B. E. S. Gunning

Formative and proliferative cell divisions, cell differentiation, and developmental changes in the meristem of Azolla roots.

The root of the water fern Azolla is a compact higher-plant organ, advantageous for studies of cell division, cell differentiation, and morphogenesis. The cell complement of A. filiculoides Lam. and A. pinnata R.Br. roots is described, and the lineages of the cell types, all derived ultimately from a tetrahedral apical cell, are characterised in terms of sites and planes of cell division within the formative zone, where the initial cells of the cell files are generated. Subsequent proliferation of the initial cells is highly specific, each cell type having its own programme of divisions prior to terminal differentiation. Both formative and proliferative divisions (but especially the former) occur in regular sequences. Two enantiomorphic forms of root develop, with the dispositions of certain types of cell correlating with the direction, dextrorse or sinistrorse, of the cell-division sequence in the apical cells. Root growth is determinate, the apical cell dividing about 55 times, and its cell-cycle duration decreasing from an initial 10 h to about 4 h during the major phase of root development. Sites of proliferation progress acropetally during aging, but do not penetrate into the zone of formative divisions. The detailed portrait of root development that was obtained is discussed with respect to genetic and epigenetic influences; quantal and non-quantal cell cycles; variation in cell-cycle durations; relationships between cell expansion and cell division: the role of the apical cell; and the limitation of the total number of mitotic cycles during root formation.

Preprophase bands, phragmoplasts, and spatial control of cytokinesis.

SUMMARY Features of preprophase bands (PPBs) of microtubules (MTs), and the spatial relationship between phragmosomes, PPB sites, and developing phragmoplasts during cytokinesis, are reviewed, setting new observations in the context of current knowledge. PPBs in onion root tip cells are present by the beginning of the G2 period of the cell cycle. They are at first wide, but later become more compact, narrower bands. MTs traverse the cytoplasm between the band at the cell cortex and the nuclear envelope. This whole assemblage of nucleus, PPB and intervening MTs remains together when the cell is ruptured during preparation for examination by immunofluorescence microscopy. Double bands are occasionally seen in early stages of PPB development, perhaps as a consequence of double induction from neighbouring cells. Calmodulin is not present in PPBs at a higher concentration than in the general cytoplasm, but it is more abundant in parts of the spindle and in the phragmoplast. The PPB MTs disappear at prophase, but nevertheless the new cell plate fuses with the parental cell walls at the PPB site. This spatial relationship can be disrupted by treatment with CIPC. Another experimental disruption of the relationship, accomplished by making minute wounds in the PPB site of mitotic cells in Tradescantia stamen hairs, is described. In other experiments on these cells the phragmoplast is shown to become tethered to the PPB site when the cell plate is half to three-quarters developed, although the telophase nuclei are free to move. Rhodamine-labelled phalloidin reveals putative F-actin in the phragmoplast of Tradescantia, but not in the gap between the extending phragmoplast and the PPB site. Rhodamine-labelled phalloidin also stains cytoplasmic strands that exist when cytoplasmic streaming occurs before and after (but not during) mitosis. Cytochalasin B treatment blocks incorporation of actin into the phragmoplast, which, however, can still develop, though usually abnormally. The F-actin of the phragmoplast may function in consolidation of the cell plate, rather than in spatial guidance of its growth toward the PPB site at the cell surface.

Embryo sac development in Arabidopsis thaliana. I. Megasporogenesis, including the microtubular cytoskeleton.

SummaryAspects of megasporogenesis in Arabidopsis thaliana have been investigated using a variety of histochemical techniques to visualize general cell organization, DNA and callose in whole ovules and sections by bright field, fluorescence, differential interference contrast and scanning electron microscopy. The microtubular cytoskeleton has been studied using immunofluorescence localization of tubulin in sections and whole cells. The observations deviate from reports of preceding studies in that the megasporocyte was found to undergo both meiotic divisions followed by simultaneous cytokinesis (i.e. without an intermediate dyad stage) to give a multiplanar tetrad of megaspores. This represents a variation of monosporic development not previously described. Polarized distribution of organelles prior to meiosis ensures that the functional megaspore receives the largest share. Aberrant wall formation is common between degenerating megaspores. Localized callose deposition in the tetrad separates these cells from the active megaspore. Their pattern of degeneration and displacement is extremely flexible within the embryo sac space. The microtubular cytoskeleton is extensive and largely cytoplasmic, as distinct from cortical, throughout megasporogenesis. In the megasporocyte, megaspores and one-nucleate embryo sac, randomly oriented microtubules throughout the cells may serve to maintain cytoplasmic integrity and position organelles. Numerous microtubules (MTs) associate closely with the nucleus and some radiate from it, perhaps functioning in nuclear positioning. During meiosis MTs are restricted to the spindle configurations and later to the phragmoplasts which form between daughter nuclei. The lack of interphase cortical arrays suggests that the role of internal influences on cell shape is small.

