Science.gov

Sample records for pombe cell wall

  1. Chromatin and Cell Wall Staining of Schizosaccharomyces pombe.

    PubMed

    Hagan, Iain M

    2016-01-01

    Fission yeasts grow by tip extension, maintaining a constant width until they reach a critical size threshold and divide. Division by medial fission-which gives these yeast their name-generates a new end that arises from the site of cytokinesis. The old end, which was produced during the previous cell cycle, initiates progression of the new cell cycle, and in G2, the new end is activated in a process termed new-end takeoff (NETO). In this protocol, the fluorescent stains calcofluor and 4',6-diamidino-2-phenylindole (DAPI) are used to give a rapid and informative assessment of morphogenesis and cell-cycle progression in the fission yeast Schizosaccharomyces pombe Calcofluor reveals the timing of NETO because it stains the birth scars that are generated at new ends by cytokinesis less efficiently than the rest of the cell wall. Intense calcofluor staining of the septum and measurement of cell length are also widely used to identify dividing cells and to gauge the timing of mitotic commitment. Staining nuclei with DAPI identifies mono- and binucleated cells and complements the calcofluor staining procedure to evaluate the stages of the cell cycle and identify mitotic errors. Equally simple DAPI staining procedures reveal chromatin structure in higher resolution, facilitating more accurate staging of mitotic progression and characterization of mitotic errors. PMID:27250942

  2. The occurrence of glucosaminoglycan in the wall of Schizosaccharomyces pombe.

    PubMed

    Sietsma, J H; Wessels, J G

    1990-11-01

    The major part of the wall of Schizosaccharomyces pombe consists of (1----3)-alpha-glucan and (1----3)-beta-glucan with some (1----6)-beta-linkages. Although in hydrolysed samples only a minute amount of glucosamine could be detected, this amino sugar may play an essential role as an integral part of a glucosaminoglycan/glucan complex. Treatment of the wall with either nitrous acid or chitinase changed the solubility properties of the beta-glucan, which suggests that the glucosaminoglycan/glucan complex is essentially similar to that found in walls of other fungi. An enzyme with properties similar to that of chitin synthase of other fungi, and probably responsible for the synthesis of the glucosaminoglycan, was detected in a mixed-membrane fraction. PMID:2079623

  3. Analysis of the Schizosaccharomyces pombe Cell Cycle.

    PubMed

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-01-01

    Schizosaccharomyces pombe cells are rod shaped, and they grow by tip elongation. Growth ceases during mitosis and cell division; therefore, the length of a septated cell is a direct measure of the timing of mitotic commitment, and the length of a wild-type cell is an indicator of its position in the cell cycle. A large number of documented stage-specific changes can be used as landmarks to characterize cell cycle progression under specific experimental conditions. Conditional mutations can permanently or transiently block the cell cycle at almost any stage. Large, synchronously dividing cell populations, essential for the biochemical analysis of cell cycle events, can be generated by induction synchrony (arrest-release of a cell cycle mutant) or selection synchrony (centrifugal elutriation or lactose-gradient centrifugation). Schizosaccharomyces pombe cell cycle studies routinely combine particular markers, mutants, and synchronization procedures to manipulate the cycle. We describe these techniques and list key landmarks in the fission yeast mitotic cell division cycle. PMID:27587785

  4. Buoyant density constancy of Schizosaccharomyces pombe cells

    SciTech Connect

    Kubitschek, H.E.; Ward, R.A.

    1985-06-01

    Buoyant densities of cells from exponentially growing cultures of the fission yeast Schizosaccharomyces pombe 972h/sup -/ with division rates from 0.14 to 0.5 per h were determined by equilibrium centrifugation in Percoll gradients. Buoyant densities were independent of growth rate, with an average value (+/- standard error) of 1.0945 (+/- 0.00037) g/ml. When cells from these cultures were separated by size, mean cell volumes were independent of buoyant density, indicating that buoyant densities also were independent of cell age during the division cycle. These results support the suggestion that most or all kinds of cells that divide by equatorial fission may have similar, evolutionarily conserved mechanisms for regulation of buoyant density.

  5. Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

    PubMed Central

    Tanaka, Naotaka; Awai, Atsuro; Bhuiyan, M. Shah Alam; Fujita, Kiyotaka; Fukui, Hiroshi; Takegawa, Kaoru

    1999-01-01

    We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe. PMID:9973368

  6. POMB/ACE chemotherapy for mediastinal germ cell tumours.

    PubMed

    Bower, M; Brock, C; Holden, L; Nelstrop, A; Makey, A R; Rustin, G J; Newlands, E S

    1997-05-01

    Mediastinal germ cell tumours (MGCT) are rare and most published series reflect the experiences of individual institutions over many years. Since 1979, we have treated 16 men (12 non-seminomatous germ cell tumours and 4 seminomas) with newly diagnosed primary MGCT with POMB/ACE chemotherapy and elective surgical resection of residual masses. This approach yielded complete remissions in 15/16 (94%) patients. The median follow-up was 6.0 years and no relapses occurred more than 2 years after treatment. The 5 year overall survival in the non-seminomatous germ cell tumours (NSGCT) is 73% (95% confidence interval 43-90%). One patient with NSGCT developed drug-resistant disease and died without achieving remission and 2 patients died of relapsed disease. In addition, 4 patients with bulky and/or metastatic seminoma were treated with POMB/ACE. One died of treatment-related neutropenic sepsis in complete remission and one died of relapsed disease. Finally, 4 patients (2 NSGCT and 2 seminomas) referred at relapse were treated with POMB/ACE and one was successfully salvaged. The combination of POMB/ACE chemotherapy and surgery is effective management for MGCT producing high long-term survival rates. PMID:9291802

  7. Fixed-Cell Imaging of Schizosaccharomyces pombe.

    PubMed

    Hagan, Iain M; Bagley, Steven

    2016-01-01

    The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. PMID:27371603

  8. Interplanetary Migration of Eucaryotic Cell, Spore of Schizosaccharomyces Pombe

    NASA Astrophysics Data System (ADS)

    Hayashi, N.; Nosaka, J.; Ando, R.; Hashimoto, H.; Yokobori, S.; Narumi, I.; Nakagawa, K.; Yamagishi, A.; Tohda, H.

    2013-11-01

    The Tanpopo mission to examine possible interplanetary migration of microbes is progressing. Spore of Schizosaccharomyces pombe are considered as the exposed samples. In this paper, results of preliminary experiments for the exposure are shown.

  9. Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

    NASA Astrophysics Data System (ADS)

    Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

    2015-11-01

    As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations.

  10. Time-lapse electrical impedance spectroscopy for monitoring the cell cycle of single immobilized S. pombe cells

    PubMed Central

    Zhu, Zhen; Frey, Olivier; Haandbaek, Niels; Franke, Felix; Rudolf, Fabian; Hierlemann, Andreas

    2015-01-01

    As a complement and alternative to optical methods, wide-band electrical impedance spectroscopy (EIS) enables multi-parameter, label-free and real-time detection of cellular and subcellular features. We report on a microfluidics-based system designed to reliably capture single rod-shaped Schizosaccharomyces pombe cells by applying suction through orifices in a channel wall. The system enables subsequent culturing of immobilized cells in an upright position, while dynamic changes in cell-cycle state and morphology were continuously monitored through EIS over a broad frequency range. Besides measuring cell growth, clear impedance signals for nuclear division have been obtained. The EIS system has been characterized with respect to sensitivity and detection limits. The spatial resolution in measuring cell length was 0.25 μm, which corresponds to approximately a 5-min interval of cell growth under standard conditions. The comprehensive impedance data sets were also used to determine the occurrence of nuclear division and cytokinesis. The obtained results have been validated through concurrent confocal imaging and plausibilized through comparison with finite-element modeling data. The possibility to monitor cellular and intracellular features of single S. pombe cells during the cell cycle at high spatiotemporal resolution renders the presented microfluidics-based EIS system a suitable tool for dynamic single-cell investigations. PMID:26608589

  11. Cell Cycle Synchronization of Schizosaccharomyces pombe by Lactose Gradient Centrifugation to Isolate Small Cells.

    PubMed

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-01-01

    Size selection of small cells from an asynchronous Schizosaccharomyces pombe culture offers a simple way to generate cultures in which progression through the mitotic cell division cycle is synchronized throughout the population. Here, we describe how density centrifugation of cells from asynchronous cultures through lactose gradients selects small G2 cells to generate synchronized cultures as large as 500 mL. The ease and simplicity of this approach makes it an accessible and attractive method for generating synchronous cultures. PMID:27250945

  12. [Comparing Cell Toxicity of Schizosaccharomyces pombe Exposure to Airborne PM2.5 from Beijing and Inert Particle SiO2].

    PubMed

    Liu, Meng-jiao; Huang, Yi; Wen, Hang; Qiu, Guo-yu

    2015-11-01

    To figure out the main factor of PM2.5 toxicity to cell, this study compared the cell toxicity of Schizosaccharomyces pombe (S. pombe), a model organism, exposed to inert ultrafine SiO2 particles, a model particle, and airborne PM2.5 collected from campus of Peking University Beijing China. Using ultraviolet spectrophotometry to measure cell proliferation ratio, and environmental scanning microscope to observe the particle adhesion on the cell surface, and detecting cellular ROS generation with DHE fluorescent dye chromogenic method, and using single cell gel electrophoresis to test cell DNA damage, the experiment results indicated that the ultrafine SiO2 particles (< 60 nm) could inhibit the cell proliferation of S. pombe, mainly through adsorbing onto the cell surface to change the permeability of the cell wall; but it could not induce cells to generate ROS to cause the oxidative damage. PM2.5, the average particle size of which was larger than that of SiO2 particles, could cause oxidative damages to cells mainly by inducing cells to generate ROS, and damage DNA simultaneously. It might illustrate that there was no direct relationship between the toxicity of PM2.5 and its physical properties such as the particle size. PMID:26910977

  13. PombeX: robust cell segmentation for fission yeast transillumination images.

    PubMed

    Peng, Jyh-Ying; Chen, Yen-Jen; Green, Marc D; Sabatinos, Sarah A; Forsburg, Susan L; Hsu, Chun-Nan

    2013-01-01

    Schizosaccharomyces pombe shares many genes and proteins with humans and is a good model for chromosome behavior and DNA dynamics, which can be analyzed by visualizing the behavior of fluorescently tagged proteins in vivo. Performing a genome-wide screen for changes in such proteins requires developing methods that automate analysis of a large amount of images, the first step of which requires robust segmentation of the cell. We developed a segmentation system, PombeX, that can segment cells from transmitted illumination images with focus gradient and varying contrast. Corrections for focus gradient are applied to the image to aid in accurate detection of cell membrane and cytoplasm pixels, which is used to generate initial contours for cells. Gradient vector flow snake evolution is used to obtain the final cell contours. Finally, a machine learning-based validation of cell contours removes most incorrect or spurious contours. Quantitative evaluations show overall good segmentation performance on a large set of images, regardless of differences in image quality, lighting condition, focus condition and phenotypic profile. Comparisons with recent related methods for yeast cells show that PombeX outperforms current methods, both in terms of segmentation accuracy and computational speed. PMID:24353754

  14. Aging and Cell Death in the Other Yeasts, Schizosaccharomyces pombe and Candida albicans

    PubMed Central

    Lin, Su-Ju; Austriaco, Nicanor

    2013-01-01

    How do cells age and die? For the past twenty years, the budding yeast, Saccharomyces cerevisiae, has been used as a model organism to uncover the genes that regulate lifespan and cell death. More recently, investigators have begun to interrogate the other yeasts, the fission yeast, Schizosaccharomyces pombe, and the human fungal pathogen, Candida albicans, to determine if similar longevity and cell death pathways exist in these organisms. After summarizing the longevity and cell death phenotypes in S. cerevisiae, this mini-review surveys the progress made in the study of both aging and programmed cell death (PCD) in the yeast models, with a focus on the biology of S. pombe and C. albicans. Particular emphasis is placed on the similarities and differences between the two types of aging, replicative aging and chronological aging, and between the three types of cell death, intrinsic apoptosis, autophagic cell death, and regulated necrosis, found in these yeasts. The development of the additional microbial models for aging and PCD in the other yeasts may help further elucidate the mechanisms of longevity and cell death regulation in eukaryotes. PMID:24205865

  15. Cell Cycle Synchronization of Schizosaccharomyces pombe by Centrifugal Elutriation of Small Cells.

    PubMed

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-01-01

    Division of Schizosaccharomyces pombe by medial fission produces identically sized daughter cells that grow by tip extension until their own division is prompted by reaching the same critical size for division as the parental cell. The fidelity of this size control in the absence of perturbation means that cells of the same size are at the same point in the cell cycle. Size selection of small cells from an asynchronous culture by centrifugal elutriation permits generation of synchronous cultures large enough for biochemical analysis. The changes observed in the synchronized cell cycle progression of such cultures are representative of those that accompany cell cycle progression of individual cells. Here, we describe how size selection with the Beckman Coulter JE-5.0 rotor can be used to generate synchronized cultures. Because of the continuous passage of medium through the rotor throughout the procedure, elutriation is considered to have less impact on the integrity of the cell cycle than other approaches. Two protocols are presented here: The first generates a 2-L culture ideal for detailed biochemical analysis, whereas the second allows rapid generation and simultaneous analysis of three smaller (200-mL) cultures. PMID:27250944

  16. Orientation of Schizosaccharomyces POMBE Nonliving Cells under Alternating Uniform and Nonuniform Electric Fields

    PubMed Central

    Iglesias, F. J.; López, M. C.; Santamaría, C.; Domínguez, A.

    1985-01-01

    When nonliving cells of Schizosaccharomyces pombe were subjected to the action of alternating uniform and nonuniform electric fields, two types of orientation were produced. The first one, with its longest axis parallel to the field lines, is similar to that obtained with living cells. The second, perpendicular to the direction of the field, is produced for relatively high frequencies and low conductivities; this probably takes place when the conductivities of the external and internal media (cell cytoplasm) become equal. A mixed cell population is produced in a discrete interval of the parameters used. Our results provide direct evidence that cell alignment does not depend on the physiological state of the cells. ImagesFIGURE 1FIGURE 3 PMID:19431597

  17. Relationships between cell cycle regulator gene copy numbers and protein expression levels in Schizosaccharomyces pombe.

    PubMed

    Chino, Ayako; Makanae, Koji; Moriya, Hisao

    2013-01-01

    We previously determined the copy number limits of overexpression for cell division cycle (cdc) regulatory genes in the fission yeast Schizosaccharomyces pombe using the "genetic tug-of-war" (gTOW) method. In this study, we measured the levels of tandem affinity purification (TAP)-tagged target proteins when their copy numbers are increased in gTOW. Twenty analyzed genes showed roughly linear correlations between increased protein levels and gene copy numbers, which suggested a general lack of compensation for gene dosage in S. pombe. Cdc16 and Sid2 protein levels but not their mRNA levels were much lower than that expected by their copy numbers, which suggested the existence of a post-transcriptional down regulation of these genes. The cyclin Cig1 protein level and its mRNA level were much higher than that expected by its copy numbers, which suggested a positive feedback mechanism for its expression. A higher Cdc10 protein level and its mRNA level, probably due to cloning its gene into a plasmid, indicated that Cdc10 regulation was more robust than that previously predicted. PMID:24019917

  18. Linear increase in cell volume during the growth cycle of Schizosaccharomyces pombe

    SciTech Connect

    Kubitschek, H.E.; Clay, K.

    1985-01-01

    Classical observations on the growth of cells of the fission yeast Schizosaccharomyces pombe indicate that cell length and volume increase to a maximum value, which is reached about three-quarters of the way through the growth cycle, followed by a constant volume plateau. The authors have reexamined the growth of these cells by phase microscopy. When growth conditions were perturbed by inoculating cells at high density, in the presence of contaminant, or upon agar slips containing the growth medium in 4% agar, all cells grew in the classical pattern. But, at smaller agar concentrations and lower cell densities, many cells grew at a constant rate throughout the entire cell cycle. Also, the frequency of this linear pattern of cell growth increased as growth perturbations were reduced. They interpret these results as evidence for a sensitivity of this microorganism to perturbations of steady-state growth. The constant rate of volume increase throughout the cell cycle in unperturbed cells, when considered along with Mitchison's earlier results for constant dry mass increase, suggests that the buoyant densities of these cells remain constant during the entire cell cycle.

  19. Stress-induced response, localization, and regulation of the Pmk1 cell integrity pathway in Schizosaccharomyces pombe.

    PubMed

    Madrid, Marisa; Soto, Teresa; Khong, Hou Keat; Franco, Alejandro; Vicente, Jero; Pérez, Pilar; Gacto, Mariano; Cansado, José

    2006-01-27

    Mitogen-activated protein kinase (MAPK) signaling pathways are critical for the sensing and response of eukaryotic cells to extracellular changes. In Schizosaccharomyces pombe, MAPK Pmk1/Spm1 has been involved in cell wall construction, morphogenesis, cytokinesis, and ion homeostasis, as part of the so-called cell integrity pathway together with MAPK kinase kinase Mkh1 and MAPK kinase Pek1. We show that Pmk1 is activated in multiple stress situations, including hyper- or hypotonic stress, glucose deprivation, presence of cell wall-damaging compounds, and oxidative stress induced by hydrogen peroxide or pro-oxidants. The stress-induced activation of Pmk1 was completely dependent on Mkh1 and Pek1 function, supporting a nonbranched pathway in the regulation of MAPK activation. Fluorescence microscopy revealed that Mkh1, Pek1, and Pmp1 (a protein phosphatase that inactivates Pmk1) are cytoplasmic proteins. Mkh1 and Pek1 were also found at the septum, whereas Pmk1 localized in both cytoplasm and nucleus as well as in the mitotic spindle and septum during cytokinesis. Interestingly, Pmk1 subcellular localization was unaffected by stress or the absence of Mkh1 and Pek1, suggesting that its activation by the Mkh1-Pek1 cascade takes place at the cytoplasm and/or septum and that the active and inactive forms of this kinase cross the nuclear membrane. Cdc42 GTPase and its effectors, p21-activated kinases Pak2 and Pak1, are not upstream elements controlling the basal level or the stress-induced activation of Pmk1. However, Sty1 MAPK was essential for proper Pmk1 deactivation after hypertonic stress in a process regulated by Atf1 transcription factor. These results provide the first evidence for the existence of cross-talk between two MAPK cascades during the stress response in fission yeast. PMID:16291757

  20. Preparation and performance of immobilized yeast cells in columns containing no inert carrier. [Schizosaccharomyces pombe

    SciTech Connect

    Hsiao, H.Y.; Chiang, L.C.; Yang, C.M.; Chen, L.F.; Tsao, G.T.

    1983-02-01

    Schizosaccharomyes pombe was cultivated in a medium of glucose (10 g/l), malt extract (3 g/l), yeast extract (3 g/l), and bactopeptone (5 g/l) to form flocs. More than 95% of the cell population were flocculated. Variation in glucose concentration (from 10 to 11 g/l) did not affect flocculation. Yeast extract helped induce flocculation. Application of the immobilized yeast for the continuous production of ethanol was tested in a column reactor. Soft yeast flocs (50-200 mesh) underwent morphological changes to heavy particles (0.1-9.3 cm diameter) after continuously being fed with fresh substrates in the column. Productivity as high as 87 g EtOH/l/hour was obtained when a 150 g/l glucose medium was fed. The performance of this yeast reactor was stable over a two-month period. The ethanol yield was 97% of the theoretical maximum based upon glucose consumed. (Refs. 16).

  1. Cell wall integrity

    PubMed Central

    Pogorelko, Gennady; Lionetti, Vincenzo; Bellincampi, Daniela; Zabotina, Olga

    2013-01-01

    The plant cell wall, a dynamic network of polysaccharides and glycoproteins of significant compositional and structural complexity, functions in plant growth, development and stress responses. In recent years, the existence of plant cell wall integrity (CWI) maintenance mechanisms has been demonstrated, but little is known about the signaling pathways involved, or their components. Examination of key mutants has shed light on the relationships between cell wall remodeling and plant cell responses, indicating a central role for the regulatory network that monitors and controls cell wall performance and integrity. In this review, we present a short overview of cell wall composition and discuss post-synthetic cell wall modification as a valuable approach for studying CWI perception and signaling pathways. PMID:23857352

  2. The Lamportian cell wall

    SciTech Connect

    Keiliszewski, M.; Lamport, D. )

    1991-05-01

    The Lamportian Warp-Weft hypothesis suggests a cellulose-extensin interpenetrating network where extensin mechanically couples the load-bearing cellulose microfibrils in a wall matrix that is best described as a microcomposite. This model is based on data gathered from the extensin-rich walls of tomato and sycamore cell suspension culture, wherein extensin precursors are insolubilized into the wall by undefined crosslinks. The authors recent work with cell walls isolated from intact tissue as well as walls from suspension cultured cells of the graminaceous monocots maize and rice, the non-graminaceous monocot asparagus, the primitive herbaceous dicot sugar beet, and the gymnosperm Douglas Fir indicate that although extensins are ubiquitous to all plant species examined, they are not the major structural protein component of most walls examined. Amino acid analyses of intact and HF-treated walls shows a major component neither an HRGP, nor directly comparable to the glycine-rich wall proteins such as those associated with seed coat walls or the 67 mole% glycine-rich proteins cloned from petunia and soybean. Clearly, structural wall protein alternatives to extensin exist and any cell wall model must take that into account. If we assume that extracellular matrices are a priori network structures, then new Hypless' structural proteins in the maize cell wall raise questions about the sort of network these proteins create: the kinds of crosslinks involved; how they are formed; and the roles played by the small amounts of HRGPs.

  3. Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Meyers, Alex; Del Rio, Zuania P; Beaver, Rachael A; Morris, Ryan M; Weiskittel, Taylor M; Alshibli, Amany K; Mannik, Jaana; Morrell-Falvey, Jennifer; Dalhaimer, Paul

    2016-06-01

    Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell. PMID:26990381

  4. A microPIXE investigation of the interaction of cells of Schizosaccharomyces pombe with the culture medium

    NASA Astrophysics Data System (ADS)

    Rombouts, P. M. M.; Gomez-Morilla, I.; Grime, G. W.; Webb, R. P.; Cuenca, L.; Rodriguez, R.; Browton, M.; Wardell, N.; Underwood, B.; Kirkby, N. F.; Kirkby, K. J.

    2007-07-01

    Schizosaccharomyces pombe ( S. pombe) is a eucaryotic cell type similar to mammalian cells but much more simple. As it also executes its cell cycle rapidly it is very useful for investigating basic processes in cells. In this paper we report a feasibility study of the applicability of microPIXE to investigate the interaction between S. pombe cells and the surrounding culture medium. Cells were cultured in various growth medium prior to preparation for analysis. 1 μl drops of medium and cells were spotted onto polypropylene foils held in contact with a polished copper block previously cooled in liquid nitrogen. The samples were dehydrated by freeze-drying. Micro PIXE analysis was carried out with the IBC microbeam facility using a beam of 2.5 MeV protons focused to 1-2 μm diameter. Initially no elemental contrast was observed between the cells and the medium, but by modifying the dilution of the cell suspension, the cells could be distinguished from the surrounding medium through an increased concentration of P and reduced concentration of Cl. The distribution of Na in the medium around the cells showed evidence of the action of the Na pump. Sporulation appears to be induced in the cells by adding Cu to the growth medium and the uptake of Cu by the cells could be clearly observed. This study shows that it is possible to analyse the mass transport of elements in and out of cells In the future this will enable concentration gradients to be analysed and allow the rate of production or consumption of individual cells to be calculated. By observing these patterns for individual cells (not populations) at various known points in the cell cycle, fundamental data can be derived.

  5. Relationship between extracellular enzymes and cell growth during the cell cycle of the fission yeast Schizosaccharomyces pombe: acid phosphatase.

    PubMed Central

    Miyata, M; Miyata, H

    1978-01-01

    By using the intact cells of the fission yeast Schizosaccharomyces pombe, the activity of acid phosphatase (EC 3.1.3.2) was compared through the cell cycle with the growth in cell length as a measure of cell growth. The cells of a growing asynchronous culture increased exponentially in number and in total enzyme activity, but remained constant in average length and in specific activity, In a synchronous culture prepared by selection or by induction, the specific activity was periodic in parallel with the increase in average cell length. When hydroxyurea was added to an asynchronous or a synchronous culture by selection, both specific and total activity followed the same continuous pattern as the growth in cell length after the stoppage of cell division. When oversized cells produced by a hydroxyurea pulse treatment to the culture previously syndronized by selection were transferred to a poor medium, they divided synchronously but could hardly grow in the total cell length. In this experimental situation, the total enzyme activity also scarcely increased through three division cycles. These results suggested that the increase in acid phosphatase in dependent on cell elongation. PMID:711673

  6. Bacterial Cell Wall Components

    NASA Astrophysics Data System (ADS)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  7. Dual functions for the Schizosaccharomyces pombe inositol kinase Ipk1 in nuclear mRNA export and polarized cell growth.

    PubMed

    Sarmah, Bhaskarjyoti; Wente, Susan R

    2009-02-01

    The inositol 1,3,4,5,6-pentakisphosphate (IP(5)) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP(6)) in eukaryotic cells. Previous studies have shown that IP(6) is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1(+) in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1(+) deleted (ipk1Delta) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP(6) production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1(+) function, encoding an IP(6) kinase downstream of Ipk1, had an effect on ipk1Delta cells. The asp1Delta mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Delta cells. Moreover, ipk1Delta asp1Delta double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Delta mutant. This suggested novel roles for asp1(+) upstream of ipk1(+). We propose that IP(6) production is a key signaling linchpin for regulating multiple essential cellular processes. PMID:19047361

  8. Dielectric energy of orientation in dead and living cells of Schizosaccharomyces pombe. Fitting of experimental results to a theoretical model.

    PubMed Central

    Asencor, F J; Santamaría, C; Iglesias, F J; Domínguez, A

    1993-01-01

    Using the experimental data obtained with killed cells of Schizosaccharomyces pombe (1), we have formulated a theoretical model that is able to predict cell orientation for microorganisms with ellipsoidal or cylindrical shapes as a function of the frequency of the electric field and of the conductivity of the external medium. In this model, comparison of the difference in potential energy for both orientations parallel-perpendicular with the thermal agitation energy allows one to interpret the intervals where these orientations occur. The model implies that the conductivity of the cytoplasm is slightly higher than that of the external medium. This assumption is easy to understand taking into account that not all the intracytoplasmic material is released to the exterior during cell death. PMID:8324197

  9. Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation. Finally, DNA is precipitated using isopropanol. At this stage, purity is usually sufficient for PCR. However, for more sensitive procedures, such as restriction enzyme digestion, additional purification steps, including proteinase K digestion and phenol-chloroform extraction, are recommended. All of these steps are described in detail here. PMID:27140918

  10. Colony Polymerase Chain Reaction with Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR. PMID:27140919

  11. Production of D-amino acid oxidase (DAO) of Trigonopsis variabilis in Schizosaccharomyces pombe and the characterization of biocatalysts prepared with recombinant cells.

    PubMed

    Isoai, Atsushi; Kimura, Hidetoshi; Reichert, Arno; Schörgendorfer, Kurt; Nikaido, Kiyokazu; Tohda, Hideki; Giga-Hama, Yuko; Mutoh, Norihiro; Kumagai, Hiromichi

    2002-10-01

    The cDNA of D-amino acid oxidase (DAO) gene isolated from Trigonopsis variabilis was expressed in Schizosaccharomyces pombe. A clone, ASP327-10, transformed with plasmid vector, pTL2M5DAO, expressed catalytically active DAO in the presence of G418, and converted Cephalosprin C to alpha-ketoadipyl-7-cephalosporanic acid (KA-7-ACA) and glutaryl-7-aminocephalosporanic acid (GL-7-ACA). Biocatalysts were prepared using ASP327-10 and T. variabilis, and evaluated to demonstrate the feasibility of recombinant S. pombe for industrial application. The cells were immobilized by crosslinking polyethylene imine after glutardialdehyde (GDA) fixation and permeabilization by alkaline treatment. Although the biocatalyst prepared from ASP327-10 exhibited DAO activity, catalase activity still remained fully even after permeabilization, under which condition, the catalase activity of T. variabilis decreased to 20-30%. Heat treatment was required before cell fixation by GDA to inactivate the catalase in S. pombe. This improved the efficiency of bioconversion to GL-7-ACA, but caused poor mechanical strength in the biocatalyst of S. pombe. To overcome this weakness, a catalase-deficient host strain was obtained by ethylmethansulfate mutagenesis. Moreover, taking economics into consideration, the integrative vector, pTL2M5DAO-8XL, with multi-copies of expression cassette was constructed to express DAO in S. pombe even in the absence of G418. The newly established integrant, ASP417-7, did not exhibit any catalase activity so that heat treatment was not required. The obtained integrant and its biocatalyst were significantly improved in GL-7ACA conversion ability and mechanical strength. This study demonstrates that the established integrant is a potential candidate as an alternative source of DAO enzyme. PMID:12209783

  12. Isolation and characterization of Schizosaccharomyces pombe fragile mutants.

    PubMed

    Belda, F; Zárate, V

    1996-05-01

    Three Schizosaccharomyces pombe fragile mutants requiring the presence of an osmotic stabilizer to grow, that lyse when transferred into hypotonic solutions and that secrete to the extracellular medium more protein than the parental strain were isolated. In the three mutants, the fragile phenotype segregated in a Mendelian fashion, indicating a single chromosomal gene mutation, and behaved as a recessive character. By complementation analysis, the three fragile mutants fell in a single complementation group, defining the same gene (SRB1). Mutations of this gene are responsible for alterations in the cells such as fragile character, increase in the cell wall porosity, changes in the cell morphology and floc-forming ability. The study of the three srb1 alleles indicated that the degree of these alterations is proportional to a significant decrease in the galactomannan fraction of the mutants cell wall. The data presented in this report suggest that the product of the SRB1 gene is critical for the maintenance of the integrity and structure of Sz. pombe cell wall. PMID:8771710

  13. Regulation of the antioxidant system in cells of the fission yeast Schizosaccharomyces pombe after combined treatment with patulin and citrinin.

    PubMed

    Papp, Gábor; Máté, Gábor; Mike, Nóra; Gazdag, Zoltán; Pesti, Miklós

    2016-03-01

    The effects of combined treatment with patulin (PAT) and citrinin (CTN) on Schizosaccharomyces pombe cells were investigated in acute toxicity tests. In comparison with the controls the exposure of fission yeast cells (10(7) cells ml(-1)) to PAT + CTN (250 μM each) for 1 h at a survival rate of 66.6% significantly elevated the concentration of total reactive oxygen species (ROS) via increased levels of peroxides without affecting the concentrations of superoxides or the hydroxyl radical. This treatment induced a 3.08-fold increase in the specific concentration of glutathione and elevated specific activities of catalase and glutathione S-transferase, while at the same time the activity of glutathione reductase decreased. The pattern of the ROS was the same as that induced by CTN (Máté et al., 2014), while the presence of PAT in the PAT + CTN combination treatment modified the activities of the antioxidant system (Papp et al., 2012) in comparison with the individual PAT or CTN treatment, suggesting toxin-specific regulation of glutathione and the enzymes of the antioxidant system and the possibility that the transcription factor (pap1 and atf1) -regulated processes might be influenced directly by ROS. PMID:26752674

  14. S. pombe CLASP needs dynein, not EB1 or CLIP170, to induce microtubule instability and slows polymerization rates at cell tips in a dynein-dependent manner

    PubMed Central

    Grallert, Agnes; Beuter, Christoph; Craven, Rachel A.; Bagley, Steve; Wilks, Deepti; Fleig, Ursula; Hagan, Iain M.

    2006-01-01

    The Schizosaccharomyces pombe CLIP170-associated protein (CLASP) Peg1 was identified in a screen for mutants with spindle formation defects and a screen for molecules that antagonized EB1 function. The conditional peg1.1 mutant enabled us to identify key features of Peg1 function. First, Peg1 was required to form a spindle and astral microtubules, yet destabilized interphase microtubules. Second, Peg1 was required to slow the polymerization rate of interphase microtubules that establish end-on contact with the cortex at cell tips. Third, Peg1 antagonized the action of S. pombe CLIP170 (Tip1) and EB1 (Mal3). Fourth, although Peg1 resembled higher eukaryotic CLASPs by physically associating with both Mal3 and Tip1, neither Tip1 nor Mal3 was required for Peg1 to destabilize interphase microtubules or for it to associate with microtubules. Conversely, neither Mal3 nor Tip1 required Peg1 to associate with microtubules or cell tips. Consistently, while mal3.Δ and tip1.Δ disrupted linear growth, corrupting peg1 + did not. Fifth, peg1.1 phenotypes resembled those arising from deletion of the single heavy or both light chains of fission yeast dynein. Furthermore, all interphase phenotypes arising from peg1 + manipulation relied on dynein function. Thus, the impact of S. pombe CLASP on interphase microtubule behavior is more closely aligned to dynein than EB1 or CLIP170. PMID:16951255

  15. Cell wall proteomics of crops

    PubMed Central

    Komatsu, Setsuko; Yanagawa, Yuki

    2012-01-01

    Cell wall proteins play key roles in cell structure and metabolism, cell enlargement, signal transduction, responses to environmental stress, and many other physiological events. Agricultural crops are often used for investigating stress tolerance because cultivars with differing degrees of tolerance are available. Abiotic and biotic stress factors markedly influence the geographical distribution and yields of many crop species. Crop cell wall proteomics is of particular importance for improving crop productivity, particularly under unfavorable environmental conditions. To better understand the mechanisms underlying stress response in crops, cell wall proteomic analyses are being increasingly utilized. In this review, the methods of purification and purity assays of cell wall protein fractions from crops are described, and the results of protein identification using gel-based and gel-free proteomic techniques are presented. Furthermore, protein composition of the cell walls of rice, wheat, maize, and soybean are compared, and the role of cell wall proteins in crops under flooding and drought stress is discussed. This review will be useful for clarifying the role of the cell wall of crops in response to environmental stresses. PMID:23403621

  16. Rho 1 GTPase activates the (1-3)beta-D-glucan synthase and is involved in Schizosaccharomyces pombe morphogenesis.

    PubMed Central

    Arellano, M; Durán, A; Pérez, P

    1996-01-01

    The Schizosaccharomyces pombe Cdc42 and Rho1 GTPases were tested for their ability to complement the cwg2-1 mutant phenotype of a decrease in (1-3)beta-D-glucan synthase activity when grown at the non-permissive temperature. Only Rho1 is able to partly complement the defect in glucan synthase associated with the cwg2-1 mutation. Moreover, overexpression of the rho1 gene in wild-type S.pombe cells causes aberrant morphology with loss of polarity and cells with several septa. Under this condition (1-3)beta-D-glucan synthase activity is increased four times, but is still dependent on GTP. When S.pombe is transformed with constitutively active rho1 mutant alleles (rho1-G15V or rho1-Q64L), cells stop growing and show a very thick cell wall with hardly any septum. Under this condition the level of (1-3)beta-D-glucan synthase activity is at least 20 times higher than wild-type and is independent of GTP. Neither cdc42+ nor the cdc42-V12G or cdc42-Q61L constitutively active mutant alleles affect (1-3)beta-D-glucan synthase activity when overexpressed in S.pombe. Cells overproducing Rho1 are hypersensitive to inhibitors of cell wall biosynthesis or to cell wall degrading enzymes. We conclude that Rho1 GTPase directly activates (1-3)beta-D-glucan synthase and regulates S.pombe morphogenesis. Images PMID:8887550

  17. Cell Lysis in S. pombe ura4 Mutants Is Suppressed by Loss of Functional Pub1, Which Regulates the Uracil Transporter Fur4

    PubMed Central

    Nishino, Kohei; Kushima, Misaki; Matsuo, Yuzy; Matsuo, Yasuhiro; Kawamukai, Makoto

    2015-01-01

    Schizosaccharomyces pombe Δura4 cells lyse when grown on YPD medium. A S. pombe non-essential gene deletion library was screened to determine suppressors of the lysis phenotype. Deletion of the pub1 gene, which encoded E3 ubiquitin ligase, strongly suppressed cell lysis in Δura4 cells. The Δpub1 cells displayed high sensitivity to 5-fluorouracil, a toxic analog of uracil, and this sensitivity was suppressed by deletion of fur4, which encoded a uracil transporter. Fur4 localized primarily to the Golgi apparatus and vacuoles in wild-type cells, but localization was predominantly at the plasma membrane in Δpub1 cells. Fur4 was necessary for the utilization of extracellular uracil, cytosine, or UMP. Uracil uptake activity increased in the Δpub1 strain in a Fur4-dependent manner. In addition, uracil starvation was critical for induction of cell lysis of Δura4 strains and uracil supplementation suppressed lysis. In summary, the increased uracil uptake ability of Δpub1 cells, where Fur4 was predominantly localized to the plasma membrane, resulted in suppression of cell lysis in the Δura4 background. PMID:26536126

  18. Plant cell walls to ethanol.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Conversion of plant cell walls to ethanol constitutes generation 2 bioethanol production. The process consists of several steps: biomass selection/genetic modification, physiochemical pretreatment, enzymatic saccharification, fermentation, and separation. Ultimately, it is desired to combine as man...

  19. The effect of the cwf14 gene of fission yeast on cell wall integrity is associated with rho1.

    PubMed

    Kim, Dong-Uk; Maeng, Shinae; Lee, Hyemi; Nam, Miyoung; Lee, Sook-Jeong; Hoe, Kwang-Lae

    2016-02-01

    In all eukaryotic organisms, a wide range of morphologies are responsible for critical cellular function and development. In particular, the Rho GTPases, which are highly conserved from yeast to mammals, are key molecules in signaling pathways that control cell polarity processes and cell wall biosynthesis, which are fundamental aspects of morphogenesis. Therefore, using haploinsufficiency deletion mutants of the fission yeast Schizosaccharomyces pombe, we screened the slow-growing mutants and their morphogenesis, specifically focusing on regulation of their Rho GTPases. Based on this screening, we found that the cwf14 mutant of S. pombe exhibited the slow growth and abnormal phenotypes with an elongated cell shape and thicker cell wall when compared with wild-type cells. In particular, cells with the cwf14 deletion showed excessive Rho1 expression. However, the wildtype strain with ectopically expressed Rho1 did not exhibited any significant change in the level of cwf14, suggesting that cwf14 may act on the upstream of Rho1. Furthermore, the cells with a cwf14 deletion also have increased sensitivity to β-glucanase, a cell wall-digesting enzyme, which is also seen in Rho1-overexpressing cells. Overall, our results suggest that the cwf14 plays a key role in fission yeast morphogenesis and cell wall biosynthesis and/or degradation possibly via the regulation of Rho1 expression. PMID:26832665

  20. Back wall solar cell

    NASA Technical Reports Server (NTRS)

    Brandhorst, H. W., Jr. (Inventor)

    1978-01-01

    A solar cell is disclosed which comprises a first semiconductor material of one conductivity type with one face having the same conductivity type but more heavily doped to form a field region arranged to receive the radiant energy to be converted to electrical energy, and a layer of a second semiconductor material, preferably highly doped, of opposite conductivity type on the first semiconductor material adjacent the first semiconductor material at an interface remote from the heavily doped field region. Instead of the opposite conductivity layer, a metallic Schottky diode layer may be used, in which case no additional back contact is needed. A contact such as a gridded contact, previous to the radiant energy may be applied to the heavily doped field region of the more heavily doped, same conductivity material for its contact.

  1. Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth▿ †

    PubMed Central

    Sarmah, Bhaskarjyoti; Wente, Susan R.

    2009-01-01

    The inositol 1,3,4,5,6-pentakisphosphate (IP5) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP6) in eukaryotic cells. Previous studies have shown that IP6 is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1+ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1+ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP6 production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1+ function, encoding an IP6 kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1+ upstream of ipk1+. We propose that IP6 production is a key signaling linchpin for regulating multiple essential cellular processes. PMID:19047361

  2. Catalysts of plant cell wall loosening

    PubMed Central

    Cosgrove, Daniel J.

    2016-01-01

    The growing cell wall in plants has conflicting requirements to be strong enough to withstand the high tensile forces generated by cell turgor pressure while selectively yielding to those forces to induce wall stress relaxation, leading to water uptake and polymer movements underlying cell wall expansion. In this article, I review emerging concepts of plant primary cell wall structure, the nature of wall extensibility and the action of expansins, family-9 and -12 endoglucanases, family-16 xyloglucan endotransglycosylase/hydrolase (XTH), and pectin methylesterases, and offer a critical assessment of their wall-loosening activity PMID:26918182

  3. Unique aspects of the grass cell wall

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Grasses are amongst the most important crops worldwide, and the composition of their cell walls is critical for uses as food, feed, and energy crops. Grass cell walls differ dramatically from dicot cell walls in terms of the major structural polysaccharides present, how those polysaccharides are lin...

  4. Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

    PubMed Central

    Zaitsevskaya-Carter, T; Cooper, J A

    1997-01-01

    A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

  5. Transformation of Schizosaccharomyces pombe: Protoplast Procedure.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-04-01

    Transformation of Schizosaccharomyces pombe with DNA requires the conditioning of cells to promote DNA uptake followed by cell growth under conditions that select and maintain the plasmid or integration event. The three main methodologies are electroporation, treatment with lithium cations, and transformation of protoplasts. The protocol for protoplast transformation, which is described here, is more complicated than those for electroporation or lithium acetate and thus less often used. However, for some strains, it remains the only reliable protocol. PMID:27037076

  6. The structure of cell wall alpha-glucan from fission yeast.

    PubMed

    Grün, Christian H; Hochstenbach, Frans; Humbel, Bruno M; Verkleij, Arie J; Sietsma, J Hans; Klis, Frans M; Kamerling, Johannis P; Vliegenthart, Johannes F G

    2005-03-01

    Morphology and structural integrity of fungal cells depend on cell wall polysaccharides. The chemical structure and biosynthesis of two types of these polysaccharides, chitin and (1-->3)-beta-glucan, have been studied extensively, whereas little is known about alpha-glucan. Here we describe the chemical structure of alpha-glucan isolated from wild-type and mutant cell walls of the fission yeast Schizosaccharomyces pombe. Wild-type alpha-glucan was found to consist of a single population of linear glucose polymers, approximately 260 residues in length. These glucose polymers were composed of two interconnected linear chains, each consisting of approximately 120 (1-->3)-linked alpha-d-glucose residues and some (1-->4)-linked alpha-D-glucose residues at the reducing end. By contrast, alpha-glucan of an alpha-glucan synthase mutant with an aberrant cell morphology and reduced alpha-glucan levels consisted of a single chain only. We propose that alpha-glucan biosynthesis involves an ordered series of events, whereby two alpha-glucan chains are coupled to create mature cell wall alpha-glucan. This mature form of cell wall alpha-glucan is essential for fission-yeast morphogenesis. PMID:15470229

  7. The cell wall of Fusarium oxysporum.

    PubMed

    Schoffelmeer, E A; Klis, F M; Sietsma, J H; Cornelissen, B J

    1999-01-01

    Sugar analysis of isolated cell walls from three formae speciales of Fusarium oxysporum showed that they contained not only glucose and (N-acetyl)-glucosamine, but also mannose, galactose, and uronic acids, presumably originating from cell wall glycoproteins. Cell wall glycoproteins accounted for 50-60% of the total mass of the wall. X-ray diffraction studies showed the presence of alpha-1, 3-glucan in the alkali-soluble cell wall fraction and of beta-1, 3-glucan and chitin in the alkali-insoluble fraction. Electron microscopy and lectin binding studies indicated that glycoproteins form an external layer covering an inner layer composed of chitin and glucan. PMID:10441453

  8. Modifying crops to increase cell wall digestibility.

    PubMed

    Jung, Hans-Joachim G; Samac, Deborah A; Sarath, Gautam

    2012-04-01

    Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants is highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-digestible. Digestibility of grasses is slowed severely by lignification of most tissues, but these cell walls remain largely digestible. Cell wall lignification creates an access barrier to potentially digestible wall material by rumen bacteria if cells have not been physically ruptured. Traditional breeding has focused on increasing total dry matter digestibility rather than cell wall digestibility, which has resulted in minimal reductions in cell wall lignification. Brown midrib mutants in some annual grasses exhibit small reductions in lignin concentration and improved cell wall digestibility. Similarly, transgenic approaches down-regulating genes in monolignol synthesis have produced plants with reduced lignin content and improved cell wall digestibility. While major reductions in lignin concentration have been associated with poor plant fitness, smaller reductions in lignin provided measurable improvements in digestibility without significantly impacting agronomic fitness. Additional targets for genetic modification to enhance digestibility and improve roughages for use as biofuel feedstocks are discussed; including manipulating cell wall polysaccharide composition, novel lignin structures, reduced lignin/polysaccharide cross-linking, smaller lignin polymers, enhanced development of non-lignified tissues, and targeting specific cell types. Greater tissue specificity of transgene expression will be needed to maximize benefits while avoiding negative impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promoter. PMID:22325867

  9. The roles of SPBC409.08 and SPAC9.02c hypothetical genes in cell cycle and stress response, in Schizosaccharomyces pombe.

    PubMed

    Güngör, I; Örs Gevrekci, A

    2016-01-01

    Polyamine molecules are known to have important roles in the cell cycle control and fighting against stress in the cell. The mechanism and modification of polyamines are regulated by the cooperation of many proteins such as polyamine transporter proteins and polyamine acetyltransferases. In this study, our aim is to characterize two hypothetical Schizosaccharomyces pombe genes, SPBC409.08 and SPAC9.02c, which show sequence similarity to spermine family transporters and polyamine N-acetyltransferases, respectively. To this end, we generated deletion mutants of SPBC409.08 and SPAC9.02c genes using Bahler method and checked the cell cycle progression and stress responses of these mutants. Our results showed that SPBC409.08Δ cells showed some defects in the cell size, while SPAC9.02cΔ cells showed some sensitivity to UV irradiation. These data support their potential roles in the cell cycle and stress response. To our knowledge our results are the first experimental characterization of these genes. PMID:27188733

  10. Moss cell walls: structure and biosynthesis

    PubMed Central

    Roberts, Alison W.; Roberts, Eric M.; Haigler, Candace H.

    2012-01-01

    The genome sequence of the moss Physcomitrella patens has stimulated new research examining the cell wall polysaccharides of mosses and the glycosyl transferases that synthesize them as a means to understand fundamental processes of cell wall biosynthesis and plant cell wall evolution. The cell walls of mosses and vascular plants are composed of the same classes of polysaccharides, but with differences in side chain composition and structure. Similarly, the genomes of P. patens and angiosperms encode the same families of cell wall glycosyl transferases, yet, in many cases these families have diversified independently in each lineage. Our understanding of land plant evolution could be enhanced by more complete knowledge of the relationships among glycosyl transferase functional diversification, cell wall structural and biochemical specialization, and the roles of cell walls in plant adaptation. As a foundation for these studies, we review the features of P. patens as an experimental system, analyses of cell wall composition in various moss species, recent studies that elucidate the structure and biosynthesis of cell wall polysaccharides in P. patens, and phylogenetic analysis of P. patens genes potentially involved in cell wall biosynthesis. PMID:22833752

  11. Architecture of dermatophyte cell Walls: Electron microscopic and biochemical analysis

    NASA Technical Reports Server (NTRS)

    Nozawa, Y.; Kitajima, Y.

    1984-01-01

    A review with 83 references on the cell wall structure of dermatophytes is presented. Topics discussed include separation and preparation of cell walls; microstructure of cell walls by electron microscopy; chemical composition of cell walls; structural model of cell walls; and morphological structure of cell walls.

  12. Natural Paradigms of Plant Cell Wall Degradation

    SciTech Connect

    Wei, H.; Xu, Q.; Taylor, L. E.; Baker, J. O.; Tucker, M. P.; Ding, S. Y.

    2009-01-01

    Natural processes of recycling carbon from plant cell walls are slow but very efficient, generally involving microbial communities and their secreted enzymes. Efficient combinations of microbial communities and enzymes act in a sequential and synergistic manner to degrade plant cell walls. Recent understanding of plant cell wall ultra-structure, as well as the carbon metabolism, ATP production, and ecology of participating microbial communities, and the biochemical properties of their cellulolytic enzymes have led to new perspectives on saccharification of biomass. Microbial communities are dynamic functions of the chemical and structural compositions of plant cell wall components. The primitive 'multicellularity' exhibited by certain cellulolytic microorganisms may play a role in facilitating cell-cell communication and cell-plant cell wall-substrate interaction.

  13. How do plant cell walls extend?

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1993-01-01

    This article briefly summarizes recent work that identifies the biophysical and biochemical processes that give rise to the extension of plant cell walls. I begin with the biophysical notion of stress relaxation of the wall and follow with recent studies of wall enzymes thought to catalyze wall extension and relaxation. Readers should refer to detailed reviews for more comprehensive discussion of earlier literature (Taiz, 1984; Carpita and Gibeaut, 1993; Cosgrove, 1993).

  14. Functional expression of the Chlorella hexose transporter in Schizosaccharomyces pombe.

    PubMed Central

    Sauer, N; Caspari, T; Klebl, F; Tanner, W

    1990-01-01

    Schizosaccharomyces pombe cells were transformed with an S. pombe expression vector containing a full-length cDNA of the Chlorella hexose transporter. The transformed cells accumulated 3-O-methylglucose up to 10-fold, whereas wild-type S. pombe and control transformants could only equilibrate this sugar analogue. In a pH-jump experiment, in which extracellular pH was lowered by 1.9 units, the accumulation ratio was increased in transformed cells but not in control cells. This result indicates that the gene product, Chlorella H+/glucose-symporter protein, and a pH gradient suffice for active sugar uptake. Km values for glucose, 6-deoxyglucose, and 3-O-methylglucose of 1.5 x 10(-5) M, 2.7 x 10(-4) M, and 1.0 x 10(-3) M, respectively, were identical in Chlorella and in S. pombe cells transformed with Chlorella cDNA and approximately 100-fold lower than those of the endogenous transport system of S. pombe. Images PMID:11607110

  15. Fluorescent Labeling of Yeast Cell Wall Components.

    PubMed

    Okada, Hiroki; Ohya, Yoshikazu

    2016-01-01

    Yeast cells stained with a fluorescent dye that specifically binds to one of the cell wall components can be observed under a fluorescent microscope. Visualization of the components 1,3-β-glucan, mannoproteins, and/or chitin not only provides information concerning the cell wall, but also reveals clues about various cellular activities such as cell polarity, vesicular transport, establishment of budding pattern, apical and isotropic bud growth, and replicative cell age. This protocol describes a standard method for visualizing different components of the yeast cell wall. PMID:27480714

  16. Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

    PubMed Central

    Ene, Iuliana V.; Walker, Louise A.; Schiavone, Marion; Lee, Keunsook K.; Martin-Yken, Hélène; Dague, Etienne; Gow, Neil A. R.; Munro, Carol A.

    2015-01-01

    ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. PMID:26220968

  17. Modifying crops to increase cell wall digestibility

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants are highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-dig...

  18. Safranine fluorescent staining of wood cell walls.

    PubMed

    Bond, J; Donaldson, L; Hill, S; Hitchcock, K

    2008-06-01

    Safranine is an azo dye commonly used for plant microscopy, especially as a stain for lignified tissues such as xylem. Safranine fluorescently labels the wood cell wall, producing green/yellow fluorescence in the secondary cell wall and red/orange fluorescence in the middle lamella (ML) region. We examined the fluorescence behavior of safranine under blue light excitation using a variety of wood- and fiber-based samples of known composition to interpret the observed color differentiation of different cell wall types. We also examined the basis for the differences in fluorescence emission using spectral confocal microscopy to examine lignin-rich and cellulose-rich cell walls including reaction wood and decayed wood compared to normal wood. Our results indicate that lignin-rich cell walls, such as the ML of tracheids, the secondary wall of compression wood tracheids, and wood decayed by brown rot, tend to fluoresce red or orange, while cellulose-rich cell walls such as resin canals, wood decayed by white rot, cotton fibers and the G-layer of tension wood fibers, tend to fluoresce green/yellow. This variation in fluorescence emission seems to be due to factors including an emission shift toward red wavelengths combined with dye quenching at shorter wavelengths in regions with high lignin content. Safranine fluorescence provides a useful way to differentiate lignin-rich and cellulose-rich cell walls without counterstaining as required for bright field microscopy. PMID:18802812

  19. Shape dynamics of growing cell walls.

    PubMed

    Banerjee, Shiladitya; Scherer, Norbert F; Dinner, Aaron R

    2016-04-14

    We introduce a general theoretical framework to study the shape dynamics of actively growing and remodeling surfaces. Using this framework we develop a physical model for growing bacterial cell walls and study the interplay of cell shape with the dynamics of growth and constriction. The model allows us to derive constraints on cell wall mechanical energy based on the observed dynamics of cell shape. We predict that exponential growth in cell size requires a constant amount of cell wall energy to be dissipated per unit volume. We use the model to understand and contrast growth in bacteria with different shapes such as spherical, ellipsoidal, cylindrical and toroidal morphologies. Coupling growth to cell wall constriction, we predict a discontinuous shape transformation, from partial constriction to cell division, as a function of the chemical potential driving cell wall synthesis. Our model for cell wall energy and shape dynamics relates growth kinetics with cell geometry, and provides a unified framework to describe the interplay between shape, growth and division in bacterial cells. PMID:26953519

  20. Molecular regulation of plant cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1998-01-01

    Gravity responses in plants often involve spatial and temporal changes in cell growth, which is regulated primarily by controlling the ability of the cell wall to extend. The wall is thought to be a cellulose-hemicellulose network embedded in a hydrated matrix of complex polysaccharides and a small amount of structural protein. The wall extends by a form of polymer creep, which is mediated by expansins, a novel group of wall-loosening proteins. Expansins were discovered during a molecular dissection of the "acid growth" behavior of cell walls. Expansin alters the rheology of plant walls in profound ways, yet its molecular mechanism of action is still uncertain. It lacks detectable hydrolytic activity against the major components of the wall, but it is able to disrupt noncovalent adhesion between wall polysaccharides. The discovery of a second family of expansins (beta-expansins) sheds light on the biological role of a major group of pollen allergens and implies that expansins have evolved for diverse developmental functions. Finally, the contribution of other processes to wall extensibility is briefly summarized.

  1. 2003 Plant Cell Walls Gordon Conference

    SciTech Connect

    Daniel J. Cosgrove

    2004-09-21

    This conference will address recent progress in many aspects of cell wall biology. Molecular, genetic, and genomic approaches are yielding major advances in our understanding of the composition, synthesis, and architecture of plant cell walls and their dynamics during growth, and are identifying the genes that encode the machinery needed to make their biogenesis possible. This meeting will bring together international scientists from academia, industry and government labs to share the latest breakthroughs and perspectives on polysaccharide biosynthesis, wood formation, wall modification, expansion and interaction with other organisms, and genomic & evolutionary analyses of wall-related genes, as well as to discuss recent ''nanotechnological'' advances that take wall analysis to the level of a single cell.

  2. Refractive index of plant cell walls

    NASA Technical Reports Server (NTRS)

    Gausman, H. W.; Allen, W. A.; Escobar, D. E.

    1974-01-01

    Air was replaced with media of higher refractive indices by vacuum infiltration in leaves of cucumber, blackeye pea, tomato, and string bean plants, and reflectance of noninfiltrated and infiltrated leaves was spectrophotometrically measured. Infiltrated leaves reflected less light than noninfiltrated leaves over the 500-2500-nm wavelength interval because cell wall-air interfaces were partly eliminated. Minimal reflectance should occur when the average refractive index of plant cell walls was matched by the infiltrating fluid. Although refractive indices that resulted in minimal reflectance differed among the four plant genera, an average value of 1.425 approximates the refractive index of plant cell walls for the four plant genera.

  3. Tensile Strength of Cell Walls of Living Cells 1

    PubMed Central

    Carpita, Nicholas C.

    1985-01-01

    A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered. PMID:16664436

  4. Secondary cell walls: biosynthesis and manipulation.

    PubMed

    Kumar, Manoj; Campbell, Liam; Turner, Simon

    2016-01-01

    Secondary cell walls (SCWs) are produced by specialized plant cell types, and are particularly important in those cells providing mechanical support or involved in water transport. As the main constituent of plant biomass, secondary cell walls are central to attempts to generate second-generation biofuels. Partly as a consequence of this renewed economic importance, excellent progress has been made in understanding how cell wall components are synthesized. SCWs are largely composed of three main polymers: cellulose, hemicellulose, and lignin. In this review, we will attempt to highlight the most recent progress in understanding the biosynthetic pathways for secondary cell wall components, how these pathways are regulated, and how this knowledge may be exploited to improve cell wall properties that facilitate breakdown without compromising plant growth and productivity. While knowledge of individual components in the pathway has improved dramatically, how they function together to make the final polymers and how these individual polymers are incorporated into the wall remain less well understood. PMID:26663392

  5. Cell wall remodeling under abiotic stress

    PubMed Central

    Tenhaken, Raimund

    2015-01-01

    Plants exposed to abiotic stress respond to unfavorable conditions on multiple levels. One challenge under drought stress is to reduce shoot growth while maintaining root growth, a process requiring differential cell wall synthesis and remodeling. Key players in this process are the formation of reactive oxygen species (ROS) and peroxidases, which initially cross-link phenolic compounds and glycoproteins of the cell walls causing stiffening. The function of ROS shifts after having converted all the peroxidase substrates in the cell wall. If ROS-levels remain high during prolonged stress, OH°-radicals are formed which lead to polymer cleavage. In concert with xyloglucan modifying enzymes and expansins, the resulting cell wall loosening allows further growth of stressed organs. PMID:25709610

  6. Differential scanning calorimetry of plant cell walls

    SciTech Connect

    Lin, Liangshiou; Varner, J.E. ); Yuen, H.K. )

    1991-03-15

    High-sensitivity differential scanning calorimetry has been used to study the phase transition of cell wall preparations of the elongating and mature regions of soybean hypocotyls and of celery epidermis and collenchyma strands. A step-like transition believed to be glass transition was observed in walls isolated from the elongating region of soybean hypocotyls at 52.9C. Addition of 1 mM CaCl{sub 2} to the cell wall preparation increased the transition temperature to 60.8C and greatly reduced the transition magnitude. In walls from the mature region, the transition was small and occurred at a higher temperature (60.1C). Addition of calcium to the mature region cell wall had little effect on the transition. Based on the known interactions between calcium and pectin, the authors propose that calcium affects the glass transition by binding to the polygalacturonate backbone of wall pectin, resulting in a more rigid wall with a smaller transition at a higher temperature. The mature region either has more calcium in the wall or has more methyl-esterified pectin, making it less responsive to added calcium.

  7. Fission yeast Schizosaccharomyces pombe in continuous culture

    SciTech Connect

    Vrana, D.

