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Sample records for arabidopsis cell suspension

  1. Plasmodesmata in Arabidopsis thaliana suspension cells.

    Science.gov (United States)

    Bayer, E; Thomas, C L; Maule, A J

    2004-06-01

    A current challenge in plant biology is to identify the structural and functional components of plasmodesmata (PDs). The use of plant tissue as a source material for plasmodesmal characterisation has had limited success, so we have explored the frequency and features of PDs occurring in suspension cell cultures of Arabidopsis thaliana. This material has the advantages of homogeneity, quantity, and ease of disruption. Using light and electron microscopy and immunostaining for callose and calreticulin, we showed that suspension cells laid down abundant PDs in division walls, and that vestiges of these structures were retained as half PDs even when the cell-to-cell contacts were disrupted during culture growth. Although callose was a reliable marker for PD distribution, which was deposited in an organised collar around the neck of PDs, it was not abundant in unstressed cells. Calreticulin and the chemical stain 3,3'-dihexyloxacarbocyanine iodide also provided useful markers when monitoring PDs in cell wall preparations by light microscopy. Purified cell walls were shown to be virtually free of contamination from cytoplasmic components, except for the presence of small amounts of cortical endoplasmic reticulum attached to PDs. Hence, clean cell walls from A. thaliana suspension cells provide a valuable resource for a proteomic approach to the analysis of plasmodesmal components.

  2. Isolation of plasmodesmata from Arabidopsis suspension culture cells.

    Science.gov (United States)

    Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

    2015-01-01

    Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

  3. Cryopreservation of transformed and wild-type Arabidopsis and tobacco cell suspension cultures.

    Science.gov (United States)

    Menges, Margit; Murray, James A H

    2004-02-01

    We have recently described Arabidopsis cell suspension cultures that can be effectively synchronised. Here, we describe procedures that allow clonal-transformed cell suspension lines to be produced using Agrobacterium-mediated transformation, and an optimised and straightforward procedure for the cryopreservation and recovery of both parental and transformed lines. Frozen cultures show 90% viability and rapid re-growth after recovery. We show that the cryopreservation procedure is equally applicable to the frequently used tobacco bright yellow (BY)2 cell suspension culture, and that cell cycle synchronisation capacity of parental lines is maintained after both transformation and recovery from cryopreservation. The techniques require no specialised equipment, and are suitable for routine laboratory use, greatly facilitating the handling and maintenance of cell cultures and providing security against both contamination and cumulative somaclonal variation. Finally, the ability to store easily large numbers of transformed lines opens the possibility of using Arabidopsis cell suspension cultures for high-throughput analysis.

  4. Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture

    DEFF Research Database (Denmark)

    Ostergaard, L; Abelskov, A K; Mattsson, O

    1996-01-01

    The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely...... sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions...

  5. Induction of cell death by graphene in Arabidopsis thaliana (Columbia ecotype) T87 cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Begum, Parvin, E-mail: parvinchy@ees.hokudai.ac.jp; Fugetsu, Bunshi

    2013-09-15

    Highlights: • This study was set up to explore potential influence of graphene on T87 cells. • Fragmented nuclei, membrane damage, mitochondrial dysfunction were observed. • ROS increased, ROS are key mediators in the cell death signaling pathway. • Translocation of graphene into cells and an endocytosis-like structure was observed. • Graphene entering into the cells by endocytosis. -- Abstract: The toxicity of graphene on suspensions of Arabidopsis thaliana (Columbia ecotype) T87 cells was investigated by examining the morphology, mitochondrial dysfunction, reactive oxygen species generation (ROS), and translocation of graphene as the toxicological endpoints. The cells were grown in Jouanneau and Péaud-Lenoel (JPL) media and exposed to graphene at concentrations 0–80 mg/L. Morphological changes were observed by scanning electron microscope and the adverse effects such as fragmented nuclei, membrane damage, mitochondrial dysfunction was observed with fluorescence microscopy by staining with Hoechst 33342/propidium iodide and succinate dehydrogenase (mitochondrial bioenergetic enzyme). Analysis of intracellular ROS by 2′,7′-dichlorofluorescein diacetate demonstrated that graphene induced a 3.3-fold increase in ROS, suggesting that ROS are key mediators in the cell death signaling pathway. Transmission electron microscopy verified the translocation of graphene into cells and an endocytosis-like structure was observed which suggested graphene entering into the cells by endocytosis. In conclusion, our results show that graphene induced cell death in T87 cells through mitochondrial damage mediated by ROS.

  6. Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

    KAUST Repository

    Turek, Ilona

    2015-06-30

    Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

  7. Characterization of transmembrane auxin transport in Arabidopsis suspension-cultured cells.

    Science.gov (United States)

    Seifertová, Daniela; Skůpa, Petr; Rychtář, Jan; Laňková, Martina; Pařezová, Markéta; Dobrev, Petre I; Hoyerová, Klára; Petrášek, Jan; Zažímalová, Eva

    2014-03-15

    Polar auxin transport is a crucial process for control and coordination of plant development. Studies of auxin transport through plant tissues and organs showed that auxin is transported by a combination of phloem flow and the active, carrier-mediated cell-to-cell transport. Since plant organs and even tissues are too complex for determination of the kinetics of carrier-mediated auxin uptake and efflux on the cellular level, simplified models of cell suspension cultures are often used, and several tobacco cell lines have been established for auxin transport assays. However, there are very few data available on the specificity and kinetics of auxin transport across the plasma membrane for Arabidopsis thaliana suspension-cultured cells. In this report, the characteristics of carrier-mediated uptake (influx) and efflux for the native auxin indole-3-acetic acid and synthetic auxins, naphthalene-1-acetic and 2,4-dichlorophenoxyacetic acids (NAA and 2,4-D, respectively) in A. thaliana ecotype Landsberg erecta suspension-cultured cells (LE line) are provided. By auxin competition assays and inhibitor treatments, we show that, similarly to tobacco cells, uptake carriers have high affinity towards 2,4-D and that NAA is a good tool for studies of auxin efflux in LE cells. In contrast to tobacco cells, metabolic profiling showed that only a small proportion of NAA is metabolized in LE cells. These results show that the LE cell line is a useful experimental system for measurements of kinetics of auxin carriers on the cellular level that is complementary to tobacco cells.

  8. Isolation of transcription factor complexes from Arabidopsis cell suspension cultures by tandem affinity purification.

    Science.gov (United States)

    Van Leene, Jelle; Eeckhout, Dominique; Persiau, Geert; Van De Slijke, Eveline; Geerinck, Jan; Van Isterdael, Gert; Witters, Erwin; De Jaeger, Geert

    2011-01-01

    Defining protein complexes is critical to virtually all aspects of cell biology because most cellular processes are regulated by stable or more dynamic protein interactions. Elucidation of the protein-protein interaction network around transcription factors is essential to fully understand their function and regulation. In the last decade, new technologies have emerged to study protein-protein interactions under near-physiological conditions. We have developed a high-throughput tandem affinity purification (TAP)/mass spectrometry (MS) platform for cell suspension cultures to analyze protein complexes in Arabidopsis thaliana. This streamlined platform follows an integrated approach comprising generic Gateway-based vectors with high cloning flexibility, the fast generation of transgenic suspension cultures, TAP adapted for plant cells, and tandem matrix-assisted laser desorption ionization MS for the identification of purified proteins. Recently, we evaluated the GS tag, originally developed to study mammalian protein complexes, that combines two IgG-binding domains of protein G with a streptavidin-binding peptide, separated by two tobacco etch virus cleavage sites. We found that this GS tag outperforms the traditional TAP tag in plant cells, regarding both specificity and complex yield. Here, we provide detailed protocols of the GS-based TAP platform that allowed us to characterize transcription factor complexes involved in signaling in response to the plant phytohormone jasmonate.

  9. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts.

    Science.gov (United States)

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T; Lorenzo, Oscar; Revuelta, José L; McCabe, Paul F; Arellano, Juan B

    2014-07-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined.

  10. Specific localization and measurement of hydrogen peroxide in Arabidopsis thaliana cell suspensions and protoplasts elicited by COS-OGA.

    Science.gov (United States)

    Ledoux, Quentin; Van Cutsem, Pierre; Markό, Istvan E; Veys, Pascal

    2014-01-01

    H2O2 acts as an important signaling molecule during plant/pathogen interactions but its study remains a challenge due to the current shortcomings in H2O2-responsive probes. In this work, ContPY1, a new molecular probe developed to specifically detect H2O2 was used to study the elicitation of Arabidopsis thaliana cells by a complex of chitosan oligomers (COS) and oligogalacturonides (OGA). The comparison of cell suspensions, protoplasts of cell suspensions and leaf protoplasts treated with different inhibitors gave indications on the potential sources of hydrogen peroxide in plant cells. The relative contribution of the cell wall, of membrane dehydrogenases and of peroxidases depended on cell type and treatment and proved to be variable. Our present protocol can be used to study hydrogen peroxide production in a large variety of plant species by simple protocol adaptation.

  11. Efficient and high-throughput vector construction and Agrobacterium-mediated transformation of Arabidopsis thaliana suspension-cultured cells for functional genomics.

    Science.gov (United States)

    Ogawa, Yoichi; Dansako, Tomoko; Yano, Kentaro; Sakurai, Nozomu; Suzuki, Hideyuki; Aoki, Koh; Noji, Masaaki; Saito, Kazuki; Shibata, Daisuke

    2008-02-01

    We established a large-scale, high-throughput protocol to construct Arabidopsis thaliana suspension-cultured cell lines, each of which carries a single transgene, using Agrobacterium-mediated transformation. We took advantage of RIKEN Arabidopsis full-length (RAFL) cDNA clones and the Gateway cloning system for high-throughput preparation of binary vectors carrying individual full-length cDNA sequences. Throughout all cloning steps, multiple-well plates were used to treat 96 samples simultaneously in a high-throughput manner. The optimal conditions for Agrobacterium-mediated transformation of 96 independent binary vector constructs were established to obtain transgenic cell lines efficiently. We evaluated the protocol by generating transgenic Arabidopsis T87 cell lines carrying individual 96 metabolism-related RAFL cDNA fragments, and showed that the protocol was useful for high-throughput and large-scale production of gain-of-function lines for functional genomics.

  12. The age-dependent epigenetic and physiological changes in an Arabidopsis T87 cell suspension culture during long-term cultivation

    Energy Technology Data Exchange (ETDEWEB)

    Kwiatkowska, Aleksandra, E-mail: A.Kwiatkows@gmail.com [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Zebrowski, Jacek [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Oklejewicz, Bernadetta [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland); Czarnik, Justyna [Department of Botany, University of Rzeszow, Kolbuszowa (Poland); Halibart-Puzio, Joanna [Department of Plant Physiology, University of Rzeszow, Kolbuszowa (Poland); Wnuk, Maciej [Department of Genetics, University of Rzeszow, Kolbuszowa (Poland)

    2014-05-02

    Highlights: • A decrease in proliferation rate during long-term cultivation of Arabidopsis cells. • Age-dependent increase in senescence-associated gene expression in Arabidopsis cells. • Age-related increase in DNA methylation, H3K9me2, and H3K27me3 in Arabidopsis cells. • High potential of photosynthetic efficiency of long-term cultured Arabidopsis cells. - Abstract: Plant cell suspension cultures represent good model systems applicable for both basic research and biotechnological purposes. Nevertheless, it is widely known that a prolonged in vitro cultivation of plant cells is associated with genetic and epigenetic instabilities, which may limit the usefulness of plant lines. In this study, the age-dependent epigenetic and physiological changes in an asynchronous Arabidopsis T87 cell culture were examined. A prolonged cultivation period was found to be correlated with a decrease in the proliferation rate and a simultaneous increase in the expression of senescence-associated genes, indicating that the aging process started at the late growth phase of the culture. In addition, increases in the heterochromatin-specific epigenetic markers, i.e., global DNA methylation, H3K9 dimethylation, and H3K27 trimethylation, were observed, suggesting the onset of chromatin condensation, a hallmark of the early stages of plant senescence. Although the number of live cells decreased with an increase in the age of the culture, the remaining viable cells retained a high potential to efficiently perform photosynthesis and did not exhibit any symptoms of photosystem II damage.

  13. Proteomic characterization of golgi membranes enriched from Arabidopsis suspension cell cultures

    DEFF Research Database (Denmark)

    Hansen, Sara Fasmer; Ebert, Berit; Rautengarten, Carsten

    2016-01-01

    The plant Golgi apparatus has a central role in the secretory pathway and is the principal site within the cell for the assembly and processing of macromolecules. The stacked membrane structure of the Golgi apparatus along with its interactions with the cytoskeleton and endoplasmic reticulum has...... historically made the isolation and purification of this organelle difficult. Density centrifugation has typically been used to enrich Golgi membranes from plant microsomal preparations, and aside from minor adaptations, the approach is still widely employed. Here we outline the enrichment of Golgi membranes...

  14. Alpha-picolinic Acid Activates Diverse Defense Responses of Salicylic Acid-, Jasmonic Acid/Ethylene- and Ca2 -dependent Pathways in Arabidopsis and Rice Suspension Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANGHai-Kuo; ZHANGXin; LIQun; HEZu-Hua

    2004-01-01

    Alpha-picolinic acid (PA) is an apoptosis inducer in animal cells, and could elicit hypersensitiv eresponse (HR) in rice, a monocotyledonous model plant. Here we report that PA is an HR inducer in plants. It induced HR in Arabidopsis, a dicotyledonous model plant, including the oxidative burst and cell death. We investigated defense signal transduction activated by PA through marker genes of particular defense pathways in Arabidopsis. The result indicated that both the salicylic acid-dependent and jasmonic acid/ethylene-dependent pathways were activated by PA, in which the marker defense genes PR-1, PR-2 and PDF 1.2 were all induced in dose-dependent and time-course manners. We also observed that the PAinduced reactive oxygen species (ROS) production in rice suspension cells was Ca2+-dependent. Together with our previous studies of PA-induced defense activation in rice, we conclude that PA acts as a nonspecific elicitor in plant defense and has a potential utilization in cellular model establishment of systemicac quired resistance (SAR) activation.

  15. An improved protocol for the preparation of protoplasts from an established Arabidopsis thaliana cell suspension culture and infection with RNA of turnip yellow mosaic tymovirus: a simple and reliable method.

    Science.gov (United States)

    Schirawski, J; Planchais, S; Haenni, A L

    2000-04-01

    An improved method for preparation of protoplasts of Arabidopsis thaliana cells grown in suspension culture is presented. This method is fast, reliable and can be used for the production of virtually an unlimited number of protoplasts at any time. These protoplasts can be transformed efficiently with RNA from turnip yellow mosaic tymovirus (TYMV) by polyethyleneglycol-mediated transfection. The simple transfection procedure has been optimized at various steps. Replication of TYMV can be monitored routinely by detection of the coat protein in as few as 2 x 10(4) infected protoplasts.

  16. Extraction and Characterization of Extracellular Proteins and Their Post-Translational Modifications from Arabidopsis thaliana Suspension Cell Cultures and Seedlings: A Critical Review

    Directory of Open Access Journals (Sweden)

    Mina Ghahremani

    2016-09-01

    Full Text Available Proteins secreted by plant cells into the extracellular space, consisting of the cell wall, apoplastic fluid, and rhizosphere, play crucial roles during development, nutrient acquisition, and stress acclimation. However, isolating the full range of secreted proteins has proven difficult, and new strategies are constantly evolving to increase the number of proteins that can be detected and identified. In addition, the dynamic nature of the extracellular proteome presents the further challenge of identifying and characterizing the post-translational modifications (PTMs of secreted proteins, particularly glycosylation and phosphorylation. Such PTMs are common and important regulatory modifications of proteins, playing a key role in many biological processes. This review explores the most recent methods in isolating and characterizing the plant extracellular proteome with a focus on the model plant Arabidopsis thaliana, highlighting the current challenges yet to be overcome. Moreover, the crucial role of protein PTMs in cell wall signalling, development, and plant responses to biotic and abiotic stress is discussed.

  17. Characterization of cell suspensions from solid tumors

    Energy Technology Data Exchange (ETDEWEB)

    Pallavicini, M.

