Microtubule organization during development of stomatal complexes inLolium rigidum

Summary Microtubule (MT) arrays in stomatal complexes ofLolium have been studied using cryosectioning and immunofluorescence microscopy. This in situ analysis reveals that the arrangement of MTs in pairs of guard cells (GCs) or subsidiary cells (SCs) within a complex is very similar, indicating that MT deployment is closely coordinated during development. In premitotic guard mother cells (GMCs), MTs of the transverse interphase MT band (IMB) are reorganized into a longitudinal array via a transitory array in which the MTs appear to radiate from the cell edges towards the centre of the walls. Following the longitudinal division of GMCs, cortical MTs are reinstated in the GCs at the edge of the periclinal and ventral walls. The MTs become organized into arrays which radiate across the periclinal walls, initially from along the length of the ventral wall and later only from the pore site. As the GCs elongate, the organization of MTs and the patterns of wall expansion differ on the internal and external periclinal walls. A final reorientation of MTs from transverse to longitudinal is associated with the elongation and constriction of GCs to produce mature complexes. During cytokinesis in the subsidiary mother cells (SMCs), MTs appear around the reforming nucleus in the daughter epidermal cells but appear in the cortex of the SC once division is complete. Our results are thus consistent with the idea that interphase MTs are nucleated in the cell cortex in all cells of the stomatal complex but not in adjacent epidermal cells.Abbreviations GMC guard mother cell - GC guard cell - IMB interphase microtubule band - MT microtubule - PPB preprophase band - SMC subsidiary mother cell - SC subsidiary cell     

The effect of taxol on centrosome function and microtubule organization in apical cells of Sphacelaria rigidula (Phaeophyceae)

Treatment of interphase apical cells of Sphacelaria rigidula Kützing with 10 μmol L^?1 taxol for 4 h induced drastic changes in microtubule (MT) organization. In normal cells these MTs converge on the centrosomes and are nucleated from the pericentriolar area. After treatment, the endoplasmic, perinuclear and centrosome‐associated MT almost disappeared, and a massive assembly of cortical/subcortical, well‐organized MT bundles was observed. The bundles tended to be axially oriented, usually following the cylindrical wall, although other orientations were not excluded. The MTs in the apical part of the cell seemed to reach the cortex of the apical dome, sometimes bending to follow its curvature, whereas those in the basal portion of the cell terminated close to the transverse wall. Mitotic cells were also highly affected. Typical metaphase stages were very rarely found, and typical anaphase arrangements of chromosomes were completely absent. The chromosomes usually appeared to be dispersed singly or in small groups. Different atypical mitotic configurations were observed, depending on the stage of the cell cycle when the treatment started. The position and the orientation of the atypical mitotic spindles was disturbed. The nuclear envelope was completely disintegrated. The separation of the duplicated centrioles, as well as their usual perinuclear position, was also disturbed. Cortical MT bundles similar to those found in interphase cells were not found in the affected mitotic cells. In contrast, numerous MTs, without definite focal points, were found in the pericentriolar areas. Cytokinesis was inhibited by taxol treatment. The perinuclear and centrosome‐associated MTs found in mitotic cells were gradually replaced by a MT system similar to that of interphase cells. When the cytokinetic diaphragm had already been initiated when taxol treatment began, MTs were found on the cytokinetic plane, a phenomenon not observed in normal untreated cells. The results show clearly that: (i) in interphase cells the ability of centrosomes to nucleate MTs is intensely disturbed by taxol; (ii) centrosome dynamics in MT nucleation vary during the cell cycle; and (iii) taxol strongly affects mitosis and cytokinesis. In addition, it seems that the cortical/subcortical cytoplasm of interphase cells assumes the capacity to form numerous MT bundles.     