    1983-08-01

    The fission yeast Schizosaccharomyces pombe was cultivated in a chemostat at dilution rates of D = 0.03, 0.05, 0.10, and 0.20/h. After steady state has been reached, the amount of dry matter, number of cells, concentration of residual sugar, yield coefficient (Y), and some morphological properties of the cells were estimated. Curves reflecting the dry mass, number of cells, and cell mean volume show a changing coordination between the growth rate and the rate of cell division, with respect of D. In addition, it could be concluded that in dividing cells the cell septum is localized asymmetrically; two nonidentical cells differing both in length and volume result. The degree of asymmetry is a function of the dilution rate. (25 Refs.)

  8. Role of cell wall deconstructing enzymes in the proanthocyanidin-cell wall adsorption-desorption phenomena.

    PubMed

    Castro-López, Liliana del Rocío; Gómez-Plaza, Encarna; Ortega-Regules, Ana; Lozada, Daniel; Bautista-Ortín, Ana Belén

    2016-04-01

    The transference of proanthocyanidins from grapes to wine is quite low. This could be due, among other causes, to proanthocyanidins being bound to grape cell wall polysaccharides, which are present in high concentrations in the must. Therefore, the effective extraction of proanthocyanidins from grapes will depend on the ability to disrupt these associations, and, in this respect, enzymes that degrade these polysaccharides could play an important role. The main objective of this work was to test the behavior of proanthocyanidin-cell wall interactions when commercial maceration enzymes are present in the solution. The results showed that cell wall polysaccharides adsorbed a high amount of proanthocyanidins and only a limited quantity of proanthocyanidins could be desorbed from the cell walls after washing with a model solution. The presence of enzymes in the solution reduced the proanthocyanidin-cell wall interaction, probably through the elimination of pectins from the cell wall network. PMID:26593523

  9. Cell-wall dynamics in growing bacteria

    NASA Astrophysics Data System (ADS)

    Furchtgott, Leon; Wingreen, Ned; Huang, Kerwyn Casey

    2010-03-01

    Bacterial cells come in a large variety of shapes, and cell shape plays an important role in the regulation of many biological functions. Cell shape in bacterial cells is dictated by a cell wall composed of peptidoglycan, a polymer made up of long, stiff glycan strands and flexible peptide crosslinks. Although much is understood about the structural properties of peptidoglycan, little is known about the dynamics of cell wall organization in bacterial cells. In particular, during cell growth, how does the bacterial cell wall continuously expand and reorganize while maintaining cell shape? In order to investigate this question quantitatively, we model the cell wall of the Gram-negative bacterium Escherichia coli using a simple elastic model, in which glycan and peptide subunits are treated as springs with different spring constants and relaxed lengths. We consider the peptidoglycan network as a single-layered network of these springs under tension due to an internal osmotic pressure. Within this model, we simulate possible hypotheses for cell growth as different combinations of addition of new springs and breakage of old springs.

  10. Vesicular transport across the fungal cell wall

    PubMed Central

    Casadevall, Arturo; Nosanchuk, Joshua D.; Williamson, Peter; Rodrigues, Marcio L.

    2014-01-01

    Recent findings indicate that fungi use vesicular transport to deliver substances across their cell walls. Fungal vesicles are similar to mammalian exosomes and could originate from cytoplasmic multivesicular bodies. Vesicular transport enables the export of large molecules across the cell wall, and vesicles contain lipids, proteins and polysaccharides, many of which are associated with virulence. Concentration of fungal products in vesicles could increase their efficiency in food acquisition and/or delivering potentially noxious substances to other cells, such as amoebae or phagocytes. The discovery of vesicular transport in fungi opens many new avenues for investigation in basic cell biology and pathogenesis. PMID:19299133

  11. Structure of Plant Cell Walls 1

    PubMed Central

    Ishii, Tadashi; Thomas, Jerry; Darvill, Alan; Albersheim, Peter

    1989-01-01

    Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na2CO3 at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ∼4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO3-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na2CO3 at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells. PMID:16666559

  12. Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

    PubMed Central

    Levin, David E.

    2011-01-01

    The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

  13. Modes of deformation of walled cells.

    PubMed

    Dumais, Jacques

    2013-11-01

    The bewildering morphological diversity found in cells is one of the starkest illustrations of life's ability to self-organize. Yet the morphogenetic mechanisms that produce the multifarious shapes of cells are still poorly understood. The shared similarities between the walled cells of prokaryotes, many protists, fungi, and plants make these groups particularly appealing to begin investigating how morphological diversity is generated at the cell level. In this review, I attempt a first classification of the different modes of surface deformation used by walled cells. Five modes of deformation were identified: inextensional bending, equi-area shear, elastic stretching, processive intussusception, and chemorheological growth. The two most restrictive modes-inextensional and equi-area deformations-are embodied in the exine of pollen grains and the wall-like pellicle of euglenoids, respectively. For these modes, it is possible to express the deformed geometry of the cell explicitly in terms of the undeformed geometry and other easily observable geometrical parameters. The greatest morphogenetic power is reached with the processive intussusception and chemorheological growth mechanisms that underlie the expansive growth of walled cells. A comparison of these two growth mechanisms suggests a possible way to tackle the complexity behind wall growth. PMID:24014868

  14. Identification of Novel Cell Wall Components

    SciTech Connect

    Michelle Momany

    2009-10-26

    Our DOE Biosciences-funded work focused on the fungal cell wall and morphogenesis. We are especially interested in how new cell wall material is targeted to appropriate areas for polar (asymmetric) growth. Polar growth is the only way that filamentous fungi explore the environment to find suitable substrates to degrade. Work funded by this grant has resulted in a total of twenty peer-reviewed publications. In work funded by this grant, we identified nine Aspergillus nidulans temperature-sensitive (ts) mutants that fail to send out a germ tube and show a swollen cell phenotype at restrictive temperature, the swo mutants. In other organisms, a swollen cell phenotype is often associated with misdirected growth or weakened cell walls. Our work shows that several of the A. nidulans swo mutants have defects in the establishment and maintenance of polarity. Cloning of several swo genes by complementation also showed that secondary modification of proteins seems is important in polarity. We also investigated cell wall biosynthesis and branching based on leads in literature from other organisms and found that branching and nuclear division are tied and that the cell wall reorganizes during development. In our most recent work we have focused on gene expression during the shift from isotropic to polar growth. Surprisingly we found that genes previously thought to be involved only in spore formation are important in early vegetative growth as well.

  15. Planctomycetes do possess a peptidoglycan cell wall

    PubMed Central

    Jeske, Olga; Schüler, Margarete; Schumann, Peter; Schneider, Alexander; Boedeker, Christian; Jogler, Mareike; Bollschweiler, Daniel; Rohde, Manfred; Mayer, Christoph; Engelhardt, Harald; Spring, Stefan; Jogler, Christian

    2015-01-01

    Most bacteria contain a peptidoglycan (PG) cell wall, which is critical for maintenance of shape and important for cell division. In contrast, Planctomycetes have been proposed to produce a proteinaceous cell wall devoid of PG. The apparent absence of PG has been used as an argument for the putative planctomycetal ancestry of all bacterial lineages. Here we show, employing multiple bioinformatic methods, that planctomycetal genomes encode proteins required for PG synthesis. Furthermore, we biochemically demonstrate the presence of the sugar and the peptide components of PG in Planctomycetes. In addition, light and electron microscopic experiments reveal planctomycetal PG sacculi that are susceptible to lysozyme treatment. Finally, cryo-electron tomography demonstrates that Planctomycetes possess a typical PG cell wall and that their cellular architecture is thus more similar to that of other Gram-negative bacteria. Our findings shed new light on the cellular architecture and cell division of the maverick Planctomycetes. PMID:25964217

  16. Genetic resources for maize cell wall biology.

    PubMed

    Penning, Bryan W; Hunter, Charles T; Tayengwa, Reuben; Eveland, Andrea L; Dugard, Christopher K; Olek, Anna T; Vermerris, Wilfred; Koch, Karen E; McCarty, Donald R; Davis, Mark F; Thomas, Steven R; McCann, Maureen C; Carpita, Nicholas C

    2009-12-01

    Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions. PMID:19926802

  17. Cell Wall Development in Maize Coleoptiles 1

    PubMed Central

    Carpita, Nicholas C.

    1984-01-01

    The physical bases for enhancement of growth rates induced by auxin involve changes in cell wall structure. Changes in the chemical composition of the primary walls during maize (Zea mays L. cv WF9 × Bear 38) coleoptile development were examined to provide a framework to study the nature of auxin action. This report documents that the primary walls of maize cells vary markedly depending on developmental state; polymers synthesized and deposited in the primary wall during cell division are substantially different from those formed during cell elongation. The embryonal coleoptile wall is comprised of mostly glucuronoarabinoxylan (GAX), xyloglucan, and polymers enriched in 5-arabinosyl linkages. During development, both GAX and xyloglucan are synthesized, but the 5-arabinosyls are not. Rapid coleoptile elongation is accompanied by synthesis of a mixed-linked glucan that is nearly absent from the embryonal wall. A GAX highly substituted with mostly terminal arabinofuranosyl units is also synthesized during elongation and, based on pulse-chase studies, exhibits turnover possibly to xylans with less substitution via loss of the arabinosyl and glucuronosyl linkages. Images Fig. 2 PMID:16663799

  18. Mitochondrial adenosine triphosphatase of the fission yeast, Schizosaccharomyces pombe 972h-. Changes in activity and oligomycin-sensitivity during the cell cycle of catabolite-repressed and -de-repressed cells.

    PubMed Central

    Edwards, S W; Lloyd, D

    1977-01-01

    1. Changes in activity of ATPase (adenosine triphosphatase) during the cell cycle of Schizosaccharomyces pombe were analysed in cell-free extracts of cells harvested from different stages of growth of synchronous cultures and also after cell-cycle fractionation. 2. Oligomycin-sensitive ATPase oscillates in both glucose-repressed synchronous cultures and shows four maxima of activity approximately equally spaced through the cell cycle. The amplitude of the oscillations accounts for between 13 and 80% of the total activity at different times in the cell cycle. 3. Oligomycin sensitivity varies over a fourfold range at different stages of the cell cycle. 4. The periodicity of maximum oligomycin sensitivity is one-quarter of a cell cycle. 5. These results were confirmed for the first three-quarters of the cell cycle by cell-cycle fractionation. 6. In cells growing synchronously with glycerol, ATPase activity increases in a stepwise pattern, with two steps per cell cycle; the first of these occurs at 0.54 of the cell cycle and the second at 0.95. 7. These results are discussed in relation to previously obtained data on the development of mitochondrial activities during the cell cycle. PMID:139890

  19. The Structure of Plant Cell Walls

    PubMed Central

    McNeil, Michael; Albersheim, Peter; Taiz, Lincoln; Jones, Russell L.

    1975-01-01

    The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls. PMID:16659029

  20. Cell Wall Metabolism in Ripening Fruit

    PubMed Central

    Ahmed, Ahmed Elrayah; Labavitch, John M.

    1980-01-01

    Mature `Bartlett' pear (Pyrus communis) fruits were ripened at 20 C. Fruits at different stages of ripeness were homogenized, and extracts of the low speed pellet (crude cell wall) were prepared. These extracts contained polygalacturonase, pectin esterase, and activity against seven p-nitrophenyl glycoside substrates. Polygalacturonase, α-galactosidase, and α-mannosidase increased in activity as the fruit ripened. Cellulase and activities against pear wall xylan and arabinan were absent from the extracts. PMID:16661276

  1. Roles of membrane trafficking in plant cell wall dynamics

    PubMed Central

    Ebine, Kazuo; Ueda, Takashi

    2015-01-01

    The cell wall is one of the characteristic components of plant cells. The cell wall composition differs among cell types and is modified in response to various environmental conditions. To properly generate and modify the cell wall, many proteins are transported to the plasma membrane or extracellular space through membrane trafficking, which is one of the key protein transport mechanisms in eukaryotic cells. Given the diverse composition and functions of the cell wall in plants, the transport of the cell wall components and proteins that are involved in cell wall-related events could be specialized for each cell type, i.e., the machinery for cell wall biogenesis, modification, and maintenance could be transported via different trafficking pathways. In this review, we summarize the recent progress in the current understanding of the roles and mechanisms of membrane trafficking in plant cells and focus on the biogenesis and regulation of the cell wall. PMID:26539200

  2. Metabolism of the phospholipid precursor inositol and its relationship to growth and viability in the natural auxotroph Schizosaccharomyces pombe.

    PubMed Central

    Fernandez, S; Homann, M J; Henry, S A; Carman, G M

    1986-01-01

    Phospholipid metabolism in the fission yeast Schizosaccharomyces pombe was examined. Three enzymes of phospholipid biosynthesis, cytidine diphosphate diacylglycerol synthase (CDP-DG), phosphatidylinositol (PI) synthase, and phosphatidylserine (PS) synthase, were characterized in extracts of S. pombe cells. Contrary to an earlier report, we were able to demonstrate that CDP-DG served as a precursor for PI and PS biosynthesis in S. pombe. S. pombe is naturally auxotrophic for the phospholipid precursor inositol. We found that S. pombe was much more resistant to loss of viability during inositol starvation than artificially generated inositol auxotrophs of Saccharomyces cerevisiae. The phospholipid composition of S. pombe cells grown in inositol-rich medium (50 microM) was similar to that of S. cerevisiae cells grown under similar conditions. However, growth of S. pombe at low inositol concentrations (below 30 microM) affected the ratio of the anionic phospholipids PI and PS, while the relative proportions of other glycerophospholipids remained unchanged. During inositol starvation, the rate of PI synthesis decreased rapidly, and there was a concomitant increase in the rate of PS synthesis. Phosphatidic acid and CDP-DG, which are precursors to these phospholipids, also increased when PI synthesis was blocked by lack of exogenous inositol. The major product of turnover of inositol-containing phospholipids in S. pombe was found to be free inositol, which accumulated in the medium and could be reused by the cell. Images PMID:3011744

  3. Cell wall peptidoglycan architecture in Bacillus subtilis

    PubMed Central

    Hayhurst, Emma J.; Kailas, Lekshmi; Hobbs, Jamie K.; Foster, Simon J.

    2008-01-01

    The bacterial cell wall is essential for viability and shape determination. Cell wall structural dynamics allowing growth and division, while maintaining integrity is a basic problem governing the life of bacteria. The polymer peptidoglycan is the main structural component for most bacteria and is made up of glycan strands that are cross-linked by peptide side chains. Despite study and speculation over many years, peptidoglycan architecture has remained largely elusive. Here, we show that the model rod-shaped bacterium Bacillus subtilis has glycan strands up to 5 μm, longer than the cell itself and 50 times longer than previously proposed. Atomic force microscopy revealed the glycan strands to be part of a peptidoglycan architecture allowing cell growth and division. The inner surface of the cell wall has a regular macrostructure with ≈50 nm-wide peptidoglycan cables [average 53 ± 12 nm (n = 91)] running basically across the short axis of the cell. Cross striations with an average periodicity of 25 ± 9 nm (n = 96) along each cable are also present. The fundamental cabling architecture is also maintained during septal development as part of cell division. We propose a coiled-coil model for peptidoglycan architecture encompassing our data and recent evidence concerning the biosynthetic machinery for this essential polymer. PMID:18784364

  4. Cell wall peptidoglycan architecture in Bacillus subtilis.

    PubMed

    Hayhurst, Emma J; Kailas, Lekshmi; Hobbs, Jamie K; Foster, Simon J

    2008-09-23

    The bacterial cell wall is essential for viability and shape determination. Cell wall structural dynamics allowing growth and division, while maintaining integrity is a basic problem governing the life of bacteria. The polymer peptidoglycan is the main structural component for most bacteria and is made up of glycan strands that are cross-linked by peptide side chains. Despite study and speculation over many years, peptidoglycan architecture has remained largely elusive. Here, we show that the model rod-shaped bacterium Bacillus subtilis has glycan strands up to 5 microm, longer than the cell itself and 50 times longer than previously proposed. Atomic force microscopy revealed the glycan strands to be part of a peptidoglycan architecture allowing cell growth and division. The inner surface of the cell wall has a regular macrostructure with approximately 50 nm-wide peptidoglycan cables [average 53 +/- 12 nm (n = 91)] running basically across the short axis of the cell. Cross striations with an average periodicity of 25 +/- 9 nm (n = 96) along each cable are also present. The fundamental cabling architecture is also maintained during septal development as part of cell division. We propose a coiled-coil model for peptidoglycan architecture encompassing our data and recent evidence concerning the biosynthetic machinery for this essential polymer. PMID:18784364

  5. Characterization of the Sclerotinia sclerotiorum cell wall proteome.

    PubMed

    Liu, Longzhou; Free, Stephen J

    2016-08-01

    We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)-anchored cell wall proteins and 30 non-GPI-anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes. PMID:26661933

  6. Cell Wall Heterogeneity in Root Development of Arabidopsis

    PubMed Central

    Somssich, Marc; Khan, Ghazanfar Abbas; Persson, Staffan

    2016-01-01

    Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes. PMID:27582757

  7. Cell Wall Heterogeneity in Root Development of Arabidopsis.

    PubMed

    Somssich, Marc; Khan, Ghazanfar Abbas; Persson, Staffan

    2016-01-01

    Plant cell walls provide stability and protection to plant cells. During growth and development the composition of cell walls changes, but provides enough strength to withstand the turgor of the cells. Hence, cell walls are highly flexible and diverse in nature. These characteristics are important during root growth, as plant roots consist of radial patterns of cells that have diverse functions and that are at different developmental stages along the growth axis. Young stem cell daughters undergo a series of rapid cell divisions, during which new cell walls are formed that are highly dynamic, and that support rapid anisotropic cell expansion. Once the cells have differentiated, the walls of specific cell types need to comply with and support different cell functions. For example, a newly formed root hair needs to be able to break through the surrounding soil, while endodermal cells modify their walls at distinct positions to form Casparian strips between them. Hence, the cell walls are modified and rebuilt while cells transit through different developmental stages. In addition, the cell walls of roots readjust to their environment to support growth and to maximize nutrient uptake. Many of these modifications are likely driven by different developmental and stress signaling pathways. However, our understanding of how such pathways affect cell wall modifications and what enzymes are involved remain largely unknown. In this review we aim to compile data linking cell wall content and re-modeling to developmental stages of root cells, and dissect how root cell walls respond to certain environmental changes. PMID:27582757

  8. Fluorescent tags to explore cell wall structure and dynamics

    PubMed Central

    Gonneau, Martine; Höfte, Herman; Vernhettes, Samantha

    2012-01-01

    Plant cell walls are highly dynamic and heterogeneous structures, which vary between cell types, growth stages but also between microdomains within a single cell wall. In this review, we summarize the imaging techniques using fluorescent tags that are currently being used and which should in the coming years revolutionize our understanding of the dynamics of cell wall architecture and the cellular processes involved in the synthesis of cell wall components. PMID:22783266

  9. An Ancient Yeast for Young Geneticists: A Primer on the Schizosaccharomyces pombe Model System

    PubMed Central

    Hoffman, Charles S.; Wood, Valerie; Fantes, Peter A.

    2015-01-01

    The fission yeast Schizosaccharomyces pombe is an important model organism for the study of eukaryotic molecular and cellular biology. Studies of S. pombe, together with studies of its distant cousin, Saccharomyces cerevisiae, have led to the discovery of genes involved in fundamental mechanisms of transcription, translation, DNA replication, cell cycle control, and signal transduction, to name but a few processes. However, since the divergence of the two species approximately 350 million years ago, S. pombe appears to have evolved less rapidly than S. cerevisiae so that it retains more characteristics of the common ancient yeast ancestor, causing it to share more features with metazoan cells. This Primer introduces S. pombe by describing the yeast itself, providing a brief description of the origins of fission yeast research, and illustrating some genetic and bioinformatics tools used to study protein function in fission yeast. In addition, a section on some key differences between S. pombe and S. cerevisiae is included for readers with some familiarity with budding yeast research but who may have an interest in developing research projects using S. pombe. PMID:26447128

  10. STREAMLINED METHOD FOR BIOMASS WHOLE-CELL-WALL STRUCTURAL PROFILING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In wide-ranging research aimed at altering plant cell wall characteristics by conventional breeding or modern genetic methods, one of the biggest problems is in delineating the effects on the cell wall. Plant cell walls are a complex conglomerate of a variety of polysaccharides and lignin. Each comp...

  11. Wall relaxation and the driving forces for cell expansive growth

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1987-01-01

    When water uptake by growing cells is prevented, the turgor pressure and the tensile stress in the cell wall are reduced by continued wall loosening. This process, termed in vivo stress relaxation, provides a new way to study the dynamics of wall loosening and to measure the wall yield threshold and the physiological wall extensibility. Stress relaxation experiments indicate that wall stress supplies the mechanical driving force for wall yielding. Cell expansion also requires water absorption. The driving force for water uptake during growth is created by wall relaxation, which lowers the water potential of the expanding cells. New techniques for measuring this driving force show that it is smaller than believed previously; in elongating stems it is only 0.3 to 0.5 bar. This means that the hydraulic resistance of the water transport pathway is small and that rate of cell expansion is controlled primarily by wall loosening and yielding.

  12. Monoclonal antibodies against plant cell wall polysaccharides

    SciTech Connect

    Hahn, M.G.; Bucheli, E.; Darvill, A.; Albersheim, P. )

    1989-04-01

    Monoclonal antibodies (McAbs) are useful tools to probe the structure of plant cell wall polysaccharides and to localize these polysaccharides in plant cells and tissues. Murine McAbs were generated against the pectic polysaccharide, rhamnogalacturonan I (RG-I), isolated from suspension-cultured sycamore cells. The McAbs that were obtained were grouped into three classes based upon their reactivities with a variety of plant polysaccharides and membrane glycoproteins. Eleven McAbs (Class I) recognize epitope(s) that appear to be immunodominant and are found in RG-I from sycamore and maize, citrus pectin, polygalacturonic acid, and membrane glycoproteins from suspension-cultured cells of sycamore, maize, tobacco, parsley, and soybean. A second group of five McAbs (Class II) recognize epitope(s) present in sycamore RG-I, but do not bind to any of the other polysaccharides or glycoproteins recognized by Class I. Lastly, one McAb (Class III) reacts with sycamore RG-I, sycamore and tamarind xyloglucan, and sycamore and rice glucuronoarabinoxylan, but does not bind to maize RG-I, polygalacturonic acid or the plant membrane glycoproteins recognized by Class I. McAbs in Classes II and III are likely to be useful in studies of the structure, biosynthesis and localization of plant cell wall polysaccharides.

  13. Calnexin Is Essential for Survival under Nitrogen Starvation and Stationary Phase in Schizosaccharomyces pombe

    PubMed Central

    Rokeach, Luis A.

    2015-01-01

    Cell fate is determined by the balance of conserved molecular mechanisms regulating death (apoptosis) and survival (autophagy). Autophagy is a process by which cells recycle their organelles and macromolecules through degradation within the vacuole in yeast and plants, and lysosome in metazoa. In the yeast Schizosaccharomyces pombe, autophagy is strongly induced under nitrogen starvation and in aging cells. Previously, we demonstrated that calnexin (Cnx1p), a highly conserved transmembrane chaperone of the endoplasmic reticulum (ER), regulates apoptosis under ER stress or inositol starvation. Moreover, we showed that in stationary phase, Cnx1p is cleaved into two moieties, L_Cnx1p and S_Cnx1p. Here, we show that the processing of Cnx1p is regulated by autophagy, induced by nitrogen starvation or cell aging. The cleavage of Cnx1p involves two vacuolar proteases: Isp6, which is essential for autophagy, and its paralogue Psp3. Blocking autophagy through the knockout of autophagy-related genes (atg) results in inhibition of both, the cleavage and the trafficking of Cnx1p from the ER to the vacuole. We demonstrate that Cnx1p is required for cell survival under nitrogen-starvation and in chronological aging cultures. The death of the mini_cnx1 mutant (overlapping S_cnx1p) cells is accompanied by accumulation of high levels of reactive-oxygen species (ROS), a slowdown in endocytosis and severe cell-wall defects. Moreover, mutant cells expressing only S_Cnx1p showed cell wall defects. Co-expressing mutant overlapping the L_Cnx1p and S_Cnx1p cleavage products reverses the death, ROS phenotype and cell wall defect to wild-type levels. As it is involved in both apoptosis and autophagy, Cnx1p could be a nexus for the crosstalk between these pro-death and pro-survival mechanisms. Ours, and observations in mammalian systems, suggest that the multiple roles of calnexin depend on its sub-cellular localization and on its cleavage. The use of S. pombe should assist in further

  14. Plant cell wall proteomics: the leadership of Arabidopsis thaliana

    PubMed Central

    Albenne, Cécile; Canut, Hervé; Jamet, Elisabeth

    2013-01-01

    Plant cell wall proteins (CWPs) progressively emerged as crucial components of cell walls although present in minor amounts. Cell wall polysaccharides such as pectins, hemicelluloses, and cellulose represent more than 90% of primary cell wall mass, whereas hemicelluloses, cellulose, and lignins are the main components of lignified secondary walls. All these polymers provide mechanical properties to cell walls, participate in cell shape and prevent water loss in aerial organs. However, cell walls need to be modified and customized during plant development and in response to environmental cues, thus contributing to plant adaptation. CWPs play essential roles in all these physiological processes and particularly in the dynamics of cell walls, which requires organization and rearrangements of polysaccharides as well as cell-to-cell communication. In the last 10 years, plant cell wall proteomics has greatly contributed to a wider knowledge of CWPs. This update will deal with (i) a survey of plant cell wall proteomics studies with a focus on Arabidopsis thaliana; (ii) the main protein families identified and the still missing peptides; (iii) the persistent issue of the non-canonical CWPs; (iv) the present challenges to overcome technological bottlenecks; and (v) the perspectives beyond cell wall proteomics to understand CWP functions. PMID:23641247

  15. Plant cell wall lignification and monolignol metabolism

    PubMed Central

    Wang, Yin; Chantreau, Maxime; Sibout, Richard; Hawkins, Simon

    2013-01-01

    Plants are built of various specialized cell types that differ in their cell wall composition and structure. The cell walls of certain tissues (xylem, sclerenchyma) are characterized by the presence of the heterogeneous lignin polymer that plays an essential role in their physiology. This phenolic polymer is composed of different monomeric units – the monolignols – that are linked together by several covalent bonds. Numerous studies have shown that monolignol biosynthesis and polymerization to form lignin are tightly controlled in different cell types and tissues. However, our understanding of the genetic control of monolignol transport and polymerization remains incomplete, despite some recent promising results. This situation is made more complex since we know that monolignols or related compounds are sometimes produced in non-lignified tissues. In this review, we focus on some key steps of monolignol metabolism including polymerization, transport, and compartmentation. As well as being of fundamental interest, the quantity of lignin and its nature are also known to have a negative effect on the industrial processing of plant lignocellulose biomass. A more complete view of monolignol metabolism and the relationship that exists between lignin and other monolignol-derived compounds thereby appears essential if we wish to improve biomass quality. PMID:23847630

  16. Cell wall sorting of lipoproteins in Staphylococcus aureus.

    PubMed Central

    Navarre, W W; Daefler, S; Schneewind, O

    1996-01-01

    Many surface proteins are thought to be anchored to the cell wall of gram-positive organisms via their C termini, while the N-terminal domains of these molecules are displayed on the bacterial surface. Cell wall anchoring of surface proteins in Staphylococcus aureus requires both an N-terminal leader peptide and a C-terminal cell wall sorting signal. By fusing the cell wall sorting of protein A to the C terminus of staphylococcal beta-lactamase, we demonstrate here that lipoproteins can also be anchored to the cell wall of S. aureus. The topology of cell wall-anchored beta-lactamase is reminiscent of that described for Braun's murein lipoprotein in that the N terminus of the polypeptide chain is membrane anchored whereas the C-terminal end is tethered to the bacterial cell wall. PMID:8550464

  17. Exploiting fungal cell wall components in vaccines

    PubMed Central

    Levitz, Stuart M.; Huang, Haibin; Ostroff, Gary R.; Specht, Charles A.

    2014-01-01

    Innate recognition of fungi leads to strong adaptive immunity. Investigators are trying to exploit this observation in vaccine development by combining antigens with evolutionarily conserved fungal cell wall carbohydrates to induce protective responses. Best studied is β-1,3-glucan, a glycan that activates complement and is recognized by Dectin-1. Administration of antigens in association with β-1,3-glucan, either by direct conjugation or complexed in glucan particles, results in robust humoral and cellular immune responses. While the host has a host of mannose receptors, responses to fungal mannoproteins generally are amplified if cells are cooperatively stimulated with an additional danger signal such as a toll-like receptor agonist. Chitosan, a polycationic homopolymer of glucosamine manufactured by the deacetylation of chitin, is being studied as an adjuvant in DNA and protein-based vaccines. It appears particularly promising in mucosal vaccines. Finally, universal and organism-specific fungal vaccines have been formulated by conjugating fungal cell wall glycans to carrier proteins. A major challenge will be to advance these experimental findings so that at risk patients can be protected. PMID:25404118

  18. Hormonal regulation of secondary cell wall formation.

    PubMed

    Didi, Vojtěch; Jackson, Phil; Hejátko, Jan

    2015-08-01

    Secondary cell walls (SCWs) have critical functional importance but also constitute a high proportion of the plant biomass and have high application potential. This is true mainly for the lignocellulosic constituents of the SCWs in xylem vessels and fibres, which form a structured layer between the plasma membrane and the primary cell wall (PCW). Specific patterning of the SCW thickenings contributes to the mechanical properties of the different xylem cell types, providing the plant with mechanical support and facilitating the transport of solutes via vessels. In the last decade, our knowledge of the basic molecular mechanisms controlling SCW formation has increased substantially. Several members of the multi-layered regulatory cascade participating in the initiation and transcriptional regulation of SCW formation have been described, and the first cellular components determining the pattern of SCW at the subcellular resolution are being uncovered. The essential regulatory role of phytohormones in xylem development is well known and the molecular mechanisms that link hormonal signals to SCW formation are emerging. Here, we review recent knowledge about the role of individual plant hormones and hormonal crosstalk in the control over the regulatory cascades guiding SCW formation and patterning. Based on the analogy between many of the mechanisms operating during PCW and SCW formation, recently identified mechanisms underlying the hormonal control of PCW remodelling are discussed as potentially novel mechanisms mediating hormonal regulatory inputs in SCW formation. PMID:26002972

  19. Exploiting fungal cell wall components in vaccines.

    PubMed

    Levitz, Stuart M; Huang, Haibin; Ostroff, Gary R; Specht, Charles A

    2015-03-01

    Innate recognition of fungi leads to strong adaptive immunity. Investigators are trying to exploit this observation in vaccine development by combining antigens with evolutionarily conserved fungal cell wall carbohydrates to induce protective responses. Best studied is β-1,3-glucan, a glycan that activates complement and is recognized by dectin-1. Administration of antigens in association with β-1,3-glucan, either by direct conjugation or complexed in glucan particles, results in robust humoral and cellular immune responses. While the host has a host of mannose receptors, responses to fungal mannoproteins generally are amplified if cells are cooperatively stimulated with an additional danger signal such as a toll-like receptor agonist. Chitosan, a polycationic homopolymer of glucosamine manufactured by the deacetylation of chitin, is being studied as an adjuvant in DNA and protein-based vaccines. It appears particularly promising in mucosal vaccines. Finally, universal and organism-specific fungal vaccines have been formulated by conjugating fungal cell wall glycans to carrier proteins. A major challenge will be to advance these experimental findings so that at risk patients can be protected. PMID:25404118

  20. [The different effects of CaM inhibitors of phenothiazines on the proliferation of Saccharomyces cerevisiae and Schizosaccharomyces pombe].

    PubMed

    Lu, L; Jiang, A Q; Yuan, S; Yin, L H; Huang, W Y; Fan, W S

    2000-06-01

    Low concentration of phenothiazines apparently stimulated the proliferation of S. pombe, the cell density incubated for 54 hours by preincubating the cells with 20 mumol/L trifluoperazine (TFP) in the EMM-Ca medium was two times more than the control. The stimulation was more obvious with lowing the concentration of calcium in the culture medium, TFP cooperated and complemented with calcium in stimulating the proliferation of S. pombe. When the original inoculated cell density was 5 x 10(6) cells/ml or during the logarithm period of growth curve, the proliferation of S. pombe wasn't affected by the low concentration of TFP. While when the concentration of TFP was increased to 100 mumol/L, the promotion effect of TFP on proliferation of S. pombe declined obviously and the proliferation of S. pombe was inhibited completely when TFP up to 200 mumol/L. The cell proliferation also could be inhibited by CaM antagonist W7 and W7-agarose, the inhibition was increased with increasing the concentration of antagonist. On the other hand, 20 mumol/L TFP used by the same method as above arrested the cell division cycle of Saccharomyces cerevisiae at a single G2 + M nuclei stage, the cells was penetrated easily by TFP, the fluorescence in cells was very obvious when TFP was 20 mumol/L, but it was difficult to penetrat by TFP in the cells of S. pombe and the Ca2+ influx of S. pombe could be induced rapidly by 20 mumol/L TFP. In this article, the cause of different effects of TFP on cell proliferation of S. pombe and S. cerevisiae was discussed, it was due to the difference of penetration of TFP and stimulation by calcium in the two kinds of cells. PMID:12548977

  1. Plant and algal cell walls: diversity and functionality

    PubMed Central

    Popper, Zoë A.; Ralet, Marie-Christine; Domozych, David S.

    2014-01-01

    Background Although plants and many algae (e.g. the Phaeophyceae, brown, and Rhodophyceae, red) are only very distantly related they are united in their possession of carbohydrate-rich cell walls, which are of integral importance being involved in many physiological processes. Furthermore, wall components have applications within food, fuel, pharmaceuticals, fibres (e.g. for textiles and paper) and building materials and have long been an active topic of research. As shown in the 27 papers in this Special Issue, as the major deposit of photosynthetically fixed carbon, and therefore energy investment, cell walls are of undisputed importance to the organisms that possess them, the photosynthetic eukaryotes (plants and algae). The complexities of cell wall components along with their interactions with the biotic and abiotic environment are becoming increasingly revealed. Scope The importance of plant and algal cell walls and their individual components to the function and survival of the organism, and for a number of industrial applications, are illustrated by the breadth of topics covered in this issue, which includes papers concentrating on various plants and algae, developmental stages, organs, cell wall components, and techniques. Although we acknowledge that there are many alternative ways in which the papers could be categorized (and many would fit within several topics), we have organized them as follows: (1) cell wall biosynthesis and remodelling, (2) cell wall diversity, and (3) application of new technologies to cell walls. Finally, we will consider future directions within plant cell wall research. Expansion of the industrial uses of cell walls and potentially novel uses of cell wall components are both avenues likely to direct future research activities. Fundamentally, it is the continued progression from characterization (structure, metabolism, properties and localization) of individual cell wall components through to defining their roles in almost every

  2. Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis.

    PubMed Central

    Imai, Y; Davey, J; Kawagishi-Kobayashi, M; Yamamoto, M

    1997-01-01

    The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h- cells but not in h+ cells. h- cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids, with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homolog of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. The isolation of a vertebrate gene encoding farnesyl cysteine carboxyl methyltransferase opens the way to in-depth studies of the role of methylation in a large body of proteins, including Ras superfamily proteins. PMID:9032282

  3. Enzymes and other agents that enhance cell wall extensibility

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1999-01-01

    Polysaccharides and proteins are secreted to the inner surface of the growing cell wall, where they assemble into a network that is mechanically strong, yet remains extensible until the cells cease growth. This review focuses on the agents that directly or indirectly enhance the extensibility properties of growing walls. The properties of expansins, endoglucanases, and xyloglucan transglycosylases are reviewed and their postulated roles in modulating wall extensibility are evaluated. A summary model for wall extension is presented, in which expansin is a primary agent of wall extension, whereas endoglucanases, xyloglucan endotransglycosylase, and other enzymes that alter wall structure act secondarily to modulate expansin action.

  4. Anthocyanins influence tannin-cell wall interactions.

    PubMed

    Bautista-Ortín, Ana Belén; Martínez-Hernández, Alejandro; Ruiz-García, Yolanda; Gil-Muñoz, Rocío; Gómez-Plaza, Encarna

    2016-09-01

    The rate of tannin extraction was studied in a vinification of red grapes and the results compared with another vinification made with white grapes fermented as for typical red wine, in the presence of skins and seeds. Even though the grapes presented a quite similar skin and seed tannin content, the differences in tannin concentration between both vinifications was very large, despite the fact that the only apparent difference between the phenolic composition of both wines was the anthocyanin content. This suggests that anthocyanins play an important role in tannin extractability, perhaps because they affect the extent of the tannin-cell wall interaction, a factor that largely controls the resulting quantity of tannins in wines. To confirm this observation, the effect of anthocyanins on the tannin extractability from grape seeds and skin and on the interaction between tannins and grape cell walls suspended in model solutions were studied. The results indicated that anthocyanins favored skin and seed tannin extraction and that there is a competition for the adsorption sites between anthocyanins and tannins that increases the tannin content when anthocyanins are present. PMID:27041322

  5. Disruption of cell walls for enhanced lipid recovery

    SciTech Connect

    Knoshaug, Eric P; Donohoe, Bryon S; Gerken, Henri; Laurens, Lieve; Van Wychen, Stefanie Rose

    2015-03-24

    Presented herein are methods of using cell wall degrading enzymes for recovery of internal lipid bodies from biomass sources such as algae. Also provided are algal cells that express at least one exogenous gene encoding a cell wall degrading enzyme and methods for recovering lipids from the cells.

  6. Plant cell wall dynamics and wall-related susceptibility in plant–pathogen interactions

    PubMed Central

    Bellincampi, Daniela; Cervone, Felice; Lionetti, Vincenzo

    2014-01-01

    The cell wall is a dynamic structure that often determines the outcome of the interactions between plants and pathogens. It is a barrier that pathogens need to breach to colonize the plant tissue. While fungal necrotrophs extensively destroy the integrity of the cell wall through the combined action of degrading enzymes, biotrophic fungi require a more localized and controlled degradation of the cell wall in order to keep the host cells alive and utilize their feeding structures. Also bacteria and nematodes need to degrade the plant cell wall at a certain stage of their infection process, to obtain nutrients for their growth. Plants have developed a system for sensing pathogens and monitoring the cell wall integrity, upon which they activate defense responses that lead to a dynamic cell wall remodeling required to prevent the disease. Pathogens, on the other hand, may exploit the host cell wall metabolism to support the infection. We review here the strategies utilized by both plants and pathogens to prevail in the cell wall battleground. PMID:24904623

  7. Growth and the Environment of Schizosaccharomyces pombe.

    PubMed

    Petersen, Janni; Russell, Paul

    2016-01-01

    Here, we summarize the composition and uses of Schizosaccharomyces pombe media and discuss key issues for consideration in the generation of S. pombe cultures. We discuss the concept of "culture memory," in which the growth state and stress experienced by a strain during storage, propagation, and starter culture preparation can alter experimental outcomes at later stages. We also describe the triggers that are widely used to manipulate signaling through the environment sensing pathways. PMID:26933253

  8. Shifting foundations: the mechanical cell wall and development.

    PubMed

    Braybrook, Siobhan A; Jönsson, Henrik

    2016-02-01

    The cell wall has long been acknowledged as an important physical mediator of growth in plants. Recent experimental and modelling work has brought the importance of cell wall mechanics into the forefront again. These data have challenged existing dogmas that relate cell wall structure to cell/organ growth, that uncouple elasticity from extensibility, and those which treat the cell wall as a passive and non-stressed material. Within this review we describe experiments and models which have changed the ways in which we view the mechanical cell wall, leading to new hypotheses and research avenues. It has become increasingly apparent that while we often wish to simplify our systems, we now require more complex multi-scale experiments and models in order to gain further insight into growth mechanics. We are currently experiencing an exciting and challenging shift in the foundations of our understanding of cell wall mechanics in growth and development. PMID:26799133

  9. Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes.

    PubMed

    Cosgrove, Daniel J

    2016-01-01

    The advent of user-friendly instruments for measuring force/deflection curves of plant surfaces at high spatial resolution has resulted in a recent outpouring of reports of the 'Young's modulus' of plant cell walls. The stimulus for these mechanical measurements comes from biomechanical models of morphogenesis of meristems and other tissues, as well as single cells, in which cell wall stress feeds back to regulate microtubule organization, auxin transport, cellulose deposition, and future growth directionality. In this article I review the differences between elastic modulus and wall extensibility in the context of cell growth. Some of the inherent complexities, assumptions, and potential pitfalls in the interpretation of indentation force/deflection curves are discussed. Reported values of elastic moduli from surface indentation measurements appear to be 10- to >1000-fold smaller than realistic tensile elastic moduli in the plane of plant cell walls. Potential reasons for this disparity are discussed, but further work is needed to make sense of the huge range in reported values. The significance of wall stress relaxation for growth is reviewed and connected to recent advances and remaining enigmas in our concepts of how cellulose, hemicellulose, and pectins are assembled to make an extensible cell wall. A comparison of the loosening action of α-expansin and Cel12A endoglucanase is used to illustrate two different ways in which cell walls may be made more extensible and the divergent effects on wall mechanics. PMID:26608646

  10. Two endogenous proteins that induce cell wall extension in plants

    NASA Technical Reports Server (NTRS)

    McQueen-Mason, S.; Durachko, D. M.; Cosgrove, D. J.

    1992-01-01

    Plant cell enlargement is regulated by wall relaxation and yielding, which is thought to be catalyzed by elusive "wall-loosening" enzymes. By employing a reconstitution approach, we found that a crude protein extract from the cell walls of growing cucumber seedlings possessed the ability to induce the extension of isolated cell walls. This activity was restricted to the growing region of the stem and could induce the extension of isolated cell walls from various dicot stems and the leaves of amaryllidaceous monocots, but was less effective on grass coleoptile walls. Endogenous and reconstituted wall extension activities showed similar sensitivities to pH, metal ions, thiol reducing agents, proteases, and boiling in methanol or water. Sequential HPLC fractionation of the active wall extract revealed two proteins with molecular masses of 29 and 30 kD associated with the activity. Each protein, by itself, could induce wall extension without detectable hydrolytic breakdown of the wall. These proteins appear to mediate "acid growth" responses of isolated walls and may catalyze plant cell wall extension by a novel biochemical mechanism.

  11. The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe

    PubMed Central

    Park, Jung M.; Evertts, Adam G.; Levin, Henry L.

    2009-01-01

    Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion. PMID:19450689

  12. The Hermes transposon of Musca domestica and its use as a mutagen of Schizosaccharomyces pombe.

    PubMed

    Park, Jung M; Evertts, Adam G; Levin, Henry L

    2009-11-01

    Transposon mutagenesis allows for the discovery and characterization of genes by creating mutations that can be easily mapped and sequenced. Moreover, this method allows for a relatively unbiased approach to isolating genes of interest. Recently, a system of transposon based mutagenesis for Schizosaccharomyces pombe became available. This mutagenesis relies on Hermes, a DNA transposon from the house fly that readily integrates into the chromosomes of S. pombe. The Hermes system is distinct from the retrotransposons of S. pombe because it efficiently integrates into open reading frames. To mutagenize S. pombe, cells are transformed with a plasmid that contains a drug resistance marker flanked by the terminal inverted repeats of Hermes. The Hermes transposase expressed from a second plasmid excises the resistance marker with the inverted repeats and inserts this DNA into chromosomal sites. After S. pombe with these two plasmids grow 25 generations, approximately 2% of the cells contain insertions. Of the cells with insertions, 68% contain single integration events. The protocols listed here provide the detailed information necessary to mutagenize a strain of interest, screen for specific phenotypes, and sequence the positions of insertion. PMID:19450689

  13. (The structure of pectins from cotton suspension culture cell walls)

    SciTech Connect

    Mort, A.

    1990-01-01

    We have made progress on several projects to do with determining the structure of pectins. These include: (1) Devising a new sensitive method to determine the degree of methyl esterification (DOM) of pectins; (2) solubilization of all of RGI from cotton cell walls; (3) solubilization of RGII from cotton cell walls; (4) characterization of xyloglucan from cotton cell walls; and (5) investigation giving an indication of a cross-link between extension and pectin.

  14. Lignin Formation in Wheat Coleoptile Cell Walls

    PubMed Central

    Whitmore, F. W.

    1971-01-01

    Four growth-influencing compounds—hydroxyproline, 2,2′-dipyridyl, 2-chloroethylphosphonic acid, and indoleacetic acid—were used to examine the relationship between lignin formation and growth of wheat coleoptile sections. Hydroxyproline and 2-chloroethylphosphonic acid, at low concentrations, inhibited growth and increased lignin content. Dipyridyl, which promoted coleoptile elongation, decreased lignin content. Indoleacetic acid caused a 300% increase in growth at 0.1 mm but resulted in lignin content no different from controls with no auxin. Chemical and anatomical evidence is given which indicates that lignin is present in the epidermal cell walls of the wheat coleoptile. It is thus possible that bonding between lignin and hemicellulose may have some influence on coleoptile growth. Images PMID:16657843

  15. An arabidopsis gene regulatory network for secondary cell wall synthesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptiona...

  16. The plant cell wall integrity maintenance mechanism--a case study of a cell wall plasma membrane signaling network.

    PubMed

    Hamann, Thorsten

    2015-04-01

    Some of the most important functions of plant cell walls are protection against biotic/abiotic stress and structural support during growth and development. A prerequisite for plant cell walls to perform these functions is the ability to perceive different types of stimuli in both qualitative and quantitative manners and initiate appropriate responses. The responses in turn involve adaptive changes in cellular and cell wall metabolism leading to modifications in the structures originally required for perception. While our knowledge about the underlying plant mechanisms is limited, results from Saccharomyces cerevisiae suggest the cell wall integrity maintenance mechanism represents an excellent example to illustrate how the molecular mechanisms responsible for stimulus perception, signal transduction and integration can function. Here I will review the available knowledge about the yeast cell wall integrity maintenance system for illustration purposes, summarize the limited knowledge available about the corresponding plant mechanism and discuss the relevance of the plant cell wall integrity maintenance mechanism in biotic stress responses. PMID:25446233

  17. Cell wall structure and biogenesis in Aspergillus species.

    PubMed

    Yoshimi, Akira; Miyazawa, Ken; Abe, Keietsu

    2016-09-01

    Aspergillus species are among the most important filamentous fungi from the viewpoints of industry, pathogenesis, and mycotoxin production. Fungal cells are exposed to a variety of environmental stimuli, including changes in osmolality, temperature, and pH, which create stresses that primarily act on fungal cell walls. In addition, fungal cell walls are the first interactions with host cells in either human or plants. Thus, understanding cell wall structure and the mechanism of their biogenesis is important for the industrial, medical, and agricultural fields. Here, we provide a systematic review of fungal cell wall structure and recent findings regarding the cell wall integrity signaling pathways in aspergilli. This accumulated knowledge will be useful for understanding and improving the use of industrial aspergilli fermentation processes as well as treatments for some fungal infections. PMID:27140698

  18. Visualization of cellulose synthases in Arabidopsis secondary cell walls.

    PubMed

    Watanabe, Y; Meents, M J; McDonnell, L M; Barkwill, S; Sampathkumar, A; Cartwright, H N; Demura, T; Ehrhardt, D W; Samuels, A L; Mansfield, S D

    2015-10-01

    Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls. PMID:26450210

  19. Increased Cell-Wall Extensibility in Elevated CO2 and O3 Indicates Modification of Leaf Cell-Wall Structure

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Soybean leaf size is increased by growth in elevated [CO2] and decreased in elevated [O3]. The mechanism likely involves changes in cell biophysical properties. Cell growth rate is a function of cell-wall extensibility, a measure of how easily the wall expands in response to turgor. Modification of...

  20. cps1+, a Schizosaccharomyces pombe gene homolog of Saccharomyces cerevisiae FKS genes whose mutation confers hypersensitivity to cyclosporin A and papulacandin B.

    PubMed Central

    Ishiguro, J; Saitou, A; Durán, A; Ribas, J C

    1997-01-01

    The Schizosaccharomyces pombe cps1-12 (for chlorpropham supersensitive) mutant strain was originally isolated as hypersensitive to the spindle poison isopropyl N-3-chlorophenyl carbamate (chlorpropham) (J. Ishiguro and Y. Uhara, Jpn. J. Genet. 67:97-109, 1992). We have found that the cps1-12 mutation also confers (i) hypersensitivity to the immunosuppressant cyclosporin A (CsA), (ii) hypersensitivity to the drug papulacandin B, which specifically inhibits 1,3-beta-D-glucan synthesis both in vivo and in vitro, and (iii) thermosensitive growth at 37 degrees C. Under any of these restrictive treatments, cells swell up and finally lyse. With an osmotic stabilizer, cells do not lyse, but at 37 degrees C they become multiseptated and multibranched. The cps1-12 mutant, grown at a restrictive temperature, showed an increase in sensitivity to lysis by enzymatic cell wall degradation, in in vitro 1,3-beta-D-glucan synthase activity (173% in the absence of GTP in the reaction), and in cell wall biosynthesis (130% of the wild-type amount). Addition of Ca2+ suppresses hypersensitivity to papulacandin B and septation and branching phenotypes. All of these data suggest a relationship between the cps1+ gene and cell wall synthesis. A DNA fragment containing the cps1+ gene was cloned, and sequence analysis indicated that it encodes a predicted membrane protein of 1,729 amino acids with 15 to 16 transmembrane domains. S. pombe cps1p has overall 55% sequence identity with Fks1p or Fks2p, proposed to be catalytic or associated subunits of Saccharomyces cerevisiae 1,3-beta-D-glucan synthase. Thus, the cps1+ product might be a catalytic or an associated copurifying subunit of the fission yeast 1,3-beta-D-glucan synthase that plays an essential role in cell wall synthesis. PMID:9401022

  1. Influence of polyethylene glycol on the size of Schizosaccharomyces pombe electropores

    SciTech Connect

    Hood, M.T.; Stachow, C. )

    1992-04-01

    The role of polyethylene glycol (PEG) in the transformation of Schizosaccharomyces pombe by electroporation is investigated by fluorescein isothiocyanate-dextran uptake and transformation studies. It is shown that when S. pombe cells are electroporated in the presence of PEG, the permeability state created is sustained until removal of PEG. In addition, the permeability of electroporated S. pombe envelopes is further increased with longer incubation times in PEG. The increased permeability is apparently a result of enlarged pores (electropores) due to the presence of PEG. Comparison of a heat pulse transformation protocol with electroporation suggests a second role for PEG in the uptake of macromolecules. Since pores are not thought to be created during a heat pulse, the PEG may be facilitating the uptake of plasmid DNA. This facilitation of uptake would also be expected to affect DNA uptake by electroporated cells.

  2. Essential Role for Schizosaccharomyces pombe pik1 in Septation

    PubMed Central

    Desautels, Michel; Hemmingsen, Sean M.

    2009-01-01

    Background Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S. Principal Findings Gene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25°C. At 36°C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1D709A was kinase-dead, but bound Cdc4. Pik1R838A did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1D709A,R838A was innocuous, pik1R838A was almost innocuous, and pik1D709A produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4G107S. Thus, D709 is essential for kinase activity and septation. Conclusions Pik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4. PMID:19587793

  3. Preparation of Cell Wall Antigens of Staphylococcus aureus

    PubMed Central

    Kowalski, J. J.; Tipper, Donald J.; Berman, David T.

    1970-01-01

    Cell walls were prepared from Staphylococcus aureus strains Copenhagen and 263 by high-speed mixing in the presence of glass beads followed by differential centrifugation. Insoluble peptidoglycan complexes were derived from cell walls by extraction of teichoic acid with 10% trichloroacetic acid. Intact teichoic acid was prepared from each strain by digestion of cell walls with lysostaphin and isolated by column chromatography. Soluble glycopeptide (peptidoglycan in which only the glycan has been fragmented) and the stable complex of teichoic acid with glycopeptide were prepared by digestion of cell walls with Chalaropsis B endo-N-acetylmuramidase and were separated by column chromatography. Amino acid and amino sugar contents of walls and subunits of walls were comparable to those reported by others. Images PMID:16557799

  4. Characterisation of Schizosaccharomyces pombe α-actinin

    PubMed Central

    Addario, Barbara; Sandblad, Linda; Persson, Karina

    2016-01-01

    The actin cytoskeleton plays a fundamental role in eukaryotic cells. Its reorganization is regulated by a plethora of actin-modulating proteins, such as a-actinin. In higher organisms, α-actinin is characterized by the presence of three distinct structural domains: an N-terminal actin-binding domain and a C-terminal region with EF-hand motif separated by a central rod domain with four spectrin repeats. Sequence analysis has revealed that the central rod domain of α-actinin from the fission yeast Schizosaccharomyces pombe consists of only two spectrin repeats. To obtain a firmer understanding of the structure and function of this unconventional α-actinin, we have cloned and characterized each structural domain. Our results show that this a-actinin isoform is capable of forming dimers and that the rod domain is required for this. However, its actin-binding and cross-linking activity appears less efficient compared to conventional α-actinins. The solved crystal structure of the actin-binding domain indicates that the closed state is stabilised by hydrogen bonds and a salt bridge not present in other α-actinins, which may reduce the affinity for actin. PMID:27069798

  5. Assembly and enlargement of the primary cell wall in plants

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1997-01-01

    Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep.

  6. Structural Studies of Complex Carbohydrates of Plant Cell Walls

    SciTech Connect

    Darvill, Alan; Hahn, Michael G.; O'Neill, Malcolm A.; York, William S.

    2015-02-17

    Most of the solar energy captured by land plants is converted into the polysaccharides (cellulose, hemicellulose, and pectin) that are the predominant components of the cell wall. These walls, which account for the bulk of plant biomass, have numerous roles in the growth and development of plants. Moreover, these walls have a major impact on human life as they are a renewable source of biomass, a source of diverse commercially useful polymers, a major component of wood, and a source of nutrition for humans and livestock. Thus, understanding the molecular mechanisms that lead to wall assembly and how cell walls and their component polysaccharides contribute to plant growth and development is essential to improve and extend the productivity and value of plant materials. The proposed research will develop and apply advanced analytical and immunological techniques to study specific changes in the structures and interactions of the hemicellulosic and pectic polysaccharides that occur during differentiation and in response to genetic modification and chemical treatments that affect wall biosynthesis. These new techniques will make it possible to accurately characterize minute amounts of cell wall polysaccharides so that subtle changes in structure that occur in individual cell types can be identified and correlated to the physiological or developmental state of the plant. Successful implementation of this research will reveal fundamental relationships between polysaccharide structure, cell wall architecture, and cell wall functions.

  7. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation

    PubMed Central

    Yoshida, Kouki; Sakamoto, Shingo; Kawai, Tetsushi; Kobayashi, Yoshinori; Sato, Kazuhito; Ichinose, Yasunori; Yaoi, Katsuro; Akiyoshi-Endo, Miho; Sato, Hiroko; Takamizo, Tadashi; Ohme-Takagi, Masaru; Mitsuda, Nobutaka

    2013-01-01

    Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications. PMID:24098302

  8. Engineering the Oryza sativa cell wall with rice NAC transcription factors regulating secondary wall formation.

    PubMed

    Yoshida, Kouki; Sakamoto, Shingo; Kawai, Tetsushi; Kobayashi, Yoshinori; Sato, Kazuhito; Ichinose, Yasunori; Yaoi, Katsuro; Akiyoshi-Endo, Miho; Sato, Hiroko; Takamizo, Tadashi; Ohme-Takagi, Masaru; Mitsuda, Nobutaka

    2013-01-01

    Plant tissues that require structural rigidity synthesize a thick, strong secondary cell wall of lignin, cellulose and hemicelluloses in a complicated bridged structure. Master regulators of secondary wall synthesis were identified in dicots, and orthologs of these regulators have been identified in monocots, but regulation of secondary cell wall formation in monocots has not been extensively studied. Here we demonstrate that the rice transcription factors SECONDARY WALL NAC DOMAIN PROTEINs (SWNs) can regulate secondary wall formation in rice (Oryza sativa) and are potentially useful for engineering the monocot cell wall. The OsSWN1 promoter is highly active in sclerenchymatous cells of the leaf blade and less active in xylem cells. By contrast, the OsSWN2 promoter is highly active in xylem cells and less active in sclerenchymatous cells. OsSWN2 splicing variants encode two proteins; the shorter protein (OsSWN2S) has very low transcriptional activation ability, but the longer protein (OsSWN2L) and OsSWN1 have strong transcriptional activation ability. In rice, expression of an OsSWN2S chimeric repressor, driven by the OsSWN2 promoter, resulted in stunted growth and para-wilting (leaf rolling and browning under normal water conditions) due to impaired vascular vessels. The same OsSWN2S chimeric repressor, driven by the OsSWN1 promoter, caused a reduction of cell wall thickening in sclerenchymatous cells, a drooping leaf phenotype, reduced lignin and xylose contents and increased digestibility as forage. These data suggest that OsSWNs regulate secondary wall formation in rice and manipulation of OsSWNs may enable improvements in monocotyledonous crops for forage or biofuel applications. PMID:24098302

  9. Methods for degrading or converting plant cell wall polysaccharides

    DOEpatents

    Berka, Randy; Cherry, Joel

    2008-08-19

    The present invention relates to methods for converting plant cell wall polysaccharides into one or more products, comprising: treating the plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into the one or more products. The present invention also relates to methods for producing an organic substance, comprising: (a) saccharifying plant cell wall polysaccharides with an effective amount of a spent whole fermentation broth of a recombinant microorganism, wherein the recombinant microorganism expresses one or more heterologous genes encoding enzymes which degrade or convert the plant cell wall polysaccharides into saccharified material; (b) fermenting the saccharified material of step (a) with one or more fermenting microoganisms; and (c) recovering the organic substance from the fermentation.

  10. Tissue-specific cell wall hydration in sugarcane stalks.

    PubMed

    Maziero, Priscila; Jong, Jennifer; Mendes, Fernanda M; Gonçalves, Adilson R; Eder, Michaela; Driemeier, Carlos

    2013-06-19

    Plant cell walls contain water, especially under biological and wet processing conditions. The present work characterizes this water in tissues of sugarcane stalks. Environmental scanning electron microscopy shows tissue deformation upon drying. Dynamic vapor sorption determines the equilibrium and kinetics of moisture uptake. Thermoporometry by differential scanning calorimetry quantifies water in nanoscale pores. Results show that cell walls from top internodes of stalks are more deformable, slightly more sorptive to moisture, and substantially more porous. These differences of top internode are attributed to less lignified walls, which is confirmed by lower infrared spectral signal from aromatics. Furthermore, cell wall nanoscale porosity, an architectural and not directly compositional characteristic, is shown to be tissue-specific. Nanoscale porosities are ranked as follows: pith parenchyma > pith vascular bundles > rind. This ranking coincides with wall reactivity and digestibility in grasses, suggesting that nanoscale porosity is a major determinant of wall recalcitrance. PMID:23738592

  11. The role of wall calcium in the extension of cell walls of soybean hypocotyls

    NASA Technical Reports Server (NTRS)

    Virk, S. S.; Cleland, R. E.

    1990-01-01

    Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozen-thawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[car boxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues.

