We have studied the timing of preprophase band (PPB) development in the division cycle of onion (Allium cepa L.) root-tip cells by combinations of immunofluorescence microscopy of microtubules, ...microspectrophotometry of nuclear DNA, and autoradiography of 3Hthymidine incorporation during pulse-chase experiments. In normally grown onion root tips, every cell with a PPB had the G2 level of nuclear DNA. Some were in interphase, prior to chromatin condensation, and some had varying degrees of chromatin condensation, up to the stage of prophase at which the PPB-prophase spindle transition occurs. In addition, autoradiography showed that PPBs can be formed in cells which have just finished their S phase, and microspectrophotometry enabled us to detect a population of cells in G2 which had no PPBs, these presumably including cells which had left the division cycle. The effects of inhibitors of DNA synthesis showed that the formation of PPBs is not fully coupled to events of the nuclear cycle. Although the mitotic index decreased 6—10-fold to less than 0.5% when roots were kept in 20 μg·ml-1 aphidicolin for more than 8 h, the percentage of cells containing PPBs did not decrease in proportion: the number of cells in interphase with PPBs increased while the number in prophase decreased. Almost the same phenomena were observed in the presence of 100 μg·ml-1 5-aminouracil and 40 μg·ml-1 hydroxyurea. In controls, all cells with PPBs were in G2 or prophase, but in the presence of aphidicolin, 5-aminouracil or hydroxyurea, some of the interphase cells with PPBs were in the S phase or even in the G1 phase. We conclude that PPB formation normally occurs in G2 (in at least some cases very early in G2) and that this timing can be experimentally uncoupled from the timing of DNA duplication in the cell-division cycle. The result accords with other evidence indicating that the cytoplasmic events of cytokinesis are controlled in parallel to the nuclear cycle, rather than in an obligatorily coupled sequence.
Using a combination of electron-microscopic and immunocytochemical techniques the behaviour of the microtubular cytoskeleton has been followed throughout microsporogenesis in Lilium henryi Thunb. ...Cells treated with colchicine at specific stages and then permitted to develop to near maturity were used to investigate any participation by microtubules in the regulation of pollen wall patterning. The microtubular cytoskeleton assumes four principal forms during the meiotic process; in pre-meiosis it resembles that characteristic of meristematic somatic cells, during meiotic prophase it becomes associated with a nuclear envelope and, perhaps, with the chromosomes and, as the nuclear and cell divisions commence, it takes the form of a normal spindle apparatus. In the young microspores, microtubules assume a radial organisation extending from sites at the nuclear envelope to the inner face of the plasma membrane. No firm evidence was found linking any one of these forms of cytoskeleton with the generation of patterning on the cell surface. Experiments with colchicine revealed that the drug would readily dislocate the colpus, but did not affect the general reticulate patterning. The radial cytoskeleton was present during the deposition of the early primexine, but evidence from these and other studies (J.M. Sheldon and H.G. Dickinson 1983, J. Cell. Sci. 63, 191—208; H.G. Dickinson and J.M. Sheldon, 1984, Planta 161, 86—90) indicates patterning to be imprinted upon the plasma membrane prior to the appearance of this type of cytoskeleton. These results are discussed in terms of a recent model proposed to explain pattern generation on the surface of Lilium pollen grains, based on the "self-assembly" of patterning determinants within the plasma membrane.
The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin ...immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0° C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 μM colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 μM colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.
