Loss of Microtubules in the Interphase Cells of Onion (Allium cepa L.) Root Tips from the Cell Cortex and Their Appearance in the Cytoplasm after Treatment with Cycloheximide.

As part of a project to investigate the mechanism of cortical microtubule (MT) alignment, we examined the effects of cycloheximide (CHM) on cortical MTs in the root tip cells of Allium cepa L. Results show that although a preprophase band of MTs remained in the cell cortex, interphase MTs disappeared from the cortical cytoplasm and then appeared concomitantly in the inner cytoplasm when the rate of de novo protein synthesis was reduced with CHM (11-360 [mu]M for 2 h)

Loosening of a preprophase band of microtubules in onion (Allium cepa L.) root tip cells by kinase inhibitors.

Effects of kinase inhibitors on the preprophase band of microtubules in onion (Allium cepa L.) root tip cells were examined. Bundled microtubules in preprophase bands were dispersed on the cell cortex when onion seedlings were incubated with 2.5-5.0 mM 6-dimethylaminopurine. Fifteen min was enough for the bundled microtubules to disappear. Although many preprophase bands remained when the seedlings were incubated with 60 microM staurosporin, these preprophase band microtubules were loosened and the width of the band became broad. These results sugget that some kinases are involved in the microtubule bundling in the preprophase band development.

Preprophase bands of microtubules and the cell cycle: Kinetics and experimental uncoupling of their formation from the nuclear cycle in onion root-tip cells.

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 [(3)H]thymidine 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.

The generation and consolidation of a radial array of cortical microtubules in developing guard cells of Allium cepa L.

The initiation and development of a radial array of microtubules (MTs) in guard cells of A. cepa was studied using immunofluorescence microscopy of tubulin in isolated epidermal layers. Soon after the completion of cytokinesis, MTs originate in the cortex adjacent to a central strip of the new, anticlinically oriented ventral wall separating the two guard cells. Cortical MTs extend from the mid-region of the central strip toward the cell edge where the ventral wall joins the inner periclinal wall. They then spread in a fan-like formation along the periclinal wall and gradually extend along the lateral and end walls as well. Many MTs criss-cross at various angles as they arc past the edge formed by the junction of the ventral and periclinal walls, but they do not terminate there, indicating that, contrary to previous report, the edge is not involved in MT initiation. Instead, the mid-region of the central strip appears to function as a planar MT-organizing zone. Initially, MTs radiate from this zone through the inner cytoplasm as well as the cortex. During cell expansion, however, the cortical MTs increasingly predominate and consolidate into relatively thick, long bundles, while the frequency of non-cortical MTs diminishes. The apparent density of MTs per unit surface area is maintained as the cells expand and gradually flex into an elliptical shape. The guard cells eventually separate completely at the pore site. The entire process is accomplished within about 12 h.

A planar microtubule-organizing zone in guard cells of Allium: experimental depolymerization and reassembly of microtubules.

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.

Development of the preprophase band from random cytoplasmic microtubules in guard mother cells of Allium cepa L.

The organization of microtubule (MT) arrays in the guard mother cells (GMCs) of A. cepa was examined, focussing on the stage at which a longitudinal preprophase band (PPB) is established perpendicular to all other division planes in the epidermis. In the majority of young GMCs, including those seen just after asymmetric division, MTs are distributed randomly throughout the cortex and inner regions of the cytoplasm. Few MTs are associated with the nuclear surface. As the GMCs continue to develop, MTs cluster around the nucleus and a PPB appears as a wide longitudinal band. Microtubules also become prominent between the nucleus and the periclinal and transverse walls, while they decrease in number along the radial longitudinal walls. The PPB progressively narrows by early prophase, and a transversely oriented spindle gradually ensheaths the nucleus. These observations indicate that the initial, broad PPB is organized by a rearrangement of the random cytoplasmic array of MTs. Additional reorganization is responsible for MTs linking the nucleus and the cortex in the future plane of the cell plate, and for narrowing of the PPB.