    1985-07-10

    The desirable features of cells in suspension will necessarily be dependent upon the use for which the cells were prepared. Adequate cell yield or recovery is defined by the measurement to be performed. Retention of cellular morphology is important for microscopic identification of cell types in a heterogenous cell suspension, and may be used to determine whether the cells in suspension are representative of those in the tumor in situ. Different dispersal protocols may yield cells with different degrees of clonogenicity, as well as altered biochemical features, such as loss of cellular proteins, surface antigens, nucleotide pools, etc. The quality of the cell suspension can be judged by the degree of cell clumping and level of cellular debris, both of which impact on flow cytometric measurements and studies in which the number of cells be known accurately. Finally, if the data measured on the cells in suspension are to be extrapolated to phenomena occurring in the tumor in situ, it is desirable that the cells in suspension are representative of those in the solid tumor in vivo. This report compares characteristics of tumor cell suspensions obtained by different types of selected disaggregation methods. 33 refs., 2 figs., 4 tabs.

  18. Cell Suspension Culture of Neem Tree

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The establishment of suspension culture system for neem (Azadirachta indica A. Juss) cells and the suspension culture condition was studied. It shows that the neem cell suspension culture system was best in B5 liquid medium, 2.0~4.0mg/L NAA with direct spill method. Based on the integrated analysis of cell biomass, Azadirachtin content and productivity, the optimum culture conditions were B5 liquid medium, 2.0-4.0 mg/L NAA, 3% sucrose at 25 ℃. The optimum rotating speed of the shaker and broth content d...

  19. Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

    Directory of Open Access Journals (Sweden)

    María Berdasco

    Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

  20. Stem cell organization in Arabidopsis

    NARCIS (Netherlands)

    Wendrich, J.R.

    2016-01-01

    Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or mer

  1. Cell Polarity Signaling in Arabidopsis

    OpenAIRE

    Yang, Zhenbiao

    2008-01-01

    Cell polarization is intimately linked to plant development, growth, and responses to the environment. Major advances have been made in our understanding of the signaling pathways and networks that regulate cell polarity in plants owing to recent studies on several model systems, e.g., tip growth in pollen tubes, cell morphogenesis in the leaf epidermis, and polar localization of PINs. From these studies we have learned that plant cells use conserved mechanisms such as Rho family GTPases to i...

  2. Evaluation of Simulated Microgravity Environments Induced by Diamagnetic Levitation of Plant Cell Suspension Cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Herranz, Raúl; van Loon, Jack J. W. A.; Christianen, Peter C. M.; Medina, F. Javier

    2016-06-01

    Ground-Based Facilities (GBF) are essetial tools to understand the physical and biological effects of the absence of gravity and they are necessary to prepare and complement space experiments. It has been shown previously that a real microgravity environment induces the dissociation of cell proliferation from cell growth in seedling root meristems, which are limited populations of proliferating cells. Plant cell cultures are large and homogeneous populations of proliferating cells, so that they are a convenient model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of the Arabidopsis thaliana cell line MM2d were exposed to four altered gravity and magnetic field environments in a magnetic levitation facility for 3 hours, including two simulated microgravity and Mars-like gravity levels obtained with different magnetic field intensities. Samples were processed either by quick freezing, to be used in flow cytometry for cell cycle studies, or by chemical fixation for microscopy techniques to measure parameters of the nucleolus. Although the trend of the results was the same as those obtained in real microgravity on meristems (increased cell proliferation and decreased cell growth), we provide a technical discussion in the context of validation of proper conditions to achieve true cell levitation inside a levitating droplet. We conclude that the use of magnetic levitation as a simulated microgravity GBF for cell suspension cultures is not recommended.

  3. Study on Cell Suspension Culture of Floribunda Rose

    Institute of Scientific and Technical Information of China (English)

    ZHANG Chun'ai; WANG Jingang; FAN Jinping; GONG Shufang; CHE Daidi

    2008-01-01

    Friable callus was induced when immature seeds of floribunda rose were inoculated on MS medium supplemented with 2,4-D 3.0 mg-L-1.When transfered onto subculture media,fi-iable callus developed into embryogenic callus,which was used to establish cell suspension lines.Cell suspensions had to be subcultured at a interval of 4-5 days at the first several culture cycles.The best subculturing cycle for the stable cell suspensions was 8-10 days.The best inoculum quantity was 1 mL PCV(Packed Cell Volume) per 40 mL culture fluid.

  4. Measuring NO Production by Plant Tissues and Suspension Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Jan Vitecek; Vilem Reinohl; Russell L.Jones

    2008-01-01

    We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants.The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap.The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors.We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources.NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected,as was the production of NO from nitrite by the incubation medium of barley aleurone.Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up.We could also measure NO production from elicitor-treated suspension cultured cells using this set-up.Further,we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM).Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues.In addition to the dramatic effects that pH can have on fluorescence from DAF-FM,the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results.Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

  5. A simple and efficient method for the long-term preservation of plant cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Boisson Anne-Marie

    2012-01-01

    Full Text Available Abstract Background The repeated weekly subculture of plant cell suspension is labour intensive and increases the risk of variation from parental cells lines. Most of the procedures to preserve cultures are based on controlled freezing/thawing and storage in liquid nitrogen. However, cells viability after unfreezing is uncertain. The long-term storage and regeneration of plant cell cultures remains a priority. Results Sycamore (Acer pseudoplatanus and Arabidopsis cell were preserved over six months as suspensions cultures in a phosphate-free nutrient medium at 5°C. The cell recovery monitored via gas exchange measurements and metabolic profiling using in vitro and in vivo 13C- and 31P-NMR took a couple of hours, and cell growth restarted without appreciable delay. No measurable cell death was observed. Conclusion We provide a simple method to preserve physiologically homogenous plant cell cultures without subculture over several months. The protocol based on the blockage of cell growth and low culture temperature is robust for heterotrophic and semi-autotrophic cells and should be adjustable to cell lines other than those utilised in this study. It requires no specialized equipment and is suitable for routine laboratory use.

  6. Assessment of drug salt release from solutions, suspensions and in situ suspensions using a rotating dialysis cell

    DEFF Research Database (Denmark)

    Parshad, Henrik; Frydenvang, Karla; Liljefors, Tommy

    2003-01-01

    A rotating dialysis cell consisting of a small (10 ml) and a large compartment (1000 ml) was used to study the release of drug salt (bupivacaine 9-anthracene carboxylate) from (i). solutions, (ii). suspensions and (iii). in situ formed suspensions. Initial release experiments from suspensions...

  7. CMOS based sensor for dielectric spectroscopy of biological cell suspension

    Science.gov (United States)

    Guha, S.; Schmalz, K.; Meliani, C.; Wenger, Ch

    2013-04-01

    In this work we investigate the use of microwave frequency range to measure the concentration of cells in a biological cell suspension. A theoretical model is discussed and the advantage of high frequency, which is to avoid dispersion mechanisms due to the cell parameters at lower frequencies (for example membrane capacitance), has been described. Interdigitated capacitor (IDC) has been proposed as the sensor for analysing the concentration of a cell species in the suspension. The read-out circuit is a VCO using the IDC and a pair of inductors as resonator. The capacitance of the IDC which is the function of the permittivity of the biological cell suspension determines the resonant frequency of the LC tank oscillator. Thus the concentration of cells in a solution, affecting its permittivity, is read out as the frequency of the oscillator.

  8. Acetylsalicylic acid induces programmed cell death in Arabidopsis cell cultures.

    Science.gov (United States)

    García-Heredia, José M; Hervás, Manuel; De la Rosa, Miguel A; Navarro, José A

    2008-06-01

    Acetylsalicylic acid (ASA), a derivative from the plant hormone salicylic acid (SA), is a commonly used drug that has a dual role in animal organisms as an anti-inflammatory and anticancer agent. It acts as an inhibitor of cyclooxygenases (COXs), which catalyze prostaglandins production. It is known that ASA serves as an apoptotic agent on cancer cells through the inhibition of the COX-2 enzyme. Here, we provide evidences that ASA also behaves as an agent inducing programmed cell death (PCD) in cell cultures of the model plant Arabidopsis thaliana, in a similar way than the well-established PCD-inducing agent H(2)O(2), although the induction of PCD by ASA requires much lower inducer concentrations. Moreover, ASA is herein shown to be a more efficient PCD-inducing agent than salicylic acid. ASA treatment of Arabidopsis cells induces typical PCD-linked morphological and biochemical changes, namely cell shrinkage, nuclear DNA degradation, loss of mitochondrial membrane potential, cytochrome c release from mitochondria and induction of caspase-like activity. However, the ASA effect can be partially reverted by jasmonic acid. Taking together, these results reveal the existence of common features in ASA-induced animal apoptosis and plant PCD, and also suggest that there are similarities between the pathways of synthesis and function of prostanoid-like lipid mediators in animal and plant organisms.

  9. Establishment of cell suspension line of Populus tomentosa Carr

    Institute of Scientific and Technical Information of China (English)

    YAO Na; ZHANG Zhi-yi; AN Xin-min; YANG Kai

    2008-01-01

    Leaves of fine Populus tomentosa genotype TC152 were used as explants to establish cell suspension lines. The effects of plant growth regulators on callus induction and establishment of cell suspension lines were studied. The callus induction rate was the highest on a MS solid medium supplemented with 1.0 mg·L-1 2,4-D. A cell suspension line could be obtained by inoculating calli which were not subeultured into a MS liquid medium supplemented with 1.5 mg·L-1 2,4-D. The best subculture medium was MS+ 0.8 mg·L-1 2,4-D + 30 g·L-1 sucrose with a subculture cycle of seven days.

  10. Proper selection of 1 g controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

    Science.gov (United States)

    Kamal, Khaled Y.; Hemmersbach, Ruth; Medina, F. Javier; Herranz, Raúl

    2015-04-01

    Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

  11. Glycosyl hydrolases of cell wall are induced by sugar starvation in Arabidopsis.

    Science.gov (United States)

    Lee, Eun-Jeong; Matsumura, Yasuhiro; Soga, Kouichi; Hoson, Takayuki; Koizumi, Nozomu

    2007-03-01

    Three Arabidopsis genes encoding a putative beta-galactosidase (At5g56870), beta-xylosidase (At5g49360) and beta-glucosidase (At3g60140) are induced by sugar starvation. The deduced proteins belong to the glycosyl hydrolase families 35, 3 and 1, respectively. They are predicted to be secretory proteins that play roles in modification of cell wall polysaccharides based on amino acid similarity. The beta-galactosidase encoded by At5g56870 was identified as a secretory protein in culture medium of suspension cells by mass spectrometry analysis. This protein was specifically detected under sugar-starved conditions with a specific antibody. Induction of these genes was repressed in suspension cells grown with galactose, xylose and glucose, as well as with sucrose. In planta, expression of the genes and protein accumulation were detected when photosynthesis was inhibited. Glycosyl hydrolase activity against galactan also increased during sugar starvation. The amount of monosaccharide in pectin and hemicellulose in detached leaves decreased in response to sugar starvation. These findings suggest that the cell wall may function as a storage reserve of carbon in addition to providing physical support for the plant body.

  12. Heparin promotes suspension adaptation process of CHO-TS28 cells by eliminating cell aggregation.

    Science.gov (United States)

    Li, Ling; Qin, Jun; Feng, Qiang; Tang, Hao; Liu, Rong; Xu, Liqing; Chen, Zhinan

    2011-01-01

    While heparin has been shown to eliminate cell aggregation in suspension adaptations of insect and HEK293 cells for virus-based cell cultures, the role of heparin in long period serum-free suspension adaptation of the anchorage-dependent Chinese hamster ovary (CHO) cell lines remains inconclusive. In this paper, we explore the potential application of heparin in suspension adaptation of CHO cell line which produces an anti-human chimeric antibody cHAb18. Heparin showed a concentration-dependent inhibition of CHO-TS28 cell-to-cell adhesion, with a significant inhibitory effect occurring when the concentration exceeded 250 μg/ml (P cell aggregation elimination role at all concentrations (P cell growth and antibody secretion, with the highest cell density ((99.83 ± 12.21) × 10(4) cells/ml, P = 0.034) and maximum antibody yield ((9.46 ± 0.94) mg/l, P cell aggregates were effectively dispersed by 250 μg/ml heparin and a single-cell suspension culture process was promoted. In suspension adapted CHO-TS28 cells, cell growth rates and specific antibody productivity were maintained; while antigen-binding activity improved slightly. Together, our results show that heparin may promote suspension adaptation of anchorage-depended CHO cells by resisting cell aggregation without reducing cell growth, antibody secretion, and antigen-binding activity.

  13. Embryo forming cells in carrot suspension cultures.

    OpenAIRE

    Toonen, M.A.J.

    1997-01-01

    Somatic cells of many plant species can be cultured in vitro and induced to form embryos that are able to develop into mature plants. This process, termed somatic embryogenesis, was originally described in carrot (Daucus carota L.). Somatic embryos develop through the same characteristic morphological stages, i.e. the globular-, heartand torpedo-stage respectively, as their zygotic counterparts. Due to the different cellular origin of somatic embryos, it is less clear to what extent the earli...

  14. Spectral Representation Theory for Dielectric Behavior of Nonspherical Cell Suspensions

    Institute of Scientific and Technical Information of China (English)

    HUANG Ji-Ping; YU Kin-Wah; LEI Jun; SUN Hong

    2002-01-01

    Recent experiments revealed that the dielectric dispersion spectrum of fission yeast cells in a suspension was mainly composed of two sub-dispersions. The low-frequency sub-dispersion depended on the cell length, while the high-frequency one was independent of it. The cell shape effect was simulated by an ellipsoidal cell model but the comparison between theory and experiment was far from being satisfactory. Prompted by the discrepancy, we proposed the use of spectral representation to analyze more realistic cell models. We adopted a shell-spheroidal model to analyze the effects of the cell membrane. It is found that the dielectric property of the cell membrane has only a minor effect on the dispersion magnitude ratio and the characteristic frequency ratio. We further included the effect of rotation of dipole induced by an external electric field, and solved the dipole-rotation spheroidal model in the spectral representation.Good agreement between theory and experiment has been obtained.

  15. Shear induced diffusion in a red blood cell suspension

    Science.gov (United States)

    Podgorski, Thomas; Grandchamp, Xavier; Srivastav, Aparna; Coupier, Gwennou

    2012-11-01

    In the microcirculation, blood exhibits an inhomogeneous structure which results in the well know Fahraeus-Lindqvist effect : the apparent viscosity decreases when the diameter of the capillary decreases due to the formation of a marginal cell depletion layer (known as plasma skimming). This structure is a consequence of several phenomena, which include i) the migration of cells aways from walls due to lift forces and gradients of shear and ii) shear induced diffusion due to collisions and interactions among cells. We investigated these phenomena through experiments in simple shear and microchannel flows, with dilute suspensions of vesicles and blood cells. Pairwise interactions between suspended objects result in non-linear and flow-dependent diffusion, whose properties have been measured in different experiments for vesicles and blood cells. The injection of a sheet of concentrated blood cell suspension in a microchannel with a rectangular cross-section allows, through the measurement of its widening along the channel, to measure the diffusivity of blood cells, both in the local plane of shear and in the vorticity direction.

  16. Rheological properties of mammalian cell culture suspensions: Hybridoma and HeLa cell lines.

    Science.gov (United States)

    Shi, Y; Ryu, D D; Ballica, R

    1993-03-25

    Data on viscous (eta') and elastic (eta'') components of the complex viscosity versus oscillatory angular frequency (0.01 to 4.0 rad/s) with increasing strains were obtained for hybridoma cell (62'D3) and HeLa cell (S3) suspensions in PBS at 0.9 (mL/mL) cell volume fraction using a Weissenberg rheogoniometer equipped with two parallel plate geometry at ambient temperature. Both cell suspensions exhibited shear thinning behavior. From the measured viscoelastic properties, the yield stress was calculated. Hybridoma cell suspension (15 microm as the mean diameter of cells) showed the yield stress at 550 dyne/cm(2) that was 1.8 times higher than the value of HeLa cell suspension (22 microm mean diameter) as measured at the oscillatory angular frequency, 4.0 rad/s. The apparent viscosities of HeLa cell suspension at four concentrations and varying steady shear rate were also determined using the Brookfield rotational viscometer. The yield stress to steady shear test was about 130 dyne/cm(2) for HeLa cell suspension at 0.9 (mL/mL) cell volume fraction. The apparent viscosity was in the range about 1 approximately 1000 Poise depending on the cell concentration and shear rate applied. A modified semiempirical Mooney equation, eta = eta(0) exp[K gamma(.)(-beta)phi(c)(1 - K'' sigmaphi(c) /D)] was derived based on the cell concentration, the cell morphology, and the steady shear rate. The beta, shear rate index, was estimated as 0.159 in the range of shear rate, 0.16 to 22.1 s(-1), for the cell volume fractions from 0.6 to 0.9 (mL/mL). In this study, the methods of determining the shear sensitivity and the viscous and the elastic components of mammalian cell suspensions are described under the steady shear field.