  12. Collenchyma: a versatile mechanical tissue with dynamic cell walls

    PubMed Central

    Leroux, Olivier

    2012-01-01

    Background Collenchyma has remained in the shadow of commercially exploited mechanical tissues such as wood and fibres, and therefore has received little attention since it was first described. However, collenchyma is highly dynamic, especially compared with sclerenchyma. It is the main supporting tissue of growing organs with walls thickening during and after elongation. In older organs, collenchyma may become more rigid due to changes in cell wall composition or may undergo sclerification through lignification of newly deposited cell wall material. While much is known about the systematic and organographic distribution of collenchyma, there is rather less information regarding the molecular architecture and properties of its cell walls. Scope and conclusions This review summarizes several aspects that have not previously been extensively discussed including the origin of the term ‘collenchyma’ and the history of its typology. As the cell walls of collenchyma largely determine the dynamic characteristics of this tissue, I summarize the current state of knowledge regarding their structure and molecular composition. Unfortunately, to date, detailed studies specifically focusing on collenchyma cell walls have not been undertaken. However, generating a more detailed understanding of the structural and compositional modifications associated with the transition from plastic to elastic collenchyma cell wall properties is likely to provide significant insights into how specific configurations of cell wall polymers result in specific functional properties. This approach, focusing on architecture and functional properties, is likely to provide improved clarity on the controversial definition of collenchyma. PMID:22933416

  13. The Interplay between Cell Wall Mechanical Properties and the Cell Cycle in Staphylococcus aureus

    PubMed Central

    Bailey, Richard G.; Turner, Robert D.; Mullin, Nic; Clarke, Nigel; Foster, Simon J.; Hobbs, Jamie K.

    2014-01-01

    The nanoscale mechanical properties of live Staphylococcus aureus cells during different phases of growth were studied by atomic force microscopy. Indentation to different depths provided access to both local cell wall mechanical properties and whole-cell properties, including a component related to cell turgor pressure. Local cell wall properties were found to change in a characteristic manner throughout the division cycle. Splitting of the cell into two daughter cells followed a local softening of the cell wall along the division circumference, with the cell wall on either side of the division circumference becoming stiffer. Once exposed, the newly formed septum was found to be stiffer than the surrounding, older cell wall. Deeper indentations, which were affected by cell turgor pressure, did not show a change in stiffness throughout the division cycle, implying that enzymatic cell wall remodeling and local variations in wall properties are responsible for the evolution of cell shape through division. PMID:25468333

  14. Characterisation of Eubacterium cell wall: peptidoglycan structure determines arthritogenicity

    PubMed Central

    Zhang, X; Rimpilainen, M; Toivanen, P

    2001-01-01

    OBJECTIVE—To elucidate factors involved in the arthritogenicity of bacterial cell walls.
METHODS—For characterisation of an arthritogenic Eubacterium aerofaciens cell wall, peptidoglycan-polysaccharide (PG-PS) polymers were isolated by removing cell wall associated proteins (CWPs), PG and PS moieties were separated, and an attempt was made to de-O-acetylate PG-PS. The cell wall of E limosum was used as a non-arthritogenic control. The chemical composition of these cell wall preparations was analysed by gas chromatography-mass spectrometry. Also, their ability to resist lysozyme degradation and to sustain experimental chronic arthritis was tested.
RESULTS—The observations made with the cell wall of E aerofaciens, an anaerobic habitant of the human intestine, were compared with those reported from a pathogenic Streptococcus, showing that in both strains a complex consisting of PG-PS is required for the induction of chronic arthritis. The PS moiety most probably protects PG from enzyme degradation, allowing prolonged tissue persistence and leading to the chronic synovial inflammation. CWPs attached to PG-PS are not necessary for this function. O-Acetylation of PG, which is required for arthritogenicity of the streptococcal cell wall, seems not to be present in the arthritogenic E aerofaciens PG or only occurs to a small degree; attempts to de-O-acylate the E aerofaciens cell wall did not affect its arthritogenicity or lysozyme resistance.
CONCLUSION—The results obtained indicate that the source of bacterial cell wall plays no part in the chemical or structural requirements for PG to induce chronic cell wall arthritis in the rats; the chemical structure of the PG moiety is decisive.

 PMID:11171690

  15. Glucuronoarabinoxylan structure in the walls of Aechmea leaf chlorenchyma cells is related to wall strength.

    PubMed

    Ceusters, Johan; Londers, Elsje; Brijs, Kristof; Delcour, Jan A; De Proft, Maurice P

    2008-09-01

    In CAM-plants rising levels of malic acid in the early morning cause elevated turgor pressures in leaf chlorenchyma cells. Under specific conditions this process is lethal for sensitive plants resulting in chlorenchyma cell burst while other species can cope with these high pressures and do not show cell burst under comparable conditions. The non-cellulosic polysaccharide composition of chlorenchyma cell walls was investigated and compared in three cultivars of Aechmea with high sensitivity for chlorenchyma cell burst and three cultivars with low sensitivity. Chlorenchyma layers were cut from the leaf and the non-cellulosic carbohydrate fraction of the cell wall fraction was analyzed by gas-liquid chromatography. Glucuronoarabinoxylans (GAXs) were the major non-cellulosic polysaccharides in Aechmea. The fine structure of these GAXs was strongly related to chlorenchyma wall strength. Chlorenchyma cell walls from cultivars with low sensitivity to cell burst were characterized by an A/X ratio of ca. 0.13 while those from cultivars with high sensitivity showed an A/X ratio of ca. 0.23. Xylose chains from cultivars with high cell burst sensitivity were ca. 40% more substituted with arabinose compared to cultivars with low sensitivity for cell burst. The results indicate a relationship in vivo between glucuronoarabinoxylan fine structure and chlorenchyma cell wall strength in Aechmea. The evidence obtained supports the hypothesis that GAXs with low degrees of substitution cross-link cellulose microfibrils, while GAXs with high degrees of substitution do not. A lower degree of arabinose substitution on the xylose backbone implies stronger cell walls and the possibility of withstanding higher internal turgor pressures without cell bursting. PMID:18632122

  16. Structure of plant cell walls: XIX. Isolation and characterization of wall polysaccharides from suspension-cultured Douglas fir cells

    SciTech Connect

    Thomas, J.R.; McNeil, M.; Darvill, A.G.; Albersheim, P.

    1987-03-01

    The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls from 1.0 M LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-..cap alpha..-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-..cap alpha..-1,4-polygalacturonase-treated walls by treatment with an endo-..beta..-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-..beta..-1,4-glucanase-treated walls by 0.5 N NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 25% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall.

  17. Method for protein tagging in Schizosaccharomyces pombe.

    PubMed

    Petrescu-Dănilă, Elena; Voicu, Pia-Manuela; Poiţelea, M; Stoica, B; Stănescu, Raluca; Rusu, M

    2006-01-01

    Tagging is a useful method for the investigation of proteins. It allows the localization of the proteins in the cell, their purification in order to investigate their function and the determination of their expression. The aim of the present study was to tag the Rad32 protein of fission yeast (which is the homologue of Mre11 protein from humans) at its N-terminus. Rad32p as well as Mre11p are involved in the repair of DNA double strand breaks and in the DNA damage checkpoint. We carried out this tagging using the Cre-loxp recombination system. In a first step, a 2 kb DNA fragment was integrated upstream of the initiating codon of rad32 gene. This fragment encoded the TAP-tag (tandem affinity purification), a loxp site, a selectable marker (sup3-5), an exogenous promoter (nmt1) and a second loxp site, in this sequence. Following transformation of this DNA fragment into S. pombe cells, rad32 was under the control of the artificial promotor, which allows a controlled expression of the gene by thiamine. In a second step, the cells were transformed with a plasmid coding for Cre recombinase, which catalyses the excision of the DNA sequence between the two loxp sites, removing the marker and the artificial promotor. Thus the tag became attached to the rad32 gene upstream of the ATG, placing the gene under the control of its native promotor. The strain thus obtained will be subsequently used for evidencing the tagged protein by Western blotting and then for its purification in order to investigate its function. PMID:17802953

  18. Architecture and Biosynthesis of the Saccharomyces cerevisiae Cell Wall

    PubMed Central

    Orlean, Peter

    2012-01-01

    The wall gives a Saccharomyces cerevisiae cell its osmotic integrity; defines cell shape during budding growth, mating, sporulation, and pseudohypha formation; and presents adhesive glycoproteins to other yeast cells. The wall consists of β1,3- and β1,6-glucans, a small amount of chitin, and many different proteins that may bear N- and O-linked glycans and a glycolipid anchor. These components become cross-linked in various ways to form higher-order complexes. Wall composition and degree of cross-linking vary during growth and development and change in response to cell wall stress. This article reviews wall biogenesis in vegetative cells, covering the structure of wall components and how they are cross-linked; the biosynthesis of N- and O-linked glycans, glycosylphosphatidylinositol membrane anchors, β1,3- and β1,6-linked glucans, and chitin; the reactions that cross-link wall components; and the possible functions of enzymatic and nonenzymatic cell wall proteins. PMID:23135325

  19. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis.

    PubMed

    De Souza, Amanda P; Alvim Kamei, Claire L; Torres, Andres F; Pattathil, Sivakumar; Hahn, Michael G; Trindade, Luisa M; Buckeridge, Marcos S

    2015-07-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  20. How cell wall complexity influences saccharification efficiency in Miscanthus sinensis

    PubMed Central

    De Souza, Amanda P.; Kamei, Claire L. Alvim; Torres, Andres F.; Pattathil, Sivakumar; Hahn, Michael G.; Trindade, Luisa M.; Buckeridge, Marcos S.

    2015-01-01

    The production of bioenergy from grasses has been developing quickly during the last decade, with Miscanthus being among the most important choices for production of bioethanol. However, one of the key barriers to producing bioethanol is the lack of information about cell wall structure. Cell walls are thought to display compositional differences that lead to emergence of a very high level of complexity, resulting in great diversity in cell wall architectures. In this work, a set of different techniques was used to access the complexity of cell walls of different genotypes of Miscanthus sinensis in order to understand how they interfere with saccharification efficiency. Three genotypes of M. sinensis displaying different patterns of correlation between lignin content and saccharification efficiency were subjected to cell wall analysis by quantitative/qualitative analytical techniques such as monosaccharide composition, oligosaccharide profiling, and glycome profiling. When saccharification efficiency was correlated negatively with lignin, the structural features of arabinoxylan and xyloglucan were found to contribute positively to hydrolysis. In the absence of such correlation, different types of pectins, and some mannans contributed to saccharification efficiency. Different genotypes of M. sinensis were shown to display distinct interactions among their cell wall components, which seem to influence cell wall hydrolysis. PMID:25908240

  1. Cell wall remodeling in mycorrhizal symbiosis: a way towards biotrophism

    PubMed Central

    Balestrini, Raffaella; Bonfante, Paola

    2014-01-01

    Cell walls are deeply involved in the molecular talk between partners during plant and microbe interactions, and their role in mycorrhizae, i.e., the widespread symbiotic associations established between plant roots and soil fungi, has been investigated extensively. All mycorrhizal interactions achieve full symbiotic functionality through the development of an extensive contact surface between the plant and fungal cells, where signals and nutrients are exchanged. The exchange of molecules between the fungal and the plant cytoplasm takes place both through their plasma membranes and their cell walls; a functional compartment, known as the symbiotic interface, is thus defined. Among all the symbiotic interfaces, the complex intracellular interface of arbuscular mycorrhizal (AM) symbiosis has received a great deal of attention since its first description. Here, in fact, the host plasma membrane invaginates and proliferates around all the developing intracellular fungal structures, and cell wall material is laid down between this membrane and the fungal cell surface. By contrast, in ectomycorrhizae (ECM), where the fungus grows outside and between the root cells, plant and fungal cell walls are always in direct contact and form the interface between the two partners. The organization and composition of cell walls within the interface compartment is a topic that has attracted widespread attention, both in ecto- and endomycorrhizae. The aim of this review is to provide a general overview of the current knowledge on this topic by integrating morphological observations, which have illustrated cell wall features during mycorrhizal interactions, with the current data produced by genomic and transcriptomic approaches. PMID:24926297

  2. Serine 26 in the PomB Subunit of the Flagellar Motor Is Essential for Hypermotility of Vibrio cholerae

    PubMed Central

    Halang, Petra; Vorburger, Thomas; Steuber, Julia

    2015-01-01

    Vibrio cholerae is motile by means of its single polar flagellum which is driven by the sodium-motive force. In the motor driving rotation of the flagellar filament, a stator complex consisting of subunits PomA and PomB converts the electrochemical sodium ion gradient into torque. Charged or polar residues within the membrane part of PomB could act as ligands for Na+, or stabilize a hydrogen bond network by interacting with water within the putative channel between PomA and PomB. By analyzing a large data set of individual tracks of swimming cells, we show that S26 located within the transmembrane helix of PomB is required to promote very fast swimming of V. cholerae. Loss of hypermotility was observed with the S26T variant of PomB at pH 7.0, but fast swimming was restored by decreasing the H+ concentration of the external medium. Our study identifies S26 as a second important residue besides D23 in the PomB channel. It is proposed that S26, together with D23 located in close proximity, is important to perturb the hydration shell of Na+ before its passage through a constriction within the stator channel. PMID:25874792

  3. A proteomic and genetic analysis of the Neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling.

    PubMed

    Ao, Jie; Aldabbous, Mash'el; Notaro, Marysa J; Lojacono, Mark; Free, Stephen J

    2016-09-01

    A proteomic analysis of the conidial cell wall identified 35 cell wall proteins. A comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. Deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. Mutants for two cell wall glycosyl hydrolases, the CGL-1 β-1,3-glucanase (NCU07523) and the NAG-1 exochitinase (NCU10852), were found to have a conidial separation phenotype. These two enzymes function in remodeling the cell wall between adjacent conidia to facilitate conidia formation and dissemination. Using promoter::RFP and promoter::GFP constructs, we demonstrated that the promoters for 15 of the conidia-specific cell wall genes, including cgl-1 and nag-1, provided for conidia-specific gene expression or for a significant increase in their expression during conidiation. PMID:27381444

  4. A Genomewide Screen in Schizosaccharomyces pombe for Genes Affecting the Sensitivity of Antifungal Drugs That Target Ergosterol Biosynthesis

    PubMed Central

    Hu, Lingling; Zhou, Xin; Jaiseng, Wurentuya; Zhang, Ben; Takami, Tomonori; Kuno, Takayoshi

    2012-01-01

    We performed a genomewide screen for altered sensitivity to antifungal drugs, including clotrimazole and terbinafine, that target ergosterol biosynthesis using a Schizosaccharomyces pombe gene deletion library consisting of 3,004 nonessential haploid deletion mutants. We identified 109 mutants that were hypersensitive and 11 mutants that were resistant to these antifungals. Proteins whose absence rendered cells sensitive to these antifungals were classified into various functional categories, including ergosterol biosynthesis, membrane trafficking, histone acetylation and deacetylation, ubiquitination, signal transduction, ribosome biosynthesis and assembly, regulation of transcription and translation, cell wall organization and biogenesis, mitochondrion function, amino acid metabolism, nucleic acid metabolism, lipid metabolism, meiosis, and other functions. Also, proteins whose absence rendered cells resistant to these antifungals were classified into functional categories including mitochondrion function, ubiquitination, membrane trafficking, cell polarity, chromatin remodeling, and some unknown functions. Furthermore, the 109 sensitive mutants were tested for sensitivity to micafungin, another antifungal drug that inhibits (1,3)-β-d-glucan synthase, and 57 hypersensitive mutants were identified, suggesting that these mutants were defective in cell wall integrity. Altogether, our findings in fission yeast have shed light on molecular pathways associated with the cellular response to ergosterol biosynthesis inhibitors and may provide useful information for developing strategies aimed at sensitizing cells to these drugs. PMID:22252817

  5. Plant expansins: diversity and interactions with plant cell walls

    PubMed Central

    Cosgrove, Daniel J.

    2015-01-01

    Expansins were discovered two decades ago as cell wall proteins that mediate acid-induced growth by catalyzing loosening of plant cell walls without lysis of wall polymers. In the interim our understanding of expansins has gotten more complex through bioinformatic analysis of expansin distribution and evolution, as well as through expression analysis, dissection of the upstream transcription factors regulating expression, and identification of additional classes of expansin by sequence and structural similarities. Molecular analyses of expansins from bacteria have identified residues essential for wall loosening activity and clarified the bifunctional nature of expansin binding to complex cell walls. Transgenic modulation of expansin expression modifies growth and stress physiology of plants, but not always in predictable and even understandable ways. PMID:26057089

  6. Measurement of pectin methylation in plant cell walls

    SciTech Connect

    McFeeters, R.F.; Armstrong, S.A.

    1984-01-01

    A procedure was developed to measure the degree of pectin methylation in small samples of isolated cell walls from nonlignified plant tissues or pectin solutions. Galacturonic acid was determined colorimetrically with the 3,5-dimethylphenol reagent. Methylation was measured by base hydrolysis of galacturonic acid methyl esters, followed by gas chromatographic determination of released methanol. Estimates of the precision of analysis of pectin and cell wall samples were made. The coefficient of variation for estimates of the pectin esterification in cell walls isolated from 10-g samples of cucumber tissue ranged from 7.7 to 13.2%.

  7. Characterization of O-mannosyltransferase family in Schizosaccharomyces pombe.

    PubMed

    Tanaka, Naotaka; Fujita, Yasuko; Suzuki, Shotaro; Morishita, Masayo; Giga-Hama, Yuko; Shimoda, Chikashi; Takegawa, Kaoru

    2005-05-13

    Protein O-glycosylation is an essential protein modification in eukaryotic cells. In Saccharomyces cerevisiae, O-mannosylation is initiated in the lumen of the endoplasmic reticulum by O-mannosyltransferase gene products (Pmt1p-7p). A search of the Schizosaccharomyces pombe genome database revealed a total of three O-glycoside mannosyltransferase homologs (ogm1+, ogm2+, and ogm4+), closely related to Saccharomyces cerevisiae PMT1, PMT2, and PMT4. Although individual ogm genes were not found to be essential, ogm1Delta and ogm4Delta mutants exhibited aberrant morphology and failed to agglutinate during mating. The phenotypes of the ogm4Delta mutant were not complemented by overexpression of ogm1+ or ogm2+, suggesting that each of the Ogm proteins does not have overlapping functions. Heterologous expression of a chitinase from S. cerevisiae in the ogm mutants revealed that O-glycosylation of chitinase had decreased in ogm1Delta cells. A GFP-tagged Fus1p from S. cerevisiae was specifically not glycosylated and accumulated in the Golgi in ogm4Delta cells. These results indicate that O-glycosylation initiated by Ogm proteins plays crucial physiological roles and can serve as a sorting determinant for protein transport of membrane glycoproteins in S. pombe. PMID:15809069

  8. On the growth of walled cells: From shells to vesicles.

    NASA Astrophysics Data System (ADS)

    Boudaoud, Arezki

    2003-03-01

    The growth of isolated walled cells is investigated. Examples of such cells range from bacteria to giant algae, and include cochlear hair, plant root hair, fungi and yeast cells. They are modeled as elastic shells inflated by a liquid. Cell growth is driven by fluid pressure and is similar to a plastic deformation of the wall. The requirement of mechanical equilibrium leads to two new scaling laws for cell size that are in quantitative agreement with the compiled biological data. Given these results, possible shapes for growing cells are computed by analogy with those of vesicle membranes.

  9. Growth of Walled Cells: From Shells to Vesicles

    NASA Astrophysics Data System (ADS)

    Boudaoud, Arezki

    2003-07-01

    The growth of isolated walled cells is investigated. Examples of such cells range from bacteria to giant algae, and include cochlear hair, plant root hair, fungi, and yeast cells. They are modeled as elastic shells containing a liquid. Cell growth is driven by fluid pressure and is is similar to a plastic deformation of the wall. The requirement of mechanical equilibrium leads to two new scaling laws for cell size that are in quantitative agreement with the compiled biological data. Given these results, possible shapes for growing cells are computed by analogy with those of vesicle membranes.

  10. An Arabidopsis Gene Regulatory Network for Secondary Cell Wall Synthesis

    PubMed Central

    Taylor-Teeples, M; Lin, L; de Lucas, M; Turco, G; Toal, TW; Gaudinier, A; Young, NF; Trabucco, GM; Veling, MT; Lamothe, R; Handakumbura, PP; Xiong, G; Wang, C; Corwin, J; Tsoukalas, A; Zhang, L; Ware, D; Pauly, M; Kliebenstein, DJ; Dehesh, K; Tagkopoulos, I; Breton, G; Pruneda-Paz, JL; Ahnert, SE; Kay, SA; Hazen, SP; Brady, SM

    2014-01-01

    Summary The plant cell wall is an important factor for determining cell shape, function and response to the environment. Secondary cell walls, such as those found in xylem, are composed of cellulose, hemicelluloses and lignin and account for the bulk of plant biomass. The coordination between transcriptional regulation of synthesis for each polymer is complex and vital to cell function. A regulatory hierarchy of developmental switches has been proposed, although the full complement of regulators remains unknown. Here, we present a protein-DNA network between Arabidopsis transcription factors and secondary cell wall metabolic genes with gene expression regulated by a series of feed-forward loops. This model allowed us to develop and validate new hypotheses about secondary wall gene regulation under abiotic stress. Distinct stresses are able to perturb targeted genes to potentially promote functional adaptation. These interactions will serve as a foundation for understanding the regulation of a complex, integral plant component. PMID:25533953

  11. Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe.

    PubMed Central

    Tsutsui, Y; Khasanov, F K; Shinagawa, H; Iwasaki, H; Bashkirov, V I

    2001-01-01

    Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved. PMID:11560889

  12. Regulation of Meristem Morphogenesis by Cell Wall Synthases in Arabidopsis.

    PubMed

    Yang, Weibing; Schuster, Christoph; Beahan, Cherie T; Charoensawan, Varodom; Peaucelle, Alexis; Bacic, Antony; Doblin, Monika S; Wightman, Raymond; Meyerowitz, Elliot M

    2016-06-01

    The cell walls of the shoot apical meristem (SAM), containing the stem cell niche that gives rise to the above-ground tissues, are crucially involved in regulating differentiation. It is currently unknown how these walls are built and refined or their role, if any, in influencing meristem developmental dynamics. We have combined polysaccharide linkage analysis, immuno-labeling, and transcriptome profiling of the SAM to provide a spatiotemporal plan of the walls of this dynamic structure. We find that meristematic cells express only a core subset of 152 genes encoding cell wall glycosyltransferases (GTs). Systemic localization of all these GT mRNAs by in situ hybridization reveals members with either enrichment in or specificity to apical subdomains such as emerging flower primordia, and a large class with high expression in dividing cells. The highly localized and coordinated expression of GTs in the SAM suggests distinct wall properties of meristematic cells and specific differences between newly forming walls and their mature descendants. Functional analysis demonstrates that a subset of CSLD genes is essential for proper meristem maintenance, confirming the key role of walls in developmental pathways. PMID:27212401

  13. Cell Wall Metabolism in Response to Abiotic Stress

    PubMed Central

    Gall, Hyacinthe Le; Philippe, Florian; Domon, Jean-Marc; Gillet, Françoise; Pelloux, Jérôme; Rayon, Catherine

    2015-01-01

    This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions. PMID:27135320

  14. Cell Wall Metabolism in Response to Abiotic Stress.

    PubMed

    Le Gall, Hyacinthe; Philippe, Florian; Domon, Jean-Marc; Gillet, Françoise; Pelloux, Jérôme; Rayon, Catherine

    2015-01-01

    This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions. PMID:27135320

  15. Replication of centromere II of Schizosaccharomyces pombe.

    PubMed Central

    Smith, J G; Caddle, M S; Bulboaca, G H; Wohlgemuth, J G; Baum, M; Clarke, L; Calos, M P

    1995-01-01

    The centromeric DNAs of Schizosaccharomyces pombe chromosomes resemble those of higher eukaryotes in being large and composed predominantly of repeated sequences. To begin a detailed analysis of the mode of replication of a complex centromere, we examined whether any sequences within S. pombe centromere II (cen2) have the ability to mediate autonomous replication. We found a high density of segments with such activity, including at least eight different regions comprising most of the repeated and unique centromeric DNA elements. A physical mapping analysis using two-dimensional gels showed that autonomous replication initiated within the S. pombe sequences in each plasmid. A two-dimensional gel analysis of replication on the chromosomes revealed that the K and L repeat elements, which occur in multiple copies at all three centromeres and comprise approximately 70% of total centromeric DNA mass in S. pombe, are both sites of replication initiation. In contrast, the unique cen2 central core, which contains multiple segments that can support autonomous replication, appears to be repressed for initiation on the chromosome. We discuss the implications of these findings for our understanding of DNA replication and centromere function. PMID:7651433

  16. Cell Wall Composition, Biosynthesis and Remodeling during Pollen Tube Growth

    PubMed Central

    Mollet, Jean-Claude; Leroux, Christelle; Dardelle, Flavien; Lehner, Arnaud

    2013-01-01

    The pollen tube is a fast tip-growing cell carrying the two sperm cells to the ovule allowing the double fertilization process and seed setting. To succeed in this process, the spatial and temporal controls of pollen tube growth within the female organ are critical. It requires a massive cell wall deposition to promote fast pollen tube elongation and a tight control of the cell wall remodeling to modify the mechanical properties. In addition, during its journey, the pollen tube interacts with the pistil, which plays key roles in pollen tube nutrition, guidance and in the rejection of the self-incompatible pollen. This review focuses on our current knowledge in the biochemistry and localization of the main cell wall polymers including pectin, hemicellulose, cellulose and callose from several pollen tube species. Moreover, based on transcriptomic data and functional genomic studies, the possible enzymes involved in the cell wall remodeling during pollen tube growth and their impact on the cell wall mechanics are also described. Finally, mutant analyses have permitted to gain insight in the function of several genes involved in the pollen tube cell wall biosynthesis and their roles in pollen tube growth are further discussed. PMID:27137369

  17. A formin-nucleated actin aster concentrates cell wall hydrolases for cell fusion in fission yeast

    PubMed Central

    Dudin, Omaya; Bendezú, Felipe O.; Groux, Raphael; Laroche, Thierry; Seitz, Arne

    2015-01-01

    Cell–cell fusion is essential for fertilization. For fusion of walled cells, the cell wall must be degraded at a precise location but maintained in surrounding regions to protect against lysis. In fission yeast cells, the formin Fus1, which nucleates linear actin filaments, is essential for this process. In this paper, we show that this formin organizes a specific actin structure—the actin fusion focus. Structured illumination microscopy and live-cell imaging of Fus1, actin, and type V myosins revealed an aster of actin filaments whose barbed ends are focalized near the plasma membrane. Focalization requires Fus1 and type V myosins and happens asynchronously always in the M cell first. Type V myosins are essential for fusion and concentrate cell wall hydrolases, but not cell wall synthases, at the fusion focus. Thus, the fusion focus focalizes cell wall dissolution within a broader cell wall synthesis zone to shift from cell growth to cell fusion. PMID:25825517

  18. Plant cell wall characterization using scanning probe microscopy techniques

    PubMed Central

    Yarbrough, John M; Himmel, Michael E; Ding, Shi-You

    2009-01-01

    Lignocellulosic biomass is today considered a promising renewable resource for bioenergy production. A combined chemical and biological process is currently under consideration for the conversion of polysaccharides from plant cell wall materials, mainly cellulose and hemicelluloses, to simple sugars that can be fermented to biofuels. Native plant cellulose forms nanometer-scale microfibrils that are embedded in a polymeric network of hemicelluloses, pectins, and lignins; this explains, in part, the recalcitrance of biomass to deconstruction. The chemical and structural characteristics of these plant cell wall constituents remain largely unknown today. Scanning probe microscopy techniques, particularly atomic force microscopy and its application in characterizing plant cell wall structure, are reviewed here. We also further discuss future developments based on scanning probe microscopy techniques that combine linear and nonlinear optical techniques to characterize plant cell wall nanometer-scale structures, specifically apertureless near-field scanning optical microscopy and coherent anti-Stokes Raman scattering microscopy. PMID:19703302

  19. Modification of cell wall polysaccharides during retting of cassava roots.

    PubMed

    Ngolong Ngea, Guillaume Legrand; Guillon, Fabienne; Essia Ngang, Jean Justin; Bonnin, Estelle; Bouchet, Brigitte; Saulnier, Luc

    2016-12-15

    Retting is an important step in traditional cassava processing that involves tissue softening of the roots to transform the cassava into flour and various food products. The tissue softening that occurs during retting was attributed to the degradation of cell wall pectins through the action of pectin-methylesterase and pectate-lyase that possibly originated from a microbial source or the cassava plant itself. Changes in cell wall composition were investigated during retting using chemical analysis, specific glycanase degradation and immuno-labelling of cell wall polysaccharides. Pectic 1,4-β-d-galactan was the main cell wall polysaccharide affected during the retting of cassava roots. This result suggested that better control of pectic galactan degradation and a better understanding of the degradation mechanism by endogenous endo-galactanase and/or exogenous microbial enzymes might contribute to improve the texture properties of cassava products. PMID:27451197

  20. Evolution and development of cell walls in cereal grains

    PubMed Central

    Burton, Rachel A.; Fincher, Geoffrey B.

    2014-01-01

    The composition of cell walls in cereal grains and other grass species differs markedly from walls in seeds of other plants. In the maternal tissues that surround the embryo and endosperm of the grain, walls contain higher levels of cellulose and in many cases are heavily lignified. This may be contrasted with walls of the endosperm, where the amount of cellulose is relatively low, and the walls are generally not lignified. The low cellulose and lignin contents are possible because the walls of the endosperm perform no load-bearing function in the mature grain and indeed the low levels of these relatively intractable wall components are necessary because they allow rapid degradation of the walls following germination of the grain. The major non-cellulosic components of endosperm walls are usually heteroxylans and (1,3;1,4)-β-glucans, with lower levels of xyloglucans, glucomannans, and pectic polysaccharides. Pectic polysaccharides and xyloglucans are the major non-cellulosic wall constituents in most dicot species, in which (1,3;1,4)-β-glucans are usually absent and heteroxylans are found at relatively low levels. Thus, the “core” non-cellulosic wall polysaccharides in grain of the cereals and other grasses are the heteroxylans and, more specifically, arabinoxylans. The (1,3;1,4)-β-glucans appear in the endosperm of some grass species but are essentially absent from others; they may constitute from zero to more than 45% of the cell walls of the endosperm, depending on the species. It is clear that in some cases these (1,3;1,4)-β-glucans function as a major store of metabolizable glucose in the grain. Cereal grains and their constituent cell wall polysaccharides are centrally important as a source of dietary fiber in human societies and breeders have started to select for high levels of non-cellulosic wall polysaccharides in grain. To meet end-user requirements, it is important that we understand cell wall biology in the grain both during development and

  1. An improved protocol to study the plant cell wall proteome

    PubMed Central

    Printz, Bruno; Dos Santos Morais, Raphaël; Wienkoop, Stefanie; Sergeant, Kjell; Lutts, Stanley; Hausman, Jean-Francois; Renaut, Jenny

    2015-01-01

    Cell wall proteins were extracted from alfalfa stems according to a three-steps extraction procedure using sequentially CaCl2, EGTA, and LiCl-complemented buffers. The efficiency of this protocol for extracting cell wall proteins was compared with the two previously published methods optimized for alfalfa stem cell wall protein analysis. Following LC-MS/MS analysis the three-steps extraction procedure resulted in the identification of the highest number of cell wall proteins (242 NCBInr identifiers) and gave the lowest percentage of non-cell wall proteins (about 30%). However, the three protocols are rather complementary than substitutive since 43% of the identified proteins were specific to one protocol. This three-step protocol was therefore selected for a more detailed proteomic characterization using 2D-gel electrophoresis. With this technique, 75% of the identified proteins were shown to be fraction-specific and 72.7% were predicted as belonging to the cell wall compartment. Although, being less sensitive than LC-MS/MS approaches in detecting and identifying low-abundant proteins, gel-based approaches are valuable tools for the differentiation and relative quantification of protein isoforms and/or modified proteins. In particular isoforms, having variations in their amino-acid sequence and/or carrying different N-linked glycan chains were detected and characterized. This study highlights how the extracting protocols as well as the analytical techniques devoted to the study of the plant cell wall proteome are complementary and how they may be combined to elucidate the dynamism of the plant cell wall proteome in biological studies. Data are available via ProteomeXchange with identifier PXD001927. PMID:25914713

  2. Construction, molecular modeling, and simulation of Mycobacterium tuberculosis cell walls.

    PubMed

    Hong, Xuan; Hopfinger, A J

    2004-01-01

    The mycobacterial cell wall is extraordinarily thick and tight consisting mainly of (1). long chain fatty acids, the mycolic acids, and (2). a unique polysaccharide, arabinogalactan (AG). These two chemical constituents are covalently linked through ester bonds. Minnikin (The Biology of the Mycobacteria; Academic: London, 1982) proposed that the mycobacterial cell wall is composed of an asymmetric lipid bilayer. The inner leaflet of the cell wall contains mycolic acids covalently linked to AG. This inner leaflet is believed to have the lowest permeability to organic compounds of the overall cell wall. Conformational search and molecular dynamics simulation were used to explore the conformational profile of AG and the conformations and structural organization of the mycolic acid-AG complex, and overall, an inner leaflet molecular model of the cell wall was constructed. The terminal arabinose residues of AG that serve as linkers between AG and mycolic acids were found to exist in four major chemical configurations. The mycolate hydrocarbon chains were determined to be tightly packed and perpendicular to the "plane" formed by the oxygen atoms of the 5-hydroxyl groups of the terminal arabinose residues. For Mycobacterium tuberculosis, the average packing distance between mycolic acids is estimated to be approximately 7.3 A. Thus, Minnikin's model is supported by this computational study. Overall, this modeling and simulation approach provides a way to probe the mechanism of low permeability of the cell wall and the intrinsic drug resistance of M. tuberculosis. In addition, monolayer models were built for both dipalmitoylphosphatidylethanolamine and dimyristoylphosphatidylcholine, two common phospholipids in bacterial and animal membranes, respectively. Structural comparisons of these cell wall phospholipid membrane models were made to the M. tuberculosis cell wall model. PMID:15132700

  3. Cell wall polysaccharides from fern leaves: evidence for a mannan-rich Type III cell wall in Adiantum raddianum.

    PubMed

    Silva, Giovanna B; Ionashiro, Mari; Carrara, Thalita B; Crivellari, Augusto C; Tiné, Marco A S; Prado, Jefferson; Carpita, Nicholas C; Buckeridge, Marcos S

    2011-12-01

    Primary cell walls from plants are composites of cellulose tethered by cross-linking glycans and embedded in a matrix of pectins. Cell wall composition varies between plant species, reflecting in some instances the evolutionary distance between them. In this work the monosaccharide compositions of isolated primary cell walls of nine fern species and one lycophyte were characterized and compared with those from Equisetum and an angiosperm dicot. The relatively high abundance of mannose in these plants suggests that mannans may constitute the major cross-linking glycan in the primary walls of pteridophytes and lycophytes. Pectin-related polysaccharides contained mostly rhamnose and uronic acids, indicating the presence of rhamnogalacturonan I highly substituted with galactose and arabinose. Structural and fine-structural analyses of the hemicellulose fraction of leaves of Adiantum raddianum confirmed this hypothesis. Linkage analysis showed that the mannan contains mostly 4-Man with very little 4,6-Man, indicating a low percentage of branching with galactose. Treatment of the mannan-rich fractions with endo-β-mannanase produced characteristic mannan oligosaccharides. Minor amounts of xyloglucan and xylans were also detected. These data and those of others suggest that all vascular plants contain xyloglucans, arabinoxylans, and (gluco)mannans, but in different proportions that define cell wall types. Whereas xyloglucan and pectin-rich walls define Type I walls of dicots and many monocots, arabinoxylans and lower proportion of pectin define the Type II walls of commelinoid monocots. The mannan-rich primary walls with low pectins of many ferns and a lycopod indicate a fundamentally different wall type among land plants, the Type III wall. PMID:21955619

  4. A Genome-Wide Screen of Genes Involved in Cadmium Tolerance in Schizosaccharomyces pombe

    PubMed Central

    Kennedy, Patrick J.; Vashisht, Ajay A.; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Russell, Paul

    2008-01-01

    Cadmium is a worldwide environmental toxicant responsible for a range of human diseases including cancer. Cellular injury from cadmium is minimized by stress-responsive detoxification mechanisms. We explored the genetic requirements for cadmium tolerance by individually screening mutants from the fission yeast (Schizosaccharomyces pombe) haploid deletion collection for inhibited growth on agar growth media containing cadmium. Cadmium-sensitive mutants were further tested for sensitivity to oxidative stress (hydrogen peroxide) and osmotic stress (potassium chloride). Of 2649 mutants screened, 237 were sensitive to cadmium, of which 168 were cadmium specific. Most were previously unknown to be involved in cadmium tolerance. The 237 genes represent a number of pathways including sulfate assimilation, phytochelatin synthesis and transport, ubiquinone (Coenzyme Q10) biosynthesis, stress signaling, cell wall biosynthesis and cell morphology, gene expression and chromatin remodeling, vacuole function, and intracellular transport of macromolecules. The ubiquinone biosynthesis mutants are acutely sensitive to cadmium but only mildly sensitive to hydrogen peroxide, indicating that Coenzyme Q10 plays a larger role in cadmium tolerance than just as an antioxidant. These and several other mutants turn yellow when exposed to cadmium, suggesting cadmium sulfide accumulation. This phenotype can potentially be used as a biomarker for cadmium. There is remarkably little overlap with a comparable screen of the Saccharomyces cerevisiae haploid deletion collection, indicating that the two distantly related yeasts utilize significantly different strategies for coping with cadmium stress. These strategies and their relation to cadmium detoxification in humans are discussed. PMID:18684775

  5. Characterization and Localization of Insoluble Organic Matrices Associated with Diatom Cell Walls: Insight into Their Roles during Cell Wall Formation

    PubMed Central

    Tesson, Benoit; Hildebrand, Mark

    2013-01-01

    Organic components associated with diatom cell wall silica are important for the formation, integrity, and function of the cell wall. Polysaccharides are associated with the silica, however their localization, structure, and function remain poorly understood. We used imaging and biochemical approaches to describe in detail characteristics of insoluble organic components associated with the cell wall in 5 different diatom species. Results show that an insoluble organic matrix enriched in mannose, likely the diatotepum, is localized on the proximal surface of the silica cell wall. We did not identify any organic matrix embedded within the silica. We also identified a distinct material consisting of glucose polymer with variable localization depending on the species. In some species this component was directly involved in the morphogenesis of silica structure while in others it appeared to be only a structural component of the cell wall. A novel glucose-rich structure located between daughter cells during division was also identified. This work for the first time correlates the structure, composition, and localization of insoluble organic matrices associated with diatom cell walls. Additionally we identified a novel glucose polymer and characterized its role during silica structure formation. PMID:23626714

  6. Live cell imaging of the cytoskeleton and cell wall enzymes in plant cells.

    PubMed

    Sampathkumar, Arun; Wightman, Raymond

    2015-01-01

    The use of live imaging techniques to visualize the dynamic changes and interactions within plant cells has given us detailed information on the function and organization of the cytoskeleton and cell wall associated proteins. This information has grown with the constant improvement in imaging hardware and molecular tools. In this chapter, we describe the procedure for the preparation and live visualization of fluorescent protein fusions associated with the cytoskeleton and the cell wall in Arabidopsis. PMID:25408450

  7. Characterizing visible and invisible cell wall mutant phenotypes.

    PubMed

    Carpita, Nicholas C; McCann, Maureen C

    2015-07-01

    About 10% of a plant's genome is devoted to generating the protein machinery to synthesize, remodel, and deconstruct the cell wall. High-throughput genome sequencing technologies have enabled a reasonably complete inventory of wall-related genes that can be assembled into families of common evolutionary origin. Assigning function to each gene family member has been aided immensely by identification of mutants with visible phenotypes or by chemical and spectroscopic analysis of mutants with 'invisible' phenotypes of modified cell wall composition and architecture that do not otherwise affect plant growth or development. This review connects the inference of gene function on the basis of deviation from the wild type in genetic functional analyses to insights provided by modern analytical techniques that have brought us ever closer to elucidating the sequence structures of the major polysaccharide components of the plant cell wall. PMID:25873661

  8. Ultrastructure and Composition of the Nannochloropsis gaditana Cell Wall

    PubMed Central

    Scholz, Matthew J.; Weiss, Taylor L.; Jinkerson, Robert E.; Jing, Jia; Roth, Robyn; Goodenough, Ursula; Posewitz, Matthew C.

    2014-01-01

    Marine algae of the genus Nannochloropsis are promising producers of biofuel precursors and nutraceuticals and are also harvested commercially for aquaculture feed. We have used quick-freeze, deep-etch electron microscopy, Fourier transform infrared spectroscopy, and carbohydrate analyses to characterize the architecture of the Nannochloropsis gaditana (strain CCMP 526) cell wall, whose recalcitrance presents a significant barrier to biocommodity extraction. The data indicate a bilayer structure consisting of a cellulosic inner wall (∼75% of the mass balance) protected by an outer hydrophobic algaenan layer. Cellulase treatment of walls purified after cell lysis generates highly enriched algaenan preparations without using the harsh chemical treatments typically used in algaenan isolation and characterization. Nannochloropsis algaenan was determined to comprise long, straight-chain, saturated aliphatics with ether cross-links, which closely resembles the cutan of vascular plants. Chemical identification of >85% of the isolated cell wall mass is detailed, and genome analysis is used to identify candidate biosynthetic enzymes. PMID:25239976

  9. A Model for Cell Wall Dissolution in Mating Yeast Cells: Polarized Secretion and Restricted Diffusion of Cell Wall Remodeling Enzymes Induces Local Dissolution

    PubMed Central

    Huberman, Lori B.; Murray, Andrew W.

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

  10. A model for cell wall dissolution in mating yeast cells: polarized secretion and restricted diffusion of cell wall remodeling enzymes induces local dissolution.

    PubMed

    Huberman, Lori B; Murray, Andrew W

    2014-01-01

    Mating of the budding yeast, Saccharomyces cerevisiae, occurs when two haploid cells of opposite mating types signal using reciprocal pheromones and receptors, grow towards each other, and fuse to form a single diploid cell. To fuse, both cells dissolve their cell walls at the point of contact. This event must be carefully controlled because the osmotic pressure differential between the cytoplasm and extracellular environment causes cells with unprotected plasma membranes to lyse. If the cell wall-degrading enzymes diffuse through the cell wall, their concentration would rise when two cells touched each other, such as when two pheromone-stimulated cells adhere to each other via mating agglutinins. At the surfaces that touch, the enzymes must diffuse laterally through the wall before they can escape into the medium, increasing the time the enzymes spend in the cell wall, and thus raising their concentration at the point of attachment and restricting cell wall dissolution to points where cells touch each other. We tested this hypothesis by studying pheromone treated cells confined between two solid, impermeable surfaces. This confinement increases the frequency of pheromone-induced cell death, and this effect is diminished by reducing the osmotic pressure difference across the cell wall or by deleting putative cell wall glucanases and other genes necessary for efficient cell wall fusion. Our results support the model that pheromone-induced cell death is the result of a contact-driven increase in the local concentration of cell wall remodeling enzymes and suggest that this process plays an important role in regulating cell wall dissolution and fusion in mating cells. PMID:25329559

  11. Bending forces plastically deform growing bacterial cell walls.

    PubMed

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B; Nelson, David R; Jun, Suckjoon

    2014-04-22

    Cell walls define a cell's shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell's responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  12. Production Model Press for the Preparation of Bacterial Cell Walls

    PubMed Central

    Perrine, T. D.; Ribi, E.; Maki, W.; Miller, B.; Oertli, E.

    1962-01-01

    A modification of the apparatus previously described permits the preparation of cell walls in quantity. This consists of a heavy duty, double-acting hydraulic press with motor-driven pump, and a superstrength alloy steel pressure cell which is corrosion resistant. Liquid cooling of the jet is substituted for the previously used gas cooling to minimize aerosol formation and to facilitate subsequent treatment of the products. The device produces cell walls of excellent quality in good yield. The pressure cell has been used satisfactorily up to about 60,000 psi. Design details are given. Images FIG. 1 FIG. 2 FIG. 6 PMID:14485524

  13. Motion of red blood cells near microvessel walls: effects of a porous wall layer

    PubMed Central

    HARIPRASAD, DANIEL S.; SECOMB, TIMOTHY W.

    2013-01-01

    A two-dimensional model is used to simulate the motion and deformation of a single mammalian red blood cell (RBC) flowing close to the wall of a microvessel, taking into account the effects of a porous endothelial surface layer (ESL) lining the vessel wall. Migration of RBCs away from the wall leads to the formation of a cell-depleted layer near the wall, which has a large effect on the resistance to blood flow in microvessels. The objective is to examine the mechanical factors causing this migration, including the effects of the ESL. The vessel is represented as a straight parallel-sided channel. The RBC is represented as a set of interconnected viscoelastic elements, suspended in plasma, a Newtonian fluid. The ESL is represented as a porous medium, and plasma flow in the layer is computed using the Brinkman approximation. It is shown that an initially circular cell positioned close to the ESL in a shear flow is deformed into an asymmetric shape. This breaking of symmetry leads to migration away from the wall. With increasing hydraulic resistivity of the layer, the rate of lateral migration increases. It is concluded that mechanical interactions of RBCs flowing in microvessels with a porous wall layer may reduce the rate of lateral migration and hence reduce the width of the cell-depleted zone external to the ESL, relative to the cell-depleted zone that would be formed if the interface between the ESL and free-flowing plasma were replaced by an impermeable boundary. PMID:23493820

  14. Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe.

    PubMed

    Brown, William R A; Thomas, Geraint; Lee, Nicholas C O; Blythe, Martin; Liti, Gianni; Warringer, Jonas; Loose, Matthew W

    2014-02-01

    Kinetochores in multicellular eukaryotes are usually associated with heterochromatin. Whether this heterochromatin simply promotes the cohesion necessary for accurate chromosome segregation at cell division or whether it also has a role in kinetochore assembly is unclear. Schizosaccharomyces pombe is an important experimental system for investigating centromere function, but all of the previous work with this species has exploited a single strain or its derivatives. The laboratory strain and most other S. pombe strains contain three chromosomes, but one recently discovered strain, CBS 2777, contains four. We show that the genome of CBS 2777 is related to that of the laboratory strain by a complex chromosome rearrangement. As a result, two of the kinetochores in CBS 2777 contain the central core sequences present in the laboratory strain centromeres, but lack adjacent heterochromatin. The closest block of heterochromatin to these rearranged kinetochores is ∼100 kb away at new telomeres. Despite lacking large amounts of adjacent heterochromatin, the rearranged kinetochores bind CENP-A(Cnp1) and CENP-C(Cnp3) in similar quantities and with similar specificities as those of the laboratory strain. The simplest interpretation of this result is that constitutive kinetochore assembly and heterochromatin formation occur autonomously. PMID:24449889

  15. Site Specific Genetic Incorporation of Azidophenylalanine in Schizosaccharomyces pombe

    PubMed Central

    Shao, Nan; Singh, N. Sadananda; Slade, Susan E.; Jones, Alexandra M. E.; Balasubramanian, Mohan K.

    2015-01-01

    The diversity of protein functions is impacted in significant part by the chemical properties of the twenty amino acids, which are used as building blocks for nearly all proteins. The ability to incorporate unnatural amino acids (UAA) into proteins in a site specific manner can vastly expand the repertoire of protein functions and also allows detailed analysis of protein function. In recent years UAAs have been incorporated in a site-specific manner into proteins in a number of organisms. In nearly all cases, the amber codon is used as a sense codon, and an orthogonal tRNA/aminoacyl-tRNA synthetase (RS) pair is used to generate amber suppressing tRNAs charged with the UAA. In this work, we have developed tools to incorporate the cross-linking amino acid azido-phenylalanine (AzF) through the use of bacterial tRNATyr and a modified version of TyrRS, AzFRS, in Schizosaccharomyces pombe, which is an attractive model organism for the study of cell behavior and function. We have incorporated AzF into three different proteins. We show that the majority of AzF is modified to amino-phenyl alanine, but protein cross-linking was still observed. These studies set the stage for exploitation of this new technology for the analysis of S. pombe proteins. PMID:26597962

  16. Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall

    PubMed Central

    Wolf, Julie M.; Espadas-Moreno, Javier; Luque-Garcia, Jose L.

    2014-01-01

    Cryptococcus neoformans produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition. PMID:24906412

  17. Another Brick in the Cell Wall: Biosynthesis Dependent Growth Model

    PubMed Central

    Barbacci, Adelin; Lahaye, Marc; Magnenet, Vincent

    2013-01-01

    Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i) a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii) new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper. PMID:24066142

  18. Another brick in the cell wall: biosynthesis dependent growth model.

    PubMed

    Barbacci, Adelin; Lahaye, Marc; Magnenet, Vincent

    2013-01-01

    Expansive growth of plant cell is conditioned by the cell wall ability to extend irreversibly. This process is possible if (i) a tensile stress is developed in the cell wall due to the coupling effect between turgor pressure and the modulation of its mechanical properties through enzymatic and physicochemical reactions and if (ii) new cell wall elements can be synthesized and assembled to the existing wall. In other words, expansive growth is the result of coupling effects between mechanical, thermal and chemical energy. To have a better understanding of this process, models must describe the interplay between physical or mechanical variable with biological events. In this paper we propose a general unified and theoretical framework to model growth in function of energy forms and their coupling. This framework is based on irreversible thermodynamics. It is then applied to model growth of the internodal cell of Chara corallina modulated by changes in pressure and temperature. The results describe accurately cell growth in term of length increment but also in term of cell pectate biosynthesis and incorporation to the expanding wall. Moreover, the classical growth model based on Lockhart's equation such as the one proposed by Ortega, appears as a particular and restrictive case of the more general growth equation developed in this paper. PMID:24066142

  19. Glycan Profiling of Plant Cell Wall Polymers using Microarrays

    PubMed Central

    Moller, Isabel E.; Pettolino, Filomena A.; Hart, Charlie; Lampugnani, Edwin R.; Willats, William G.T.; Bacic, Antony

    2012-01-01

    Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging. Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4. In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification. Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments

  20. Cell wall proteomics of the green alga Haematococcus pluvialis (Chlorophyceae).

    PubMed

    Wang, Sheng-Bing; Hu, Qiang; Sommerfeld, Milton; Chen, Feng

    2004-03-01

    The green microalga Haematococcus pluvialis can synthesize and accumulate large amounts of the ketocarotenoid astaxanthin, and undergo profound changes in cell wall composition and architecture during the cell cycle and in response to environmental stresses. In this study, cell wall proteins (CWPs) of H. pluvialis were systematically analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with peptide mass fingerprinting (PMF) and sequence-database analysis. In total, 163 protein bands were analyzed, which resulted in positive identification of 81 protein orthologues. The highly complex and dynamic composition of CWPs is manifested by the fact that the majority of identified CWPs are differentially expressed at specific stages of the cell cycle along with a number of common wall-associated 'housekeeping' proteins. The detection of cellulose synthase orthologue in the vegetative cells suggested that the biosynthesis of cellulose occurred during primary wall formation, in contrast to earlier observations that cellulose was exclusively present in the secondary wall of the organism. A transient accumulation of a putative cytokinin oxidase at the early stage of encystment pointed to a possible role in cytokinin degradation while facilitating secondary wall formation and/or assisting in cell expansion. This work represents the first attempt to use a proteomic approach to investigate CWPs of microalgae. The reference protein map constructed and the specific protein markers obtained from this study provide a framework for future characterization of the expression and physiological functions of the proteins involved in the biogenesis and modifications in the cell wall of Haematococcus and related organisms. PMID:14997492

  1. The composition of the cell wall of Aspergillus niger

    PubMed Central

    Johnston, I. R.

    1965-01-01

    1. The cell-wall composition of Aspergillus niger has been investigated. Analysis shows the presence of six sugars, glucose, galactose, mannose, arabinose, glucosamine and galactosamine, all in the d-configuration, except that a small amount of l-galactose may be present. Sixteen common amino acids are also present. 2. The wall consists chiefly of neutral carbohydrate (73–83%) and hexosamine (9–13%), with smaller amounts of lipid (2–7%), protein (0·5–2·5%) and phosphorus (less than 0·1%). The acetyl content (3·0–3·4%) corresponds to 1·0mole/mole of hexosamine nitrogen. 3. A fractionation of the cell-wall complex was achieved, with or without a preliminary phenol extraction, by using n-sodium hydroxide. Though this caused some degradation, 30–60% of the wall could be solubilized (depending on the preparation). Analyses on several fractions suggest that fractionation procedures bring about some separation of components although not in a clear-cut fashion. 4. Cell-wall preparations were shown to yield a fraction having [α]D approx. +240° (in n-sodium hydroxide) and consisting largely of glucose. This was separated into two subfractions, one of which had [α]D+281° (in n-sodium hydroxide) and had properties resembling the polysaccharide nigeran; the other had [α]D +231° (in n-sodium hydroxide). It is suggested that nigeran is a cell-wall component. PMID:5862404

  2. Control of cell wall extensibility during pollen tube growth.

    PubMed

    Hepler, Peter K; Rounds, Caleb M; Winship, Lawrence J

    2013-07-01

    In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity. PMID:23770837

  3. Control of Cell Wall Extensibility during Pollen Tube Growth

    PubMed Central

    Hepler, Peter K.

    2013-01-01

    In this review, we address the question of how the tip-growing pollen tube achieves its rapid rate of elongation while maintaining an intact cell wall. Although turgor is essential for growth to occur, the local expansion rate is controlled by local changes in the viscosity of the apical wall. We focus on several different structures and underlying processes that are thought to be major participants including exocytosis, the organization and activity of the actin cytoskeleton, calcium and proton physiology, and cellular energetics. We think that the actin cytoskeleton, in particular the apical cortical actin fringe, directs the flow of vesicles to the apical domain, where they fuse with the plasma membrane and contribute their contents to the expanding cell wall. While pH gradients, as generated by a proton-ATPase located on the plasma membrane along the side of the clear zone, may regulate rapid actin turnover and new polymerization in the fringe, the tip-focused calcium gradient biases secretion towards the polar axis. The recent data showing that exocytosis of new wall material precedes and predicts the process of cell elongation provide support for the idea that the intussusception of newly secreted pectin contributes to decreases in apical wall viscosity and to cell expansion. Other prime factors will be the localization and activity of the enzyme pectin methyl-esterase, and the chelation of calcium by pectic acids. Finally, we acknowledge a role for reactive oxygen species in the control of wall viscosity. PMID:23770837

  4. Bending forces plastically deform growing bacterial cell walls

    PubMed Central

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B.; Nelson, David R.; Jun, Suckjoon

    2014-01-01

    Cell walls define a cell’s shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell’s responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  5. Microfabricated alkali vapor cell with anti-relaxation wall coating

    SciTech Connect

    Straessle, R.; Pétremand, Y.; Briand, D.; Rooij, N. F. de; Pellaton, M.; Affolderbach, C.; Mileti, G.

    2014-07-28

    We present a microfabricated alkali vapor cell equipped with an anti-relaxation wall coating. The anti-relaxation coating used is octadecyltrichlorosilane and the cell was sealed by thin-film indium-bonding at a low temperature of 140 °C. The cell body is made of silicon and Pyrex and features a double-chamber design. Depolarizing properties due to liquid Rb droplets are avoided by confining the Rb droplets to one chamber only. Optical and microwave spectroscopy performed on this wall-coated cell are used to evaluate the cell's relaxation properties and a potential gas contamination. Double-resonance signals obtained from the cell show an intrinsic linewidth that is significantly lower than the linewidth that would be expected in case the cell had no wall coating but only contained a buffer-gas contamination on the level measured by optical spectroscopy. Combined with further experimental evidence this proves the presence of a working anti-relaxation wall coating in the cell. Such cells are of interest for applications in miniature atomic clocks, magnetometers, and other quantum sensors.

  6. Casein kinase 2 inhibits HomolD-directed transcription by Rrn7 in Schizosaccharomyces pombe.

    PubMed

    Moreira-Ramos, Sandra; Rojas, Diego A; Montes, Matías; Urbina, Fabiola; Miralles, Vicente J; Maldonado, Edio

    2015-02-01

    In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA analogue element called the HomolD box. The HomolD-binding protein Rrn7 forms a complex with the RNA polymerase II machinery. Despite the importance of ribosome biogenesis to cell survival, the mechanisms involved in the regulation of transcription of eukaryotic RPGs are unknown. In this study, we identified Rrn7 as a new substrate of the pleiotropic casein kinase 2 (CK2), which is a regulator of basal transcription. Recombinant Rrn7 from S. pombe, which is often used as a model organism for studying eukaryotic transcription, interacted with CK2 in vitro and in vivo. Furthermore, CK2-mediated phosphorylation of Rrn7 inhibited its HomolD-directed transcriptional activity and ability to bind to an oligonucleotide containing a HomolD box in vitro. Mutation of Rrn7 at Thr67 abolished these effects, indicating that this residue is a critical CK2 phosphorylation site. Finally, Rrn7 interacted with the regulatory subunit of CK2 in vivo, inhibition of CK2 in vivo potentiated ribosomal protein gene transcription, and chromatin immunoprecipitation analyses identified that the catalytic subunit of CK2 was associated with the rpk5 gene promoter in S. pombe. Taken together, these data suggest that CK2 inhibits ribosomal protein gene transcription in S. pombe via phosphorylation of Rrn7 at Thr67. PMID:25410910

  7. Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

    PubMed

    Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

    2012-11-01

    Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA. PMID:22898792

  8. Co-delivery of cell-wall-forming enzymes in the same vesicle for coordinated fungal cell wall formation.

    PubMed

    Schuster, Martin; Martin-Urdiroz, Magdalena; Higuchi, Yujiro; Hacker, Christian; Kilaru, Sreedhar; Gurr, Sarah J; Steinberg, Gero

    2016-01-01

    Fungal cells are surrounded by an extracellular cell wall. This complex matrix of proteins and polysaccharides protects against adverse stresses and determines the shape of fungal cells. The polysaccharides of the fungal wall include 1,3-β-glucan and chitin, which are synthesized by membrane-bound synthases at the growing cell tip. A hallmark of filamentous fungi is the class V chitin synthase, which carries a myosin-motor domain. In the corn smut fungus Ustilago maydis, the myosin-chitin synthase Mcs1 moves to the plasma membrane in secretory vesicles, being delivered by kinesin-1 and myosin-5. The myosin domain of Mcs1 enhances polar secretion by tethering vesicles at the site of exocytosis. It remains elusive, however, how other cell-wall-forming enzymes are delivered and how their activity is coordinated post secretion. Here, we show that the U. maydis class VII chitin synthase and 1,3-β-glucan synthase travel in Mcs1-containing vesicles, and that their apical secretion depends on Mcs1. Once in the plasma membrane, anchorage requires enzyme activity, which suggests co-synthesis of chitin and 1,3-β-glucan polysaccharides at sites of exocytosis. Thus, delivery of cell-wall-forming enzymes in Mcs1 vesicles ensures local foci of fungal cell wall formation. PMID:27563844

  9. Characterization of rhamnogalacturonan I from cotton suspension culture cell walls

    SciTech Connect

    Not Available

    1991-01-01

    Progress has been made on the project of determining the structure of pectins. From recent progress, a covalent crosslink between rhamnogalacturonan I (RGI) and xyloglucan was hypothesized and a structure for RGI was proposed. The development of a method to determine the distribution of methyl esterification with pectins also progressed. The degree of methyl esterification of cotton cotyledon cell walls was compared to that of cotton suspension cultures. Cotyledon wall were found to have {approximately}55% of the galacturonic acid esterified whereas suspension culture wall were only about 14% methyl esterified. 10 refs. (SM)

  10. Simulated microgravity inhibits cell wall regeneration of Penicillium decumbens protoplasts

    NASA Astrophysics Data System (ADS)

    Zhao, C.; Sun, Y.; Yi, Z. C.; Rong, L.; Zhuang, F. Y.; Fan, Y. B.

