A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.     

Taxol maintains organized microtubule patterns in protoplasts which lead to the resynthesis of organized cell wall microfibrils

Summary We have investigated the effects of microtubule stabilizing conditions upon microtubule patterns in protoplasts and developed a new method for producing protoplasts which have non-random cortical microtubule arrays. Segments of elongating pea epicotyl tissue were treated with the microtubule stabilizing drug taxol for 1 h before enzymatic digestion of the cell walls in the presence of the drug. Anti-tubulin immunofluorescence showed that 40 M taxol preserved regions of ordered microtubules. The microtubules in these regions were arranged in parallel arrays, although the arrays did not always show the transverse orientation seen in the intact tissue. Protoplasts prepared without taxol had microtubules which were random in distribution. Addition of taxol to protoplasts with random microtubule arrangements did not result in organized microtubule arrays. Taxol-treated protoplasts were used to determine whether or not organized microtubule arrays would affect the organization of cell wall microfibrils as new walls were regenerated. We found that protoplasts from taxol-treated tissue which were allowed to regenerate cell walls produced organized arrays of microfibrils whose patterns matched those of the underlying microtubules. Protoplasts from untreated tissue synthesized microfibrils which were disordered. The synthesis of organized microfibrils by protoplasts with ordered microtubules arrays shows that microtubule arrangements in protoplasts influence the arrangement of newly synthesized microfibrils.Abbreviations DIC differential interference contrast - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - IgG immunoglobulin G - PIPES piperazine-N,N-bis[2-ethane-sulfonic acid] - PBS phosphate buffered saline     

Orientation of cortical microtubules correlates with cell shape and division direction

Summary Cortical microtubules in the epidermis of regeneratingGraptopetalum plants were examined by in situ immunofluorescence. Paradermal slices of tissue were prepared by a method that preserves microtubule arrays and also maintains cell junctions. To test the hypothesis that cortical microtubule arrays align perpendicular to the direction of organ growth, arrays were visualized and their orientation quantified. A majority of microtubules are in transverse orientation with respect to the organ axis early in shoot development when the growth habit is uniform. Later in development, when growth habit is non-uniform and the tissue is contoured, cortical microtubules are increasingly longitudinal and oblique in orientation. Microtubules show only a minor change in orientation at the site of greatest curvature, the transition zone of a developing leaf. To assess the role of the division plane on orientation of arrays, the pattern of microtubules was examined in individual cells of common shape. Cells derived from transverse divisions have predominately transverse cortical arrays, whereas cells derived from oblique and longitudinal divisions have non-transverse arrays. The results show that, regardless of the stage of development, microtubules orient with respect to cell shape and plane of division. The results suggest that cytoskeletal function is best considered in small domains of growth within an organ.Abbrevations DMSO dimethylsulfoxide - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetra acetic acid - FITC fluorescein isothiocyanate - MTSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Cellulose microfibril alignment recovers from DCB-induced disruption despite microtubule disorganization

Cellulose microfibril deposition patterns define the direction of plant cell expansion. To better understand how microfibril alignment is controlled, we examined microfibril orientation during cortical microtubule disruption using the temperature-sensitive mutant of Arabidopsis thaliana, mor1-1. In a previous study, it was shown that at restrictive temperature for mor1-1, cortical microtubules lose transverse orientation and cells lose growth anisotropy without any change in the parallel arrangement of cellulose microfibrils. In this study, we investigated whether a pre-existing template of well-ordered microfibrils or the presence of well-organized cortical microtubules was essential for the cell to resume deposition of parallel microfibrils. We first transiently disrupted the parallel order of microfibrils in mor1-1 using a brief treatment with the cellulose synthesis inhibitor 2,6-dichlorobenzonitrile (DCB). We then analysed the alignment of recently deposited cellulose microfibrils (by field emission scanning electron microscopy) as cellulose synthesis recovered and microtubules remained disrupted at the mor1-1 mutant's non-permissive culture temperature. Despite the disordered cortical microtubules and an initially randomized wall texture, new cellulose microfibrils were deposited with parallel, transverse orientation. These results show that transverse cellulose microfibril deposition requires neither accurately transverse cortical microtubules nor a pre-existing template of well-ordered microfibrils. We also demonstrated that DCB treatments reduced the ability of cortical microtubules to form transverse arrays, supporting a role for cellulose microfibrils in influencing cortical microtubule organization.     