  17. Arabidopsis CAP regulates the actin cytoskeleton necessary for plant cell elongation and division.

    Science.gov (United States)

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2002-01-01

    An Arabidopsis cDNA (AtCAP1) that encodes a predicted protein of 476 amino acids highly homologous with the yeast cyclase-associated protein (CAP) was isolated. Expression of AtCAP1 in the budding yeast CAP mutant was able to rescue defects such as abnormal cell morphology and random budding pattern. The C-terminal domain, 158 amino acids of AtCAP1 possessing in vitro actin binding activity, was needed for the regulation of cytoskeleton-related defects of yeast. Transgenic plants overexpressing AtCAP1 under the regulation of a glucocorticoid-inducible promoter showed different levels of AtCAP1 accumulation related to the extent of growth abnormalities, in particular size reduction of leaves as well as petioles. Morphological alterations in leaves were attributable to decreased cell size and cell number in both epidermal and mesophyll cells. Tobacco suspension-cultured cells (Bright Yellow 2) overexpressing AtCAP1 exhibited defects in actin filaments and were unable to undergo mitosis. Furthermore, an immunoprecipitation experiment suggested that AtCAP1 interacted with actin in vivo. Therefore, AtCAP1 may play a functional role in actin cytoskeleton networking that is essential for proper cell elongation and division.

  18. Auxin requirements of sycamore cells in suspension culture.

    Science.gov (United States)

    Moloney, M M; Hall, J F; Robinson, G M; Elliott, M C

    1983-04-01

    Sycamore (Acer pseudoplatanus L.) cell suspension cultures (strain OS) require 2,4-dichlorophenoxyacetic acid (2,4-D) in their culture medium for normal growth. If the 2,4-D is omitted, rates of cell division are dramatically reduced and cell lysis may occur. Despite this ;auxin requirement,' it has been shown by gas chromatography-mass spectrometry that the cells synthesize indol-3yl-acetic acid (IAA). Changes in free 2,4-D and IAA in the cells during a culture passage have been monitored.There is a rapid uptake of 2,4-D by the cells during the lag phase leading to a maximum concentration per cell (125 nanograms per 10(6) cells) on day 2 followed by a decline to 45 nanograms per 10(6) cells by day 9 (middle of linear phase). The initial concentration of IAA (0.08 nanograms per 10(6) cells) rises slowly to a peak of 1.4 nanograms per 10(6) cells by day 9 then decreases rapidly to 0.2 nanograms per 10(6) cells by day 15 (early declining phase) and 0.08 nanograms per 10(6) cells by day 23 (early stationary phase).

  19. Axial dispersion in flowing red blood cell suspensions

    Science.gov (United States)

    Podgorski, Thomas; Losserand, Sylvain; Coupier, Gwennou

    2016-11-01

    A key parameter in blood microcirculation is the transit time of red blood cells (RBCs) through an organ, which can influence the efficiency of gas exchange and oxygen availability. A large dispersion of this transit time is observed in vivo and is partly due to the axial dispersion in the flowing suspension. In the classic Taylor-Aris example of a solute flowing in a tube, the combination of molecular diffusion and parabolic velocity profile leads to enhanced axial dispersion. In suspensions of non-Brownian deformable bodies such as RBCs, axial dispersion is governed by a combination of shear induced migration and shear-induced diffusion arising from hydrodynamic interactions. We revisit this problem in the case of RBC pulses flowing in a microchannel and show that the axial dispersion of the pulse eventually saturates with a final extension that depends directly on RBC mechanical properties. The result is especially interesting in the dilute limit since the final pulse length depends only on the channel width, exponent of the migration law and dimensionless migration velocity. In continuous flow, the dispersion of transit times is the result of complex cell-cell and cell-wall interactions and is strongy influenced by the polydispersity of the blood sample. The authors acknowledge support from LabEx TEC21 and CNES.

  20. Signal transduction events in aluminum-induced cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Woltering, E.J.

    2007-01-01

    In this study, some of the signal transduction events involved in AlCl3-induced cell death in tomato (Lycopersicon esculentum Mill.) suspension cells were elucidated. Cells treated with 100 ¿M AlCl3 showed typical features of programmed cell death (PCD) such as nuclear and cytoplasmic condensation.

  1. Importance of Interaction between Integrin and Actin Cytoskeleton in Suspension Adaptation of CHO cells.

    Science.gov (United States)

    Walther, Christa G; Whitfield, Robert; James, David C

    2016-04-01

    The biopharmaceutical production process relies upon mammalian cell technology where single cells proliferate in suspension in a chemically defined synthetic environment. This environment lacks exogenous growth factors, usually contributing to proliferation of fibroblastic cell types such as Chinese hamster ovary (CHO) cells. Use of CHO cells for production hence requires a lengthy 'adaptation' process to select clones capable of proliferation as single cells in suspension. The underlying molecular changes permitting proliferation in suspension are not known. Comparison of the non-suspension-adapted clone CHO-AD and a suspension-adapted propriety cell line CHO-SA by flow cytometric analysis revealed a highly variable bi-modal expression pattern for cell-to-cell contact proteins in contrast to the expression pattern seen for integrins. Those have a uni-modal expression on suspension and adherent cells. Integrins showed a conformation distinguished by regularly distributed clusters forming a sphere on the cell membrane of suspension-adapted cells. Actin cytoskeleton analysis revealed reorganisation from the typical fibrillar morphology found in adherent cells to an enforced spherical subcortical actin sheath in suspension cells. The uni-modal expression and specific clustering of integrins could be confirmed for CHO-S, another suspension cell line. Cytochalasin D treatment resulted in breakdown of the actin sheath and the sphere-like integrin conformation demonstrating the link between integrins and actin in suspension-adapted CHO cells. The data demonstrates the importance of signalling changes, leading to an integrin rearrangement on the cell surface, and the necessity of the reinforcement of the actin cytoskeleton for proliferation in suspension conditions.

  2. Enhancement of heat transfer in red cell suspensions in vitro experiments.

    Science.gov (United States)

    Carr, R T; Tiruvaloor, N R

    1989-05-01

    New data on laminar heat convection with red cell suspensions have been gathered for both heating and cooling. When compared to data for the suspending medium alone, it is apparent that the red cells enhance laminar heat transfer when Pe greater than 4. This is probably due to particle movements. These new data disagree with earlier studies which indicated no enhancement of heat transfer for blood cell suspensions. The data do agree with previous correlations for enhanced thermal transport in sheared suspensions.

  3. Somatic Embryogenesis from Cell Suspension Cultures of Aspen Clone

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Suspension cultures initiated from callus derived from petiole explants of aspen hybrid (Populus tremuloides × P.tremula) produced somatic embryos. Callus was induced on a MS medium supplemented with 5 mg·L-1 2,4-D and 0.05 mg·L-1 zeatin under light conditions. Embryogenic calli were obtained when a subsequent subculture of calli was suspended in the same basal medium with 10 mg·L-1 2,4-D. The highest number of globular embryos were induced from embryogenic calli by cell suspension culture in a MS liquid medium supplemented with 10 mg·L-1 2,4-D. Genotype and 2,4-D concentration were vital to the induction of embryogenic calli producing competent cells. Embryogenic calli for each genotype were heterogeneous. Green calli with gel-like consistency could yield more competent cells than light yellow embryogenic calli. However, some globular embryos broke into slices and some developed abnormally after one month of culture under the same or other hormonal conditions.

  4. Chromatin Remodeling in Stem Cell Maintenance in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    Lin Xu; Wen-Hui Shen

    2009-01-01

    Pluripotent stem cells are able to both self-renew and generate undifferentiated cells for the formation of new tissues and organs.In higher plants,stem cells found in the shoot apical meristem (SAM) and the root apical meristem (RAM) are origins of organogenesis occurring post-embryonically.It is important to understand how the regulation of stem cell fate is coordinated to enable the meristem to constantly generate different types of lateral organs.Much knowledge has accumulated on specific transcription factors controlling SAM and RAM activity.Here,we review recent evidences for a role of chromatin remodeling in the maintenance of stable expression states of transcription factor genes and the control of stem cell activity in Arabidopsis.

  5. ROS production and scavenging under anoxia and re-oxygenation in Arabidopsis cells: a balance between redox signaling and impairment.

    Directory of Open Access Journals (Sweden)

    Annalisa Paradiso

    2016-12-01

    Full Text Available Plants can frequently experience low oxygen concentrations due to environmental factors such as flooding or waterlogging. It has been reported that both anoxia and the transition from anoxia to re-oxygenation determine a strong imbalance in the cellular redox state involving the production of reactive oxygen species (ROS and nitric oxide (NO. Plant cell cultures can be a suitable system to study the response to oxygen deprivation stress since a close control of physicochemical parameters is available when using bioreactors. For this purpose, Arabidopsis cell suspension cultures grown in a stirred bioreactor were subjected to a severe anoxic stress and analyzed during anoxia and re-oxygenation for alteration in ROS and NO as well as in antioxidant enzymes and metabolites. The results obtained by confocal microscopy showed the dramatic increase of ROS, H2O2 and NO during the anoxic shock. All the ascorbate-glutathione related parameters were altered during anoxia but restored during re-oxygenation. Anoxia also induced a slight but significant increase of α-tocopherol levels measured at the end of the treatment. Overall, the evaluation of cell defenses during anoxia and re-oxygenation in Arabidopsis cell cultures revealed that the immediate response involving the overproduction of reactive species activated the antioxidant machinery including ascorbate-glutathione system, α-tocopherol and the ROS-scavenging enzymes ascorbate peroxidase, catalase and peroxidase making cells able to counteract the stress towards cell survival.

  6. Rheological characteristics of cell suspension and cell culture of Perilla frutescens.

    Science.gov (United States)

    Zhong, J J; Seki, T; Kinoshita, S; Yoshida, T

    1992-12-01

    Physical properties such as viscosity, fluid dynamic behavior of cell suspension, and size distribution of cell aggregates of a plant, Perilla frustescens, cultured in a liquid medium were studied. As a result of investigations using cells harvester after 12 days of cultivation in a flask, it was found that the apparent viscosity of the cell suspension did not change with any variation of cell concentration below 5 g dry cell/L but markedly increased when the cell concentration increased over 12.8 g dry cell/L. The cell suspension exhibited the characteristics of a Bingham plastic fluid with a small yield stress. The size of cell aggregates in the range 74 to 500 mum did not influence the rheological characteristics of the cell suspension. The rheological characteristics of cultivation mixtures of P. frutescens cultivated in a flask and in a bioreactor were also investigated. The results showed that the flow characteristics of the cell culture could be described by a Bingham plastic model. At the later stage of cultivation, the apparent viscosity increased steadily, even though the biomass concentration (by dry weight) decreased, due to the increase of individual cell size.

  7. Suspension culture of pluripotent stem cells: effect of shear on stem cell fate.

    Science.gov (United States)

    Keller, Kevin C; Rodrigues, Beatriz; zur Nieden, Nicole I

    2014-01-01

    Despite significant promise, the routine usage of suspension cell culture to manufacture stem cell-derived differentiated cells has progressed slowly. Suspension culture is an innovative way of either expanding or differentiating cells and sometimes both are combined into a single bioprocess. Its advantages over static 2D culturing include a homogeneous and controllable culture environment and producing a large quantity of cells in a fraction of time. This feature makes suspension cell culture ideal for use in stem cell research and eventually ideal in the large-scale production of differentiated cells for regenerative medicine. Because of their tremendous differentiation capacities and unlimited growth properties, pluripotent stem cells (PSCs) in particular are considered potential sources for future cell-replacement therapies. Currently, expansion of PSCs is accomplished in 2D, which only permits a limited amount of cell growth per culture flask before cells need to be passaged. However, before stem cells can be applied clinically, several aspects of their expansion, such as directed growth, but also differentiation, need to be better controlled. This review will summarize recent advantages in suspension culture of PSCs, while at the same time highlighting current challenges.

  8. Isolation and culture of Celosia cristata L cell suspension protoplasts

    Directory of Open Access Journals (Sweden)

    Retno Mastuti

    2003-06-01

    Full Text Available Developmental competence of Celosia cristata L. cell suspension-derived protoplasts was investigated. The protoplasts were isolatedfrom 3- to 9-d old cultures in enzyme solution containing 2% (w/v Cellulase YC and 0.5% (w/v Macerozyme R-10 which was dissolvedin washing solution (0.4 M mannitol and 10 mM CaCl2 at pH 5.6 for 3 hours. The highest number of viable protoplasts was releasedfrom 5-d old culture of a homogenous cell suspension. Subsequently, three kinds of protoplast culture media were simultaneously examinedwith four kinds of concentration of gelling agent. Culturing the protoplasts on KM8p medium solidified with 1.2% agarose significantlyenhanced plating efficiency as well as microcolony formation. Afterwards, the microcalli actively proliferated into friable watery calluswhen they were subcultured on MS medium supplemented with 0.3 mg/l 2,4-D and 1.0 mg/l kinetin. Although the plant regenerationfrom the protoplasts-derived calli has not yet been obtained, the reproducible developmental step from protoplasts to callus in thisstudy may facilitate the establishment of somatic hybridization using C. cristata as one parent.

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

    Directory of Open Access Journals (Sweden)

    Cécile eALBENNE

    2013-05-01

    Full Text Available 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, cells 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 ten 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.

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

    Science.gov (United States)

    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.

  11. Comparative Transcriptomics of Arabidopsis thaliana Sperm Cells

    Science.gov (United States)

    In flowering plants the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part...

  12. Transcriptional Wiring of Cell Wall-Related Genes in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Marek Mutwil; Colin Ruprecht; Federico M. Giorgi; Martin Bringmann; Bj(o)rn Usadel; Staffan Persson

    2009-01-01

    Transcriptional coordination, or co-expression, of genes may signify functional relatedness of the correspond-ing proteins. For example, several genes involved in secondary cell wall cellulose biosynthesis are co-expressed with genes engaged in the synthesis of xylan, which is a major component of the secondary cell wall. To extend these types of anal-yses, we investigated the co-expression relationships of all Carbohydrate-Active enZYmes (CAZy)-related genes for Arabidopsis thaliana. Thus, the intention was to transcriptionally link different cell wall-related processes to each other, and also to other biological functions. To facilitate easy manual inspection, we have displayed these interactions as networks and matrices, and created a web-based interface (http://aranet.mpimp-golm.mpg.de/corecarb) containing downloadable files for all the transcriptional associations.

  13. Influence of Flow Behavior of Alginate-Cell Suspensions on Cell Viability and Proliferation.

    Science.gov (United States)

    Ning, Liqun; Guillemot, Arthur; Zhao, Jingxuan; Kipouros, Georges; Chen, Xiongbiao

    2016-07-01

    Tissue scaffolds with living cells fabricated by three-dimensional bioprinting/plotting techniques are becoming more prevalent in tissue repair and regeneration. In the bioprinting process, cells are subject to process-induced forces (such as shear force) that can result in cell damage and loss of cell function. The flow behavior of the biomaterial solutions that encapsulate living cells in this process plays an important role. This study used a rheometer to examine the flow behavior of alginate solution and alginate-Schwann cell (RSC96), alginate-fibroblast cell (NIH-3T3), and alginate-skeletal muscle cell (L8) suspensions during shearing with respect to effects on cell viability and proliferation. The flow behavior of all the alginate-cell suspensions varied with alginate concentration and cell density and had a significant influence on the viability and proliferation of the cells once sheared as well as on the recovery of the sheared cells. These findings provide a mean to preserve cell viability and/or retain cell proliferation function in the bioprinting process by regulating the flow behavior of cell-biomaterial suspensions and process parameters.