Relationship between microtubule orientation and patterns of cell expansion in developing cortical cells of Lemna minor roots

In actively growing cortical cells in the elongation zone of Lemna minor L. roots, both longitudinal (radial and tangential) and transverse walls expand in both length and width. The longitudinal walls of the three types of cortical cells in the root (i.e. outer, middle and inner) showed the largest expansion in the longitudinal axis. In contrast, the inner cortical cells exhibited the least expansion in width, whereas the middle cortical cells displayed the largest expansion in width. Thus, the profiles of the expansion of longitudinal walls were characteristic for the three types of cortical cells. In this study, both the orientation of cortical microtubule (MT) arrays and their dynamic reorientation, and the density of cortical MTs, were documented and correlated to the patterns of cell wall expansion. Significantly, transverse arrays of cortical MTs were most prominent in the radial walls of the inner cortical cells, and least so in those of the middle cortical cells. Toward the base of roots, beyond the elongation zone, the orientation of cortical MTs shifted continuously from transverse to oblique and then to longitudinal. In this case, the rate of shift in the orientation of cortical MTs along the root axis was appreciably faster in the middle cortical cells than in the other two types of cortical cells. Interestingly, the continuous change in cortical MT orientation was not confirmed in the transverse walls which showed much smaller two-dimensional expansion than the radial walls. Additionally, the presence of fragmented or shortened cortical MTs rapidly increased concomitantly with the decrease of transversely oriented cortical MTs. This relationship was especially prominent in the transverse walls of the inner cortical cells, which displayed the least expansion among the three types of cortical cells investigated. In the root elongation zone, the density of cortical MTs in the inner cortical cells was about three times higher than that in the other two cortical cell types. These results indicate that in the early stage of cell expansion, the orientation of cortical MTs determines a preferential direction of cell expansion and both the shifting orientation and density of cortical MTs affect the magnitude of expansion in width of the cell wall.     

Reorganization of the actin and microtubule cytoskeleton throughout blue-light-induced differentiation of characean protonemata into multicellular thalli

Summary The reorganization of the actin and microtubule (MT) cytoskeleton was immunocytochemically visualized by confocal laser scanning microscopy throughout the photomorphogenetic differentiation of tip-growing characean protonemata into multicellular green thalli. After irradiating dark-grown protonemata with blue or white light, decreasing rates of gravitropic tip-growth were accompanied by a series of events leading to the first cell division: the nucleus migrated towards the tip; MTs and plastids invaded the apical cytoplasm; the polar zonation of cytoplasmic organelles and the prominent actin patch at the cell tip disappeared and the tip-focused actin microfilaments (MFs) were reorganized into a homogeneous network. During prometaphase and metaphase, extranuclear spindle microtubules formed between the two spindle poles. Cytoplasmic MTs associated with the apical spindle pole decreased in number but did not disappear completely during mitosis. The basal cortical MTs represent a discrete MT population that is independent from the basal spindle poles and did not redistribute during mitosis and cytokinesis. Preprophase MT bands were never detected but cytokinesis was characterized by higher-plant-like phragmoplast MT arrays. Cytoplasmic actin MFs persisted as a dense network in the apical cytoplasm throughout the first cell division. They were not found in close contact with spindle MTs, but actin MFs were clearly coaligned along the MTs of the early phragmoplast. The later belt-like phragmoplast was completely depleted of MFs close to the time of cell plate fusion except for a few actin MF bundles that extended to the margin of the growing cell plate. The cell plate itself and young anticlinal cell walls showed strong actin immunofluorescence. After several anticlinal cell divisions, basal cells of the multicellular protonema produced nodal cell complexes by multiple periclinal divisions. The apical-dome cell of the new shoot which originated from a nodal cell becomes the meristem initial that regularly divides to produce a segment cell. The segment cell subsequently divides to produce a single file of alternating internodal cells and multicellular nodes which together form the complexly organized characean thallus. The actin and MT distribution of nodal cells resembles that of higherplant meristem cells, whereas the internodal cells exhibit a highly specialized cortical system of MTs and streaming-generating actin bundles, typical of highly vacuolated plant cells. The transformation from the asymmetric mitotic spindle of the polarized tip-growing protonema cell to the symmetric, higher-plant-like spindle of nodal thallus cells recapitulates the evolutionary steps from the more primitive organisms to higher plants.Abbreviations FITC fluorescein isothiocyanate - MF microfilament - MT microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline     

