Advertisement

The Histochemical Journal

, Volume 13, Issue 3, pp 461–480 | Cite as

Immunofluorescent localization of two water-soluble glycoproteins including the major allergen from the pollen of ryegrass,Lolium perenne

  • B. J. Howlett
  • H. I. M. V. Vithanage
  • R. B. Knox
Papers

Summary

Two major glycoproteins have been localized in sectioned grains of ryegrass pollen by direct and indirect immunofluorescence methods using Fluorescein isothiocyanate (FITC)-labelled IgC fractions of antisera. These glycoproteins are the major allergen Group 1 allergen, and a principal antigen Antigen A. Four methods of fixation were employed: freeze-drying, methanol, 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h at 4°C. The post-embedding staining technique of immunocytochemistry was used: anthers were embedded directly, or after dehydration, in JB-4 plastic resin and antibody reacted with sectioned pollen.

The effects of these fixatives on the antibody combining sites of the antigens were quantified by a solid phase radioimmunoassay using [125I]protein A to measure antibody binding. Glutaraldehyde was the only fixative to significantly depress antibody binding of both Antigen A and Group 1 allergen to their homologous antisera. This radioimmunoassay was modified to reyeal that FITC conjugation to either antibody did not impair antigen binding. In mature pollen, these antigens were located in the cytoplasm and in the complex wall. In developing grains early in the maturation period, specific fluorescence was concentrated at the periphery of the cytoplasm.

