Dynamics of microtubule reassembly and reorganization in the coenocytic green alga Ernodesmis verticillata (Kützing) Børgesen

The dynamics of microtubule (MT) disassembly and reassembly were studied in the green alga Ernodesmis verticillata, using indirect immunofluorescent localization of tubulin. This alga possesses two distinct MT arrays: highly-ordered, longitudinally-oriented cortical MTs, and shorter perinuclear MTs radiating from nuclear surfaces. Perinuclear MTs are very labile, completely disassembling in the cold (cells on ice) within 5–10 min or in 25 μM amiprophos-methyl (APM) within 15–30 min. Although cortical MTs are generally absent after 3 h in APM, it takes 45–60 min before any cold-induced depolymerization is apparent, and some cortical MTs persist after 6 h of cold treatment. The extent of immunofluorescence of cytoplasmic (depolymerized?) tubulin is inversely proportional to the abundance of cortical MTs. Recovery of MT arrays upon warming or upon removal of APM occurs within 30–60 min for the perinuclear MTs, but the cortical arrays take much longer to regain their normal patterns. The cortical MTs initially reappear in a random distribution with respect to the cell axis, but within 3–4 d of warming (or 24–36 h of removing APM) they are nearly parallel to each other and to the cell's longitudinal axis. Thus, although the timing differs, the actual patterns of depolymerization and recovery are similar, irrespective of whether physical or chemical agents are used. Longer-term treatments in 1 μM APM indicate that despite the rapid disappearance of perinuclear MTs, a loss of the uniform nuclear spacing occurs gradually over 1–6 d. Similar disorganization of nuclei is obtained with long-term treatment with 1 μM taxol, where a gradual loss of perinuclear MTs is accompanied by an increased abundance of mitotic spindles. This implies that perinuclear MTs can disassemble in vivo in the presence of taxol, and that they are not the sole components involved in maintaining nuclear spacing in these coenocytes. The results indicate that both nuclear and cortical sites of MT nucleation may exist in this organism, and that MT reassembly and re-organization are temporally distinct events in cells that have highly-ordered arrays of long MTs.     

Microtubule cytoskeleton in intact and wounded coenocytic green algae

Microtubule (MT) arrangements were investigated, with immunofluorescence and electron microscopy, in two related species of coenocytic green algae. Intact cells of both Ernodesmis verticillata (Kützing) Boergesen and Boergesenia forbesii (Harvey) Feldmann have two morphologically distinct populations of MTs: a highly regular cortical array consisting of a single layer of parallel, longitudinal MTs; and perinuclear MTs radiating from the surface of the envelope of each interphase nucleus. In both algae, mitotic figures lack perinuclear MTs around them. Pre-incubation with taxol does not alter the appearance of these arrays. The cortical and nuclear MTs appear to coexist throughout the nuclear cycle, unlike the condition in most plant cells. At the cut/contracting ends of wounded Ernodesmis cells, cortical MTs exhibit bundling and marked convolution, with some curvature and slight bundling of MTs throughout the cell cortices. In Boergesenia, wound-induced reticulation and separation of the protoplasm into numerous spheres also involves a fasciation of MTs within the attenuating regions of the cytoplasm. Although some cortical MTs are fairly resistant to cold and amiprophos-methyl-induced depolymerization, the perinuclear ones are very labile, depolymerizing in 5–10 min in the cold. The MT cytoskeleton is not believed to be directly involved in wound-induced motility in these plants because amiprophos-methyl and cold depolymerize most cortical MTs without inhibiting motility. Also, the identical MT distributions in intact cells of these two algae belie the very different patterns of cytoplasmic motility. Although certain roles of the MT arrays may be ruled out, their exact functions in these plants are not known.Abbreviations APM amiprophos-methyl - DIC differential interference contrast - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MT(s) microtubule(s) - PBS phosphate-buffered saline     

The microtubule cytoskeleton does not integrate auxin transport and gravitropism in maize roots