  14. Induction of Apoptosis in Protoplasts and Suspension Cultures of Plant Cells

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Many studies have showed that apoptosis exists in plants. Our study shows that (1) menadione(VK3) induces apoptosis in suspension cultures of carrot cells; (2) heat shock induces apoptosis in suspension cultures of tobacco cells; and (3) ethrel induces apoptosis in carrot protoplasts. Some important indications of apoptosis were observed, including DNA laddering, TUNEL-positive reaction, condensation and degradation of nuclei.

  15. Culture of isolated single cells from Taxus suspensions for the propagation of superior cell populations.

    Science.gov (United States)

    Naill, Michael C; Roberts, Susan C

    2005-11-01

    Single cells isolated from aggregated Taxus cuspidata cultures via enzymatic digestion were grown in suspension culture. High seeding density (4 x 10(5 )cells/ml) and the addition of cell-free conditioned medium were essential for growth. Doubling the concentration of the nutrients [ascorbic acid (150 g/l), glutamine (6.25 mM: ), and citric acid (150 g/l)] had no effect on single cell growth or viability. A specific growth rate of 0.11 days(-1) was achieved, which is similar to the observed growth rate of aggregated Taxus suspensions. The biocide, Plant Preservative Mixture, added at 0.2% (v/v) to all single cell cultures to prevent microbial contamination, had no significant effect on growth or viability. Following cell sorting, single cell cultures can be used to establish new cell lines for biotechnology applications or provide cells for further study.

  16. Molecule mechanism of stem cells in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Wenjin Zhang

    2014-01-01

    Full Text Available Plants possess the ability to continually produce new tissues and organs throughout their life. Unlike animals, plants are exposed to extreme variations in environmental conditions over the course of their lives. The vitality of plants is so powerful that they can survive several hundreds of years or even more making it an amazing miracle that comes from plant stem cells. The stem cells continue to divide to renew themselves and provide cells for the formation of leaves, stems, and flowers. Stem cells are not only quiescent but also immortal, pluripotent and homeostatic. Stem cells are the magic cells that repair tissues and regenerate organs. During the past decade, scholars around the world have paid more and more attention toward plant stem cells. At present, the major challenge is in relating molecule action mechanism to root apical meristem, shoot apical meristem and vascular system. The coordination between stem cells maintenance and differentiation is critical for normal plant growth and development. Elements such as phytohormones, transcription factors and some other known or unknown genes cooperate to balance this process. In this review, Arabidopsis thaliana as a pioneer system, we highlight recent developments in molecule modulating, illustrating how plant stem cells generate new mechanistic insights into the regulation of plants growth and development.

  17. The FRIABLE1 gene product affects cell adhesion in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Lutz Neumetzler

    Full Text Available Cell adhesion in plants is mediated predominantly by pectins, a group of complex cell wall associated polysaccharides. An Arabidopsis mutant, friable1 (frb1, was identified through a screen of T-DNA insertion lines that exhibited defective cell adhesion. Interestingly, the frb1 plants displayed both cell and organ dissociations and also ectopic defects in organ separation. The FRB1 gene encodes a Golgi-localized, plant specific protein with only weak sequence similarities to known proteins (DUF246. Unlike other cell adhesion deficient mutants, frb1 mutants do not have reduced levels of adhesion related cell wall polymers, such as pectins. Instead, FRB1 affects the abundance of galactose- and arabinose-containing oligosaccharides in the Golgi. Furthermore, frb1 mutants displayed alteration in pectin methylesterification, cell wall associated extensins and xyloglucan microstructure. We propose that abnormal FRB1 action has pleiotropic consequences on wall architecture, affecting both the extensin and pectin matrices, with consequent changes to the biomechanical properties of the wall and middle lamella, thereby influencing cell-cell adhesion.

  18. Profilin Plays a Role in Cell Elongation, Cell Shape Maintenance, and Flowering in Arabidopsis

    DEFF Research Database (Denmark)

    Ramachandran, S.; Christensen, Hans Erik Mølager; Ishimaru, Y.

    2000-01-01

    carrying a 35S-PFN-1 or 35S-antisense PFN-1 transgene. Etiolated seedlings underexpressing PFN (PFN-U) displayed an overall dwarf phenotype with short hypocotyls whose lengths were 20% to 25% that of wild type (WT) at low temperatures. Light-grown PFN-U plants were smaller in stature and flowered early......Profilin (PFN) is an ubiquitous, low-M-r, actin-binding protein involved in the organization of the cytoskeleton of eukaryotes including higher plants. PFNs are encoded by a multigene family in Arabidopsis. We have analyzed in vivo functions of Arabidopsis PFN by generating transgenic plants...... expressed in the vascular bundles of cotyledons and leaves. Our results show that Arabidopsis PFNs play a role in cell elongation, cell shape maintenance, polarized growth of root hair, and unexpectedly, in determination of flowering time....

  19. Convective flows of colloidal suspension in an inclined closed cell

    Science.gov (United States)

    Smorodin, Boris; Cherepanov, Ivan; Ishutov, Sergey

    2016-12-01

    The nonlinear spatiotemporal evolution of convective flows is numerically investigated in the case of colloidal suspension filling an inclined closed cell heated from below. The bifurcation diagram (the dependency of the Nusselt number on the Rayleigh number) is obtained. The characteristics of the wave and steady patterns are investigated depending on heat intensity. The travelling wave changing travel direction and the non-regular oscillatory flow are found to be stable solutions within a certain interval of the Rayleigh number. Temporal Fourier decomposition is used together with other diagnostic tools to analyse the complex bifurcation and spatiotemporal properties caused by the interplay of the gravity-induced gradient of concentration and convective mixing of the fluid. It is shown that a more complex flow structure exists at a lower heating intensity (Rayleigh number).

  20. Comparison of the oxygen exchange between photosynthetic cell suspensions and detached leaves of Euphorbia characias L

    Energy Technology Data Exchange (ETDEWEB)

    Carrier, P.; Chagvardieff, P.; Tapie, P. (C.E.N. Cadarache, Saint-Paul lez Durance (France))

    1989-11-01

    Using a mass-spectrometric {sup 16}O{sub 2}/{sup 18}O{sub 2}-isotope technique, we compared the nature and the relative importance of oxygen exchange in photomixotrophic (PM) and photoautotrophic (PA) suspensions of Euphorbia characias L. with those in intact leaves of the same species. Young and mature leaves, dividing and nondividing cell suspensions were characterized in short-term experiments. On chlorophyll basis, the gross photosynthetic activities at CO{sub 2} saturating concentration of PA and PM suspensions varied little from those of leaves. On dry weight basis, gross photosynthesis of PA suspensions was equal to that of leaves because of their similar chlorophyll content. This was not the case in PM suspensions where gross photosynthesis was lower and largely varied during the growth cycle. The CO{sub 2} compensation point of PA cells was much higher than that of leaves. Oxygen uptakes were analyzed in terms of mitochondrial respiration, photorespiration and light stimulation of oxygen uptake (LSOU), often identified to Mehler-type reactions. In Pa and PM suspensions, mitochondrial respiration rates were higher than in leaves by a factor of 1.5 to 4.5. In PM suspensions, photorespiration and LSOU were observed only in nondividing cells. Photorespiration and LSOU rates were comparable in PA suspensions and leaves. Our results demonstrate that photorespiration of PA suspensions has not been affected by the 2% CO{sub 2} concentration imposed during 2 years of culture.

  1. Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

    OpenAIRE

    Bauwens, Celine L.; Toms, Derek; Ungrin, Mark

    2016-01-01

    Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization l...

  2. Fine-mapping of an Arabidopsis cell death mutation locus

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    An Arabidopsis cell death mutation locus was mapped to chromosome 2 between IGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.

  3. Fine-mapping of an Arabidopsis cell death mutation locus

    Institute of Scientific and Technical Information of China (English)

    牟中林; 戴亚; 李家洋

    2000-01-01

    An Arabidopsis cell death mutation locus was mapped to chromosome 2 between lGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.

  4. Gibberellins accumulate in the elongating endodermal cells of Arabidopsis root.

    Science.gov (United States)

    Shani, Eilon; Weinstain, Roy; Zhang, Yi; Castillejo, Cristina; Kaiserli, Eirini; Chory, Joanne; Tsien, Roger Y; Estelle, Mark

    2013-03-19

    Plant hormones are small-molecule signaling compounds that are collectively involved in all aspects of plant growth and development. Unlike animals, plants actively regulate the spatial distribution of several of their hormones. For example, auxin transport results in the formation of auxin maxima that have a key role in developmental patterning. However, the spatial distribution of the other plant hormones, including gibberellic acid (GA), is largely unknown. To address this, we generated two bioactive fluorescent GA compounds and studied their distribution in Arabidopsis thaliana roots. The labeled GAs specifically accumulated in the endodermal cells of the root elongation zone. Pharmacological studies, along with examination of mutants affected in endodermal specification, indicate that GA accumulation is an active and highly regulated process. Our results strongly suggest the presence of an active GA transport mechanism that would represent an additional level of GA regulation.

  5. The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses

    Directory of Open Access Journals (Sweden)

    Asako eUchiyama

    2014-11-01

    Full Text Available Synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. Thought to be exclusive to animals, synaptotagmins have recently been characterized in Arabidopsis thaliana, in which they comprise a five gene family. Using infectivity and leaf-based functional assays, we have shown that Arabidopsis SYTA regulates endocytosis and marks an endosomal vesicle recycling pathway to regulate movement protein-mediated trafficking of the Begomovirus Cabbage leaf curl virus (CaLCuV and the Tobamovirus Tobacco mosaic virus (TMV through plasmodesmata (Lewis and Lazarowitz, 2010. To determine whether SYTA has a central role in regulating the cell-to-cell trafficking of a wider range of diverse plant viruses, we extended our studies here to examine the role of SYTA in the cell-to-cell movement of additional plant viruses that employ different modes of movement, namely the Potyvirus Turnip mosaic virus (TuMV, the Caulimovirus Cauliflower mosaic virus (CaMV and the Tobamovirus Turnip vein clearing virus (TVCV, which in contrast to TMV does efficiently infect Arabidopsis. We found that both TuMV and TVCV systemic infection, and the cell-to-cell trafficking of the their movement proteins, were delayed in the Arabidopsis Col-0 syta-1 knockdown mutant. In contrast, CaMV systemic infection was not inhibited in syta-1. Our studies show that SYTA is a key regulator of plant virus intercellular movement, being necessary for the ability of diverse cell-to-cell movement proteins encoded by Begomoviruses (CaLCuV MP, Tobamoviruses (TVCV and TMV 30K protein and Potyviruses (TuMV P3N-PIPO to alter PD and thereby mediate virus cell-to-cell spread.

  6. A numerical model of localized convection cells of Euglena suspensions

    Science.gov (United States)

    Iima, Makoto; Shoji, Erika; Yamaguchi, Takayuki

    2014-11-01

    Suspension of Euglena gracilis shows localized convection cells when it is illuminated form below with strong light intensity. Experiments in an annular container shows that there are two elementary localized structures. One consists of a pair of convection cells and a single region where number density of Euglena is high. The other consists a localized traveling wave. Based on the measurements of the flux of number density, we propose a model of bioconvection incorporating lateral phototaxis effect proportional to the light intensity gradient. Using pseudo spectral method, we performed numerical simulation of this model. We succeed in reproducing one of the localized structures, a convection pair with single region of high number density. Also, when the aspect ratio is large, there are a parameter region where the localized structure and conductive state are both stable, which is suggested by experiments. Spatial distribution of the number density implies that the accumulation of microorganism due to the convective flow causes such bistability. CREST(PJ74100011) and KAKENHI(26400396).

  7. Microchannel-free collection and single-cell isolation of yeast cells in a suspension using liquid standing wave

    Science.gov (United States)

    Matsutani, Akihiro; Takada, Ayako

    2016-11-01

    We demonstrate a microchannel-free collection method at nodes of liquid standing waves by the vertical vibration of a suspension including yeast cells. The pattern formation of the collection of cells using standing waves in a suspension was investigated by varying the frequency and waveform of vibrations. The single-cell isolation of yeast cells was achieved using a microenclosure array set at the nodes. In addition, we succeeded in the microchannel-free collection of yeast cells in a suspension, where patterns were formed by tapping vibration. The proposed technique is very simple and we believe that it will be useful for single-cell analysis and investigation.

  8. Cytokinin signaling regulates pavement cell morphogenesis in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Hongjiang Li; Tongda Xu; Deshu Lin; Mingzhang Wen; Mingtang Xie; Jér(o)me Duclercq; Agnieszka Bielach

    2013-01-01

    The puzzle piece-shaped Arabidopsis leaf pavement cells (PCs) with interdigitated lobes and indents is a good model system to investigate the mechanisms that coordinate cell polarity and shape formation within a tissue.Auxin has been shown to coordinate the interdigitation by activating ROP GTPase-dependent signaling pathways.To identify additional components or mechanisms,we screened for mutants with abnormal PC morphogenesis and found that cytokinin signaling regulates the PC interdigitation pattern.Reduction in cytokinin accumulation and defects in cytokinin signaling (such as in ARR7-over-expressing lines,the ahk3cre1 cytokinin receptor mutant,and the ahp12345 cytokinin signaling mutant) enhanced PC interdigitation,whereas over-production of cytokinin and over-activation of cytokinin signaling in an ARR20 over-expression line delayed or abolished PC interdigitation throughout the cotyledon.Genetic and biochemical analyses suggest that cytokinin signaling acts upstream of ROPs to suppress the formation of interdigitated pattern.Our results provide novel mechanistic understanding of the pathways controlling PC shape and uncover a new role for cytokinin signaling in cell morphogenesis.

  9. Exploring Arabidopsis thaliana Root Endophytes via Single-Cell Genomics

    Energy Technology Data Exchange (ETDEWEB)

    Lundberg, Derek; Woyke, Tanja; Tringe, Susannah; Dangl, Jeff

    2014-03-19

    Land plants grow in association with microbial communities both on their surfaces and inside the plant (endophytes). The relationships between microbes and their host can vary from pathogenic to mutualistic. Colonization of the endophyte compartment occurs in the presence of a sophisticated plant immune system, implying finely tuned discrimination of pathogens from mutualists and commensals. Despite the importance of the microbiome to the plant, relatively little is known about the specific interactions between plants and microbes, especially in the case of endophytes. The vast majority of microbes have not been grown in the lab, and thus one of the few ways of studying them is by examining their DNA. Although metagenomics is a powerful tool for examining microbial communities, its application to endophyte samples is technically difficult due to the presence of large amounts of host plant DNA in the sample. One method to address these difficulties is single-cell genomics where a single microbial cell is isolated from a sample, lysed, and its genome amplified by multiple displacement amplification (MDA) to produce enough DNA for genome sequencing. This produces a single-cell amplified genome (SAG). We have applied this technology to study the endophytic microbes in Arabidopsis thaliana roots. Extensive 16S gene profiling of the microbial communities in the roots of multiple inbred A. thaliana strains has identified 164 OTUs as being significantly enriched in all the root endophyte samples compared to their presence in bulk soil.

  10. A phytochemical study of lignans in whole plants and cell suspension cultures of Anthriscus sylvestris

    NARCIS (Netherlands)

    Koulman, A; Kubbinga, M.E.; Batterman, S; Woerdenbag, H.J.; Pras, N.; Woolley, J.G.; Quax, Wim

    2003-01-01

    In the roots of Anthriscus sylvestris 12 different lignans were detected. Arctigenin, dimethylmatairesinol, dimethylthujaplicatin, podophyllotoxin, 7-hydroxyyatein and 7-hydroxyanhydropodorhizol have not been previously reported to be present in A. sylvestris. In the cell suspension cultures, which

  11. Transplantation of autologous noncultured epidermal cell suspension in treatment of patients with stable vitiligo

    Institute of Scientific and Technical Information of China (English)

    XU Ai-e; WEI Xiao-dong; CHENG Dong-qing; ZHOU He-fen; QIAN Guo-pei

    2005-01-01

    @@ Treatment of vitiligo by transplantation of noncultured melanocytes containing keratino-cytes has been successful since 1992,1 We report the encouraging results of autologous epidermal cell suspension in the treatment of 24 patients with stable vitiligo since 1998.

  12. A Novel Function for Arabidopsis CYCLASE1 in Programmed Cell Death Revealed by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) Analysis of Extracellular Matrix Proteins.

