Plastid apportionment and preprophase microtubule bands in monoplastidic root meristem cells ofIsoetes andSelaginella

Summary Ultrastructural observations on monoplastidic root tip cells ofIsoetes andSelaginella demonstrate two important phenomena associated with preprophasic preparation for mitotic cell division, 1. the preprophase band and 2. precise orientation of the dividing plastid relative to the preprophase band. Both of these phenomena accurately predict the future plane of cell division. The plastid divides in a plane parallel to the spindle and each cell inherits a single plastid which caps the telophase nucleus. When succesive transverse divisions occur, the plastid migrates prior to prophase from a position near an old transverse wall to a lateral position in the cell. The plastid is oriented with its median constriction precisely intersected by the plane of the preprophase band. When a longitudinal division follows a transverse division, the plastid remains in its position adjacent to an old transverse wall where it is bisected by the plane of the longitudinally oriented preprophase band microtubules.     

A cytoskeletal system predicts division plane in meiosis ofSelaginella

Summary An ultrastructural investigation of the monoplastidic microsporocytes ofSelaginella arenicola revealed a unique cytoskeletal array that predicts the future division plane before nuclear division takes place. By midprophase of the first meiotic division, the single plastid has divided once and the two plastids lie on opposite sides of the nucleus which is elongated in the plane of the incipient metaphase I spindle. A cytoplasmic structure, the procytokinetic plate (PCP), predicts the division plane of of both plastid and cytoplasm. The PCP consists of a distinct concentration of vesicles lying in the future division plane and an elaborate system of microtubules aligned parallel to the long axis of plastids and nucleus. Microtubules of the axially aligned system appear to terminate in clusters of vesicles in the central zone of the PCP. The PCP with axially aligned microtubules is as predictive of the division plane in these meiotic cells as is the girdling preprophase band of microtubules in mitotic cells.     

Preprophasic establishment of division polarity in monoplastidic mitosis of hornworts

Summary Preprophase in the monoplastidic mitotic cells ofPhaeoceros andNotothylas is characterized by the establishment of a division site in the absence of a typical preprophase band. The future cytokinetic plane is predicted by plastid orientation and development of an elaborate preprophasic microtubule system perpendicular to the division plane. Division of the single plastid is initiated early in preprophase and the constricting plastid migrates to a position perpendicular to the future plane of division. Plastid orientation assures that division of the plastid by mid-constriction will result in distribution of a plastid to each daughter cell. Microtubules parallel the long axis of the plastid and are most numerous adjacent to the nucleus which becomes elongated in the future spindle axis. We conclude that the division site is a fundamental component of the cytokinetic apparatus involved in the determination of cleavage plane prior to nuclear division.     

Morphogenetic plastid migration and microtubule organization during megasporogenesis inIsoetes

Summary The large megasporocytes ofIsoetes provide an exceptional system for studying microtubule dynamics in monoplastidic meiosis where plastid polarity assures coordination of plastid and nuclear division by the intimate association of MTOCs with plastids. Division and migration of the plastid in prophase establishes the tetrahedrally arranged cytoplasmic domains of the future spore tetrad and the four plastid-MTOCs serve as focal points of a unique quadripolar microtubule system (QMS). The QMS is a dynamic structure which functions in plastid deployment and contributes directly to development of both first and second division spindles. The nucleation of microtubules at discrete plastid-MTOCs is compared with centrosomal nucleation of microtubules in animal cells where growth of microtubules involves dynamic instability.Abbreviations AMS axial microtubule system - MTOC microtubule organizing center - N nucleus - QMS quadripolar microtubule system - P plastid - PPB preprophase band of microtubules     

DEVELOPMENT OF STOMATA IN SELAGINELLA: DIVISION POLARITY AND PLASTID MOVEMENTS

The young guard cell of Selaginella inherits a single plastid from the division of the stomatal guard mother cell (GMC). During early stomatal development the single plastid undergoes a complex series of migrations and divisions. The regular pattern of plastid behavior appears to be an expression of the genetic program controlling division plane and cytomorphogenesis. The plastid in the GMC becomes precisely aligned with its midconstriction intersected by the plane of a preprophase band of microtubules (PPB) oriented parallel to the long axis of the leaf. This alignment with respect to the future division plane of the cytoplasm ensures equal plastid distribution to the daughter cells. Cytokinesis occurs in the plane previously marked by the PPB and the plastid in each daughter cell lies between the lateral wall and the newly formed nucleus. Following cytokinesis the plastid in each young guard cell develops a median constriction and migrates to the common ventral wall where the isthmus is associated with a system of microtubules in the vicinity of the developing pore region. Plastid division is completed while the plastid is adjacent to the common ventral wall. Following division, the two daughter plastids move back toward the lateral wall. Each plastid may divide again during guard cell maturation but no further migrations occur.     

