Field emission scanning electron microscopy of microtubule arrays in higher plant cells

Summary Specimen preparation protocols that allow field emission scanning electron microscope imaging of microtubules in plant cells were developed, involving simultaneous permeabilization with saponin and stabilization of microtubules with taxol. All categories of microtubule array were observed in onion root tip cells and in tobacco BY-2 cells grown in suspension culture and synchronized to provide high frequencies of mitotic stages. Cortical arrays consist of overlapping microtubules with free ends; individual microtubules directly overlie individual microfibrils in the cell wall. Preprophase bands and spindle microtubule bundles were also imaged. Phragmoplasts revealed early stages of wall deposition in the included cell plates and features interpreted as relating to high rates of microtubule turnover at the growing margins. It was possible to combine high resolution three-dimensional imaging with immunogold labelling of microtubules. Individual gold particles were readily distinguished decorating microtubules in the preparations; the method should be vaulable for studying many features of plant cell microtubules and their associated macromolecules.Abbreviations FESEM field emission gun scanning electron microscope - MTSB microtubule stabilising buffer Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Microfilaments in epidermal cancer cells

The occurrence and structure of microfilaments in epidermal cancers induced in mice by treatment with 3,4-benzpyrene were investigated with the electron microscope. With malignant change, pleomorphic, undifferentiated cells with a cortical zone of microfilaments became increasingly abundant. The microfilaments were 40 Å in diameter and occupied the cortex of the cells beneath the plasma membrane, extended into cell processes, and were situated in the cores of microvilli. At high magnification, the filamentous areas were formed by an interconnected meshwork of filaments which in favorable planes had a polygonal arrangement. When exposed to high concentrations of cytochalasin B, the microfilaments became clumped and moderately disrupted. At the same time, the processes and microvilli of the cells were blunted. The structure of these filaments and their sensitivity to cytochalasin B place them in a class of microfilaments believed to be related to cell motility. Their presence in malignant cells may be correlated with the motile, invasive properties of these cells.     

Low-light-induced formation of semicrystalline photosystem II arrays in higher plant chloroplasts

Remodeling of photosynthetic machinery induced by growing spinach plants under low light intensities reveals an up-regulation of light-harvesting complexes and down-regulation of photosystem II and cytochrome b6f complexes in intact thylakoids and isolated grana membranes. The antenna size of PSII increased by 40-60% as estimated by fluorescence induction and LHCII/PSII stoichiometry. These low-light-induced changes in the protein composition were accompanied by the formation of ordered particle arrays in the exoplasmic fracture face in grana thylakoids detected by freeze-fracture electron microscopy. Most likely these highly ordered arrays consist of PSII complexes. A statistical analysis of the particles in these structures shows that the distance of neighboring complexes in the same row is 18.0 nm, the separation between two rows is 23.7 nm, and the angle between the particle axis and the row is 26 degrees . On the basis of structural information on the photosystem II supercomplex, a model on the supramolecular arrangement was generated predicting that two neighboring complexes share a trimeric light-harvesting complex. It was suggested that the supramolecular reorganization in ordered arrays in low-light grana thylakoids is a strategy to overcome potential diffusion problems in this crowded membrane. Furthermore, the occurrence of a hexagonal phase of the lipid monogalactosyldiacylglycerol in grana membranes of low-light-adapted plants could trigger the rearrangement by changing the lateral membrane pressure.     

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.     

Modelling dynamic plant cells

A major challenge in plant biology is to understand how functions in plant cells emerge from interactions between molecular components. Computational and mathematical modelling can encapsulate the relationships between molecular components and reveal how biological functions emerge. We review recent progress in modelling in metabolism, growth, signalling and circadian rhythms in plant cells. We discuss challenges and opportunities for future directions.     

Mobile arrays of vacuole ripples are common in plant cells

In tobacco (Nicotiana tabacum L.), a structure frequently interpreted as endoplasmic reticulum, was clearly identified as a set of ripple-shaped protrusions of the vacuole into the surrounding cytoplasm. The occurrence in other species suggests that these ripples might be common in vacuolated plant cells. The apparent mobility of the ripples depends on the integrity of the F-actin network. This raises questions concerning the precise composition and architecture of the cytoplasm/vacuole border. The stereotypic concept of the central vacuole as a kind of inflated balloon with a smooth surface has to be debated. Received: 7 November 1997 / Revision received: 27 April 1998 / Accepted: 28 April 1998     

