (kdalton) was recognized in the symbionts of all three Azolla species and that the polyclonal sera used were monospecific. In all symbionts, nitrogenase was immunologically recognizable within heterocysts. It was absent from vegetative cells, and also from the akinetes of the A. caroliniana and A. pinnata symbionts. The differentiation of vegetative cells into heterocysts in all three symbionts was initiated by formation of additional external cell-wall layers and narrowing of the neck followed by loss of glycogen, mild vesiculation of thylakoid membranes, and the appearance of polar nodules. No nitrogenase was detected at these early stages, but it appeared in the intermediate proheterocyst stage concomitantly with the formation of contorted membranes, and reached the strongest labeling in mature heterocysts, containing extensive tightly packed membranes. Nitrogenase was evenly distributed throughout heterocysts except at the polar regions, which contained honey-comb configurations and large polar nodules. With increased age of the A. caroliniana and A. pinnata symbionts, heterocysts became highly vesiculated, with a concomitant decrease in the amount of nitrogenase detected.
Similar content being viewed by others
Abbreviations
- IgG:
-
Immunoglobulin G
- PAGE:
-
polyacrylamide gel electrophoresis
- SDS:
-
sodium dodecyl sulfate
- TEM:
-
transmission electron micrograph
References
Baker, D., Lending, C., Dean, D. (1984) Localization of nitrogenase using monoclonal antibodies. In: Advances in nitrogen fixation research. p. 249, Veeger, C., Newton, W.E., eds. M. Nijhoff/Dr. W. Junk Publ., The Hague, and Pudoc, Wageningen, The Netherlands
Bergman, B., Lindblad, P., Pettersson, A., Renström, E., Tiberg, E. (1985) Immuno-gold localization of glutamine synthetase in a nitrogen-fixing cyanobacterium (Anabaena cylindrica). Planta 166, 329–334
Bergman, B., Lindblad, P., Rai, A.N. (1986) Nitrogenase in free-living and symbiotic cyanobacteria: immunoelectron microscopic localization. FEMS Microbiol. Lett. 35, 75–78
Bothe, H., Neuer, G., Kalby, I., Eisbrenner, G. (1980) Electron donors and hydrogenase in nitrogen-fixing microorganisms. In: Nitrogen fixation, pp. 83–112, Stewart, W.D.P., Gallon, J.R., eds. Academic Press, London
Fleming, H., Haselkorn, R. (1974) The program of protein synthesis during heterocyst differentiation in nitrogen-fixing blue-green algae. Cell 3, 159–170
Franche, C., Cohen-Bazire, G. (1987) Evolutionary divergence in the nif HDK gene region among nine symbiotic Anabaena azollae and between Anabaena azollae and some free-living heterocystous cyanobacteria. Symbiosis 3, 159–178
Gallon, J.R., La Rue, T.A., Kurz, W.G.W. (1972) Characteristics of nitrogenase activity in broken cell preparations of the blue-green alga Gloeocapsa sp. LB 795. Can. J. Microbiol. 18, 327–332
Golden, J.W., Robinson, S.J., Haselkorn, R. (1985) Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena. Nature 314, 419–423
Haaker, H., Veeger, C. (1977) Involvement of the cytoplasmic membrane in nitrogen fixation by Azotobacter vinelandii. Eur. J. Biochem. 77, 1–10
Hallenbeck, P.C. (1987) Molecular aspects of nitrogen fixation by photosynthetic prokaryotes. CRC Crit. Rev. Microbiol. 14, 1–48
Hallenbeck, P.C., Kostel, P.J., Benemann, J.R. (1979) Purification and properties of nitrogenase from the cyanobacterium, Anabaena cylindrica. Eur. J. Biochem. 98, 275–284
Haury, J.F., Wolk, C.P. (1978) Classes of Anabaena variabilis mutants with oxygen-sensitive nitrogenase activity. J. Bacteriol. 136, 688–692
Jensen, B.B., Cox, R.P., Burris, R.H. (1986) Isolation of cyanobacterial heterocysts with high and sustained dinitrogenfixation capacity supported by endogenous reductants. Arch. Microbiol. 145, 241–247
Kallas, T., Rippka, R., Coursin, T., Rebiére, M.C., Tandeau de Marrsac, N., Cohen-Bazire, G. (1983) Aerobic nitrogen fixation by non-heterocystous cyanobacteria. In: Photosynthetic procaryotes: Cell differentiation and function, pp. 281–303, Papageorgiou, C., Packer, L., eds. Elsevier Publ. Co., Limerick, Ireland
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
Lang, N.J. (1965) Electron microscopic study of heterocyst development in Anabaena azollae Strasburger. J. Phycol. 1, 127–134
Lang, N.J., Fay, P. (1971) The heterocysts of blue-green algae. II. Details of ultrastructure. Proc. R. Soc. London Ser. B 178, 193–203
Lumpkin, T.A., Plucknett, D.L. (1982) Azolla as a green manure: Use and management in crop production. Westview Press, Boulder, Colo., USA
McCowen, S.M., MacArthur, L., Gates, J.E. (1987) Azolla fern lectins that specifically recognize endosymbiotic cyanobacteria. Curr. Microbiol. 14, 329–333
Meeks, J.C., Joseph, C.M., Haselkorn, R. (1988) Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros. Arch. Microbiol. 150, 61–71
