Skip to main content Accessibility help
×
Home

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L.

  • Uday K. Tirlapur (a1), Monica Scali (a1), Alessandra Moscatelli (a1), Cecilia Del Casino (a1), Gianpiero Cai (a1), Antonio Tiezzi (a1) and Mauro Cresti (a1)...

Summary

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

Copyright

Corresponding author

M. Cresti, Dipartimento di Biologia Ambientale, Università di Siena, Via P.A. Mattioli 4, Sienna, Italy. Telephone: 0039 577 298854. Fax: 0039 577 298860.

References

Hide All
Bershadsky, A.D. & Vasiliev, J.M. (1988). Cytoskeleton. New York: Plenum Press.
Biro, R.L., Daye, S., Serlin, B.S., Terry, E.M., Datta, N., Sopory, S.K. & Roux, S.J. (1984). Characterization of oat calmodulin and radio immuoassay of its subcellular distribution. Plant Physiol. 75, 382–6.
Brewbaker, J.L. & Kwack, B.H. (1963). The essential role of calcium in pollen germination and pollen tube growth. Am. J. Bot. 50, 859–65.
Brockerhoff, S.E. & Davis, T.N. (1992). Calmodulin concentrates at regions of cell growth in Saccharomyces cerevisiae. J. Cell Biol. 118, 619–29.
Cai, G.Bartalesi, A., Del Casino, C., Moscatelli, A., Tiezzi, A. & Cresti, M. (1993). The kinesin immunoreactive homologue from Nicotiana tabacum pollen tube: biochemical properties and subcellular localization. Planta. 191, 496506.
Condeelis, J.S. (1974). The identification of F-actin in the pollen tube and protoplast of Amaryllis belladonna. Exp. Cell Res. 88, 435–9.
Cyr, R.J. (1991). Calcium/calmodulin affects microtubule stability in lysed protoplasts. J. Cell Sci. 100, 311–17.
Dauwalder, M., Roux, S.J. & Hardison, L. (1986). Distribution of calmodulin in pea seedling: immunocytochemical localization in plumules and root apices. Planta 168, 461–70.
Evans, D.E., Briars, S.A. & Williams, L.E. (1991). Active calcium transport by plant cell membranes. J. Exp. Bot. 42, 285303.
Franke, W.W., Herth, W., Van Der Woude, W.J. & Morré, D.J. (1972). Tubular and filamentous structures in pollen tubes: Possible involvement as guide elements in protoplasmic streaming and vectorial migration of secretory vesicles. Planta 105, 317–41.
Hausser, I., Herth, W. & Reiss, H.-D. (1984). Calmodulin in tip-growing plant cells, visualized by fluorescing calmodulin-binding phenothiazines. Planta 162, 33–9.
Herth, W., Reiss, H.-D. & Hartmann, E. (1990). Role of calcium ions in tip growth of pollen tubes and moss protonema cells. In Tip Growth in Plant and Fungal Cells, ed. Heath, IB, pp. 91113. San Diego: Academic Press.
Heslop-Harrison, J. & Heslop-Harrison, Y. (1989 a). Conformation and movement of the vegetative nucleus of the angiosperm pollen tube: association with the actin cytoskeleton. J. Cell Sci. 93, 299308.
Heslop-Harrison, J. & Heslop-Harrison, Y. (1989 b). Actomyosin and movement in the angiosperm pollen tube: an interpretation of some recent results. Sex. Plant Reprod. 2, 199207.
Heslop-Harrison, J. & Heslop-Harrison, Y. (1989 c). Myosin associated with the surface of organelles, vegetative nuclei and generative cells in angiosperm pollen grains and tubes. J. Cell Sci. 94, 319–25.
Heslop-Harrison, J. & Heslop-Harrison, Y. (1989 d). Cytochalasin effects on structure and movement in the pollen tube of Iris. Sex. Plant Reprod. 2, 2737.
Heslop-Harrison, J. & Heslop-Harrison, Y. (1990). Dynamic aspects of apical zonation in the angiosperm pollen tube. Sex. Plant Reprod. 3, 187–94.
Hulen, D., Baron, A., Salisbury, J. & Clarke, M. (1991). Production and specificity of monoclonal antibodies against calmodulin from Dictyostelium discoideum. Cell Motil. Cytoskel. 18, 113–22.
Jablonsky, P.P., Grolig, F., Perkin, J.L. & Williamson, R.E. (1991). Properties of monoclonal antibodies to plant calmodulin. Plant Sci. 76, 175–84.
Kohno, T., Chaen, S. & Shimmen, T. (1990). Characterization of the translocator associated with pollen tube organelles. Protoplasma 161, 75–7.
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–5.
Lancelle, S.A. & Hepler, P.K. (1989). Cytochalasin-induced ultrastructural alteration in Nicotiana pollen tubes. Protoplasma (Suppl. 2), 6775.
Mascarenhas, J.P. & Lafountain, J. (1972). Protoplasmic streaming, cytochalasin B, and growth of the pollen tube. Tissue Cell. 4, 1114.
Miller, D.D., Callaham, D.A., Gross, D.J. & Hepler, P.K. (1992). Free Ca2+ gradient in growing pollen tubes of Lilium. J. Cell Sci. 101, 712.