Microtubules and morphogenesis in stomata of the water fernAzolla: An unusual mode of guard cell and pore development

Summary A mature stomate of the water fernAzolla consists of a single apparently unspecialized annular guard cell (GC) with two nuclei surrounding an elongated pore aligned longitudinally in the leaf. During development, the guard mother cell develops a preprophase band (PPB) of microtubules (MTs) oriented transverse to the leaf axis. This is followed by a cell plate which fuses with the parental walls at the PPB site. Subsequently only the central part of the cell plate is consolidated, while the parts to either side become perforated and tenuous and may disperse completely, forming a single composite GC.Meanwhile, a dense array of MTs appears along both faces of the central part of the new wall, oriented normal to the leaf surface. Further MT arrays radiate out across the periclinal walls from the region of the consolidated cell plate. Putative MT nucleating sites are seen along the cell edges between these anticlinal and periclinal arrays. Polarized light microscopy reveals cellulose deposition parallel to the periclinal MT arrays. At the same time lamellar material is deposited within the new anticlinal wall. As the GC complex elongates, a split appears in these lamellae creating an initially transverse slit which then opens up to become first circular and ultimately an elongated pore aligned in the long axis of the leaf,i.e., at right angles to the wall in which it originated. The radiating pattern of cellulose microfibrils in the periclinal walls contributes to the shaping of the pore. Elongation at the apical and basal ends of the GC is restricted by longitudinal microfibril orientation, while that at the sides is facilitated by transverse alignment.     

Severing at sites of microtubule crossover contributes to microtubule alignment in cortical arrays

The cortical microtubule (MT) array and its organization is important in defining the growth axes of plant cells. In roots, the MT array exhibits a net-like configuration in the division zone, and a densely-packed transverse alignment in the elongation zone. This transition is essential for anisotropic cell expansion and consequently has been the subject of intense study. Cotyledons exhibit a net-like array in pavement cells and a predominantly aligned array in the petioles, and provide an excellent system for determining the basis of plant MT organization. We show that in both kinds of MT array, growing MTs frequently encounter existing MTs. Although some steep-angled encounters result in catastrophes, the most frequent outcome of these encounters is successful negotiation of the existing MT by the growing MT to form an MT crossover. Surprisingly, the outcome of such encounters is similar in both aligned and net-like arrays. In contrast, aligned arrays exhibit a much higher frequency of MT severing events compared with net-like arrays. Severing events occur almost exclusively at sites where MTs cross over one another. This process of severing at sites of MT crossover results in the removal of unaligned MTs, and is likely to form the basis for the difference between a net-like and an aligned MT array.     

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.Abbreviations DIC differential interference contrast - GC guard cell - MT microtubule To whom correspondence should be addressed.     

Reorientation of microtubules at the outer epidermal wall of maize coleoptiles by phytochrome,blue-light photoreceptor,and auxin

Summary The effects of red and blue light on the orientation of cortical microtubules (MTs) underneath the outer epidermal wall of maize (Zea mays L.) coleoptiles were investigated with immunofluorescent techniques. The epidermal cells of dark-grown coleoptiles demonstrated an irregular pattern of regions of parallel MTs with a random distribution of orientations. This pattern could be changed into a uniformly transverse MT alignment with respect to the long cell axis by 1 h of irradiation with red light. This response was transient as the MTs spontaneously shifted into a longitudinal orientation after 1–2 h of continued irradiation. Induction/reversion experiments with short red and far-red light pulses demonstrated the involvement of phytochrome in this response. In contrast to red light, irradiation with blue light induced a stable longitudinal MT alignment which was established within 10 min. The blue-light response could not be affected by subsequent irradiations with red or far-red light indicating the involvement of a separate blue-light photoreceptor which antagonizes the effect of phytochrome. In mixed light treatments with red and blue light, the blue-light photoreceptor always dominated over phytochrome which exhibited an apparently less stable influence on MT orientation. Long-term irradiations with red or blue light up to 6 h did not reveal any rhythmic changes of MT orientation that could be related to the rhythmicity of helicoidal cell-wall structure. Subapical segments isolated from dark-grown coleoptiles maintained a longitudinal MT arrangement even in red light indicating that the responsiveness to phytochrome was lost upon isolation. Conversely auxin induced a transverse MT arrangement in isolated segments even in blue light, indicating that the responsiveness to blue-light photoreceptor was eliminated by the hormone. These complex interactions are discussed in the context of current hypotheses on the functional significance of MT reorientations for cell development.Abbreviations MT cortical microtubule - P_r, P_fr red and far-red absorbing form of phytochrome     