    Science.gov (United States)

    Smith, Sarah J; Kroon, Johan T M; Simon, William J; Slabas, Antoni R; Chivasa, Stephen

    2015-06-01

    Programmed cell death is essential for plant development and stress adaptation. A detailed understanding of the signal transduction pathways that regulate plant programmed cell death requires identification of the underpinning protein networks. Here, we have used a protagonist and antagonist of programmed cell death triggered by fumonisin B1 as probes to identify key cell death regulatory proteins in Arabidopsis. Our hypothesis was that changes in the abundance of cell death-regulatory proteins induced by the protagonist should be blocked or attenuated by concurrent treatment with the antagonist. We focused on proteins present in the mobile phase of the extracellular matrix on the basis that they are important for cell-cell communications during growth and stress-adaptive responses. Salicylic acid, a plant hormone that promotes programmed cell death, and exogenous ATP, which can block fumonisin B1-induced cell death, were used to treat Arabidopsis cell suspension cultures prior to isobaric-tagged relative and absolute quantitation analysis of secreted proteins. A total of 33 proteins, whose response to salicylic acid was suppressed by ATP, were identified as putative cell death-regulatory proteins. Among these was CYCLASE1, which was selected for further analysis using reverse genetics. Plants in which CYCLASE1 gene expression was knocked out by insertion of a transfer-DNA sequence manifested dramatically increased cell death when exposed to fumonisin B1 or a bacterial pathogen that triggers the defensive hypersensitive cell death. Although pathogen inoculation altered CYCLASE1 gene expression, multiplication of bacterial pathogens was indistinguishable between wild type and CYCLASE1 knockout plants. However, remarkably severe chlorosis symptoms developed on gene knockout plants in response to inoculation with either a virulent bacterial pathogen or a disabled mutant that is incapable of causing disease in wild type plants. These results show that CYCLASE1, which

  13. Diadenosine triphosphate is a novel factor which in combination with cyclodextrins synergistically enhances the biosynthesis of trans-resveratrol in Vitis vinifera cv. Monastrell suspension cultured cells.

    Science.gov (United States)

    Pietrowska-Borek, Małgorzata; Czekała, Lukasz; Belchí-Navarro, Sarai; Pedreño, María Angeles; Guranowski, Andrzej

    2014-11-01

    Dinucleoside polyphosphates are considered as signal molecules that may evoke response of plant cells to stress. Other compounds whose biological effects have been recognized are cyclodextrins. They are cyclic oligosaccharides that chemically resemble the alkyl-derived pectic oligosaccharides naturally released from the cell walls during fungal attack, and they act as true elicitors, since, when added to plant cell culture, they induce the expression of genes involved in some secondary metabolism pathways. Previously, we demonstrated that some dinucleoside polyphosphates triggered the biosynthesis of enzymes involved in the phenylpropanoid pathway in Arabidopsis thaliana. In Vitis vinifera suspension cultured cells, cyclodextrins were shown to enhance the accumulation of trans-resveratrol, one of the basic units of the stilbenes derived from the phenylpropanoid pathway. Here, we show that diadenosine triphosphate, applied alone or in combination with cyclodextrins to the grapevine suspension-cultured cells, increased the transcript level of genes encoding key phenylpropanoid-pathway enzymes as well as the trans-resveratrol production inside cells and its secretion into the extracellular medium. In the latter case, these two compounds acted synergistically. However, the accumulation of trans-resveratrol and its glucoside trans-piceid inside cells were stimulated much better by diadenosine triphosphate than by cyclodextrins.

  14. Disposable orbitally shaken TubeSpin bioreactor 600 for Sf9 cell cultivation in suspension.

    Science.gov (United States)

    Monteil, Dominique T; Shen, Xiao; Tontodonati, Giulia; Baldi, Lucia; Hacker, David L; Wurm, Florian M

    2016-07-15

    Disposable orbitally shaken TubeSpin bioreactor 600 tubes (TS600s) were recently developed for the bench-scale cultivation of animal cells in suspension. Here we compared batch cultures of Sf9 insect cells in TS600s, spinner flasks, and shake flasks. Superior cell growth was observed in TS600s and shake flasks as compared with spinner flasks, and more favorable oxygen-enriched cell culture conditions were observed in TS600s as compared with either spinner or shake flasks. The results demonstrated the suitability of TS600s as a disposable vessel for the cultivation of Sf9 cells in suspension.

  15. Growth and Plating of Cell Suspension Cultures of Datura Innoxia

    DEFF Research Database (Denmark)

    Engvild, Kjeld Christensen

    1974-01-01

    Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did...

  16. Xyloglucan Endotransglycosylase Activity in Carrot Cell Suspensions during cell Elongation and Somatic Embryogenesis.

    Science.gov (United States)

    Hetherington, P. R.; Fry, S. C.

    1993-11-01

    Xyloglucan endotransglycosylase (XET) has been proposed to contribute to cell elongation through wall loosening. To explore this relationship further, we assayed this enzyme activity in suspensions of carrot (Daucus carota L.) cells exhibiting various rates of cell elongation. In one cell line, elongation was induced by dilution into dichlorophenoxyacetic acid (2,4-D)-free medium. During this elongation, 93% of the XET activity was found in the culture medium; in nonelongating controls, by contrast, 68% was found in the cell extracts even though the specific activity of these extracts was lower than in the elongating cells. By far the highest rates of XET secretion per cell were in the elongating cells. A second cell line was induced to undergo somatic embryogenesis by dilution into 2,4-D-free medium. During the first 6 d, numerous globular embryoids composed of small, isodiametric cells were formed in the absence of cell elongation; extracellular XET activity was almost undetectable, and intracellular specific activity markedly declined. After 6 d, heart, torpedo, and cotyledonary embryoids began to appear (i.e. cell elongation resumed); the intracellular specific activity of XET rose rapidly and >80% of the XET activity accumulated in the medium. Thus, nonexpanding cell suspensions (whether or not they were rapidly dividing) produced and secreted less XET activity than did expanding cells. We propose that a XET molecule has an ephemeral wall-loosening role while it passes through the load-bearing layer of the wall on its way from the protoplast into the culture medium.

  17. Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum.

    Science.gov (United States)

    Natori, Tomomi; Fujiyoshi, Masachika; Uchida, Masashi; Abe, Natsuki; Kanaki, Tatsuro; Fukumoto, Yasunori; Ishii, Itsuko

    2017-03-01

    The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27(Kip1) expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.

  18. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis

    NARCIS (Netherlands)

    Gouget, A.; Senchou, V.; Govers, F.; Sanson, A.; Barre, A.; Rougé, P.; Pont-Lezica, R.; Canut, H.

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsi

  19. SHORT-ROOT and SCARECROW regulate leaf growth in Arabidopsis by stimulating S-phase progression of the cell cycle.

    NARCIS (Netherlands)

    Dhondt, S.; Coppens, F.; Winter, F. de; Swarup, K.; Merks, R.M.H.; Inze, D.; Bennett, M.J.; Beemster, G.T.S.

    2010-01-01

    SHORT-ROOT (SHR) and SCARECROW (SCR) are required for stem cell maintenance in the Arabidopsis (Arabidopsis thaliana) root meristem, ensuring its indeterminate growth. Mutation of SHR and SCR genes results in disorganization of the quiescent center and loss of stem cell activity, resulting in the ce

  20. Metabolomic, proteomic and biophysical analyses of Arabidopsis thaliana cells exposed to a caesium stress. Influence of potassium supply.

    Science.gov (United States)

    Le Lay, P; Isaure, M-P; Sarry, J-E; Kuhn, L; Fayard, B; Le Bail, J-L; Bastien, O; Garin, J; Roby, C; Bourguignon, J

    2006-11-01

    The incorporation and localisation of 133Cs in a plant cellular model and the metabolic response induced were analysed as a function of external K concentration using a multidisciplinary approach. Sucrose-fed photosynthetic Arabidopsis thaliana suspension cells, grown in a K-containing or K-depleted medium, were submitted to a 1 mM Cs stress. Cell growth, strongly diminished in absence of K, was not influenced by Cs. In contrast, the chlorophyll content, affected by a Cs stress superposed to K depletion, did not vary under the sole K depletion. The uptake of Cs was monitored in vivo using 133Cs NMR spectroscopy while the final K and Cs concentrations were determined using atomic absorption spectrometry. Cs absorption rate and final concentration increased in a K-depleted external medium; in vivo NMR revealed that intracellular Cs was distributed in two kinds of compartment. Synchrotron X-ray fluorescence microscopy indicated that one could be the chloroplasts. In parallel, the cellular response to the Cs stress was analysed using proteomic and metabolic profiling. Proteins up- and down-regulated in response to Cs, in presence of K+ or not, were analysed by 2D gel electrophoresis and identified by mass spectrometry. No salient feature was detected excepting the overexpression of antioxidant enzymes, a common response of Arabidopsis cells stressed whether by Cs or by K-depletion. 13C and 31P NMR analysis of acid extracts showed that the metabolome impact of the Cs stress was also a function of the K nutrition. These analyses suggested that sugar metabolism and glycolytic fluxes were affected in a way depending upon the medium content in K+. Metabolic flux measurements using 13C labelling would be an elegant way to pursue on this line. Using our experimental system, a progressively stronger Cs stress might point out other specific responses elicited by Cs.

  1. Ultrasound-mediated gene transfection: A comparison between cells irradiated in suspension and attachment status

    Science.gov (United States)

    Zhang, Yiwei; Azuma, Takashi; Sasaki, Akira; Yoshinaka, Kiyoshi; Takagi, Shu; Matsumoto, Yoichiro

    2012-10-01

    Sonoporation, in the presence of microbubbles, is a promising nonviral gene transfection method. Although the mechanism is not yet fully understood, shock waves emitted by cavitation bubbles have been known to play an important role in creating pores on cell membranes. This work investigates the gene transfection efficiency and influencing parameters of cells in two different statuses: attachment and suspension based on the fact that cells in suspension have more bubbles surrounding them and that shock wave has distinct effects on hit objects whether the object is attached to a rigid wall or not. Fibroblast cells (NIH3T3), both in attachment and suspension, and green fluorescent protein (GFP) plasmid were exposed to variations in acoustic pressure (0.6-1.2 MPa) and 10% duty cycle at fixed settings of 2 MHz central frequency, 5 kHz pulse repetition frequency and 1 minute insonation time, in the presence of 10% v/v microbubbles (Sonazoid, a commercialized product of ultrasound contrast agent). The transfection efficiency and cell viability are compared for two statuses and a distribution map of GFP transfected cells as well as viable cells over the well bottom is given for attachment status. The results show that cells irradiated in suspension status has higher transfection ratio as well as viability than those irradiated in attachment status with the same intensity and that the transfected cells of attachment status experiment are highly concentrated near the center of the well.

  2. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Iakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.; Harren, F.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 2¿3 days which indicates the existence

  3. Involvement of ethylene and lipid signalling in cadmium-induced programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Yakimova, E.T.; Kapchina-Toteva, V.M.; Laarhoven, L.J.J.; Harren, F.J.M.; Woltering, E.J.

    2006-01-01

    Cadmium-induced cell death was studied in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells (line MsK8) treated with CdSO4. Within 24 h, cadmium treatment induced cell death in a concentration-dependent manner. Cell cultures showed recovery after 23 days which indicates the existence

  4. Automatic Quantification of the Number of Intracellular Compartments in Arabidopsis thaliana Root Cells

    Science.gov (United States)

    Bayle, Vincent; Platre, Matthieu Pierre; Jaillais, Yvon

    2017-01-01

    In the era of quantitative biology, it is increasingly required to quantify confocal microscopy images. If possible, quantification should be performed in an automatic way, in order to avoid bias from the experimenter, to allow the quantification of a large number of samples, and to increase reproducibility between laboratories. In this protocol, we describe procedures for automatic counting of the number of intracellular compartments in Arabidopsis root cells, which can be used for example to study endocytosis or secretory trafficking pathways and to compare membrane organization between different genotypes or treatments. While developed for Arabidopsis roots, this method can be used on other tissues, cell types and plant species. PMID:28255574

  5. Cell fate in the Arabidopsis root epidermis is determined by competition between WEREWOLF and CAPRICE.

    Science.gov (United States)

    Song, Sang-Kee; Ryu, Kook Hui; Kang, Yeon Hee; Song, Jae Hyo; Cho, Young-Hee; Yoo, Sang-Dong; Schiefelbein, John; Lee, Myeong Min

    2011-11-01

    The root hair and nonhair cells in the Arabidopsis (Arabidopsis thaliana) root epidermis are specified by a suite of transcriptional regulators. Two of these are WEREWOLF (WER) and CAPRICE (CPC), which encode MYB transcription factors that are required for promoting the nonhair cell fate and the hair cell fate, respectively. However, the precise function and relationship between these transcriptional regulators have not been fully defined experimentally. Here, we examine these issues by misexpressing the WER gene using the GAL4-upstream activation sequence transactivation system. We find that WER overexpression in the Arabidopsis root tip is sufficient to cause epidermal cells to adopt the nonhair cell fate through direct induction of GLABRA2 (GL2) gene expression. We also show that GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), two closely related bHLH proteins, are required for the action of the overexpressed WER and that WER interacts with these bHLHs in plant cells. Furthermore, we find that CPC suppresses the WER overexpression phenotype quantitatively. These results show that WER acts together with GL3/EGL3 to induce GL2 expression and that WER and CPC compete with one another to define cell fates in the Arabidopsis root epidermis.

  6. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells

    Directory of Open Access Journals (Sweden)

    Vincent C. Chen

    2015-09-01

    Full Text Available To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2 × 109 CM/L at scales up to 1 L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  7. Development of a scalable suspension culture for cardiac differentiation from human pluripotent stem cells.

    Science.gov (United States)

    Chen, Vincent C; Ye, Jingjing; Shukla, Praveen; Hua, Giau; Chen, Danlin; Lin, Ziguang; Liu, Jian-chang; Chai, Jing; Gold, Joseph; Wu, Joseph; Hsu, David; Couture, Larry A

    2015-09-01

    To meet the need of a large quantity of hPSC-derived cardiomyocytes (CM) for pre-clinical and clinical studies, a robust and scalable differentiation system for CM production is essential. With a human pluripotent stem cells (hPSC) aggregate suspension culture system we established previously, we developed a matrix-free, scalable, and GMP-compliant process for directing hPSC differentiation to CM in suspension culture by modulating Wnt pathways with small molecules. By optimizing critical process parameters including: cell aggregate size, small molecule concentrations, induction timing, and agitation rate, we were able to consistently differentiate hPSCs to >90% CM purity with an average yield of 1.5 to 2×10(9) CM/L at scales up to 1L spinner flasks. CM generated from the suspension culture displayed typical genetic, morphological, and electrophysiological cardiac cell characteristics. This suspension culture system allows seamless transition from hPSC expansion to CM differentiation in a continuous suspension culture. It not only provides a cost and labor effective scalable process for large scale CM production, but also provides a bioreactor prototype for automation of cell manufacturing, which will accelerate the advance of hPSC research towards therapeutic applications.

  8. Use of sulfate reducing cell suspension bioreactors for the treatment of SO2 rich flue gases

    NARCIS (Netherlands)

    Lens, P.N.L.; Gastesi, R.; Lettinga, G.

    2003-01-01

    This paper describes a novel bioscrubber concept for biological flue gas desulfurization, based on the recycling of a cell suspension of sulfite/sulfate reducing bacteria between a scrubber and a sulfite/sulfate reducing hydrogen fed bioreactor. Hydrogen metabolism in sulfite/sulfate reducing cell s

  9. Computer Simulation Study of Collective Phenomena in Dense Suspensions of Red Blood Cells under Shear

    CERN Document Server

    Krüger, Timm

    2012-01-01

    The rheology of dense red blood cell suspensions is investigated via computer simulations based on the lattice Boltzmann, the immersed boundary, and the finite element methods. The red blood cells are treated as extended and deformable particles immersed in the ambient fluid. In the first part of the work, the numerical model and strategies for stress evaluation are discussed. In the second part, the behavior of the suspensions in simple shear flow is studied for different volume fractions, particle deformabilities, and shear rates. Shear thinning behavior is recovered. The existence of a shear-induced transition from a tumbling to a tank-treading motion is demonstrated. The transition can be parameterized by a single quantity, namely the effective capillary number. It is the ratio of the suspension stress and the characteristic particle membrane stress. At the transition point, a strong increase in the orientational order of the red blood cells and a significant decrease of the particle diffusivity are obser...