Evidence for initiation of microtubules in discrete regions of the cell cortex in Azolla root-tip cells, and an hypothesis on the development of cortical arrays of microtubules

Complexes of microtubules, vesicles, and (to varying degrees) dense matrix material around the microtubules were seen along the edges of cells in root apices of Azolla pinnata R.Br. (viewing the cells as polyhedra with faces, vertices and edges). They are best developed after cytokinesis has been completed, when the daughter cells are reinstating their interphase arrays of microtubules. They are not confined to edges made by the junction of new cell plates with parental walls, but occur also along older edges. Similar matrices and vesicles are seen amongst phragmoplast microtubules and where pre-prophase bands intersect the edges of cells. It is suggested that the complexes participate in the development of cortical arrays of microtubules. The observations are combined with others, made on pre-prophase bands and on the substructure of cortical arrays lying against the faces of cells, to develop an hypothesis on the development of cortical microtubules, summarised below: Microtubules are nucleated along the edges of cells, at first growing in unspecified orientations and then becoming bridged to the plasma membrane. Parallelism of microtubules in the arrays arises by inter-tubule cross-bridging. Lengths of microtubule are released from, or break off, the nucleating centres and are moved out onto the face of the cell by intertubule and tubule-membrane sliding, thus accounting for the presence there of short tubules with randomly placed terminations. The nucleating zones along cell edges might have vectorial properties, and thus be able to control the orientation of the microtubules on the different faces of the cell. Also, localised activation could generate localised arrays, especially pre-prophase bands in specified sites and planes. Two possible reasons for the spatial restriction of nucleation to cell edges are considered. One is that the geometry of an edge is itself important; the other is that along most cell edges there is a persistent specialised zone, inherited at cytokinesis by the daughter cells when the cell plate bisects the former pre-prophase-band zone.

Intercellular communication inAzolla roots: I. Ultrastructure of plasmodesmata

SummaryA model is proposed for the structure of the plasmodesmata ofAzolla root primordia, based on micrographs obtained by a combination of fixation in glutaraldehyde/p-formaldehyde/tannic acid/ferric chloride, digestion of cell walls and the use of stereo pairs. Unlike the model for plasmodesmatal structure proposed byRobards (1971), the desmotubule is depicted as a virtually closed cylindrical bilayer providing little or no open pathway for transport. In this respect it is similar to the model ofLópez-Sáezet al. (1966). An analysis of the molecular packing of types of lipids found in endoplasmic reticulum (of which the desmotubule is an extension) indicates that the model is geometrically feasible. Details cannot be discerned with accuracy, but material, possibly particulate, occupies much of the space between desmotubule and plasma membrane, the cytoplasmic lumen being reduced to inter-particle spaces of cross-sectional area comparable to that of the bore in a gap junction connexon. Implications for intercellular transport are discussed.

Confocal fluorescence microscopy of plant cells

SummaryThe confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the “z” axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.

Pre-prophase bands of microtubules in all categories of formative and proliferative cell division in Azolla roots.