Reorganization of cortical microtubules and cellulose deposition during leaf formation in Graptopetalum paraguayense

In the regeneration of a shoot from a leaf of the succulent, Graptopetalum paraguayense E. Walther the first new organs are leaf primordia. The original arrangement of cellulose microfibrils and of microtubules (MTs) in the epidermis of the leaf-forming site is one of parallel, straight lines. In the new primordium both structures still have a congruent arrangement but it is roughly in the form of concentric circles that surround the new cylindrical organ. The regions which undergo the greatest shift in orientation (90°) were studied in detail. Departures from the original cellulose alignment are detected in changes in the polarized-light image. Departures from the original cortical MT arrangement are detected using electron microscopy. The over-all reorganization of the MT pattern is followed by the tally of MT profiles, the various regions being studied in two perpendicular planes of section. This corrects for the difference in efficiency in counting transverse versus longitudinal profiles of MTs. Reorientation takes place sporadically, cell by cell, for both the cellulose microfibrils and the MTs, indicating a coordinated reorientation of the two structures. That MTs and cellulose microfibrils reorient jointly in individual cells was shown by reconstruction of the arrays of cortical MTs in paradermal sections of individual cells whose recent change in the orientation of cellulose deposition had been detected with polarized light. Closeness of the two alignments was also indicated by images where the MT and microfibril alignments co-varied within a single cell. The change-over in alignment of the MTs appears to involve stages where arrays of contrasting orientation co-exist to give a criss-cross image. During this critical reorganization, the frequency of the MTs is high. It falls during subsequent enlargement of the organ. It was found that the rearrangement of the cortical MTs to approximate a series of concentric circles on the residual meristem occurred before the emergence of leaf primordia. Through their apparent influence on microfibril alignments, the changes in MT disposition, described here, have the potential to generate major biophysical changes that accompany organogenesis.Abbreviation MT(s) microtubule(s)     

BOTERO1 is required for normal orientation of cortical microtubules and anisotropic cell expansion in Arabidopsis

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.     

Ultrastructural effects of the herbicide chlorpropham (CIPC) in root tip cells of wheat

The present ultrastructural investigation on the effects of 50 M chlorpropham (previously called CIPC) on growing roots of wheat (Triticum aestivum (L.) Thell cv. Vergina) was undertaken to clarify the mechanism of a carbamate herbicide action in plant cells, since the wide range of responses of plant cells to carbamate herbicides is based mainly on immunofluorescence studies. Cells of control roots contained abundant microtubules both in interphase and mitotic arrays. In chlorpropham-treated roots, however, no microtubules could be detected at all, neither in dividing nor in differentiating cells. Cycling cells became binucleate, polyploid or contained incomplete cell walls, the result of inhibition of cytokinesis. In long-term drug treatments (24 h or more) the affected cells entered a new cycle, which, however, did not progress beyond mid-metaphase. The nuclei of binucleate cells initiated prophase synchronously. Small vacuoles and Golgi vesicles were trapped within the nucleoplasm of the multilobed nuclei. In roots recovering from 8 h chlorpropham treatment, cells continued to exhibit polyploid nuclei, intranuclear vacuoles and incomplete walls. Microtubules reappeared but they were sparse and lacked a definite orientation. Preprophase cells did not form normal preprophase bands of microtubules, while mitotic cells occasionally contained microtubules bound to chromosomes and converged to minipoles. It is concluded that chlorpropham disorganized directly microtubules in addition to irreversibly affecting microtubule organizing centres, which failed to further support microtubule arrays.     