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     

Growth and microtubule orientation of Zea mays roots subjected to osmotic stress

Previous work has shown that microtubule (MT) reorientation follows the onset of growth inhibition on the lower side of graviresponding roots, indicating that growth reduction can occur independently of MT reorientation. To test this observation further, we examined whether the reduction in growth in response to osmotic stress is correlated with MT reorientation. The distribution and rate of growth in maize roots exposed to 350 mOsm sorbitol and KCl or 5 mM Mes/Tris buffer were measured with a digitizer. After various times roots were processed for indirect immunofluorescence microscopy. Application of sorbitol or KCl had no effect on the organization of MTs in the apical 2 mm of the root but resulted in striking and different effects in the basal region of the root. Sorbitol treatment caused rapid appearance of oval to circular holes in the microtubular array that persisted for at least 9 h. Between 30 min and 4 h of submersion in KCl, MTs in cortical cells 4 mm and farther from the quiescent center began to reorient oblique to the longitudinal axis. After 9 h, the alignment of MTs had shifted to parallel to the root axis but MTs of the epidermal cells remained transverse. In KCl-treated roots MT reorientation appeared to follow a pattern of development similar to that in controls but without elongation. Our data provide additional evidence that MT reorientation is not the cause but a consequence of growth inhibition.     

Organization of cortical microtubules and microfibril deposition in response to blue-light-induced apical swelling in a tip-growing Adiantum protonema cell

The arrangements of cortical microtubules (MTs) in a tip-growing protonemal cell of Adiantum capillus-veneris L. and of cellulose microfibrils (MFs) in its wall were examined during blue-light (BL)-induced apical swelling. In most protonemal cells which had been growing in the longitudinal direction under red light, apical swelling was induced within 2 h of the onset of BL irradiation, and swelling continued for at least 8 h. During the longitudinal growth under red light, the arrangement of MFs around the base of the apical hemisphere (the subapical region) was perpendicular to the cell axis, while a random arrangement of MFs was found at the very tip, and a roughly axial arrangement was observed in the cylindrical region of most cells. This orientation of MFs corresponds to that of the cortical MTs reported previously (Murata et al. 1987, Protoplasma 141, 135–138). In cells irradiated with BL, a random rather than transverse arrangement of both MTs and MFs was found in the subapical region. Time-course studies showed that this reorientation occurred within 1 h after the onset of the BL irradiation, i.e. it preceded the change in growth pattern. These results indicate that the orientation of cortical MTs and of cellulose MFs is involved in the regulation of cell diameter in a tip-growing Adiantum protonemal cell.Abbreviations BL blue light - MF(s) microfibril(s) - MT(s) microtubule(s)     

Root contraction in hyacinth III. Orientation of cortical microtubules visualized by immunofluorescence microscopy

Summary Cortical microtubules (MTs) were visualized in root cortex cells ofHyacinthus orientalis L. using immunofluorescence techniques. Cellular MT orientation was determined adjacent to radial longitudinal and transverse walls of root tip, uncontracted, contracting, and fully contracted regions. As seen in longitudinal views, MTs formed parallel, apparently helical arrays which were oriented transversely, axially or obliquely depending upon the region. Transverse sectional views showed that MTs adjacent to transverse cell walls formed a variety of patterns which varied with developmental stage and cell location. Microtubules were oriented in crisscross or parallel arrays. The parallel arrays were oriented either parallel, perpendicular or oblique to the radius of the root. There was an apparent temporal progression in MT reorientation from outer cortical to inner cortical cell layers. A resultant progression of reoriented cell growth could account for root contraction. These findings corroborate earlier electron microscopic observations of changing MT orientation accompanying root contraction, and provide cytological evidence to test mathematical and biophysical models of the mechanics of cell expansion.Abbreviations MT microtubule - MF microfibril - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