    2010-09-01

    This work compares cell wall regeneration from protoplasts of the fungus Penicillium decumbens under rotary culture (simulated microgravity) and stationary cultures. Using an optimized lytic enzyme mixture, protoplasts were successfully released with a yield of 5.3 × 10 5 cells/mL. Under simulated microgravity conditions, the protoplast regeneration efficiency was 33.8%, lower than 44.9% under stationary conditions. Laser scanning confocal microscopy gave direct evidence for reduced formation of polysaccharides under simulated conditions. Scanning electron microscopy showed the delayed process of cell wall regeneration by simulated microgravity. The delayed regeneration of P. decumbens cell wall under simulated microgravity was likely caused by the inhibition of polysaccharide synthesis. This research contributes to the understanding of how gravitational loads affect morphological and physiological processes of fungi.

  11. Modification of glass cell walls by rubidium vapor

    NASA Astrophysics Data System (ADS)

    Ma, J.; Kishinevski, A.; Jau, Y.-Y.; Reuter, C.; Happer, W.

    2009-04-01

    It has long been known that the inner walls of freshly manufactured glass cells filled with a few droplets of alkali metal undergo a “curing” process, where the properties of the cell wall change over a period of days to weeks. We report quantitative studies of “curing” in Pyrex cells filled with rubidium metal. Our experiment shows that at 94°C , the surface of Pyrex glass adsorbs about 3×1015 rubidium atoms per cm2 , which is equivalent to 6-7 monolayers of liquid rubidium.

  12. Chromosome and cell wall segregation in Streptococcus faecium ATCC 9790

    SciTech Connect

    Higgins, M.L.; Glaser, D.; Dicker, D.T.; Zito, E.T.

    1989-01-01

    Segregation was studied by measuring the positions of autoradiographic grain clusters in chains formed from single cells containing on average less than one radiolabeled chromosome strand. The degree to which chromosomal and cell wall material cosegregated was quantified by using the methods of S. Cooper and M. Weinberger, dividing the number of chains labeled at the middle. This analysis indicated that in contrast to chromosomal segregation in Escherichia coli and, in some studies, to that in gram-positive rods, chromosomal segregation in Streptococcus faecium was slightly nonrandom and did not vary with growth rate. Results were not significantly affected by strand exchange. In contrast, labeled cell wall segregated predominantly nonrandomly.

  13. Molecular Rigidity in Dry and Hydrated Onion Cell Walls.

    PubMed Central

    Ha, M. A.; Apperley, D. C.; Jarvis, M. C.

    1997-01-01

    Solid-state nuclear magnetic resonance relaxation experiments can provide information on the rigidity of individual molecules within a complex structure such as a cell wall, and thus show how each polymer can potentially contribute to the rigidity of the whole structure. We measured the proton magnetic relaxation parameters T2 (spin-spin) and T1p (spin-lattice) through the 13C-nuclear magnetic resonance spectra of dry and hydrated cell walls from onion (Allium cepa L.) bulbs. Dry cell walls behaved as rigid solids. The form of their T2 decay curves varied on a continuum between Gaussian, as in crystalline solids, and exponential, as in more mobile materials. The degree of molecular mobility that could be inferred from the T2 and T1p decay patterns was consistent with a crystalline state for cellulose and a glassy state for dry pectins. The theory of composite materials may be applied to explain the rigidity of dry onion cell walls in terms of their components. Hydration made little difference to the rigidity of cellulose and most of the xyloglucan shared this rigidity, but the pectic fraction became much more mobile. Therefore, the cellulose/xyloglucan microfibrils behaved as solid rods, and the most significant physical distinction within the hydrated cell wall was between the microfibrils and the predominantly pectic matrix. A minor xyloglucan fraction was much more mobile than the microfibrils and probably corresponded to cross-links between them. Away from the microfibrils, pectins expanded upon hydration into a nonhomogeneous, but much softer, almost-liquid gel. These data are consistent with a model for the stress-bearing hydrated cell wall in which pectins provide limited stiffness across the thickness of the wall, whereas the cross-linked microfibril network provides much greater rigidity in other directions. PMID:12223827

  14. Molecular Rigidity in Dry and Hydrated Onion Cell Walls.

    PubMed

    Ha, M. A.; Apperley, D. C.; Jarvis, M. C.

    1997-10-01

    Solid-state nuclear magnetic resonance relaxation experiments can provide information on the rigidity of individual molecules within a complex structure such as a cell wall, and thus show how each polymer can potentially contribute to the rigidity of the whole structure. We measured the proton magnetic relaxation parameters T2 (spin-spin) and T1p (spin-lattice) through the 13C-nuclear magnetic resonance spectra of dry and hydrated cell walls from onion (Allium cepa L.) bulbs. Dry cell walls behaved as rigid solids. The form of their T2 decay curves varied on a continuum between Gaussian, as in crystalline solids, and exponential, as in more mobile materials. The degree of molecular mobility that could be inferred from the T2 and T1p decay patterns was consistent with a crystalline state for cellulose and a glassy state for dry pectins. The theory of composite materials may be applied to explain the rigidity of dry onion cell walls in terms of their components. Hydration made little difference to the rigidity of cellulose and most of the xyloglucan shared this rigidity, but the pectic fraction became much more mobile. Therefore, the cellulose/xyloglucan microfibrils behaved as solid rods, and the most significant physical distinction within the hydrated cell wall was between the microfibrils and the predominantly pectic matrix. A minor xyloglucan fraction was much more mobile than the microfibrils and probably corresponded to cross-links between them. Away from the microfibrils, pectins expanded upon hydration into a nonhomogeneous, but much softer, almost-liquid gel. These data are consistent with a model for the stress-bearing hydrated cell wall in which pectins provide limited stiffness across the thickness of the wall, whereas the cross-linked microfibril network provides much greater rigidity in other directions. PMID:12223827

  15. 15. View of interior, north wall of hot cell featuring ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    15. View of interior, north wall of hot cell featuring radioactive materials containment box, facing east - Nevada Test Site, Reactor Maintenance & Disassembly Complex, Junior Hot Cell, Jackass Flats, Area 25, South of intersection of Roads F & G, Mercury, Nye County, NV

  16. 47. ARAI. Interior view of operating wall of hot cell ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    47. ARA-I. Interior view of operating wall of hot cell in ARA-626. Note stands for operators at viewing windows. Manipulators with hand grips extend cables and other controls into hot cell through ducts above windows. Ineel photo no. 81-27. - Idaho National Engineering Laboratory, Army Reactors Experimental Area, Scoville, Butte County, ID

  17. Alterations in auxin homeostasis suppress defects in cell wall function.

    PubMed

    Steinwand, Blaire J; Xu, Shouling; Polko, Joanna K; Doctor, Stephanie M; Westafer, Mike; Kieber, Joseph J

    2014-01-01

    The plant cell wall is a highly dynamic structure that changes in response to both environmental and developmental cues. It plays important roles throughout plant growth and development in determining the orientation and extent of cell expansion, providing structural support and acting as a barrier to pathogens. Despite the importance of the cell wall, the signaling pathways regulating its function are not well understood. Two partially redundant leucine-rich-repeat receptor-like kinases (LRR-RLKs), FEI1 and FEI2, regulate cell wall function in Arabidopsis thaliana roots; disruption of the FEIs results in short, swollen roots as a result of decreased cellulose synthesis. We screened for suppressors of this swollen root phenotype and identified two mutations in the putative mitochondrial pyruvate dehydrogenase E1α homolog, IAA-Alanine Resistant 4 (IAR4). Mutations in IAR4 were shown previously to disrupt auxin homeostasis and lead to reduced auxin function. We show that mutations in IAR4 suppress a subset of the fei1 fei2 phenotypes. Consistent with the hypothesis that the suppression of fei1 fei2 by iar4 is the result of reduced auxin function, disruption of the WEI8 and TAR2 genes, which decreases auxin biosynthesis, also suppresses fei1 fei2. In addition, iar4 suppresses the root swelling and accumulation of ectopic lignin phenotypes of other cell wall mutants, including procuste and cobra. Further, iar4 mutants display decreased sensitivity to the cellulose biosynthesis inhibitor isoxaben. These results establish a role for IAR4 in the regulation of cell wall function and provide evidence of crosstalk between the cell wall and auxin during cell expansion in the root. PMID:24859261

  18. The role of the cell wall in plant immunity

    PubMed Central

    Malinovsky, Frederikke G.; Fangel, Jonatan U.; Willats, William G. T.

    2014-01-01

    The battle between plants and microbes is evolutionarily ancient, highly complex, and often co-dependent. A primary challenge for microbes is to breach the physical barrier of host cell walls whilst avoiding detection by the plant’s immune receptors. While some receptors sense conserved microbial features, others monitor physical changes caused by an infection attempt. Detection of microbes leads to activation of appropriate defense responses that then challenge the attack. Plant cell walls are formidable and dynamic barriers. They are constructed primarily of complex carbohydrates joined by numerous distinct connection types, and are subject to extensive post-synthetic modification to suit prevailing local requirements. Multiple changes can be triggered in cell walls in response to microbial attack. Some of these are well described, but many remain obscure. The study of the myriad of subtle processes underlying cell wall modification poses special challenges for plant glycobiology. In this review we describe the major molecular and cellular mechanisms that underlie the roles of cell walls in plant defense against pathogen attack. In so doing, we also highlight some of the challenges inherent in studying these interactions, and briefly describe the analytical potential of molecular probes used in conjunction with carbohydrate microarray technology. PMID:24834069

  19. Effects of spaceflight on polysaccharides of Saccharomyces cerevisiae cell wall.

    PubMed

    Liu, Hong-Zhi; Wang, Qiang; Liu, Xiao-Yong; Tan, Sze-Sze

    2008-12-01

    Freeze-dried samples of four Saccharomyces cerevisiae strains, namely, FL01, FL03, 2.0016, and 2.1424, were subjected to spaceflight. After the satellite's landing on Earth, the samples were recovered and changes in yeast cell wall were analyzed. Spaceflight strains of all S. cerevisiae strains showed significant changes in cell wall thickness (P < 0.05). One mutant of S. cerevisiae 2.0016 with increased biomass, cell wall thickness, and cell wall glucan was isolated (P < 0.05). The spaceflight mutant of S. cerevisiae 2.0016 showed 46.7%, 62.6%, and 146.0% increment in biomass, cell wall thickness and beta-glucan content, respectively, when compared to the ground strain. Moreover, growth curve analysis showed spaceflight S. cerevisiae 2.0016 had a faster growth rate, shorter lag phase periods, higher final biomass, and higher content of beta-glucan. Genetic stability analysis showed that prolonged subculturing of spaceflight strain S. cerevisiae 2.0016 did not lead to the appearance of variants, indicating that the genetic stability of S. cerevisiae 2.0016 mutant could be sufficient for its exploitation of beta-glucan production. PMID:18797865

  20. Purification and characterization of a soybean cell wall protein

    SciTech Connect

    San Francisco, S.; Tierney, M.L. )

    1989-04-01

    Plant cell wall composition is thought to reflect cellular responses to developmental and environmental signals. We have purified a 33 kDa protein from cell wall extracts of soybean seedlings which is most abundant in extracts from the hook region of the hypocotyl and is rich in proline and hydroxypyroline. In vivo {sup 3}H-proline labelling of hypocotyl tissues indicates that the hook tissue is the predominant site for synthesis of this protein. In unwounded hook, label is incorporated into a 33 kDa protein, while in wounded hook this and additional proteins rich in proline are synthesized. Similarly treated cell wall extracts analyzed by Western blot analysis, using a polyclonal antibody raised against this 33kD protein, showed that the 33 kDa protein is most abundant in cell wall extracts from the hook region of unwounded seedlings and does not increase upon wounding. An immunologically related 35kD protein is also apparent in extracts from wounded hooks and appears to co-migrate with one of the labelled proteins extractable from this tissue. These data indicate that there are two related, proline-rich cell wall proteins in the hook region of soybean seedlings, one of which (33 kDa) is prominent during seedling development and another (35 kDa) which is wound inducible.

  1. Structure, function, and biosynthesis of plant cell walls: proceedings of the seventh annual symposium in botany

    SciTech Connect

    Dugger, W.M.; Bartnicki-Garcia, S.

    1984-01-01

    Papers in the following areas were included in these symposium proceedings: (1) cell wall chemistry and biosynthesis; (2) cell wall hydrolysis and associated physiology; (3) cellular events associated with cell wall biosynthesis; and (4) interactions of plant cell walls with pathogens and related responses. Papers have been individually abstracted for the data base. (ACR)

  2. TERRA promotes telomerase-mediated telomere elongation in Schizosaccharomyces pombe.

    PubMed

    Moravec, Martin; Wischnewski, Harry; Bah, Amadou; Hu, Yan; Liu, Na; Lafranchi, Lorenzo; King, Megan C; Azzalin, Claus M

    2016-07-01

    Telomerase-mediated telomere elongation provides cell populations with the ability to proliferate indefinitely. Telomerase is capable of recognizing and extending the shortest telomeres in cells; nevertheless, how this mechanism is executed remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, shortened telomeres are highly transcribed into the evolutionarily conserved long noncoding RNA TERRA A fraction of TERRA produced upon telomere shortening is polyadenylated and largely devoid of telomeric repeats, and furthermore, telomerase physically interacts with this polyadenylated TERRA in vivo We also show that experimentally enhanced transcription of a manipulated telomere promotes its association with telomerase and concomitant elongation. Our data represent the first direct evidence that TERRA stimulates telomerase recruitment and activity at chromosome ends in an organism with human-like telomeres. PMID:27154402

  3. Ultrastructure of organic cell walls in Proterozoic microalgae

    NASA Astrophysics Data System (ADS)

    Moczydlowska-Vidal, M.

    2009-04-01

    The antiquity of life has been well appreciated since the discoveries of microfossils and confirmation of their authenticity, as well as the recognition of geochemical signs of biogenicity in the Archean successions. Resolving the biological affinities of early biota is essential for the unravelling the changes that led to modern biodiversity, but also for the detection of possible biogenic records outside of the terrestrial biosphere. Advanced techniques in microscopy, tomography and spectroscopy applied to examine individual microfossils at the highest attainable spatial resolution have provided unprecedented insights into micro- and nano-scale structure and composition of organic matter. Transmission and scanning electron microscopy studies of the wall ultrastructure of sphaeromorphic and ornamented acritarchs have revealed complex, single to multilayered walls, having a unique texture in sub-layers and an occasionally preserved trilaminar sheath structure (TLS) of the cell wall. A variety of optical characteristics, the electron density and texture of fabrics of discrete layers, and the properties of biopolymers may indicate the polyphyletic affiliations of such microfossils and/or the preservation of various stages (vegetative, resting) in their life cycle. I evaluate the morphological features of organic-walled unicellular microfossils in conjunction with their cell wall ultrastructure to infer their life cycle and to recognize various developmental stages represented among microfossils attributed to a single form-taxon. Several cases of fine wall ultrastructure in microfossils have been documented and have had a conclusive influence on understanding their affinities. Some Proterozoic and Cambrian leiosphaerids are of algal affinities. Certain specimens represent chlorophyceaens, having the multilayered composite wall with TLS structure known from vegetative and resting cells in modern genera of the Chlorococcales and Volvocales. The wall ultrastructure of

  4. Cellulose synthesis in two secondary cell wall processes in a single cell type

    PubMed Central

    Mendu, Venugopal; Stork, Jozsef; Harris, Darby; DeBolt, Seth

    2011-01-01

    Plant cells have a rigid cell wall that constrains internal turgor pressure yet extends in a regulated and organized manner to allow the cell to acquire shape. The primary load-bearing macromolecule of a plant cell wall is cellulose, which forms crystalline microfibrils that are organized with respect to a cell's function and shape requirements. A primary cell wall is deposited during expansion whereas secondary cell wall is synthesized post expansion during differentiation. A complex form of asymmetrical cellular differentiation occurs in Arabidopsis seed coat epidermal cells, where we have recently shown that two secondary cell wall processes occur that utilize different cellulose synthase (CESA) proteins. One process is to produce pectinaceous mucilage that expands upon hydration and the other is a radial wall thickening that reinforced the epidermal cell structure. Our data illustrate polarized specialization of CESA5 in facilitating mucilage attachment to the parent seed and CESA2, CESA5 and CESA9 in radial cell wall thickening and formation of the columella. Herein, we present a model for the complexity of cellulose biosynthesis in this highly differentiated cell type with further evidence supporting each cellulosic secondary cell wall process. PMID:22057330

  5. Cotton fiber: a powerful single-cell model for cell wall and cellulose research

    PubMed Central

    Haigler, Candace H.; Betancur, Lissete; Stiff, Michael R.; Tuttle, John R.

    2012-01-01

    Cotton fibers are single-celled extensions of the seed epidermis. They can be isolated in pure form as they undergo staged differentiation including primary cell wall synthesis during elongation and nearly pure cellulose synthesis during secondary wall thickening. This combination of features supports clear interpretation of data about cell walls and cellulose synthesis in the context of high throughput modern experimental technologies. Prior contributions of cotton fiber to building fundamental knowledge about cell walls will be summarized and the dynamic changes in cell wall polymers throughout cotton fiber differentiation will be described. Recent successes in using stable cotton transformation to alter cotton fiber cell wall properties as well as cotton fiber quality will be discussed. Futurec prospects to perform experiments more rapidly through altering cotton fiberwall properties via virus-induced gene silencing will be evaluated. PMID:22661979

  6. Cell wall integrity signalling in human pathogenic fungi.

    PubMed

    Dichtl, Karl; Samantaray, Sweta; Wagener, Johannes

    2016-09-01

    Fungi are surrounded by a rigid structure, the fungal cell wall. Its plasticity and composition depend on active regulation of the underlying biosynthesis and restructuring processes. This involves specialised signalling pathways that control gene expression and activities of biosynthetic enzymes. The cell wall integrity (CWI) pathway is the central signalling cascade required for the adaptation to a wide spectrum of cell wall perturbing conditions, including heat, oxidative stress and antifungals. In the recent years, great efforts were made to analyse the CWI pathway of diverse fungi. It turned out that the CWI signalling cascade is mostly conserved in the fungal kingdom. In this review, we summarise as well as compare the current knowledge on the canonical CWI pathway in the human pathogenic fungi Candida albicans, Candida glabrata, Aspergillus fumigatus and Cryptococcus neoformans. Understanding the differences and similarities in the stress responses of these organisms could become a key to improving existing or developing new antifungal therapies. PMID:27155139

  7. Fluorescent probes for exploring plant cell wall deconstruction: a review.

    PubMed

    Paës, Gabriel

    2014-01-01

    Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction. PMID:24995923

  8. A new method for extraction of pectin from cell walls

    SciTech Connect

    Maness, N.O.; Mort, A.J. )

    1991-05-01

    Pectin is often extracted from plant tissues using the Ca{sup ++} chelators ethylenediamine tetraacetate (EDTA) or cyclohexane-trans-1,2 diamine tetraacetate (CDTA). While these chelators are effective in solubilizing pectin, even after extensive dialysis against distilled water, EDTA or CDTA remains associated with the pectin. The authors have found that if 500 mM imidazole buffer, pH 7.0 is substituted for 50 mM CDTA, pH 6.5, and for equivalent extraction periods, an equivalent amount of pectin with the same sugar composition is extracted. But, the imidazole buffer can be dialyzed away completely into distilled water. Their alternative procedure for extraction of pectin from cell walls will be presented. Utilization of the procedure for extraction of whole cell walls or cell walls pretreated with liquid hydrogen fluoride is discussed.

  9. Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors

    PubMed Central

    Fernandes, Chantal; Anjos, Jorge; Walker, Louise A.; Silva, Branca M. A.; Cortes, Luísa; Mota, Marta; Munro, Carol A.; Gow, Neil A. R.

    2014-01-01

    The present work reports the effects of caspofungin, a β-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting β-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the β-glucan synthase inhibitor against this fungus. PMID:24614372

  10. Merkel cell carcinoma of the abdominal wall

    PubMed Central

    Gaopande, Vandana L.; Joshi, Avinash R.; Khandeparkar, Siddhi G. S.; Deshmukh, Sanjay D.

    2015-01-01

    Merkel cell carcinoma also known as neuroendocrine carcinoma of the skin is a very rare skin tumor. It commonly presents in the old age and the common sites are head, neck and extremities. The diagnosis requires histopathological examination with immunohistochemical correlation. We report a case of Merkel cell carcinoma stage IIIB with bilateral inguinal lymphadenopathy that on FNAB showed metastatic deposits of the tumor. PMID:26225333

  11. A zoom into the nanoscale texture of secondary cell walls

    PubMed Central

    2014-01-01

    Background Besides classical utilization of wood and paper, lignocellulosic biomass has become increasingly important with regard to biorefinery, biofuel production and novel biomaterials. For these new applications the macromolecular assembly of cell walls is of utmost importance and therefore further insights into the arrangement of the molecules on the nanolevel have to be gained. Cell wall recalcitrance against enzymatic degradation is one of the key issues, since an efficient degradation of lignocellulosic plant material is probably the most crucial step in plant conversion to energy. A limiting factor for in-depth analysis is that high resolution characterization techniques provide structural but hardly chemical information (e.g. Transmission Electron Microscopy (TEM), Atomic Force Microscopy (AFM)), while chemical characterization leads to a disassembly of the cell wall components or does not reach the required nanoscale resolution (Fourier Tranform Infrared Spectroscopy (FT-IR), Raman Spectroscopy). Results Here we use for the first time Scanning Near-Field Optical Microscopy (SNOM in reflection mode) on secondary plant cell walls and reveal a segmented circumferential nanostructure. This pattern in the 100 nm range was found in the secondary cell walls of a softwood (spruce), a hardwood (beech) and a grass (bamboo) and is thus concluded to be consistent among various plant species. As the nanostructural pattern is not visible in classical AFM height and phase images it is proven that the contrast is not due to changes in surfaces topography, but due to differences in the molecular structure. Conclusions Comparative analysis of model substances of casted cellulose nanocrystals and spin coated lignin indicate, that the SNOM signal is clearly influenced by changes in lignin distribution or composition. Therefore and based on the known interaction of lignin and visible light (e.g. fluorescence and resonance effects), we assume the elucidated nanoscale

  12. Distribution of cell wall components in Sphagnum hyaline cells and in liverwort and hornwort elaters.

    PubMed

    Kremer, Celeste; Pettolino, Filomena; Bacic, Antony; Drinnan, Andrew

    2004-10-01

    Spiral secondary walls are found in hyaline cells of Sphagnum, in the elaters of most liverworts, and in elaters of the hornwort Megaceros. Recent studies on these cells suggest that cytoskeletal and ultrastructural processes involved in cell differentiation and secondary wall formation are similar in bryophytes and vascular plant tracheary elements. To examine differences in wall structure, primary and secondary wall constituents of the hyaline cells of Sphagnum novo-zelandicum and elaters of the liverwort Radula buccinifera and the hornwort Megaceros gracilis were analyzed by immunohistochemical and chemical methods. Anti-arabinogalactan-protein antibodies, JIM8 and JIM13, labeled the central fibrillar secondary wall layer of Megaceros elaters and the walls of Sphagnum leaf cells, but did not label the walls of Radula elaters. The CCRC-M7 antibody, which detects an arabinosylated (1-->6)-linked beta-galactan epitope, exclusively labeled hyaline cells in Sphagnum leaves and the secondary walls of Radula elaters. Anti-pectin antibodies, LM5 and JIM5, labeled the primary wall in Megaceros elaters. LM5 also labeled the central layer of the secondary wall but only during formation. In Radula elaters, JIM5 and another anti-pectin antibody, JIM7, labeled the primary wall. The distribution of arabinogalactan-proteins and pectic polysaccharides restricted to specific wall types and stages of development provides evidence for the developmental and functional regulation of cell wall composition in bryophytes. Monosaccharide-linkage analysis of Sphagnum leaf cell walls suggests they contain polysaccharides similar to those of higher plants. The most abundant linkage was 4-Glc, typical of cellulose, but there was also evidence for xyloglucans, 4-linked mannans, 4-linked xylans and rhamnogalacturonan-type polysaccharides. PMID:15290291

  13. Particle Trajectories in Rotating Wall Cell Culture Devices

    NASA Technical Reports Server (NTRS)

    Ramachandran N.; Downey, J. P.

    1999-01-01

    Cell cultures are extremely important to the medical community since such cultures provide an opportunity to perform research on human tissue without the concerns inherent in experiments on individual humans. Development of cells in cultures has been found to be greatly influenced by the conditions of the culture. Much work has focused on the effect of the motions of cells in the culture relative to the solution. Recently rotating wall vessels have been used with success in achieving improved cellular cultures. Speculation and limited research have focused on the low shear environment and the ability of rotating vessels to keep cells suspended in solution rather than floating or sedimenting as the primary reasons for the improved cellular cultures using these devices. It is widely believed that the cultures obtained using a rotating wall vessel simulates to some degree the effect of microgravity on cultures. It has also been speculated that the microgravity environment may provide the ideal acceleration environment for culturing of cellular tissues due to the nearly negligible levels of sedimentation and shear possible. This work predicts particle trajectories of cells in rotating wall vessels of cylindrical and annular design consistent with the estimated properties of typical cellular cultures. Estimates of the shear encountered by cells in solution and the interactions with walls are studied. Comparisons of potential experiments in ground and microgravity environments are performed.

  14. Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

    PubMed

    Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

    2015-09-01

    Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components. PMID:26223789

  15. Xlf1 is required for DNA repair by nonhomologous end joining in Schizosaccharomyces pombe.

    PubMed

    Cavero, Santiago; Chahwan, Charly; Russell, Paul

    2007-02-01

    The accurate repair of DNA double-strand breaks is essential for cell survival and maintenance of genome integrity. Here we describe xlf1+, a gene in the fission yeast Schizosaccharomyces pombe that is required for repair of double-strand breaks by nonhomologous end joining during G1 phase of the cell cycle. Xlf1 is the ortholog of budding yeast Nej1 and human XLF/Cernunnos proteins. PMID:17151234

  16. The structure of secondary cell wall polymers: how Gram-positive bacteria stick their cell walls together.

    PubMed

    Schäffer, Christina; Messner, Paul

    2005-03-01

    The cell wall of Gram-positive bacteria has been a subject of detailed chemical study over the past five decades. Outside the cytoplasmic membrane of these organisms the fundamental polymer is peptidoglycan (PG), which is responsible for the maintenance of cell shape and osmotic stability. In addition, typical essential cell wall polymers such as teichoic or teichuronic acids are linked to some of the peptidoglycan chains. In this review these compounds are considered as 'classical' cell wall polymers. In the course of recent investigations of bacterial cell surface layers (S-layers) a different class of 'non-classical' secondary cell wall polymers (SCWPs) has been identified, which is involved in anchoring of S-layers to the bacterial cell surface. Comparative analyses have shown considerable differences in chemical composition, overall structure and charge behaviour of these SCWPs. This review discusses the progress that has been made in understanding the structural principles of SCWPs, which may have useful applications in S-layer-based 'supramolecular construction kits' in nanobiotechnology. PMID:15758211

  17. Anammox Planctomycetes have a peptidoglycan cell wall

    PubMed Central

    van Teeseling, Muriel C.F.; Mesman, Rob J.; Kuru, Erkin; Espaillat, Akbar; Cava, Felipe; Brun, Yves V.; VanNieuwenhze, Michael S.; Kartal, Boran; van Niftrik, Laura

    2015-01-01

    Planctomycetes are intriguing microorganisms that apparently lack peptidoglycan, a structure that controls the shape and integrity of almost all bacterial cells. Therefore, the planctomycetal cell envelope is considered exceptional and their cell plan uniquely compartmentalized. Anaerobic ammonium-oxidizing (anammox) Planctomycetes play a key role in the global nitrogen cycle by releasing fixed nitrogen back to the atmosphere as N2. Here using a complementary array of state-of-the-art techniques including continuous culturing, cryo-transmission electron microscopy, peptidoglycan-specific probes and muropeptide analysis, we show that the anammox bacterium Kuenenia stuttgartiensis contains peptidoglycan. On the basis of the thickness, composition and location of peptidoglycan in K. stuttgartiensis, we propose to redefine Planctomycetes as Gram-negative bacteria. Our results demonstrate that Planctomycetes are not an exception to the universal presence of peptidoglycan in bacteria. PMID:25962786

  18. Quantitative Fitness Analysis Identifies exo1∆ and Other Suppressors or Enhancers of Telomere Defects in Schizosaccharomyces pombe

    PubMed Central

    Narayanan, Siddharth; Dubarry, Marion; Lawless, Conor; Banks, A. Peter; Wilkinson, Darren J.; Whitehall, Simon K.; Lydall, David

    2015-01-01

    Synthetic genetic array (SGA) has been successfully used to identify genetic interactions in S. cerevisiae and S. pombe. In S. pombe, SGA methods use either cycloheximide (C) or heat shock (HS) to select double mutants before measuring colony size as a surrogate for fitness. Quantitative Fitness Analysis (QFA) is a different method for determining fitness of microbial strains. In QFA, liquid cultures are spotted onto solid agar and growth curves determined for each spot by photography and model fitting. Here, we compared the two S. pombe SGA methods and found that the HS method was more reproducible for us. We also developed a QFA procedure for S. pombe. We used QFA to identify genetic interactions affecting two temperature sensitive, telomere associated query mutations (taz1Δ and pot1-1). We identify exo1∆ and other gene deletions as suppressors or enhancers of S. pombe telomere defects. Our study identifies known and novel gene deletions affecting the fitness of strains with telomere defects. The interactions we identify may be relevant in human cells. PMID:26168240

  19. Effect of human immunodeficiency virus type 1 protein R (vpr) gene expression on basic cellular function of fission yeast Schizosaccharomyces pombe.

    PubMed Central

    Zhao, Y; Cao, J; O'Gorman, M R; Yu, M; Yogev, R

    1996-01-01

    The human immunodeficiency virus type 1 (HIV-1) Vpr protein affects cell morphology and prevents proliferation of human cells by induction of cell cycle G2 arrest. In this study, we used the fission yeast Schizosaccharomyces pombe as a model system to investigate the cellular effects of HIV-1 vpr gene expression. The vpr gene was cloned into an inducible fission yeast gene expression vector and expressed in wild-type S. pombe cells, and using these cells, we were able to demonstrate the specific Vpr-induced effects by induction and suppression of vpr gene expression. Induction of HIV-1 vpr gene expression affected S. pombe at the colonial, cellular, and molecular levels. Specifically, Vpr induced small-colony formation, polymorphic cells, growth delay, and cell cycle G2 arrest. Additionally, Vpr-induced G2 arrest appeared to be independent of cell size and morphological changes. The cell cycle G2 arrest correlated with increased phosphorylation of p34cdc2, suggesting negative regulation of mitosis by HIV-1 Vpr. Treatment of Vpr-induced cell with a protein phosphatase inhibitor, okadaic acid, transiently suppressed cell cycle arrest and morphological changes. This observation implicates possible involvement of protein phosphatase(s) in the effects of Vpr. Together, these data showed that the HIV-1 Vpr-induced cellular changes in S. pombe are similar to those observed in human cells. Therefore, the S. pombe system is suited for further investigation of the HIV-1 vpr gene functions. PMID:8709199

  20. The Cellulose System in the Cell Wall of Micrasterias

    PubMed

    Kim; Herth; Vuong; Chanzy

    1996-11-01

    The cellulose system of the cell wall of Micrasterias denticulata and Micrasterias rotata was analyzed by diffraction contrast transmission electron microscopy, electron diffraction, and X-ray analysis. The studies, achieved on disencrusted cell ghosts, confirmed that the cellulose microfibrils occurred in crisscrossed bands consisting of a number of parallel ribbon-like microfibrils. The individual microfibrils had thicknesses of 5 nm for a width of around 20 nm, but in some instances, two or three microfibrils merged into one another to yield larger monocrystalline domains reaching up to 60 nm in lateral size. The orientation of the cellulose of Micrasterias is very unusual, as it was found that in the cell wall, the equatorial crystallographic planes of cellulose having a d-spacing of 0.60 nm [(11;0) in the Ibeta cellulose unit cell defined by Sugiyama et al., 1991, Macromolecules 24, 4168-4175] were oriented perpendicular to the cell wall surface. Up to now, such orientation has been found only in Spirogyra, another member of the Zygnemataceae group. The unusual structure of the secondary wall cellulose of Micrasterias may be tentatively correlated with the unique organization of the terminal complexes, which in this alga occur as hexagonal arrays of rosettes. PMID:8986649

  1. Sexual reproduction as a response to H sub 2 O sub 2 damage in Schizosaccharomyces pombe

    SciTech Connect

    Bernstein, C.; Johns, V. )

    1989-04-01

    Although sexual reproduction is widespread, its adaptive advantage over asexual reproduction is unclear. One major advantage of sex may be its promotion of recombinational repair of DNA damage during meiosis. This idea predicts that treatment of the asexual form of a facultatively sexual-asexual eucaryote with a DNA-damaging agent may cause it to enter the sexual cycle more frequently. Endogenous hydrogen peroxide is a major natural source of DNA damage. Thus, the authors treated vegetative cells of Schizosaccharomyces pombe with hydrogen peroxide to test if sexual reproduction increases. Among untreated stationary-phase S. pombe populations the sexual spores produced by meiosis represented about 1% of the total cells. However, treatment of late-exponential-phase vegetative cells with hydrogen peroxide increased the percentage of meiotic spores in the stationary phase by 4- to 18-fold. Oxidative damage therefore induces sexual reproduction in a facultatively sexual organism, a result expected by the hypothesis that sex promotes DNA repair.

  2. A model of cell wall expansion based on thermodynamics of polymer networks

    NASA Technical Reports Server (NTRS)

    Veytsman, B. A.; Cosgrove, D. J.

    1998-01-01

    A theory of cell wall extension is proposed. It is shown that macroscopic properties of cell walls can be explained through the microscopic properties of interpenetrating networks of cellulose and hemicellulose. The qualitative conclusions of the theory agree with the existing experimental data. The dependence of the cell wall yield threshold on the secretion of the wall components is discussed.

  3. Synchronizing Progression of Schizosaccharomyces pombe Cells from G2 through Repeated Rounds of Mitosis and S Phase with cdc25-22 Arrest Release.

    PubMed

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-01-01

    Transient inactivation of the cdc25(+) gene product by manipulation of the culture temperature for cdc25-22 cells is the most commonly exploited approach to mitotic synchronization in fission yeast. Because Cdc25 removes the inhibitory phosphate placed on Cdk1 by Wee1, inactivation of Cdc25 arrests cells at the G2/M boundary. Incubation at the restrictive temperature of 36°C for just over one generation time forces all cells in the culture to accumulate at the G2/M boundary. Restoration of Cdc25 function via a return to the permissive temperature or chemical inhibition of Wee1 activity at 36°C can then promote a highly synchronous wave of cell division throughout the culture. These approaches can be performed on any scale and thus support simultaneous assessment of numerous events within a single culture. After describing this simple and widely applicable procedure, we discuss frequently overlooked issues that can have a considerable impact on the interpretation of data from cdc25-22 induction-synchronized cultures. PMID:27480720

  4. The yin and yang of cell wall integrity control: brassinosteroid and FERONIA signaling.

    PubMed

    Höfte, Herman

    2015-02-01

    Understanding how developmental and environmental signals control plant cell expansion requires an intimate knowledge of the architecture of the primary cell wall and the chemo-rheological processes that underlie cell wall relaxation. In this review I discuss recent findings that reveal a more prominent role than previously suspected for covalent bonds and pectin cross-links in primary cell wall architecture. In addition, genetic studies have uncovered a role for receptor kinases in the control of cell wall homeostasis in growing cells. The emerging view is that, upon cell wall disruption, compensatory changes are induced in the cell wall through the interplay between the brassinosteroid signaling module, which positively regulates wall extensibility and receptor kinases of the CrRLKL1 family, which may act as negative regulators of cell wall stiffness. These findings lift the tip of the veil of a complex signaling network allowing normal homeostasis in walls of growing cells but also crisis management under stress conditions. PMID:25481004

  5. Influence of the Cell Wall on Intracellular Delivery to Algal Cells by Electroporation and Sonication

    PubMed Central

    Azencott, Harold R.; Peter, Gary F.; Prausnitz, Mark R.

    2007-01-01

    To assess the cell wall’s role as a barrier to intracellular delivery, wild-type Chlamydomonas reinhardtii algal cells and mutant cells lacking a cell wall were exposed to electroporation or sonication. Flow cytometry determined intracellular uptake of calcein and bovine serum albumin (BSA) and loss of cell viability as functions of electroporation transmembrane potential and acoustic energy. Electroporation of wild-type cells increased calcein uptake with increasing transmembrane potential, but delivered much less BSA. Electroporation of wall-deficient cells had similar effects on calcein uptake, but increased BSA uptake as much as 7.5-fold relative to wild-type cells, which indicated that the cell wall was a significant barrier to BSA delivery during electroporation. Sonication of wild-type cells caused calcein and BSA uptake at similar levels. This suggests that the cell wall barrier to BSA delivery can be overcome by sonication. Increased electroporation transmembrane potential or acoustic energy also caused increased loss of cell viability, where wall-deficient cells were especially susceptible to lysis. Overall, we believe this is the first study to compare the effects of electroporation and sonication in a direct fashion in any cell type. Specifically, these findings suggest that electroporation primarily transports molecules across the plasma membrane, because its mechanism is specific to lipid bilayer disruption, whereas sonication transports molecules across both the plasma membrane and cell wall, because it non-specifically disrupts cell-surface barriers. PMID:17602827

  6. Cell wall structure and function in lactic acid bacteria

    PubMed Central

    2014-01-01

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts. PMID:25186919

  7. Cell wall structure and function in lactic acid bacteria.

    PubMed

    Chapot-Chartier, Marie-Pierre; Kulakauskas, Saulius

    2014-08-29

    The cell wall of Gram-positive bacteria is a complex assemblage of glycopolymers and proteins. It consists of a thick peptidoglycan sacculus that surrounds the cytoplasmic membrane and that is decorated with teichoic acids, polysaccharides, and proteins. It plays a major role in bacterial physiology since it maintains cell shape and integrity during growth and division; in addition, it acts as the interface between the bacterium and its environment. Lactic acid bacteria (LAB) are traditionally and widely used to ferment food, and they are also the subject of more and more research because of their potential health-related benefits. It is now recognized that understanding the composition, structure, and properties of LAB cell walls is a crucial part of developing technological and health applications using these bacteria. In this review, we examine the different components of the Gram-positive cell wall: peptidoglycan, teichoic acids, polysaccharides, and proteins. We present recent findings regarding the structure and function of these complex compounds, results that have emerged thanks to the tandem development of structural analysis and whole genome sequencing. Although general structures and biosynthesis pathways are conserved among Gram-positive bacteria, studies have revealed that LAB cell walls demonstrate unique properties; these studies have yielded some notable, fundamental, and novel findings. Given the potential of this research to contribute to future applied strategies, in our discussion of the role played by cell wall components in LAB physiology, we pay special attention to the mechanisms controlling bacterial autolysis, bacterial sensitivity to bacteriophages and the mechanisms underlying interactions between probiotic bacteria and their hosts. PMID:25186919

  8. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis

    PubMed Central

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. DOI: http://dx.doi.org/10.7554/eLife.14590.001 PMID:27304077

  9. Environmental stability of stem cell wall traits in alfalfa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The concentration of stem cell wall constituents in alfalfa, Medicago sativa L., herbage can affect dry matter intake and energy availability in dairy and beef production systems and impact energy conversion efficiency when alfalfa is used to produce biofuels. Stem Klason lignin, glucose, xylose, an...

  10. A cytoplasmic peptidoglycan amidase homologue controls mycobacterial cell wall synthesis.

    PubMed

    Boutte, Cara C; Baer, Christina E; Papavinasasundaram, Kadamba; Liu, Weiru; Chase, Michael R; Meniche, Xavier; Fortune, Sarah M; Sassetti, Christopher M; Ioerger, Thomas R; Rubin, Eric J

    2016-01-01

    Regulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in Mycobacterium tuberculosis (Mtb). However, little is known about how the cell wall is regulated in stress. We found that CwlM, a protein homologous to peptidoglycan amidases, coordinates peptidoglycan synthesis with nutrient availability. Surprisingly, CwlM is sequestered from peptidoglycan (PG) by localization in the cytoplasm, and its enzymatic function is not essential. Rather, CwlM is phosphorylated and associates with MurA, the first enzyme in PG precursor synthesis. Phosphorylated CwlM activates MurA ~30 fold. CwlM is dephosphorylated in starvation, resulting in lower MurA activity, decreased cell wall metabolism, and increased tolerance to multiple antibiotics. A phylogenetic analysis of cwlM implies that localization in the cytoplasm drove the evolution of this factor. We describe a system that controls cell wall metabolism in response to starvation, and show that this regulation contributes to antibiotic tolerance. PMID:27304077

  11. Cell wall proteome of Clostridium thermocellum and detection of glycoproteins.

    PubMed

    Yu, Tingting; Xu, Xinping; Peng, Yanfeng; Luo, Yuanming; Yang, Keqian

    2012-06-20

    Clostridium thermocellum, a thermophilic anaerobe, has the unusual capacity to convert cellulosic biomass into ethanol and hydrogen. In this work, the cell wall proteome of C. thermocellum was investigated. The proteins in the cell wall fraction of C. thermocellum prepared by the boiling SDS method were released by mutanolysin digestion and resolved on two-dimensional (2D) gel. One hundred and thirty-two proteins were identified by mass spectrometry, among which the extracellular solute-binding protein (CbpB/cthe_1020), enolase, glyceraldehyde-3-phosphate dehydrogenase and translation elongation factor EF-Tu were detected as highly abundant proteins. Besides the known surface localized proteins, including FtsZ, MinD, GroEL, DnaK, many enzymes involved in bioenergetics, such as alcohol dehydrogenases and hydrogenases were also detected. By glycan stain and MS analysis of glycopeptides, we identified CbpB as a glycoprotein, which is the second glycoprotein from C. thermocellum characterized. The fact that CbpB was highly abundant in the cell wall region and glycosylated, reflects its importance in substrate assimilation. Our results indicate cell wall proteins constitute a significant portion of cellular proteins and may play important physiological roles (i.e. bioenergetics) in this bacterium. The insights described are relevant for the development of C. thermocellum as a biofuel producer. PMID:22494898

  12. Titration of Isolated Cell Walls of Lemna minor L 1

    PubMed Central

    Morvan, Claudine; Demarty, Maurice; Thellier, Michel

    1979-01-01

    A theoretical model has been built to bypass the equation of titration of the cell wall. This equation, which is an extension of the Henderson-Hasselbach equation, underlines the importance of the exchange constant, the ionic strength as well as the rate of neutralization. The model is restricted to the case when the ionization degree is equal to the neutralization degree. The shape of the titration curve is shown to be strongly dependent on the valency of the base used. Experimental results have shown that isolated cell walls bear at least two kinds of sites. The first sites which are titrated after a short time of equilibration are attributed to polyuronic acids (capacity: 0.3 milliequivalents per gram fresh cell walls). The second sites, are obtained after a long time of equilibration (capacity: 1.2 to 1.3 milliequivalents per gram, fresh cell walls). Titrations have been performed with different bases [KOH, NaOH, and Ca(OH)2] and under different ionic strengths. The results obtained with NaOH and KOH do not exhibit any difference of selectivity. Conversely, the sites have a much bigger affinity for the Ca2+ ions than for the monovalent ones. The apparent pKa of the uronic acids was estimated to lie between 3.0 and 3.4; this is consistent with the values obtained with polyuronic acid solutions. PMID:16660868

  13. Hetero-oligomeric cell wall channels (porins) of Nocardia farcinica.

    PubMed

    Kläckta, Christian; Knörzer, Philipp; Riess, Franziska; Benz, Roland

    2011-06-01

    The cell wall of Nocardia farcinica contains a cation-selective cell wall channel, which may be responsible for the limited permeability of the cell wall of N. farcinica for negatively charged antibiotics. Based on partial sequencing of the protein responsible for channel formation derived from N. farcinica ATTC 3318 we were able to identify the corresponding genes (nfa15890 and nfa15900) within the known genome of N. farcinica IFM 10152. The corresponding genes of N. farcinica ATTC 3318 were separately expressed in the Escherichia coli BL21DE3Omp8 strain and the N-terminal His10-tagged proteins were purified to homogeneity using immobilized metal affinity chromatography. The pure proteins were designated NfpANHis and NfpBNHis, for N. farcinica porin A and N. farcinica porin B. The two proteins were checked separately for channel formation in lipid bilayers. Our results clearly indicate that the proteins NfpANHis and NfpBNHis expressed in E. coli could only together form a channel in lipid bilayer membranes. This means that the cell wall channel of N. farcinica is formed by a heterooligomer. NfpA and NfpB form together a channel that may structurally be related to MspA of Mycobacterium smegmatis based on amino acid comparison and renaturation procedure. PMID:21092733

  14. Polymer mobility in cell walls of cucumber hypocotyls

    NASA Technical Reports Server (NTRS)

    Fenwick, K. M.; Apperley, D. C.; Cosgrove, D. J.; Jarvis, M. C.

    1999-01-01

    Cell walls were prepared from the growing region of cucumber (Cucumis sativus) hypocotyls and examined by solid-state 13C NMR spectroscopy, in both enzymically active and inactivated states. The rigidity of individual polymer segments within the hydrated cell walls was assessed from the proton magnetic relaxation parameter, T2, and from the kinetics of cross-polarisation from 1H to 13C. The microfibrils, including most of the xyloglucan in the cell wall, as well as cellulose, behaved as very rigid solids. A minor xyloglucan fraction, which may correspond to cross-links between microfibrils, shared a lower level of rigidity with some of the pectic galacturonan. Other pectins, including most of the galactan side-chain residues of rhamnogalacturonan I, were much more mobile and behaved in a manner intermediate between the solid and liquid states. The only difference observed between the enzymically active and inactive cell walls, was the loss of a highly mobile, methyl-esterified galacturonan fraction, as the result of pectinesterase activity.

  15. Medicago truncatula as a Model for Dicot Cell Wall Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Strong interest in renewable energy has promoted an upsurge of research on plant cell wall traits that influence the availability of lignocellulosic-derived sugars for fermentation in production of biofuels. We have initiated a genome-wide transcript profiling study using the model legume Medicago t...

  16. Determination of carbohydrate profile in sugarbeet (Beta vulgaris) cell walls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sugarbeet germplasms USH20, C869, EL55, EL54 were used, and different tissues at different developmental stages were sampled, including dry seeds, germinating seedlings, developing leaves, mature leaves, petioles, hypocotyls, mature roots, flowering stems and inflorescences. Cell Wall Composition An...

  17. Undressing the fungal cell wall/cell membrane--the antifungal drug targets.

    PubMed

    Tada, Rui; Latgé, Jean-Paul; Aimanianda, Vishukumar

    2013-01-01

    Being external, the fungal cell wall plays a crucial role in the fungal life. By covering the underneath cell, it offers mechanical strength and acts as a barrier, thus protecting the fungus from the hostile environment. Chemically, this cell wall is composed of different polysaccharides. Because of their specific composition, the fungal cell wall and its underlying plasma membrane are unique targets for the development of drugs against pathogenic fungal species. The objective of this review is to consolidate the current knowledge on the antifungal drugs targeting the cell wall and plasma membrane, mainly of Aspergillus and Candida species - the most prevalent fungal pathogens, and also to present challenges and questions conditioning the development of new antifungal drugs targeting the cell wall. PMID:23278542

  18. Synchronizing Progression of Schizosaccharomyces pombe Cells from Prophase through Mitosis and into S Phase with nda3-KM311 Arrest Release.

    PubMed

    Hagan, Iain M; Grallert, Agnes; Simanis, Viesturs

    2016-01-01

    Here, we describe how the rapid reversibility of the nda3-KM311 cold-sensitive β-tubulin mutation was optimized by Mitsuhiro Yanagida's laboratory to synchronize mitotic progression in an entire cell population. The inability to form microtubules following the loss of β-tubulin function at 20°C triggers the spindle assembly checkpoint, which arrests mitotic progression. Restoration of β-tubulin function by rewarming to 30°C (or higher) releases the arrest, generating a highly synchronous progression through mitosis. The viability of nda3-KM311 strains at 30°C makes it feasible to generate double mutants between nda3-KM311 and any temperature-sensitive mutant that can also grow at 30°C. These double mutants can be used in reciprocal shift analyses, in which cold-induced early mitotic arrest is relieved by a shift to 36°C, which then inactivates the product of the second mutant gene. The addition of microtubule depolymerizing drugs before the return to 36°C will maintain checkpoint signaling at 36°C transiently, permitting analysis of the impact of temperature-sensitive mutations on checkpoint function. Silencing the checkpoint of nda3-KM311-arrested cells at 20°C through chemical inhibition of aurora kinase is a powerful way to study checkpoint recovery pathways and mitotic exit without anaphase. PMID:27480719

  19. Partial Chemical Characterization of Corn Root Cell Walls 1

    PubMed Central

    Dever, John E.; Bandurski, Robert S.; Kivilaan, A.

    1968-01-01

    The present study reports on chemical changes which occur in the cell wall of Zea mays during early phases of growth. Roots of seedling corn plants were divided into a meristematic zone, the zone of elongation, and the maturation zone, and the cell wall isolated from each of these zones. The wall preparations were then extracted sequentially to obtain pectin, hemicellulose, cellulose, and lignin fractions. Each of these, except for the lignin fraction, was hydrolyzed and the resultant sugars isolated, identified, and estimated quantitatively. Quantitative analysis of the products of hydrolysis of these fractions demonstrated that the classical scheme of fractionation is a valuable indicator of the changes in solubility properties which the various polysaccharide components for the wall undergo. It does not however yield definite chemical entities. For example, the “pectin” fraction contains only about 3% galacturonic acid; the bulk of it being composed of glucose, xylose, and galactose. By summation of analysis of these various fractions, it was found that substances yielding glucose and xylose upon hydrolysis increase with advancing age of the tissue. Galactose- and arabinose-yielding compounds decrease and mannose appears during maturation. Anhydrouronic acids first decrease, then increase. Most interestingly, of the total dry weight of the cell wall, only 24, 45, and 50% of the meristematic, elongation, and maturation zones respectively are accounted for as simple sugars in the acid hydrolysates. Oligosaccharides were not encountered in large amounts so that the 50 to 75% of the wall weight unaccounted for would consist of polysaccharides or oligosaccharides not precipitated by ethanol from the extracting solutions employed and by polysaccharides in the hemicellulose fraction which are resistant to acid hydrolysis. PMID:16656735

  20. Response to Arsenate Treatment in Schizosaccharomyces pombe and the Role of Its Arsenate Reductase Activity

    PubMed Central

    Matia-González, Ana M.; Sotelo, Jael; Zarco-Fernández, Sonia; Muñoz-Olivas, Riansares; Cámara, Carmen; Rodríguez-Gabriel, Miguel A.

    2012-01-01

    Arsenic toxicity has been studied for a long time due to its effects in humans. Although epidemiological studies have demonstrated multiple effects in human physiology, there are many open questions about the cellular targets and the mechanisms of response to arsenic. Using the fission yeast Schizosaccharomyces pombe as model system, we have been able to demonstrate a strong activation of the MAPK Spc1/Sty1 in response to arsenate. This activation is dependent on Wis1 activation and Pyp2 phosphatase inactivation. Using arsenic speciation analysis we have also demonstrated the previously unknown capacity of S. pombe cells to reduce As (V) to As (III). Genetic analysis of several fission yeast mutants point towards the cell cycle phosphatase Cdc25 as a possible candidate to carry out this arsenate reductase activity. We propose that arsenate reduction and intracellular accumulation of arsenite are the key mechanisms of arsenate tolerance in fission yeast. PMID:22912829

  1. Uncleavable Nup98–Nup96 is functional in the fission yeast Schizosaccharomyces pombe

    PubMed Central

    Asakawa, Haruhiko; Mori, Chie; Ohtsuki, Chizuru; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2015-01-01

    Essential nucleoporins Nup98 and Nup96 are coded by a single open reading frame, and produced by autopeptidase cleavage. The autocleavage site of Nup98–Nup96 is highly conserved in a wide range of organisms. To understand the importance of autocleavage, we examined a mutant that produces the Nup98–Nup96 joint molecule as a sole protein product of the nup189+ gene in the fission yeast Schizosaccharomyces pombe. Cells expressing only the joint molecule were found to be viable. This result indicates that autocleavage of Nup98–Nup96 is dispensable for cell growth, at least under normal culture conditions in S. pombe. PMID:26137436

  2. Cell-Wall Polysaccharides of Developing Flax Plants.

    PubMed Central

    Gorshkova, T. A.; Wyatt, S. E.; Salnikov, V. V.; Gibeaut, D. M.; Ibragimov, M. R.; Lozovaya, V. V.; Carpita, N. C.

    1996-01-01

    Flax (Linum usitatissimum L.) fibers originate from procambial cells of the protophloem and develop in cortical bundles that encircle the vascular cylinder. We determined the polysaccharide composition of the cell walls from various organs of the developing flax plant, from fiber-rich strips peeled from the stem, and from the xylem. Ammonium oxalate-soluble polysaccharides from all tissues contained 5-linked arabinans with low degrees of branching, rhamnogalacturonans, and polygalacturonic acid. The fiber-rich peels contained, in addition, substantial amounts of a buffer-soluble, 4-linked galactan branched at the 0-2 and 0-3 positions with nonreducing terminal-galactosyl units. The cross-linking glycans from all tissues were (fucogalacto)xyloglucan, typical of type-I cell walls, xylans containing (1->)-[beta]-D-xylosyl units branched exclusively at the xylosyl O-2 with t-(4-O-methyl)-glucosyluronic acid units, and (galacto)glucomannans. Tissues containing predominantly primary cell wall contained a larger proportion of xyloglucan. The xylem cells were composed of about 60% 4-xylans, 32% cellulose, and small amounts of pectin and the other cross-linking polysaccharides. The noncellulosic polysaccharides of flax exhibit an uncommonly low degree of branching compared to similar polysaccharides from other flowering plants. Although the relative abundance of the various noncellulosic polysaccharides varies widely among the different cell types, the linkage structure and degree of branching of several of the noncellulosic polysaccharides are invariant. PMID:12226214

  3. Crushing Strength of Aluminum Honeycomb with Thinning Cell Wall

    NASA Astrophysics Data System (ADS)

    Ogasawara, Nagahisa; Chiba, Norimasa; Kobayashi, Eiji; Kikuchi, Yuji

    To evaluate the crash safety of automobiles, various collision tests are performed by the auto industry. In the offset frontal collision test and the side collision test, the target is an aluminum honeycomb material which has thinning cell walls. In this study, based on the analyses of the shock absorption mechanism, a new crushing strength formula is proposed. First, load-displacement curves obtained from compression tests in quasi-static condition showed an almost linear relation between a thinning rate of cell walls and a crushing strength. Second, based on Wierzbicki's theory, a new formula was proposed, which can estimate a crushing strength of a honeycomb material with thinning wall. In addition, a correcting equation which considered an elastic deformation was also proposed. Third, parametric analyses were carried out with a FE model which can simulate a delamination between cell walls. The results obtained from the theory and FEM almost corresponded to each other for a wide range of the thinning rate. Fourth, impact tests were carried out, in which the weight was dropped freely at the speed used for the automobile tests. Those results almost agreed well with the sum of the theoretical crush strength and the inside air pressure.