Biochemical changes during sucrose deprivation in higher plant cells

The mobilization of stored carbohydrates (sucrose and starch) during sucrose starvation was studied with sycamore (Acer pseudoplatanus) cells. When sucrose was omitted from the nutrient medium, vacuolar sucrose was first consumed. When a threshold of intracellular sucrose concentration was attained the cytoplasmic phosphorylated compounds decreased whereas cytoplasmic Pi increased symmetrically. Such a situation triggered starch breakdown. When almost all the intracellular sucrose pool had disappeared, the cell respiration rates (normal and uncoupled) declined progressively. The decrease in the rate of respiration triggered by sucrose starvation was attributable neither to the availability of substrate for mitochondrial respiration nor to a decrease in the maximal rate of O2 consumption by mitochondria expressed in terms of nanomole of O2 consumed per min/mg of mitochondrial protein. In fact, the uncoupled respiration rates decreased in parallel with the decrease in total intracellular cardiolipin or cytochrome aa3. These results demonstrate therefore that after a long period of sucrose starvation the progressive decrease in the uncoupled rate of O2 consumption by sycamore cells was attributable to a progressive diminution of the number of mitochondria/cell.     

Characterization of a cytosolic aconitase in higher plant cells

Protoplasts obtained from sycamore (Acer pseudoplatanus) cell suspensions were found to be highly intact. If the protoplasts were taken up and expelled through a fine nylon mesh, all the protoplasts were ruptured leaving the fragile amyloplasts largely intact. Aconitase hydratase (citrate [isocitrate] hydro-lyase, EC 4.2.1.3) activity of sycamore cells was associated with two protein fractions, one present in the cytosol while the second is of mitochondrial origin. Chromatography on DEAE-trisacryl did not separate the aconitase hydratase isoenzymes. EPR studies established that both isoenzymes exhibited an EPR signal at g = 2.03 once oxidized.     

Temperature dependence of intracellular pH in higher plant cells

The recent introduction of ³¹P nuclear magnetic resonance spectroscopy offers a new approach to the problem of obtaining a simultaneous and direct evaluation of both the cytoplasmic and vacuolar pH in higher plant cells (J. K. M. Roberts, P.M. Ray, N. Waderlardetzky and O. Sardetzky, 1980, Nature 283, 870–872; 1981, Planta 152, 74–78). Using this method we have been able to detect a selective pH decrease of about 0.5 units at the level of the cytoplasmic compartment of maize root tips when the temperature was increased from 4 to 28°C. This effect was completely reversible with temperature. No pH variation could be detected at the level of the vacuolar compartment.     

Microfilaments (F-actin) in generative cells and sperm: an evaluation

Summary Disagreement has arisen over the presence of actin-containing microfilaments (Mfs) in angiosperm generative cells and sperm (GSP). In order to address this issue, we subjected GSP of Tradescantia virginiana, Nicotiana tabacum and Rhododendron laetum to a series of localizations using different antiactins, rhodamine phalloidin and antimyosin. Coordinate staining with antitubulin and Hoechst 33258 defined the status of the microtubule (Mt) cytoskeleton and stages of generative cell division. Additional experiments utilized cytochalasin D (CD). In no instance could Mfs be detected in GSP of the three species. Instead, Mfs seen at the periphery of GSP appear to be continuous with vegetative Mfs and thus are in the vegetative cytoplasm. Mfs are not seen in the constriction zone of dividing T. virginiana generative cells, nor are they indicated in the phragmoplast of N. tabacum and R. laetum. Myosin localizations reveal punctate staining in the vegetative cytoplasm and a thin line of fluorescence around the the outside of the generative cell. While CD seems to delay generative cell division, cytokinesis still takes place. CD-induced Mf fragments are evident in the vegetative cytoplasm but not in GSP. The weight of evidence therefore indicates that GSP do not contain Mfs. The implications of this conclusion for the behavior of GSP and the mechanism of cytokinesis in dividing generative cells are considerable.     

Microfilaments in regenerating cells of rabbit cornea: immunological and ultrastructural observations

The role of myosin-like protein in regenerating and proliferating corneal cells following a standard alkali-injury in the rabbit eye has been studied. Microfilaments were observed by conventional transmission electron microscopy (T.E.M.) in injured epithelium and in fibroblasts. Typical microfilament bundles with electron dense structures and with stress fibers were evident. The presence of myosin-like proteins was demonstrated by means of immunochemical and autoradiographical techniques. Both epithelial cells and fibroblasts bind antimyosin-like antibodies (AMA). The same cells studied with the E.M. showed bundles of microfilaments in the cortical areas of their cytoplasm in correspondence with the same side of fluorescent or labelled AMA. The immunochemical and ultrastructural results suggest that both cells are able to produce in vivo movements involved in morphogenetic phenomena. Therefore, these structures play a role in post-traumatic corneal regeneration.     