  10. Arabidopsis  SABRE and CLASP interact to stabilize cell division plane orientation and planar polarity

    OpenAIRE

    2013-01-01

    The orientation of cell division and the coordination of cell polarity within the plane of the tissue layer (planar polarity) contribute to shape diverse multicellular organisms. The root of Arabidopsis thaliana displays regularly oriented cell divisions, cell elongation and planar polarity providing a plant model system to study these processes. Here we report that the SABRE protein, which shares similarity with proteins of unknown function throughout eukaryotes, has important roles in orien...

  11. Establishment of forskolin yielding transformed cell suspension cultures of Coleus forskohlii as controlled by different factors.

    Science.gov (United States)

    Mukherjee, S; Ghosh, B; Jha, S

    2000-01-07

    Suspension cultures derived from gall calli which were obtained following infection with Agrobacterium tumefaciens (C58) were established in Coleus forskohlii. Cell line selection following single cell cloning or cell aggregate cloning was carried out to select cell lines capable of fast growth and for producing high level of forskolin. A fast growing cell line (GSO-5/7) thus selected was found to accumulate 0.021% forskolin in 42 days. The effect of cultural conditions on cell growth was studied to identify factors influencing biomass yield. Cell growth in suspension was found to be influenced significantly by carbon source, initial cell density and light or dark condition. Optimal cell growth (20 fold increase in biomass in a 42 day period) was obtained when the cells were grown in dark condition in B5O media containing 3% sucrose as sole carbon source with an initial cell density of 1.5 x 10(5) cells per ml. Forskolin accumulation was maximum (0.021%) in the stationary phase of cell growth. These suspension cultures showed continuous and stable production of forskolin.

  12. Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia an tobacco

    NARCIS (Netherlands)

    Kater, M.M.; Franken, J.; Aelst, van A.; Angenent, G.C.

    2000-01-01

    Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ov

  13. A critical role for ethylene in hydrogen peroxide release during programmed cell death in tomato suspension cells

    NARCIS (Netherlands)

    Jong, de A.J.; Yakimova, E.T.; Kapchina, V.M.; Woltering, E.J.

    2002-01-01

    Camptothecin, a topo isomerase-I inhibitor used in cancer therapy, induces apoptosis in animal cells. In tomato (Lycopersicon esculentum Mill.) suspension cells, camptothecin induces cell death that is accompanied by the characteristic nuclear morphological changes such as chromatin condensation and

  14. Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Shafa Mehdi

    2011-12-01

    Full Text Available Abstract Background Embryonic stem cells (ESCs can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs. However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Results Murine D3-MHC-neor ESCs formed embyroid bodies (EBs and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. Conclusions This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC

  15. LESION SIMULATING DISEASE1 interacts with catalases to regulate hypersensitive cell death in Arabidopsis.

    Science.gov (United States)

    Li, Yansha; Chen, Lichao; Mu, Jinye; Zuo, Jianru

    2013-10-01

    LESION SIMULATING DISEASE1 (lsd1) is an important negative regulator of programmed cell death (PCD) in Arabidopsis (Arabidopsis thaliana). The loss-of-function mutations in lsd1 cause runaway cell death triggered by reactive oxygen species. lsd1 encodes a novel zinc finger protein with unknown biochemical activities. Here, we report the identification of CATALASE3 (CAT3) as an lsd1-interacting protein by affinity purification and mass spectrometry-based proteomic analysis. The Arabidopsis genome contains three homologous catalase genes (CAT1, CAT2, and CAT3). Yeast two-hybrid and coimmunoprecipitation analyses demonstrated that lsd1 interacted with all three catalases both in vitro and in vivo, and the interaction required the zinc fingers of lsd1. We found that the catalase enzymatic activity was reduced in the lsd1 mutant, indicating that the catalase enzyme activity was partially dependent on lsd1. Consistently, the lsd1 mutant was more sensitive to the catalase inhibitor 3-amino-1,2,4-triazole than the wild type, suggesting that the interaction between lsd1 and catalases is involved in the regulation of the reactive oxygen species generated in the peroxisome. Genetic studies revealed that lsd1 interacted with CATALASE genes to regulate light-dependent runaway cell death and hypersensitive-type cell death. Moreover, the accumulation of salicylic acid was required for PCD regulated by the interaction between lsd1 and catalases. These results suggest that the lsd1-catalase interaction plays an important role in regulating PCD in Arabidopsis.

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

    Institute of Scientific and Technical Information of China (English)

    Hugo Melida; Antonio Encina; Asier Largo-Gosens; Esther Novo-Uzal; Rogelio Santiago; Federico Pomar; Pedro Garca; Penelope Garca-Angulo; Jose Luis Acebes; Jesus Alvarez

    2015-01-01

    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.

  17. Comparative metabolite profiling of the insecticide thiamethoxam in plant and cell suspension culture of tomato.

    Science.gov (United States)

    Karmakar, Rajib; Bhattacharya, Ramcharan; Kulshrestha, Gita

    2009-07-22

    The metabolism of thiamethoxam [(EZ)-3-(2-chloro-1,3-thiazol-5-yl-methyl)-5-methyl-1,3,5-oxadiazinan-4-ylidene (nitro) amine] was investigated in whole plant, callus, and heterotrophic cell suspension culture of aseptically and field grown tomato (Lycopersicon esculentum Mill.) plants. The structure of the metabolites was elucidated by chromatographic (HPLC) and spectroscopic (IR, NMR, and MS) methods. Thiamethoxam metabolism proceeded by the formation of a urea derivative, a nitroso product, and nitro guanidine. Both urea and nitro guanidine metabolites further degraded in plants, and a mechanism has been proposed. In the plant, organ-specific differences in thiamethoxam metabolism were observed. Only one metabolite was formed in whole plant against four in callus and eight metabolites in cell suspension culture under aseptic conditions. Out of six metabolites of thiamethoxam in tomato fruits in field conditions, five were similar to those formed in the cell suspension culture. In the cell suspension culture, thiamethoxam degraded to maximum metabolites within 72 h, whereas in plants, such extensive conversion could only be observed after 10 days.

  18. DIGLUCOSYLATION OF SALICYL ALCOHOL BY CELL SUSPENSION CULTURES OF SOLANUM LACINIATUM

    Institute of Scientific and Technical Information of China (English)

    ACHMAD SYAHRANI; FRANSISCA HARTUTI; GUNAWAN INDRAYANTO; ALISTAIR L.WILKINS

    2001-01-01

    A new biotransformation product, salicyl alcohol-7-O-β-D-(β-l,6-D-glucopyranosyl)-gluco pyranoside was isolated from cell suspension cultures of Solanum laciniatum, following administration of salicyl alcohol, and its structure was elucidated using a combination of one and two-dimensional 1H and 13C-NMR data, and positive and negative ion ESMS data.

  19. Accumulation of podophyllotoxin and related lignans in cell suspension cultures of Linum album

    NARCIS (Netherlands)

    Smollny, T.; Wichers, H.; Kalenberg, S.; Shahsavari, A.; Petersen, M.; Alfermann, A.W.

    1998-01-01

    Cell suspension cultures of Linum album were established, which were able to synthesize and accumulate lignans. Podophyllotoxin and 5-methoxypodophyllotoxin were the main products and were present as glycosides, together with small amounts of deoxypodophyllotoxin, 5′-demethoxy-5-methoxypodophyllotox

  20. Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions.

    Science.gov (United States)

    Broggi, Achille; Cigni, Clara; Zanoni, Ivan; Granucci, Francesca

    2016-04-20

    The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

  1. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    Science.gov (United States)

    Yuan, Hengguang; Hu, Shanglian; Huang, Peng; Song, Hua; Wang, Kan; Ruan, Jing; He, Rong; Cui, Daxiang

    2011-12-01

    Herein we are the first to report that single-walled carbon nanotubes (SWCNTs) exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS) generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  2. Single Walled Carbon Nanotubes Exhibit Dual-Phase Regulation to Exposed Arabidopsis Mesophyll Cells

    Directory of Open Access Journals (Sweden)

    Huang Peng

    2011-01-01

    Full Text Available Abstract Herein we are the first to report that single-walled carbon nanotubes (SWCNTs exhibit dual-phase regulation to Arabidopsis mesophyll cells exposed to different concentration of SWCNTs. The mesophyll protoplasts were prepared by enzyme digestion, and incubated with 15, 25, 50, 100 μg/ml SWCNTs for 48 h, and then were observed by optical microscopy and transmission electron microscopy, the reactive oxygen species (ROS generation was measured. Partial protoplasts were stained with propidium iodide and 4'-6- diamidino-2-phenylindole, partial protoplasts were incubated with fluorescein isothiocyanate-labeled SWCNTs, and observed by fluorescence microscopy. Results showed that SWCNTs could traverse both the plant cell wall and cell membrane, with less than or equal to 50 μg/ml in the culture medium, SWCNTs stimulated plant cells to grow out trichome clusters on their surface, with more than 50 μg/ml SWCNTs in the culture medium, SWCNTs exhibited obvious toxic effects to the protoplasts such as increasing generation of ROS, inducing changes of protoplast morphology, changing green leaves into yellow, and inducing protoplast cells' necrosis and apoptosis. In conclusion, single walled carbon nanotubes can get through Arabidopsis mesophyll cell wall and membrane, and exhibit dose-dependent dual-phase regulation to Arabidopsis mesophyll protoplasts such as low dose stimulating cell growth, and high dose inducing cells' ROS generation, necrosis or apoptosis.

  3. Analysis of impedance measurements of a suspension of microcapsules using a variable length impedance measurement cell

    Directory of Open Access Journals (Sweden)

    Dejan Krizaj

    2012-10-01

    Full Text Available Electrical impedance measurements of the suspensions have to take into account the double layer impedance that is due to a very thin charged layer formed at the electrode-electrolite interface. A dedicated measuring cell that enables variation of the distance between the electrodes was developed for investigation of electrical properties of suspensions using two electrode impedance measurements. By varying the distance between the electrodes it is possible to separate the double layer and the suspension impedance from the measured data. From measured and extracted impedances electrical lumped models have been developed. The error of non inclusion of the double layer impedance has been analyzed. The error depends on the frequency of the measurements as well as on the distance between the electrodes.

  4. LACHESIS restricts gametic cell fate in the female gametophyte of Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Rita Gross-Hardt

    2007-03-01

    Full Text Available In flowering plants, the egg and sperm cells form within haploid gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg cell and the central cell, which are flanked by five accessory cells. Both gametic and accessory cells are vital for fertilization; however, the mechanisms that underlie the formation of accessory versus gametic cell fate are unknown. In a screen for regulators of egg cell fate, we isolated the lachesis (lis mutant which forms supernumerary egg cells. In lis mutants, accessory cells differentiate gametic cell fate, indicating that LIS is involved in a mechanism that prevents accessory cells from adopting gametic cell fate. The temporal and spatial pattern of LIS expression suggests that this mechanism is generated in gametic cells. LIS is homologous to the yeast splicing factor PRP4, indicating that components of the splice apparatus participate in cell fate decisions.

  5. Regulation of Arabidopsis Early Anther Development by Putative Cell-Cell Signaling Molecules and Transcriptional Regulators

    Institute of Scientific and Technical Information of China (English)

    Yu-Jin Sun; Carey LH Hord; Chang-Bin Chen; Hong Ma

    2007-01-01

    Anther development in flowering plants involves the formation of several cell types, including the tapetal and pollen mother cells. The use of genetic and molecular tools has led to the identification and characterization of genes that are critical for normal cell division and differentiation in Arabidopsis early anther development. We review here several recent studies on these genes, including the demonstration that the putative receptor protein kinases BAM1 and BAM2 together play essential roles in the control of early cell division and differentiation. In addition, we discuss the hypothesis that BAM1/2 may form a positive-negative feedback regulatory loop with a previously identified key regulator, SPOROCYTELESS (also called NOZZLE),to control the balance between sporogenous and somatic cell types in the anther. Furthermore, we summarize the isolation and functional analysis of the DYSFUNCTIONAL TAPETUM1 (DYT1) gene in promoting proper tapetal cell differentiation. Our finding that DYT1 encodes a putative transcription factor of the bHLH family, as well as relevant expression analyses, strongly supports a model that DYT1 serves as a critical link between upstream factors and downstream target genes that are critical for normal tapetum development and function. These studies, together with other recently published works, indicate that cell-cell communication and transcriptional control are key processes essential for cell fate specification in anther development.

  6. Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm

    Science.gov (United States)

    An Arabidopsis pollen grain (male gametophyte) consists of three cells: the vegetative cell, which forms the pollen tube, and two sperm cells enclosed within the vegetative cell. It is still unclear if there is intercellular communication between the vegetative cell and the sperm cells. Here we show...

  7. Programmed cell death features in apple suspension cells under low oxygen culture.

    Science.gov (United States)

    Xu, Chang-jie; Chen, Kun-song; Ferguson, Ian B

    2004-02-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  8. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    XU Chang-jie(徐昌杰); CHEN Kun-song(陈昆松); FERGUSON Ian B.

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple cell death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  9. Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche

    Science.gov (United States)

    Willis, Lisa; Refahi, Yassin; Wightman, Raymond; Landrein, Benoit; Teles, José; Huang, Kerwyn Casey; Meyerowitz, Elliot M.

    2016-01-01

    Cell size and growth kinetics are fundamental cellular properties with important physiological implications. Classical studies on yeast, and recently on bacteria, have identified rules for cell size regulation in single cells, but in the more complex environment of multicellular tissues, data have been lacking. In this study, to characterize cell size and growth regulation in a multicellular context, we developed a 4D imaging pipeline and applied it to track and quantify epidermal cells over 3–4 d in Arabidopsis thaliana shoot apical meristems. We found that a cell size checkpoint is not the trigger for G2/M or cytokinesis, refuting the unexamined assumption that meristematic cells trigger cell cycle phases upon reaching a critical size. Our data also rule out models in which cells undergo G2/M at a fixed time after birth, or by adding a critical size increment between G2/M transitions. Rather, cell size regulation was intermediate between the critical size and critical increment paradigms, meaning that cell size fluctuations decay by ∼75% in one generation compared with 100% (critical size) and 50% (critical increment). Notably, this behavior was independent of local cell–cell contact topologies and of position within the tissue. Cells grew exponentially throughout the first >80% of the cell cycle, but following an asymmetrical division, the small daughter grew at a faster exponential rate than the large daughter, an observation that potentially challenges present models of growth regulation. These growth and division behaviors place strong constraints on quantitative mechanistic descriptions of the cell cycle and growth control. PMID:27930326

  10. Effects of Selected Physicochemical Parameters on Zerumbone Production of Zingiber zerumbet Smith Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Mahanom Jalil

    2015-01-01

    Full Text Available Zingiber zerumbet Smith is an important herb that contains bioactive phytomedicinal compound, zerumbone. To enhance cell growth and production of this useful compound, we investigated the growth conditions of cell suspension culture. Embryogenic callus generated from shoot bud was used to initiate cell suspension culture. The highest specific growth rate of cells was recorded when it was cultured in liquid Murashige and Skoog basal medium containing 3% sucrose with pH 5.7 and incubated under continuous shaking condition of 70 rpm for 16 h light and 8 h dark cycle at 24°C. Our results also revealed that the type of carbohydrate substrate, light regime, agitation speed, and incubation temperature could affect the production of zerumbone. Although the zerumbone produced in this study was not abundant compared to rhizome of Z. zerumbet, the possibility of producing zerumbone during early stage could serve as a model for subsequent improvement.

  11. Optimization of Lycopene Extraction from Tomato Cell Suspension Culture by Response Surface Methodology

    OpenAIRE

    Lu, Chi-Hua; Engelmann, Nancy J.; Lila, Mary Ann; Erdman, John W

    2008-01-01

    Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combi...