Keywords

Fluorescein Isothiocyanate Antibody Binding Mature Pollen Antigen Binding Maturation Period 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Ashford, A. E. &Knox, R. B. (1981) characteristics of pollen diffusates and pollen wall cytochemistry in poplars,J. Cell Sci. 44, 1–17.Google Scholar
  2. Axen, R., Heilbronn, E. &Winter, A. (1969) Preparation and properties of cholinesterases covalently bound to Sepharose.Biochem. Biophys. Acta 191, 478–81.Google Scholar
  3. Barlow, K. (1978).A study of chromosomal male sterility in wheat. Ph.D. thesis, University of New South Wales, Kensington, New South Wales.Google Scholar
  4. Christensen, J. E. &Horner, H. T. (1974) Pollen pore development and its spatial orientation during microsporogenesis in the grassSorghum bicolor, Am. J. Bot. 61, 604–23.Google Scholar
  5. Craig, S., Goodchild, D. &Millerd, A. (1979) Immunofluorescent localization of pea storage proteins in glycolmethacrylate embedded tissue.J. Histochem. Cytochem. 27, 1312–6.Google Scholar
  6. Carnegie, J. A., McCully, M. E. &Robertson, H. H. (1980) Embedment in glycol methacrylate at low temperature allows immunofluorescent localisation of a labile tissue protein.J. Histochem. Cytochem. 28, 308–10.Google Scholar
  7. Ewijk, W. Van, Coffman, R. C. &Weissman, L. L. (1980) Immunoelectron microscopy of cell surface antigens: a quantitative analysis of antibody binding after fixation protocols.Histochem. J. 12, 349–61.Google Scholar
  8. Feder, N. &O'Brien, T. P. (1968) Plant microtechnique: some principles and new methods.Am. J. Bot. 55, 123–34.Google Scholar
  9. Fraker, P. J. &Speck, J. C. Jr (1968) Protein and cell membrane iodinations with a sparingly soluble chloroanide 1,3,4,6 tetrachloro-3a,6a diphenyl glycoluril.Biochem. Biophys. Res. Commun. 80, 849–57.Google Scholar
  10. Goldman, M. (1968)Fluorescent Antibody Methods. New York: Academic Press.Google Scholar
  11. Heslop-Harrison, J. (1976) A new look at pollination.Rep. E. Malling Res. Stn for 1975, 141–57.Google Scholar
  12. Heslop-Harrison, J. (1979) Aspects of the structure, cytochemistry and germination of the pollen of rye, (Secale cereale L.).Ann. Bot. Suppl. 1, 1–42.Google Scholar
  13. Heslop-Harrison, J., Heslop-Harrison, Y., Knox, R. B. &Howlett, B. (1973) Pollen-wall proteins: ‘gametophytic’ and ‘sporophytic’ fractions in the pollen walls of Malvaceae.Ann. Bot. 37, 402–12.Google Scholar
  14. Hielm, H., Hjelm, K. &Sjoquist, J. (1972) Protein A fromStaphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins,FEBS Lett. 28, 73–6.Google Scholar
  15. Howlett, B. J., Knox, R. B. &Heslop-Harrison, J. (1973) Pollen-wall proteins: release of the allergen Antigen E from intine and exine sites in the pollen grains of ragweed and Cosmos.J. Cell. Sci. 13, 603–19.Google Scholar
  16. Howlett, B. J., Knox, R. B., Paxton, J. D. &Heslop-Harrison, J. (1975) Pollen-wall proteins: physicochemical characterization and role in self-incompatability inCosmos bipinnatus.Proc. R. Soc., Ser. B 188, 166–82.Google Scholar
  17. Howlett, B. J. & Clarke, A. E. (1981) Isolation and partial characterization of two antigenic glycoproteins from rye grass (Lolium perenne) pollen.Biochem. J. (in press).Google Scholar
  18. Howlett, B. J., Vithanage, H. I. M. V. &Knox, R. B. (1979) Pollen antigens, allergens and enzymes.Curr. Adv. Plant Sci.,35, 1–170.Google Scholar
  19. Johnson, P. &Marsh, D. G. (1965) The isolation and characterisation of allergens from the pollen of rye grass (Lolium perenne).Eur. Polymer J. 1, 63–77.Google Scholar
  20. Knox, R. B. (1971) Pollen-wall proteins: localization, enzymic and antigenic activity during development inGladiolus (Iridaceae)J. Cell Sci. 9, 209–37.Google Scholar
  21. Knox, R. B. (1973) Localization of proteins in plant cells.Zeiss Information Oberkochen 81, 52–5.Google Scholar
  22. Knox, R. B. &Heslop-Harrison, J. (1970) Pollen-wall proteins: localization and enzymic activity.J. Cell Sci. 6, 1–27.Google Scholar
  23. Knox, R. B. &Heslop-Harrison, J. (1971) Pollen-wall proteins: fate of intine-held antigens in compatible and incompatible pollinations ofPhalaris tuberosa L.J. Cell. Sci. 9, 239–51.Google Scholar
  24. Knox, R. B. Heslop-Harrison, J. &Heslop-Harrison, Y. (1975) Pollen-wall proteins: localization and characterization of gametophytic and sporophytic fractions. InThe Biology of the Male Gamete (edited byDuckett, I. G. andRacey, P. A.)Biol. J. Linn. Soc. Vol. 7., Suppl. 1, pp. 177–87 London: Academic Press.Google Scholar
  25. Knox, R. B., Vithanage, H. I. M. V. &Howlett, B. J. (1980) Botanical immunocytochemistry: a review with special reference to pollen antigens and allergens.Histochem. J. 12, 247–72.Google Scholar
  26. Livingston, D. M. (1974) Immunoaffinity chromatography of proteins. InMethods in Enzymology (edited byJacoby, W. B. andWilchek, M.), Vol. 34, pp. 723–731. New York, London: Academic Press.Google Scholar
  27. Maede, H. &Ishida, N. (1967) Specific binding of hexopyranosyl polysaccharides with fluorescent brightener.J. Biochem. 62, 276–8.Google Scholar
  28. Marier, R., Jansen, M. &Andriole, V. T. (1979) A new method of measuring antibody using radio-labelled Protein A in a solid phase radioimmunoassay.J. Immun. Meth. 28, 41–9.Google Scholar
  29. McLean, I. W. &Nakane, P. K. (1974) Periodate-lysine-paraformaldehyde fixative-a new fixative for immuno electron microscopy.J. Histochem. Cytochem. 22, 1077–83.Google Scholar
  30. Marsh, D. G. (1975) Allergens and the genetics of allergy. InThe Antigens (edited bySela, M.), vol. 3, pp. 271–359. London: Academic Press.Google Scholar
  31. Ouchterlony, O. (1958) Diffusion-in-gel methods for immunological, analysis. InProgress in Allergy (edited byKallos, P.), Vol. 5, pp. 1–78 Basel: Karger.Google Scholar
  32. Rollens, T. E. &Smith, W. L. (1980) Subcellular localisation of prostaglandin-forming cyclo oxygenase in Swiss Mouse 3T3 fibroblasis by electron microscopic immunocytochemistry.J. biol. Chem. 255, 4872–5.Google Scholar
  33. Rowley, J. R. (1964) Formation of the spore in pollen ofPoa annua. InPollen Physiology and Fertilization (edited byLinskens, H. F.), pp. 59–69. Amsterdam: North Holland.Google Scholar
  34. Rowley, J. R. &Dahl, A. O. (1977) Pollen development inArtemisia vulgaris with special reference to glycocalyx material.Poll. Spores 19, 169–284.Google Scholar
  35. Ruddel, C. L. (1971) Embedding media for 1–2 micron sectioning. 3-Hydroxyethyl methacrylate-benzoyl peroxide activated with pyridine.Stain Technol. 46, 77–84.Google Scholar
  36. Smart, I. J. &Knox, R. B. (1979) Aerobiology of grass pollen in the city atmosphere of Melbourne: quantitative analysis of seasonal and diurnial changes.Aust. J. Bot. 27, 317–31.Google Scholar
  37. Smart, I. J. &Knox, R. B. (1980) Rapid batch fractionation of ryegrass pollen allergens.Int. Arch. Allergy Appl. Immun. 62, 179–87.Google Scholar
  38. Skvarla, J. &Larson, D. A. (1966) Fine structural studies ofZea mays pollen. 1. Cell membranes and exine ontogeny.Am. J. Bot. 53, 1112–25.Google Scholar
  39. Southworth, D. (1973) Cytochemical reactivity of pollen walls.J. Histochem. Cytochem. 21, 73–8.Google Scholar
  40. Vithanage, H. I. M. V. &Knox, R. B. (1979) Periodicity of pollen development and quantitative cytochemistry of exine and intine enzymes in the grasses,Lolium perenne andPhalaris tuberosa.Ann Bot. 45, 131–41.Google Scholar
  41. Vithanage, H. I. M. V., Howlett, B. J. &Knox, R. B. (1981) Localization of grass pollen allergen by immunocytochemistry.Micron 11, 411–2.Google Scholar
  42. Young, B. A., Schultz-Schaeffer, R. &Carroll, T. W. (1979) Anther and pollen development in male sterile intermediate wheatgrass derived from wheat and wheatgrass hybrids.Can. J. Bot. 57, 602–18.Google Scholar

Copyright information

© Chapman and Hall Ltd 1981

Authors and Affiliations

  • B. J. Howlett
    • 1
  • H. I. M. V. Vithanage
    • 1
  • R. B. Knox
    • 1
  1. 1.School of BotanyUniversity of MelbourneParkvilleAustralia

Personalised recommendations