Preprophasic microtubule systems and development of the mitotic spindle in hornworts (Bryophyta)

Summary Studies of monoplastidic mitosis in hornworts (Bryophyta) using transmission electron microscopy and indirect immunofluorescence staining of microtubules have revealed that two mutually perpendicular microtubule systems predict division polarity in preprophase. Events of cytoplasmic reorganization in preparation for division occur in the following order: migration of the single plastid to a position perpendicular to the division site, constriction of the plastid where its midpoint intersects the division site, development of an axial system of microtubules parallel to the elongating plastid isthmus, and appearance of an atypical preprophase band of microtubules (PPB). The PPB is asymmetrical with a tight band of microtubules on the side over the plastid isthmus and a broad band of widely spaced microtubules over the nucleus. The axial system contributes directly to development of the spindle. In prometaphase, the axial system separates at the equator and additional microtubule bundles project from polar regions, creating two opposing halfspindles. The PPB is still present during asymmetrical organization of the spindle and microtubules extending from the broad portion of the PPB to poles appear to be incorporated into the developing spindle. Dynamic changes in the microtubular cytoskeleton demonstrate (1) intimate relationship of plastid and nuclear division, (2) contribution of preprophase/prophase microtubule systems to spindle development in monoplastidic cells, and (3) dynamic reorientation of microtubules from one system to another.     

Polar organizers mark division axis prior to preprophase band formation in mitosis of the hepaticReboulia hemisphaerica (Bryophyta)

Summary Changes in the pattern of microtubules during the cell cycle of the hepaticReboulia hemisphaerica (Bryophyta) were studied by indirect immunofluorescence using conventional and confocal laser scanning microscopy (CLSM). The first indication that a cell is preparing for division is fusiform shaping of the nucleus accompanied by the appearance of well-defined polar organizers (POs) at the future spindle poles. Microtubules emanating from the POs ensheath the nucleus and eventually develop into the half-spindles of mitosis. Some of the microtubules from each PO pass tangential to the nucleus and interact in the region of the future mitotic equator. A preprophase band (PPB) forms in this region later in prophase and coexists with the prophase spindle. Thus, the plane of division appears to be determined by interaction of opposing arrays of microtubules emanating from POs. Prometaphase is marked by disappearance of the POs, loss of astral microtubules, and conversion of the fusiform spindle of prophase to a truncated, barrel-shaped spindle more typical of higher plants. Restoration of cortical microtubules in daughter cell occurs on the cell side distal to the new cell plate, but nucleation of microtubules is associated with the nuclear envelope and not with organized POs. At the next division POs appear at opposite poles of preprophase nuclei with no evidence of division and migration that is characteristic of cells with centriolar centrosomes. These data lend additional support for the view that mitosis in hepatics is transitional between green algae and higher plants.Abbreviations AMS axial microtubule system - CLSM confocal laser scanning microscopy - MTOC microtubule organizing center - PO polar organizer - PPB preprophase band of microtubules - QMS quadripolar microtubule system - TEM transmission electron microscopy     

Re-organization of microtubules during preprophase band development inAdiantum protonemata

Summary Microtubule organization during preprophase band development was investigated using immunofluorescence microscopy in filamentous protonemal cells (approx. 600 m in length, 20 m in width) ofAdiantum capillus-veneris L. Protonemata pre-cultured under red light were transferred to continuous blue light or total darkness to induce synchronous cell division. Preprophase bands were found under both light conditions. In an early stage of development, the preprophase band which is transverse to the cell axis overlapped with an interphase cortical array of microtubules which is random or parallel to the cell axis. The interphase cortical array disappeared thereafter. While the width of the preprophase band became narrow during development under dark conditions, under blue light conditions it did not.Spatial and temporal aspects of the disappearance of the interphase cortical array of microtubules were also investigated. The interphase cortical array began to disappear at nearly the same time as the beginning of preprophase band formation. Under blue light, the disruption of cortical microtubules started at approx. 150 m from the tip (approx. 120 m from the nucleus), and spread toward the tip as far as the nuclear region and toward the base to an area approx. 300–400 m from the tip. Cortical microtubules remained in the basal part of the protonema. The pattern of disappearance between the tip and nucleus could not be determined. Under dark conditions, the pattern of the disappearance of cortical microtubules was somewhat different in many cells from that encountered with exposure to blue light. Microtubules first re-oriented from longitudinal to transverse, and then gradually disappeared. In some cells, the pattern of disappearance was similar to that observed under blue light.Abbreviations DAPI 4, 6-diamidino-2-phenylindole - ICM interphase cortical microtubules - PBS phosphate buffered saline - PPB preprophase band - MT microtubule     