The morphogenesis of lobed plant cells in the mesophyll and epidermis: organization and distinct roles of cortical microtubules and actin filaments

The morphogenesis of lobed plant cells has been considered to be controlled by microtubule (MT) and/or actin filament (AF) organization. In this article, a comprehensive mechanism is proposed, in which distinct roles are played by these cytoskeletal components. First, cortical MT bundles and, in the case of pavement cells, radial MT arrays combined with MT bundles determine the deposition of local cell wall thickenings, the cellulose microfibrils of which copy the orientation of underlying MTs. Cell growth is thus locally prevented and, consequently, lobes and constrictions are formed. Arch-like tangential expansion is locally imposed at the external periclinal wall of pavement cells by the radial arrangement of cellulose microfibrils at every wall thickening. Whenever further elongation of the original cell lobes occurs, AF patches assemble at the tips of growing lobes. Intercellular space formation is promoted or prevented by the opposite or alternate, respectively, arrangement of cortical MT arrays between neighboring cells. The genes that are possibly involved in the molecular regulation of the above morphogenetic procedure by MT and AF array organization are reviewed.     

Microtubule orientation and dynamics in elongating characean internodal cells following cytosolic acidification, induction of pH bands, or premature growth arrest

Summary Cortical microtubules (MTs) at indifferent zones in immatureNitella internodes were investigated by injection of fluorescently tagged sheep brain tubulin into living cells and by immunofluorescence on fixed material. Nearly identical MT patterns and numbers were detected with the two techniques, indicating that sheep brain tubulin incorporated into all cortical MTs. MTs were aligned transversely to the long axis of the cell and approximately one MT was present every micrometer of longitudinal cell distance. Treatment of internodes with propionic acid to acidify cytosolic pH caused depolymerization of MTs and an increase in the unpolymerized tubulin pool. Transfer of young, vigorously elongating cells to media inducing premature growth cessation resulted in a slight decrease in microtubule numbers but did not significantly alter microtubule orientation patterns or microtubule lifespans. MTs remained transverse for days following growth cessation before finally assuming a more random alignment characteristic of mature, non-growing internodes. No differences in MT numbers, orientation, or dynamics were detected between acid and alkaline bands in internodes incubated in a band-inducing medium. Thus, properties of cortical MT arrays were not closely coupled to growth status or to regional differences in cellular physiology associated with pH banding.Abbrevations BIM band-inducing medium - CCM Chara culture medium - CF carboxyfluorescein - FRAP fluorescence redistribution after photobleaching - MT microtubule     

Studies on the development of the air pores and air chambers ofMarchantia paleacea

Summary The preprophase-prophase initial aperture (IA) cells ofMarchantia paleacea undergo a particular sequence of protoplasmic changes, which reflects the establishment of an unusual premitotic polarization. The marking feature of preprophase-prophase thallus cells is the shape of the nucleus which becomes spindle-shaped. This phenomenon accompanies the organization of an extranuclear microtubule (MT) sheath, nucleated and/or organized by distinct polar MT organizing centres (MTOCs).The interphase MTs disappear after activation of polar MTOCs. In preprophase IA cells incomplete preprophase MT bands (PMBs) are organized. They consist of PMB portions which traverse only small portions of the cell cortex at the level of the future cytokinesis and do not form a complete ring. In the same cells other MT bundles, independent of the incomplete PMBs terminate in the cortical cytoplasm abutting on the lower part of the intercellular spaces (ISs) or the surface cavities (SCs). Almost complete or complete PMBs are organized in IA cells in which the plane of PMB formation coincides with that passing through ISs of the same growth.The observations suggest that in preprophase-prophase IA cells ofMarchantia paleacea cortical MTOCs function in regions distant from each other: One region is the PMB cortical cytoplasm, probably that covering the wall edges, and the other is the one adjacent to the lower part of the wall facing the IS(s) or that underlying the SCs. The competition between the cortical MTOCs as well as between them and the polar ones may be responsible for the organization of incomplete PMBs.     