  4. Nucleated assembly of Chlamydomonas and Volvox cell walls.

    PubMed

    Adair, W S; Steinmetz, S A; Mattson, D M; Goodenough, U W; Heuser, J E

    1987-11-01

    The Chlamydomonas reinhardtii cell wall is made up of hydroxyproline-rich glycoproteins, arranged in five distinct layers. The W6 (crystalline) layer contains three major glycoproteins (GP1, GP2, GP3), selectively extractable with chaotropic agents, that self-assemble into crystals in vitro. A system to study W6 assembly in a quantitative fashion was developed that employs perchlorate-extracted Chlamydomonas cells as nucleating agents. Wall reconstitution by biotinylated W6 monomers was monitored by FITC-streptavidin fluorescence and quick-freeze/deep-etch electron microscopy. Optimal reconstitution was obtained at monomer concentrations (0.2-0.3 mg/ml) well below those required for nonnucleated assembly. Assembly occurred from multiple nucleation sites, and faithfully reflected the structure of the intact W6 layer. Specificity of nucleated assembly was demonstrated using two cell-wall mutants (cw-2 and cw-15); neither served as a substrate for assembly of wild-type monomers. In addition, W6 sublayers were assembled from purified components: GP2 and GP3 coassembled to form the inner (W6A) sublayer; this then served as a substrate for self-assembly of GP1 into the outer (W6B) sublayer. Finally, evolutionary relationships between C. reinhardtii and two additional members of the Volvocales (Chlamydomonas eugametos and Volvox carteri) were explored by performing interspecific reconstitutions. Hybrid walls were obtained between C. reinhardtii and Volvox but not with C. eugametos, confirming taxonomic assignments based on structural criteria. PMID:3680387

  5. Cell wall staining with Trypan blue enables quantitative analysis of morphological changes in yeast cells

    PubMed Central

    Liesche, Johannes; Marek, Magdalena; Günther-Pomorski, Thomas

    2015-01-01

    Yeast cells are protected by a cell wall that plays an important role in the exchange of substances with the environment. The cell wall structure is dynamic and can adapt to different physiological states or environmental conditions. For the investigation of morphological changes, selective staining with fluorescent dyes is a valuable tool. Furthermore, cell wall staining is used to facilitate sub-cellular localization experiments with fluorescently-labeled proteins and the detection of yeast cells in non-fungal host tissues. Here, we report staining of Saccharomyces cerevisiae cell wall with Trypan Blue, which emits strong red fluorescence upon binding to chitin and yeast glucan; thereby, it facilitates cell wall analysis by confocal and super-resolution microscopy. The staining pattern of Trypan Blue was similar to that of the widely used UV-excitable, blue fluorescent cell wall stain Calcofluor White. Trypan Blue staining facilitated quantification of cell size and cell wall volume when utilizing the optical sectioning capacity of a confocal microscope. This enabled the quantification of morphological changes during growth under anaerobic conditions and in the presence of chemicals, demonstrating the potential of this approach for morphological investigations or screening assays. PMID:25717323

  6. Resistance to antibiotics targeted to the bacterial cell wall

    PubMed Central

    Nikolaidis, I; Favini-Stabile, S; Dessen, A

    2014-01-01

    Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed. PMID:24375653

  7. Messenger Functions of the Bacterial Cell Wall-derived Muropeptides

    PubMed Central

    Boudreau, Marc A.; Fisher, Jed. F.; Mobashery, Shahriar

    2012-01-01

    Bacterial muropeptides are soluble peptidoglycan structures central to recycling of the bacterial cell wall, and messengers in diverse cell-signaling events. Bacteria sense muropeptides as signals that antibiotics targeting cell-wall biosynthesis are present, and eukaryotes detect muropeptides during the innate immune response to bacterial infection. This review summarizes the roles of bacterial muropeptides as messengers, with a special emphasis on bacterial muropeptide structures and the relationship of structure to the biochemical events that the muropeptides elicit. Muropeptide sensing and recycling in both Gram-positive and Gram-negative bacteria is discussed, followed by muropeptide sensing by eukaryotes as a crucial event to the innate immune response of insects (via peptidoglycan-recognition proteins) and mammals (through Nod-like receptors) to bacterial invasion. PMID:22409164

  8. Identification of Cell Wall Synthesis Regulatory Genes Controlling Biomass Characteristics and Yield in Rice (Oryza Sativa)

    SciTech Connect

    Peng, Zhaohua PEng; Ronald, Palmela; Wang, Guo-Liang

    2013-04-26

    This project aims to identify the regulatory genes of rice cell wall synthesis pathways using a cell wall removal and regeneration system. We completed the gene expression profiling studies following the time course from cell wall removal to cell wall regeneration in rice suspension cells. We also completed, total proteome, nuclear subproteome and histone modification studies following the course from cell wall removal and cell wall regeneration process. A large number of differentially expressed regulatory genes and proteins were identified. Meanwhile, we generated RNAi and over-expression transgenic rice for 45 genes with at least 10 independent transgenic lines for each gene. In addition, we ordered T-DNA and transposon insertion mutants for 60 genes from Korea, Japan, and France and characterized the mutants. Overall, we have mutants and transgenic lines for over 90 genes, exceeded our proposed goal of generating mutants for 50 genes. Interesting Discoveries a) Cell wall re-synthesis in protoplasts may involve a novel cell wall synthesis mechanism. The synthesis of the primary cell wall is initiated in late cytokinesis with further modification during cell expansion. Phragmoplast plays an essential role in cell wall synthesis. It services as a scaffold for building the cell plate and formation of a new cell wall. Only one phragmoplast and one new cell wall is produced for each dividing cell. When the cell wall was removed enzymatically, we found that cell wall re-synthesis started from multiple locations simultaneously, suggesting that a novel mechanism is involved in cell wall re-synthesis. This observation raised many interesting questions, such as how the starting sites of cell wall synthesis are determined, whether phragmoplast and cell plate like structures are involved in cell wall re-synthesis, and more importantly whether the same set of enzymes and apparatus are used in cell wall re-synthesis as during cytokinesis. Given that many known cell wall

  9. Ectopic lignification in primary cellulose-deficient cell walls of maize cell suspension cultures.

    PubMed

    Mélida, Hugo; Largo-Gosens, Asier; Novo-Uzal, Esther; Santiago, Rogelio; Pomar, Federico; García, Pedro; García-Angulo, Penélope; Acebes, José Luis; Álvarez, Jesús; Encina, Antonio

    2015-04-01

    Maize (Zea mays L.) suspension-cultured cells with up to 70% less cellulose were obtained by stepwise habituation to dichlobenil (DCB), a cellulose biosynthesis inhibitor. Cellulose deficiency was accompanied by marked changes in cell wall matrix polysaccharides and phenolics as revealed by Fourier transform infrared (FTIR) spectroscopy. Cell wall compositional analysis indicated that the cellulose-deficient cell walls showed an enhancement of highly branched and cross-linked arabinoxylans, as well as an increased content in ferulic acid, diferulates and p-coumaric acid, and the presence of a polymer that stained positive for phloroglucinol. In accordance with this, cellulose-deficient cell walls showed a fivefold increase in Klason-type lignin. Thioacidolysis/GC-MS analysis of cellulose-deficient cell walls indicated the presence of a lignin-like polymer with a Syringyl/Guaiacyl ratio of 1.45, which differed from the sensu stricto stress-related lignin that arose in response to short-term DCB-treatments. Gene expression analysis of these cells indicated an overexpression of genes specific for the biosynthesis of monolignol units of lignin. A study of stress signaling pathways revealed an overexpression of some of the jasmonate signaling pathway genes, which might trigger ectopic lignification in response to cell wall integrity disruptions. In summary, the structural plasticity of primary cell walls is proven, since a lignification process is possible in response to cellulose impoverishment. PMID:25735403

  10. Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Senoo, Takanori; Yamanaka, Mayumi; Nakamura, Atori; Terashita, Tomoki; Kawano, Shinji; Ikeda, Shogo

    2016-08-01

    Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging. PMID:27236021

  11. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation

    PubMed Central

    Hayot, Céline M.; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A.

    2012-01-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  12. Viscoelastic properties of cell walls of single living plant cells determined by dynamic nanoindentation.

    PubMed

    Hayot, Céline M; Forouzesh, Elham; Goel, Ashwani; Avramova, Zoya; Turner, Joseph A

    2012-04-01

    Plant development results from controlled cell divisions, structural modifications, and reorganizations of the cell wall. Thereby, regulation of cell wall behaviour takes place at multiple length scales involving compositional and architectural aspects in addition to various developmental and/or environmental factors. The physical properties of the primary wall are largely determined by the nature of the complex polymer network, which exhibits time-dependent behaviour representative of viscoelastic materials. Here, a dynamic nanoindentation technique is used to measure the time-dependent response and the viscoelastic behaviour of the cell wall in single living cells at a micron or sub-micron scale. With this approach, significant changes in storage (stiffness) and loss (loss of energy) moduli are captured among the tested cells. The results reveal hitherto unknown differences in the viscoelastic parameters of the walls of same-age similarly positioned cells of the Arabidopsis ecotypes (Col 0 and Ws 2). The technique is also shown to be sensitive enough to detect changes in cell wall properties in cells deficient in the activity of the chromatin modifier ATX1. Extensive computational modelling of the experimental measurements (i.e. modelling the cell as a viscoelastic pressure vessel) is used to analyse the influence of the wall thickness, as well as the turgor pressure, at the positions of our measurements. By combining the nanoDMA technique with finite element simulations quantifiable measurements of the viscoelastic properties of plant cell walls are achieved. Such techniques are expected to find broader applications in quantifying the influence of genetic, biological, and environmental factors on the nanoscale mechanical properties of the cell wall. PMID:22291130

  13. SUMO Chain Formation Is Required for Response to Replication Arrest in S. pombe

    PubMed Central

    Skilton, Andrew; Ho, Jenny C. Y.; Mercer, Brenda; Outwin, Emily; Watts, Felicity Z.

    2009-01-01

    SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved ββαββαβ fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe. PMID:19707600

  14. (Rapid regulatory control of plant cell expansion and wall relaxation)

    SciTech Connect

    Cosgrove, D.J.

    1990-01-01

    This section presents a brief overview of accomplishments related to this project in the past 3-year period. Our work has focused on the basic mechanisms of plant cell expansion, particularly on the interrelations of water and solute transport with cell wall relaxation and expansion. To study these processes, we have developed new methods and used these methods to analyze the dynamic behavior of growth processes and to examine how various agents (GA, drought, light, genetic lesions) alter the growth machinery of the cell.

  15. Orbital wall infarction in child with sickle cell disease.

    PubMed

    Janssens, C; Claeys, L; Maes, P; Boiy, T; Wojciechowski, M

    2015-12-01

    We present the case of a 17-year-old boy, known with homozygous sickle cell disease, who was admitted because of generalised pain. He developed bilateral periorbital oedema and proptosis, without pain or visual disturbances. In addition to hyperhydration, oxygen and analgesia IV antibiotics were started, to cover a possible osteomyelitis. Patients with sickle cell disease are at risk for vaso-occlusive crises, when the abnormally shaped red blood cells aggregate and block the capillaries. Such a crisis typically presents at a location with high bone marrow activity, as the vertebrae and long bones. At an early age, the bone marrow is still active at other sites, for example the orbital wall, and thus infarction can also occur there. Thus, in young persons with sickle cell disease, it is important to consider orbital wall infarction in the differential diagnosis, since the approach is different from osteomyelitis. If the disease is complicated by an orbital compression syndrome, corticosteroids or surgical intervention may be necessary to preserve the vision. In our patient, an MRI of the orbitae demonstrated periorbital oedema with bone anomalies in the orbital and frontal bones, confirming orbital wall infarction. Ophthalmological examination revealed no signs of pressure on the nervus opticus. The patient recovered gradually with conservative treatment. PMID:26790559

  16. Immunofluorescence Microscopy of Schizosaccharomyces pombe Using Chemical Fixation.

    PubMed

    Hagan, Iain M

    2016-01-01

    Establishing the subcellular distribution of molecules of interest and the dynamics of their spatial control underpins all areas of cell and developmental biology. Although the ability to monitor the distribution of fluorescent fusion proteins has revolutionized cell and developmental biology, indirect immunofluorescence microscopy of fixed samples remains an essential complement to this approach. Immunofluorescence is often a more appropriate approach for the study of subcellular architecture. It avoids potential artifacts caused by studying fusion proteins, which might show altered function under stressful imaging conditions. Furthermore, the quantitative analysis of multiple cells in an unperturbed population by immunofluorescence invariably provides a more accurate assessment of the spatial and temporal control of a particular process than does the analysis of individual cells that is the hallmark of live-cell imaging. Parallel studies of living and fixed cells often provide complementary data sets, both of which can be considered necessary for a comprehensive understanding of molecular function. This protocol provides a method for the visualization of the Schizosaccharomyces pombe microtubule cytoskeleton by indirect immunofluorescence microscopy following chemical fixation with formaldehyde and glutaraldehyde. It includes discussion of common modifications used to monitor the distribution of other fission yeast antigens and forms a basis from which to develop protocols to localize new molecules of interest. PMID:27371599

  17. Dynamics of cell wall elasticity pattern shapes the cell during yeast mating morphogenesis.

    PubMed

    Goldenbogen, Björn; Giese, Wolfgang; Hemmen, Marie; Uhlendorf, Jannis; Herrmann, Andreas; Klipp, Edda

    2016-09-01

    The cell wall defines cell shape and maintains integrity of fungi and plants. When exposed to mating pheromone, Saccharomyces cerevisiae grows a mating projection and alters in morphology from spherical to shmoo form. Although structural and compositional alterations of the cell wall accompany shape transitions, their impact on cell wall elasticity is unknown. In a combined theoretical and experimental approach using finite-element modelling and atomic force microscopy (AFM), we investigated the influence of spatially and temporally varying material properties on mating morphogenesis. Time-resolved elasticity maps of shmooing yeast acquired with AFM in vivo revealed distinct patterns, with soft material at the emerging mating projection and stiff material at the tip. The observed cell wall softening in the protrusion region is necessary for the formation of the characteristic shmoo shape, and results in wider and longer mating projections. The approach is generally applicable to tip-growing fungi and plants cells. PMID:27605377

  18. Effect of Yeast Cell Morphology, Cell Wall Physical Structure and Chemical Composition on Patulin Adsorption.

    PubMed

    Luo, Ying; Wang, Jianguo; Liu, Bin; Wang, Zhouli; Yuan, Yahong; Yue, Tianli

    2015-01-01

    The capability of yeast to adsorb patulin in fruit juice can aid in substantially reducing the patulin toxic effect on human health. This study aimed to investigate the capability of yeast cell morphology and cell wall internal structure and composition to adsorb patulin. To compare different yeast cell morphologies, cell wall internal structure and composition, scanning electron microscope, transmission electron microscope and ion chromatography were used. The results indicated that patulin adsorption capability of yeast was influenced by cell surface areas, volume, and cell wall thickness, as well as 1,3-β-glucan content. Among these factors, cell wall thickness and 1,3-β-glucan content serve significant functions. The investigation revealed that patulin adsorption capability was mainly affected by the three-dimensional network structure of the cell wall composed of 1,3-β-glucan. Finally, patulin adsorption in commercial kiwi fruit juice was investigated, and the results indicated that yeast cells could adsorb patulin from commercial kiwi fruit juice efficiently. This study can potentially simulate in vitro cell walls to enhance patulin adsorption capability and successfully apply to fruit juice industry. PMID:26295574

  19. Cell wall bound anionic peroxidases from asparagus byproducts.

    PubMed

    Jaramillo-Carmona, Sara; López, Sergio; Vazquez-Castilla, Sara; Jimenez-Araujo, Ana; Rodriguez-Arcos, Rocio; Guillen-Bejarano, Rafael

    2014-10-01

    Asparagus byproducts are a good source of cationic soluble peroxidases (CAP) useful for the bioremediation of phenol-contaminated wastewaters. In this study, cell wall bound peroxidases (POD) from the same byproducts have been purified and characterized. The covalent forms of POD represent >90% of the total cell wall bound POD. Isoelectric focusing showed that whereas the covalent fraction is constituted primarily by anionic isoenzymes, the ionic fraction is a mixture of anionic, neutral, and cationic isoenzymes. Covalently bound peroxidases were purified by means of ion exchange chromatography and affinity chromatography. In vitro detoxification studies showed that although CAP are more effective for the removal of 4-CP and 2,4-DCP, anionic asparagus peroxidase (AAP) is a better option for the removal of hydroxytyrosol (HT), the main phenol present in olive mill wastewaters. PMID:25195693

  20. Compounds active against cell walls of medically important fungi.

    PubMed Central

    Hector, R F

    1993-01-01

    A number of substances that directly or indirectly affect the cell walls of fungi have been identified. Those that actively interfere with the synthesis or degradation of polysaccharide components share the property of being produced by soil microbes as secondary metabolites. Compounds specifically interfering with chitin or beta-glucan synthesis have proven effective in studies of preclinical models of mycoses, though they appear to have a restricted spectrum of coverage. Semisynthetic derivatives of some of the natural products have offered improvements in activity, toxicology, or pharmacokinetic behavior. Compounds which act on the cell wall indirectly or by a secondary mechanism of action, such as the azoles, act against diverse fungi but are usually fungistatic in nature. Overall, these compounds are attractive candidates for further development. PMID:8457977

  1. A simplified vector system for visualization of localized RNAs in Schizosaccharomyces pombe.

    PubMed

    Takeuchi-Andoh, Tomoko; Ohba, Sayaka; Shinoda, Yu; Fuchita, Ayako; Hayashi, Sachiko; Nishiyoshi, Emi; Terouchi, Nobuyuki; Tani, Tokio

    2016-07-01

    RNA localization is an important event that is essential for the polarization and differentiation of a cell. Although several methods are currently used to detect localized RNAs, a simplified detection system has not yet been developed for Schizosaccharomyces pombe. In the present study, we describe a new vector system for the visualization of localized RNAs in S. pombe using a U1A-tag-GFP system. A pREP1-U1A-tag vector plasmid to express U1A-tagged RNA and a pREP2-U1AGFP plasmid to produce a U1A-GFP fusion protein were constructed for this system. Since the U1A-GFP protein binds U1A-tagged RNA, fluorescence is observed at the location of U1A-tagged RNA in cells expressing both of these. The nucleolar localization of U3 snoRNA was successfully detected using this system, and a novel RNA localized at the DNA region of the nucleus was found by screening localized RNAs. This system will accelerate the study of localized RNAs in S. pombe. PMID:26979837

  2. A Geographically Diverse Collection of Schizosaccharomyces pombe Isolates Shows Limited Phenotypic Variation but Extensive Karyotypic Diversity

    PubMed Central

    Brown, William R. A.; Liti, Gianni; Rosa, Carlos; James, Steve; Roberts, Ian; Robert, Vincent; Jolly, Neil; Tang, Wen; Baumann, Peter; Green, Carter; Schlegel, Kristina; Young, Jonathan; Hirchaud, Fabienne; Leek, Spencer; Thomas, Geraint; Blomberg, Anders; Warringer, Jonas

    2011-01-01

    The fission yeast Schizosaccharomyces pombe has been widely used to study eukaryotic cell biology, but almost all of this work has used derivatives of a single strain. We have studied 81 independent natural isolates and 3 designated laboratory strains of Schizosaccharomyces pombe. Schizosaccharomyces pombe varies significantly in size but shows only limited variation in proliferation in different environments compared with Saccharomyces cerevisiae. Nucleotide diversity, π, at a near neutral site, the central core of the centromere of chromosome II is approximately 0.7%. Approximately 20% of the isolates showed karyotypic rearrangements as detected by pulsed field gel electrophoresis and filter hybridization analysis. One translocation, found in 6 different isolates, including the type strain, has a geographically widespread distribution and a unique haplotype and may be a marker of an incipient speciation event. All of the other translocations are unique. Exploitation of this karyotypic diversity may cast new light on both the biology of telomeres and centromeres and on isolating mechanisms in single-celled eukaryotes. PMID:22384373

  3. Enzyme Amplified Detection of Microbial Cell Wall Components

    NASA Technical Reports Server (NTRS)

    Wainwright, Norman R.

    2004-01-01

    This proposal is MBL's portion of NASA's Johnson Space Center's Astrobiology Center led by Principal Investigator, Dr. David McKay, entitled: 'Institute for the Study of Biomarkers in Astromaterials.' Dr. Norman Wainwright is the principal investigator at MBL and is responsible for developing methods to detect trace quantities of microbial cell wall chemicals using the enzyme amplification system of Limulus polyphemus and other related methods.

  4. Bacterial Cell Wall Polymer-Induced Granulomatous Inflammation

    PubMed

    Sartor; Herfarth; Van Tol EAF

    1996-04-01

    Local or systemic injection of peptidoglycan-polysaccharide polymers, which are the primary structural components of cell walls of nearly all bacteria, leads to acute inflammation, which can develop into chronic, spontaneously relapsing, granulomatous inflammation in a number of organs. Evolution into chronic granulomatous inflammation is dependent upon persistence of poorly biodegradable cell wall polymers within tissues, genetically determined host susceptibility, and generation of a T-lymphocyte-mediated immune response. Intraperitoneal injection of peptidoglycan-polysaccharide fragments from group A streptococci or selected intestinal bacteria into susceptible Lewis rats leads to chronic, spontaneously reactivating erosive arthritis and hepatic granulomas. Subserosal (intramural) injection of poorly biodegradable cell wall fragments into the distal intestine of Lewis rats induces chronic, spontaneously relapsing granulomatous enterocolitis with associated arthritis, hepatic granulomas, anemia, and leukocytosis. Chronic inflammation does not occur in T-lymphocyte-deficient rats and is prevented by cyclosporin-A therapy and degradation of peptidoglycan by the muralytic enzyme, mutanolysin. Moreover, resistant Buffalo and Fischer F344 rats, the latter sharing identical MHC antigens with Lewis rats, develop only acute inflammation with no chronic granulomatous response. Peptidoglycan-polysaccharide polymers activate almost every limb of the inflammatory response. Blockade of specific pathways suggests that interleukin-1, transforming growth factor-beta, plasma kallikrein, and T lymphocytes are dominant mediators of peptidoglycan-polysaccharide-induced arthritis, hepatic granulomas, and enterocolitis. Because of the similarity of immune mechanisms of these rat models to human disease, bacterial cell wall-induced inflammation provides unique opportunities to study pathogenic mechanisms of granuloma formation in response to ubiquitous microbial agents and to test

  5. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli.

    PubMed

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2015-08-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  6. Proteomic Analysis to Identify Tightly-Bound Cell Wall Protein in Rice Calli

    PubMed Central

    Cho, Won Kyong; Hyun, Tae Kyung; Kumar, Dhinesh; Rim, Yeonggil; Chen, Xiong Yan; Jo, Yeonhwa; Kim, Suwha; Lee, Keun Woo; Park, Zee-Yong; Lucas, William J.; Kim, Jae-Yean

    2015-01-01

    Rice is a model plant widely used for basic and applied research programs. Plant cell wall proteins play key roles in a broad range of biological processes. However, presently, knowledge on the rice cell wall proteome is rudimentary in nature. In the present study, the tightly-bound cell wall proteome of rice callus cultured cells using sequential extraction protocols was developed using mass spectrometry and bioinformatics methods, leading to the identification of 1568 candidate proteins. Based on bioinformatics analyses, 389 classical rice cell wall proteins, possessing a signal peptide, and 334 putative non-classical cell wall proteins, lacking a signal peptide, were identified. By combining previously established rice cell wall protein databases with current data for the classical rice cell wall proteins, a comprehensive rice cell wall proteome, comprised of 496 proteins, was constructed. A comparative analysis of the rice and Arabidopsis cell wall proteomes revealed a high level of homology, suggesting a predominant conservation between monocot and eudicot cell wall proteins. This study importantly increased information on cell wall proteins, which serves for future functional analyses of these identified rice cell wall proteins. PMID:26194822

  7. Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

    PubMed Central

    de Vries, Ronald P.; Visser, Jaap

    2001-01-01

    Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded. PMID:11729262

  8. Phosphate-containing cell wall polymers of bacilli.

    PubMed

    Potekhina, N V; Streshinskaya, G M; Tul'skaya, E M; Kozlova, Yu I; Senchenkova, S N; Shashkov, A S

    2011-07-01

    Anionic phosphate-containing cell wall polymers of bacilli are represented by teichoic acids and poly(glycosyl 1-phosphates). Different locations of phosphodiester bonds in the main chain of teichoic acids as well as the nature and combination of the constituent structural elements underlie their structural diversity. Currently, the structures of teichoic acids of bacilli can be classified into three types, viz. poly(polyol phosphates) with glycerol or ribitol as the polyol; poly(glycosylpolyol phosphates), mainly glycerol-containing polymers; and poly(acylglycosylglycerol phosphate), in which the components are covalently linked through glycosidic, phosphodiester, and amide bonds. In addition to teichoic acids, poly(glycosyl 1-phosphates) with mono- and disaccharide residues in the repeating units have been detected in cell walls of several Bacillus subtilis and Bacillus pumilus strains. The known structures of teichoic acids and poly(glycosyl 1-phosphates) of B. subtilis, B. atrophaeus, B. licheniformis, B. pumilus, B. stearothermophilus, B. coagulans, B. cereus as well as oligomers that link the polymers to peptidoglycan are surveyed. The reported data on the structures of phosphate-containing polymers of different strains of B. subtilis suggest heterogeneity of the species and may be of interest for the taxonomy of bacilli to allow differentiation of closely related organisms according to the "structures and composition of cell wall polymers" criterion. PMID:21999535

  9. Modeling of thin, back-wall silicon solar cells

    NASA Technical Reports Server (NTRS)

    Baraona, C. R.

    1979-01-01

    The performance of silicon solar cells with p-n junctions on the nonilluminated surface (i.e., upside-down or back-wall cells) was calculated. These structures consisted of a uniformly shaped p-type substrate layer, a p(+)-type field layer on the front (illuminated) surface, and a shallow, n-type junction on the back (nonilluminated) surface. A four-layer solar cell model was used to calculate efficiency, open-circuit voltage, and short-circuit current. The effect on performance of p-layer thickness and resistivity was determined. The diffusion length was varied to simulate the effect of radiation damage. The results show that peak initial efficiencies greater than 15 percent are possible for cell thicknesses or 100 micrometers or less. After 10 years of radiation damage in geosynchronous orbit, thin (25 to 50 micrometers thick) cells made from 10 to 100 ohm cm material show the smallest decrease (approximately 10 percent) in performance.

  10. The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

    SciTech Connect

    Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

    2010-08-20

    Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

  11. Evaluating fundamental position-dependent differences in wood cell wall adhesion using nanoindentation

    PubMed Central

    Obersriebnig, Michael; Konnerth, Johannes; Gindl-Altmutter, Wolfgang

    2013-01-01

    Spruce wood specimens were bonded with one-component polyurethane (PUR) and urea-formaldehyde (UF) adhesive, respectively. The adhesion of the adhesives to the wood cell wall was evaluated at two different locations by means of a new micromechanical assay based on nanoindentation. One location tested corresponded to the interface between the adhesive and the natural inner cell wall surface of the secondary cell wall layer 3 (S3), whereas the second location corresponded to the interface between the adhesive and the freshly cut secondary cell wall layer 2 (S2). Overall, a trend towards reduced cell wall adhesion was found for PUR compared to UF. Position-resolved examination revealed excellent adhesion of UF to freshly cut cell walls (S2) but significantly diminished adhesion to the inner cell wall surface (S3). In contrast, PUR showed better adhesion to the inner cell wall surface and less adhesion to freshly cut cell walls. Atomic force microscopy revealed a less polar character for the inner cell wall surface (S3) compared to freshly cut cell walls (S2). It is proposed that differences in the polarity of the used adhesives and the surface chemistry of the two cell wall surfaces examined account for the observed trends.

  12. Proteomic analysis of cell walls of two developmental stages of alfalfa stems

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the ...

  13. Stress analysis for wall structure in mobile hot cell design

    NASA Astrophysics Data System (ADS)

    Bahrin, Muhammad Hannan; Rahman, Anwar Abdul; Hamzah, Mohd Arif; Mamat, Mohd Rizal; Azman, Azraf; Hasan, Hasni

    2016-01-01

    Malaysian Nuclear Agency is developing a Mobile Hot Cell (MHC) in order to handle and manage Spent High Activity Radioactive Sources (SHARS) such as teletherapy heads and irradiators. At present, there are only two units of MHC in the world, in South Africa and China. Malaysian Mobile Hot cell is developed by Malaysian Nuclear Agency with the assistance of IAEA expert, based on the design of South Africa and China, but with improved features. Stress analysis has been performed on the design in order to fulfil the safety requirement in operation of MHC. This paper discusses the loading analysis effect from the sand to the MHC wall structure.

  14. Characterization of nuclear pore complex components in fission yeast Schizosaccharomyces pombe

    PubMed Central

    Asakawa, Haruhiko; Yang, Hui-Ju; Yamamoto, Takaharu G; Ohtsuki, Chizuru; Chikashige, Yuji; Sakata-Sogawa, Kumiko; Tokunaga, Makio; Iwamoto, Masaaki; Hiraoka, Yasushi; Haraguchi, Tokuko

    2014-01-01

    The nuclear pore complex (NPC) is an enormous proteinaceous complex composed of multiple copies of about 30 different proteins called nucleoporins. In this study, we analyzed the composition of the NPC in the model organism Schizosaccharomyces pombe using strains in which individual nucleoporins were tagged with GFP. We identified 31 proteins as nucleoporins by their localization to the nuclear periphery. Gene disruption analysis in previous studies coupled with gene disruption analysis in the present study indicates that 15 of these nucleoporins are essential for vegetative cell growth and the other 16 nucleoporins are non-essential. Among the 16 non-essential nucleoporins, 11 are required for normal progression through meiosis and their disruption caused abnormal spore formation or poor spore viability. Based on fluorescence measurements of GFP-fused nucleoporins, we estimated the composition of the NPC in S. pombe and found that the organization of the S. pombe NPC is largely similar to that of other organisms; a single NPC was estimated as being 45.8–47.8 MDa in size. We also used fluorescence measurements of single NPCs and quantitative western blotting to analyze the composition of the Nup107-Nup160 subcomplex, which plays an indispensable role in NPC organization and function. Our analysis revealed low amounts of Nup107 and Nup131 and high amounts of Nup132 in the Nup107-Nup160 subcomplex, suggesting that the composition of this complex in S. pombe may differ from that in S. cerevisiae and humans. Comparative analysis of NPCs in various organisms will lead to a comprehensive understanding of the functional architecture of the NPC. PMID:24637836

  15. Essential roles of class E Vps proteins for sorting into multivesicular bodies in Schizosaccharomyces pombe

    PubMed Central

    Iwaki, Tomoko; Onishi, Masayuki; Ikeuchi, Masaru; Kita, Ayako; Sugiura, Reiko; Giga-Hama, Yuko; Fukui, Yasuhisa; Takegawa, Kaoru

    2007-01-01

    The multivesicular body (MVB) sorting pathway is required for a number of biological processes, including downregulation of cell-surface proteins and protein sorting into the vacuolar lumen. The function of this pathway requires endosomal sorting complexes required for transport (ESCRT) composed of class E vacuolar protein sorting (Vps) proteins in Saccharomyces cerevisiae, many of which are conserved in Schizosaccharomyces pombe. Of these, sst4/vps27 (homologous to VPS27) and sst6 (similar to VPS23) have been identified as suppressors of sterility in ste12Δ (sst), although their functions have not been uncovered to date. In this report, these two sst genes are shown to be required for vacuolar sorting of carboxypeptidase Y (CPY) and an MVB marker, the ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein, despite the lack of the ubiquitin E2 variant domain in Sst6p. Disruption mutants of a variety of other class E vps homologues also had defects in sorting of CPY and Ub–GFP–CPS. Sch. pombe has a mammalian AMSH homologue, sst2. Phenotypic analyses suggested that Sst2p is a class E Vps protein. Taken together, these results suggest that sorting into multivesicular bodies is dependent on class E Vps proteins, including Sst2p, in Sch. pombe. PMID:17660439

  16. A Wd Repeat Protein, Rec14, Essential for Meiotic Recombination in Schizosaccharomyces Pombe

    PubMed Central

    Evans, D. H.; Li, Y. F.; Fox, M. E.; Smith, C. R.

    1997-01-01

    Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III. A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb. The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts. The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein. Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination. The pattern of expression of rec14 differs from that of previously analyzed S. pombe rec genes. Based upon mutant phenotypes and amino acid sequence similarities, we propose that S. pombe Rec14 is a functional homologue of S. cerevisiae Rec103. PMID:9258671

  17. Nitric oxide signaling and its role in oxidative stress response in Schizosaccharomyces pombe.

    PubMed

    Astuti, Rika Indri; Watanabe, Daisuke; Takagi, Hiroshi

    2016-01-30

    In the fission yeast Schizosaccharomyces pombe, we found that the putative NO dioxygenase SPAC869.02c (named Yhb1) and the S-nitrosoglutathione reductase Fmd2 cooperatively reduced intracellular NO levels as NO-detoxification enzymes. Although both mRNA and protein levels were increased with exogenous NO, their expression patterns were different during growth phases. While treatment with an NO synthase inhibitor in the log phase abrogated both NO production and Yhb1 expression, induction of Fmd2 in the stationary phase was correlated with elevated mitochondrial respiratory chain (MRC) activity, confirmed by the fact that inhibition of MRC complex III led to a decrease in Fmd2 and NO levels. Moreover, NO was localized in the mitochondria in the stationary phase, suggesting that there are two distinctive types of NO signaling in S. pombe. For mitochondria, pretreatment with an NO donor rescued cell growth by repressing generation of reactive oxygen species (ROS) under oxidative stress. DNA microarray analysis revealed that exogenous NO contributes to tolerance to hydrogen peroxide (H2O2) by (i) inhibition of Fe(3+) to Fe(2+) conversion, (ii) upregulation of the H2O2-detoxifying enzymes, and (iii) downregulation of the MRC genes, suggesting that NO plays a pivotal role in the negative feedback system to regulate ROS levels in S. pombe. PMID:26645666

  18. Local Nanomechanical Motion of the Cell Wall of Saccharomyces cerevisiae

    NASA Astrophysics Data System (ADS)

    Pelling, Andrew E.; Sehati, Sadaf; Gralla, Edith B.; Valentine, Joan S.; Gimzewski, James K.

    2004-08-01

    We demonstrate that the cell wall of living Saccharomyces cerevisiae (baker's yeast) exhibits local temperature-dependent nanomechanical motion at characteristic frequencies. The periodic motions in the range of 0.8 to 1.6 kHz with amplitudes of ~3 nm were measured using the cantilever of an atomic force microscope (AFM). Exposure of the cells to a metabolic inhibitor causes the periodic motion to cease. From the strong frequency dependence on temperature, we derive an activation energy of 58 kJ/mol, which is consistent with the cell's metabolism involving molecular motors such as kinesin, dynein, and myosin. The magnitude of the forces observed (~10 nN) suggests concerted nanomechanical activity is operative in the cell.

  19. Impact of processing on the noncovalent interactions between procyanidin and apple cell wall.

    PubMed

    Le Bourvellec, Carine; Watrelot, Aude A; Ginies, Christian; Imberty, Anne; Renard, Catherine M G C

    2012-09-19

    Procyanidins can bind cell wall material in raw product, and it could be supposed that the same mechanism of retention of procyanidins by apple cell walls takes place in cooked products. To evaluate the influence of cell wall composition and disassembly during cooking on the cell walls' capacity to interact with procyanidins, four cell wall materials differing in their protein contents and physical characteristics were prepared: cell wall with proteins, cell wall devoid of protein, and two processed cell walls differing by their drying method. Protein contents varied from 23 to 99 mg/g and surface areas from 1.26 to 3.16 m(2)/g. Apple procyanidins with an average polymerization degree of 8.7 were used. The adsorption of apple procyanidins on solid cell wall material was quantified using the Langmuir isotherm formulation. The protein contents in cell wall material had no effect on procyanidin/cell wall interactions, whereas modification of the cell wall material by boiling, which reduces pectin content, and drying decreased the apparent affinity and increased the apparent saturation levels when constants were expressed relative to cell wall weight. However, boiling and drying increased apparent saturation levels and had no effect on apparent affinity when the same data were expressed per surface units. Isothermal titration calorimetry indicated strong affinity (K(a) = 1.4 × 10(4) M(-1)) between pectins solubilized by boiling and procyanidins. This study higllights the impact of highly methylated pectins and drying, that is, composition and structure of cell wall in the cell wall/procyanidin interactions. PMID:22861056

  20. Seed coat mucilage cells of Arabidopsis thaliana as a model for plant cell wall research.

    PubMed

    Arsovski, Andrej A; Haughn, George W; Western, Tamara L

    2010-07-01

    Plant cells are encased within a complex polysaccharide wall that strengthens the cell and has key roles in all aspects of plant cell growth, differentiation, and interaction with the environment. This dynamic structure is under continual modification during plant development, and its synthesis and modification require the activity of a myriad of enzymes. The mucilage secretory cells (MSCs) of the Arabidopsis thaliana seed coat provide a model for the discovery of novel genes involved in the synthesis, secretion and modification of cell wall components, particularly pectin. These cells synthesize copious amounts of pectinaceous mucilage during development and, upon hydration of the desiccated seed, the mucilage rapidly swells, bursts from the MSCs and surrounds the seed in a gelatinous capsule. Several genes affecting MSC differentiation, pectin synthesis, and mucilage release have been identified and additional genes involved in these and related processes including pectin secretion and the mechanical alteration of cell walls await to be discovered. PMID:20505351

  1. Cell wall pH and auxin transport velocity

    NASA Technical Reports Server (NTRS)

    Hasenstein, K. H.; Rayle, D.

    1984-01-01

    According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport.

  2. Cell wall pH and auxin transport velocity.

    PubMed Central

    Hasenstein, K H; Rayle, D

    1984-01-01

    According to the chemiosmotic polar diffusion hypothesis, auxin pulse velocity and basal secretion should increase with decreasing cell wall pH. Experiments were designed to test this prediction. Avena coleoptile sections were preincubated in either fusicoccin (FC), cycloheximide, pH 4.0, or pH 8.0 buffer and subsequently their polar transport capacities were determined. Relative to controls, FC enhanced auxin (IAA) uptake while CHI and pH 8.0 buffer reduced IAA uptake. Nevertheless, FC reduced IAA pulse velocity while cycloheximide increased velocity. Additional experiments showed that delivery of auxin to receivers is enhanced by increased receiver pH. This phenomenon was overcome by a pretreatment of the tissue with IAA. Our data suggest that while acidic wall pH values facilitate cellular IAA uptake, they do not enhance pulse velocity or basal secretion. These findings are inconsistent with the chemiosmotic hypothesis for auxin transport. PMID:11540807

  3. Enzymology and molecular biology of cell wall biosynthesis. Progress report

    SciTech Connect

    Ray, P.M.

    1993-03-20

    In order to be able to explore the control of cell wall polysaccharide synthesis at the molecular level, which inter alia might eventually lead to means for useful modification of plant biomass polysaccharide production, the immediate goals of this project are to identify polypeptides responsible for wall polysaccharide synthase activities and to obtain clones of the genes that encode them. We are concentrating on plasma membraneassociated (1,3)-{beta}-glucan synthase (glucan synthase-II or GS-II) and Golgi-associated (1,4)-{beta}-glucan synthase (glucan synthase-I or GS-I), of growing pea stem tissue. Our progress has been much more rapid with respect to GS-II than regarding GS-I.

  4. Scattering properties of microalgae: the effect of cell size and cell wall

    NASA Astrophysics Data System (ADS)

    Svensen, Øyvind; Frette, Øyvind; Rune Erga, Svein

    2007-08-01

    The main objective of this work was to investigate how the cell size and the presence of a cell wall influence the scattering properties of the green microalgae Chlamydomonas reinhardtii. The growth cycle of two strains, one with a cell wall and one without, was synchronized to be in the same growth phase. Measurements were conducted at two different phases of the growth cycle on both strains of the algae. It was found that the shape of the scattering phase function was very similar for both strains at both growth phases, but the regular strain with a cell wall scatters more strongly than the wall-less mutant. It was also found that the mutant strain has a stronger increase in scattering than the regular strain, as the algae grow, and that the scattering from the regular strain is more wavelength dependent than from the mutant strain.

  5. Measuring the Mechanical Properties of Plant Cell Walls.

    PubMed

    Vogler, Hannes; Felekis, Dimitrios; Nelson, Bradley J; Grossniklaus, Ueli

    2015-01-01

    The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM). PMID:27135321

  6. Measuring the Mechanical Properties of Plant Cell Walls

    PubMed Central

    Vogler, Hannes; Felekis, Dimitrios; Nelson, Bradley J.; Grossniklaus, Ueli

    2015-01-01

    The size, shape and stability of a plant depend on the flexibility and integrity of its cell walls, which, at the same time, need to allow cell expansion for growth, while maintaining mechanical stability. Biomechanical studies largely vanished from the focus of plant science with the rapid progress of genetics and molecular biology since the mid-twentieth century. However, the development of more sensitive measurement tools renewed the interest in plant biomechanics in recent years, not only to understand the fundamental concepts of growth and morphogenesis, but also with regard to economically important areas in agriculture, forestry and the paper industry. Recent advances have clearly demonstrated that mechanical forces play a crucial role in cell and organ morphogenesis, which ultimately define plant morphology. In this article, we will briefly review the available methods to determine the mechanical properties of cell walls, such as atomic force microscopy (AFM) and microindentation assays, and discuss their advantages and disadvantages. But we will focus on a novel methodological approach, called cellular force microscopy (CFM), and its automated successor, real-time CFM (RT-CFM). PMID:27135321

  7. [Hydroxyproline: Rich glycoproteins of the plant and cell wall

    SciTech Connect

    Varner, J.E.

    1993-01-01

    Since xylem tissue includes the main cell types which are lignified, we are interested in gene expression of glycine-rich proteins and proline-rich proteins, and other proteins which are involved in secondary cell wall thickening during xylogenesis. Since the main feature of xylogenesis is the deposition of additional wall components, study of the mechanism of xylogenesis will greatly advance our knowledge of the synthesis and assembly of wall macromolecules. We are using the in vitro xylogenesis system from isolated Zinnia mesophyll cells to isolate genes which are specifically expressed during xylogenesis. We have used subtractive hybridization methods to isolate a number of cDNA clones for differentially regulated genes from the cells after hormonal induction. So far, we have partially characterized 18 different cDNA clones from 239 positive clones. These differentially regulated genes can be divided into three sets according to the characteristics of gene expression in the induction medium and the control medium. The first set is induced in both the induction medium and the control medium without hormones. The second set is induced mainly in the induction medium and in the control medium with the addition of NAA alone. Two of thesegenes are exclusively induced by auxin. The third set of genes is induced mainly in the induction medium. Since these genes are not induced by either auxin or cytokinin alone, they may be directly involved in the process of xylogenesis. Our experiments on the localization of H[sub 2]O[sub 2] production reinforce the earlier ideas of others that H[sub 2]O[sub 2] is involved in normal lignification.

  8. Differences in osmotolerant and cell-wall properties of two Zygosaccharomyces rouxii strains.

    PubMed

    Pribylová, L; Farkas, V; Slaninová, I; de Montigny, J; Sychrová, H

    2007-01-01

    The osmotolerant and cell wall properties of the two most studied wild-type Zygosaccharomyces rouxii strains (CBS 732 and ATCC 42981) were examined. Differences in their (1) tolerance to high salt content in the medium, (2) resistance to the lysing enzymes Lyticase and Zymolyase, (3) cell-wall polymer content and (4) cell wall micromorphology suggested that the less osmotolerant CBS 732 strain possesses a more rigid cell wall than the more osmotolerant ATCC 42981, whose cell wall seems to be more flexible and elastic. PMID:17702462

  9. Comparative structure and biomechanics of plant primary and secondary cell walls

    PubMed Central

    Cosgrove, Daniel J.; Jarvis, Michael C.

    2012-01-01

    Recent insights into the physical biology of plant cell walls are reviewed, summarizing the essential differences between primary and secondary cell walls and identifying crucial gaps in our knowledge of their structure and biomechanics. Unexpected parallels are identified between the mechanism of expansion of primary cell walls during growth and the mechanisms by which hydrated wood deforms under external tension. There is a particular need to revise current “cartoons” of plant cell walls to be more consistent with data from diverse approaches and to go beyond summarizing limited aspects of cell walls, serving instead as guides for future experiments and for the application of new techniques. PMID:22936943

  10. Architecture-based multiscale computational modeling of plant cell wall mechanics to examine the hydrogen-bonding hypothesis of cell wall network structure model

    SciTech Connect

    Yi, Hojae; Puri, Virendra M.

    2012-11-01

    A primary plant cell wall network was computationally modeled using the finite element approach to study the hypothesis of hemicellulose (HC) tethering with the cellulose microfibrils (CMFs) as one of the major load-bearing mechanisms of the growing cell wall. A computational primary cell wall network fragment (10 × 10 μm) comprising typical compositions and properties of CMFs and HC was modeled with well-aligned CMFs. The tethering of HC to CMFs is modeled in accordance with the strength of the hydrogen bonding by implementing a specific load-bearing connection (i.e. the joint element). The introduction of the CMF-HC interaction to the computational cell wall network model is a key to the quantitative examination of the mechanical consequences of cell wall structure models, including the tethering HC model. When the cell wall network models with and without joint elements were compared, the hydrogen bond exhibited a significant contribution to the overall stiffness of the cell wall network fragment. When the cell wall network model was stretched 1% in the transverse direction, the tethering of CMF-HC via hydrogen bonds was not strong enough to maintain its integrity. When the cell wall network model was stretched 1% in the longitudinal direction, the tethering provided comparable strength to maintain its integrity. This substantial anisotropy suggests that the HC tethering with hydrogen bonds alone does not manifest sufficient energy to maintain the integrity of the cell wall during its growth (i.e. other mechanisms are present to ensure the cell wall shape).

  11. Proteomic Analysis of Cell Walls of Two Developmental Stages of Alfalfa Stems

    PubMed Central

    Verdonk, Julian C.; Hatfield, Ronald D.; Sullivan, Michael L.

    2012-01-01

    Cell walls are important for the growth and development of all plants. They are also valuable resources for feed and fiber, and more recently as a potential feedstock for bioenergy production. Cell wall proteins comprise only a fraction of the cell wall, but play important roles in establishing the walls and in the chemical interactions (e.g., crosslinking) of cell wall components. This crosslinking provides structure, but restricts digestibility of cell wall complex carbohydrates, limiting available energy in animal and bioenergy production systems. Manipulation of cell wall proteins could be a strategy to improve digestibility. An analysis of the cell wall proteome of apical alfalfa stems (less mature, more digestible) and basal alfalfa stems (more mature, less digestible) was conducted using a recently developed low-salt/density gradient method for the isolation of cell walls. Walls were subsequently subjected to a modified extraction utilizing EGTA to remove pectins, followed by a LiCl extraction to isolate more tightly bound proteins. Recovered proteins were identified using shotgun proteomics. We identified 272 proteins in the alfalfa stem cell wall proteome, 153 of which had not previously been identified in cell wall proteomic analyses. Nearly 70% of the identified proteins were predicted to be secreted, as would be expected for most cell wall proteins, an improvement over previously published studies using traditional cell wall isolation methods. A comparison of our and several other cell wall proteomic studies indicates little overlap in identified proteins among them, which may be largely due to differences in the tissues used as well as differences in experimental approach. PMID:23248635

  12. Bacterial cell wall-induced arthritis: chemical composition and tissue distribution of four Lactobacillus strains.

    PubMed

    Simelyte, E; Rimpiläinen, M; Lehtonen, L; Zhang, X; Toivanen, P

    2000-06-01

    To study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus. Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei. The cell wall of Lactobacillus fermentum did not induce chronic arthritis. All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L. fermentum was different. Likewise, all arthritogenic cell walls were resistant to lysozyme degradation, whereas the L. fermentum cell wall was lysozyme sensitive. Muramic acid was observed in the liver, spleen, and lymph nodes in considerably larger amounts after injection of an arthritogenic L. casei cell wall than following injection of a nonarthritogenic L. fermentum cell wall. The L. casei cell wall also persisted in the tissues longer than the L. fermentum cell wall. The present results, taken together with those published previously, underline the possibility that the chemical structure of peptidoglycan is important in determining the arthritogenicity of the bacterial cell wall. PMID:10816508

  13. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata

    PubMed Central

    Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  14. KRE5 Suppression Induces Cell Wall Stress and Alternative ER Stress Response Required for Maintaining Cell Wall Integrity in Candida glabrata.

    PubMed

    Tanaka, Yutaka; Sasaki, Masato; Ito, Fumie; Aoyama, Toshio; Sato-Okamoto, Michiyo; Takahashi-Nakaguchi, Azusa; Chibana, Hiroji; Shibata, Nobuyuki

    2016-01-01

    The maintenance of cell wall integrity in fungi is required for normal cell growth, division, hyphae formation, and antifungal tolerance. We observed that endoplasmic reticulum stress regulated cell wall integrity in Candida glabrata, which possesses uniquely evolved mechanisms for unfolded protein response mechanisms. Tetracycline-mediated suppression of KRE5, which encodes a predicted UDP-glucose:glycoprotein glucosyltransferase localized in the endoplasmic reticulum, significantly increased cell wall chitin content and decreased cell wall β-1,6-glucan content. KRE5 repression induced endoplasmic reticulum stress-related gene expression and MAP kinase pathway activation, including Slt2p and Hog1p phosphorylation, through the cell wall integrity signaling pathway. Moreover, the calcineurin pathway negatively regulated cell wall integrity, but not the reduction of β-1,6-glucan content. These results indicate that KRE5 is required for maintaining both endoplasmic reticulum homeostasis and cell wall integrity, and that the calcineurin pathway acts as a regulator of chitin-glucan balance in the cell wall and as an alternative mediator of endoplasmic reticulum stress in C. glabrata. PMID:27548283

  15. Degradation of isolated tomato cell walls by purified polygalacturonase in vitro.

    PubMed

    Themmen, A P; Tucker, G A; Grierson, D

    1982-01-01

    Cell wall preparations from green pericarp of normal and mutant Neverripe (Nr) and ripening inhibitor (rin) tomato (Lycopersicon esculentum Mill.) fruit were all equally degraded in vitro by a cell wall-bound protein extract from ripe normal tomatoes.Similar cell wall-bound protein extracts from ripe Nr fruit were not as effective and those from ripe rin fruit gave no cell wall degradation at all in vitro. This was correlated with the absence of polygalacturonase in rin and low activity of Nr extracts.Purified polygalacturonase was capable of in vitro cell wall degradation and it seems that this enzyme can account for the cell wall degradation observed with the total cell wall-bound protein extracts from ripe fruit. PMID:16662142

  16. Profiling the Hydrolysis of Isolated Grape Berry Skin Cell Walls by Purified Enzymes.

    PubMed

    Zietsman, Anscha J J; Moore, John P; Fangel, Jonatan U; Willats, William G T; Vivier, Melané A

    2015-09-23

    The unraveling of crushed grapes by maceration enzymes during winemaking is difficult to study because of the complex and rather undefined nature of both the substrate and the enzyme preparations. In this study we simplified both the substrate, by using isolated grape skin cell walls, and the enzyme preparations, by using purified enzymes in buffered conditions, to carefully follow the impact of the individual and combined enzymes on the grape skin cell walls. By using cell wall profiling techniques we could monitor the compositional changes in the grape cell wall polymers due to enzyme activity. Extensive enzymatic hydrolysis, achieved with a preparation of pectinases or pectinases combined with cellulase or hemicellulase enzymes, completely removed or drastically reduced levels of pectin polymers, whereas less extensive hydrolysis only opened up the cell wall structure and allowed extraction of polymers from within the cell wall layers. Synergistic enzyme activity was detectable as well as indications of specific cell wall polymer associations. PMID:26309153

  17. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-11-30

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

  18. Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Dupres, Vincent; McEvoy, Kevin; Wildling, Linda; Gruber, Hermann J.; Dufrêne, Yves F.

    2008-09-01

    Although the chemical composition of yeast cell walls is known, the organization, assembly, and interactions of the various macromolecules remain poorly understood. Here, we used in situ atomic force microscopy (AFM) in three different modes to probe the ultrastructure, cell wall elasticity and polymer properties of two brewing yeast strains, i.e. Saccharomyces carlsbergensis and S. cerevisiae. Topographic images of the two strains revealed smooth and homogeneous cell surfaces, and the presence of circular bud scars on dividing cells. Nanomechanical measurements demonstrated that the cell wall elasticity of S. carlsbergensis is homogeneous. By contrast, the bud scar of S. cerevisiae was found to be stiffer than the cell wall, presumably due to the accumulation of chitin. Notably, single molecule force spectroscopy with lectin-modified tips revealed major differences in polysaccharide properties of the two strains. Polysaccharides were clearly more extended on S. cerevisiae, suggesting that not only oligosaccharides, but also polypeptide chains of the mannoproteins were stretched. Consistent with earlier cell surface analyses, these findings may explain the very different aggregation properties of the two organisms. This study demonstrates the power of using multiple complementary AFM modalities for probing the organization and interactions of the various macromolecules of microbial cell walls.

  19. Cellulose-hemicellulose interaction in wood secondary cell-wall

    NASA Astrophysics Data System (ADS)

    Zhang, Ning; Li, Shi; Xiong, Liming; Hong, Yu; Chen, Youping

    2015-12-01

    The wood cell wall features a tough and relatively rigid fiber reinforced composite structure. It acts as a pressure vessel, offering protection against mechanical stress. Cellulose microfibrils, hemicellulose and amorphous lignin are the three major components of wood. The structure of secondary cell wall could be imagined as the same as reinforced concrete, in which cellulose microfibrils acts as reinforcing steel bar and hemicellulose-lignin matrices act as the concrete. Therefore, the interface between cellulose and hemicellulose/lignin plays a significant role in determine the mechanical behavior of wood secondary cell wall. To this end, we present a molecular dynamics (MD) simulation study attempting to quantify the strength of the interface between cellulose microfibrils and hemicellulose. Since hemicellulose binds with adjacent cellulose microfibrils in various patterns, the atomistic models of hemicellulose-cellulose composites with three typical binding modes, i.e. bridge, loop and random binding modes are constructed. The effect of the shape of hemicellulose chain on the strength of hemicellulose-cellulose composites under shear loadings is investigated. The contact area as well as hydrogen bonds between cellulose and hemicellulose, together with the covalent bonds in backbone of hemicellulose chain are found to be the controlling parameters which determine the strength of the interfaces in the composite system. For the bridge binding model, the effect of shear loading direction on the strength of the cellulose material is also studied. The obtained results suggest that the shear strength of wood-inspired engineering composites can be optimized through maximizing the formations of the contributing hydrogen bonds between cellulose and hemicellulose.

  20. Nutrient depletion modifies cell wall adsorption activity of wine yeast.

    PubMed

    Sidari, R; Caridi, A

    2016-06-01

    Yeast cell wall is a structure that helps yeasts to manage and respond to many environmental stresses. The mannosylphosphorylation is a modification in response to stress that provides the cell wall with negative charges able to bind compounds present in the environment. Phenotypes related to the cell wall modification such as the filamentous growth in Saccharomyces cerevisiae are affected by nutrient depletion. The present work aimed at describing the effect of carbon and/or nitrogen limitation on the aptitude of S. cerevisiae strains to bind coloured polyphenols. Carbon- and nitrogen-rich or deficient media supplemented with grape polyphenols were used to simulate different grape juice conditions-early, mid, 'adjusted' for nitrogen, and late fermentations. In early fermentation condition, the R+G+B values range from 106 (high adsorption, strain Sc1128) to 192 (low adsorption, strain Σ1278b), in mid-fermentation the values range from 111 (high adsorption, strain Sc1321) to 258 (low adsorption, strain Sc2306), in 'adjusted' for nitrogen conditions the values range from 105 (high adsorption, strain Sc1321) to 194 (low adsorption, strain Sc2306) while in late fermentation conditions the values range from 101 (high adsorption, strain Sc384) to 293 (low adsorption, strain Sc2306). The effect of nutrient availability is not univocal for all the strains and the different media tested modified the strains behaviour. In all the media the strains show significant differences. Results demonstrate that wine yeasts decrease/increase their parietal adsorption activity according to the nutrient availability. The wide range of strain variability observed could be useful in selecting wine starters. PMID:27116955

  1. Immunochemistry of the streptococcal group R cell wall polysaccharide antigen.

    PubMed

    Soprey, P; Slade, H D

    1972-01-01

    The group R streptococcal group antigen has been shown to be a polysaccharide located at the surface of the cell wall of the organism. The antigen was extracted from cell walls in 0.05 n HCl or 5% trichloracetic acid at 100 C, from whole cells at room temperature in 0.85% NaCl or 0.1 m acetate (pH 5.0), and by sonic oscillation. The antigen is largely destroyed when extracted from whole cells in 0.05 n HCl at 100 C. Acetate is recommended for routine extraction. The antigen extracted by sonic treatment was separated into six immunologically active fractions on diethylaminoethyl-Sephadex. The fractions were found to possess a common antigen which exhibited similar properties on immunodiffusion and immunoelectrophoresis. The purified antigen did not react with any other streptococcal group antisera. Adsorption of group R serum with the antigen removed all antibodies against whole cell antigen extracts of R cells. Chemical and enzymatic analysis of three fractions showed that the antigen was composed of d-glucose, d-galactose, rhamnose, and glucosamine. No significant quantities of phosphorus, glycerol, ribitol, or muramic acid were present. Significant inhibition of the quantitative precipitin determination by d-galactose and stachyose indicated that galactose in terminal alpha linkage was the immunodominant hexose in the antigen. d-Glucose and d-glucosamine possessed a partial inhibitory activity. N-acetyl-d-glucosamine and l-rhamnose did not produce significant inhibition. The results indicate that the R antigen is an immunologically specific structure which serves as a reliable means of identification of these streptococci as a serological group. PMID:4632470

  2. A radioimmunoassay for lignin in plant cell walls

    SciTech Connect

    Dawley, R.M.

    1989-01-01

    Lignin detection and determination in herbaceous tissue requires selective, specific assays which are not currently available. A radioimmunoassay (RIA) was developed to study lignin metabolism in these tissues. A {beta}-aryl ether lignin model compound was synthesized, linked to keyhole limpet hemocyanin using a water-soluble carbodiimide, and injected into rabbits. The highest titer of the antiserum obtained was 34 {eta}g/mL of model derivatized BSA. An in vitro system was developed to characterize the RIA. The model compound was linked to amino activated polyacrylamide beads to mimic lignin in the cell walls. {sup 125}I Radiolabelled protein A was used to detect IgG antibody binding. The RIA was shown in the in vitro system to exhibit saturable binding. The amount of antibody bound decreased when the serum was diluted. Immunoelectrophoresis and competitive binding experiments confirmed that both aromatic rings of the lignin model compound had been antigenic. Chlorogenic acid, a phenolic known to be present in plant cells, did not compete for antibody binding. The RIA was used to measure lignin in milled plant samples and barley seedlings. Antiserum binding to wheat cell walls and stressed barley segments was higher than preimmune serum binding. Antibody binding to stressed barley tissue decreased following NaClO{sub 2} delignification. The RIA was found to be less sensitive than expected, so several avenues for improving the method are discussed.

  3. Lignification in poplar tension wood lignified cell wall layers.

    PubMed

    Yoshinaga, Arata; Kusumoto, Hiroshi; Laurans, Françoise; Pilate, Gilles; Takabe, Keiji

    2012-09-01

    The lignification process in poplar tension wood lignified cell wall layers, specifically the S(1) and S(2) layers and the compound middle lamella (CML), was analysed using ultraviolet (UV) and transmission electron microscopy (TEM). Variations in the thickness of the gelatinous layer (G-layer) were also measured to clarify whether the lignified cell wall layers had completed their lignification before the deposition of G-layers, or, on the contrary, if lignification of these layers was still active during G-layer formation. Observations using UV microscopy and TEM indicated that both UV absorbance and the degree of potassium permanganate staining increased in the CML and S(1) and S(2) layers during G-layer formation, suggesting that the lignification of these lignified layers is still in progress during G-layer formation. In the context of the cell-autonomous monolignol synthesis hypothesis, our observations suggest that monolignols must go through the developing G-layer during the lignification of CML and the S(1) and S(2) layers. The alternative hypothesis of external synthesis (in the rays) does not require that monolignols go through the G-layer before being deposited in the CML, or the S(1) and S(2) layers. Interestingly, the previous observation of lignin in the poplar G-layer was not confirmed with the microscopy techniques used in the present study. PMID:22933655

  4. Functional analysis of putative phosphoenolpyruvate transporters localized to the Golgi apparatus in Schizosaccharomyces pombe.

    PubMed

    Yoritsune, Ken-ichi; Higuchi, Yujiro; Matsuzawa, Tomohiko; Takegawa, Kaoru

    2014-11-01

    The cell surface of Schizosaccharomyces pombe is negatively charged due to the presence of pyruvylated oligosaccharides, which is important for cell-cell recognition. However, the mechanism of pyruvate supply to oligosaccharides is not clearly understood. Here, we analyzed three putative phosphoenolpyruvate (PEP) transporter genes (pet1(+) , pet2(+) , and pet3(+) ) in S. pombe, identified by sequence homology search against the Arabidopsis thaliana PEP transporter AtPPT1. Schizosaccharomyces pombe strain carrying a disruption in pet1(+) (pet1Δ) or in pet2(+) (pet2Δ), but not the strain carrying a disruption in pet3(+) (pet3Δ), showed reduced pyruvate level on the cell surface. This reduction in pyruvate level was restored to the control level by expressing green fluorescent protein (GFP)-tagged Pet1p and Pet2p in respective disruptants. Fluorescence microscope studies revealed that GFP-tagged Pet1p and Pet2p were localized to the Golgi apparatus. Although expression of neither AtPPT1 nor AtPPT2 suppressed the pet1Δ phenotype, that of chimeric constructs, where the N-terminal regions of AtPPT1 and AtPPT2 were replaced by the N-terminal region of Pet1p, partially suppressed the pet1Δ phenotype. Furthermore, the reduction in cell surface negative charge in pet1Δ cells was restored by incubating these cells with recombinant Pvg1p and PEP. Thus, Pet1p and Pet2p are likely involved in transporting PEP from the cytoplasm into the Golgi. PMID:25195688

  5. Forage digestibility: the intersection of cell wall lignification and plant tissue anatomy

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellulose and the other polysaccharides present in forage cell walls can be completely degraded by the rumen microflora but only when these polysaccharides have been isolated from the wall and all matrix structures eliminated. Understanding how cell wall component interactions limit microbial degrad...