F-actin redistributions at the division site in livingTradescantia stomatal complexes as revealed by microinjection of rhodamine-phalloidin

Summary Microinjection of rhodamine-phalloidin into living cells of isolatedTradescantia leaf epidermis and visualisation by confocal microscopy has extended previous results on the distribution of actin in mitotic cells of higher plants and revealed new aspects of actin arrays in stomatal cells and their initials. Divisions in the stomatal guard mother cells and unspecialised epidermal cells are symmetrical. Asymmetrical divisions occur in guard mother precursor cells and subsidiary mother cells. Each asymmetrical division is preceded by migration of the nucleus and the subsequent accumulation of thick bundles of anticlinally oriented actin filaments localised to the area of the anticlinal wall closest to the polarised nucleus. During prophase, in all cell types, a subset of cortical actin filaments coaligns to form a band, which, like the preprophase band of microtubules, accurately delineates the site of insertion of the future cell wall. Following the breakdown of the nuclear envelope, F-actin in these bands disassembles but persists elsewhere in the cell cortex. Thus, cortical F-actin marks the division site throughout mitosis, firstly as an appropriately positioned band and then by its localised depletion from the same region of the cell cortex. This sequence has been detected in all classes of division inTradescantia leaf epidermis, irrespective of whether the division is asymmetrical or symmetrical, or whether the cell is vacuolate or densely cytoplasmic. Taken together with earlier observations on stamen hair cells and root tip cells it may therefore be a general cytoskeletal feature of division in cells of higher plants.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band - Rh rhodamine - SMC subsidiary mother cell     

Microtubule reorganization accompanying preprophase band formation in guard mother cells ofAvena sativa L.

Summary In order to study developmental changes in microtubule organization attending the formation of a longitudinally oriented preprophase band, the guard mother cells ofAvena were examined using a new procedure for anti-tubulin immunocytochemistry on large epidermal segments. We found that the interphase band (IMB) of transverse cortical microtubules present in these cells following asymmetric division is replaced after subsidiary cell formation by mesh-like to radial microtubules that extend throughout the cytoplasm. Many of the Mts are also grouped in bundles. Gradually, this intermediate array is succeeded by longitudinal elements of the PPB. Thus, preprophase band formation is accompanied by a 90° shift in Mt orientation, with a radial arrangement serving as an intermediate stage. The micrographs are most consistent with the rearrangement of intact Mts, although changes in Mt assembly are possible as well. The role of the IMB in guard mother cells is also discussed.Abbreviations GMC guard mother cell - IMB interphase microtubule band - Mt microtubule - PPB preprophase band     

The electronmicroscopical study of the division of highly vacuolised grapevine callus cells

The ultrastructural aspects of the cell division in the grapevine(Vitis riparia × V.labrusca) calli were studied. A large central vacuole plays a noticeable part in this process. Before its division the nucleus with some encircling cytosol moves into the central vacuole where the small, round-shaped portion of cytosol (phragmosome) originates. In this central mass of cytosol connected with the peripheral one by thin cytosolic strands karyokinesis is carried out and the cell plate formation starts. Before karyokinesis the phragmosome, however, does not exhibit the form of the cytosolic layer completely traversing the cell. No preprophase band of microtubules has been observed in the cells either. The polarity of the mitotic spindle designating the orientation of the new cell wall is random then and it is not determined by the position of the preprophase band of microtubules or by the orientation of phragmosome. The unorganized growth of the grapevine callus reflects this fact.     

Pollen development in orchids 4. Cytoskeleton and ultrastructure of the unequal pollen mitosis inPhalaenopsis