The effects of microtubule and microfilament disrupting agents on cytoskeletal arrays and wall deposition in developing cotton fibers

Summary The effects of various cytoskeletal disrupting agents (cholchicine, oryzalin, trifluralin, taxol, cytochalasins B and D) on microtubules, microfilaments and wall microfibril deposition were monitored in developing cotton fibers, using immunocytochemical and fluorescence techniques. Treatment with 10^–4 M colchicine, 10^–6 M trifluralin or 10^–6 M oryzalin resulted in a reduction in the number of microtubules, however, the drug-stable microtubules still appear to influence wall deposition. Treatment with 10^–5 M taxol increased the numbers of microtubules present within 15 minutes of application. New microtubules were aligned parallel to the existing ones, however, some evidence of random arrays was observed. Microtubules stabilized with taxol appeared to function in wall organization but do not undergo normal re-orientations during development. Microtubule disrupting agent had no detectable affect on the microfilament population. Exposure to either 4×10^–5 M cytochalasin B or 2×10^–6M cytochalasin D resulted in a disruption of microfilaments and a re-organization of microtubule arrays. Treatment with either cytochalasin caused a premature shift in the orientation of microtubules in young fibers, whereas in older fibers the microtubule arrays became randomly organized. These observations indicate that microtubule populations during interphase are heterogeneous, differing at least in their susceptibility to disruption by depolymerizing agents. Changes in microtubule orientation (induced by cytochalasin) indicate that microfilaments may be involved in regulating microtubule orientation during development.     

Mutation or drug-dependent microtubule disruption causes radial swelling without altering parallel cellulose microfibril deposition in Arabidopsis root cells

As critical determinants of growth anisotropy in plants, cortical microtubules are thought to constrain the movement of cellulose synthase complexes and thus align newly deposited cellulose microfibrils. We tested this cellulose synthase constraint model using the temperature-sensitive mor1-1 mutant of Arabidopsis. Contrary to predictions, the disruption of cortical microtubules in mor1-1 root epidermal cells led to left-handed root twisting and radial swelling but did not alter the transverse orientation of cellulose microfibrils. We also found that drug-dependent disassembly or hyperstabilization of cortical microtubules did not alter the parallel order of cellulose microfibrils. By measuring cellulose content in mor1-1 seedlings, we verified that cellulose synthesis is not reduced at the restrictive temperature. The independence of cortical microtubule organization and cellulose microfibril alignment was supported by the observation that double mutants of mor1-1 and rsw1-1, the cellulose-deficient mutant with misaligned microfibrils, had additive phenotypes. Our results suggest that cortical microtubules regulate growth anisotropy by some mechanism other than cellulose microfibril alignment or synthesis.     

The missing link: do cortical microtubules define plasma membrane nanodomains that modulate cellulose biosynthesis?

Cellulose production is a crucial aspect of plant growth and development. It is functionally linked to cortical microtubules, which self-organize into highly ordered arrays often situated in close proximity to plasma membrane-bound cellulose synthase complexes (CSCs). Although most models put forward to explain the microtubule–cellulose relationship have considered mechanisms by which cortical microtubule arrays influence the orientation of cellulose microfibrils, little attention has been paid to how microtubules affect the physicochemical properties of cellulose. A recent study using the model system Arabidopsis, however, indicates that microtubules can modulate the crystalline and amorphous content of cellulose microfibrils. Microtubules are required during rapid growth for reducing crystalline content, which is predicted to increase the degree to which cellulose is tethered by hemicellulosic polysaccharides. Such tethering is, in turn, critical for maintaining unidirectional cell expansion. In this article, we hypothesize that cortical microtubules influence the crystalline content of cellulose either by controlling plasma membrane fluidity or by modulating the deposition of noncellulosic wall components in the vicinity of the CSCs. We discuss the current limitations of imaging technology to address these hypotheses and identify the image acquisition and processing strategies that will integrate live imaging with super resolution three-dimensional information.     

