, Volume 152, Issue 3, pp 272–281 | Cite as

Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

  • Mary Jane Saunders
  • Peter K. Hepler


We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca2+-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca2+ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca2+ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca2+ indicate a localized rise in intracellular free Ca2+ concentration brought about by cytokinin action.

Key words

Bryophyta Bud formation (moss) Calcium Chlorotetracycline Cytokinin Funaria 







endoplasmic reticulum




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  1. Ballard, S.G., Barker, R.W., Barrett Bee, K.J., Dwak, R.A., Radda, G.K., Smith, D.S., Taylor, J.A. (1972) The location and response of probes in membranes. In: Biochemistry and biophysics of mitochondrial membranes, pp. 257–275, Azzone, G.F., Carafoli, E., Lehninger, A.L., Quagliariello, E., Siliprandi, N., eds. Academic Press, New York LondonGoogle Scholar
  2. Brandes, H., Kende, H. (1968) Studies on cytokinin-controlled bud formation in moss protonemata. Plant Physiol. 43, 827–837Google Scholar
  3. Burrows, W.J. (1975) Mechanisms of action of cytokinins. Curr. Adv. Plant Sci. 7, 837–845Google Scholar
  4. Caswell, A.H. (1979) Methods of measuring intracellular calcium. Int. Rev. Cytol. 56, 145–181Google Scholar
  5. Caswell, A.H., Hutchison, J.D. (1971) Selectivity of cation chelation to tetracyclines: evidence for special conformation of calcium chelate. Biochem. Biophys. Res. Commun. 43, 625–630PubMedGoogle Scholar
  6. Fulton, B.P., Whittingham, D.G. (1978) Activation of mammalian oocytes by intracellular injection of calcium. Nature 273, 149–151Google Scholar
  7. Hepler, P.K. (1980) Membranes in the mitotic apparatus of barley cells. J. Cell Biol. 86, 490–499Google Scholar
  8. Hepler, P.K., Palevitz, B.A. (1974) Microtubules and microfilaments. Annu. Rev. Plant Physiol. 25, 309–362Google Scholar
  9. Hepler, P.K., Wick, S.M., Wolniak, S.M. (1981) The structure and role of membranes in the mitotic apparatus. In: International cell biology (1980–1981), pp. 673–687, Schweiger, H.-G., ed. Springer-Verlag, Berlin Heidelberg New YorkGoogle Scholar
  10. Izzard, C.S., Izzard, S.L. (1975) Calcium regulation of the contractile state of isolated mammalian fibroblast cytoplasm. J. Cell Sci. 18, 241–256Google Scholar
  11. Jaffe, L.F. (1979) Control of development by ionic currents. In: Membrane transduction mechanisms, pp. 199–231, Cone, R.A., Dowling, J.E., eds. Raven Press, New YorkGoogle Scholar
  12. Johnson, G.S., D'Armiento, M., Carchman, R.A. (1974) N6-substituted adenines induce cell elongation irrespective of the intracellular cyclic AMP levels. Exp. Cell Res. 85, 47–56Google Scholar
  13. Kanatani, H. (1973) Maturation-inducing substance in starfishes. Int. Rev. Cytol. 35, 253–298Google Scholar
  14. Kiehart, D.P. (1981) Studies on the in vivo sensitivity of spindle microtubules to calcium ions and evidence for a vesicular calcium sequestering system. J. Cell Biol. 88, 601–617CrossRefGoogle Scholar
  15. Laetsch, W.M. (1967) Ferns. In: Methods in developmental biology, pp. 319–328, Wilt, F.H., Wessells, N.K., eds. Thomas Y. Crowell Co., New YorkGoogle Scholar
  16. Lau, O.-L., Yang, S.F. (1975) Interaction of kinetin and calcium in relation to their effect on stimulation of ethylene production. Plant Physiol. 55, 738–740Google Scholar
  17. LeJohn, H.B., Cameron, L.E. (1973) Cytokinins regulate calcium binding to a glycoprotein from fungal cells. Biochem. Biophys. Res. Commun. 54, 1053–1060Google Scholar
  18. LeJohn, H.B., Stevenson, R.M. (1973) Cytokinins and magnesium ions may control the flow of metabolites and calcium ions through fungal cell membranes. Biochem. Biophys. Res. Commun. 54, 1061–1066PubMedGoogle Scholar
  19. Letham, D.S. (1978) Cytokinins. In: Phytohormones and related compounds—a comprehensive treatise, vol. I, The biochemistry of phytohormones and related compounds, pp. 205–263, Letham, D.S., Goodwin, P.B., Higgins, T.J.V., eds. Elsevier/North-Holland Biomedical Press, Amsterdam Oxford New YorkGoogle Scholar
  20. Mazia, D. (1937) The release of calcium in Arbacia eggs upon fertilization. J. Cell Comp. Physiol. 10, 291–304Google Scholar
  21. Moreau, M., Guerrier, P., Doree, M., Ashley, C.C. (1978) Hormone-induced release of intracellular Ca2+ triggers meiosis in starfish oocytes. Nature 272, 251–252Google Scholar
  22. Poovaiah, B.W., Leopold, A.C. (1973) Deferral of leaf senescence with calcium. Plant Physiol. 52, 236–239Google Scholar
  23. Quader, H., Robinson, D.G. (1979) Structure, synthesis and orientation of microfibrils. VI. The role of ions in microfibril deposition in Oosystis solitaria. Eur. J. Cell Biol. 20, 51–56Google Scholar
  24. Reiss, H.D., Herth, W. (1979) Calcium gradients in tip growing plant cells visualized by chlorotetracycline fluorescence. Planta 146, 615–621Google Scholar
  25. Rose, B., Loewenstein, W.R. (1975) Calcium ion distribution in cytoplasm visualized by aequorin: diffusion in cytosol restricted by energized sequestering. Science 190, 1204–1206Google Scholar
  26. Salmon, E.D., Segall, R.R. (1980) Calcium-labile mitotic spindles isolated from sea urchin eggs. J. Cell Biol. 86, 355–365Google Scholar
  27. Schmiedel, G., Schnepf, E. (1979) Side branch formation and orientation in the caulonema of the moss Funaria hygrometrica: experiments with inhibitors and with centrifugation. Protoplasma 101, 47–59Google Scholar
  28. Schmiedel, G., Schnepf, E. (1980) Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica. Planta 147, 405–413Google Scholar
  29. Steinhardt, R.A., Epel, D. (1974) Activation of sea urchin eggs by a calcium ionophore. Proc. Natl. Acad. Sci. USA 71, 1915–1919Google Scholar
  30. Träuble, H., Overath, P. (1973) The structure of Escherichia coli membranes studied by fluorescence measurements of lipid phase transitions. Biochim. Biophys. Acta 307, 491–512Google Scholar

Copyright information

© Springer-Verlag 1981

Authors and Affiliations

  • Mary Jane Saunders
    • 1
  • Peter K. Hepler
    • 1
  1. 1.Botany DepartmentUniversity of MassachusettsAmherstUSA

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