  12. Morphological characterization of cells in concentrated suspensions using multispectral diffuse optical tomography.

    Science.gov (United States)

    Hajihashemi, Mohammad Reza; Li, Xiaoqi; Jiang, Huabei

    2012-10-01

    Based on a non-spherical model of particle scattering, we investigate the capabilities and limitations of a T-matrix based inverse algorithm to morphologically characterize cells in concentrated suspensions. Here the cells are modeled as randomly orientated spheroidal particles with homogenous dielectric properties and suspended in turbid media. The inverse algorithm retrieves the geometrical parameters and the concentration of cells simultaneously by inverting the reduced scattering coefficient spectra obtained from multispectral diffuse optical tomography (MS-DOT). Both round and spheroidal cells are tested and the role of multiple and higher order scattering of particles on the performance of the algorithm is evaluated using different concentrations of cells.

  13. Programmed cell death features in apple suspension cells under low oxygen culture

    Institute of Scientific and Technical Information of China (English)

    徐昌杰; 陈昆松; FERGUSONIanB

    2004-01-01

    Suspension-cultured apple fruit cells (Malus pumila Mill. cv. Braeburn) were exposed to a low oxygen atmosphere to test whether programmed cell death (PCD) has a role in cell dysfunction and death under hypoxic conditions. Protoplasts were prepared at various times after low oxygen conditions were established, and viability tested by triple staining with fluorescein diacetate (FDA), propidium iodide (PI) and Hoechst33342 (HO342). DNA breakdown and phosphatidylserine exposure on the plasma membrane were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL), and annexin V binding. About 30% of protoplasts from cells after 48 h under low oxygen showed an increased accumulation of HO342, indicating increased membrane permeability. Positive TUNEL and annexin V results were also only obtained with protoplasts from cells under low oxygen. The results suggest that apple celi death under low oxygen is at least partially PCD mediated, and may explain tissue breakdown under controlled atmosphere (low oxygen) conditions in apple fruit.

  14. Origination of turbulence in dense suspensions of sperm cells

    Science.gov (United States)

    Denissenko, Petr; Kirkman-Brown, Jackson; Smith, David; Kantsler, Vasily

    2014-11-01

    Motile micro-organisms with pushing flagella, such as sperm cells, can be directed by ``one way'' microchannels with ratchet teeth-like wall configuration. We use an array of such micro-channels to gradually concentrate human spermatozoa in a circular arena of 1 mm diameter and 200 micron depth. Velocities of individual cells are measured by particle tracking and velocity of cell-carrying fluid is measured using PIV. At high concentrations, fluid velocities and the velocity fluctuations of individual cells exceeding that of individual swimmers in the dilute regime by an order of magnitude have been measured. Velocity correlations are calculated to study evolution of characteristic length scales as the cell concentration increases. Results are discussed in the context of self-organisation phenomena in active fluids and cooperation of sperm cells.

  15. The development of cat testicular sperm cryopreservation protocols: Effects of tissue fragments or sperm cell suspension.

    Science.gov (United States)

    Chatdarong, Kaywalee; Thuwanut, Paweena; Morrell, Jane M

    2016-01-15

    In endangered animals that have been found dead or sterilized for medical reasons, testis is the ultimate source of haploid DNA or sperm. Thus, preservation of testicular sperm may be performed to rescue their genetics. The aim of this study was to evaluate protocols for testicular sperm freezing: as tissue fragments or cell suspension in domestic cats as a model. A pair of testes from each cat (n = 9) were cut into eight equal pieces. Four randomly selected pieces were cryopreserved as: (1) tissue pieces using two-step freezing; (2) tissue pieces using a slow passive cooling device (CoolCell); (3) sperm suspension after single-layer centrifugation (SLC) through colloids; and (4) sperm suspension without being processed through SLC. A testicular piece from each cat served as fresh control. Testicular sperm membrane and DNA integrity were evaluated before, and after, the cryopreservation process. In addition, spermatogenic cell types (testicular sperm, spermatogonia, spermatocyte, and spermatid) present in the suspension samples were counted before and after SLC. The results found that testicular sperm membrane integrity in the suspension after SLC process was higher than that in the fragment form neither using the two-step nor CoolCell freezing, both before and after freezing (before freezing: 92.3 ± 3.4 vs. 81 ± 4.5 and 80.0 ± 7.0; after freezing: 84.5 ± 4.6 vs. 71.2 ± 12 and 76.2 ± 4.6; P ≤ 0.05). Testicular sperm DNA integrity was, however, not different among groups. Furthermore, the samples processed through the SLC had higher ration of sperm cells: other spermatogenic cells than those were not processed through the SLC (88.9 ± 3.8 vs. 30 ± 7.9; P ≤ 0.05). In summary, testicular sperm cryopreserved as a minced suspension is considered suitable in terms of preventing sperm membrane integrity, and SLC is considered a selection tool for enriching haploid sperm cells from castrated or postmortem cats.

  16. A dynamic model for stem cell homeostasis and patterning in Arabidopsis meristems.

    Directory of Open Access Journals (Sweden)

    Tim Hohm

    Full Text Available Plants maintain stem cells in their meristems as a source for new undifferentiated cells throughout their life. Meristems are small groups of cells that provide the microenvironment that allows stem cells to prosper. Homeostasis of a stem cell domain within a growing meristem is achieved by signalling between stem cells and surrounding cells. We have here simulated the origin and maintenance of a defined stem cell domain at the tip of Arabidopsis shoot meristems, based on the assumption that meristems are self-organizing systems. The model comprises two coupled feedback regulated genetic systems that control stem cell behaviour. Using a minimal set of spatial parameters, the mathematical model allows to predict the generation, shape and size of the stem cell domain, and the underlying organizing centre. We use the model to explore the parameter space that allows stem cell maintenance, and to simulate the consequences of mutations, gene misexpression and cell ablations.

  17. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant

    KAUST Repository

    Hudik, Elodie

    2014-07-18

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  18. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant.

    Science.gov (United States)

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-09-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants.

  19. On the dielectric relaxation of biological cell suspensions: the effect of the membrane electrical conductivity.

    Science.gov (United States)

    Di Biasio, A; Cametti, C

    2011-06-01

    Due to the mismatch of the electrical parameters (the permittivity ϵ' and the electrical conductivity σ) of the membrane of a biological cell with the ones of the cytosol and the extracellular medium, biological cell suspensions are the site, under the influence of an external electric field, of large dielectric relaxations in the radiowave frequency range. However, a point still remains controversial, i.e., whether or not the value of membrane conductivity σ(s) might be extracted from the de-convolution of the dielectric spectra or otherwise if it would be more reasonable to assign to the membrane conductivity a value equal to zero. This point is not to be considered with superficiality since it concerns an a priori choice which ultimately influences the values of the electrical parameters deduced from this technique. As far as this point is concerned, the opinion of the researchers in this field diverges. We believe that, at least within certain limits, the membrane conductivity can be deduced from the shape of the relaxation spectra. We substantiate this thesis with two different examples concerning the first a suspension of human normal erythrocyte cells and the second a suspension of human lymphocyte cells. In both cases, by means of an accurate fitting procedure based on the Levenberg-Marquardt method for complex functions, we can evaluate the membrane conductivity σ(s) with its associated uncertainty. The knowledge of the membrane electrical conductivity will favor the investigation of different ion transport mechanisms across the cell membrane.

  20. High affinity RGD-binding sites at the plasma membrane of Arabidopsis thaliana links the cell wall.

    Science.gov (United States)

    Canut, H; Carrasco, A; Galaud, J P; Cassan, C; Bouyssou, H; Vita, N; Ferrara, P; Pont-Lezica, R

    1998-10-01

    The heptapeptide Tyr-Gly-Arg-Gly-Asp-Ser-Pro containing the sequence Arg-Gly-Asp (RGD--the essential structure recognised by animal cells in substrate adhesion molecules) was tested on epidermal cells of onion and cultured cells of Arabidopsis upon plasmolysis. Dramatic changes were observed on both types of cells following treatment: on onion cells, Hechtian strands linking the cell wall to the membrane were lost, while Arabidopsis cells changed from concave to convex plasmolysis. A control heptapeptide Tyr-Gly-Asp-Gly-Arg-Ser-Pro had no effect on the shape of plasmolysed cells. Protoplasts isolated from Arabidopsis cells agglutinate in the presence of ProNectinF, a genetically engineered protein of 72 kDa containing 13 RGD sequences: several protoplasts may adhere to a single molecule of ProNectinF. The addition of the RGD-heptapeptide disrupted the adhesion between the protoplasts. Purified plasma membrane from Arabidopsis cells exhibits specific binding sites for the iodinated RGD-heptapeptide. The binding is saturable, reversible, and two types of high affinity sites (Kd1 approximately 1 nM, and Kd2 approximately 40 nM) can be discerned. Competitive inhibition by several structurally related peptides and proteins noted the specific requirement for the RGD sequence. Thus, the RGD-binding activity of Arabidopsis fulfils the adhesion features of integrins, i.e. peptide specificity, subcellular location, and involvement in plasma membrane-cell wall attachments.

  1. Root border-like cells of Arabidopsis. Microscopical characterization and role in the interaction with rhizobacteria.

    Science.gov (United States)

    Vicré, Maïté; Santaella, Catherine; Blanchet, Sandrine; Gateau, Aurélien; Driouich, Azeddine

    2005-06-01

    Plant roots of many species produce thousands of cells that are released daily into the rhizosphere. These cells are commonly termed border cells because of their major role in constituting a biotic boundary layer between the root surface and the soil. In this study, we investigated the occurrence and ultrastructure of such cells in Arabidopsis (Arabidopsis thaliana) using light and electron microscopy coupled to high-pressure freezing. The secretion of cell wall molecules including pectic polysaccharides and arabinogalactan-proteins (AGPs) was examined also using immunofluorescence microscopy and a set of anticarbohydrate antibodies. We show that root tips of Arabidopsis seedlings released cell layers in an organized pattern that differs from the rather randomly dispersed release observed in other plant species studied to date. Therefore, we termed such cells border-like cells (BLC). Electron microscopical results revealed that BLC are rich in mitochondria, Golgi stacks, and Golgi-derived vesicles, suggesting that these cells are actively engaged in secretion of materials to their cell walls. Immunocytochemical data demonstrated that pectins as well as AGPs are among secreted material as revealed by the high level of expression of AGP-epitopes. In particular, the JIM13-AGP epitope was found exclusively associated with BLC and peripheral cells in the root cap region. In addition, we investigated the function of BLC and root cap cell AGPs in the interaction with rhizobacteria using AGP-disrupting agents and a strain of Rhizobium sp. expressing a green fluorescent protein. Our findings demonstrate that alteration of AGPs significantly inhibits the attachment of the bacteria to the surface of BLC and root tip.

  2. Inhibition of cell proliferation, cell expansion and differentiation by the Arabidopsis SUPERMAN gene in transgenic tobacco plants.

    Science.gov (United States)

    Bereterbide, A; Hernould, M; Castera, S; Mouras, A

    2001-11-01

    Plant development depends upon the control of growth, organization and differentiation of cells derived from shoot and root meristems. Among the genes involved in flower organ determination, the cadastral gene SUPERMAN controls the boundary between whorls 3 and 4 and the growth of the adaxial outer ovule integument by down-regulating cell divisions. To determine the precise function of this gene we overexpressed ectopically the Arabidopsis thaliana (L.) Heynh. SUPERMAN gene in tobacco (Nicotiana tabacum L.). The transgenic plants exhibited a dwarf phenotype. Histologically and cytologically detailed analyses showed that dwarfism is correlated with a reduction in cell number, which is in agreement with the SUPERMAN function in Arabidopsis. Furthermore, a reduction in cell expansion and an impairment of cell differentiation were observed in tobacco organs. These traits were observed in differentiated vegetative and floral organs but not in meristem structures. A potential effect of the SUPERMAN transcription factor in the control of gibberellin biosynthesis is discussed.

  3. Induced accumulation of oleanolic acid and ursolic acid in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Feria-Romero, Iris; Lazo, Elizabeth; Ponce-Noyola, Teresa; Cerda-García-Rojas, Carlos M; Ramos-Valdivia, Ana C

    2005-06-01

    Increasing sucrose from 20 to 50 g l(-1) in Uncaria tomentosa cell suspension cultures enhanced ursolic acid and oleanolic acid production from 129 +/- 61 to 553 +/- 193 microg g(-1) cell dry wt. The maximal concentration of both triterpenes (1680 +/- 39 microg g(-1) cell dry wt) was 8 days after elicitation by jasmonic acid, while yeast extract or citrus pectin treatments produced 1189 +/- 20 or 1120 +/- 26 microg g(-1) cell dry wt, respectively. The ratio of ursolic acid:oleanolic acid was constant at 70:30.

  4. Survival of Suspension-cultured Sycamore Cells Cooled to the Temperature of Liquid Nitrogen.

    Science.gov (United States)

    Sugawara, Y; Sakai, A

    1974-11-01

    Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) which were immersed in liquid nitrogen after prefreezing to the temperatures from -30 to -50 C in the presence of dimethylsulfoxide and glucose as cryoprotective additive could proliferate vigorously when rewarmed rapidly in water at 40 C. For maintaining high viability of the cells after immersion in liquid nitrogen, it seems to be essential to use the cells at the later lag phase or the early cell division phase. This study provides a possibility for long term preservation in liquid nitrogen of plant-cultured lines.

  5. Extracellular Hydrolysis of Starch in Sugarcane Cell Suspensions 12

    Science.gov (United States)

    Maretzki, A.; dela Cruz, A.; Nickell, L. G.

    1971-01-01

    Evidence is presented for the increased excretion of amylolytic enzymes into a sugarcane cell culture medium when starch was substituted for sucrose as an energy source. The excretion was further enhanced by the inclusion of 1 μm gibberellic acid in the nutrient medium. The growth rate of the cells increased after they became adapted to starch relative to cells grown on sucrose, but the rate of amylolytic enzyme excretion remained unaltered. Amylolytic enzymes in the medium included α-amylase but the identity of one or more other enzymes related to starch hydrolysis remains in doubt. PMID:16657831

  6. Inter-microcarrier transfer and phenotypic stability of stem cell-derived Schwann cells in stirred suspension bioreactor culture.

    Science.gov (United States)

    Shakhbazau, Antos; Mirfeizi, Leila; Walsh, Tylor; Wobma, Holly M; Kumar, Ranjan; Singh, Bhagat; Kallos, Michael S; Midha, Rajiv

    2016-02-01

    Emerging bioreactor technologies offer an effective way for scaled-up production of large numbers of cells for cell therapy applications. One of the clinical paradigms where cell therapy can be an asset is restorative neurosciences. Nerve repair can benefit from the injections of stem cells and/or Schwann cells, acting as a source for axon myelination, myelin debris clearance, and trophic support. We have adapted microcarrier-based suspension bioreactor culture for Schwann cells (SCs) differentiated from a new stem cell source - skin-derived precursors (SKPs). SKP-derived SCs attach and grow on different types of microcarriers in both static and stirred culture, with Cytodex 3 and CultiSpher-S found most effective. Inter-microcarrier migration of SKP-SCs represents a key mechanism for rapid expansion and colonization in stirred suspension culture. We have shown that microcarrier-expanded SKP-SCs cells express Schwann cell markers p75-NTR, GFAP and S100 and retain their key ability to myelinate axons both in vitro and in vivo. Scaled-up microcarrier-based production of SKP-SCs in suspension bioreactors appears feasible for timely generation of sufficient cell numbers for nerve repair strategies.

  7. Collision rates for rare cell capture in periodic obstacle arrays strongly depend on density of cell suspension.

    Science.gov (United States)

    Cimrák, I

    2016-11-01

    Recently, computational modelling has been successfully used for determination of collision rates for rare cell capture in periodic obstacle arrays. The models were based on particle advection simulations where the cells were advected according to velocity field computed from two dimensional Navier-Stokes equations. This approach may be used under the assumption of very dilute cell suspensions where no mutual cell collisions occur. We use the object-in-fluid framework to demonstrate that even with low cell-to-fluid ratio, the optimal geometry of the obstacle array significantly changes. We show computational simulations for ratios of 3.5, 6.9 and 10.4% determining the optimal geometry of the periodic obstacle arrays. It was already previously demonstrated that cells in periodic obstacle arrays follow trajectories in two modes: the colliding mode and the zig-zag mode. The colliding mode maximizes the cell-obstacle collision frequency. Our simulations reveal that for dilute suspensions and for suspensions with cell-to-fluid ratio 3.5%, there is a range of column shifts for which the cells follow colliding trajectories. However we showed, that for 6.9 and 10.4%, the cells never follow colliding trajectories.