Summary The unequal first mitosis in pollen ofPhalaenopsis results in a small generative cell cut off at the distal surface of the microspore and a large vegetative cell. No preprophase band of microtubules is present, but polarization of the microspore prior to this critical division is well marked. A generative pole microtubule system (GPMS) marks the path of nuclear migration to the distal surface, and the organelles become unequally distributed. Mitochondria, plastids and dictyosomes are concentrated around the vegetative pole in the center of the microspore and are almost totally excluded from the generative pole. The prophase spindle is multipolar with a dominant convergence center at the GPMS site. The metaphase spindle is disc-shaped with numerous minipoles terminating in broad polar regions. In anaphase, the spindle becomes cone-shaped as the spindle elongates and the vegetative pole narrows. These changes in spindle architecture are reflected in the initial shaping of the telophase chromosome groups. F-actin is coaligned with microtubules in the spindle and is also seen as a network in the cytoplasm. An outstanding feature of orchid pollen mitosis is the abundance of endoplasmic reticulum (ER) associated with the spindle. ER extends along the kinetochore fibers, and the numerous foci of spindle fibers at the broad poles terminate in a complex of ER.Abbreviations CLSM confocal laser scanning microscope/microscopy - DMSO dimethyl sulfoxide - ER endoplasmic reticulum - FITC fluorescein isothiocyanate - GPMS generative pole microtubule system - MBS m-maleimidobenzoic acidN-hydroxysuccinimide ester - PPB preprophase band of microtubules - RhPh rhodamine palloidin - TEM transmission electron microscope/microscopy     

Plastid polarity and meiotic spindle development in microsporogenesis ofSelaginella

Summary Microsporogenesis inSelaginella was studied by fluorescence light microscopy and transmission electron microscopy. As in other examples of monoplastidic meiosis the plastids are involved in determination of division polarity and organization of microtubules. However, there are important differences: (1) the meiotic spindle develops from a unique prophase microtubule system associated with two plastids rather than from a typical quadripolar microtubule system associated with four plastids; (2) the division axes for first and second meiotic division are established sequentially, whereas as in all other cases the poles of second division are established before those of first division; and (3) the plastids remain in close contact with the nucleus throughout meiotic prophase and provide clues to the early determination of spindle orientation. In early prophase the single plastid divides in the plane of the future division and the two daughter plastids rotate apart until they lie on opposite sides of the nucleus. The procytokinetic plate (PCP) forms in association with the two slender plastids; it consists of two spindle-shaped microtubule arrays focused on the plastid tips with a plate of vesicles at the equatorial region and a picket row of microtubules around one side of the nucleus. Second plastid division occurs just before metaphase and the daughter plastids remain together at the spindle poles during first meiotic division. The meiotic spindle develops from merger of the component arrays of the PCP and additional microtubules emanating from the pair of plastid tips located at the poles. After inframeiotic interphase the plastids migrate to tetrahedral arrangement where they serve as poles of second division.Abbreviations AMS axial microtubule system - FITC fluorescein isothiocyanate - MTOC microtubule organizing center - PCP procytokinetic plate - QMS quadripolar microtubule system - TEM transmission electron microscope (microscopy)     

Cell division in leaves ofNicotiana

Summary Young leaves ofNicotiana tabacum were fixed in glutaraldehyde-formaldehyde followed by osmium tetroxide. The fine structure of dividing cells was studied. Before prophase a band of microtubules was observed between the nucleus and the cell wall at a position judged as the future plane of division. The microtubules in the band are 4–6 units deep and relatively closely packed, giving sections of the band a characteristic appearance. Micro-tubules of the mitotic spindle, the phragmoplast, and the preprophase band are morphologically similar. Some of the microtubules of the mitotic spindle and the phragmoplast have an undulate appearance. It is suggested that the undulate microtubules may have been fixed at a time when microwaves were traveling along them. The cell plate is formed by a fusion of small smooth surfaced vesicles and small coated vesicles. Fusion of small vesicles results first in larger vesicles and then in a meshwork of new cell-wall material surrounded by new regions of plasma membrane. Most of the vesicles are derived from dictyosomes and may be produced before and during prophase as well as during later stages of division. The ER may also contribute some vesicles to the cell plate.     

Associations between membranes and microtubules during mitosis and cytokinesis in caulonema tip cells of the mossFunaria hygrometrica

Summary The interphase nucleus in theFunaria caulonema tip cells is associated with many non-cortical microtubules (Mts). In prophase, the cortical Mts disappear in the nuclear region; in contrast to moss leaflets, a preprophase band of Mts is not formed in the caulonema. The Mts of the early spindle are associated with the fragments of the nuclear envelope. Remnants of the nucleolus remain in the form of granular bodies till interphase. The metaphase chromosomes have distinct kinetochores; the kinetochore Mts are intermingled with non-kinetochore Mts running closely along the chromatin. Each kinetochore is associated with an ER cisterna. ER cisternae also accompany the spindle fibers in metaphase and anaphase. In telophase, Golgi vesicles accumulate in the periphery of the developing cell plate where no Mts are found. The reorientation of the cell plate into an oblique position can be inhibited by colchicine. It is concluded that the ER participates in controlling the Mt system, perhaps via calcium ions (membrane-bound calcium ions have been visualized by staining with chlorotetracycline) but that, on the other hand, the Mt system also influences the distribution of the ER. The occurrence and function of the preprophase band of Mts is discussed.     