Cortical Microtubule Arrays Are Initiated from a Nonrandom Prepattern Driven by Atypical Microtubule Initiation

The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-γ-tubulin complex protein2-tagged γ-nucleation complexes (γ-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving γ-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.Higher plant cells feature ordered arrays of microtubules at the cell cortex (Ledbetter and Porter, 1963) that are essential for cell and tissue morphogenesis, as revealed by disruption of cortical arrays by drugs that cause microtubule depolymerization (Green, 1962) or stabilization (Weerdenburg and Seagull, 1988) and by loss-of-function mutations in a wide variety of microtubule-associated proteins (Baskin, 2001; Whittington et al., 2001; Buschmann and Lloyd, 2008; Lucas et al., 2011). The structure of these arrays is thought to control the pattern of cell growth primarily by its role in the deposition of cellulose microfibrils, the load-bearing component of the cell wall (Somerville, 2006). Functional relations between cortical microtubules and cellulose microfibrils have been proposed since the early sixties, even before cortical microtubules had been visualized (Green, 1962). Recent live-cell imaging studies have confirmed that cortical microtubules indeed guide the movement of cellulose synthase complexes that produce cellulose microfibrils (Paredez et al., 2006) and have shown further that microtubules position the insertion of most cellulose synthase complexes into the plasma membrane (Gutierrez et al., 2009). These activities of ordered cortical microtubules are proposed to facilitate the organization of cell wall structure, creating material properties that underlie cell growth anisotropy.While organization of the interphase cortical array appears to be essential for cell morphogenesis, this organization is disrupted during the cell cycle as microtubules are rearranged to create the preprophase band, spindle, and phragmoplast during mitosis and cytokinesis (for review, see Wasteneys, 2002). Upon completion of cytokinesis, an organized interphase cortical array is regenerated, but the pathway for this reassembly is not well understood.The plant interphase microtubule array is organized and maintained without centrosomes as organizing centers (for review, see Wasteneys, 2002; Bartolini and Gundersen, 2006; Ehrhardt and Shaw, 2006), and microtubule self-organization is proposed to play an important role in cortical microtubule array ordering (Dixit and Cyr, 2004). In electron micrographs, microtubules have been observed to be closely associated with the plasma membrane (Hardham and Gunning, 1978), and live-cell imaging provides evidence for attachment of microtubules to the cell cortex (Shaw et al., 2003; Vos et al., 2004). The close association to the plasma membrane restricts the cortical microtubules to a quasi two-dimensional plane where they interact through polymerization-driven collisions (Shaw et al., 2003; Dixit and Cyr, 2004). Microtubule encounters at shallow angles (<40°) have a high probability of leading to bundling, while microtubule encounters at steeper angles most likely result in induced catastrophes or microtubule crossovers (Dixit and Cyr, 2004). Several computational modeling studies have since shown that these types of interactions between surface-bound dynamical microtubules can indeed explain spontaneous coalignment of microtubules (Allard et al., 2010; Eren et al., 2010; Hawkins et al., 2010; Tindemans et al., 2010).The question of how the orientation of the cortical array is established with respect to the cell axis is less well understood. One possibility is that microtubules are selectively destabilized with respect to cellular coordinates (Ehrhardt and Shaw, 2006). Indeed, recent results from biological observations and modeling suggest that catastrophic collisions induced at the edges between cell faces or heighted catastrophe rates in cell caps could be sufficient to selectively favor microtubules in certain orientation and hence determine the final orientation of the array (Allard et al., 2010; Eren et al., 2010; Ambrose et al., 2011; Dhonukshe et al., 2012).To date, all models of cortical array assembly assume random initial conditions. However, experimental work by Wasteneys and Williamson (1989a, 1989b) in Nitella tasmanica showed that, during array reassembly after drug-induced disruption, microtubules were initially transverse. This was followed by a less ordered phase and later by the acquisition of the final transverse order. A nonrandom initial ordering was also observed in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells by Kumagai et al. (2001), who concluded that the process of transverse array establishment starts with longitudinal order but did not provide quantitative data for the process of array assembly. The initial conditions for the cortical microtubule array formation are important to consider, as they may strongly influence the speed at which order is established and could even prevent it from being established over a biologically relevant time scale.In this study, we used live-cell imaging to follow and record the whole transition from the cortical microtubule-free state to the final transverse array and used digital tracking algorithms to quantify the microtubule order. Nucleation stands out as a central process to characterize during array initiation. Lacking a central body to organize microtubule nucleations, the higher plant cell has dispersed nucleation complexes (Wasteneys and Williamson, 1989a, 1989b; Chan et al., 2003; Shaw et al., 2003; Murata et al., 2005; Pastuglia et al., 2006; Nakamura et al., 2010). Therefore, we performed high time resolution observations to quantify nucleation complex recruitment, nucleation rates, and microtubule nucleation angles. We found evidence for a highly nonrandom initial ordering state that features diagonal microtubule orientation and an atypical microtubule initiation mechanism. Simulation analysis indicates that these atypical nucleations have the potential to accelerate the recovery of cortical array density.     