  6. The Mechanisms of Plant Cell Wall Deconstruction during Enzymatic Hydrolysis

    PubMed Central

    Thygesen, Lisbeth G.; Thybring, Emil E.; Johansen, Katja S.; Felby, Claus

    2014-01-01

    Mechanical agitation during enzymatic hydrolysis of insoluble plant biomass at high dry matter contents is indispensable for the initial liquefaction step in biorefining. It is known that particle size reduction is an important part of liquefaction, but the mechanisms involved are poorly understood. Here we put forward a simple model based on mechanical principles capable of capturing the result of the interaction between mechanical forces and cell wall weakening via hydrolysis of glucosidic bonds. This study illustrates that basic material science insights are relevant also within biochemistry, particularly when it comes to up-scaling of processes based on insoluble feed stocks. PMID:25232741

  7. Theoretical investigation on breaking plant cell wall by laser

    NASA Astrophysics Data System (ADS)

    Chen, Liang-cai; Wang, Jin-ji; Ma, Peng; Zuo, Du-luo; Wang, Xin-bing; Cheng, Zu-hai

    2012-03-01

    The experiment collected some spinach leaves which were irradiated by pulsed CO2 laser with energy 5.6J, 8.0J and 9.5J respectively. Each of them was soaked in three kinds of solvents (water, ethanol, the mixture of ethanol and petroleum ether) respectively. The experiment shows that the ethanol solution which contains the irradiated leaves turn dark green than the ethanol solution which contains the intact leaves and the color of solution with the leaves irradiated by CO2 laser with 9.5J changes the most significantly. Further, selective excitation on the molecular level of the cell wall were used to explain the phenomenon.

  8. Theoretical investigation on breaking plant cell wall by laser

    NASA Astrophysics Data System (ADS)

    Chen, Liang-cai; Wang, Jin-ji; Ma, Peng; Zuo, Du-luo; Wang, Xin-bing; Cheng, Zu-hai

    2011-11-01

    The experiment collected some spinach leaves which were irradiated by pulsed CO2 laser with energy 5.6J, 8.0J and 9.5J respectively. Each of them was soaked in three kinds of solvents (water, ethanol, the mixture of ethanol and petroleum ether) respectively. The experiment shows that the ethanol solution which contains the irradiated leaves turn dark green than the ethanol solution which contains the intact leaves and the color of solution with the leaves irradiated by CO2 laser with 9.5J changes the most significantly. Further, selective excitation on the molecular level of the cell wall were used to explain the phenomenon.

  9. The Metabolic Enzyme ManA Reveals a Link between Cell Wall Integrity and Chromosome Morphology

    PubMed Central

    Elbaz, Maya; Ben-Yehuda, Sigal

    2010-01-01

    Synchronizing cell growth, division and DNA replication is an essential property of all living cells. Accurate coordination of these cellular events is especially crucial for bacteria, which can grow rapidly and undergo multifork replication. Here we show that the metabolic protein ManA, which is a component of mannose phosphotransferase system, participates in cell wall construction of the rod shaped bacterium Bacillus subtilis. When growing rapidly, cells lacking ManA exhibit aberrant cell wall architecture, polyploidy and abnormal chromosome morphologies. We demonstrate that these cellular defects are derived from the role played by ManA in cell wall formation. Furthermore, we show that ManA is required for maintaining the proper carbohydrate composition of the cell wall, particularly of teichoic acid constituents. This perturbed cell wall synthesis causes asynchrony between cell wall elongation, division and nucleoid segregation. PMID:20862359

  10. Comparison of Post-Germination Mobilization of Cell Wall Polysaccharides and Non-Cell Wall Carbohydrates in Soybean (Glycine max L.) Cotyledons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cell wall polysaccharides (CWP) and non-cell wall carbohydrates (NCWC) (sucrose, raffinose, stachyose, starch) were measured in cotyledons of germinating soybean [Glycine max (L.) Merr. cv. Lambert] seedlings grown for 14 d in darkness or under a 16-h photoperiod. Ungerminated seeds contained equiva...

  11. Yeast and fungal cell-wall polysaccharides can self-assemble in vitro into an ultrastructure resembling in vivo yeast cell walls.

    PubMed

    Kopecká, Marie

    2013-06-01

    Polysaccharides account for more than 90% of the content of fungal cell walls, but the mechanism underlying the formation of the architecture of the cell walls, which consist of microfibrils embedded in an amorphous wall matrix, remains unknown. We used electron microscopy to investigate ten different fungal cell-wall polysaccharides to determine whether they could self-assemble into the fibrillar or amorphous component of fungal cell walls in a test tube without enzymes. The ultrastructures formed by precipitating β-1,3-glucan and β-1,6-glucan are different depending on the existence of branching in the molecule. Linear β-1,3-glucan and linear β-1,6-glucan precipitate into a fibrillar ultrastructure. Branched β-1,6-glucan, mannan and glycogen precipitates are amorphous. Branched β-1,3-glucan forms a fibrillar plus amorphous ultrastructure. Self-assembly among combinations of different linear and branched cell-wall polysaccharides results in an ultrastructure that resembles that of a yeast cell wall, which suggests that self-assembly of polysaccharides may participate in the development of the three-dimensional architecture of the yeast cell wall. PMID:23160360

  12. The cell-wall glycoproteins of the green alga Scenedesmus obliquus. The predominant cell-wall polypeptide of Scenedesmus obliquus is related to the cell-wall glycoprotein gp3 of Chlamydomonas reinhardtii.

    PubMed

    Voigt, Jürgen; Stolarczyk, Adam; Zych, Maria; Malec, Przemysław; Burczyk, Jan

    2014-02-01

    The green alga Scenedesmus obliquus contains a multilayered cell wall, ultrastructurally similar to that of Chlamydomonas reinhardtii, although its proportion of hydroxyproline is considerably lower. Therefore, we have investigated the polypeptide composition of the insoluble and the chaotrope-soluble wall fractions of S. obliquus. The polypeptide pattern of the chaotrope-soluble wall fraction was strongly modified by chemical deglycosylation with anhydrous hydrogen fluoride (HF) in pyridine indicating that most of these polypeptides are glycosylated. Polypeptide constituents of the chaotrope-soluble cell-wall fraction with apparent molecular masses of 240, 270, 265, and 135 kDa cross-reacted with a polyclonal antibody raised against the 100 kDa deglycosylation product of the C. reinhardtii cell-wall glycoprotein GP3B. Chemical deglycosylation of the chaotrope-soluble wall fraction resulted in a 135 kDa major polypeptide and a 106 kDa minor component reacting with the same antibody. This antibody recognized specific peptide epitopes of GP3B. When the insoluble wall fraction of S. obliquus was treated with anhydrous HF/pyridine, three polypeptides with apparent molecular masses of 144, 135, and 65 kDa were solubilized, which also occured in the deglycosylated chaotrope-soluble wall fraction. These findings indicate that theses glycoproteins are cross-linked to the insoluble wall fraction via HF-sensitive bonds. PMID:24388513

  13. Biosynthesis of the Plant Cell Wall Matrix Polysaccharide Xyloglucan.

    PubMed

    Pauly, Markus; Keegstra, Kenneth

    2016-04-29

    Xyloglucan (XyG) is a matrix polysaccharide that is present in the cell walls of all land plants. It consists of a β-1,4-linked glucan backbone that is further substituted with xylosyl residues. These xylosyl residues can be further substituted with other glycosyl and nonglycosyl substituents that vary depending on the plant family and specific tissue. Advances in plant mutant isolation and characterization, functional genomics, and DNA sequencing have led to the identification of nearly all transferases and synthases necessary to synthesize XyG. Thus, in terms of the molecular mechanisms of plant cell wall polysaccharide biosynthesis, XyG is the most well understood. However, much remains to be learned about the molecular mechanisms of polysaccharide assembly and the regulation of these processes. Knowledge of the XyG biosynthetic machinery allows the XyG structure to be tailored in planta to ascertain the functions of this polysaccharide and its substituents in plant growth and interactions with the environment. PMID:26927904

  14. Chemical Profiling of the Plant Cell Wall through Raman Microspectroscopy

    SciTech Connect

    Han, Ju; Singh, Seema; Sun, Lan; Simmons, Blake; Auer, Manfred; Parvin, Bahram

    2010-03-02

    This paper presents a computational framework for chemical pro.ling of the plant cell wall through the Raman spectroscopy. The system enables query of known spectral signatures and clustering of spectral data based on intrinsic properties. As a result, presence and relative concentration of speci.c chemical bonds can be quanti.ed. The primary contribution of this paper is in representation of raman pro.le in terms of .uorescence background and multiscale peak detection at each grid point (voxel). Such a representation allows ef.cient spatial segmentation based on the coupling between high-level salient properties and low-level symbolic representation at each voxel. The high-level salient properties refer to preferred peaks and their attributes for the entire image. The low-level symbolic representations are based on .uorescence background, spectral peak locations, and their attributes. We present results on a corn stover tissue section that is imaged through Raman microscopy, and the results are consistent with the literature. In addition, automatic clustering indicates several distinct layers of the cell walls with different spectral signatures.

  15. The toughness of secondary cell wall and woody tissue

    PubMed Central

    Lucas, P. W.; Tan, H. T. W.; Cheng, P. Y.

    1997-01-01

    The 'across grain' toughness of 51 woods has been determined on thin wet sections using scissors. The moisture content of sections and the varying sharpness of the scissor blades had little effect on the results. In thin sections (less than 0.6mm), toughness rose linearly with section thickness. The intercept toughness at zero thickness, estimated from regression analysis, was proportional to relative density, consistent with values reported for non-woody plant tissues. Extrapolation of the intercept toughness of these woods and other plant tissues/materials to a relative density of 1.0 predicted a toughness of 3.45kJ m-2 , which we identify with the intrinsic toughness of the cell wall. This quantity appears to predict published results from KIC tests on woods and is related to the propensity for crack deflection. The slope of the relationship between section thickness and toughness, describing the work of plastic buckling of cells, was not proportional to relative density, the lightest (balsa) and heaviest (lignum vitae) woods fracturing with less plastic work than predicted. The size of the plastic zone around the crack tip was estimated to be 0.5mm in size. From this, the hypothetical overall toughness of a thick (greater than 1 mm) block of solid cell wall material was calculated as 39.35 kJ m-2, due to both cell wall resistance (10 per cent) and the plastic buckling of cells (90 per cent). This value successfully predicts the toughness of most commercial woods (of relative densities between 0.2 and 0.8) from 'work area' tests in tension and bending. Though density was the most important factor, both fibre width/fibre length (in hardwoods) and lignin/cellulose ratios were negatively correlated with the work of plastic buckling, after correcting for density. At low densities the work of plastic buckling in the longitudinal radial (LR) direction exceeded that in longitudinal tangential (LT), but the reverse was true for relative densities above 0.25. This could

  16. Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation

    PubMed Central

    Yang, Jung Hyun; Wang, Huanzhong

    2016-01-01

    Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses. PMID:27047525

  17. Cell wall proteins of Sporothrix schenckii as immunoprotective agents.

    PubMed

    Alba-Fierro, Carlos A; Pérez-Torres, Armando; López-Romero, Everardo; Cuéllar-Cruz, Mayra; Ruiz-Baca, Estela

    2014-01-01

    Sporothrix schenckii is the etiological agent of sporotrichosis, an endemic subcutaneous mycosis in Latin America. Cell wall (CW) proteins located on the cell surface are inducers of cellular and humoral immune responses, potential candidates for diagnosis purposes and to generate vaccines to prevent fungal infections. This mini-review emphasizes the potential use of S. schenckii CW proteins as protective and therapeutic immune response inducers against sporotrichosis. A number of pathogenic fungi display CW components that have been characterized as inducers of protective cellular and humoral immune responses against the whole pathogen from which they were originally purified. The isolation and characterization of immunodominant protein components of the CW of S. schenckii have become relevant because of their potential in the development of protective and therapeutic immune responses against sporotrichosis. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012). PMID:24257472

  18. Rapid regulatory control of plant cell expansion and wall relaxation

    SciTech Connect

    Cosgrove, D.J.

    1991-08-14

    The aim of this project is to elucidate the biophysical and cellular mechanisms that control plant cell expansion. At present we are attempting to characterize the kinetics of the system(s) responsible for regulatory and compensatory behavior of growing cells and tissues. This work is significantly because it indicates that biochemical loosening and biophysical stress relaxation of the wall are part of a feedback loop controlling growth. This report briefly summarizes the efforts and results of the past 12 months. In large part, we have been trying to analyze the nature of growth rate noise,'' i.e. spontaneous and often erratic variations in growth rate. We are obtaining evidence that such noise'' is not random, but rather reveals an underlying growth mechanism with complex dynamics.

  19. Molecular Mechanisms for Vascular Development and Secondary Cell Wall Formation.

    PubMed

    Yang, Jung Hyun; Wang, Huanzhong

    2016-01-01

    Vascular tissues are important for transporting water and nutrients throughout the plant and as physical support of upright growth. The primary constituents of vascular tissues, xylem, and phloem, are derived from the meristematic vascular procambium and cambium. Xylem cells develop secondary cell walls (SCWs) that form the largest part of plant lignocellulosic biomass that serve as a renewable feedstock for biofuel production. For the last decade, research on vascular development and SCW biosynthesis has seen rapid progress due to the importance of these processes to plant biology and to the biofuel industry. Plant hormones, transcriptional regulators and peptide signaling regulate procambium/cambium proliferation, vascular patterning, and xylem differentiation. Transcriptional regulatory pathways play a pivot role in SCW biosynthesis. Although most of these discoveries are derived from research in Arabidopsis, many genes have shown conserved functions in biofuel feedstock species. Here, we review the recent advances in our understanding of vascular development and SCW formation and discuss potential biotechnological uses. PMID:27047525

  20. Construction of the first compendium of chemical-genetic profiles in the fission yeast Schizosaccharomyces pombe and comparative compendium approach

    SciTech Connect

    Han, Sangjo; Lee, Minho; Chang, Hyeshik; Nam, Miyoung; Park, Han-Oh; Kwak, Youn-Sig; Ha, Hye-jeong; Kim, Dongsup; Hwang, Sung-Ook; Hoe, Kwang-Lae; Kim, Dong-Uk

    2013-07-12

    Highlights: •The first compendium of chemical-genetic profiles form fission yeast was generated. •The first HTS of drug mode-of-action in fission yeast was performed. •The first comparative chemical genetic analysis between two yeasts was conducted. -- Abstract: Genome-wide chemical genetic profiles in Saccharomyces cerevisiae since the budding yeast deletion library construction have been successfully used to reveal unknown mode-of-actions of drugs. Here, we introduce comparative approach to infer drug target proteins more accurately using two compendiums of chemical-genetic profiles from the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe. For the first time, we established DNA-chip based growth defect measurement of genome-wide deletion strains of S. pombe, and then applied 47 drugs to the pooled heterozygous deletion strains to generate chemical-genetic profiles in S. pombe. In our approach, putative drug targets were inferred from strains hypersensitive to given drugs by analyzing S. pombe and S. cerevisiae compendiums. Notably, many evidences in the literature revealed that the inferred target genes of fungicide and bactericide identified by such comparative approach are in fact the direct targets. Furthermore, by filtering out the genes with no essentiality, the multi-drug sensitivity genes, and the genes with less eukaryotic conservation, we created a set of drug target gene candidates that are expected to be directly affected by a given drug in human cells. Our study demonstrated that it is highly beneficial to construct the multiple compendiums of chemical genetic profiles using many different species. The fission yeast chemical-genetic compendium is available at (http://pombe.kaist.ac.kr/compendium)

  1. The architecture of the Schizosaccharomyces pombe CCR4-NOT complex

    PubMed Central

    Ukleja, Marta; Cuellar, Jorge; Siwaszek, Aleksandra; Kasprzak, Joanna M.; Czarnocki-Cieciura, Mariusz; Bujnicki, Janusz M.; Dziembowski, Andrzej; M. Valpuesta, Jose

    2016-01-01

    CCR4-NOT is a large protein complex present both in cytoplasm and the nucleus of eukaryotic cells. Although it is involved in a variety of distinct processes related to expression of genetic information such as poly(A) tail shortening, transcription regulation, nuclear export and protein degradation, there is only fragmentary information available on some of its nine subunits. Here we show a comprehensive structural characterization of the native CCR4-NOT complex from Schizosaccharomyces pombe. Our cryo-EM 3D reconstruction of the complex, combined with techniques such as immunomicroscopy, RNA-nanogold labelling, docking of the available high-resolution structures and models of different subunits and domains, allow us to propose its full molecular architecture. We locate all functionally defined domains endowed with deadenylating and ubiquitinating activities, the nucleus-specific RNA-interacting subunit Mmi1, as well as surfaces responsible for protein–protein interactions. This information provides insight into cooperation of the different CCR4-NOT complex functions. PMID:26804377

  2. Centromeric barrier disruption leads to mitotic defects in Schizosaccharomyces pombe.

    PubMed

    Gaither, Terilyn L; Merrett, Stephanie L; Pun, Matthew J; Scott, Kristin C

    2014-04-01

    Centromeres are cis-acting chromosomal domains that direct kinetochore formation, enabling faithful chromosome segregation and preserving genome stability. The centromeres of most eukaryotic organisms are structurally complex, composed of nonoverlapping, structurally and functionally distinct chromatin subdomains, including the specialized core chromatin that underlies the kinetochore and pericentromeric heterochromatin. The genomic and epigenetic features that specify and preserve the adjacent chromatin subdomains critical to centromere identity are currently unknown. Here we demonstrate that chromatin barriers regulate this process in Schizosaccharomyces pombe. Reduced fitness and mitotic chromosome segregation defects occur in strains that carry exogenous DNA inserted at centromere 1 chromatin barriers. Abnormal phenotypes are accompanied by changes in the structural integrity of both the centromeric core chromatin domain, containing the conserved CENP-A(Cnp1) protein, and the flanking pericentric heterochromatin domain. Barrier mutant cells can revert to wild-type growth and centromere structure at a high frequency after the spontaneous excision of integrated exogenous DNA. Our results reveal a previously undemonstrated role for chromatin barriers in chromosome segregation and in the prevention of genome instability. PMID:24531725

  3. The Schizosaccharomyces pombe rad3 checkpoint gene.

    PubMed Central

    Bentley, N J; Holtzman, D A; Flaggs, G; Keegan, K S; DeMaggio, A; Ford, J C; Hoekstra, M; Carr, A M

    1996-01-01

    The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution. Images PMID:8978690

  4. The connection of cytoskeletal network with plasma membrane and the cell wall

    PubMed Central

    Liu, Zengyu; Persson, Staffan; Zhang, Yi

    2015-01-01

    The cell wall provides external support of the plant cells, while the cytoskeletons including the microtubules and the actin filaments constitute an internal framework. The cytoskeletons contribute to the cell wall biosynthesis by spatially and temporarily regulating the transportation and deposition of cell wall components. This tight control is achieved by the dynamic behavior of the cytoskeletons, but also through the tethering of these structures to the plasma membrane. This tethering may also extend beyond the plasma membrane and impact on the cell wall, possibly in the form of a feedback loop. In this review, we discuss the linking components between the cytoskeletons and the plasma membrane, and/or the cell wall. We also discuss the prospective roles of these components in cell wall biosynthesis and modifications, and aim to provide a platform for further studies in this field. PMID:25693826

  5. ENZYMATIC HYDROLYSIS OF YEAST CELL WALLS. I. ISOLATION OF WALL-DECOMPOSING ORGANISMS AND SEPARATION AND PURIFICATION OF LYTIC ENZYMES.

    PubMed

    TANAKA, H; PHAFF, H J

    1965-06-01

    Tanaka, Hirosato (University of California, Davis), and Herman J. Phaff. Enzymatic hydrolysis of yeast cell walls. I. Isolation of wall-decomposing organisms and separation and purification of lytic enzymes. J. Bacteriol. 89:1570-1580. 1965.-A number of microorganisms, able to decompose and grow on yeast cell walls, were isolated from soil. These isolates demonstrated various types of attack on yeast walls. A bacterium, identified as Bacillus circulans, and a species of Streptomyces produced clear, lysed zones when grown on an agar medium containing baker's yeast cell walls. The streptomycete formed glucanase, mannanase, and protease, but B. circulans produced only glucanases. Purified mannan could be prepared from the culture fluid of B. circulans grown on baker's yeast cell walls. In a liquid, mineral medium, extracellular lytic enzyme production by B. circulans was optimal after 3 days of aerobic growth at 30 C with 0.5% baker's yeast cell walls as the carbon source. Twelve other carbon sources were ineffective as inducers. Among a number of polysaccharides tested, the crude enzymes of B. circulans hydrolyzed only beta-1-->3 glucan (laminarin) and beta-1-->6 glucan (pustulan), both by a random mechanism, to a mixture of dimer and glucose. The beta-1-->3 and beta-1-->6 glucanases were separated from each other by diethylaminoethyl cellulose column chromatography. Water-soluble oat glucan, which contains in the linear chain both beta-1-->3 and beta-1-->4 bonds, was also hydrolyzed by the bacterial beta-1-->3 glucanase. The products of this reaction indicated that this enzyme hydrolyzes beta-1-->3 or beta-1-->4 glucosidic linkages, provided the beta-glucopyranosyl units composing these bonds are substituted in the 3 position by another glucose unit. PMID:14291597

  6. Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms.

    PubMed Central

    Neiman, A M; Stevenson, B J; Xu, H P; Sprague, G F; Herskowitz, I; Wigler, M; Marcus, S

    1993-01-01

    We present genetic evidence that three presumptive protein kinases of Schizosaccharomyces pombe, byr2, byr1, and spk1 that are structurally related to protein kinases of Saccharomyces cerevisiae, STE11, STE7, and FUS3, respectively, are also functionally related. In some cases, introduction of the heterologous protein kinase into a mutant was sufficient for complementation. In other cases (as in a ste11- mutant of S. cerevisiae), expression of two S. pombe protein kinases (byr2 and byr1) was required to observe complementation, suggesting that byr2 and byr1 act cooperatively. Complementation in S. pombe mutants is observed as restoration of sporulation and conjugation and in S. cerevisiae as restoration of conjugation, pheromone-induced cell cycle arrest, and pheromone-induced transcription of the FUS1 gene. We also show that the S. pombe kinases bear a similar relationship to the mating pheromone receptor apparatus as do their S. cerevisiae counterparts. Our results indicate that pheromone-induced signal transduction employs a conserved set of kinases in these two evolutionarily distant yeasts despite an apparently significant difference in function of the heterotrimeric G proteins. We suggest that the STE11/byr2, STE7/byr1, and FUS3/spk1 kinases comprise a signal transduction module that may be conserved in higher eukaryotes. Consistent with this hypothesis, we show that a mammalian mitogen-activated protein (MAP) kinase, ERK2, can partially replace spk1 function in S. pombe. Images PMID:8443406

  7. The plant cell wall in the feeding sites of cyst nematodes.

    PubMed

    Bohlmann, Holger; Sobczak, Miroslaw

    2014-01-01

    Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium. PMID:24678316

  8. The plant cell wall in the feeding sites of cyst nematodes

    PubMed Central

    Bohlmann, Holger; Sobczak, Miroslaw

    2014-01-01

    Plant parasitic cyst nematodes (genera Heterodera and Globodera) are serious pests for many crops. They enter the host roots as migratory second stage juveniles (J2) and migrate intracellularly toward the vascular cylinder using their stylet and a set of cell wall degrading enzymes produced in the pharyngeal glands. They select an initial syncytial cell (ISC) within the vascular cylinder or inner cortex layers to induce the formation of a multicellular feeding site called a syncytium, which is the only source of nutrients for the parasite during its entire life. A syncytium can consist of more than hundred cells whose protoplasts are fused together through local cell wall dissolutions. While the nematode produces a cocktail of cell wall degrading and modifying enzymes during migration through the root, the cell wall degradations occurring during syncytium development are due to the plants own cell wall modifying and degrading proteins. The outer syncytial cell wall thickens to withstand the increasing osmotic pressure inside the syncytium. Furthermore, pronounced cell wall ingrowths can be formed on the outer syncytial wall at the interface with xylem vessels. They increase the surface of the symplast-apoplast interface, thus enhancing nutrient uptake into the syncytium. Processes of cell wall degradation, synthesis and modification in the syncytium are facilitated by a variety of plant proteins and enzymes including expansins, glucanases, pectate lyases and cellulose synthases, which are produced inside the syncytium or in cells surrounding the syncytium. PMID:24678316

  9. Nanoindentation techniques for the cell walls of wood

    NASA Astrophysics Data System (ADS)

    Jakes, Joseph Eugene

    There is a recognized need in forest products research to better understand how the mechanical properties of wood derive from the basic polymer components that make up the wood. For development of new engineered wood products there is the need to understand how chemical additives and adhesives interact with wood polymers and influence properties at the cellular level. To meet these needs I have developed nanoindentation techniques for probing the mechanical properties of the cell walls in wood. There are two, key results of this research. The first is a newly invented structural compliance method for isolating the properties of local regions within materials and excluding artifacts brought about by neighboring edges including free edges and interfaces between dissimilar cell wall layers. The second consists of methods to obtain viscoplastic and viscoelastic data over as wide a range of deformation rate as possible. The broadband nanoindentation creep (BNC) technique assesses the viscoplastic properties over 5 orders of magnitude in deformation rate (-10-4 to 10 s-1). Viscoelastic measurements can be made with unloading times ranging from 0.01 to 100 s, resulting in viscoelastic data that span four orders of magnitude in frequency or inverse time (˜10-3 to 10 s-1). To demonstrate the efficacy of these techniques, experiments are performed on a range of materials including fused silica, silicon, molybdenum, siliconon-insulator layered specimen, poly (methylmetacrylate), polycarbonate, polystyrene, wood cells in loblolly pine (Pinus taeda ), and a polypropylene-wood composite. Finally, the structural compliance method and BNC are combined to explore polymeric methylene diphenyl diisocyanate (pMDI)-wood interactions. The data suggest that pMDI polymerizes in situ to create an interpenetrating polymer network.

  10. Cell wall invertase in tobacco crown gall cells : enzyme properties and regulation by auxin.

    PubMed

    Weil, M; Rausch, T

    1990-12-01

    The cell wall invertase from an Agrobacterium tumefaciens-transformed Nicotiana tabacum cell line (SR1-C58) was purified. The heterogeneously glycosylated enzyme has the following properties: M(r) 63,000, pH optimum at 4.7, K(m sucrose) 0.6 millimolar (at pH 4.7), pl 9.5. Enzyme activity is inhibited by micromolar concentrations of HgCl(2) but is insensitive to H(2)O(2), N-ethylmaleimide and dithiothreitol. Upon transfer of transformed cells from the stationary phase to fresh medium, a cycloheximide- and tunicamycin-sensitive de novo formation of cell wall invertase is demonstrated in the absence or presence of sucrose. While in an auxin mutant (lacking gene 1;SR1-3845) 1 micromolar 1-naphthaleneacetic acid led to a further increased activity, the wild-type transformed cell line (SR1-C58) responded with a decreased activity compared to the control. An analysis of cell wall invertase in and around tumors initiated with Agrobacterium tumefaciens (strain C58) on Nicotiana tabacum stem and Kalanchoë daigremontiana leaves revealed gradients of activity. The results indicate that the auxin-stimulated cell wall invertase is essential for the establishment of the tumor sink. PMID:16667892

  11. Role of calcium in the mechanical strength of soybean hypocotyl cell walls

    SciTech Connect

    Virk, S.S.; Cleland, R.E.

    1986-04-01

    Calcium ions inhibit auxin-induced growth in both dicot stems and coleoptiles. In coleoptiles calcium does not directly stiffen cell walls. The authors have tested here whether calcium might alter the mechanical strength of a dicot cell wall, the soybean hypocotyl. Sections were longitudinally bisected, boiled or frozen-thawed, incubated in solutions and then the mechanical strength was determined with an Instron. The calcium content was also measured. Removal of calcium by EGTA or by acidic buffers such as K-Pi-citrate resulted in a proportional increase in wall extensibility. Addition of calcium, on the other hand, stiffened the walls. These changes were reversible. It was concluded that calcium crosslinks make a significant contribution to the strength of dicot stem cell walls, and that in vivo, removal of calcium from the wall by uptake into the cell could result in wall loosening and thus enhanced growth.

  12. Relating the mechanics of the primary plant cell wall to morphogenesis.

    PubMed

    Bidhendi, Amir J; Geitmann, Anja

    2016-01-01

    Regulation of the mechanical properties of the cell wall is a key parameter used by plants to control the growth behavior of individual cells and tissues. Modulation of the mechanical properties occurs through the control of the biochemical composition and the degree and nature of interlinking between cell wall polysaccharides. Preferentially oriented cellulose microfibrils restrict cellular expansive growth, but recent evidence suggests that this may not be the trigger for anisotropic growth. Instead, non-uniform softening through the modulation of pectin chemistry may be an initial step that precedes stress-induced stiffening of the wall through cellulose. Here we briefly review the major cell wall polysaccharides and their implication for plant cell wall mechanics that need to be considered in order to study the growth behavior of the primary plant cell wall. PMID:26689854

  13. Approaches to understanding the functional architecture of the plant cell wall.

    PubMed

    McCann, M C; Bush, M; Milioni, D; Sado, P; Stacey, N J; Catchpole, G; Defernez, M; Carpita, N C; Hofte, H; Ulvskov, P; Wilson, R H; Roberts, K

    2001-07-01

    Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins. PMID:11423133

  14. Plant biomass recalcitrance: effect of hemicellulose composition on nanoscale forces that control cell wall strength.

    PubMed

    Silveira, Rodrigo L; Stoyanov, Stanislav R; Gusarov, Sergey; Skaf, Munir S; Kovalenko, Andriy

    2013-12-26

    Efficient conversion of lignocellulosic biomass to second-generation biofuels and valuable chemicals requires decomposition of resilient plant cell wall structure. Cell wall recalcitrance varies among plant species and even phenotypes, depending on the chemical composition of the noncellulosic matrix. Changing the amount and composition of branches attached to the hemicellulose backbone can significantly alter the cell wall strength and microstructure. We address the effect of hemicellulose composition on primary cell wall assembly forces by using the 3D-RISM-KH molecular theory of solvation, which provides statistical-mechanical sampling and molecular picture of hemicellulose arrangement around cellulose. We show that hemicellulose branches of arabinose, glucuronic acid, and especially glucuronate strengthen the primary cell wall by strongly coordinating to hydrogen bond donor sites on the cellulose surface. We reveal molecular forces maintaining the cell wall structure and provide directions for genetic modulation of plants and pretreatment design to render biomass more amenable to processing. PMID:24274712

  15. Area Expansivity Moduli of Regenerating Plant Protoplast Cell Walls Exposed to Shear Flows

    NASA Astrophysics Data System (ADS)

    Fujimura, Yuu; Iino, Masaaki; Watanabe, Ugai

    2005-05-01

    To control the elasticity of the plant cell wall, protoplasts isolated from cultured Catharanthus roseus cells were regenerated in shear flows of 115 s-1 (high shear) and 19.2 s-1 (low shear, as a control). The surface area expansivity modulus and the surface breaking strength of these regenerating protoplasts were measured by a micropipette aspiration technique. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye. High shear exposure for 3 h doubled both the surface area modulus and breaking strength observed under low shear, significantly decreased cell wall synthesis, and roughly quadrupled the moduli of the cell wall. Based on the cell wall synthesis data, we estimated the three-dimensional modulus of the cell wall to be 4.1± 1.2 GPa for the high shear, and 0.35± 0.2 GPa for the low shear condition, using the surface area expansivity modulus divided by the cell wall thickness, which is identical with the Young’s modulus divided by 2(1-σ), where σ is Poisson's ratio. We concluded that high shear exposure considerably strengthens the newly synthesized cell wall.

  16. Starting to gel: how Arabidopsis seed coat epidermal cells produce specialized secondary cell walls.

    PubMed

    Voiniciuc, Cătălin; Yang, Bo; Schmidt, Maximilian Heinrich-Wilhelm; Günl, Markus; Usadel, Björn

    2015-01-01

    For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls. PMID:25658798

  17. Starting to Gel: How Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls

    PubMed Central

    Voiniciuc, Cătălin; Yang, Bo; Schmidt, Maximilian Heinrich-Wilhelm; Günl, Markus; Usadel, Björn

    2015-01-01

    For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls. PMID:25658798

  18. Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

    PubMed Central

    Chardwiriyapreecha, Soracom; Manabe, Kunio; Iwaki, Tomoko; Kawano-Kawada, Miyuki; Sekito, Takayuki; Lunprom, Siriporn; Akiyama, Koichi; Takegawa, Kaoru; Kakinuma, Yoshimi

    2015-01-01

    In Saccharomyces cerevisiae, Avt3p and Avt4p mediate the extrusion of several amino acids from the vacuolar lumen into the cytosol. SpAvt3p of Schizosaccharomyces pombe, a homologue of these vacuolar amino acid transporters, has been indicated to be involved in spore formation. In this study, we confirmed that GFP-SpAvt3p localized to the vacuolar membrane in S. pombe. The amounts of various amino acids increased significantly in the vacuolar pool of avt3Δ cells, but decreased in that of avt3+-overexpressing avt3Δ cells. These results suggest that SpAvt3p participates in the vacuolar compartmentalization of amino acids in S. pombe. To examine the export activity of SpAvt3p, we expressed the avt3+ gene in S. cerevisiae cells. We found that the heterologously overproduced GFP-SpAvt3p localized to the vacuolar membrane in S. cerevisiae. Using the vacuolar membrane vesicles isolated from avt3+-overexpressing S. cerevisiae cells, we detected the export activities of alanine and tyrosine in an ATP-dependent manner. These activities were inhibited by the addition of a V-ATPase inhibitor, concanamycin A, thereby suggesting that the activity of SpAvt3p is dependent on a proton electrochemical gradient generated by the action of V-ATPase. In addition, the amounts of various amino acids in the vacuolar pools of S. cerevisiae cells were decreased by the overproduction of SpAvt3p, which indicated that SpAvt3p was functional in S. cerevisiae cells. Thus, SpAvt3p is a vacuolar transporter that is involved in the export of amino acids from S. pombe vacuoles. PMID:26083598

  19. Two cationic peroxidases from cell walls of Araucaria araucana seeds.

    PubMed

    Riquelme, A; Cardemil, L

    1995-05-01

    We have previously reported the purification and partial characterization of two cationic peroxidases from the cell walls of seeds and seedlings of the South American conifer, Araucaria araucana. In this work, we have studied the amino acid composition and NH2-terminal sequences of both enzymes. We also compare the data obtained from these analyses with those reported for other plant peroxidases. The two peroxidases are similar in their amino acid compositions. Both are particularly rich in glycine, which comprises more than 30% of the amino acid residues. The content of serine is also high, ca 17%. The two enzymes are different in their content of arginine, alanine, valine, phenylalanine and threonine. Both peroxidases have identical NH2-terminal sequences, indicating that the two proteins are genetically related and probably are isoforms of the same kind of peroxidase. The amino acid composition and NH2-terminal sequence analyses showed marked differences from the cationic peroxidases from turnip and horseradish. PMID:7786490

  20. Single Wall Carbon Nanotube-polymer Solar Cells

    NASA Technical Reports Server (NTRS)

    Bailey, Sheila G.; Castro, Stephanie L.; Landi, Brian J.; Gennett, Thomas; Raffaelle, Ryne P.

    2005-01-01

    Investigation of single wall carbon nanotube (SWNT)-polymer solar cells has been conducted towards developing alternative lightweight, flexible devices for space power applications. Photovoltaic devices were constructed with regioregular poly(3-octylthiophene)-(P3OT) and purified, >95% w/w, laser-generated SWNTs. The P3OT composites were deposited on ITO-coated polyethylene terapthalate (PET) and I-V characterization was performed under simulated AM0 illumination. Fabricated devices for the 1.0% w/w SWNT-P3OT composites showed a photoresponse with an open-circuit voltage (V(sub oc)) of 0.98 V and a short-circuit current density (I(sub sc)) of 0.12 mA/sq cm. Optimization of carrier transport within these novel photovoltaic systems is proposed, specifically development of nanostructure-SWNT complexes to enhance exciton dissociation.

  1. Pectinous cell wall thickenings formation - A common defense strategy of plants to cope with Pb.

    PubMed

    Krzesłowska, Magdalena; Rabęda, Irena; Basińska, Aneta; Lewandowski, Michał; Mellerowicz, Ewa J; Napieralska, Anna; Samardakiewicz, Sławomir; Woźny, Adam

    2016-07-01

    Lead, one of the most abundant and hazardous trace metals affecting living organisms, has been commonly detected in plant cell walls including some tolerant plants, mining ecotypes and hyperaccumulators. We have previously shown that in tip growing Funaria sp. protonemata cell wall is remodeled in response to lead by formation of thickenings rich in low-methylesterified pectins (pectin epitope JIM5 - JIM5-P) able to bind metal ions, which accumulate large amounts of Pb. Hence, it leads to the increase of cell wall capacity for Pb compartmentalization. Here we show that diverse plant species belonging to different phyla (Arabidopsis, hybrid aspen, star duckweed), form similar cell wall thickenings in response to Pb. These thickenings are formed in tip growing cells such as the root hairs, and in diffuse growing cells such as meristematic and root cap columella cells of root apices in hybrid aspen and Arabidopsis and in mesophyll cells in star duckweed fronds. Notably, all analyzed cell wall thickenings were abundant in JIM5-P and accumulated high amounts of Pb. In addition, the co-localization of JIM5-P and Pb commonly occurred in these cells. Hence, cell wall thickenings formed the extra compartment for Pb accumulation. In this way plant cells increased cell wall capacity for compartmentalization of this toxic metal, protecting protoplast from its toxicity. As cell wall thickenings occurred in diverse plant species and cell types differing in the type of growth we may conclude that pectinous cell wall thickenings formation is a widespread defense strategy of plants to cope with Pb. Moreover, detection of natural defense strategy, increasing plant cell walls capacity for metal accumulation, reveals a promising direction for enhancing plant efficiency in phytoremediation. PMID:27107260

  2. In-vitro fermentability of cell walls as influenced by lignin composition and cross-linking.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We assessed how diverse modifications in lignin composition and reductions in ferulate-lignin cross-linking influence the degradability of cell walls. Cell walls from nonlignified maize cell suspensions were artificially lignified with varying ratios of normal monolignols (coniferyl and sinapyl alco...

  3. Composition and architecture of the cell walls of grasses and the mechanisms of synthesis of cell wall polysaccharides. Final report for period September 1, 1988 - April 30, 2001

    SciTech Connect

    Carpita, Nicholas C.

    2001-10-18

    This program was devoted toward complete understanding of the polysaccharide structure and architecture of the primary cell walls grasses and cereals, and the biosynthesis of the mixed-linkage beta-glucane, a cellulose interacting polymer that is synthesized uniquely by grass species and close relatives. With these studies as focal point, the support from DOE was instrumental in the development of new analytical means that enabled us to characterize carbohydrate structure, to reveal new features of cell wall dynamics during cell growth, and to apply these techniques in other model organisms. The support by DOE in these basic studies was acknowledged on numerous occasions in review articles covering current knowledge of cell wall structure, architecture, dynamics, biosynthesis, and in all genes related to cell wall biogenesis.

  4. Lead sulfide nanoparticles increase cell wall chitin content and induce apoptosis in Saccharomyces cerevisiae.

    PubMed

    Sun, Meiqing; Yu, Qilin; Hu, Mengyuan; Hao, Zhenwei; Zhang, Chengdong; Li, Mingchun

    2014-05-30

    Although there have been numerous studies on bacterial toxicity, the cytotoxicity of nanoparticles toward fungi remains poorly understood. We investigated the toxicity of various sizes of lead sulfide particles against the important model fungus, Saccharomyces cerevisiae. The smallest particle exerted the highest toxicity, inhibiting cell growth and decreasing cell viability, likely reflecting reduced sedimentation and persistent cell wall attack. In response to cell wall stress, S. cerevisiae showed an increase in the cell wall chitin content and the overexpression of FKS2 and PRM5, two genes of the cell wall integrity signaling pathway. Cell wall stress increased the concentration of intracellular reactive oxygen species, leading to mitochondrial dysfunction and cell apoptosis. The contribution of dissolved lead ions to the overall toxicity was negligible. These findings provide the first demonstration of the physiological protective response of a fungus toward nanoparticles, thereby contributing useful information to the assessment of the environmental impact of metal nanoparticles. PMID:24704549

  5. Binding of nascent glucuronoxylan to the cell walls of pea seedlings.

    PubMed

    Brett, C T; Healy, S A; McDonald, M S; Macgregor, C; Baydoun, E A

    1997-08-01

    Glucuronoxylan synthesised in vitro by membrane-bound enzymes from etiolated pea epicotyls was found to bind to isolated cell walls from the same tissue in a pH-dependant manner. The binding was maximum at pH 3.5-4.0, and decreased to zero at pH 6. The bound glucuronoxylan could be dissociated from the cell walls by washing at pH 6, and the binding appeared to be non-covalent. Extraction experiments indicated that the glucuronoxylan was binding to hemicellulose in the cell-wall. The observed binding may be significant in the process of cell-wall assembly in vivo. PMID:9283032

  6. Detecting Cellulase Penetration Into Corn Stover Cell Walls by Immuno-Electron Microscopy

    SciTech Connect

    Donohoe, B. S.; Selig, M. J.; Viamajala, S.; Vinzant, T. B.; Adney, W. S.; Himmel, M. E.

    2009-06-15

    In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild-, moderate- and high-severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme- and polymer-specific antibodies. Low severity dilute-acid pretreatment (20 min at 100 C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120 C) allowed the enzymes to penetrate {approx}20% of the cell wall, and the high severity (20 min pretreatment at 150 C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute-acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high-resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes.

  7. Plant cell walls throughout evolution: towards a molecular understanding of their design principles

    SciTech Connect

    Sarkar, Purbasha; Bosneaga, Elena; Auer, Manfred

    2009-02-16

    Throughout their life, plants typically remain in one location utilizing sunlight for the synthesis of carbohydrates, which serve as their sole source of energy as well as building blocks of a protective extracellular matrix, called the cell wall. During the course of evolution, plants have repeatedly adapted to their respective niche,which is reflected in the changes of their body plan and the specific design of cell walls. Cell walls not only changed throughout evolution but also are constantly remodelled and reconstructed during the development of an individual plant, and in response to environmental stress or pathogen attacks. Carbohydrate-rich cell walls display complex designs, which together with the presence of phenolic polymers constitutes a barrier for microbes, fungi, and animals. Throughout evolution microbes have co-evolved strategies for efficient breakdown of cell walls. Our current understanding of cell walls and their evolutionary changes are limited as our knowledge is mainly derived from biochemical and genetic studies, complemented by a few targeted yet very informative imaging studies. Comprehensive plant cell wall models will aid in the re-design of plant cell walls for the purpose of commercially viable lignocellulosic biofuel production as well as for the timber, textile, and paper industries. Such knowledge will also be of great interest in the context of agriculture and to plant biologists in general. It is expected that detailed plant cell wall models will require integrated correlative multimodal, multiscale imaging and modelling approaches, which are currently underway.

  8. The cell-wall phosphatase of cotton (Gossypium) is inhibited by kelthane.

    PubMed Central

    Daley, L S; Carroll, P; Mussell, H

    1979-01-01

    Kelthane [4,4'-dichloro-alpha-(trichloromethyl)benzhydrol] was previously shown to decrease the limited tolerance of susceptible varieties of cotton (Gossypium) to Verticillium wilt. Kelthane was shown in the present study to inhibit the cell-wall p-nitrophenyl phosphatase of cotton. In view of information already establishing the cell wall as a primary site of action of Verticillium wilt, the data are interpreted as suggesting an as yet undefined interaction between Kelthane, cell-wall phosphatase and verticillium-resistance mechanisms of the cell wall. PMID:224864

  9. Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals.

    PubMed

    TAKEYA, K; HISATSUNE, K

    1963-01-01

    Takeya, Kenji (Kyushu University, Fukuoka, Japan) and Kazuhito Hisatsune. Mycobacterial cell walls. I. Methods of preparation and treatment with various chemicals. J. Bacteriol. 85:16-23. 1963.-Several methods of preparation of mycobacterial cell walls were examined, and the grinding method with glass powder, using Dry Ice, was found to give fairly good cell-wall preparations. "Paired fibrous structures" were clearly seen on the purified cell wall. The appearance of the cell wall as revealed by the electron microscope was not altered by digestion with trypsin, pronase, or pronase in 5% alcoholic solution, nor by treatment with 95% alcohol, acetone-alcohol mixture, or ether-alcohol mixture. By treatment with alcoholic KOH solution, the fibrous structure was removed. The remaining thin layer of the cell wall was tentatively designated the "basal layer" of the mycobacterial cell wall. The fibers appeared also to be removed by chloroform treatment. Nagarse digestion seemed to solubilize some constituents of the cell wall. The cell wall lost its shape and rigidity after lysozyme digestion. PMID:13984703

  10. Production of Bacteriolytic Enzymes by Streptomyces globisporus Regulated by Exogenous Bacterial Cell Walls.

    PubMed

    Brönneke, V; Fiedler, F

    1994-03-01

    Mutanolysin biosynthesis and pigment production in Streptomyces globisporus ATCC 21553 were stimulated by adding bacterial cell walls to the medium. The increased bacteriolytic activity in the supernatant correlated with an increased de novo synthesis of mutanolysin and was between 4- and 20-fold higher than in cultures grown without bacterial cell walls. The increase in mutanolysin synthesis was brought about by enhanced transcription of the mutanolysin gene. The stimulation was only observed in medium which contained dextrin or starch as the carbon source. Glucose abolished the stimulation and also inhibited the low constitutive synthesis of mutanolysin. The induction of lytic activity was observed to require minimally 0.4 mg of bacterial cell walls per ml, whereas 0.6 mg of bacterial cell walls per ml yielded maximal lytic activity. Further supplements of bacterial cell walls did not result in enhanced lytic activity. The stimulation could be achieved independently of the phase of growth of the Streptomyces strain. Cultures grown in the presence of bacterial cell walls exhibited a higher growth yield. However, the accelerated growth was not the reason for the increased amount of mutanolysin produced. The growth of cultures with peptidoglycan monomers added to the medium instead of cell walls was similarly increased, but an effect on the biosynthesis of mutanolysin was not observed. All bacterial cell walls tested were capable of eliciting the stimulation of lytic activity, including cell walls of archaea, which contained pseudomurein. PMID:16349213

  11. Sequence conservation of the rad21 Schizosaccharomyces pombe DNA double-strand break repair gene in human and mouse

    SciTech Connect

    McKay, M.J.; Troelstra, C.; Kanaar, R.

    1996-09-01

    The rad21 gene of Schizosaccharomyces pombe is involved in the repair of ionizing radiation-induced DNA double-strand breaks. The isolation of mouse and human putative homologs of rad21 is reported here. Alignment of the predicted amino acid sequence of Rad21 with the mammalian proteins showed that the similarity was distributed across the length of the proteins, with more highly conserved regions at both termini. The mHR21{sup sp} (mouse homolog of Rad21, S. pombe) and hHR21{sup sp} (human homolog of Rad21, S. pombe) predicted proteins were 96% identical, whereas the human and S. pombe proteins were 25% identical and 47% similar. RNA blot analysis showed that mHR21{sup sp} mRNA was abundant in all adult mouse tissues examined, with highest expression in testis and thymus. In addition to a 3.1-kb constitutive mRNA transcript, a 2.2-kb transcript was present at a high level in postmeiotic spermatids, while expression of the 3.1-kb mRNA in testis was confined to the meiotic compartment. hHR21{sup sp} mRNA was cell-cycle regulated in human cells, increasing in late S phase to a peak in G2 phase. The level of hHR21{sup sp} transcripts was not altered by exposure of normal diploid fibroblasts to 10 Gy ionizing radiation. In situ hybridization showed that mHR21{sup sp} resided on chromosome 15D3, whereas hHR21{sup sp} localized to the syntenic 8q24 region. Elevated expression of mHR21{sup sp} in testis and thymus supports a possible role for the rad21 mammalian homologs in V(D)J and meiotic recombination, respectively. Cell cycle regulation of rad21, retained from S. pombe to human, is consistent with a conservation of function between S. pombe and human rad21 genes. 62 refs., 8 figs., 1 tab.

  12. A phosphorylated pseudokinase complex controls cell wall synthesis in mycobacteria.

    PubMed

    Gee, Christine L; Papavinasasundaram, Kadamba G; Blair, Sloane R; Baer, Christina E; Falick, Arnold M; King, David S; Griffin, Jennifer E; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K; Crick, Dean C; Rubin, Eric J; Sassetti, Christopher M; Alber, Tom

    2012-01-24

    Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase-FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks. PMID:22275220

  13. A Phosphorylated Pseudokinase Complex Controls Cell Wall Synthesis in Mycobacteria

    PubMed Central

    Gee, Christine L.; Papavinasasundaram, Kadamba G.; Blair, Sloane R.; Baer, Christina E.; Falick, Arnold M.; King, David S.; Griffin, Jennifer E.; Venghatakrishnan, Harene; Zukauskas, Andrew; Wei, Jun-Rong; Dhiman, Rakesh K.; Crick, Dean C.; Rubin, Eric J.; Sassetti, Christopher M.; Alber, Tom

    2013-01-01

    Prokaryotic cell wall biosynthesis is coordinated with cell growth and division, but the mechanisms regulating this dynamic process remain obscure. Here, we describe a phosphorylation-dependent regulatory complex that controls peptidoglycan (PG) biosynthesis in Mycobacterium tuberculosis. We found that PknB, a PG-responsive Ser-Thr protein kinase (STPK), initiates complex assembly by phosphorylating a kinase-like domain in the essential PG biosynthetic protein, MviN. This domain was structurally diverged from active kinases and did not mediate phosphotransfer. Threonine phosphorylation of the pseudokinase domain recruited the FhaA protein through its forkhead-associated (FHA) domain. The crystal structure of this phosphorylated pseudokinase–FHA domain complex revealed the basis of FHA domain recognition, which included unexpected contacts distal to the phosphorylated threonine. Conditional degradation of these proteins in mycobacteria demonstrated that MviN was essential for growth and PG biosynthesis and that FhaA regulated these processes at the cell poles and septum. Controlling this spatially localized PG regulatory complex is only one of several cellular roles ascribed to PknB, suggesting that the capacity to coordinate signaling across multiple processes is an important feature conserved between eukaryotic and prokaryotic STPK networks. PMID:22275220

  14. Wall extensibility: its nature, measurement and relationship to plant cell growth

    NASA Technical Reports Server (NTRS)

    Cosgrove, D. J.

    1993-01-01

    Expansive growth of plant cells is controlled principally by processes that loosen the wall and enable it to expand irreversibly. The central role of wall relaxation for cell expansion is reviewed. The most common methods for assessing the extension properties of plant cell walls ( wall extensibility') are described, categorized and assessed critically. What emerges are three fundamentally different approaches which test growing cells for their ability (a) to enlarge at different values of turgor, (b) to induce wall relaxation, and (c) to deform elastically or plastically in response to an applied tensile force. Analogous methods with isolated walls are similarly reviewed. The results of these different assays are related to the nature of plant cell growth and pertinent biophysical theory. I argue that the extensibilities' measured by these assays are fundamentally different from one another and that some are more pertinent to growth than others.

  15. DBIO Best Thesis Award: Mechanics, Dynamics, and Organization of the Bacterial Cytoskeleton and Cell Wall

    NASA Astrophysics Data System (ADS)

    Wang, Siyuan

    2012-02-01

    Bacteria come in a variety of shapes. While the peptidoglycan (PG) cell wall serves as an exoskeleton that defines the static cell shape, the internal bacterial cytoskeleton mediates cell shape by recruiting PG synthesis machinery and thus defining the pattern of cell-wall synthesis. While much is known about the chemistry and biology of the cytoskeleton and cell wall, much of their biophysics, including essential aspects of the functionality, dynamics, and organization, remain unknown. This dissertation aims to elucidate the detailed biophysical mechanisms of cytoskeleton guided wall synthesis. First, I find that the bacterial cytoskeleton MreB contributes nearly as much to the rigidity of an Escherichia coli cell as the cell wall. This conclusion implies that the cytoskeletal polymer MreB applies meaningful force to the cell wall, an idea favored by theoretical modeling of wall growth, and suggests an evolutionary origin of cytoskeleton-governed cell rigidity. Second, I observe that MreB rotates around the long axis of E. coli, and the motion depends on wall synthesis. This is the first discovery of a cell-wall assembly driven molecular motor in bacteria. Third, I prove that both cell-wall synthesis and the PG network have chiral ordering, which is established by the spatial pattern of MreB. This work links the molecular structure of the cytoskeleton and of the cell wall with organismal-scale behavior. Finally, I develop a mathematical model of cytoskeleton-cell membrane interactions, which explains the preferential orientation of different cytoskeleton components in bacteria.

  16. De Novo Biosynthesis of Vanillin in Fission Yeast (Schizosaccharomyces pombe) and Baker's Yeast (Saccharomyces cerevisiae) ▿

    PubMed Central

    Hansen, Esben H.; Møller, Birger Lindberg; Kock, Gertrud R.; Bünner, Camilla M.; Kristensen, Charlotte; Jensen, Ole R.; Okkels, Finn T.; Olsen, Carl E.; Motawia, Mohammed S.; Hansen, Jørgen

    2009-01-01

    Vanillin is one of the world's most important flavor compounds, with a global market of 180 million dollars. Natural vanillin is derived from the cured seed pods of the vanilla orchid (Vanilla planifolia), but most of the world's vanillin is synthesized from petrochemicals or wood pulp lignins. We have established a true de novo biosynthetic pathway for vanillin production from glucose in Schizosaccharomyces pombe, also known as fission yeast or African beer yeast, as well as in baker's yeast, Saccharomyces cerevisiae. Productivities were 65 and 45 mg/liter, after introduction of three and four heterologous genes, respectively. The engineered pathways involve incorporation of 3-dehydroshikimate dehydratase from the dung mold Podospora pauciseta, an aromatic carboxylic acid reductase (ACAR) from a bacterium of the Nocardia genus, and an O-methyltransferase from Homo sapiens. In S. cerevisiae, the ACAR enzyme required activation by phosphopantetheinylation, and this was achieved by coexpression of a Corynebacterium glutamicum phosphopantetheinyl transferase. Prevention of reduction of vanillin to vanillyl alcohol was achieved by knockout of the host alcohol dehydrogenase ADH6. In S. pombe, the biosynthesis was further improved by introduction of an Arabidopsis thaliana family 1 UDP-glycosyltransferase, converting vanillin into vanillin β-d-glucoside, which is not toxic to the yeast cells and thus may be accumulated in larger amounts. These de novo pathways represent the first examples of one-cell microbial generation of these valuable compounds from glucose. S. pombe yeast has not previously been metabolically engineered to produce any valuable, industrially scalable, white biotech commodity. PMID:19286778

  17. Suppression of ricinoleic acid toxicity by ptl2 overexpression in fission yeast Schizosaccharomyces pombe.

    PubMed

    Yazawa, Hisashi; Ogiso, Masayo; Kumagai, Hiromichi; Uemura, Hiroshi

    2014-11-01

    We previously succeeded to obtain a high content of ricinoleic acid (RA), a hydroxylated fatty acid with great values as a petrochemical replacement, in fission yeast Schizosaccharomyces pombe by introducing Claviceps purpurea oleate Δ12-hydroxylase gene (CpFAH12). Although the production was toxic to S. pombe cells, we identified plg7, encoding phospholipase A2, as a multicopy suppressor that restored the growth defect by removing RA from phospholipids and induced secretion of a part of the released free RA into culture media. In this study, we extended our analysis and examined the effect of triglyceride (TG) lipase overexpression on the tolerance to RA toxicity and RA productivity. S. pombe has three TG lipase genes, ptl1, ptl2, and ptl3, which have high protein sequence similarities to each other and to Saccharomyces cerevisiae counterparts TGL3, TGL4, and TGL5, but only ptl2 overexpression suppressed the growth defect induced by RA production, and the culture grown at 20 °C secreted free RA into media like plg7 overexpression. Suppression by ptl2 was independent of plg7, and a large amount of free RA was accumulated in the cells concomitant with the decrease in RA moieties in phospholipids. Furthermore, the suppression by ptl2 was attenuated by bromoenol lactone (BEL), a phospholipase A2 specific inhibitor, suggesting that Ptl2p may have phospholipase activity. Simultaneous overexpression of ptl2 and plg7 in the FAH12 integrant increased secretion and intracellular accumulation of RA 1.2- and 1.3-fold, respectively, compared to those with single overexpression of plg7 on day 10 at 20 °C. PMID:25109267

  18. Protein diffusion in plant cell plasma membranes: the cell-wall corral

    PubMed Central

    Martinière, Alexandre; Runions, John

    2013-01-01

    Studying protein diffusion informs us about how proteins interact with their environment. Work on protein diffusion over the last several decades has illustrated the complex nature of biological lipid bilayers. The plasma membrane contains an array of membrane-spanning proteins or proteins with peripheral membrane associations. Maintenance of plasma membrane microstructure can be via physical features that provide intrinsic ordering such as lipid microdomains, or from membrane-associated structures such as the cytoskeleton. Recent evidence indicates, that in the case of plant cells, the cell wall seems to be a major player in maintaining plasma membrane microstructure. This interconnection / interaction between cell-wall and plasma membrane proteins most likely plays an important role in signal transduction, cell growth, and cell physiological responses to the environment. PMID:24381579

  19. Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae.

