, Volume 178, Issue 1–2, pp 11–17 | Cite as

Capacity for microtubule reorganization and cell wall synthesis in cytoplasts of the green algaMougeotia

  • Moira E. Galway
  • G. J. Hyde
  • Adrienne R. Hardham


A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoplasts (cytoplasts) are formed during the preparation of protoplasts from the filamentous green algaMougeotia. Regeneration ofMougeotia protoplasts is an orderly process known to involve reorganisation of cortical microtubules into polar arrays centered upon two opposing foci, synthesis of new cell walls and elongation to reform cylindrical cells. The ability of cytoplasts to carry out microtubule reorganisation and cell wall synthesis was investigated by combining Hoechst staining, to distinguish cytoplasts from karyoplasts and protoplasts, with immunofluorescent staining of microtubules and Calcofluor or Tinopal staining of cell walls. Cytoplasts survived at least 20 h in culture, but did not elongate. However, cytoplasts did participate in the first steps of protoplast regeneration. The majority of cytoplasts synthesized some cell wall material, while a small proportion was able to form ordered arrays of cortical microtubules indistinguishable from those in regenerating nucleate protoplasts. These results demonstrate the ability of plant microtubules to form new, orderly arrays in the absence of a nucleus, and suggest that the reestablishment of axiality in the protoplasts does not require a nucleus or nuclear DNA transcription.