Microfibrillar Structure, Cortical Microtubule Arrangement and the Effect of Amiprophos-methyl on Microfibril Orientation in the Thallus Cells of the Filamentous Green Alga, Chamaedoris orientalis

Microfibrillar structure, cortical microtubule orientation andthe effect of amiprophos-methyl (APM) on the arrangement ofthe most recently deposited cellulose microfibrils were investigatedin the marine filamentous green alga, Chamaedoris orientalis.The thallus cells of Chamaedoris showed typical tip growth.The orientation of microfibrils in the thick cell wall showedorderly change in longitudinal, transverse and oblique directionsin a polar dependent manner. Microtubules run parallel to thelongitudinally arranged microfibrils in the innermost layerof the wall but they are never parallel to either transverseor obliquely arranged microfibrils. The ordered change in microfibrilorientation is altered by the disruption of the microtubuleswith APM. The walls, deposited in the absence of the microtubules,showed typical helicoidal pattern. However, the original crossedpolylamellate pattern was restored by the removal of APM. Thissuggests that cortical microtubules in this alga do not controlthe direction of microfibril orientation but control the orderedchange of microfibril orientation. Amiprophos-methyl, Chamaedoris orientalis, coenocytic green alga, cortical microtubule, microfibrillar structure, tip growth     

Colchicine and microfibril orientation

Summary Investigations on the mechanism of orientation of the cellulose microfibrils of the green algaOocystis solitaria have been carried out. This organism demonstrates easily observable and highly ordered microfibrils in its wall, which are arranged parallel to one another and regularly alternate at 90 from layer to layer of which there are approximately 30. During the entire wall development, and always parallel to one of the microfibril directions, are microtubules lying in the cortical cytoplasm. In the presence of 10^–2 M colchicine, microtubules are no longer detected and the typical cell wall pattern is not developed. The possible role of microtubules in the orientation of cellulose microfibrils is briefly discussed.     

Structure of cortical microtubule arrays in plant cells

Serial sectioning was used to track the position and measure the lengths of cortical microtubules in glutaraldehyde-osmium tetroxide-fixed root tip cells. Microtubules lying against the longitudinal walls during interphase, those overlying developing xylem thickenings, and those in pre-prophase bands are oriented circumferentially but on average are only about one-eighth of the cell circumference in length, i.e., 2-4 micrometer. The arrays consist of overlapping component microtubules, interconnected by cross bridges where they are grouped and also connected to the plasma membrane. Microtubule lengths vary greatly in any given array, but the probability that any pass right around the cell is extremely low. The majority of the microtubule terminations lie in statistically random positions in the arrays, but nonrandomness in the form of groups of terminations and terminations in short lines parallel to the axis of cell elongation has been observed. Low temperature induces microtubule shortening and increases the frequency of C-shaped terminations over the 1.7% found under normal conditions; colchicine and high pressures produce abnormally large proportions of very short microtubules amongst those that survive the treatments. Deuterium oxide (D2O) treatment probably induces the formation of additional microtubules as distinct from increasing the length of those already present. The distribution of C-shaped terminations provides evidence for at least local polarity in the arrays. The validity of the findings is discussed, along with implications for the development, maintenance, and orientation of the arrays and their possible relationship to the orientation of cellulose deposition.     