Meesters, T.M. (1987) Localization of nitrogenase in vesicles of Frankia sp. Cc1.17 by immunogold labeling on ultra thin cryosections. Arch. Microbiol. 146, 327–331
Meesters, T.M., Van Vliet, W.M., Akkermans, A.D.L. (1987) Nitrogenase is restricted to the vesicles in Frankia strain EAN1pec. Physiol. Plant. 70, 267–271
Murry, M.A., Hallenbeck, P.C., Beneman, J.R. (1984) Immunochemical evidence that nitrogenase is restricted to the heterocysts in Anabaena cylindrica. Arch. Microbiol. 137, 194–199
Neumuller, M., Bergman, B. (1981) The ultrastructure of Anabaena azollae in Azolla pinnata. Physiol. Plant. 51, 69–76
Nierzwicki-Bauer, S.A., Balkwill, D.L., Stevens, S.E., Jr. (1984) Heterocyst differentiation in the cyanobacterium Mastigocladus laminosus. J. Bacteriol. 157, 514–525
Nierzwicki-Bauer, S.A., Haselkorn, R. (1986) Differences in mRNA levels in Anabaena living freely or in symbiotic association with Azolla. EMBO J. 5, 29–35
Peters, G.A., Kaplan, D., Meeks, J.C., Buzby, K.M., Marsh, B.H., Corbin, J.L. (1985) Aspects of nitrogen and carbon exchange in the Azolla-Anabaena symbiosis. In: Nitrogen fixation and CO2-metabolism, pp. 213–222, Ludden, P.W., Burris, J.E., eds. Elsevier Science Publ. Co., New York
Peterson, R.B., Burris, R.H. (1976) Properties of heterocysts isolated with colloidal silica. Arch. Microbiol. 108, 35–40
Potts, M. (1986) The protein index of Nostoc commune UTEX 584 (cyanobacteria): changes induced in immobilized cells by water stress. Arch. Microbiol. 146, 87–95
Reed, D.W., Toia, R.E., Jr., Raveed, D. (1974) Purification of azotophore membranes containing the nitrogenase from Azotobacter vinelandii. Biochem. Biophys. Res. Comm. 58, 20–26
Reynolds, E.S. (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17, 208–212
Rippka, R., Stanier, R.Y. (1978) The effects of anaerobiosis on nitrogenase synthesis and heterocyst development by Nostocacean cyanobacteria. J. Gen. Microbiol. 105, 83–94
Rippka, R., Waterbury, J.B. (1977) The synthesis of nitrogenase by non-heterocystous cyanobacteria. FEMS Microbiol. Lett. 2, 83–86
Robson, R.L., Eady, R.R., Richardson, T.H., Miller, R.W., Hawkins, M., Postgate, J.R. (1986) The alternative nitrogenase of Azotobacter chrococceum is a vanadium enzyme. Nature 322, 388–390
Roussard-Jacquemin, M. (1983) Étude ultrastructuraie de la différentiation des heterocystes chez la cyanobacterie, Anabaena cylindrica Lemm. Can. J. Bot. 29, 1564–1575
Smith, R.L., Van Baalen, C., Tabita, F.R. (1987) Alteration of the Fe protein of nitrogenase by oxygen in the cyanobacterium Anabaena sp. strain CA. J. Bacteriol. 169, 2537–2542
Spence, D.W., Stewart, W.D.P. (1987) Heterocystless mutants of Anabaena PCC7120 with nitrogenase activity. FEMS Microbiol. Lett. 40, 112–119
Stanier, R.Y., Kunisawa, R., Mandel, M., Cohen-Bazire, G. (1971) Purification and properties of unicellular blue-green algae (Order Chroococcaeles). Bacteriol. Rev. 35, 171–205
Stewart, W.D.P., Rowell, P., Hawkesford, M., Sampaio, M.T.A.M., Ernst, A. (1982) Nitrogenase and aspects of its regulation in cyanobacteria. Isr. J. Bot. 31, 168–189
Tel-Or, E., Stewart, W.D.P. (1977) Photosynthetic components and activities of nitrogen-fixing isolated heterocysts of Anabaena cylindrica. Proc. R. Soc. London Ser. B 198, 61–86
Wallace, W.H., Gates, J.E. (1986) Identification of eubacteria isolated from leaf cavities of four species of the N-fixing Azolla fern as Arthrobacter Conn and Dimmick. Appl. Environ. Microbiol. 52, 425–429
Watanabe, I., Espiñas, C.R., Berja, N.S., Alimaguo, B.V. (1977) The utilization of the Azolla-Anabaena complex as a nitrogen fertilizer for rice. Int. Rice Res. Paper Ser. No. 11, 1–15
Wilcox, M.G., Mitchison, G.J., Smith, R.J. (1973) Pattern formation in the blue-green alga Anabaena II. Controlled proheterocyst regression. J. Cell Sci. 13, 637–649
Wildon, D.C., Mercer, F.V. (1963) The ultrastructure of the vegetative cell of blue-green algae. Aust. J. Biol. Sci. 16, 585–596
Winkenbach, F., Wolk, C.P. (1973) Activities of enzymes of the oxidative and the reductive pentose phosphate pathways in heterocysts of a blue-green alga. Plant Physiol. 52, 480–483
Wolk, C.P. (1982) Heterocysts. In: The biology of cyanobacteria, Bot. Monog. vol. 19, pp. 359–386, Carr, N.G., Whitton, B.A., eds. Blackwell Scientific Publishers, London, UK
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Braun-Howland, E.B., Lindblad, P., Nierzwicki-Bauer, S.A. et al. Dinitrogenase reductase (Fe-protein) of nitrogenase in the cyanobacterial symbionts of three Azolla species: Localization and sequence of appearance during heterocyst differentiation. Planta 176, 319–322 (1988). https://doi.org/10.1007/BF00395412
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00395412