The Cholodny-Went hypothesis of gravitropism suggests that the graviresponse is controlled by the distribution of auxin. However, the mechanism of auxin transport during the graviresponse of roots is still unresolved. To determine whether the microtubule (MT) cytoskeleton is participating in auxin transport, the cytoskeleton was examined and the movement of 3 H-IAA measured in intact and excised taxol, oryzalin, and naphthylphthalamic acid (NPA)-treated roots of Zea mays cv. Merit. Taxol and oryzalin did not inhibit the graviresponse of roots but the auxin transport inhibitor NPA greatly inhibited both auxin transport and graviresponse. NPA had no effect on MT organization in vertical roots, but caused MT reorientation in horizontally placed roots. Regardless of treatment, the organization of MTs in intact roots differed from that in root segments. The MT inhibitors, taxol and oryzalin had opposite effects on the MTs, namely, depolymerization (oryzalin) and stabilization and thickening (taxol), but both treatments caused swelling of the roots. The data indicate that the MT cytoskeleton does not directly interfere with auxin transport or auxin-mediated growth responses in maize roots.     

Complete disintegration of the microtubular cytoskeleton precedes its auxin-mediated reconstruction in postmitotic maize root cells

The inhibitory action of 0.1 microM auxin (IAA) on maize root growth was closely associated with a rapid and complete disintegration of the microtubular (MT) cytoskeleton, as visualized by indirect immunofluorescence of tubulin, throughout the growth region. After 30 min of this treatment, only fluorescent spots were present in root cells, accumulating either around nuclei or along cell walls. Six h later, in addition to some background fluorescence, dense but partially oriented oblique or longitudinal arrays of cortical MTs (CMTs) were found in most growing cells of the root apex. After 24 h of treatment, maize roots had adapted to the auxin, as inferred from the slowly recovering elongation rate and from the reassembly of a dense and well-ordered MT cytoskeleton which showed only slight deviations from that of the control root cells. Taxol pretreatment (100 microM, 24 h) prevented not only the rapid auxin-mediated disintegration of the MT cytoskeleton but also a reorientation of the CMT arrays, from transversal to longitudinal. The only tissue to show MTs in their cells throughout the auxin treatment was the epidermis. Significant resistance of transverse CMT arrays in these cells towards auxin was confirmed using a higher auxin concentration (100 microM, 24 h). The latter auxin dose also revealed inter-tissue-specific responses to auxin: outer cortical cell files reoriented their CMTs from the transversal to longitudinal orientation, whereas inner cortical cell files lost their MTs. This high auxin-mediated response, associated with the swelling of root apices, was abolished with the pretreatment of maize root with taxol.     

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.     

Nod factors alter the microtubule cytoskeleton in Medicago truncatula root hairs to allow root hair reorientation

The microtubule (MT) cytoskeleton is an important part of the tip-growth machinery in legume root hairs. Here we report the effect of Nod factor (NF) on MTs in root hairs of Medicago truncatula. In tip-growing hairs, the ones that typically curl around rhizobia, NF caused a subtle shortening of the endoplasmic MT array, which recovered within 10 min, whereas cortical MTs were not visibly affected. In growth-arresting root hairs, endoplasmic MTs disappeared shortly after NF application, but reformed within 20 min, whereas cortical MTs remained present in a high density. After NF treatment, growth-arresting hairs were swelling at their tips, after which a new outgrowth formed that deviated with a certain angle from the former growth axis. MT depolymerization with oryzalin caused a growth deviation similar to the NF; whereas, combined with NF, oryzalin increased and the MT-stabilizing drug taxol suppressed NF-induced growth deviation. The NF-induced disappearance of the endoplasmic MTs correlated with a loss of polar cytoarchitecture and straight growth directionality, whereas the reappearance of endoplasmic MTs correlated with the new set up of polar cytoarchitecture. Drug studies showed that MTs are involved in determining root hair elongation in a new direction after NF treatment.     

Isolation of microtubule-associated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus

A method for biochemically isolating microtubule-associated proteins (MAPs) from the detergent-extracted cytoskeletons of carrot suspension cells has been devised. The advantage of cytoskeletons is that filamentous proteins are enriched and separated from vacuolar contents. Depolymerization of cytoskeletal microtubules with calcium at 4°C releases MAPs which are then isolated by association with taxol stabilized neurotubules. Stripped from microtubules (MTs) by salt, then dialysed, the resulting fraction contains a limited number of high molecular weight proteins. Turbidimetric assays demonstrate that this MAP fraction stimulates polymerization of tubulin at concentrations at which it does not self-assemble. By adding it to rhodamine-conjugated tubulin, the fraction can be seen to form radiating arrays of long filaments, unlike MTs induced by taxol. In the electron microscope, these arrays are seen to be composed of mainly single microtubules. Blot-affinity purified antibodies confirm that two of the proteins decorate cellular microtubules and fulfil the criteria for MAPs. Antibodies to an antigenically related triplet of proteins about 60–68 kDa (MAP 65) stain interphase, preprophase band, spindle and phragmoplast microtubules. Antibodies to the 120 kDa MAP also stain all of the MT arrays but labelling of the cortical MTs is more punctate and, unlike anti-MAP 65, the nuclear periphery is also stained. Both the anti-65 kDa and the anti-120 kDa antibodies stain cortical MTs in detergent-extracted, substrate-attached plasma membrane disks ('footprints'). Since the 120 kDa protein is detected at two surfaces (nucleus and plasma membrane) known to support MT growth in plants, it is hypothesized that it may function there in the attachment or nucleation of MTs.     

Isolated Plant Nuclei Nucleate Microtubule Assembly: The Nuclear Surface in Higher Plants Has Centrosome-like Activity

In most eukaryotic cells, microtubules (MTs) are assembled at identified nucleating sites, such as centrosomes or spindle pole bodies. Higher plant cells do not possess such centrosome-like structures. Thus, the fundamental issues of where and how the intracellular plant MTs are nucleated remain highly debatable. A large body of evidence indicates that plant MTs emerge from the nuclear periphery. In this study, we developed an in vitro assay in which isolated maize nuclei nucleate MT assembly at a tubulin concentration (14 [mu]M of neurotubulin) that is not efficient for spontaneous MT assembly. No MT-stabilizing agents, such as taxol or dimethyl sulfoxide, were used. Our model provides evidence that the nuclear surface functions as a MT-nucleating site in higher plant cells. A monoclonal antibody raised against a pericentriolar antigen immunostained the surface of isolated nuclei, and a 100-kD polypeptide in 4 M urea-treated nuclear extracts was detected.     

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

The generation of the unique radial array of microtubules (MTs) in stomatal guard cells raises questions about the location and activities of relevant MT-organizing centers. By using tubulin immunofluorescence microscopy, we studied the pattern of depolymerization and reassembly of MTs in guard cells of Allium cepa L. Chilling at 0°C reduces the MTs to small remnants that surround the nuclear surface of cells in the early postcytokinetic stage, or form a dense layer along the central portion of the ventral wall in older guard cells. A rapid reassembly on rewarming restores either MTs extending from the nuclear surface randomly throughout the cytoplasm in very young cells, or an array of MTs radiating from the dense layer at the ventral wall later in development. A similar pattern of depolymerization and reassembly is achieved by incubation with 100 M colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 M colchicine leads to a complete depolymerization that leaves only a uniform, diffuse cytoplasmic fluorescence. Nonetheless, UV irradiation of developing guard cells induces the regeneration of a dense layer of MTs at the ventral wall. The layer is again positioned centrally along the wall, even if the nucleus has been displaced by centrifugation in the presence of cytochalasin D. Neither the regenerated layer nor the perinuclear MTs seen earlier are related to the staining pattern of serum 5051, which reportedly binds to centrosomal material in animal and plant cells. The results support the view that, soon after cytokinesis, a planar MT-organizing zone is established in the cortex along the central portion of the ventral wall, which then generates the radial MT array.Abbreviations GC guard cell - MT microtubule - MTOC microtubule-organizing center - UV ultraviolet To whom correspondence should be addressed.     

Taxol induces microtubule-rough endoplasmic reticulum complexes and microtubule-bundles in cultured chondroblasts

Taxol induces a vast increase in the number of microtubules (MTs) in functional chondroblasts. The drug also induces a marked change in MT distribution. In control cultures, anti-tubulin stains long, fine, sinuous filaments radiating from a perinuclear center. In taxol-treated cells, anti-tubulin stains stubby, straight, chevron-like structures that assume a striking antipodal distribution. Such MT-bundles are relatively stable: they persist for over 48 h after removal of taxol, and even for 16-24 h in Colcemid. Many of these supernumerary MTs bind to, and align on, the cytoplasmic face of the rough endoplasmic reticulum (RER). In binding, the MTs displace the numerous ribosomes that normally stud the surface of the cisternae of the RER. The bound MTs form a remarkably uniform layer with center-to-center spacings of 40 nm. The attached parallel arrays of MTs achieve lengths of over 10 microns. These bound MTs not only dislodge ribosomes from the RER surface, but they also zip together adjacent ER complexes, forming tiers of two to eight cisternae. Numerous cytoplasmic bundles of hexagonally-ordered MTs are also induced. When closely aligned, the MTs assume a crystalline configuration with a six-fold symmetry, a central MT being surrounded by six equidistant MTs. A single cell can have over 100 MT-bundles and the number of MTs per bundles varies from 2-30. The forces aggregating cytoplasmic MT-bundles probably differ from those that bind MTs to the RER. Taxol also fragments the prominent Golgi complex that characterizes actively secreting chondroblasts. No obvious morphologic relationship has yet been detected between these induced MTs and other organelles such as intermediate-sized filaments, microfilaments, mitochondria, Golgi cisternae, or secretory vesicles.     

Reinstatement of Microtubule Arrays from Cortical Nucleating Sites in Stomatal Complexes of Lolium rigidum Following Depolymerization of Microtubules by Oryzalin and High Pressure

Immunofluorescence visualization of microtubule (MT) arraysin stomatal complexes of Lolium rigidum shows that disassemblyof the arrays can be successfully achieved using oryzalin orhigh pressure treatments. Under conditions allowing for MT recovery,MTs reappear within an hour after oryzalin or within 5 min afterhigh pressure treatment. During recovery guard mother cells(GMCs) nucleate MTs at sites distributed randomly in the cellcortex. Even after 22 h of recovery the MTs are not arrangedinto any configuration found in untreated tissue. This inabilityto reorganize their MTs after treatment makes GMCs more sensitiveto the loss of MTs than are other cells of the leaf. In guardcells (GCs) MTs reappear around the pore at the junction ofthe periclinal and ventral walls. They subsequently appear throughoutmost of the cell cortex and the majority of stomatal complexesrecover fully organized MT arrays indistinguishable from thosein untreated cells. The results support and extend ultrastructuraland immunofluorescence observations that suggest that MTs inGCs of developing stomata are nucleated in the cell cortex. ²Present address: Department of Biology, The University of SouthwesternLouisiana, Lafayette, Louisiana 70504-2451, U.S.A. (Received April 24, 1990; Accepted July 7, 1990)     

Taxol Induces Microtubule-Rough Endoplasmic Reticulum Complexes and Microtubule-Bundles in Cultured Chondroblasts

Abstract. Taxol induces a vast increase in the number of microtubules (MTs) in functional chondroblasts. The drug also induces a marked change in MT distribution. In control cultures, anti-tubulin stains long, fine, sinuous filaments radiating from a perinuclear center. In taxol-treated cells, anti-tubulin stains stubby, straight, chevron-like structures that assume a striking antipodal distribution. Such MT-bundles are relatively stable: they persist for over 48 h after removal of taxol, and even for 16–24 h in Colcemid. Many of these supernumerary MTs bind to, and align on, the cytoplasmic face of the rough endoplasmic reticulum (RER). In binding, the MTs displace the numerous ribosomes that normally stud the surface of the cisternae of the RER. The bound MTs form a remarkably uniform layer with center-to-center spacings of 40 nm. The attached parallel arrays of MTs achieve lengths of over 10 μm. These bound MTs not only dislodge ribosomes from the RER surface, but they also zip together adjacent ER complexes, forming tiers of two to eight cisternae. Numerous cytoplasmic bundles of hexagonally-ordered MTs are also induced. When closely aligned, the MTs assume a crystalline configuration with a six-fold symmetry, a central MT being surrounded by six equidistant MTs. A single cell can have over 100 MT-bundles and the number of MTs per bundles varies from 2–30. The forces aggregating cytoplasmic MT-bundles probably differ from those that bind MTs to the RER. Taxol also fragments the prominent Golgi complex that characterizes actively secreting chondroblasts. No obvious morphologic relationship has yet been detected between these induced MTs and other organelles such as intermediate-sized filaments, microfilaments, mitochondria, Golgi cisternae, or secretory vesicles.     

Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells

Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of beta-l,3-glucan (callose) and fibrils of beta-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.     

Tissue- and development-specific distributions of cytoskeletal elements in growing cells of the maize root apex

ABSTRACT

Indirect immunofluorescence performed using sections of actively growing maize root apices fixed and then embedded in low-melting-point Steedman's wax has proved efficient in revealing the arrangements and reorganizations of motility-related cytoskeletal elements which are associated with root cell development and tissue differentiation. This powerful, yet relatively simple, technique shows that specific rearrangements of both microtubular (MT) and actin microfilament (MF) arrays occur in cells as they leave the meristem and traverse the transitional region interpolated between meristem and elongation region. Cytoskeletal and growth analyses have identified the transition zone as critical for both cell and root development; it is in this zone that cell growth is channelled, by the cytoskeleton, into a strictly polarized mode which enables root tips to extend rapidly through the soil in search of water and nutrients. An integrated cytoskeletal network is crucial for both the cytomorphogenesis of individual cells and the overall morphogenesis of the plant body. The latter process can be viewed as a reflection of the tight control which cytoskeletal networks exert not only over cell division planes in the cells within meristematic apices but also over the orientation of cell growth in the meristem and elsewhere. Endoplasmic MTs interconnecting the plasma membrane with the nucleus are suggested to be involved in cell division control; they may also act as a two-way cytoskeletal communication channel for signals passing to and fro between the extracellular environment and the genome. Moreover, the dynamism of endoplasmic MTs exerts direct effects on chromatin structure and the accompanying nuclear architecture and hence can help exert a cellular level of control over cell growth and cell cycle progression. Because the inherent dynamic instability of MTs depends on the concentration of tubulin dimers within the cytoplasm, we propose that when asymmetric cell division occurs, it will result in two daughter cells which differ in the turnover rates of their MTs. This phenomenon could be responsible for different cell fates of daughter plant cells produced by such cell divisions.     

Halogenated Auxins Affect Microtubules and Root Elongation in Lactuca sativa

We studied the effect of 4,4,4-trifluoro-3-(indole-3-)butyric acid (TFIBA), a recently described root growth stimulator, and 5,6-dichloro-indole-3-acetic acid (DCIAA) on growth and microtubule (MT) organization in roots of Lactuca sativa L. DCIAA and indole-3-butyric acid (IBA) inhibited root elongation and depolymerized MTs in the cortex of the elongation zone, inhibited the elongation of stele cells, and promoted xylem maturation. Both auxins caused the plane of cell division to shift from anticlinal to periclinal. In contrast, TFIBA (100 micromolar) promoted elongation of primary roots by 40% and stimulated the elongation of lateral roots, even in the presence of IBA, the microtubular inhibitors oryzalin and taxol, or the auxin transport inhibitor naphthylphthalamic acid. However, TFIBA inhibited the formation of lateral root primordia. Immunostaining showed that TFIBA stabilized MTs orientation perpendicular to the root axis, doubled the cortical cell length, but delayed xylem maturation. The data indicate that the auxin-induced inhibition of elongation and swelling of roots results from reoriented phragmoplasts, the destabilization of MTs in elongating cells, and promotion of vessel formation. In contrast, TFIBA induced promotion of root elongation by enhancing cell length, prolonging transverse MT orientation, delaying cell and xylem maturation.     

Role of cortical microtubules in the orientation of cellulose microfibril deposition in higher-plant cells

Summary Cortical microtubules (MTs) have been implicated in the morphogenesis of plant cells by regulating the orientation of newly deposited cellulose microfibrils (CMFs). However, the role of MTs in oriented CMF deposition is still unclear. We have investigated the mechanism of CMF deposition with cultured tobacco protoplasts derived from taxol-treated BY-2 cells (taxol protoplasts). The BY-2 protoplasts regenerated patches of β-l,3-glucan (callose) and fibrils of β-l,4-glucan (cellulose). Taxol protoplasts possessed the same ordered MT arrays as material cells and regenerated CMFs with patterns almost coincidental with MTs. Electron microscopy revealed that, on the surface of cultured taxol protoplasts, each CMF bundle appeared to be deposited on each cortical MT. These results suggest that MTs may attach directly to the cellulose-synthesizing complexes, by some form of linkage, and regulate the movement of these complexes in higher-plant cells.     

Disruption of microtubular cytoskeleton induced by cryptogein, an elicitor of hypersensitive response in tobacco cells

The dynamics of microtubular cytoskeleton were studied in tobacco (Nicotiana tabacum cv Xanthi) cells in response to two different plant defense elicitors: cryptogein, a protein secreted by Phytophthora cryptogea and oligogalacturonides (OGs), derived from the plant cell wall. In tobacco plants cryptogein triggers a hypersensitive-like response and induces systemic resistance against a broad spectrum of pathogens, whereas OGs induce defense responses, but fail to trigger cell death. The comparison of the microtubule (MT) dynamics in response to cryptogein and OGs in tobacco cells indicates that MTs appear unaffected in OG-treated cells, whereas cryptogein treatment caused a rapid and severe disruption of microtubular network. When hyperstabilized by the MT depolymerization inhibitor, taxol, the MT network was still disrupted by cryptogein treatment. On the other hand, the MT-depolymerizing agent oryzalin and cryptogein had different and complementary effects. In addition to MT destabilization, cryptogein induced the death of tobacco cells, whereas OG-treated cells did not die. We demonstrated that MT destabilization and cell death induced by cryptogein depend on calcium influx and that MT destabilization occurs independently of active oxygen species production. The molecular basis of cryptogein-induced MT disruption and its potential significance with respect to cell death are discussed.     

Amiprophos-methyl (APM): A rapid,reversible, anti-microtuble agent for plant cell cultures

Summary In plant cell suspension cultures sensitive to the herbicide amiprophos-methyl (APM), 1 to 3 M APM completely depolymerized both cortical and mitotic microtubule (MT) arrays in 1 hour. In comparison, a 2 hour application of 3 mM colchicine had no effect on MT arrays. Recovery from APM treatment occurred as early as 5 minutes after removal of APM. Short, cortical MTs were visible in 3 hours and complete MT arrays were found within 22 hours after drug removal.Sensitivity to APM-induced MT depolymerization varied according to species but was increased or decreased by varying the mitotic rate in cultures. The results indicated APM sensitivity was related to lowered stability of MT arrays in rapidly cycling cells. APM treatment may help distinguish stabilized cortical MTs in elongating cells and nonstabilized cortical MTs in rapidly dividing cells.Abbreviations MT microtubule - APM amiprophos-methyl - DMSO dimethyl sulfoxide - PBS phosphate buffered saline     

Microtubule dynamics are involved in stomatal movement ofVicia faba L.

Summary To obtain a full picture of microtubule (MT) behavior during the opening and closure of guard cells we have microinjected living guard cells ofVicia faba with fluorescent tubulin, examined fine detail by freeze shattering fixed cells, and used drug treatments to confirm aspects of MT dynamics. Cortical MTs in fully opened guard cells are transversely oriented from the ventral wall to the dorsal wall. When the stomatal aperture was decreased by darkness, these MTs became twisted and patched and broken down into diffuse fragments when stomata were closed. When the closed stomata were opened in response to light, the MTs in guard cells changed from the diffused, transitional pattern back to one in which MTs are transversely oriented from stomatal pore to dorsal wall. This observation indicates a linkage between these MT changes and stomatal movement. To confirm this, we used the MT-stabilizing agent taxol and the MT-depolymerizing herbicide oryzalin and observed their effects on the stomatal aperture and MT dynamics. Both drugs suppressed light-induced stomatal opening and dark-induced closure. MTs are known to be necessary for maintaining the static kidney shape of guard cells; the present data now show that the dynamic properties of polymeric tubulin accompany changes in shape with stomatal movement and may be functionally involved in stomatal movement.