Rearrangement of the actin filament and microtubule cytoskeleton during induction of microspore embryogenesis inBrassica napus L. cv. Topas

Summary Changes in the actin filament and microtubule cytoskeleton were examined during heat- and cytochalasin D-induced embryogenesis in microspores ofBrassica napus cv. Topas by rhodamine phalloidin and immunofluorescence labelling respectively. The nucleus was displaced from its peripheral to a more central position in the cell, and perinuclear actin microfilaments and microtubules extended onto the cytoplasm. Heat treatment induced the formation of a preprophase band of microtubules in microspores; preprophase bands are not associated with the first pollen mitosis. Actin filament association with the preprophase band was not observed. The orientation and position of the mitotic spindle were altered, and it was surrounded with randomly oriented microfilaments. The phragmoplast contained microfilaments and microtubules, as in pollen mitosis I, but it assumed a more central position. Cytoskeletal reorganisation also occurred in microspores subjected to a short cytochalasin D treatment, in the absence of a heat treatment. Cytochalasin D treatment of microspores resulted in dislocated mitotic spindles, disrupted phragmoplasts, and symmetric divisions and led to embryogenesis, confirming that a normal actin cytoskeleton has a role in preventing the induction of embryogenesis.Abbreviations CD cytochalasin D - MF actin microfilament - MT microtubule - PPB preprophase band     

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.Abbreviations GMC guard mother cell - MT microtubule - PPB preprophase band     

The presence of extended phragmosomes containing cytoskeletal elements in fusiform cambial cells ofFraxinus excelsior L.

Summary Fusiform cambial cells of the ash (Fraxinus excelsior L.), which are strongly elongated and vacuolated, contain a phragmosome which traverses the whole length of the cells during preprophase and karyokinesis and which remains present during cytokinesis until it is integrated in cell plate with adjacent cytoplasm.The phragmosome consists of a thin perforated cytoplasmic layer located in the plane of the future cell plate. Otherwise oriented transvacuolar cytoplasmic layers or strands are not present in these cells.The phragmosome contains cytoskeletal elements, namely microtubules and also microfilament bundles both of which are oriented mainly in longitudinal direction.The phragmosomal microtubules are a new category of microtubules associated with cell division; presumably they guide the centrifugally growing cell plate to the parental cell wall site previously marked by the preprophase band of microtubules.     

Presence and absence of the preprophase band of microtubules in moss protonemata: a clue to understanding its function?

Summary InFunaria protonemata, preprophase bands (PPBs) of microtubules do not develop when the tip cell divides, when side branches are initiated or in intercalary regeneration divisions. We report here that PPBs do, however, develop when a tmema cell is formed. In the former cases, cell division is not coupled with an expansion of the mother cell wall at the site where the cell plate will attach. In the latter case, the mother cell wall ruptures at that site and the tmema cell elongates. This observation and the findings on presence and absence of the PPB in other cell types indicate a connection between PPB occurrence and mother cell wall expansion. They support the idea that the PPB might be involved in the local secretion of cell wall material. We extend this notion, suggesting that the microtubules of the PPB control the oriented deposition of a thin layer of cellulose microfibrils at the mother cell wall which supports the firm attachment of the cell plate when the mother cell wall expands.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - MT microtubule - PPB preprophase band of microtubules - TC tmema cell     

Spatio-temporal relationship between nuclear-envelope breakdown and preprophase band disappearance in cultured tobacco cells

Cell division involves the coordinated progression of karyokinesis and cytokinesis, which is accomplished by communication between the nucleus and the cytoplasm. We have utilized green-fluorescent-protein technology to generate a line of tobacco 'Bright Yellow 2' (BY-2) cells labeled for both microtubules and the nuclear envelope. This cell line allowed us to use living cells to investigate the relationship between nuclear-envelope breakdown and preprophase band disappearance with high spatial and temporal resolution. Our observations demonstrate that nuclear-envelope breakdown always precedes preprophase band disappearance in BY-2 cells. In addition, the rate of preprophase band disappearance, and the attenuation of perinuclear microtubule fluorescence, correlates with the proximity of the nucleus to the preprophase band site. These results indicate the presence of communication between the nucleus and the preprophase band and suggest a causal relationship between nuclear-envelope breakdown and preprophase band disappearance.