Pre-prophase bands of microtubules were found in every category of cell division, symmetrical and asymmetrical, in the cell lineages of the root apex of Azolla pinnata R.Br. and A. filiculoides Lam., and in the transverse divisions in the cell files of the roots. They are also found in the asymmetrical cell division that gives rise to trichoblasts in roots of Hydrocharis dubia (B1). Backer. It is possible, in a variety of cell types in roots of Azolla, to predict within a fraction of a micrometre where a new cell wall will be located. In every such case the midline of the 1.5–3-μm-wide pre-prophase band anticipates this location. Each of the daughter cells thus inherits approximately half of the former pre-prophase band site. Images interpreted as stages of formation of the band were obtained, its microtubules replacing the interphase cortical arrays. In one highly asymmetrical division, band formation precedes migration of the nucleus to the site of mitosis. The asymmetrical division that gives rise to root hairs passes acropetally along every cell in the dermatogen layer, and preprophase bands were seen up to 8 cells in advance of the last completed division. Here, and in the zone of formative divisions, the band is present for much longer than the duration of mitosis. The ubiquity of the band in the Azolla root tip is discussed in relation to the literature, and a working hypothesis is presented that takes into account current knowledge of occurrence, development and function of the band.

Plastid stromules: video microscopy of their outgrowth, retraction, tensioning, anchoring, branching, bridging and tip-shedding

Summary.Stromules are stroma-containing tubules which can grow from the surface of plastids, most commonly leucoplasts and chromoplasts, but also chloroplasts in some tissues. Their functions are obscure. Stills from video rate movies are presented here. They illustrate interaction of stromules with cytoskeletal strands and the anchoring of stromules to unidentified components at the cell surface. Anchoring leads to stretching and relaxation of stromules when forces arising from cytoplasmic streaming act on the attached, freely suspended plastid bodies. Data on stromule growth, retraction, and regrowth rates are provided. Formation and movement of stromular branches and bridges between plastids are described. The shedding of a tip region into the streaming cytoplasm is recorded in frame-by-frame detail, in accord with early observations.

The microtubular cytoskeleton during development of the zygote, proemhryo and free-nuclear endosperm in Arabidopsis thaliana (L.) Heynh

The microtubular cytoskeleton has been studied during development of the zygote, proembryo and free-nuclear endosperm in A. thaliana using immunofluorescence localization of tubulin in enzymatically isolated material. Abundant micro tubules (MTs) are found throughout proembryogenesis. Microtubules in the coenocytic endosperm are mainly internal. By contrast, there is a re-orientation of MTs to a transverse cortical distribution during zygote development, predominantly in a subapical band which accompanies a phase of apical extension. The presence of these cortical arrays coincides with the elongation of the zygote. Cortical arrays also accompany elongation of the cylindrical suspensor. Extensive networks of MTs ramify throughout the cytoplasm of cells in the proembryo proper. Perinuclear arrays are detected in a number of cell types and MTs contribute to typical mitotic configurations during nuclear divisions. Preprophase bands of MTs are absent throughout megasporogenesis and embryo-sac development and do not occur in endosperm cell divisions. We have observed MTs throughout the first division cycle of the zygote. By placing the observed stages in a most probable sequence, we have identified this cell cycle as the point during embryogenesis at which a preprophase band is reinstated as a regular feature of cell division. Preprophase bands were observed to predict planes of cytokinesis in cell divisions up to the octant stage.

Age-related and Origin-related Control of the Numbers of Plasmodesmata in Cell Walls of Developing Azolla Roots

Plasmodesmata were counted in the longitudinal and transverse walls in developmental sequences of merophytes in roots of Azolla pinnata R.Br. The differences between certain categories of longitudinal wall were traced to factors that govern the surface area of the cell plates, the density of plasmodesmata (number per unit area of cell plate), and the amount by which each type of plate expands. No evidence for secondary augmentation of plasmodesmatal numbers after the cell-plate stage of development was found, but plasmodesmata are lost from the walls of sieve and xylem elements during their differentiation. Losses caused by cell separation occur in other tissues. The relatively high density of plasmodesmata in transverse walls is based not so much on a high density in the cell plates as on the relatively low expansion that these walls undergo. There appears to be a compensatory mechanism that relates initial plasmodesmatal density to the future expansion of the cell plate. The root shows determinate growth, the apical cell dividing about 55 times. Beginning at about the 35th division there is a progressive failure to maintain the plasmodesmatal frequencies that were developed in earlier cell divisions in the apical cell. The divisions that occur within the later-produced merophytes also show progressive diminution of plasmodesmatal numbers. The result is that the apex of the root, and particularly the apical cell, becomes more and more isolated symplastically, a phenomenon which could account for its limited lifespan and the determinate growth pattern of the root.