    PubMed Central

    López-Ribot, J L; Chaffin, W L

    1996-01-01

    Western blot (immunoblot) analysis of cell wall and cytosolic extracts obtained from parental and ssa1 and ssa2 single- and double-mutant strains of Saccharomyces cerevisiae showed that the heat shock protein 70 (Hsp70) products of these genes, previously thought to be restricted to the cell interior, are also present in the cell wall. A cell wall location was further confirmed by indirect immunofluorescence with intact cells and biotinylation of extracellular Hsp70. Hsp70s have been implicated in translocation across the membrane and as molecular chaperones, and changes in the profile of cell wall proteins suggested that these proteins may have a similar role in the cell wall. PMID:8755907

  20. Heteromannan and Heteroxylan Cell Wall Polysaccharides Display Different Dynamics During the Elongation and Secondary Cell Wall Deposition Phases of Cotton Fiber Cell Development

    PubMed Central

    Hernandez-Gomez, Mercedes C.; Runavot, Jean-Luc; Guo, Xiaoyuan; Bourot, Stéphane; Benians, Thomas A.S.; Willats, William G.T.; Meulewaeter, Frank; Knox, J. Paul

    2015-01-01

    The roles of non-cellulosic polysaccharides in cotton fiber development are poorly understood. Combining glycan microarrays and in situ analyses with monoclonal antibodies, polysaccharide linkage analyses and transcript profiling, the occurrence of heteromannan and heteroxylan polysaccharides and related genes in developing and mature cotton (Gossypium spp.) fibers has been determined. Comparative analyses on cotton fibers at selected days post-anthesis indicate different temporal and spatial regulation of heteromannan and heteroxylan during fiber development. The LM21 heteromannan epitope was more abundant during the fiber elongation phase and localized mainly in the primary cell wall. In contrast, the AX1 heteroxylan epitope occurred at the transition phase and during secondary cell wall deposition, and localized in both the primary and the secondary cell walls of the cotton fiber. These developmental dynamics were supported by transcript profiling of biosynthetic genes. Whereas our data suggest a role for heteromannan in fiber elongation, heteroxylan is likely to be involved in the regulation of cellulose deposition of secondary cell walls. In addition, the relative abundance of these epitopes during fiber development varied between cotton lines with contrasting fiber characteristics from four species (G. hirsutum, G. barbadense, G. arboreum and G. herbaceum), suggesting that these non-cellulosic polysaccharides may be involved in determining final fiber quality and suitability for industrial processing. PMID:26187898

  1. Heteromannan and Heteroxylan Cell Wall Polysaccharides Display Different Dynamics During the Elongation and Secondary Cell Wall Deposition Phases of Cotton Fiber Cell Development.

    PubMed

    Hernandez-Gomez, Mercedes C; Runavot, Jean-Luc; Guo, Xiaoyuan; Bourot, Stéphane; Benians, Thomas A S; Willats, William G T; Meulewaeter, Frank; Knox, J Paul

    2015-09-01

    The roles of non-cellulosic polysaccharides in cotton fiber development are poorly understood. Combining glycan microarrays and in situ analyses with monoclonal antibodies, polysaccharide linkage analyses and transcript profiling, the occurrence of heteromannan and heteroxylan polysaccharides and related genes in developing and mature cotton (Gossypium spp.) fibers has been determined. Comparative analyses on cotton fibers at selected days post-anthesis indicate different temporal and spatial regulation of heteromannan and heteroxylan during fiber development. The LM21 heteromannan epitope was more abundant during the fiber elongation phase and localized mainly in the primary cell wall. In contrast, the AX1 heteroxylan epitope occurred at the transition phase and during secondary cell wall deposition, and localized in both the primary and the secondary cell walls of the cotton fiber. These developmental dynamics were supported by transcript profiling of biosynthetic genes. Whereas our data suggest a role for heteromannan in fiber elongation, heteroxylan is likely to be involved in the regulation of cellulose deposition of secondary cell walls. In addition, the relative abundance of these epitopes during fiber development varied between cotton lines with contrasting fiber characteristics from four species (G. hirsutum, G. barbadense, G. arboreum and G. herbaceum), suggesting that these non-cellulosic polysaccharides may be involved in determining final fiber quality and suitability for industrial processing. PMID:26187898

  2. The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair

    SciTech Connect

    Lehmann, A.R.; Walicka, M.; Griffiths, D.J.F.; Carr, A.M.

    1995-12-01

    This report describes the cloning and sequencing of the rad18 gene of Schizosaccharomyces pombe and its essential role in cell proliferation. It also describes the isolation and sequencing of its homolog from Saccharomyces cerevisiae, designated RHC18. Genetic radiation effects were explored and results indicate the gene product`s importance in a DNA repair pathway that is distinct from classical nucleotide excision repair. 57 refs., 20 figs., 1 tab.

  3. The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host-Cell Interaction.

    PubMed

    Gil-Bona, Ana; Reales-Calderon, Jose A; Parra-Giraldo, Claudia M; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

    2016-01-01

    Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a "veil growth," never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain. PMID:26870022

  4. The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host–Cell Interaction

    PubMed Central

    Gil-Bona, Ana; Reales-Calderon, Jose A.; Parra-Giraldo, Claudia M.; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

    2016-01-01

    Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a “veil growth,” never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain. PMID:26870022

  5. Germ tube-specific antigens of Candida albicans cell walls

    SciTech Connect

    Sundstrom, P.R.

    1986-01-01

    Studies were performed to characterize the surface differences between blastospores and germ tubes of the pathogenic, dimorphic yeast, Candida albicans, and to identify components of yeast cells responsible for these differences. Investigation of surfaces differences of the two growth forms was facilitated by the production of rabbit antiserum prepared against Formalin-treated yeast possessing germ tubes. To prepare antiserum specific for germ tubes, this serum was adsorbed with stationary phase blastospores. Whereas the unadsorbed antiserum reacted with both blastospore and germ tube forms by immunofluorescence and Enzyme-Linked Immunosorbent Assay, the adsorbed antiserum did not react with blastospores but detected germ tube-specific antigens in hyphal forms. The differences between blastospores and germ tubes of Candida albicans, were further studied by comparing enzymatic digests of cell walls of both growth forms in radiolabeled organisms. Organisms were labeled either on the surface with /sup 125/I, or metabolically with (/sup 35/S) methionine or (/sup 3/H) mannose. Three-surface-located components (as shown by antibody adsorption and elution experiments) were precipitated from Zymolase digests. All three components were mannoproteins as shown by their ability to bind Concanavalin A, and to be labeled in protein labeling procedures, and two of these (200,000 and 155,000 molecular weight) were germ tube specific, as shown by their ability to be precipitated by germ tube-specific antiserum. Monoclonal antibodies were prepared to C. albicans, using blastospores bearing germ tubes as immunogen.

  6. Structural studies of complex carbohydrates of plant cell walls: Progress report, December 1986--June 1989

    SciTech Connect

    Darvill, A.

    1989-06-01

    Characterizing the polysaccharides that constitute the primary cell walls of plants has been a major research goal of our laboratory for many years. During the past three years we have made considerable progress towards this goal. In particular, we have emphasized the structural studies of three of the major non-cellulosic polysaccharides; namely, the pectic polysaccharides rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II), and the hemicellulosic polysaccharide xyloglucan. We have identified and partially characterized the major pectic polysaccharides present in the primary cell walls of suspension-cultured monocot (maize and rice) and gymnosperm (Douglas fir) cells. We have come to realize that RG-I, RG-II and xyloglucan, together with homogalacturonan are arabinoxylan, constitute the predominant non-cellulosic polysaccharides of primary cell walls. In fact, we have accumulated considerable evidence to suggest that these are the predominant and perhaps only non-cellulosic polysaccharides of primary cell walls. While writing a comprehensive review of the pectic polysaccharides of primary cell walls we came to realize that all of the pectic polysaccharide fragments described in the literature may have originated from homogalacturonan, RG-I, or RG-II. Indeed, we have been quite surprised by the remarkable structural conservation of these very complicated polysaccharides in cell walls originating from such evolutionarily diverse plants. Furthermore, our structural studies of cell wall hemicellulosic xyloglucan have reinforced the concept that cell wall polysaccharides have extremely complicated and evolutionarily-conserved structures.

  7. Building and degradation of secondary cell walls: are there common patterns of lamellar assembly of cellulose microfibrils and cell wall delamination?

    PubMed

    De Micco, Veronica; Ruel, Katia; Joseleau, Jean-Paul; Aronne, Giovanna

    2010-08-01

    During cell wall formation and degradation, it is possible to detect cellulose microfibrils assembled into thicker and thinner lamellar structures, respectively, following inverse parallel patterns. The aim of this study was to analyse such patterns of microfibril aggregation and cell wall delamination. The thickness of microfibrils and lamellae was measured on digital images of both growing and degrading cell walls viewed by means of transmission electron microscopy. To objectively detect, measure and classify microfibrils and lamellae into thickness classes, a method based on the application of computerized image analysis combined with graphical and statistical methods was developed. The method allowed common classes of microfibrils and lamellae in cell walls to be identified from different origins. During both the formation and degradation of cell walls, a preferential formation of structures with specific thickness was evidenced. The results obtained with the developed method allowed objective analysis of patterns of microfibril aggregation and evidenced a trend of doubling/halving lamellar structures, during cell wall formation/degradation in materials from different origin and which have undergone different treatments. PMID:20532796

  8. Cell wall as a target for bacteria inactivation by pulsed electric fields

    PubMed Central

    Pillet, Flavien; Formosa-Dague, Cécile; Baaziz, Houda; Dague, Etienne; Rols, Marie-Pierre

    2016-01-01

    The integrity and morphology of bacteria is sustained by the cell wall, the target of the main microbial inactivation processes. One promising approach to inactivation is based on the use of pulsed electric fields (PEF). The current dogma is that irreversible cell membrane electro-permeabilisation causes the death of the bacteria. However, the actual effect on the cell-wall architecture has been poorly explored. Here we combine atomic force microscopy and electron microscopy to study the cell-wall organization of living Bacillus pumilus bacteria at the nanoscale. For vegetative bacteria, exposure to PEF led to structural disorganization correlated with morphological and mechanical alterations of the cell wall. For spores, PEF exposure led to the partial destruction of coat protein nanostructures, associated with internal alterations of cortex and core. Our findings reveal for the first time that the cell wall and coat architecture are directly involved in the electro-eradication of bacteria. PMID:26830154

  9. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls

    PubMed Central

    Marcus, Susan E; Verhertbruggen, Yves; Hervé, Cécile; Ordaz-Ortiz, José J; Farkas, Vladimir; Pedersen, Henriette L; Willats, William GT; Knox, J Paul

    2008-01-01

    Background Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Results Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. Conclusion These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation

  10. Miniature1-encoded cell wall invertase is essential for assembly and function of wall-in-growth in the maize endosperm transfer cell

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The miniature1 (mn1) seed phenotype in maize is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase, INCW2, which localizes exclusively to the basal endosperm transfer cells (BETC) of developing seeds. A common feature of all transfer cells is the labyrinth-like wa...

  11. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

    PubMed Central

    Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; Budziszek, Michael J.; Scavuzzo-Duggan, Tess R.; Roberts, Alison W.

    2016-01-01

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants. PMID:27014284

  12. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens

    DOE PAGESBeta

    Berry, Elizabeth A.; Tran, Mai L.; Dimos, Christos S.; Budziszek, Michael J.; Scavuzzo-Duggan, Tess R.; Roberts, Alison W.

    2016-03-08

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into severalmore » different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants.« less

  13. Immuno and Affinity Cytochemical Analysis of Cell Wall Composition in the Moss Physcomitrella patens.

    PubMed

    Berry, Elizabeth A; Tran, Mai L; Dimos, Christos S; Budziszek, Michael J; Scavuzzo-Duggan, Tess R; Roberts, Alison W

    2016-01-01

    In contrast to homeohydric vascular plants, mosses employ a poikilohydric strategy for surviving in the dry aerial environment. A detailed understanding of the structure, composition, and development of moss cell walls can contribute to our understanding of not only the evolution of overall cell wall complexity, but also the differences that have evolved in response to selection for different survival strategies. The model moss species Physcomitrella patens has a predominantly haploid lifecycle consisting of protonemal filaments that regenerate from protoplasts and enlarge by tip growth, and leafy gametophores composed of cells that enlarge by diffuse growth and differentiate into several different types. Advantages for genetic studies include methods for efficient targeted gene modification and extensive genomic resources. Immuno and affinity cytochemical labeling were used to examine the distribution of polysaccharides and proteins in regenerated protoplasts, protonemal filaments, rhizoids, and sectioned gametophores of P. patens. The cell wall composition of regenerated protoplasts was also characterized by flow cytometry. Crystalline cellulose was abundant in the cell walls of regenerating protoplasts and protonemal cells that developed on media of high osmolarity, whereas homogalactuonan was detected in the walls of protonemal cells that developed on low osmolarity media and not in regenerating protoplasts. Mannan was the major hemicellulose detected in all tissues tested. Arabinogalactan proteins were detected in different cell types by different probes, consistent with structural heterogneity. The results reveal developmental and cell type specific differences in cell wall composition and provide a basis for analyzing cell wall phenotypes in knockout mutants. PMID:27014284

  14. Structure of the Schizosaccharomyces pombe cytochrome c gene

    SciTech Connect

    Russell, P.R.; Hall, B.D.

    1982-02-01

    The cytochrome c gene of the fission yeast Schizosaccharomyces pombe has been cloned by using the Saccharomyces cerevisiae iso-1-cytochrome c gene as a molecular hybridization probe. The DNA sequence and the 5' termini of the mRNA transcripts of the gene have been determined. The DNA sequence has confirmed, with two exceptions, the previously determined protein sequence. The nonrandom distribution of silent third base differences which was observed between the two cytochrome c genes of S. cerevisiae does not extend to the S. pombe cytochrome c gene, suggesting that there are no constraints other than protein function and codon usage which have acted to conserve the cytochrome c DNA sequences of the two yeasts. Introduction of the S. pombe cytochrome c gene on a yeast plasmid into a S. cerevisiae mutant which lacked functional cytochrome c transformed that recipient strain for the ability to grow on a nonfermentable carbon source. This implies that the S. pombe cytochrome c gene has all the regulatory signals which are required for its expression in S. cerevisiae, and that none of the amino acid differences between the cytochrome c proteins of the two yeasts has a drastic effect on the function of the protein in vivo.

  15. The Spontaneous Mutation Rate in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Farlow, Ashley; Long, Hongan; Arnoux, Stéphanie; Sung, Way; Doak, Thomas G; Nordborg, Magnus; Lynch, Michael

    2015-10-01

    The rate at which new mutations arise in the genome is a key factor in the evolution and adaptation of species. Here we describe the rate and spectrum of spontaneous mutations for the fission yeast Schizosaccharomyces pombe, a key model organism with many similarities to higher eukaryotes. We undertook an ∼1700-generation mutation accumulation (MA) experiment with a haploid S. pombe, generating 422 single-base substitutions and 119 insertion-deletion mutations (indels) across the 96 replicates. This equates to a base-substitution mutation rate of 2.00 × 10(-10) mutations per site per generation, similar to that reported for the distantly related budding yeast Saccharomyces cerevisiae. However, these two yeast species differ dramatically in their spectrum of base substitutions, the types of indels (S. pombe is more prone to insertions), and the pattern of selection required to counteract a strong AT-biased mutation rate. Overall, our results indicate that GC-biased gene conversion does not play a major role in shaping the nucleotide composition of the S. pombe genome and suggest that the mechanisms of DNA maintenance may have diverged significantly between fission and budding yeasts. Unexpectedly, CpG sites appear to be excessively liable to mutation in both species despite the likely absence of DNA methylation. PMID:26265703

  16. HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe

    PubMed Central

    Benko, Zsigmond; Elder, Robert T.; Li, Ge; Liang, Dong; Zhao, Richard Y.

    2016-01-01

    Background HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a fission yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. Results A fission yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in fission yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in fission yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. Conclusions This is the first report to show that HIV-1 protease is functional as an enzyme in fission yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in fission yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings. PMID:26982200

  17. Ethanol yields and cell wall properties in divergently bred switchgrass genotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genetic modification of herbaceous plant cell walls to increase biofuels yields from harvested biomass is a primary bioenergy research goal. The focus of much of this research has been on cell wall lignin concentration. Using switchgrass genotypes developed by divergent breeding for ruminant diges...

  18. A summary of staphylococcal C-terminal SH3b_5 cell wall binding domains.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Staphylococcal peptidoglycan hydrolases are a potential new source of antimicrobials. A large subset of these proteins contain a C-terminal SH3b_5 cell wall binding domain that has been shown for some to be essential for accurate cell wall recognition and subsequent staphylolytic activity, propert...

  19. De-esterified Pectins in the Cell Walls of Cotton Fiber: A Study of Fiber Mutants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In the wild-type cotton (DP 5690), the cell walls of elongating cotton fibers are bilayered, with the outer layer enriched in de-esterified homogalacturonan (HGA), and an inner layer enriched in xyloglucans and cellulose. This bilayer is conspicuously absent in the cell walls of the ovule epidermal...

  20. A novel technique to evaluate interactions between Saccharomyces cerevisiae cell wall and mycotoxins: application to zearalenone.

    PubMed

    Yiannikouris, Alexandros; Poughon, Laurent; Cameleyre, Xavier; Dussap, Claude-Gilles; François, Jean; Bertin, Gérard; Jouany, Jean-Pierre

    2003-05-01

    Three models based on sigmoidal plotting were tested for their ability to describe zearalenone adsorption on Saccharomyces cerevisiae cell walls in vitro. All three models closely fitted the experimental data, but Hill's equation gave the most accurate parameters, and provided information on the physical and chemical mechanisms involved in the adsorption of mycotoxin on yeast cell walls. PMID:12882008

  1. Comparison of stem morphology and anatomy of two alfalfa clonal lines exhibiting divergent cell wall composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In previous research, two alfalfa clonal lines (252, 1283) were identified that exhibited environmentally stable differences in stem cell walls. Compared to stems of 1283, stems of 252 have a higher cell wall concentration and greater amounts of lignin and cellulose but reduced levels of pectic suga...

  2. Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes

    DOEpatents

    Raab, R. Michael; Zhang, Dongcheng; Bougri, Oleg

    2016-02-02

    Methods for consolidated pretreatment and hydrolysis of genetically engineered plants expressing cell wall degrading enzymes are provided. Expression cassettes and vectors for making transgenic plants are described. Plants engineered to express one or more cell wall degrading enzymes using expression cassettes and vectors of the invention are also provided.

  3. Experimental approaches to study plant cell walls during plant-microbe interactions

    PubMed Central

    Xia, Ye; Petti, Carloalberto; Williams, Mark A.; DeBolt, Seth

    2014-01-01

    Plant cell walls provide physical strength, regulate the passage of bio-molecules, and act as the first barrier of defense against biotic and abiotic stress. In addition to providing structural integrity, plant cell walls serve an important function in connecting cells to their extracellular environment by sensing and transducing signals to activate cellular responses, such as those that occur during pathogen infection. This mini review will summarize current experimental approaches used to study cell wall functions during plant-pathogen interactions. Focus will be paid to cell imaging, spectroscopic analyses, and metabolic profiling techniques. PMID:25352855

  4. Histology and cell wall biochemistry of stone cells in the physical defence of conifers against insects.

    PubMed

    Whitehill, Justin G A; Henderson, Hannah; Schuetz, Mathias; Skyba, Oleksandr; Yuen, Macaire Man Saint; King, John; Samuels, A Lacey; Mansfield, Shawn D; Bohlmann, Jörg

    2016-08-01

    Conifers possess an array of physical and chemical defences against stem-boring insects. Stone cells provide a physical defence associated with resistance against bark beetles and weevils. In Sitka spruce (Picea sitchensis), abundance of stone cells in the cortex of apical shoots is positively correlated with resistance to white pine weevil (Pissodes strobi). We identified histological, biochemical and molecular differences in the stone cell phenotype of weevil resistant (R) or susceptible (S) Sitka spruce genotypes. R trees displayed significantly higher quantities of cortical stone cells near the apical shoot node, the primary site for weevil feeding. Lignin, cellulose, xylan and mannan were the most abundant components of stone cell secondary walls, respectively. Lignin composition of stone cells isolated from R trees contained a higher percentage of G-lignin compared with S trees. Transcript profiling revealed higher transcript abundance in the R genotype of coumarate 3-hydroxylase, a key monolignol biosynthetic gene. Developing stone cells in current year apical shoots incorporated fluorescent-tagged monolignol into the secondary cell wall, while mature stone cells of previous year apical shoots did not. Stone cell development is an ephemeral process, and fortification of shoot tips in R trees is an effective strategy against insect feeding. PMID:26474726

  5. Degradation of 14C-labeled streptococcal cell walls by egg white lysozyme and lysosomal enzymes.

    PubMed Central

    Gallis, H A; Miller, S E; Wheat, R W

    1976-01-01

    The resistance of native and trypsin-treated [14C] glucose-labeled cell walls to degradation by lysozyme and human lysosomal enzymes was confirmed. In contrast, chemically N-acetylated cell walls undergo significant degradation by these enzymes in the pH range of 4.5 to 5.5 without prior removal of the group-specific carbohydrate. N-acetylation after removal of the group A carbohydrate by formamide extraction renders the cell walls considerably more susceptible to these enzymes than by formamaide extraction alone. It appears, therefore, that unless N-acetylation can occur in vivo, streptococcal cell walls are minimally degraded, if at all, by human peripheral blood leukocytes or lysozyme. Examination of leukocyte extracts from normal subjects and patients with post-streptococcal syndromes revealed no qualitative differences in ability to dissolve streptococcal cell walls. Images PMID:773836

  6. Bacterial cell wall research in Tübingen: a brief historical account.

    PubMed

    Braun, Volkmar

    2015-02-01

    Research in Tübingen on bacterial cell walls began in 1951 and continues to this day. The studies over the decades reflect the development in the field, which was strongly influenced by the design of suitable biochemical and genetic methods used to unravel the highly complex envelope structure. At the beginning of this period, improper crude extraction and solubilization methods were employed in an attempt to isolate pure components. Nevertheless, progress was steady and culminated in major insights into the structure and function of individual cell wall components and the cell wall as a whole. The "cell wall" has various definitions. In this short overview, the term includes the cell wall of gram-positive bacteria in the strict sense, and also the outer membrane, the murein (peptidoglycan) and the outer membrane of gram-negative bacteria and the cytoplasmic membranes. PMID:25583455

  7. Fuel from plant cell walls: recent developments in second generation bioethanol research.

    PubMed

    Cook, Charis; Devoto, Alessandra

    2011-08-15

    As bioethanol from sugarcane and wheat falls out of favour due to concerns about food security, research is ongoing into genetically engineering model plants and microorganisms to find the optimum cell wall structure for the ultimate second generation bioethanol crop. Charis Cook and Alessandra Devoto highlight here the progress made to tailor the plant cell wall to improve the accessibility of cellulose by acting on the regulation, the structure or the relative composition of other cell wall components to ultimately improve saccharification efficiency. They also consider possible side effects of cell wall modification and focus on the latest advances made to improve the efficiency of digestion of lignocellulosic materials by cell wall degrading microorganisms. PMID:21681755

  8. Amyloid-like properties of Saccharomyces cerevisiae cell wall glucantransferase Bgl2p

    PubMed Central

    Plotnikova, Tatyana A; Gorkovskii, Anton A; Selyakh, Irina O; Galzitskaya, Oxana V; Bezsonov, Evgeniy E; Gellissen, Gerd; Kulaev, Igor S

    2008-01-01

    Glucantransferase Bgl2p is a major conserved cell wall constituent described for a wide range of yeast species. In the baker's yeast Saccharomyces cerevisiae it is the only non-covalently bound cell wall protein that cannot be released from cell walls by sequential SDS and trypsin treatment. It contains seven amyloidogenic determinants. Circular dichroism analysis and fluorescence spectroscopy with thioflavin T indicate the presence of β-sheet structures in Bgl2p isolates. Bgl2p forms fibrils, a process that is enforced in the presence of other cell wall components. Thus the data obtained is the first evidence for amyloid-like properties of yeast cell wall protein—glucantransferase Bgl2p. PMID:19098439

  9. The three-dimensional structure of the cell wall glycoprotein of Chlorogonium elongatum.

    PubMed

    Shaw, P J; Hills, G J

    1984-06-01

    The green alga Chlorogonium elongatum, a member of the Volvocales, possesses a crystalline cell wall composed of hydroxyproline-rich glycoprotein similar to the primary cell wall glycoproteins of higher plants. Electron microscopy and computer image processing have been used to determine the crystal structure of the Chlorogonium cell wall in three dimensions to a resolution of 2.0 nm. The structure is composed of heterologous dimers. Each subunit of the dimer comprises a long, thin spacer domain and a large globular domain, which is the site of the intra- and inter-dimer interactions. There are also sites of intersubunit interactions at the opposite ends of the rod domains. We suggest that the rods are composed predominantly of glycosylated polyproline helix, as has been suggested for higher plant cell wall glycoproteins and has been shown for the cell wall glycoprotein of Chlamydomonas reinhardtii, which is closely related to Chlorogonium. PMID:6490737

  10. Dressed to impress: impact of environmental adaptation on the C andida albicans cell wall

    PubMed Central

    2015-01-01

    Summary C andida albicans is an opportunistic fungal pathogen of humans causing superficial mucosal infections and life‐threatening systemic disease. The fungal cell wall is the first point of contact between the invading pathogen and the host innate immune system. As a result, the polysaccharides that comprise the cell wall act as pathogen associated molecular patterns, which govern the host–pathogen interaction. The cell wall is dynamic and responsive to changes in the external environment. Therefore, the host environment plays a critical role in regulating the host–pathogen interaction through modulation of the fungal cell wall. This review focuses on how environmental adaptation modulates the cell wall structure and composition, and the subsequent impact this has on the innate immune recognition of C . albicans. PMID:25846717

  11. Clinostation influence on regeneration of cell wall in Solanum Tuberosum L. protoplasts

    NASA Astrophysics Data System (ADS)

    Nedukha, Elena M.; Sidorov, V. A.; Samoylov, V. M.

    1994-08-01

    Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solanum tuberosum leaves and were cultivated on the horizontal low rotating clinostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in an regeneration inhibition of cell wall. With electron-microscopical and electro-cytochemical methods (staining with alcianum blue) dynamics of the regeneration of cell walls in protoplasts was studied; carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants.

  12. Non-invasive imaging of cellulose microfibril orientation within plant cell walls by polarized Raman microspectroscopy.

    PubMed

    Sun, Lan; Singh, Seema; Joo, Michael; Vega-Sanchez, Miguel; Ronald, Pamela; Simmons, Blake A; Adams, Paul; Auer, Manfred

    2016-01-01

    Cellulose microfibrils represent the major scaffold of plant cell walls. Different packing and orientation of the microfibrils at the microscopic scale determines the macroscopic properties of cell walls and thus affect their functions with a profound effect on plant survival. We developed a polarized Raman microspectroscopic method to determine cellulose microfibril orientation within rice plant cell walls. Employing an array of point measurements as well as area imaging and subsequent Matlab-assisted data processing, we were able to characterize the distribution of cellulose microfibril orientation in terms of director angle and anisotropy magnitude. Using this approach we detected differences between wild type rice plants and the rice brittle culm mutant, which shows a more disordered cellulose microfibril arrangement, and differences between different tissues of a wild type rice plant. This novel non-invasive Raman imaging approach allows for quantitative assessment of cellulose fiber orientation in cell walls of herbaceous plants, an important advancement in cell wall characterization. PMID:26137889

  13. Involvement of small heat shock proteins, trehalose, and lipids in the thermal stress management in Schizosaccharomyces pombe.

    PubMed

    Glatz, Attila; Pilbat, Ana-Maria; Németh, Gergely L; Vince-Kontár, Katalin; Jósvay, Katalin; Hunya, Ákos; Udvardy, Andor; Gombos, Imre; Péter, Mária; Balogh, Gábor; Horváth, Ibolya; Vígh, László; Török, Zsolt

    2016-03-01

    Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast. PMID:26631139

  14. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

    PubMed

    Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

    2016-07-01

    The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

  15. Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

    PubMed Central

    Mistou, Michel-Yves; Sutcliffe, Iain C.; van Sorge, Nina M.

    2016-01-01

    The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

  16. Breakdown of Cell Wall Nanostructure in Dilute Acid Pretreated Biomass

    SciTech Connect

    Pingali, Sai Venkatesh; Urban, Volker S; Heller, William T; McGaughey, Joseph; O'Neill, Hugh Michael; Foston, Marcus B; Myles, Dean A A; Ragauskas, Arthur J; Evans, Barbara R

    2010-01-01

    The generation of bioethanol from lignocellulosic biomass holds great promise for renewable and clean energy production. A better understanding of the complex mechanisms of lignocellulose breakdown during various pretreatment methods is needed to realize this potential in a cost and energy efficient way. Here, we use small-angle neutron scattering (SANS) to characterize morphological changes in switchgrass lignocellulose across molecular to sub-micron length scales resulting from the industrially-relevant dilute acid pretreatment method. Our results demonstrate that dilute acid pretreatment increases the cross-sectional radius of the crystalline cellulose fibril. This change is accompanied by removal of hemicellulose and the formation of Rg ~ 135 lignin aggregates. The structural signature of smooth cell wall surfaces is observed at length scales larger than 1000 , and it remains remarkably invariable during pretreatment. This study elucidates the interplay of the different biomolecular components in the break down process of switchgrass by dilute acid pretreatment. The results are important for the development of efficient strategies of biomass to biofuel conversion.

  17. Single-Wall Carbon Nanotube Anodes for Lithium Cells

    NASA Technical Reports Server (NTRS)

    Hepp, Aloysius F.; Raffaelle, Ryne; Gennett, Tom; Kumta, Prashant; Maranchi, Jeff; Heben, Mike

    2006-01-01

    In recent experiments, highly purified batches of single-wall carbon nanotubes (SWCNTs) have shown promise as superior alternatives to the graphitic carbon-black anode materials heretofore used in rechargeable thin-film lithium power cells. The basic idea underlying the experiments is that relative to a given mass of graphitic carbon-black anode material, an equal mass of SWCNTs can be expected to have greater lithium-storage and charge/discharge capacities. The reason for this expectation is that whereas the microstructure and nanostructure of a graphitic carbon black is such as to make most of the interior of the material inaccessible for intercalation of lithium, a batch of SWCNTs can be made to have a much more open microstructure and nanostructure, such that most of the interior of the material is accessible for intercalation of lithium. Moreover, the greater accessibility of SWCNT structures can be expected to translate to greater mobilities for ion-exchange processes and, hence, an ability to sustain greater charge and discharge current densities.

  18. Cotton fiber tips have diverse morphologies and show evidence of apical cell wall synthesis.

    PubMed

    Stiff, Michael R; Haigler, Candace H

    2016-01-01

    Cotton fibers arise through highly anisotropic expansion of a single seed epidermal cell. We obtained evidence that apical cell wall synthesis occurs through examining the tips of young elongating Gossypium hirsutum (Gh) and G. barbadense (Gb) fibers. We characterized two tip types in Gh fiber (hemisphere and tapered), each with distinct apical diameter, central vacuole location, and distribution of cell wall components. The apex of Gh hemisphere tips was enriched in homogalacturonan epitopes, including a relatively high methyl-esterified form associated with cell wall pliability. Other wall components increased behind the apex including cellulose and the α-Fuc-(1,2)-β-Gal epitope predominantly found in xyloglucan. Gb fibers had only one narrow tip type featuring characters found in each Gh tip type. Pulse-labeling of cell wall glucans indicated wall synthesis at the apex of both Gh tip types and in distal zones. Living Gh hemisphere and Gb tips ruptured preferentially at the apex upon treatment with wall degrading enzymes, consistent with newly synthesized wall at the apex. Gh tapered tips ruptured either at the apex or distantly. Overall, the results reveal diverse cotton fiber tip morphologies and support primary wall synthesis occurring at the apex and discrete distal regions of the tip. PMID:27301434

  19. Cotton fiber tips have diverse morphologies and show evidence of apical cell wall synthesis

    PubMed Central

    Stiff , Michael R.; Haigler, Candace H.

    2016-01-01

    Cotton fibers arise through highly anisotropic expansion of a single seed epidermal cell. We obtained evidence that apical cell wall synthesis occurs through examining the tips of young elongating Gossypium hirsutum (Gh) and G. barbadense (Gb) fibers. We characterized two tip types in Gh fiber (hemisphere and tapered), each with distinct apical diameter, central vacuole location, and distribution of cell wall components. The apex of Gh hemisphere tips was enriched in homogalacturonan epitopes, including a relatively high methyl-esterified form associated with cell wall pliability. Other wall components increased behind the apex including cellulose and the α-Fuc-(1,2)-β-Gal epitope predominantly found in xyloglucan. Gb fibers had only one narrow tip type featuring characters found in each Gh tip type. Pulse-labeling of cell wall glucans indicated wall synthesis at the apex of both Gh tip types and in distal zones. Living Gh hemisphere and Gb tips ruptured preferentially at the apex upon treatment with wall degrading enzymes, consistent with newly synthesized wall at the apex. Gh tapered tips ruptured either at the apex or distantly. Overall, the results reveal diverse cotton fiber tip morphologies and support primary wall synthesis occurring at the apex and discrete distal regions of the tip. PMID:27301434

  20. A Katanin-like Protein Regulates Normal Cell Wall Biosynthesis and Cell Elongation

    PubMed Central

    Burk, David H.; Liu, Bo; Zhong, Ruiqin; Morrison, W. Herbert; Ye, Zheng-Hua

    2001-01-01

    Fibers are one of the mechanical tissues that provide structural support to the plant body. To understand how the normal mechanical strength of fibers is regulated, we isolated an Arabidopsis fragile fiber (fra2) mutant defective in the mechanical strength of interfascicular fibers in the inflorescence stems. Anatomical and chemical analyses showed that the fra2 mutation caused a reduction in fiber cell length and wall thickness, a decrease in cellulose and hemicellulose contents, and an increase in lignin condensation, indicating that the fragile fiber phenotype of fra2 is a result of alterations in fiber cell elongation and cell wall biosynthesis. In addition to the effects on fibers, the fra2 mutation resulted in a remarkable reduction in cell length and an increase in cell width in all organs, which led to a global alteration in plant morphology. The FRA2 gene was shown to encode a protein with high similarity to katanin (hence FRA2 was renamed AtKTN1), a protein shown to be involved in regulating microtubule disassembly by severing microtubules. Consistent with the putative function of AtKTN1 as a microtubule-severing protein, immunolocalization demonstrated that the fra2 mutation caused delays in the disappearance of perinuclear microtubule array and in the establishment of transverse cortical microtubule array in interphase and elongating cells. Together, these results suggest that AtKTN1, a katanin-like protein, is essential not only for normal cell wall biosynthesis and cell elongation in fiber cells but also for cell expansion in all organs. PMID:11283338

  1. Mechanical Consequences of Cell-Wall Turnover in the Elongation of a Gram-Positive Bacterium

    PubMed Central

    Misra, Gaurav; Rojas, Enrique R.; Gopinathan, Ajay; Huang, Kerwyn Casey

    2013-01-01

    A common feature of walled organisms is their exposure to osmotic forces that challenge the mechanical integrity of cells while driving elongation. Most bacteria rely on their cell wall to bear osmotic stress and determine cell shape. Wall thickness can vary greatly among species, with Gram-positive bacteria having a thicker wall than Gram-negative bacteria. How wall dimensions and mechanical properties are regulated and how they affect growth have not yet been elucidated. To investigate the regulation of wall thickness in the rod-shaped Gram-positive bacterium Bacillus subtilis, we analyzed exponentially growing cells in different media. Using transmission electron and epifluorescence microscopy, we found that wall thickness and strain were maintained even between media that yielded a threefold change in growth rate. To probe mechanisms of elongation, we developed a biophysical model of the Gram-positive wall that balances the mechanical effects of synthesis of new material and removal of old material through hydrolysis. Our results suggest that cells can vary their growth rate without changing wall thickness or strain by maintaining a constant ratio of synthesis and hydrolysis rates. Our model also indicates that steady growth requires wall turnover on the same timescale as elongation, which can be driven primarily by hydrolysis rather than insertion. This perspective of turnover-driven elongation provides mechanistic insight into previous experiments involving mutants whose growth rate was accelerated by the addition of lysozyme or autolysin. Our approach provides a general framework for deconstructing shape maintenance in cells with thick walls by integrating wall mechanics with the kinetics and regulation of synthesis and turnover. PMID:23746506

  2. The effect of magnesium on mitotic spindle formation in Schizosaccharomyces pombe

    PubMed Central

    Uz, Gulsen; Sarikaya, Aysegul Topal

    2016-01-01

    Abstract Magnesium (Mg2+), an essential ion for cells and biological systems, is involved in a variety of cellular processes, including the formation and breakdown of microtubules. The results of a previous investigation suggested that as cells grow the intracellular Mg2+ concentration falls, thereby stimulating formation of the mitotic spindle. In the present work, we used a Mg2+-deficient Schizosaccharomyces pombe strain GA2, in which two essential membrane Mg2+ transporter genes (homologs of ALR1 and ALR2 in Saccharomyces cerevisae) were deleted, and its parental strain Sp292, to examine the extent to which low Mg2+ concentrations can affect mitotic spindle formation. The two S. pombe strains were transformed with a plasmid carrying a GFP-α2-tubulin construct to fluorescently label microtubules. Using the free Mg2+-specific fluorescent probe mag-fura-2, we confirmed that intracellular free Mg2+ levels were lower in GA2 than in the parental strain. Defects in interphase microtubule organization, a lower percentage of mitotic spindle formation and a reduced mitotic index were also observed in the GA2 strain. Although there was interphase microtubule polymerization, the lower level of mitotic spindle formation in the Mg2+-deficient strain suggested a greater requirement for Mg2+ in this phenomenon than previously thought. PMID:27560651

  3. The effect of magnesium on mitotic spindle formation in Schizosaccharomyces pombe.

    PubMed

    Uz, Gulsen; Sarikaya, Aysegul Topal

    2016-01-01

    Magnesium (Mg2+), an essential ion for cells and biological systems, is involved in a variety of cellular processes, including the formation and breakdown of microtubules. The results of a previous investigation suggested that as cells grow the intracellular Mg2+ concentration falls, thereby stimulating formation of the mitotic spindle. In the present work, we used a Mg2+-deficient Schizosaccharomyces pombe strain GA2, in which two essential membrane Mg2+ transporter genes (homologs of ALR1 and ALR2 in Saccharomyces cerevisae) were deleted, and its parental strain Sp292, to examine the extent to which low Mg2+ concentrations can affect mitotic spindle formation. The two S. pombe strains were transformed with a plasmid carrying a GFP-α2-tubulin construct to fluorescently label microtubules. Using the free Mg2+-specific fluorescent probe mag-fura-2, we confirmed that intracellular free Mg2+ levels were lower in GA2 than in the parental strain. Defects in interphase microtubule organization, a lower percentage of mitotic spindle formation and a reduced mitotic index were also observed in the GA2 strain. Although there was interphase microtubule polymerization, the lower level of mitotic spindle formation in the Mg2+-deficient strain suggested a greater requirement for Mg2+ in this phenomenon than previously thought. PMID:27560651

  4. Shared catalysis in virus entry and bacterial cell wall depolymerization.

    PubMed

    Cohen, Daniel N; Sham, Yuk Y; Haugstad, Greg D; Xiang, Ye; Rossmann, Michael G; Anderson, Dwight L; Popham, David L

    2009-04-01

    Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and a structural pattern of beta-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage varphi29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a d,d-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn(2)(+) metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn(2)(+) binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn(2)(+) binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action. PMID:19361422

  5. Functional control of the Candida albicans cell wall by catalytic protein kinase A subunit Tpk1

    PubMed Central

    Fanning, S; Xu, W; Beaurepaire, C; Suhan, JP; Nantel, A; Mitchell, AP

    2014-01-01

    SUMMARY The cyclic AMP-protein kinase A pathway governs numerous biological features of the fungal pathogen Candida albicans. The catalytic protein kinase A subunits, Tpk1 (orf19.4892) and Tpk2 (orf19.2277), have divergent roles, and most studies indicate a more pronounced role for Tpk2. Here we dissect two Tpk1-responsive properties: adherence and cell wall integrity. Homozygous tpk1/tpk1 mutants are hyperadherent, and a Tpk1 defect enables biofilm formation in the absence of Bcr1, a transcriptional regulator of biofilm adhesins. A quantitative gene expression-based assay reveals that tpk1/tpk1 and bcr1/bcr1 genotypes show mixed epistasis, as expected if Tpk1 and Bcr1 act mainly in distinct pathways. Overexpression of individual Tpk1-repressed genes indicates that cell surface proteins Als1, Als2, Als4, Csh1, and Csp37 contribute to Tpk1-regulated adherence. Tpk1 is also required for cell wall integrity, but has no role in the gene expression response to cell wall inhibition by caspofungin. Interestingly, increased expression of the adhesin gene ALS2 confers a cell wall defect, as manifested in hypersensitivity to the cell wall inhibitor caspofungin and a shallow cell wall structure. Our findings indicate that Tpk1 governs C. albicans cell wall properties through repression of select cell surface protein genes. PMID:22882910

  6. Immunogold Localization of Xyloglucan and Rhamnogalacturonan I in the Cell Walls of Suspension-Cultured Sycamore Cells 1

    PubMed Central

    Moore, Patricia J.; Darvill, Alan G.; Albersheim, Peter; Staehelin, L. Andrew

    1986-01-01

    Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:16665111

  7. Wall Extensibility and Cell Hydraulic Conductivity Decrease in Enlarging Stem Tissues at Low Water Potentials 1

    PubMed Central

    Nonami, Hiroshi; Boyer, John S.

    1990-01-01

    Measurements with a guillotine psychrometer (H Nonami, JS Boyer [1990] Plant Physiol 94: 1601-1609) indicate that the inhibition of stem growth at low water potentials (low ψw) is accompanied by decreases in cell wall extensibility and tissue hydraulic conductance to water that eventually limit growth rate in soybean (Glycine max L. Merr.). To check this conclusion, we measured cell wall properties and cell hydraulic conductivities with independent techniques in soybean seedlings grown and treated the same way, i.e. grown in the dark and exposed to low ψw by transplanting dark grown seedlings to vermiculite of low water content. Wall properties were measured with an extensiometer modified for intact plants, and conductances were measured with a cell pressure probe in intact plants. Theory was developed to relate the wall measurements to those with the psychrometer. In the elongation zone, the plastic deformability of the walls decreased when measured with the extensiometer while growth was inhibited at low ψw. It increased during a modest growth recovery. This behavior was the same as that for the wall extensibility observed previously with the psychrometer. Tissue that was killed before measurement with the extensiometer also showed a similar response, indicating that changes in wall extensibility represented changes in wall physical properties and not rates of wall biosynthesis. The elastic compliance (reciprocal of bulk elastic modulus) did not change in the elongating or mature tissue. The hydraulic conductivity of cortical cells decreased in the elongating tissue and increased slightly during growth recovery in a response similar to that observed with the psychrometer. We conclude that the plastic properties of the cell walls and the conductance of the cells to water were decreased at low ψw but that the elastic properties of the walls were of little consequence in this response. PMID:16667664

  8. Spectral Snapshots of Bacterial Cell-Wall Composition and the Influence of Antibiotics by Whole-Cell NMR

    PubMed Central

    Nygaard, Rie; Romaniuk, Joseph A.H.; Rice, David M.; Cegelski, Lynette

    2015-01-01

    Gram-positive bacteria surround themselves with a thick cell wall that is essential to cell survival and is a major target of antibiotics. Quantifying alterations in cell-wall composition are crucial to evaluating drug modes of action, particularly important for human pathogens that are now resistant to multiple antibiotics such as Staphylococcus aureus. Macromolecular and whole-cell NMR spectroscopy allowed us to observe the full panel of carbon and nitrogen pools in S. aureus cell walls and intact whole cells. We discovered that one-dimensional 13C and 15N NMR spectra, together with spectroscopic selections based on dipolar couplings as well as two-dimensional spin-diffusion measurements, revealed the dramatic compositional differences between intact cells and cell walls and allowed the identification of cell-wall signatures in whole-cell samples. Furthermore, the whole-cell NMR approach exhibited the sensitivity to detect distinct compositional changes due to treatment with the antibiotics fosfomycin (a cell-wall biosynthesis inhibitor) and chloramphenicol (a protein synthesis inhibitor). Whole cells treated with fosfomycin exhibited decreased peptidoglycan contributions while those treated with chloramphenicol contained a higher percentage of peptidoglycan as cytoplasmic protein content was reduced. Thus, general antibiotic modes of action can be identified by profiling the total carbon pools in intact whole cells. PMID:25809251

  9. Isolation and solubilization of gram-positive bacterial cell wall-associated proteins.

    PubMed

    Cole, Jason N; Djordjevic, Steven P; Walker, Mark J

    2008-01-01

    This chapter describes a simple, rapid and reproducible method to prepare bacterial cell wall extracts for two-dimensional gel electrophoresis (2DE). The extraction process uses mutanolysin, an N-acetylmuramidase, to gently solubilize cell wall-associated proteins from Gram-positive prokaryotes. The cells are first washed with buffer and resuspended in a solution containing mutanolysin. Following incubation at 37 degrees C, the sample is centrifuged and the supernatant containing the soluble cell wall-associated proteins is harvested. Following a brief precipitation step, the pellet is solubilized in sample buffer ready for isoelectric focusing and 2DE analysis. PMID:18369905

  10. Atomic Force Microscopy Measurements of the Mechanical Properties of Cell Walls on Living Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Bailey, Richard; Mullin, Nic; Turner, Robert; Foster, Simon; Hobbs, Jamie

    2014-03-01

    Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved.

  11. Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression

    PubMed Central

    Chaffin, W. Lajean; López-Ribot, José Luis; Casanova, Manuel; Gozalbo, Daniel; Martínez, José P.

    1998-01-01

    The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis. PMID:9529890

  12. Genetic modification of plant cell walls to enhance biomass yield and biofuel production in bioenergy crops.

    PubMed

    Wang, Yanting; Fan, Chunfen; Hu, Huizhen; Li, Ying; Sun, Dan; Wang, Youmei; Peng, Liangcai

    2016-01-01

    Plant cell walls represent an enormous biomass resource for the generation of biofuels and chemicals. As lignocellulose property principally determines biomass recalcitrance, the genetic modification of plant cell walls has been posed as a powerful solution. Here, we review recent progress in understanding the effects of distinct cell wall polymers (cellulose, hemicelluloses, lignin, pectin, wall proteins) on the enzymatic digestibility of biomass under various physical and chemical pretreatments in herbaceous grasses, major agronomic crops and fast-growing trees. We also compare the main factors of wall polymer features, including cellulose crystallinity (CrI), hemicellulosic Xyl/Ara ratio, monolignol proportion and uronic acid level. Furthermore, the review presents the main gene candidates, such as CesA, GH9, GH10, GT61, GT43 etc., for potential genetic cell wall modification towards enhancing both biomass yield and enzymatic saccharification in genetic mutants and transgenic plants. Regarding cell wall modification, it proposes a novel groove-like cell wall model that highlights to increase amorphous regions (density and depth) of the native cellulose microfibrils, providing a general strategy for bioenergy crop breeding and biofuel processing technology. PMID:27269671

  13. Impact of Cell Wall Composition on Maize Resistance to Pests and Diseases

    PubMed Central

    Santiago, Rogelio; Barros-Rios, Jaime; Malvar, Rosa A.

    2013-01-01

    In cereals, the primary cell wall is built of a skeleton of cellulosic microfibrils embedded in a matrix of hemicelluloses and smaller amounts of pectins, glycoproteins and hydroxycinnamates. Later, during secondary wall development, p-coumaryl, coniferyl and sinapyl alcohols are copolymerized to form mixed lignins. Several of these cell wall components show a determinative role in maize resistance to pest and diseases. However, defense mechanisms are very complex and vary among the same plant species, different tissues or even the same tissue at different developmental stages. Thus, it is important to highlight that the role of the cell wall components needs to be tested in diverse genotypes and specific tissues where the feeding or attacking by the pathogen takes place. Understanding the role of cell wall constituents as defense mechanisms may allow modifications of crops to withstand pests and diseases. PMID:23535334

  14. Response of Paracoccidioides brasiliensis Pb01 to stressor agents and cell wall osmoregulators.

    PubMed

    Tomazett, Patrícia Kott; Castro, Nadya da Silva; Lenzi, Henrique Leonel; de Almeida Soares, Célia Maria; Pereira, Maristela

    2011-01-01

    In its attempt to survive, the fungal cell can change the cell wall composition and/or structure in response to environmental stress. The molecules involved in these compensatory mechanisms are a possible target for the development of effective antifungal agents. In the thermodimorphic fungus Paracoccidioides brasiliensis Pb01, the main polymers that compose the cell wall are chitin and glucans. These polymers form a primary barrier that is responsible for the structural integrity and formation of the cell wall. In this study the behaviour of P. brasiliensis was evaluated under incubation with cell wall stressor agents such as Calcofluor White (CFW), Congo Red (CR), Sodium Dodecyl Sulphate (SDS), NaCl, KCl, and Sorbitol. Use of concentrations at which the fungus is visually sensitive to those agents helped to explain some of the adaptive mechanisms used by P. brasiliensis in response to cell wall stress. Our results show that 1,3-β-D-glucan synthase (PbFKS1), glucosamine-6-phosphate synthase (PbGFA1) and β-1,3-glucanosyltransferase (PbGEL3)as well as 1,3-β-D-glucan and N-acetylglucosamine (GlcNAc) residues in the cell wall are involved in compensatory mechanisms against cell wall damage. PMID:21215956

  15. Measurements and models of synchronous growth of fission yeast induced by temperature oscillations. [Schizosaccharomyces pombe

    SciTech Connect

    Agar, D.W.; Bailey, J.E.

    1982-01-01

    Pulsing of temperature in a fermentor at intervals coincident with cell generation time was used to induce synchrony in a population of the fission yeast Schizosaccharomyces pombe. Measurements of culture protein, RNA, and DNA during synchronous growth confirm continuous synthesis of protein and RNA and discontinuous synthesis of DNA as previously reported. Flow microfluorometry of populations at different times during the synchrony cycle was used to monitor the changes in single-cell protein, RNA, and DNA frequency functions. These measurements illustrate very clearly the degree of synchrony and patterns of macromolecular synthesis and also confirm previous estimates of the cellular protein contents characteristic of dividing cells. Additional insights into single-cell kinetics and division controls are provided by two-parameter flow microfluorometry measurements and by mathematical modeling of population dynamics. Such data are necessary foundations for robust population balance models of microbial processes. (Refs. 31).

  16. The Basic Leucine Zipper Domain Transcription Factor Atf1 Directly Controls Cdc13 Expression and Regulates Mitotic Entry Independently of Wee1 and Cdc25 in Schizosaccharomyces pombe

    PubMed Central

    Bandyopadhyay, Sushobhana; Dey, Isha; Suresh, Megalakshmi

    2014-01-01

    Progression into mitosis is a major point of regulation in the Schizosaccharomyces pombe cell cycle, and its proper control is essential for maintenance of genomic stability. Investigation of the G2/M progression event in S. pombe has revealed the existence of a complex regulatory process that is responsible for making the decision to enter mitosis. Newer aspects of this regulation are still being revealed. In this paper, we report the discovery of a novel mode of regulation of G2/M progression in S. pombe. We show that the mitogen-activated protein kinase (MAPK)-regulated transcription factor Atf1 is a regulator of Cdc13 (mitotic cyclin) transcription and is therefore a prominent player in the regulation of mitosis in S. pombe. We have used genetic approaches to study the effect of overexpression or deletion of Atf1 on the cell length and G2/M progression of S. pombe cells. Our results clearly show that Atf1 overexpression accelerates mitosis, leading to an accumulation of cells with shorter lengths. The previously known major regulators of entry into mitosis are the Cdc25 phosphatase and the Wee1 kinase, which modulate cyclin-dependent kinase (CDK) activity. The significantly striking aspect of our discovery is that Atf1-mediated G2/M progression is independent of both Cdc25 and Wee1. We have shown that Atf1 binds to the Cdc13 promoter, leading to activation of Cdc13 expression. This leads to enhanced nuclear localization of CDK Cdc2, thereby promoting the G2/M transition. PMID:24728197

  17. Critical cell wall hole size for lysis in Gram-positive bacteria

    NASA Astrophysics Data System (ADS)

    Mitchell, Gabriel; Wiesenfeld, Kurt; Nelson, Daniel; Weitz, Joshua

    2013-03-01

    Gram-positive bacteria transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here we develop and analyze a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range 15-24nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insight into the range of cell wall hole sizes compatible with bacterial homeostasis.

  18. Anhydrobiosis in yeast: cell wall mannoproteins are important for yeast Saccharomyces cerevisiae resistance to dehydration.

    PubMed

    Borovikova, Diana; Teparić, Renata; Mrša, Vladimir; Rapoport, Alexander

    2016-08-01

    The state of anhydrobiosis is linked with the reversible delay of metabolism as a result of strong dehydration of cells, and is widely distributed in nature. A number of factors responsible for the maintenance of organisms' viability in these conditions have been revealed. This study was directed to understanding how changes in cell wall structure may influence the resistance of yeasts to dehydration-rehydration. Mutants lacking various cell wall mannoproteins were tested to address this issue. It was revealed that mutants lacking proteins belonging to two structurally and functionally unrelated groups (proteins non-covalently attached to the cell wall, and Pir proteins) possessed significantly lower cell resistance to dehydration-rehydration than the mother wild-type strain. At the same time, the absence of the GPI-anchored cell wall protein Ccw12 unexpectedly resulted in an increase of cell resistance to this treatment; this phenomenon is explained by the compensatory synthesis of chitin. The results clearly indicate that the cell wall structure/composition relates to parameters strongly influencing yeast viability during the processes of dehydration-rehydration, and that damage to cell wall proteins during yeast desiccation can be an important factor leading to cell death. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27510749

  19. Trifluoromethanesulfonic acid-based proteomic analysis of cell wall and secreted proteins of the ascomycetous fungi Neurospora crassa and Candida albicans.

    PubMed

    Maddi, Abhiram; Bowman, Shaun M; Free, Stephen J

    2009-10-01

    Cell wall proteins from purified Candida albicans and Neurospora crassa cell walls were released using trifluoromethanesulfonic acid (TFMS) which cleaves the cell wall glucan/chitin matrix and deglycosylates the proteins. The cell wall proteins were then characterized by SDS-PAGE and identified by proteomic analysis. The analyses for C. albicans identified 15 cell wall proteins and six secreted proteins. For N. crassa, the analyses identified 26 cell wall proteins and nine secreted proteins. Most of the C. albicans cell wall proteins are found in the cell walls of both yeast and hyphae cells, but some cell type-specific cell wall proteins were observed. The analyses showed that the pattern of cell wall proteins present in N. crassa vegetative hyphae and conidia (asexual spores) are quite different. Almost all of the cell wall proteins identified in N. crassa have close homologs in the sequenced fungal genomes, suggesting that these proteins have important conserved functions within the cell wall. PMID:19555771

  20. Nic1 Inactivation Enables Stable Isotope Labeling with 13C615N4-Arginine in Schizosaccharomyces pombe*

    PubMed Central

    Carpy, Alejandro; Patel, Avinash; Tay, Ye Dee; Hagan, Iain M.; Macek, Boris

    2015-01-01

    Stable Isotope Labeling by Amino Acids (SILAC) is a commonly used method in quantitative proteomics. Because of compatibility with trypsin digestion, arginine and lysine are the most widely used amino acids for SILAC labeling. We observed that Schizosaccharomyces pombe (fission yeast) cannot be labeled with a specific form of arginine, 13C615N4-arginine (Arg-10), which limits the exploitation of SILAC technology in this model organism. We hypothesized that in the fission yeast the guanidinium group of 13C615N4-arginine is catabolized by arginase and urease activity to 15N1-labeled ammonia that is used as a precursor for general amino acid biosynthesis. We show that disruption of Ni2+-dependent urease activity, through deletion of the sole Ni2+ transporter Nic1, blocks this recycling in ammonium-supplemented EMMG medium to enable 13C615N4-arginine labeling for SILAC strategies in S. pombe. Finally, we employed Arg-10 in a triple-SILAC experiment to perform quantitative comparison of G1 + S, M, and G2 cell cycle phases in S. pombe. PMID:25368411

  1. Transcription of the Schizosaccharomyces pombe U2 gene in vivo and in vitro is directed by two essential promoter elements

    PubMed Central

    Zhou, Dewang; Lobo-Ruppert, Susan M.

    2001-01-01

    As compared to the metazoan small nuclear RNAs (snRNAs), relatively little is known about snRNA synthesis in unicellular organisms. We have analyzed the transcription of the Schizosaccharomyces pombe U2 snRNA gene in vivo and in the homologous in vitro system. Deletion and linker-scanning analyses show that the S.pombe U2 promoter contains at least two elements: the spUSE centered at –55, which functions as an activator, and a TATA box at –26, which is essential for basal transcription. These data point to a similar architecture among S.pombe, plant and invertebrate snRNA promoters. Factors recognizing the spUSE can be detected in whole cell extracts by DNase I footprinting and competition studies show that the binding of these factors correlates with transcriptional activity. Electrophoretic mobility shift assays and gel-filtration chromatography revealed a native molecular mass of ∼200 kDa for the spUSE binding activity. Two polypeptides of molecular masses 25 and 65 kDa were purified by virtue of their ability to specifically bind the spUSE. PMID:11353068

  2. Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing

    PubMed Central

    Tong, Kevin; Keller, Thomas; Hoffman, Charles S.

    2012-01-01

    The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast. PMID:22771823

  3. Schizosaccharomyces pombe Hat1 (Kat1) is associated with Mis16 and is required for telomeric silencing.

    PubMed

    Tong, Kevin; Keller, Thomas; Hoffman, Charles S; Annunziato, Anthony T

    2012-09-01

    The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast. PMID:22771823

  4. Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit.

