CAPACITY FOR MICROTUBULE REORGANIZATION AND CELL-WALL SYNTHESIS IN CYTOPLASTS OF THE GREEN-ALGA MOUGEOTIA

A small proportion of nucleate subprotoplasts (karyoplasts) and enucleate subprotoplasts (cytoplasts) are formed during the preparation of protoplasts from the filamentous green alga Mougeotia. Regeneration of Mougeotia 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 20h 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.