The assembly of keratins from higher plant cells

Summary In vitro assembly and morphological characteristics of purified 58 kDa, 52 kDa, 50 kDa, and 45 kDa polypeptides in the leaves and the cotyledons of the cabbage (Brassica pekinensis Rupt.) were investigated by electron microscopy and scanning tunneling microscopy. The three or four purified intermediate filament (IF) polypeptides can spontaneously assemble into intermediate filaments in vitro with a 23–24 nm axial repeat, which indicates that keratin IFs in higher plant cells have the same molecular arrangement as in animal cells. STM images suggest that the plant keratin filaments display a pronounced structural polymorphism, which can be composed of 3 nm, 4.5 nm, or 6 nm wide keratin protofilaments.Abbreviation IF intermediate filament - STM scanning tunneling microscopy - SDS sodium dodecyl sulfate - BCIP 5-bromo-4-chloro-3-indolyl phosphate-toluidine - NBC p-nitroblue tetrazolium chloride - PMSF phenylmethyl sulfonylfluoride - HOPG high oriented pyrolytic graphite     

Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Microfilaments were isolated from cultured mammalian cells, utilizing procedures similar to those for isolation of "native" thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK- 21), and Swiss mouse 3T3 cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin, and a high molecular weight actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS-polyacrylamide gels with both muscle and nonmuscle types of tropomyosin. In order to identify more specifically these subunits, we isolated and partially characterized tropomyosin from three cell types. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. However, tropomyosin isolated from rate embryo and 3T3 cells contained subunits that comigrated with both skeletal muscle and nonmuscle types of myosin, whereas the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.     

Microtubule-nucleation sites on nuclei of higher plant cells

Summary The nucleation and the elongation of microtubules from isolated nuclei of higher plant cells were investigated. Isolated intact nuclei failed to nucleate microtubules at their surface when they were incubated with purified tubulin from plant or animal sources. However, frozen and thawed nuclei or nuclear particles obtained by gentle nuclei homogenization nucleated microtubules and nucleated microtubules elongated radially from the surface of nuclei or from the nuclear particles. Microtubules radiating from the nuclear particles were very much shorter than those radiating from frozen and thawed nuclei. The washing of the nuclear particles diminished the ability of the particles to nucleate microtubules. The ability of the washed nuclear particles to nucleate microtubules was restored by the addition of the soluble fraction of a nuclear homogenate. The complexes of radiating microtubules could easily be observed under a phasecontrast microscope. Electron microscopy demonstrated that microtubules in the complexes formed bundles. The staining with a monoclonal antibody specific for plant tubulin of the complexes of radiating microtubules, prepared by successive polymerization of animal tubulin and plant tubulin, revealed that microtubules in the complex incorporated tubulin at their proximal ends. This result indicates that the mode of incorporation of tubulin onto frozen and thawed nuclei or onto the nuclear particles is different from that in pericentriolar bodies in animal cells. Mg²⁺ seems to participate in the regulatory mechanism that determines the length of microtubules on the complexes.Abbreviations MTOC microtubule-organizing center - MES 2-(N-morpholino) ethane-sulfonic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF phenylmethyl sulfonyl fluoride - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - GTP guanosine triphosphate - NP-40 Nonidet P-40 - DMSO dimethylsulfoxide - EPC ethyl N-phenylcarbamate - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - DAPI 4,6-diamidiho-2-phenylindole     

高等植物根细胞高亲和性吸收钾的机制

K^ 是高等植物所必需的大量元素,它在植物的膨压调节、电荷平衡、叶片运动和蛋白质合成中都具有重要的作用。高等植物根细胞吸收K^ 通过高亲和性K^ 吸收系统和低亲和性K^ 吸收系统和低亲和性K^ 吸收系统两条途径。高新和性K^ 吸收系统,是在微摩尔浓度的外界K^ 水平时起作用,K^ 的吸收必须消耗能量。近年来,随着分子生物学技术和电生理技术的飞速发展,植物根细胞吸收K^ 的机制取得了较大进展。本文对高等植物根细胞高亲和性吸收K^ 的机制的研究进展进行了综述。