Alga Cell wall Cytoplast Microtubule Mougeotia Protoplast 





microtubule associated protein


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bajer AS, Molè-Bajer J (1982) Asters, poles, and transport properties within spindlelike microtubule arrays. Cold Spring Harbor Symp Quant Biol 46: 263–283PubMedGoogle Scholar
  2. — — (1986) Reorganization of microtubules in endosperm cells and cell fragments of the higher plantHaemanthus in vivo. J Cell Biol 102: 263–281PubMedGoogle Scholar
  3. Bershadsky AD, Vasiliv JM (1988) Cytoskeleton. Plenum, New YorkGoogle Scholar
  4. Bilkey PC, Davey MR, Cocking EC (1982) Isolation, origin and properties of enucleate plant microplasts. Protoplasma 110: 147–152Google Scholar
  5. Bunn CL (1982) The influence of cytoplast-to-cell ratio on cybrid formation. In: Shay JW (ed) Techniques in somatic cell genetics. Plenum, New York, pp 189–201Google Scholar
  6. Burridge K, Path K, Kelly T, Nuckolls G, Turner C (1988) Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol 4: 487–525PubMedGoogle Scholar
  7. Chang-Jie J, Sonobe S, Shibaoka H (1992) Assembly of microtubules in a cytoplasmic extract of tobacco BY-2 miniprotoplasts in the absence of microtubule-stabilizing agents. Plant Cell Physiol 33: 497–501Google Scholar
  8. Cyr RJ (1991) Microtubule-associated proteins in higher plants. In: Lloyd CW (ed) The cytoskeletal basis of plant growth and form. Academic Press, London, pp 57–67Google Scholar
  9. Galway ME, Hardham AR (1986) Microtubule reorganization, cell wall synthesis and establishment of the axis of elongation in regenerating protoplasts of the algaMougeotia. Protoplasma 135: 130–143Google Scholar
  10. — — (1989) Oryzalin-induced microtubule disassembly and recovery in regenerating protoplasts of the algaMougeotia. J Plant Physiol 135: 337–345Google Scholar
  11. Gelfand VI, Glushankova NA, Ivanova OYu, Mittelman LA, Pletyushkina OYu, Vasiliev JM, Gelfand IM (1985) Polarization of cytoplasmic fragments microsurgically detached from mouse fibroblasts. Cell Biol Int Rep 9: 883–892PubMedGoogle Scholar
  12. Guha Roy S, Bhisey AN (1992) Cytoplasmic microtubule assembly is altered in cytoplasts. Cell Biol Int Rep 16: 269–281PubMedGoogle Scholar
  13. Gunning BES, Hardham AR (1982) Microtubules. Annu Rev Plant Physiol 33: 651–698Google Scholar
  14. Hughes J, McCully ME (1975) The use of an optical brightener in the study of plant structure. Stain Technol 50: 319–329PubMedGoogle Scholar
  15. Ingber DE (1993) Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci 104: 613–627PubMedGoogle Scholar
  16. Itoh T, Legge RL, Brown RM Jr (1986) The effects of selected inhibitors on cellulose microfibril assembly inBoergesenia forbesii (Chlorophyta) protoplasts. J Phycol 22: 224–233Google Scholar
  17. Karsenti E, Kobayashi S, Mitchison T, Kirschner M (1984) Role of the centrosome in organizing the interphase microtubule array: properties of cytoplasts containing or lacking centrosomes. J Cell Biol 98: 1763–1776PubMedGoogle Scholar
  18. Kennedy FGR, Hoshaw RW (1978) Culture study of reproductive cycles and systematics inMougeotia transeaui (Chlorophyta). J Phycol 14: 445–450Google Scholar
  19. Kopecká M, Gabriel M, Farkas V (1987) Some enucleated yeast protoplasts synthesize β-(1,3)-D-glucan microfibrils of the cell wall. Naturwissenschaften 74: 389–391Google Scholar
  20. Kroh M, Knuiman B (1988) Development of subprotoplasts from in vitro-grown tobacco pollen tubes. Sex Plant Reprod 1: 103–113Google Scholar
  21. Maeda H, Ishida N (1967) Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J Biochem 62: 276–278PubMedGoogle Scholar
  22. Marchant HJ (1979) Microtubules, cell wall deposition and the determination of plant cell shape. Nature 278: 167–168Google Scholar
  23. —, Fowke LC (1977) Preparation, culture, and regeneration of protoplasts from filamentous green algae. Can J Bot 55: 3080–3086Google Scholar
  24. —, Hines ER (1979) The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts ofMougeotia. Planta 146: 41–48Google Scholar
  25. Osborn M, Weber K (1982) Immunofluorescence and immunocytochemical procedures with affinity purified antibodies: tubulincontaining structures. In: Wilson L (ed) Methods in cell biology, vol 24, the cytoskeleton, part A. Academic Press, New York, pp 97–132Google Scholar
  26. Raghow R (1987) Regulation of messenger RNA turnover in eukaryotes. Trends Biochem Sci 12: 358–60Google Scholar
  27. Reinert J, Binding H (1986) Protoplast fusion and early development of fusants. In: Reinert J, Binding H (eds) Differentiation of protoplasts and of transformed plant cells. Springer, Berlin Heidelberg New York Tokyo, pp 37–66 [Hennig W, Nover L, Scheer U (eds) Problems in cell differentiation, vol 12]Google Scholar
  28. Roberts K (1989) The plant extracellular matrix. Curr Opin Cell Biol 1: 1020–1027PubMedGoogle Scholar
  29. — (1990) Structures at the plant cell surface. Curr Opin Cell Biol 2: 920–928PubMedGoogle Scholar
  30. Rutten TLM, Derksen J (1990) Organization of actin filaments in regenerating and outgrowing subprotoplasts from pollen tubes ofNicotiana tabacum L. Planta 180: 471–479Google Scholar
  31. — — (1992) Microtubules in pollen tube subprotoplasts: organization during protoplasts formation and protoplast outgrowth. Protoplasma 167: 231–237Google Scholar
  32. —, Kroh M, Knuiman B (1991) Cell-wall regeneration in pollentube subprotoplasts. Acta Bot Neerl 40: 211–216Google Scholar
  33. Schweiger H-G, Berger S (1979) Nucleocytoplasmic interrelationships inAcetabularia and some other Dasycladaceae. Int Rev Cytol [Suppl 9]: pp 11–44Google Scholar
  34. White RG, Hyde GJ, Overall RL (1990) Microtubule arrays in regeneratingMougeotia protoplasts may be oriented by electric fields. Protoplasma 158: 73–85Google Scholar
  35. Williamson RE (1991) Orientation of cortical microtubules in interphase plant cells. Int Rev Cytol 129: 135–206Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Moira E. Galway
    • 1
  • G. J. Hyde
    • 1
  • Adrienne R. Hardham
    • 1
  1. 1.Plant Cell Biology Group, Research School of Biological SciencesThe Australian National UniversityCanberraAustralia

Personalised recommendations