The cyclic reorientation of cortical microtubules on walls with a crossed polylamellate structure: effects of plant hormones and an inhibitor of protein kinases on the progression of the cycle

Summary The outer tangential wall (OTW) of epidermal cells of azuki bean epicotyls has a crossed polylamellate structure, in which lamellae of longitudinal cellulose microfibrils alternate with lamellae of transverse cellulose microfibrils. This implies that the cyclic reorientation of cortical microtubules (MTs) from longitudinal to transverse and from transverse to longitudinal occurs on the OTW. Treatment with a solution that contained no auxin caused the accumulation of cells with longitudinal MTs, suggesting that auxin is required for the reorientation of MTs from longitudinal to transverse during the reorientation cycle. Treatment with 6-dimethylaminopurine (DMAP), an inhibitor of protein kinases that promoted the reorientation of MTs from transverse to longitudinal, resulted in the accumulation of cells with longitudinal MTs. Subsequent treatment with auxin caused a marked increase in the percentage of cells with transverse MTs and then a decrease in the percentage, indicating that the reorientation of MTs from longitudinal to transverse and then from transverse to longitudinal occurred during treatment with auxin. The percentage of cells with transverse MTs decreased more slowly in segments that had been pretreated with gibberellin A₃ (GA) than in segments that had been pretreated without GA, suggesting that GA, in cooperation with auxin, caused the suppression of the reorientation of MTs from transverse to longitudinal.Abbreviations BL brassinolide - BSA bovine serum albumin - GA gibberellin A₃ - DMAP 6-dimethylaminopurine - DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - IAA indoleacetic acid - MT microtubule - OTW outer tangential wall - PBS phosphate-buffered saline Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Reorganization of microfilaments in protonemal tip cells of the mossCeratodon purpureus during the phototropic response

Summary The F-actin distribution in caulonemal tip cells of the mossCeratodon purpureus was examined by rhodamine-phalloidin staining. Gravitropically-growing caulonemal tip cells of the moss possess a distinct alignment of microfilaments (MFs) in their apices. Axially oriented actin bundles run from subapical regions to the apex where they converge towards a central area of the tip, although bundles are absent from the central area itself thus forming a collar-like structure. During a unilateral red light irradiation the actin strands of the apical dome become reoriented towards the irradiated apical flank and still surround an area free of MFs, the point of prospective outgrowth. This process is closely correlated with the morphological effect of bulging and precedes the light-directed outgrowth. The collar structure is essential for the tubular growth form. In darkness, under the influence of antimicrotubule agents the structure is decomposed, the actin strands drift along the cell flanks and finally accumulate in randomly distributed areas where further growth takes place. The microtubules (MTs) are not involved in the phytochromemediated reorientation of the microfilaments. Unilateral red light suppresses the distorting effect of antimicrotubule drugs and restores the collar structure with a pronounced light-directed orientation. Instead, the MTs seem to be responsible for restricting the reorientation to the cell tip. This notion is based on the observation that the small area in the apical dome, which is normally the exclusive location of the light-regulated MF rearrangement, extends towards the cell base when MT inhibitors are applied before the unilateral red light irradiation. This in turn leads to a non-tubular expansion of the light-directed cell flank.Abbreviations DIG differential interference contrast - DMSO dimethyl sulfoxide - EGTA ethyleneglycol-bis-(beta-aminoethylether) N,N,N,N-tetraacetic acid - MF microfilament - MT microtubule - MTSB microtubule stabilizing buffer - MBS 3-maleimidobenzoic     

Cytoskeletal changes during resumption of tip growth in nongrowing protonemal cells of the fernAdiantum capillus-veneris L.