    PubMed

    Kong, D; DePamphilis, M L

    2001-12-01

    The mechanism by which origin recognition complexes (ORCs) identify replication origins was investigated using purified Orc proteins from Schizosaccharomyces pombe. Orc4p alone bound tightly and specifically to several sites within S. pombe replication origins that are genetically required for origin activity. These sites consisted of clusters of A or T residues on one strand but were devoid of either alternating A and T residues or GC-rich sequences. Addition of a complex consisting of Orc1, -2, -3, -5, and -6 proteins (ORC-5) altered neither Orc4p binding to origin DNA nor Orc4p protection of specific sequences. ORC-5 alone bound weakly and nonspecifically to DNA; strong binding required the presence of Orc4p. Under these conditions, all six subunits remained bound to chromatin isolated from each phase of the cell division cycle. These results reveal that the S. pombe ORC binds to multiple, specific sites within replication origins and that site selection, at least in vitro, is determined solely by the Orc4p subunit. PMID:11689699

  5. Cell Wall Nonlinear Elasticity and Growth Dynamics: How Do Bacterial Cells Regulate Pressure and Growth?

    NASA Astrophysics Data System (ADS)

    Deng, Yi

    In my thesis, I study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. I find strong evidence of power--law stress--stiffening in the E. coli cell wall, with an exponent of 1.22±0.12, such that the wall is significantly stiffer in intact cells (E = 23±8 MPa and 49±20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29±3 kPa. The nonlinearity in cell elasticity serves as a plausible mechanism to balance the mechanical protection and tension measurement sensitivity of the cell envelope. I also study the growth dynamics of the Bacillus subtilis cell wall to help understand the mechanism of the spatiotemporal order of inserting new cell wall material. High density fluorescent markers are used to label the entire cell surface to capture the morphological changes of the cell surface at sub-cellular to diffraction-limited spatial resolution and sub-minute temporal resolution. This approach reveals that rod-shaped chaining B. subtilis cells grow and twist in a highly heterogeneous fashion both spatially and temporally. Regions of high growth and twisting activity have a typical length scale of 5 μm, and last for 10-40 minutes. Motivated by the quantification of the cell wall growth dynamics, two microscopy and image analysis techniques are developed and applied to broader applications beyond resolving bacterial growth. To resolve densely distributed quantum dots, we present a fast and efficient image analysis algorithm, namely Spatial Covariance Reconstruction (SCORE) microscopy that takes into account the blinking statistics of the fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging, which is at least an order of magnitude faster than single-particle localization based methods

  6. Xyloglucan and its interactions with other components of the growing cell wall.

    PubMed

    Park, Yong Bum; Cosgrove, Daniel J

    2015-02-01

    The discovery of xyloglucan and its ability to bind tightly to cellulose has dominated our thinking about primary cell wall structure and its connection to the mechanism of cell enlargement for 40 years. Gene discovery has advanced our understanding of the synthesis of xyloglucan in the past decade, and at the same time new and unexpected results indicate that xyloglucan's role in wall structure and wall extensibility is more subtle than commonly believed. Genetic deletion of xyloglucan synthesis does not greatly disable cell wall functions. Nuclear magnetic resonance studies indicate that pectins, rather than xyloglucans, make the majority of contacts with cellulose surfaces. Xyloglucan binding may be selective for specific (hydrophobic) surfaces on the cellulose microfibril, whose structure is more complex than is commonly portrayed in cell wall cartoons. Biomechanical assessments of endoglucanase actions challenge the concept of xyloglucan tethering. The mechanically important xyloglucan is restricted to a minor component that appears to be closely intertwined with cellulose at limited sites ('biomechanical hotspots') of direct microfibril contact; these may be the selective sites of cell wall loosening by expansins. These discoveries indicate that wall extensibility is less a matter of bulk viscoelasticity of the matrix polymers and more a matter of selective control of slippage and separation of microfibrils at specific and limited sites in the wall. PMID:25613914

  7. Navigating the transcriptional roadmap regulating plant secondary cell wall deposition

    PubMed Central

    Hussey, Steven G.; Mizrachi, Eshchar; Creux, Nicky M.; Myburg, Alexander A.

    2013-01-01

    The current status of lignocellulosic biomass as an invaluable resource in industry, agriculture, and health has spurred increased interest in understanding the transcriptional regulation of secondary cell wall (SCW) biosynthesis. The last decade of research has revealed an extensive network of NAC, MYB and other families of transcription factors regulating Arabidopsis SCW biosynthesis, and numerous studies have explored SCW-related transcription factors in other dicots and monocots. Whilst the general structure of the Arabidopsis network has been a topic of several reviews, they have not comprehensively represented the detailed protein–DNA and protein–protein interactions described in the literature, and an understanding of network dynamics and functionality has not yet been achieved for SCW formation. Furthermore the methodologies employed in studies of SCW transcriptional regulation have not received much attention, especially in the case of non-model organisms. In this review, we have reconstructed the most exhaustive literature-based network representations to date of SCW transcriptional regulation in Arabidopsis. We include a manipulable Cytoscape representation of the Arabidopsis SCW transcriptional network to aid in future studies, along with a list of supporting literature for each documented interaction. Amongst other topics, we discuss the various components of the network, its evolutionary conservation in plants, putative modules and dynamic mechanisms that may influence network function, and the approaches that have been employed in network inference. Future research should aim to better understand network function and its response to dynamic perturbations, whilst the development and application of genome-wide approaches such as ChIP-seq and systems genetics are in progress for the study of SCW transcriptional regulation in non-model organisms. PMID:24009617

  8. Cell Wall Changes in Nectarines (Prunus persica) 1

    PubMed Central

    Dawson, Debra M.; Melton, Laurence D.; Watkins, Christopher B.

    1992-01-01

    Nectarine fruit (Prunus persica L. Batsch var nectarina [Ait] maxim) cultivar Fantasia were either ripened immediately after harvest at 20°C or stored for 5 weeks at 2°C prior to ripening. Fruit ripened after 5 weeks of storage did not soften to the same extent as normally ripened fruit, they lacked juice, and had a dry, mealy texture. Pectic and hemicellulosic polysaccharides were solubilized from the mesocarp of the fruit using phenol:acetic acid:water (PAW) treatment to yield PAW-soluble material and cell wall material (CWM). The carbohydrate composition and relative molecular weight (Mr) of polysaccharide fractions released from the CWM by sequential treatment with cyclohexane-trans-1,2-diamine tetra-acetate, 0.05 m Na2CO3, 6 m guanidinium thiocyanate, and 4 m KOH were determined. Normal ripening of nectarines resulted in solubilization of pectic polymers of high Mr from CWM during the first 2 d at ripening temperatures. Concurrently, galactan side chains were removed from pectic polymers. Solubilized pectic polymers were depolymerized to lower Mr species during the latter stages of ripening. Upon removal from cool storage, fruit had undergone some pectic polymer solubilization, and after ripening, pectins were not depolymerized and were of high Mr. Side chains did not appear to be removed from insoluble pectic polymers and branched pectins accumulated in the CWM. The molecular weight profiles obtained by gel filtration of the hemicellulosic fractions from normally ripening and mealy fruit were similar. The results suggest that mealiness results as a consequence of altered pectic polymer breakdown, including that associated with neutral side chains. PMID:16653106

  9. Xyloglucans from flaxseed kernel cell wall: Structural and conformational characterisation.

    PubMed

    Ding, Huihuang H; Cui, Steve W; Goff, H Douglas; Chen, Jie; Guo, Qingbin; Wang, Qi

    2016-10-20

    The structure of ethanol precipitated fraction from 1M KOH extracted flaxseed kernel polysaccharides (KPI-EPF) was studied for better understanding the molecular structures of flaxseed kernel cell wall polysaccharides. Based on methylation/GC-MS, NMR spectroscopy, and MALDI-TOF-MS analysis, the dominate sugar residues of KPI-EPF fraction comprised of (1,4,6)-linked-β-d-glucopyranose (24.1mol%), terminal α-d-xylopyranose (16.2mol%), (1,2)-α-d-linked-xylopyranose (10.7mol%), (1,4)-β-d-linked-glucopyranose (10.7mol%), and terminal β-d-galactopyranose (8.5mol%). KPI-EPF was proposed as xyloglucans: The substitution rate of the backbone is 69.3%; R1 could be T-α-d-Xylp-(1→, or none; R2 could be T-α-d-Xylp-(1→, T-β-d-Galp-(1→2)-α-d-Xylp-(1→, or T-α-l-Araf-(1→2)-α-d-Xylp-(1→; R3 could be T-α-d-Xylp-(1→, T-β-d-Galp-(1→2)-α-d-Xylp-(1→, T-α-l-Fucp-(1→2)-β-d-Galp-(1→2)-α-d-Xylp-(1→, or none. The Mw of KPI-EPF was calculated to be 1506kDa by static light scattering (SLS). The structure-sensitive parameter (ρ) of KPI-EPF was calculated as 1.44, which confirmed the highly branched structure of extracted xyloglucans. This new findings on flaxseed kernel xyloglucans will be helpful for understanding its fermentation properties and potential applications. PMID:27474598

  10. Structure of cellulose-deficient secondary cell walls from the irx3 mutant of Arabidopsis thaliana.

    PubMed

    Ha, Marie-Ann; MacKinnon, Iain M; Sturcová, Adriana; Apperley, David C; McCann, Maureen C; Turner, Simon R; Jarvis, Michael C

    2002-09-01

    In the Arabidopsis mutant irx3, truncation of the AtCesA7 gene encoding a xylem-specific cellulose synthase results in reduced cellulose synthesis in the affected xylem cells and collapse of mature xylem vessels. Here we describe spectroscopic experiments to determine whether any cellulose, normal or abnormal, remained in the walls of these cells and whether there were consequent effects on other cell-wall polysaccharides. Xylem cell walls from irx3 and its wild-type were prepared by anatomically specific isolation and were examined by solid-state NMR spectroscopy and FTIR microscopy. The affected cell walls of irx3 contained low levels of crystalline cellulose, probably associated with primary cell walls. There was no evidence that crystalline cellulose was replaced by less ordered glucans. From the molecular mobility of xylans and lignin it was deduced that these non-cellulosic polymers were cross-linked together in both irx3 and the wild-type. The disorder previously observed in the spatial pattern of non-cellulosic polymer deposition in the secondary walls of irx3 xylem could not be explained by any alteration in the structure or cross-linking of these polymers and may be attributed directly to the absence of cellulose microfibrils which, in the wild-type, scaffold the organisation of the other polymers into a coherent secondary cell wall. PMID:12165296

  11. Chemical and enzymatic extraction of heavy metal binding polymers from isolated cell walls of Saccharomyces cerevisiae

    SciTech Connect

    Brady, D.; Stoll, A.D.; Starke, L.; Duncan, J.R. . Dept. of Biochemistry and Microbiology)

    1994-07-01

    Isolated cell walls of the yeast Saccharomyces cerevisiae were treated by either chemical (alkali and acid) or enzymatic (protease, mannanase or [beta]-glucuronidase) processes to yield partially purified products. These products were partially characterized by infrared analysis. They were subsequently reacted with heavy metal cation solutions and the quantity of metal accumulated by the cell wall material determined. The Cu[sup 2+] ion (0.24, 0.36, 1.12, and 0.60 [mu]mol/mg) was accumulated to a greater extent than either Co[sup 2+] (0.13, 0.32, 0.43, and 0.32 [mu]mol/mg) or Cd[sup 2+] (0.17, 0.34, 0.39, and 0.46 [mu]mol/mg) by yeast cell walls, glucan, mannan, and chitin, respectively. The isolated components each accumulated greater quantities of the cations than the intact cell wall. Removal of the protein component of the yeast cell wall by Pronase caused a 29.5% decrease in metal accumulation by yeast cell walls per mass, indicating that protein is a heavy metal accumulating component. The data indicate that the outer mannan-protein layer of the yeast cell wall is more important than the inner glucan-chitin layer in heavy metal cation accumulation.

  12. Altered cell wall properties are responsible for ammonium-reduced aluminium accumulation in rice roots.

    PubMed

    Wang, Wei; Zhao, Xue Qiang; Chen, Rong Fu; Dong, Xiao Ying; Lan, Ping; Ma, Jian Feng; Shen, Ren Fang

    2015-07-01

    The phytotoxicity of aluminium (Al) ions can be alleviated by ammonium (NH4(+)) in rice and this effect has been attributed to the decreased Al accumulation in the roots. Here, the effects of different nitrogen forms on cell wall properties were compared in two rice cultivars differing in Al tolerance. An in vitro Al-binding assay revealed that neither NH4(+) nor NO3(-) altered the Al-binding capacity of cell walls, which were extracted from plants not previously exposed to N sources. However, cell walls extracted from NH4(+)-supplied roots displayed lower Al-binding capacity than those from NO3(-)-supplied roots when grown in non-buffered solutions. Fourier-transform infrared microspectroscopy analysis revealed that, compared with NO3(-)-supplied roots, NH4(+)-supplied roots possessed fewer Al-binding groups (-OH and COO-) and lower contents of pectin and hemicellulose. However, when grown in pH-buffered solutions, these differences in the cell wall properties were not observed. Further analysis showed that the Al-binding capacity and properties of cell walls were also altered by pHs alone. Taken together, our results indicate that the NH4(+)-reduced Al accumulation was attributed to the altered cell wall properties triggered by pH decrease due to NH4(+) uptake rather than direct competition for the cell wall binding sites between Al(3+) and NH4(+). PMID:25444246

  13. Recent advances in the understanding of the Aspergillus fumigatus cell wall.

    PubMed

    Lee, Mark J; Sheppard, Donald C

    2016-03-01

    Over the past several decades, research on the synthesis and organization of the cell wall polysaccharides of Aspergillus fumigatus has expanded our knowledge of this important fungal structure. Besides protecting the fungus from environmental stresses and maintaining structural integrity of the organism, the cell wall is also the primary site for interaction with host tissues during infection. Cell wall polysaccharides are important ligands for the recognition of fungi by the innate immune system and they can mediate potent immunomodulatory effects. The synthesis of cell wall polysaccharides is a complicated process that requires coordinated regulation of many biosynthetic and metabolic pathways. Continuous synthesis and remodeling of the polysaccharides of the cell wall is essential for the survival of the fungus during development, reproduction, colonization and invasion. As these polysaccharides are absent from the human host, these biosynthetic pathways are attractive targets for antifungal development. In this review, we present recent advances in our understanding of Aspergillus fumigatus cell wall polysaccharides, including the emerging role of cell wall polysaccharides in the host-pathogen interaction. PMID:26920883

  14. Expression of Arabidopsis callose synthase 5 results in callose accumulation and cell wall permeability alteration.

    PubMed

    Xie, Bo; Deng, Yunfei; Kanaoka, Masahiro M; Okada, Kiyotaka; Hong, Zonglie

    2012-02-01

    Callose is the major polysaccharide present in the callose wall of developing microspores and the growing pollen tube wall. It is also an essential component of other specialized cell walls and its synthesis can be induced by pathogen infection, wounding and environmental cues. Among the 12 callose synthase genes (CalS) present in the Arabidopsis genome, CalS5 plays the predominant role in the synthesis of the callose wall, callose plugs and pollen tube wall. When expressed as a GFP-tagged protein in cultured tobacco BY-2 cells, CalS5 was found to be present in the plasma membrane and the Golgi-related endomembranes. Unlike the cell plate-specific CalS1 isozyme, CalS5 was not concentrated to the cell plate at cytokinesis. Expression of CalS5 resulted in callose accumulation only in the cell wall of BY-2 cells. The fact that no callose was found in the endomembranes suggests that CalS5 is not functional in that compartment. These cells exhibited a decreased plasmolysis rate in hypotonic solutions and an increased cytolysis rate in hypertonic conditions. This study demonstrates that an artificial callose wall could be synthesized by expressing a callose synthase enzyme. PMID:22195570

  15. Electrical Potentials of Plant Cell Walls in Response to the Ionic Environment1

    PubMed Central

    Shomer, Ilan; Novacky, Anton J.; Pike, Sharon M.; Yermiyahu, Uri; Kinraide, Thomas B.

    2003-01-01

    Electrical potentials in cell wallsWall) and at plasma membrane surfaces (ψPM) are determinants of ion activities in these phases. The ψPM plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of ψWall. ψWall from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of ψWall with effectiveness in the order Al3+ > La3+ > H+ > Cu2+ > Ni2+ > Ca2+ > Co2+ > Cd2+ > Mg2+ > Zn2+ > hexamethonium2+ > Rb+ > K+ > Cs+ > Na+. This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of ψWall, and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for ψWall were in close agreement, usually, and we consider ψWall to be at least proportional to the actual Donnan potentials. ψWall and ψPM display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. ψWall is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion. PMID:12970506

  16. Electrical potentials of plant cell walls in response to the ionic environment.

    PubMed

    Shomer, Ilan; Novacky, Anton J; Pike, Sharon M; Yermiyahu, Uri; Kinraide, Thomas B

    2003-09-01

    Electrical potentials in cell walls (psi(Wall)) and at plasma membrane surfaces (psi(PM)) are determinants of ion activities in these phases. The psi(PM) plays a demonstrated role in ion uptake and intoxication, but a comprehensive electrostatic theory of plant-ion interactions will require further understanding of psi(Wall). psi(Wall) from potato (Solanum tuberosum) tubers and wheat (Triticum aestivum) roots was monitored in response to ionic changes by placing glass microelectrodes against cell surfaces. Cations reduced the negativity of psi(Wall) with effectiveness in the order Al(3+) > La(3+) > H(+) > Cu(2+) > Ni(2+) > Ca(2+) > Co(2+) > Cd(2+) > Mg(2+) > Zn(2+) > hexamethonium(2+) > Rb(+) > K(+) > Cs(+) > Na(+). This order resembles substantially the order of plant-root intoxicating effectiveness and indicates a role for both ion charge and size. Our measurements were combined with the few published measurements of psi(Wall), and all were considered in terms of a model composed of Donnan theory and ion binding. Measured and model-computed values for psi(Wall) were in close agreement, usually, and we consider psi(Wall) to be at least proportional to the actual Donnan potentials. psi(Wall) and psi(PM) display similar trends in their responses to ionic solutes, but ions appear to bind more strongly to plasma membrane sites than to readily accessible cell wall sites. psi(Wall) is involved in swelling and extension capabilities of the cell wall lattice and thus may play a role in pectin bonding, texture, and intercellular adhesion. PMID:12970506

  17. Mal3 masks catastrophe events in Schizosaccharomyces pombe microtubules by inhibiting shrinkage and promoting rescue.

    PubMed

    Katsuki, Miho; Drummond, Douglas R; Osei, Michael; Cross, Robert A

    2009-10-23

    Schizosaccharomyces pombe Mal3 is a member of the EB family of proteins, which are proposed to be core elements in a tip-tracking network that regulates microtubule dynamics in cells. How Mal3 itself influences microtubule dynamics is unclear. We tested the effects of full-length recombinant Mal3 on dynamic microtubules assembled in vitro from purified S. pombe tubulin, using dark field video microscopy to avoid fluorescent tagging and data-averaging techniques to improve spatiotemporal resolution. We find that catastrophe occurs stochastically as a fast (<2.2 s) transition from constant speed growth to constant speed shrinkage with a constant probability that is independent of the Mal3 concentration. This implies that Mal3 neither stabilizes nor destabilizes microtubule tips. Mal3 does, however, stabilize the main part of the microtubule lattice, inhibiting shrinkage and increasing the frequency of rescues, consistent with recent models in which Mal3 on the lattice forms stabilizing lateral links between neighboring protofilaments. At high concentrations, Mal3 can entirely block shrinkage and induce very rapid rescue, making catastrophes impossible to detect, which may account for the apparent suppression of catastrophe by Mal3 and other EBs in vivo. Overall, we find that Mal3 stabilizes microtubules not by preventing catastrophe at the microtubule tip but by inhibiting lattice depolymerization and enhancing rescue. We argue that this implies that Mal3 binds microtubules in different modes at the tip and on the lattice. PMID:19740752

  18. Dimeric tRNA gene arrangement in Schizosaccharomyces pombe allows increased expression of the downstream gene.

    PubMed Central

    Hottinger-Werlen, A; Schaack, J; Lapointe, J; Mao, J; Nichols, M; Söll, D

    1985-01-01

    Three Schizosaccharomyces pombe dimeric tRNA genes, consisting of a tRNASer gene encoding a minor species with an intervening sequence followed by a tRNAMeti gene, have been described [Mao et al. (1980) Cell 21, 509-516; Hottinger et al. (1982) Mol. Gen. Genet. 188, 219-224; Willis et al. (1984) EMBO J. 3, 1573-1580]. We have examined the reason for the dimeric structure by comparing the transcriptional efficiencies and competitive abilities of the genes subcloned from the dimeric arrangement. Both of the subcloned genes are active in vivo in Saccharomyces cerevisiae, but only the tRNASer gene is efficiently transcribed in vitro. The tRNASer gene competes efficiently for transcription factors, while the tRNAMeti gene does so only weakly. Thus, it appears that the dimeric arrangement is required to support expression of the tRNAMeti gene. S. pombe genes encoding major species of tRNASer are transcribed considerably less efficiently than are the minor genes from the dimers, so coupling of the tRNAMeti gene to the minor species genes should lead to efficient production of tRNAMeti. Images PMID:3936021

  19. Identification of nuclear genes affecting 2-Deoxyglucose resistance in Schizosaccharomyces pombe.

    PubMed

    Vishwanatha, Akshay; Rallis, Charalampos; Bevkal Subramanyaswamy, Shubha; D'Souza, Cletus Joseph Michael; Bähler, Jürg; Schweingruber, Martin Ernst

    2016-09-01

    2-Deoxyglucose (2-DG) is a toxic glucose analog. To identify genes involved in 2-DG toxicity in Schizosaccharomyces pombe, we screened a wild-type overexpression library for genes which render cells 2-DG resistant. A gene we termed odr1, encoding an uncharacterized hydrolase, led to strong resistance and altered invertase expression when overexpressed. We speculate that Odr1 neutralizes the toxic form of 2-DG, similar to the Saccharomyces cerevisiae Dog1 and Dog2 phosphatases which dephosphorylate 2-DG-6-phosphate synthesized by hexokinase. In a complementary approach, we screened a haploid deletion library to identify 2-DG-resistant mutants. This screen identified the genes snf5, ypa1, pas1 and pho7 In liquid medium, deletions of these genes conferred 2-DG resistance preferentially under glucose-repressed conditions. The deletion mutants expressed invertase activity more constitutively than the control strain, indicating defects in the control of glucose repression. No S. cerevisiae orthologs of the pho7 gene is known, and no 2-DG resistance has been reported for any of the deletion mutants of the other genes identified here. Moreover, 2-DG leads to derepressed invertase activity in S. pombe, while in S. cerevisiae it becomes repressed. Taken together, these findings suggest that mechanisms involved in 2-DG resistance differ between budding and fission yeasts. PMID:27481777

  20. 'Strengthening the fungal cell wall through chitin-glucan cross-links: effects on morphogenesis and cell integrity'.

    PubMed

    Arroyo, Javier; Farkaš, Vladimír; Sanz, Ana Belén; Cabib, Enrico

    2016-09-01

    The cross-linking of polysaccharides to assemble new cell wall in fungi requires transglycosylation mechanisms by which preexisting glycosidic linkages are broken and new linkages are created between the polysaccharides. The molecular mechanisms for these processes, which are essential for fungal cell biology, are only now beginning to be elucidated. Recent development of in vivo and in vitro biochemical approaches has allowed characterization of important aspects about the formation of chitin-glucan covalent cell wall cross-links by cell wall transglycosylases of the CRH family and their biological function. Covalent linkages between chitin and glucan mediated by Crh proteins control morphogenesis and also play important roles in the remodeling of the fungal cell wall as part of the compensatory responses necessary to counterbalance cell wall stress. These enzymes are encoded by multigene families of redundant proteins very well conserved in fungal genomes but absent in mammalian cells. Understanding the molecular basis of fungal adaptation to cell wall stress through these and other cell wall remodeling enzymatic activities offers an opportunity to explore novel antifungal treatments and to identify potential fungal virulence factors. PMID:27185288

  1. Identification of a Streptococcus salivarius Cell Wall Component Mediating Coaggregation with Veillonella alcalescens VI

    PubMed Central

    Weerkamp, Anton H.; McBride, Barry C.

    1981-01-01

    Cell walls of Streptococcus salivarius HB aggregated Veillonella alcalescens V1, but cell walls of the mutant S. salivarius HB-V5 did not. We found no correlation between the presence of fimbriae on streptococcal walls and the ability to aggregate Veillonella strains. Treatment of the walls with lysozyme solubilized a fraction which possessed Veillonella-aggregating activity. Solubilized cell wall preparations of strain HB contained three major (glyco)proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at least four antigens as determined by immunoelectrophoresis with antiserum prepared against strain HB walls. A specific antiserum, which was obtained by adsorption of anti-HB serum on strain HB-V5 cells, contained monospecific antibody that reacted with the solubilized strain HB wall preparation. Similar fractions prepared from strain HB-V5 cell walls did not possess aggregating activity and lacked one protein band (protein I) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and one antigen (antigen b) after immunoelectrophoresis. The same antigen was absent when lysozyme-solubilized wall preparations of strain HB were reacted with anti-HB-V5 serum. Crossed-immunoisoelectric focusing indicated that this specific (glyco)protein and this antigen were identical and had an isoelectric point of 4.60. Protein I and antigen b were specifically adsorbed when solubilized strain HB cell walls were incubated with V. alcalescens V1 but were not adsorbed by nonaggregating Veillonella parvula ATCC 10790 cells. Culture supernatants of strain HB contained V. alcalescens V1-aggregating activity. Antigen b was present in the culture supernatant, but was not found in cultures of strain HB-V5. A total of 18 S. salivarius isolates possessing the streptococcal group K antigen released aggregating activity and antigen b into the culture medium, but 11 strains which lacked the K-antigen did not. Images PMID:7251145

  2. Studies on the control of cell wall extension. Progress report, September 1, 1979-August 31, 1980

    SciTech Connect

    Cleland, R. E.

    1980-09-24

    The growth rate of plant cells can be defined in terms of five parameters: the hydraulic conductivity of the tissue (L'p), the rate of export from the cell to the wall of a wall loosening factor (dW/dt), the capacity of the wall to be loosened in response to the wall loosening factor (WLC), the osmotic potential of the cells and outside solution (..pi../sub c/ and ..pi../sub o/, respectively), and the yield threshold for wall expansion (Y). The study assesses each of these parameters under conditions where growth is being enhanced or inhibited, with the purpose of explaining at the cellular level just how growth is being controlled. (ACR)

  3. Membrane organization and cell fusion during mating in fission yeast requires multipass membrane protein Prm1.

    PubMed

    Curto, M-Ángeles; Sharifmoghadam, Mohammad Reza; Calpena, Eduardo; De León, Nagore; Hoya, Marta; Doncel, Cristina; Leatherwood, Janet; Valdivieso, M-Henar

    2014-04-01

    The involvement of Schizosaccharomyces pombe prm1(+) in cell fusion during mating and its relationship with other genes required for this process have been addressed. S. pombe prm1Δ mutant exhibits an almost complete blockade in cell fusion and an abnormal distribution of the plasma membrane and cell wall in the area of cell-cell interaction. The distribution of cellular envelopes is similar to that described for mutants devoid of the Fig1-related claudin-like Dni proteins; however, prm1(+) and the dni(+) genes act in different subpathways. Time-lapse analyses show that in the wild-type S. pombe strain, the distribution of phosphatidylserine in the cytoplasmic leaflet of the plasma membrane undergoes some modification before an opening is observed in the cross wall at the cell-cell contact region. In the prm1Δ mutant, this membrane modification does not take place, and the cross wall between the mating partners is not extensively degraded; plasma membrane forms invaginations and fingers that sometimes collapse/retract and that are sometimes strengthened by the synthesis of cell-wall material. Neither prm1Δ nor prm1Δ dniΔ zygotes lyse after cell-cell contact in medium containing and lacking calcium. Response to drugs that inhibit lipid synthesis or interfere with lipids is different in wild-type, prm1Δ, and dni1Δ strains, suggesting that membrane structure/organization/dynamics is different in all these strains and that Prm1p and the Dni proteins exert some functions required to guarantee correct membrane organization that are critical for cell fusion. PMID:24514900

  4. Temperature Gradients on the Cell Wall in the Critical Viscosity Experiment

    NASA Technical Reports Server (NTRS)

    Berg, Robert F.; Moldover, Michael R.

    1993-01-01

    Because of the diverging susceptibility delta rho/delta Tau near the liquid-vapor critical point, temperature gradients must be kept small to maintain adequate sample homogeneity. In our Science Requirements Document we paid particular attention to radial density gradients caused by equilibration of the xenon sample. Axial density gradients were addressed through the requirement that the cell's copper wall have a gradient less than 22 microK/m. This report re-examines the cell wall's temperature distribution in more detail by estimating all known significant contributions to temperature differences on the cell's wall.

  5. Influence of cell wall shape and density on the mechanical behaviour of 2D foam structures

    SciTech Connect

    Harders, Harald . E-mail: h.harders@tu-bs.de; Hupfer, Knut; Roesler, Joachim

    2005-03-01

    This article describes simulations on the influence of the cell wall shape as well as the density on the elastic stiffness of regular and stochastic honeycomb structures. Starting from an equation by Gibson and Ashby for regular honeycombs, an analytical model is developed that describes the influence of different cell wall shapes on the elastic response of these structures. In addition, this analytical model is modified in order to use free parameters that can be fitted to finite element simulation results of stochastic Voronoi honeycomb structures. The model describes the results well. Young's modulus depends strongly on the cell wall shape, achieving a maximum for slightly concave shapes.

  6. [Lysis of the cell walls of streptococcus group A by Streptomyces griseus pronase].

    PubMed

    Savel'ev, E P; Petrov, G I

    1978-01-01

    The effect of Streptomyces griseus pronase on Streptococcus group A cell walls was studied. Cell walls were shown to be lysed by pronase, the lysis level being dependent on the molarity of the potassium-phosphate buffer used. With an increase in the buffer molarity from 0.005 M to 0.05 M lysis of cell walls decreased from 70-80% to 30%. By DEAE-cellulose chromatography lysates were separated into two fractions the first of which contained a group specific polysaccharide. A preparative method of obtaining a group specific polysaccharide of Streptococcus group A using Streptomyces griseus pronase under mild conditions is described. PMID:416431

  7. A Novel Isoform of Sucrose Synthase Is Targeted to the Cell Wall during Secondary Cell Wall Synthesis in Cotton Fiber[C][W][OA

    PubMed Central

    Brill, Elizabeth; van Thournout, Michel; White, Rosemary G.; Llewellyn, Danny; Campbell, Peter M.; Engelen, Steven; Ruan, Yong-Ling; Arioli, Tony; Furbank, Robert T.

    2011-01-01

    Sucrose (Suc) synthase (Sus) is the major enzyme of Suc breakdown for cellulose biosynthesis in cotton (Gossypium hirsutum) fiber, an important source of fiber for the textile industry. This study examines the tissue-specific expression, relative abundance, and temporal expression of various Sus transcripts and proteins present in cotton. A novel isoform of Sus (SusC) is identified that is expressed at high levels during secondary cell wall synthesis in fiber and is present in the cell wall fraction. The phylogenetic relationships of the deduced amino acid sequences indicate two ancestral groups of Sus proteins predating the divergence of monocots and dicots and that SusC sequences form a distinct branch in the phylogeny within the dicot-specific clade. The subcellular location of the Sus isoforms is determined, and it is proposed that cell wall-localized SusC may provide UDP-glucose for cellulose and callose synthesis from extracellular sugars. PMID:21757635

  8. The role of the secondary cell wall in plant resistance to pathogens.

    PubMed

    Miedes, Eva; Vanholme, Ruben; Boerjan, Wout; Molina, Antonio

    2014-01-01

    Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process. PMID:25161657

  9. Detection of wood cell wall porosity using small carbohydrate molecules and confocal fluorescence microscopy.

    PubMed

    Donaldson, L A; Kroese, H W; Hill, S J; Franich, R A

    2015-09-01

    A novel approach to nanoscale detection of cell wall porosity using confocal fluorescence microscopy is described. Infiltration of cell walls with a range of nitrophenyl-substituted carbohydrates of different molecular weights was assessed by measuring changes in the intensity of lignin fluorescence, in response to the quenching effect of the 4-nitrophenyl group. The following carbohydrates were used in order of increasing molecular weight; 4-nitrophenyl β-D-glucopyrano-side (monosaccharide), 4-nitrophenyl β-D-lactopyranoside (disaccharide), 2-chloro-4-nitrophenyl β-D-maltotrioside (trisaccharide), and 4-nitrophenyl α-D-maltopentaoside (pentasaccharide). This technique was used to compare cell wall porosity in wood which had been dewatered to 40% moisture content using supercritical CO2, where cell walls remain fully hydrated, with kiln dried wood equilibrated to 12% moisture content. Infiltration of cell walls as measured by fluorescence quenching, was found to decrease with increasing molecular weight, with the pentasaccharide being significantly excluded compared to the monosaccharide. Porosity experiments were performed on blocks and sections to assess differences in cell wall accessibility. Dewatered and kiln dried wood infiltrated as blocks showed similar results, but greater infiltration was achieved by using sections, indicating that not all pores were easily accessible by infiltration from the lumen surface. In wood blocks infiltrated with 4-nitrophenyl α-D-maltopentaoside, quenching of the secondary wall was quite variable, especially in kiln dried wood, indicating limited connectivity of pores accessible from the lumen surface. PMID:25925133

  10. The role of the secondary cell wall in plant resistance to pathogens

    PubMed Central

    Miedes, Eva; Vanholme, Ruben; Boerjan, Wout; Molina, Antonio

    2014-01-01

    Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process. PMID:25161657

  11. Cell wall changes in nisin-resistant variants of Listeria innocua grown in the presence of high nisin concentrations.

    PubMed

    Maisnier-Patin, S; Richard, J

    1996-06-15

    Two nisin-resistant variants of a strain of Listeria innocua were isolated after growth in the presence of 500 and 4000 IU ml-1 of nisin A showed increased cell wall hydrophobicity, resistance to phage attack and three different cell wall-acting antibiotics, as well as to the peptidoglycan hydrolytic enzymes lysozyme and mutanolysin, as compared to the parental strain. Transmission electron microscopy revealed marked thickening of the wall of nisin-resistant cells with an irregular surface. Differences in thickness were lost after cell wall purification and no significant difference in gross wall composition was observed between the parental and resistant variants. Cell wall changes in nisin-resistant listeriae are attributed to abnormal cell wall synthesis and autolysin inhibition, the latter possibly associated with subtle changes in cell wall structures and function. PMID:8666198

  12. Biochemical and Immunocytological Characterizations of Arabidopsis Pollen Tube Cell Wall1[C][W][OA

    PubMed Central

    Dardelle, Flavien; Lehner, Arnaud; Ramdani, Yasmina; Bardor, Muriel; Lerouge, Patrice; Driouich, Azeddine; Mollet, Jean-Claude

    2010-01-01

    During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1→5)-α-l-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics. PMID:20547702

  13. DISSOLUTION OF THE ENTIRE PLANT CELL WALL FRACTION FOR SOLUTION-STATE NMR (AND CHEMOMETRICS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although dissolution of intact cell walls is problematic, finely divided (ball-milled) walls are amenable to dissolution in two useful solvent systems, DMSO/N-methyl imidazole and DMSO/tetrabutyl ammonium fluoride. In the former system, acetylation is trivially accomplished, producing acetylated cel...

  14. CHARACTERIZATION OF NON-DERIVATIZED PLANT CELL WALLS USING HIGH-RESOLUTION SOLUTION-STATE NMR SPECTROSCOPY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A recently described plant cell wall dissolution system has been logically modified to utilize perdeuterated solvents to allow direct in-nmr-tube dissolution and high-resolution solution-state NMR of the whole cell wall without derivatization. Finely ground cell wall material dissolves in a solvent ...

  15. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility

    PubMed Central

    Eudes, Aymerick; Zhao, Nanxia; Sathitsuksanoh, Noppadon; Baidoo, Edward E. K.; Lao, Jeemeng; Wang, George; Yogiswara, Sasha; Lee, Taek Soon; Singh, Seema; Mortimer, Jenny C.; Keasling, Jay D.; Simmons, Blake A.; Loqué, Dominique

    2016-01-01

    Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet). In this study, we demonstrate in Arabidopsis stems that targeted expression of AdoMet hydrolase (AdoMetase, E.C. 3.3.1.2) in secondary cell wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H) units and a reduction of dimethylated syringyl (S) units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild-type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock. PMID:27486577

  16. Expression of S-adenosylmethionine Hydrolase in Tissues Synthesizing Secondary Cell Walls Alters Specific Methylated Cell Wall Fractions and Improves Biomass Digestibility.

    PubMed

    Eudes, Aymerick; Zhao, Nanxia; Sathitsuksanoh, Noppadon; Baidoo, Edward E K; Lao, Jeemeng; Wang, George; Yogiswara, Sasha; Lee, Taek Soon; Singh, Seema; Mortimer, Jenny C; Keasling, Jay D; Simmons, Blake A; Loqué, Dominique

    2016-01-01

    Plant biomass is a large source of fermentable sugars for the synthesis of bioproducts using engineered microbes. These sugars are stored as cell wall polymers, mainly cellulose and hemicellulose, and are embedded with lignin, which makes their enzymatic hydrolysis challenging. One of the strategies to reduce cell wall recalcitrance is the modification of lignin content and composition. Lignin is a phenolic polymer of methylated aromatic alcohols and its synthesis in tissues developing secondary cell walls is a significant sink for the consumption of the methyl donor S-adenosylmethionine (AdoMet). In this study, we demonstrate in Arabidopsis stems that targeted expression of AdoMet hydrolase (AdoMetase, E.C. 3.3.1.2) in secondary cell wall synthesizing tissues reduces the AdoMet pool and impacts lignin content and composition. In particular, both NMR analysis and pyrolysis gas chromatography mass spectrometry of lignin in engineered biomass showed relative enrichment of non-methylated p-hydroxycinnamyl (H) units and a reduction of dimethylated syringyl (S) units. This indicates a lower degree of methylation compared to that in wild-type lignin. Quantification of cell wall-bound hydroxycinnamates revealed a reduction of ferulate in AdoMetase transgenic lines. Biomass from transgenic lines, in contrast to that in control plants, exhibits an enrichment of glucose content and a reduction in the degree of hemicellulose glucuronoxylan methylation. We also show that these modifications resulted in a reduction of cell wall recalcitrance, because sugar yield generated by enzymatic biomass saccharification was greater than that of wild-type plants. Considering that transgenic plants show no important diminution of biomass yields, and that heterologous expression of AdoMetase protein can be spatiotemporally optimized, this novel approach provides a valuable option for the improvement of lignocellulosic biomass feedstock. PMID:27486577

  17. Villosiclava virens infects specifically rice and barley stamen filaments due to the unique host cell walls.

    PubMed

    Yong, Ming-Li; Fan, Lin-Lin; Li, Dan-Yang; Liu, Yi-Jia; Cheng, Fang-Min; Xu, Ying; Wang, Zheng-Yi; Hu, Dong-Wei

    2016-09-01

    Rice false smut, caused by the fungal pathogen Villosiclava virens, is one of the most important rice diseases in the world. Previous studies reported that the pathogen has less number of cell wall-degraded genes and attacks dominantly rice stamen filaments and extends intercellularly. To reveal why the fungus infects plant stamen filaments, inoculation test on barley was carried out with the similar protocol to rice. The experimental results showed that the fungus could penetrate quickly into barley stamen filaments and extends both intracellularly and intercellularly, usually resulting in severe damage of the stamen filament tissues. It also attacked young barley lodicules and grew intercellularly by chance. The light microscopic observations found that the epidermal and cortex cells in barley stamen filaments arranged loosely with very thick cell walls and large cell gaps. Cellulose microfibrils in barley stamen filament cell walls arranged very sparsely so that the cell walls looked like transparent. The cell walls were very soft and flexible, and often folded. However, V. virens extended dominantly in the noncellulose regions and seemed never to degrade microfibrils in barley and rice cell walls. This suggested that the unique structures of rice and barley stamen filaments should be fit for their function of elongation in anthesis, and also endow with the susceptibility to the fungus, V. virens. PMID:27357263

  18. The Genomic and Phenotypic Diversity of Schizosaccharomyces pombe

    PubMed Central

    Jeffares, Daniel C.; Rallis, Charalampos; Rieux, Adrien; Speed, Doug; Převorovský, Martin; Mourier, Tobias; Marsellach, Francesc X.; Iqbal, Zamin; Lau, Winston; Cheng, Tammy M.K.; Pracana, Rodrigo; Mülleder, Michael; Lawson, Jonathan L.D.; Chessel, Anatole; Bala, Sendu; Hellenthal, Garrett; O’Fallon, Brendan; Keane, Thomas; Simpson, Jared T.; Bischof, Leanne; Tomiczek, Bartlomiej; Bitton, Danny A.; Sideri, Theodora; Codlin, Sandra; Hellberg, Josephine E.E.U.; van Trigt, Laurent; Jeffery, Linda; Li, Juan-Juan; Atkinson, Sophie; Thodberg, Malte; Febrer, Melanie; McLay, Kirsten; Drou, Nizar; Brown, William; Hayles, Jacqueline; Carazo Salas, Rafael E.; Ralser, Markus; Maniatis, Nikolas; Balding, David J.; Balloux, Francois; Durbin, Richard; Bähler, Jürg

    2015-01-01

    Natural variation within species reveals aspects of genome evolution and function. The fission yeast Schizosaccharomyces pombe is an important model for eukaryotic biology, but researchers typically use one standard laboratory strain. To extend the utility of this model, we surveyed the genomic and phenotypic variation in 161 natural isolates. We sequenced the genomes of all strains, revealing moderate genetic diversity (π = 3 ×10−3) and weak global population structure. We estimate that dispersal of S. pombe began within human antiquity (~340 BCE), and ancestors of these strains reached the Americas at ~1623 CE. We quantified 74 traits, revealing substantial heritable phenotypic diversity. We conducted 223 genome-wide association studies, with 89 traits showing at least one association. The most significant variant for each trait explained 22% of variance on average, with indels having higher effects than SNPs. This analysis presents a rich resource to examine genotype-phenotype relationships in a tractable model. PMID:25665008

  19. Differential expressions of essential and nonessential alpha-tubulin genes in Schizosaccharomyces pombe.

    PubMed

    Adachi, Y; Toda, T; Niwa, O; Yanagida, M

    1986-06-01

    The fission yeast Schizosaccharomyces pombe has two alpha-tubulin genes and one beta-tubulin gene. Gene disruption experiments showed that the alpha 1-tubulin gene (NDA2) is essential whereas the alpha 2 gene is dispensable. The alpha 2-disrupted cells missing alpha 2 transcript and alpha 2 polypeptide could grow and sporulate normally. The alpha 2 gene, however, was expressed in the wild type and the alpha 1 mutant. Alpha 2-Tubulin was distinguished as an electrophoretic band and was assembled into microtubules. The alpha 2-disrupted cells had an increased sensitivity to an antimicrotubule drug thiabendazole, and the alpha 1(cold-sensitive [cs]) alpha 2 (disrupted) cells became not only cs but also temperature sensitive. Northern blot experiments indicated that alpha 2 transcription was minor and constitutive whereas alpha 1 transcription was major and modulated, depending on the gene copy number of the alpha 2 gene. The amounts of alpha 1 and alpha 2 polypeptides estimated by beta-galactosidase activities of the lacZ-fused genes integrated on the chromosome and by intensities of the electrophoretic bands in crude tubulin fractions, however, were comparable, indicating that alpha 2 tubulin is not a minor subtype. We assume that the cells of Schizosaccharomyces pombe have no excess tubulin pool. alpha 1 mutants would then be blocked in the cell cycle because only half the amount of functional alpha-tubulin required for growth can be produced by the alpha 2 gene. On the other hand, the alpha 2-disrupted cells became viable because the synthesis of alpha 1 tubulin was increased by transcriptional or translational modulation or both. The real cause for essential alpha 1 and dispensable alpha 2 genes seems to be in their regulatory sequences instead of the coding sequences. PMID:3785193

  20. Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

    DOE PAGESBeta

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G.; Ragauskas, Arthur J.; Kieliszewski, Marcia J.

    2014-12-23

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increasedmore » wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. In conclusion, these data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.« less

  1. Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

    SciTech Connect

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G.; Ragauskas, Arthur J.; Kieliszewski, Marcia J.

    2014-12-23

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. In conclusion, these data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

  2. Changes in Cell Wall Properties Coincide with Overexpression of Extensin Fusion Proteins in Suspension Cultured Tobacco Cells

    PubMed Central

    Tan, Li; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G.; Ragauskas, Arthur J.; Kieliszewski, Marcia J.

    2014-01-01

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production. PMID:25536327

  3. Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

    PubMed

    Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G; Ragauskas, Arthur J; Kieliszewski, Marcia J

    2014-01-01

    Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production. PMID:25536327

  4. A cell wall protein down-regulated by auxin suppresses cell expansion in Daucus carota (L.).

    PubMed

    Holk, A; Klumpp, L; Scherer, G F E

    2002-09-01

    We investigated the function of the auxin-regulated cell wall gene DC 2.15, a member of a small gene family, present in Daucus carota (L.) and other plants. Cultured cells derived from carrot hypocotyls transformed by the DC 2.15 cDNA in antisense direction were ten-fold longer than wild-type cells, indicating a function of the corresponding protein in suppression of cell expansion. The analysis of carrot plants expressing the DC2.15 gene in antisense direction showed that the corresponding protein and/or related proteins probably are involved in leaf and vascular bundle development. The antisense plants generally displayed a retarded growth phenotype and delayed greening in comparison to wild-type plants. The asymmetric architecture of the wild-type leaves was degenerated in the DC 2.15 antisense plants and the leaves showed a torsion within and along their major vein. The vascular bundles showed a lowered ratio of the phloem/xylem area in cross sections of the leaf middle vein whereas the bundle sheath and the cambium showed no obvious phenotype. Expression of a promoter-GUS construct was found primarily in vascular bundles of stems, leaves and in the nectar-producing flower discs. The observed pleiotropic antisense phenotype indicates, by loss of function, that one or several related cell wall proteins of this gene family are necessary to realize several complex developmental processes. PMID:12175021

  5. Primary Cell Wall Composition of Bryophytes and Charophytes

    PubMed Central

    POPPER, ZOË A.; FRY, STEPHEN C.

    2003-01-01

    Major differences in primary cell wall (PCW) components between non‐vascular plant taxa are reported. (1) Xyloglucan: driselase digestion yielded isoprimeverose (the diagnostic repeat unit of xyloglucan) from PCW‐rich material of Anthoceros (a hornwort), mosses and both leafy and thalloid liverworts, as well as numerous vascular plants, showing xyloglucan to be a PCW component in all land plants tested. In contrast, charophycean green algae (Klebsormidium flaccidium, Coleochaete scutata and Chara corallina), thought to be closely related to land plants, did not contain xyloglucan. They did not yield isoprimeverose; additionally, charophyte material was not digestible with xyloglucan‐specific endoglucanase or cellulase to give xyloglucan‐derived oligosaccharides. (2) Uronic acids: acid hydrolysis of PCW‐rich material from the charophytes, the hornwort, thalloid and leafy liverworts and a basal moss yielded higher concentrations of glucuronic acid than that from the remaining land plants including the less basal mosses and all vascular plants tested. Polysaccharides of the hornwort Anthoceros contained an unusual repeat‐unit, glucuronic acid‐α(1→3)‐galactose, not found in appreciable amounts in any other plants tested. Galacturonic acid was consistently the most abundant PCW uronic acid, but was present in higher concentrations in acid hydrolysates of bryophytes and charophytes than in those of any of the vascular plants. Mannuronic acid was not detected in any of the species surveyed. (3) Mannose: acid hydrolysis of charophyte and bryophyte PCW‐rich material also yielded appreciably higher concentrations of mannose than are found in vascular plant PCWs. (4) Mixed‐linkage glucan (MLG) was absent from all algae and bryophytes tested; however, upon digestion with licheninase, PCW‐rich material from the alga Ulva lactuca and the leafy liverwort Lophocolea bidentata yielded penta‐ to decasaccharides, indicating the presence of MLG

  6. Cell wall polysaccharides are mislocalized to the Vacuole in echidna mutants.

    PubMed

    McFarlane, Heather E; Watanabe, Yoichiro; Gendre, Delphine; Carruthers, Kimberley; Levesque-Tremblay, Gabriel; Haughn, George W; Bhalerao, Rishikesh P; Samuels, Lacey

    2013-11-01

    During cell wall biosynthesis, the Golgi apparatus is the platform for cell wall matrix biosynthesis and the site of packaging, of both matrix polysaccharides and proteins, into secretory vesicles with the correct targeting information. The objective of this study was to dissect the post-Golgi trafficking of cell wall polysaccharides using echidna as a vesicle traffic mutant of Arabidopsis thaliana and the pectin-secreting cells of the seed coat as a model system. ECHIDNA encodes a trans-Golgi network (TGN)-localized protein, which was previously shown to be required for proper structure and function of the secretory pathway. In echidna mutants, some cell wall matrix polysaccharides accumulate inside cells, rather than being secreted to the apoplast. In this study, live cell imaging of fluorescent protein markers as well as transmission electron microscopy (TEM)/immunoTEM of cryofixed seed coat cells were used to examine the consequences of TGN disorganization in echidna mutants under conditions of high polysaccharide production and secretion. While in wild-type seed coat cells, pectin is secreted to the apical surface, in echidna, polysaccharides accumulate in post-Golgi vesicles, the central lytic vacuole and endoplasmic reticulum-derived bodies. In contrast, proteins were partially mistargeted to internal multilamellar membranes in echidna. These results suggest that while secretion of both cell wall polysaccharides and proteins at the TGN requires ECHIDNA, different vesicle trafficking components may mediate downstream events in their secretion from the TGN. PMID:24058145

  7. Enzymatic cell wall degradation of Chlorella vulgaris and other microalgae for biofuels production.

    PubMed

    Gerken, Henri G; Donohoe, Bryon; Knoshaug, Eric P

    2013-01-01

    Cell walls of microalgae consist of a polysaccharide and glycoprotein matrix providing the cells with a formidable defense against its environment. We characterized enzymes that can digest the cell wall and weaken this defense for the purpose of protoplasting or lipid extraction. A growth inhibition screen demonstrated that chitinase, lysozyme, pectinase, sulfatase, β-glucuronidase, and laminarinase had the broadest effect across the various Chlorella strains tested and also inhibited Nannochloropsis and Nannochloris strains. Chlorella is typically most sensitive to chitinases and lysozymes, both enzymes that degrade polymers containing N-acetylglucosamine. Using a fluorescent DNA stain, we developed rapid methodology to quantify changes in permeability in response to enzyme digestion and found that treatment with lysozyme in conjunction with other enzymes has a drastic effect on cell permeability. Transmission electron microscopy of enzymatically treated Chlorella vulgaris indicates that lysozyme degrades the outer surface of the cell wall and removes hair-like fibers protruding from the surface, which differs from the activity of chitinase. This action on the outer surface of the cell causes visible protuberances on the cell surface and presumably leads to the increased settling rate when cells are treated with lysozyme. We demonstrate radical ultrastructural changes to the cell wall in response to treatment with various enzyme combinations which, in some cases, causes a greater than twofold increase in the thickness of the cell wall. The enzymes characterized in this study should prove useful in the engineering and extraction of oils from microalgae. PMID:23011569

  8. Topochemical and morphological characterization of wood cell wall treated with the ionic liquid, 1-ethylpyridinium bromide.

    PubMed

    Kanbayashi, Toru; Miyafuji, Hisashi

    2015-09-01

    MAIN CONCLUSION : [EtPy][Br] is more reactive toward lignin than toward the PSs in wood cell walls, and [EtPy][Br] treatment results in inhomogenous changes to the cell wall's ultrastructural and chemical components. The effects of the ionic liquid 1-ethylpyridinium bromide ([EtPy][Br]), which prefers to react with lignin rather than cellulose on the wood cell walls of Japanese cedar (Cryptomeria japonica), were investigated from a morphology and topochemistry point of view. The [EtPy][Br] treatment induced cell wall swelling, the elimination of warts, and the formation of countless pores in the tracheids. However, many of the pit membranes and the cellulose crystalline structure remained unchanged. Raman microscopic analyses revealed that chemical changes in the cell walls were different for different layers and that the lignin in the compound middle lamella and the cell corner resists interaction with [EtPy][Br]. Additionally, the interaction of [EtPy][Br] with the wood cell wall is different to that of other types of ionic liquid. PMID:25556160

  9. Turnover of galactans and other cell wall polysaccharides during development of flax plants

    SciTech Connect

    Gorshkova, T.A.; Chemikosova, S.B.; Lozovaya, V.V.; Carpita, N.C.

    1997-06-01

    We investigated the synthesis and turnover of cell wall polysaccharides of the flax (Linum usitatissimum L.) plant during development of the phloem fibers. One-month-old flax plants were exposed to a 40-min pulse with {sup 14}CO{sub 2} followed by 8-h, 24-h, and 1-month periods of chase with ambient CO{sub 2}, and radioactivity in cell wall sugars was determined in various plant parts. The relative radioactivity of glucose in noncellulosic polysaccharides was the highest compared with all other cell wall sugars immediately after the pulse and decreased substantially during the subsequent chase. The relative radioactivities of the other cell wall sugars changed with differing rates, indicating turnover of specific polysaccharides. Notably, after 1 month of chase there was a marked decrease in the proportional mass and total radioactivity in cell wall galactose, indicating a long-term turnover of the galactans enriched in the fiber-containing tissues. The ratio of radiolabeled xylose to arabinose also increased during the chase, indicating a turnover of arabinose-containing polymers and interconversion to xylose. The pattern of label redistribution differed between organs, indicating that the cell wall turnover processes are tissue- and cell-specific.

  10. Non-lignified helical cell wall thickenings in root cortical cells of Aspleniaceae (Polypodiales): histology and taxonomical significance

    PubMed Central

    Leroux, O.; Bagniewska-Zadworna, A.; Rambe, S. K.; Knox, J. P.; Marcus, S. E.; Bellefroid, E.; Stubbe, D.; Chabbert, B.; Habrant, A.; Claeys, M.; Viane, R. L. L.

    2011-01-01

    Background and Aims Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae. Methods Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed. Key Results The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100 % bootstrap value and composed of mainly epiphytic species. Conclusions This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species. PMID:21118842

  11. Abundance of mixed linkage glucan in mature tissues and secondary cell walls of grasses.

    PubMed

    Vega-Sánchez, Miguel E; Verhertbruggen, Yves; Scheller, Henrik V; Ronald, Pamela C

    2013-02-01

    (1,3; 1,4)-β-D-glucan, also known as mixed linkage glucan (MLG), is a polysaccharide that in flowering plants is unique to the cell walls of grasses and other related members of Poales. MLG is highly abundant in endosperm cell walls, where it is considered a storage carbohydrate. In vegetative tissues, MLG transiently accumulates in the primary cell walls of young, elongating organs. In evolutionary distant species such as Equisetum, MLG accumulates predominantly in old tissues in the stems. Similarly, we have recently shown that rice accumulates a large amount of MLG in mature stems, which prompted us to re-evaluate the hypothesis that MLG is solely related to growth in grass vegetative tissues. Here, we summarize data that confirms the presence of MLG in secondary cell walls and mature tissues in rice and other grasses. Along with these results, we discuss additional evidence indicating a broader role for MLG than previously considered. PMID:23299432

  12. Coniferyl Ferulate Incorporation into Lignin Enhances the Alkaline Delignification and Enzymatic Degradation of Cell Walls

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Incorporating ester interunit linkages into lignin could facilitate fiber delignification and utilization. In model studies with maize cell walls, we examined how partial substitution of coniferyl alcohol (a normal monolignol) with coniferyl ferulate (an ester conjugate from lignan biosynthesis) alt...

  13. Wall Analyses of Lophocolea Seta Cells (Bryophyta) Before and After Elongation 1

    PubMed Central

    Thomas, Robert J.

    1977-01-01

    Lophocolea heterophylla (Schrad.) Dum. (a leafy liverwort) produces sporophytes with seta cells that elongate 50-fold in 3 to 4 days. Wall components of these cells have been characterized by microscopic histochemistry, colorimetry, and gas chromatography of neutral sugars. Seta cell walls are qualitatively similar to primary cell walls of higher plants. The pectic fraction, however, responds differently to standard histochemical staining and extraction. Quantitatively, mannose, fucose, and rhamnose are in higher percentage, and arabinose and xylose are lower than typically found in vascular plants. Hexuronic acids increase on a percentage basis during elongation; pentoses decrease slightly, while hexose levels remain about the same. Increase in total wall carbohydrate after 2,400% elongation of setae was 1.8-fold. Images PMID:16659846

  14. 2012 PLANT CELL WALLS GORDON RESEARCH CONFERENCE AND GORDON RESEARCH SEMINAR, AUGUST 4-10, 2012

    SciTech Connect

    Rose, Jocelyn

    2012-08-10

    The sub-theme of this year’s meeting, ‘Cell Wall Research in a Post-Genome World’, will be a consideration of the dramatic technological changes that have occurred in the three years since the previous cell wall Gordon Conference in the area of DNA sequencing. New technologies are providing additional perspectives of plant cell wall biology across a rapidly growing number of species, highlighting a myriad of architectures, compositions, and functions in both "conventional" and specialized cell walls. This meeting will focus on addressing the knowledge gaps and technical challenges raised by such diversity, as well as our need to understand the underlying processes for critical applications such as crop improvement and bioenergy resource development.

  15. Regulation of auxin on secondary cell wall cellulose biosynthesis in developing cotton fibers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton (Gossypium hirsutum L.) fibers are unicellular trichomes that differentiate from epidermal cells of developing cotton ovules. Mature fibers exhibit thickened secondary walls composed of nearly pure cellulose. Cotton fiber development is divided into four overlapping phases, 1) initiation sta...

  16. Cell wall compounds of red grapes skins and their grape marcs from three different winemaking techniques.

    PubMed

    Apolinar-Valiente, Rafael; Romero-Cascales, Inmaculada; Gómez-Plaza, Encarna; López-Roca, José María; Ros-García, José María

    2015-11-15

    Different winemaking practices are aimed at increasing cell wall degradation to facilitate extraction of valuables molecules into the wine. However, little attention has been paid to the composition of marcs from different cultivars according to the influence of the winemaking procedures. We provide information on skin cell walls from Cabernet Sauvignon, Syrah and Monastrell grapes and examine how different winemaking practices (addition of enzymatic preparation and β-galactosidase separately and dry ice addition) may affect the composition of marc skin cell wall material (CWM). The efficiency of CWM isolation from the grape skin and also its composition is influenced by the cultivar. A similar cultivar influence has been detected on CWM from the marc, being the differences also due to the enological technique. Our results help to increase our knowledge on the degradation of cell walls during vinification, while providing a valuable guideline to upgrade the value of these by-products. PMID:25977002

  17. In Situ Chemical Imaging of Plant Cell Walls Using CARS/SRS Microscopy (Poster)

    SciTech Connect

    Zeng, Y.; Liu, Y. S.; Saar, B. G.; Xie, X. S.; Chen, F.; Dixon, R. A.; Himmel, M. E.; Ding S. Y.

    2009-06-01

    This poster demonstrates coherent anti-Stokes Raman scattering and stimulated Raman scattering of plant cell walls. It includes simultaneous chemical imaging of lignin and cellulose (corn stover) during acidic pretreatment.

  18. Identification of polysaccharide hydrolases involved in autolytic degradation of Zea cell walls

    SciTech Connect

    Nock, L.P.; Smith, C.J. )

    1987-08-01

    Cell walls of Zea mays (cv L.G.11) seedlings labeled with {sup 14}C were treated with {alpha}-amylase from Bacillus subtilis to remove starch and mixed linkage glucans. These walls released arabinose, xylose, galactose, and galacturonic acid in addition to glucose when they were allowed to autolyze. Methylation analysis was performed on samples of wall which had been incubated autolytically and the results indicated that degradation of the major polymer of the wall, the glucoarabinoxylan, had occurred. A number of glycanases could be dissociated from the wall by use of 3 M LiCL. The proteins which were released were found to contain a number of exoglycosidase activities in addition to being effective in degrading the polysaccharide substrates, araban, xylan, galactan, laminarin, mannan, and polygalacturonic acid. The effects of these enzymes on the wall during autolysis appear to result from endo-activity in addition to exo-activity. The structural changes that occurred in the cell walls during autolysis were found to be related to the changes previous