Electron microscopy of mitosis in amebae. 3. Cold and urea treatments: a basis for tests of direct effects of mitotic inhibitors on microtubule formation

The mitotic apparatus (MA) of the giant ameba, Chaos carolinensis, has characteristic sequences of microtubule arrays and deployment of nuclear envelope fragments. If mitotic organisms are subjected to 2°C for 5 min, the MA microtubules are completely degraded, and the envelope fragments are released from the chromosomes which remain condensed but lose their metaphase-plate orientation. On warming, microtubules reform but show partial loss of their parallel alignment; displacement of the envelope fragments persists or is increased by microtubule reformation. This study demonstrates that cooling causes destruction of microtubules and intermicrotubular cross-bonds and further shows that such controlled dissolution and reformation can provide an in vivo test sequence for studies on the effects of inhibitor-compounds on microtubule subunit aggregation. Urea, at the comparatively low concentration of 0.8 M, inhibited reformation following cooling and rewarming but was ineffective in altering microtubules that had formed before treatment.     

Electric fields affect the orientation of cortical microtubules and cell expansion in pea callus

Summary Cortical microtubules in callus derived fromPisum sativum roots form parallel arrays within cells but are randomly oriented across the tissue. These arrays align perpendicular to the direction of an applied electric field of 6 mV per cell. Application of a field of 6 mV per cell for 4 days resulted in the co-ordinated expansion of cells parallel to the field direction. Cortical microtubule arrays were still aligned perpendicular to the applied field 24 h after removal of the field. The imposition of a field to callus after the removal of cortical microtubules by oryzalin and in the presence of the herbicide resulted in the orientation of recovering microtubules perpendicular to the direction of the field, indicating that microtubules are not directly involved in the detection of the field.Abbreviations EGTA ethylene glycol-bis (-aminoethyl ether) N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule stabilising buffer - PIPES piperazine-N,N-bis(2-ethanesulphonic acid) - oryzalin 3,5-dinitro-N⁴,N⁴ dipropylsulphanil-amide     

Cell proliferation,cell shape,and microtubule and cellulose microfibril organization of tobacco BY-2 cells are not altered by exposure to near weightlessness in space

The microtubule cytoskeleton and the cell wall both play key roles in plant cell growth and division, determining the plant’s final stature. At near weightlessness, tubulin polymerizes into microtubules in vitro, but these microtubules do not self-organize in the ordered patterns observed at 1g. Likewise, at near weightlessness cortical microtubules in protoplasts have difficulty organizing into parallel arrays, which are required for proper plant cell elongation. However, intact plants do grow in space and therefore should have a normally functioning microtubule cytoskeleton. Since the main difference between protoplasts and plant cells in a tissue is the presence of a cell wall, we studied single, but walled, tobacco BY-2 suspension-cultured cells during an 8-day space-flight experiment on board of the Soyuz capsule and the International Space Station during the 12S mission (March–April 2006). We show that the cortical microtubule density, ordering and orientation in isolated walled plant cells are unaffected by near weightlessness, as are the orientation of the cellulose microfibrils, cell proliferation, and cell shape. Likely, tissue organization is not essential for the organization of these structures in space. When combined with the fact that many recovering protoplasts have an aberrant cortical microtubule cytoskeleton, the results suggest a role for the cell wall, or its production machinery, in structuring the microtubule cytoskeleton.     

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     

Is the recovery of microtubule orientation in pea roots dependent on the cell wall?

This study tested several aspects of a model proposed by Williamson (1990, 1991) in which stresses in plant cell walls, detected by stress-receptive portions of inelastic cellulose microfibrils, orient microtubules via interactions with cell wall-linked transmembrane proteins. Young expanding cells of pea root tips have highly ordered transverse arrays of microtubules oriented perpendicular to the direction of cell expansion. The recovery of these ordered MT arrays after depolymerisation with oryzalin was assessed. It was shown that treating roots with disruptors of microfibril synthesis (2,6-dichlorobenzonitrile and calcofluor white) or the disruption of Arg-Gly-Asp (RGD)-mediated wall-membrane links did not affect the orientation of recovering microtubule arrays. Furthermore, cell wall stresses themselves appeared unnecessary for regeneration of transverse arrays. The relevance of these findings to Williamson's hypothesis is discussed.     

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