Summary Nongrowing, two-celled protonemata of the fernAdiantum capillus-veneris L. resume tip growth within the apical cell upon irradiation with red light. In this study, the phenomenon of growth resumption was analyzed with reference to changes in cytoskeletal organization. Continuous observations of apical cells with time lapse video-microscopy revealed that the nucleus migrated toward the tip ca. 1.9 h after the onset of red light, much earlier than the initiation of tip growth, which took place ca. 8.5 h after irradiation. Cytoskeletal organization was observed at various time points during growth resumption by fluorescent staining of microfilaments (MFs) and microtubules (MTs) with rhodamine-phalloidin and anti-tubulin antibodies. At 2 h after red-light irradiation, endoplasmic MF and MT strands appeared at the apical end of nucleus. These strands extended into the apical endoplasm, where filaments were rare prior to irradiation. Many fine filaments branched from the strands to the cell periphery, including the cortex of the apical-dome region. At this time, cortical circular arrays of MTs and MFs, normally found in the growing apex of protonemal cells, were absent. Both MT and MF circular arrays appeared during the resumption of tip growth concomitantly. The half-maximum appearance of MT and MF circular arrays within a population occurred at 5.4 h and 5.8 h after red-light irradiation, respectively. Thus, the process of red-light-induced resumption of tip growth in fern protonemal cell is composed of a series of events. These events include: (1) the appearance of strands extending from the nucleus toward the apical cortex and the migration of nucleus toward the apex; (2) the formation of circular MT and MF arrays at the sub-apical cortex; and (3) the initiation of cell growth at the apex. These results reflect the significant roles of MF and MT cytoskeleton in the resumption of tip growth.Abbreviations MBS m-maleimidobenzoic acid N-hydroxysuccinimide ester - MF microfilament - MT microtubule     

Microtubule organization in root cortical cells ofHyacinthus orientalis

Summary Overall cellular arrangement of cortical microtubules (MTs) is studied by reconstruction of MT images on serial thin sections. The mature root cortex ofHyacinthus orientalis L. cv. Delft blue is composed of elongate, highly vacuolate nondividing parenchyma cells. In longitudinal sections in these cells, MTs generally form parallel arrays at oblique angles to longitudinal cell axes. These MTs extend towards the transverse face of the cell where they appear in localized parallel arrays as well as in crisscross patterns. Repeated observations of oblique parallel arrays of MTs along the length of the cell and the continuity of MT bundles in serial sections suggest that MTs form a single helix in the cell. MTs in neighboring cells appear in sections either as parallel or as herringbone patterns, suggesting that the MT helices in these cells may spiral in the same or the opposite directions.Abbreviations MT Microtubule - MF microfibil - EM electron microscopy     

Organization of cortical microtubules in graviresponding maize roots

Immunofluorescence labeling of cortical microtubules (MTs) was used to investigate the relationship between MT arrangement and changes in growth rate of the upper and lower sides of horizontally placed roots of maize (Zea mays L. cv. Merit). Cap cells and cells of the elongation zone of roots grown vertically in light or darkness showed MT arrangements that were transverse (perpendicular) to the growth direction. Microtubules of cells basal to the elongation zone typically showed oblique orientation. Two hours after horizontal reorientation, cap cells of gravicompetent, light-grown and curving roots contained MTs parallel to the gravity vector. The MT arrangement on the upper side of the elongation zone remained transverse but the MTs of the outer four to five layers of cortical cells along the lower side of the elongation zone showed reorientation parallel to the axis of the root. The MTs of the lower epidermis retained their transverse orientation. Dark-grown roots did not curve and did not show reorientation of MTs in cells of the root cap or elongation zone. The data indicate that MT depolymerization and reorientation is correlated with reduction in growth rate, and that MT reorientation is one of the steps of growth control of graviresponding roots.Abbreviations MT microtubule - QC quiescent center This work was supported by National Science Foundation grant IBN-9118094.