Nelson, G.A., Andrews, M.L. & Karnovsky, M.J. (1982). Participation of calmodulin in immunoglobulin capping. J. Cell Biol. 95, 771–80.
Obermeyer, G. & Weisenseel, M.H. (1991). Calcium channel blocker and calmodulin antagonists affect the gradient of free calcium ions in lily pollen tubes. Eur. J. Cell Biol. 56, 319–27.
Picton, J.M. & Steer, M.W. (1981). Determination of secretory vesicle production rates by dictyosomes in pollen tubes of Tradescantia using cytochalasin D. J. Cell Sci. 49, 261–72.
Picton, J.M. & Steer, M.W. (1985). The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport, vesicle fusion and tip extension in pollen tubes. Planta 163, 20–6.
Pierson, E.S. & Cresti, M. (1992). Cytoskeleton and cytoplasmic organization of pollen and pollen tubes. Int. Rev. Cytol. 140, 73125.
Polito, V.S. (1983). Membrane-associated calcium during pollen grain germination: a microfluorometric analysis. Protoplasma 117, 226–32.
Rathore, K.S., Cork, R.J. & Robinson, K.R. (1991). A cytoplasmic gradient of Ca2+ is correlated with the growth of lily pollen tubes. Dev. Biol. 148, 612–19.
Reiss, H.-D. & Herth, W. (1979). Calcium gradients in tip growing plant cells visualized by CTC-fluorescence. Planta 146, 615–21.
Reiss, H.-D., Herth, W., Schnepf, E. & Nobiling, R. (1983). The tip-to-base calcium gradient in pollen tubes of Lilium longiflorum measured by proton-induced X-ray emission (PIXE). Protoplasma 115, 153–9.
Sloat, B.F., Adams, A. & Pringle, J.R. (1981). Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J. Cell Biol. 89, 395405.
Staiger, C.J. & Schliwa, M. (1987). Actin localization and function in higher plants. Protoplasma 141, 112.
Steer, M.W. & Steer, J.M. (1989). Pollen tube tip growth. New Phytol. 111, 323–58.
Sun, G.-H., Ohya, Y. & Anraku, Y. (1992). Yeast calmodulin localizes to sites of cell growth. Protoplasma 166, 110–13.
Tang, X.J., Lancelle, S.A. & Hepler, P.K. (1989 a). Fluorescence microscopic localization of actin in pollen tubes: comparison of actin antibody and phalloidin staining. Cell Motil. Cytoskel. 12, 216–24.
Tang, X.J., Hepler, P.K. & Scordilis, S.P. (1989 b). Immunochemical and immunocytochemical identification of a myosin heavy chain polypeptide in Nicotiana pollen tubes. J. Cell Sci. 92, 569–74.
Tiezzi, A. (1991). The pollen tube cytoskeleton. Electron Microsc. Rev. 4, 205–19.
Tiezzi, A., Moscatelli, A., Cai, G., Bartalesi, A. & Cresti, M. (1992). An immunoreactive homolog of mammalian kinesin in Nicotiana tabacum pollen tubes. Cell Motil. Cytoskel. 21, 132–7.
Tirlapur, U.K. (1987). The role of calmodulin in reproductive physiology of higher plants. In XIV Int. Bot. Congress,24 July-1 August 1987,Berlin, Germany, abstract 1–110, p. 63.
Tirlapur, U.K. & Cresti, M. (1992). Computer-assisted video image analysis of spatial variations in membrane-associated Ca2+ and calmodulin during pollen hydration, germination and tip growth in Nicotiana tabacum L. Ann. Bot. 69, 503–8.
Tirlapur, U.K. & Willemse, M.T.M. (1992). Changes in calcium and calmodulin levels during microsporogenesis, pollen development and germination in Gasteria verrucosa (Mill.) H. Duval. Sex. Plant Reprod. 5, 214–23.
Tirlapur, U.K., Häder, D.P. & Scheuerlein, R. (1992). UV-B mediated damage in the photosynthetic flagellate, Euglena gracilis, studied by image analysis. Biol. Pflanz. 67, 305–17.
Wick, S.M., Muto, S. & Duniec, J. (1985). Double immunofluorescence labeling of calmodulin and tubulin in dividing plant cells. Protoplasma 126, 198206.
Williamson, R.E. (1986). Organelle movements along actin filaments and microtubules. Plant Physiol. 82, 631–4.
Williamson, R.E. (1993). Organelle movements. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 181202.
Yen, L.F., Wang, X.Z., Teng, X.Y., Ma, Y.Z. & Liu, G.Q. (1986). Actin and myosin in pollens and their role in the growth of pollen tubes. Chin. Sci. Bull. 31, 267–78.

Keywords

Confocal image analysis of spatial variations in immunocytochemically identified calmodulin during pollen hydration, germination and pollen tube tip growth in Nicotiana tabacum L.

  • Uday K. Tirlapur (a1), Monica Scali (a1), Alessandra Moscatelli (a1), Cecilia Del Casino (a1), Gianpiero Cai (a1), Antonio Tiezzi (a1) and Mauro Cresti (a1)...

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed