Gamma-tubulin in Chlamydomonas: characterization of the gene and localization of the gene product in cells.

In addition to their role in nucleating the assembly of axonemal microtubules, basal bodies often are associated with a microtubule organizing center (MTOC) for cytoplasmic microtubules. In an effort to define molecular components of the basal body apparatus in Chlamydomonas reinhardtii, genomic and cDNA clones encoding gamma-tubulin were isolated and sequenced. The gene, present in a single copy in the Chlamydomonas genome, encodes a protein with a predicted molecular mass of 52,161 D and 73% and 65% conservation with gamma-tubulin from higher plants and humans, respectively. To examine the distribution of gamma-tubulin in cells, a polyclonal antibody was raised against two peptides contained within the protein. Immunoblots of Chlamydomonas proteins show a major cross-reaction with a protein of Mr 53,000. In Chlamydomonas cells, the antibody stains the basal body apparatus as two or four spots at the base of the flagella and proximal to the microtubule rootlets. During cell division, two groups of fluorescent dots separate and localize to opposite ends of the mitotic apparatus. They then migrate during cleavage to positions known to be occupied by basal bodies. Changes in gamma-tubulin localization during the cell cycle are consistent with a role for this protein in the nucleation of microtubules of both the interphase cytoplasmic array and the mitotic spindle. Immunogold labeling of cell sections showed that gamma-tubulin is closely associated with the basal bodies. The flagellar transition region was also labeled, possibly indicating a role for gamma-tubulin in assembly of the central pair microtubules of the axoneme.

gamma-Tubulin and microtubule organization in plants.

In animal cells, microtubule assembly is usually initiated at one specialized structure, the centrosome. By contrast, in plant cells, microtubule assembly begins at a variety of locations within the cell. A member of the tubulin gene family, gamma-tubulin, is localized to the centrosome in animal cells and is important in the assembly of microtubules in vivo. Recent reports have identified gamma-tubulin genes in plants and have described the complex intracellular distribution of the encoded polypeptides. Here, Harish Joshi and Barry Palevitz comment upon how this information may help elucidate the organizing principles of the complex arrays of microtubules in plant cells.

A kinesin-like protein, KatAp, in the cells of arabidopsis and other plants.

The kinesin-like proteins (KLPs) are a large family of plus- or minus-end-directed microtubule motors important in intracellular transport, mitosis, meiosis, and development. However, relatively little is known about plant KLPs. We prepared an antibody against two peptides in the microtubule binding domain of an Arabidopsis KLP (KatAp) encoded by the KatA gene, one of a family of genes encoding KLPs whose motor domain is located near the C terminus of the polypeptide. Such KLPs typically move materials toward the minus end of microtubules. An immunoreactive band (Mr of 140,000) corresponding to KatAp was demonstrated with this antibody on immunoblots of Arabidopsis seedling extracts. During immunofluorescence localizations, the antibody produced weak, variable staining in the cytoplasm and nucleus of interphase Arabidopsis suspension cells but much stronger staining of the mitotic apparatus during division. Staining was concentrated near the midzone during metaphase and was retained there during anaphase. The phragmoplast was also stained. Similar localization patterns were seen in tobacco BY-2 cells. The antibody produced a single band (Mr of 130,000) in murine brain fractions prepared according to procedures that enrich for KLPs (binding to microtubules in the presence of AMP-PNP but not ATP). A similar fraction from carrot suspension cells yielded a cross-reacting polypeptide of similar apparent molecular mass. When dividing BY-2 cells were lysed in the presence of taxol and ATP, antibody staining moved rapidly toward the poles, supporting the presence of a minus-end motor. Movement did not occur without ATP, with AMP-PNP, or with ATP plus antibody. Our results indicate that the protein encoded by KatA, KatAp, is expressed in Arabidopsis and is specifically localized to the midzone of the mitotic apparatus and phragmoplast. A similar protein is also present in other species.

Organization of cortical microtubules in plant cells.

Cortical microtubule arrays in plants are involved in many morphogenetically important processes. Recent analog cytochemical and immunolocalization experiments have provided new insights into the temporal and spatial dynamics of cortical microtubules. Current data suggest that the arrangement of these arrays is modulated by cell cycle and signal transduction elements, including calcium.

Experimental manipulation of gamma-tubulin distribution in Arabidopsis using anti-microtubule drugs.

gamma-Tubulin-specific antibodies stain the microtubule (Mt) arrays of Arabidopsis suspension cells in a punctate or patchy manner. During division, staining of kinetochore fibers and the phragmoplast is extensive, except in the vicinity of the plus ends at the metaphase plate and cell plate. gamma-Tubulin localization responds to low levels of colchicine, with staining receding farther toward the minus (pole) ends of kinetochore fibers. At higher drug concentrations, gamma-tubulin also associates with abnormal Mt foci as well as with the surface of the daughter nuclei facing the phragmoplast. During UV-induced recovery from colchicine, gamma-tubulin increases along the presumptive minus ends of mitotic Mts as well as the phragmoplast near the daughter nuclei. With CIPC, immunostaining is concentrated around the centers of focal Mt arrays in multipolar spindles. In the presence of taxol, Mts are more prominent but the mitotic apparatus and phragmoplast are abnormal. As with CIPC, gamma-tubulin is concentrated at focal arrays. Increased punctate staining is also present in interphase arrays, with fluorescent dots often located at the ends of Mts. These results support a preferential association between gamma-tubulin and Mt minus ends, but are also consistent with more general binding along the walls of Mts. Thus, minus ends (and Mt nucleation sites) may be present throughout plant Mt arrays, but gamma-tubulin may also serve another function, such as in structural stabilization.

gamma-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies.

gamma-Tubulin is a protein associated with microtubule (Mt)-organizing centers in a variety of eukaryotic cells. Unfortunately, little is known about such centers in plants. Genomic and partial cDNA clones encoding two gamma-tubulins of Arabidopsis were isolated and sequenced. Comparisons of genomic and cDNA sequences showed that both genes, TubG1 and TubG2, contain nine introns at conserved locations. The sequences of the two genes both predict proteins containing 474 amino acids, with molecular masses of 53,250 and 53,280 D, respectively. The predicted gamma 1- and gamma 2-tubulins exhibit 98% amino acid identity with each other and approximately 70% amino acid identity with the gamma-tubulins of animals and fungi. RNA gel blot results demonstrated that both genes are transcribed in suspension culture cells, seedlings, and roots and flowers of mature plants. Immunoblots of Arabidopsis proteins using an antibody specific to a conserved peptide of gamma-tubulin showed a major cross-reacting polypeptide with an M(r) of 58,000. The same antibody stained all Mt arrays in tissue and suspension culture cells of this species. Binding was inhibited by the homologous oligopeptide in the gamma-tubulins predicted by the two Arabidopsis gene sequences. Antibody staining avoided the plus ends of Mts at the kinetochores and cell plate, but unlike the case in animal cells, seemed to be localized over broad stretches of the kinetochore fibers and phragmoplast toward the minus ends. We concluded that at least two gamma-tubulin protein homologs are present in Arabidopsis and that at least one of them is localized along Mt arrays. Its distribution is correlated with and may help explain unique characteristics of Mt organization in plants.

A gamma-tubulin-related protein associated with the microtubule arrays of higher plants in a cell cycle-dependent manner.

An antibody specific for a conserved gamma-tubulin peptide identifies a plant polypeptide of 58 kDa. gamma-Tubulin antibody affinity purified from this polypeptide recognizes the centrosome in mammalian cells. Using immunofluorescence microscopy, we determined the distribution of this gamma-tubulin-related polypeptide during the complex changes in microtubule arrays that occur throughout the plant cell cycle. We report a punctate association of gamma-tubulin-related polypeptide with the cortical microtubule array and the preprophase band. As cells enter prophase, gamma-tubulin-related polypeptide accumulates around the nucleus and forms a polar cap from which early spindle microtubules radiate. During metaphase and anaphase, gamma-tubulin-related polypeptide preferentially associates with kinetochore fibers and eventually accumulates at the poles. In telophase, localization occurs over the phragmoplast. gamma-Tubulin-related polypeptide appears to be excluded from the plus ends of microtubules at the metaphase plate and cell plate. Its distribution during the cell cycle may be significant in light of differences in the behavior and organization of plant microtubules. The identification of gamma-tubulin-related polypeptide could help characterize microtubule organizing centers in these organisms.

Gamma-tubulin is associated with a cortical-microtubule-organizing zone in the developing guard cells of Allium cepa L.

A key event in the differentiation of elliptically shaped guard cells such as those in Allium is the formation of a radial array of cortical microtubules (Mts) which, by controlling the orientation of wall microfibrils, plays an important role in cell shaping. Previous experiments strongly indicated that the array is nucleated in a zone adjacent to the new ventral wall soon after cytokinesis. In order to further clarify the function of this zone, we performed dual immunolocalizations on Allium guard cells with anti-beta-tubulin, to detect Mts, and an antibody to gamma-tubulin, a protein known to be present at Mt-organizing centers in other species and recently identified in plants as well. gamma-Tubulin antibody stained the cortical zone adjacent to the ventral wall, while little or no fluorescence was present elsewhere along the radial Mt array or at other sites in the cell. The antibody also stained the mitotic poles and phragmoplast in guard mother cells, as it does in other material. No staining was seen when the primary antibody was omitted. The results are consistent with nucleation of the radial array at a cortical-Mt-organizing zone next to the ventral wall, and set the stage for more in-depth studies on the spatial and temporal control of Mt formation in differentiating cells.

Regulation of the spatial order of cortical microtubules in developing guard cells ofAllium.

The organization of microtubules (MTs) in the cortex of cells at interphase is an important element in morphogenesis. Mechanisms controlling the initiation of MTs and their spatial ordering, however, are largely unknown. Our recent study concerning the generation of a radial array of MTs in stomatal guard cells inAllium showed that the MTs initiate in a cortical MT-organizing zone adjacent to the ventral wall separating the two young guard cells (Marc, Mineyuki and Palevitz, 1989, Planta179, 516, 530). In an attempt to detect MT-ordering mechanisms separate from the sites of MT initiation, we now employ various drugs to manipulate the geometry and integrity of the ventral wall and thereby also the associated MT-organizing zone. In the presence of cytochalasin D the ventral wall is tilted away from its normal mid-longitudinal anticlinal alignment, while treatments with the herbicide chloroisopropyl-N-phenylcarbamate (CIPC) induce the formation of a branched ventral wall. Nonetheless, in either case the MTs still form a radial array, although this is asymmetric as it is centered in accordance with the misaligned or branched ventral wall. Since the MTs maintain their original course undisturbed as they extend beyond the abnormal ventral wall, there is no evidence for the presence of an inherent MT-ordering mechanism at locations remote from MT-initiation sites. Following treatments with caffeine, which abolishes the formation of the ventral wall, the MTs revert to a transversely oriented cylindrical array as in normal epidermal cells. Thus the presence of the ventral wall, and presumably also the associated MT-organizing zone, is essential for the establishment of the radial array. The MT-organizing zone is therefore involved not only in the initiation of MTs, but also in determining their spatial order throughout the cell cortex.

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.

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 µM colchicine followed by a brief irradiation with ultraviolet (UV) light. Incubation with 200 µM 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.

Microtubule-binding proteins from carrot : I. Initial characterization and microtubule bundling.

Microtubules (MTs) participate in several processes of fundamental importance to growth and development in higher plants, yet little is known about the proteins with which they associate. Information about these molecules is important because they probably play a role in mediating functional and structural differences between various MT arrays. As a first step in gaining insight into this problem, we have isolated, from suspension-cultured cells of carrot (Daucus carota L.), non-tubulin proteins which bind to and affect microtubules (MTs) in vitro. These proteins were isolated using taxol-stabilized neuronal MTs as an affinity substrate. They cause MT bundling at substoichiometric concentrations, support the assembly of tubulin in vitro, and at low concentrations, decorate single MTs in a periodic fashion. The bundled MTs formed in vitro share similarities with those seen in situ in a variety of plant cells, including a center-center spacing of 34 nm, cold stability, resistance to anti-microtubule drugs, and sensitivity to calcium. The bundling activity is specific; other cationic proteins, as well as poly-L-lysine, do not behave in a similar manner. The bundling activity is insensitive to ATP. By assaying bundling activity with dark-field microscopy and employing standard biochemical procedures, a small number of polypeptides involved in the bundling process were identified. Affinity-isolated antibodies to one of these polypeptides (Mr=76000) were found to co-localize with MTs in the cortical array of protoplasts. Our findings are discussed with reference to the importance of these proteins in the cell and to their relationship to microtubule-associated proteins in other eukaryotes.

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.

Dissociation and reassembly of soybean clathrin.

Coated vesicles (CVs), approximately 85 nanomolar in diameter, were obtained from etiolated soybean hypocotyls using discontinuous sucrose gradients. The CVs were treated with 4 molar urea and the vesicle membranes were then removed by centrifugation. When the supernatant subsequently was dialyzed against isolation buffer, sedimentable complexes were obtained. Electron microscopic examination of the pelleted complexes shows many spherical, 65 nanometer baskets consisting of a polygonal lattice free of internal membrane. LDS-PAGE reveals that a 185 kilodalton clathrin heavy chain is enriched in both the basket and CV pellets. However, the overall protein pattern is more complex than that of comparable brain fractions. The results are discussed in terms of the similarities between soybean and brain clathrins and the function of CVs in vivo.

Actin in the preprophase band of Allium cepa.

F-actin has been identified in the preprophase band of Allium cepa. Cells attached to subbed slides were obtained from formaldehyde-fixed root tips digested in EGTA and Cellulysin. The air-dried cells were extracted in Triton X-100, treated with rhodamine-phalloidin, rinsed briefly in PBS, and viewed in the fluorescence microscope. Interphase cells contain a network of actin fibers that extends into all areas of the cytoplasm. During preprophase, the network is replaced by a band of fibers aligned in the position of the preprophase band. Colocalization of F-actin with rhodamine-phalloidin and microtubules with tubulin immunocytochemistry confirms that the two bands are coincident. The actin appears to comprise a thin layer of fibers next to the plasmalemma. Like the microtubule preprophase band, the actin band narrows as preprophase progresses and disappears by midprophase. Fluorescent actin bands are not seen in fixed cells pretreated with excess unlabeled phalloidin before staining. They are also absent in roots exposed to cytochalasins B and D before fixation, but preprophase band microtubules at all stages of aggregation are still present. Colchicine treatment leads to the loss of both preprophase band microtubules and actin. The possible function of preprophase band actin is discussed.

Metabolic inhibitors block anaphase A in vivo.

Anaphase in dividing guard mother cells of Allium cepa and stamen hair cells of Tradescantia virginiana consists almost entirely of chromosome-to-pole motion, or anaphase A. Little or no separation of the poles (anaphase B) occurs. Anaphase is reversibly blocked at any point by azide or dinitrophenol, with chromosome motion ceasing 1-10 min after application of the drugs. Motion can be stopped and restarted several times in the same cell. Prometaphase, metaphase, and cytoplasmic streaming are also arrested. Carbonyl cyanide m-chlorophenyl hydrazone also stops anaphase, but its effects are not reversible. Whereas the spindle collapses in the presence of colchicine, the chromosomes seem to "freeze" in place when cells are exposed to respiratory inhibitors. Electron microscope examination of dividing guard mother cells fixed during azide and dinitrophenol treatment reveals that spindle microtubules are still present. Our results show that chromosome-to-pole motion in these cells is sensitive to proton ionophores and electron transport inhibitors. They therefore disagree with recent reports that anaphase A does not require a continuous supply of energy. It is possible, however, that anaphase does not directly use ATP but instead depends on the energy of chemical and/or electrical gradients generated by cellular membranes.

Abscisic Acid Control of Lectin Accumulation in Wheat Seedlings and Callus Cultures : Effects of Exogenous ABA and Fluridone.

Wheat germ agglutinin is found in wheat embryos and a similar lectin is present in the roots of older plants. We report here that 10 micromolar abscisic acid (ABA) produces an average two to three-fold enhancement in the amount of lectin in the shoot base and the terminal portion of the root system of hydroponically grown wheat seedlings. Although ABA stunts seedling growth, a similar growth inhibition produced by ancymidol is not accompanied by elevated lectin levels. To further clarify the role of ABA, wheat callus cultures were employed. Callus derived from immature embryos was grown on growth medium containing various combinations of ABA and 2,4-dichlorophenoxyacetic acid. Those grown in the presence of 10 micromolar ABA exhibit the largest increases in lectin compared to material grown on other regimes. The involvement of ABA in lectin accumulation was further probed with fluridone, an inhibitor of carotenoid synthesis which has also been linked to depressed levels of endogenous ABA. Wheat seedlings grown in the presence of 1 or 10 milligrams per liter fluridone have few or no carotenoids, and wheat germ agglutinin levels in the shoot base and roots are lower compared to controls. The greatest effect (a 39% reduction in the shoot base) is produced at an herbicide concentration of 10 milligrams per liter. Exogenous 10 micromolar ABA greatly stimulates lectin accumulation in the presence of fluridone, but the levels are not as high as those produced by ABA alone. These results indicate that lectin synthesis is under ABA control in both wheat embryos and adult plants.

Changes in dye coupling of stomatal cells of Allium and Commelina demonstrated by microinjection of Lucifer yellow.

Lucifer yellow has been microinjected into stomatal cells of Allium cepa L. epidermal slices and Commelina communis L. epidermal peels and the symplastic spread of dye to neighboring cells monitored by fluorescence microscopy. Dye does not move out of injected mature guard cells, nor does it spread into the guard cells when adjacent epidermal or subsidiary cells are injected. Dye does spread from injected subsidiary cells to other subsidiary cells. These results are consistent with the reported absence of plasmodesmata in the walls of mature guard cells. Microinjection was also used to ascertain when dye coupling ceases during stomatal differentiation in Allium. Dye rapidly moves into and out of guard mother cells and young guard cells. Hovewer, dye movement ceases midway through development as the guard cells begin to swell but well before a pore first opens. Since plasmodesmata are still present at this stage, the loss of symplastic transport may result from changes in these structures well in advance of their actual disappearance from the guard cell wall.

The endoplasmic reticulum in the cortex of developing guard cells: coordinate studies with chlorotetracycline and osmium ferricyanide.

The distribution of endoplasmic reticulum (ER) was investigated in young guard cells of Vicia faba and Allium cepa in order to gain more information on the control of guard cell development. Young, living guard cells of V. faba fluoresce when exposed to 25-100 microM chlorotetracycline (CTC). Intense fluorescence is restricted to the cytoplasm between the nucleus and adjacent regions of the ventral and paradermal walls. Much of the fluorescence is fibrillar in appearance and seems to arise from endomembranes, but not from particulate organelles such as mitochondria and plastids. A similar fluorescence pattern is produced by the membrane probes oxytetracycline and N-phenyl-1-napthylamine. Procaine and dibucaine render the fluorescence highly prone to photobleaching. Fluorescence appears near the ventral wall during early stages of cell development but declines when the guard cells mature. Epidermal tissue of V. faba and A. cepa was examined in the electron microscope with the aid of osmium ferricyanide staining. ER appears to be concentrated in regions of the guard cell that exhibit intense CTC fluorescence, while no other organelles (e.g., mitochondria) are similarly distributed. Much of the ER consists of a tubular network in close proximity to the plasmalemma. Our results indicate that the ER becomes asymmetrically distributed in young guard cells adjacent to those regions of the cell wall that undergo extensive thickening during cell differentiation. Furthermore, these membranes appear to sequester divalent cations such as Ca2+.