  8. Meiotic and Mitotic Cell Cycle Mutants Involved in Gametophyte Development in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Jingjing Liu; Li-Jia Qu

    2008-01-01

    The alternation between diploid and haploid generations is fundamentalin the life cycles of both animals and plants.The meiotic cell cycle is common to both animals and plants gamete formation, but in animals the products of meiosis are gametes,whereas for most plants,subsequent mitotic cell cycles are needed for their formation. Clarifying the regulatory mechanisms of mitotic cell cycle progression during gametophyte development will help understanding of sexual reproduction in plants.Many mutants defective in gametophyte development and,in particular,many meiotic and mitotic cell cycle mutants in Arabidopsis male and female gametophyte development were identified through both forward and reverse genetics approaches.

  9. Calcium dynamics in root cells of Arabidopsis thaliana visualized with selective plane illumination microscopy.

    Directory of Open Access Journals (Sweden)

    Alex Costa

    Full Text Available Selective Plane Illumination Microscopy (SPIM is an imaging technique particularly suited for long term in-vivo analysis of transparent specimens, able to visualize small organs or entire organisms, at cellular and eventually even subcellular resolution. Here we report the application of SPIM in Calcium imaging based on Förster Resonance Energy Transfer (FRET. Transgenic Arabidopsis plants expressing the genetically encoded-FRET-based Ca(2+ probe Cameleon, in the cytosol or nucleus, were used to demonstrate that SPIM enables ratiometric fluorescence imaging at high spatial and temporal resolution, both at tissue and single cell level. The SPIM-FRET technique enabled us to follow nuclear and cytosolic Ca(2+ dynamics in Arabidopsis root tip cells, deep inside the organ, in response to different stimuli. A relevant physiological phenomenon, namely Ca(2+ signal percolation, predicted in previous studies, has been directly visualized.

  10. Secondary metabolite production in Hypericum perforatum L. cell suspensions upon elicitation with fungal mycelia from Aspergillus flavus

    Directory of Open Access Journals (Sweden)

    Gadzovska-Simic Sonja

    2012-01-01

    Full Text Available We investigated the production of phenylpropanoids (phenolic compounds, flavanols, flavonols and anthocyanins and naphtodianthrones (hypericins in elicited Hypericum perforatum L. cell suspensions. To determine whether secondary metabolite production could be enhanced, Hypericum cell suspensions were exposed to mycelia extract from the fungus Aspergillus flavus. Elicited Hypericum cell suspension cultures displayed reduced growth and viability and a modification of secondary metabolites production. Anthocyanins were only stimulated in fungal-elicited cell suspensions. Secondary metabolite production in elicited Hypericum cells revealed an antagonism between the flavonoid/naphtodianthrone and anthocyanin pathways. The data suggest a modification of the channeling of the phenylpropanoid compounds. Together, these results represent useful data for monitoring the channeling in different secondary metabolite pathways during the scaled-up production of naphtodianthrones for medicinal uses.

  11. A photonic crystal hydrogel suspension array for the capture of blood cells from whole blood

    Science.gov (United States)

    Zhang, Bin; Cai, Yunlang; Shang, Luoran; Wang, Huan; Cheng, Yao; Rong, Fei; Gu, Zhongze; Zhao, Yuanjin

    2016-02-01

    Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells.Diagnosing hematological disorders based on the separation and detection of cells in the patient's blood is a significant challenge. We have developed a novel barcode particle-based suspension array that can simultaneously capture and detect multiple types of blood cells. The barcode particles are polyacrylamide (PAAm) hydrogel inverse opal microcarriers with characteristic reflection peak codes that remain stable during cell capture on their surfaces. The hydrophilic PAAm hydrogel scaffolds of the barcode particles can entrap various plasma proteins to capture different cells in the blood, with little damage to captured cells. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06368j

  12. Research on Electric Impedance Spectroscopy of Living Cell Suspensions by a Chip with Microelectrodes

    Institute of Scientific and Technical Information of China (English)

    Xing Yang; Zhaoying Zhou; Mingfei Xiao; Ying Wu; Shangfeng Liu

    2006-01-01

    A microfabricated electrical impedance spectroscopy (EIS) chip with microelectrodes was developed. The substrate and the electrodes of the chip were made of glass and gold, respectively. The experimental results demonstrated that the EIS-chip could distinguish different solutions (physiological saline, culture medium, living cell suspension etc.) by scanning from 10Hz to 45kHz. A 6-element circuit model was used for fitting the real part and the imaginary part admittance curves of the living cell suspension. An actual circuit was also built and tested to verify the 6-element circuit model proposed. The micro-EIS chip has several advantages including the use of small sample volumes, high resolution and ease of operation. It shows good application prospects in the areas of cellular electrophysiology, drug screening and bio-sensors etc.

  13. Acyl chains of phospholipase D transphosphatidylation products in Arabidopsis cells: a study using multiple reaction monitoring mass spectrometry.

    Directory of Open Access Journals (Sweden)

    Dominique Rainteau

    Full Text Available BACKGROUND: Phospholipases D (PLD are major components of signalling pathways in plant responses to some stresses and hormones. The product of PLD activity is phosphatidic acid (PA. PAs with different acyl chains do not have the same protein targets, so to understand the signalling role of PLD it is essential to analyze the composition of its PA products in the presence and absence of an elicitor. METHODOLOGY/PRINCIPAL FINDINGS: Potential PLD substrates and products were studied in Arabidopsis thaliana suspension cells treated with or without the hormone salicylic acid (SA. As PA can be produced by enzymes other than PLD, we analyzed phosphatidylbutanol (PBut, which is specifically produced by PLD in the presence of n-butanol. The acyl chain compositions of PBut and the major glycerophospholipids were determined by multiple reaction monitoring (MRM mass spectrometry. PBut profiles of untreated cells or cells treated with SA show an over-representation of 160/18:2- and 16:0/18:3-species compared to those of phosphatidylcholine and phosphatidylethanolamine either from bulk lipid extracts or from purified membrane fractions. When microsomal PLDs were used in in vitro assays, the resulting PBut profile matched exactly that of the substrate provided. Therefore there is a mismatch between the acyl chain compositions of putative substrates and the in vivo products of PLDs that is unlikely to reflect any selectivity of PLDs for the acyl chains of substrates. CONCLUSIONS: MRM mass spectrometry is a reliable technique to analyze PLD products. Our results suggest that PLD action in response to SA is not due to the production of a stress-specific molecular species, but that the level of PLD products per se is important. The over-representation of 160/18:2- and 16:0/18:3-species in PLD products when compared to putative substrates might be related to a regulatory role of the heterogeneous distribution of glycerophospholipids in membrane sub-domains.

  14. Validation of Flow Cytometry and Magnetic Bead-Based Methods to Enrich CNS Single Cell Suspensions for Quiescent Microglia.

    Science.gov (United States)

    Volden, T A; Reyelts, C D; Hoke, T A; Arikkath, J; Bonasera, S J

    2015-12-01

    Microglia are resident mononuclear phagocytes within the CNS parenchyma that intimately interact with neurons and astrocytes to remodel synapses and extracellular matrix. We briefly review studies elucidating the molecular pathways that underlie microglial surveillance, activation, chemotaxis, and phagocytosis; we additionally place these studies in a clinical context. We describe and validate an inexpensive and simple approach to obtain enriched single cell suspensions of quiescent parenchymal and perivascular microglia from the mouse cerebellum and hypothalamus. Following preparation of regional CNS single cell suspensions, we remove myelin debris, and then perform two serial enrichment steps for cells expressing surface CD11b. Myelin depletion and CD11b enrichment are both accomplished using antigen-specific magnetic beads in an automated cell separation system. Flow cytometry of the resultant suspensions shows a significant enrichment for CD11b(+)/CD45(+) cells (perivascular microglia) and CD11b(+)/CD45(-) cells (parenchymal microglia) compared to starting suspensions. Of note, cells from these enriched suspensions minimally express Aif1 (aka Iba1), suggesting that the enrichment process does not evoke significant microglial activation. However, these cells readily respond to a functional challenge (LPS) with significant changes in the expression of molecules specifically associated with microglia. We conclude that methods employing a combination of magnetic-bead based sorting and flow cytometry produce suspensions highly enriched for microglia that are appropriate for a variety of molecular and cellular assays.

  15. Solid oxide fuel cell electrolytes produced via very low pressure suspension plasma spray and electrophoretic deposition

    OpenAIRE

    Fleetwood, James D

    2014-01-01

    Solid oxide fuel cells (SOFCs) are a promising element of comprehensive energy policies due to their direct mechanism for converting the oxidization of fuel, such as hydrogen, into electrical energy. Both very low pressure plasma spray and electrophoretic deposition allow working with high melting temperature SOFC suspension based feedstock on complex surfaces, such as in non-planar SOFC designs. Dense, thin electrolytes of ideal composition for SOFCs can be fabricated with each of these proc...

  16. Putrescine facilitated enhancement of capsaicin production in cell suspension cultures of Capsicum frutescens.

    Science.gov (United States)

    Sudha, Govindaswamy; Ravishankar, Gokare A

    2003-04-01

    Putrescine treatment (0.1 mmol/L) influenced enhancement of growth and capsaicin production in the cell suspension cultures of C. frutescens. The administration of polyamine inhibitor DFMA (alpha-DL-difluoromethylarginine) resulted in a reduction of the growth, capsaicin content and the endogenous titres of polyamines (PAs). The capsaicin synthase activity was also higher in the putrescine (Put) treated cultures. Ethylene levels were lower in the cultures treated with putrescine. This study suggested that Put facilitates growth and capsaicin production.

  17. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract

    OpenAIRE

    Liangyun Zhou; Jian Yang; Guang Yang; Chuanzhi Kang; Wenjuan Xiao; Chaogeng Lv; Sheng Wang; Jinfu Tang; Lanping Guo

    2016-01-01

    Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphe...

  18. Isolation of fatty acids and aromatics from cell suspension cultures of Lavandula angustifolia.

    Science.gov (United States)

    Topçu, Gülaçti; Herrmann, Gabriele; Kolak, Ufuk; Gören, C; Porzel, Andrea; Kutchan, Toni M

    2007-02-01

    Cell suspension cultures of Lavandula angustifolia Mill. ssp. angustifolia (syn.: L. officinalis Chaix.) afforded a fatty acid composition, cis and trans p-coumaric acids (=p-hydroxy cinnamic acids), and beta-sitosterol. The fatty acid composition was analyzed by GC-MS, and the structures of the isolated three compounds were determined by 1H- and 13C-NMR, and MS spectroscopic techniques.

  19. Establishment of cell suspension cultures of two Costa Rican Jatropha species (Euphorbiaceae)

    OpenAIRE

    Solís Ramos, Laura Yesenia; Miranda Carballo, Laura; Valdez Melara, Marta

    2013-01-01

    J. curcas has been studied in different countries and some interesting agronomic, pharmacological and industrial properties have been reported. More recently, it has been considered an important alternative source for biofuel production. The objective of this study was to establish a long-term method for the maintenance of calli and cell suspension cultures of the local species J. curcas and J. gossypifolia, in order to allow future studies for novel compounds with pharmaceutical or industria...

  20. C-27 AND C-3 GLUCOSYLATION OF DIOSGENIN BY CELL SUSPENSION CULTURES OF COSTUS SPECIOSUS

    Institute of Scientific and Technical Information of China (English)

    GUNAWAN INDRAYANTO; SITI ZUMAROH; ACHMAD SYAHRANI; ALISTAIR L. WILKINS

    2001-01-01

    3-O-[β-D-glucopyranosyl-(l″→ 2′)-β-D-glucopyranosyl], 27-O-β-D-glucopyranosyl-(25R)-spir ost-5-ene-3β,27-diol was isolated from cell suspension cultures of Costus speciosus, following incubation with diosgenin, and its structure was elucidated using a combination of one- and two-dimensional 1H and 13C NMR spectral data, and positive and negative ion ESMS spectral data.

  1. Ce4+-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    葛志强; 元英进; 王艳东; 马振毅; 胡宗定

    2002-01-01

    The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged "DNA ladder" on agarose gel electrophoresis. TdT-mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′-OH termini. These results suggest that Ce4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product-Taxol.

  2. Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata).

    Science.gov (United States)

    Cheema, G S

    1989-02-01

    Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.

  3. Nanometer-scale sizing accuracy of particle suspensions on an unmodified cell phone using elastic light scattering.

    Directory of Open Access Journals (Sweden)

    Zachary J Smith

    Full Text Available We report on the construction of a Fourier plane imaging system attached to a cell phone. By illuminating particle suspensions with a collimated beam from an inexpensive diode laser, angularly resolved scattering patterns are imaged by the phone's camera. Analyzing these patterns with Mie theory results in predictions of size distributions of the particles in suspension. Despite using consumer grade electronics, we extracted size distributions of sphere suspensions with better than 20 nm accuracy in determining the mean size. We also show results from milk, yeast, and blood cells. Performing these measurements on a portable device presents opportunities for field-testing of food quality, process monitoring, and medical diagnosis.

  4. Translation of cell therapies to the clinic: characteristics of cell suspensions in large-diameter injection cannulae.

    Science.gov (United States)

    Torres, Eduardo M; Trigano, Matthieu; Dunnett, Stephen B

    2015-01-01

    With the use of cell replacement therapies as a realistic prospect for conditions such as Parkinson's and Huntington's diseases, the logistics of the delivery of cell suspensions to deep brain targets is a topic for consideration. Because of the large cannulae required for such procedures, we need to consider the behavior of cell suspensions within the cannulae if we are to ensure that the injected cells are distributed as intended within the target tissue. We have investigated the behavior of primary embryonic cell suspensions of neural tissue, in cannulae of different diameters, using a protocol designed to mimic the handling and injection of cells during clinical application. Internal cannula diameter had a large effect on the distribution of cells during their dispensation from the syringe. In vertical or near vertical cannulae, cells settled toward the tip of the needle, and were dispensed unevenly, with the majority of cells emerging in the first 10-20% of the injectate. In horizontal or near-horizontal cannulae, we observed the opposite effect, such that few cells were dispensed in the first 80% of the injectate, and the majority emerged in the final 10-20%. Use of a glass cannula showed that the results obtained using the horizontal cannula were caused by settling and adherence of the cells on the side of the cannulae, such that during dispensation, the overlying, cell-free solution was dispensed first, prior to the emergence of the cells. We show that the behavior of cells in such cannulae is affected by the cannula diameter, and by the material of the cannula itself. In horizontal cannulae, uneven expulsion of cells from the needle can be ameliorated by regular rotation of the cannula during the procedure. We discuss the potential impact of these observations on the translation of cell therapies to the clinic.

  5. [Determination of Azospirillum Brasilense Cells With Bacteriophages via Electrooptical Analysis of Microbial Suspensions].

    Science.gov (United States)

    Gulii, O I; Karavayeva, O A; Pavlii, S A; Sokolov, O I; Bunin, V D; Ignatov, O V

    2015-01-01

    The dependence-of changes in the electrooptical properties of Azospirillum brasilense cell suspension Sp7 during interaction with bacteriophage ΦAb-Sp7 on the number and time of interactions was studied. Incubation of cells with bacteriophage significantly changed the electrooptical signal within one minute. The selective effect of bacteriophage ΦAb on 18 strains of bacteria of the genus Azospirillum was studied: A. amazonense Ami4, A. brasilense Sp7, Cd, Sp107, Sp245, Jm6B2, Brl4, KR77, S17, S27, SR55, SR75, A. halopraeferans Au4, A. irakense KBC1, K A3, A. lipoferum Sp59b, SR65 and RG20a. We determined the limit of reliable determination of microbial cells infected with bacteriophage: - 10(4) cells/mL. The presence of foreign cell cultures of E. coli B-878 and E. coli XL-1 did not complicate the detection of A brasilense Sp7 cells with the use of bacteriophage ΦAb-Sp7. The results demonstrated that bacteriophage (ΦAb-Sp7 can be used for the detection of Azospirillum microbial cells via t electrooptical analysis of cell suspensions.

  6. The role of cell replacement in benthic–pelagic coupling by suspension feeders

    Science.gov (United States)

    2016-01-01

    Benthic–pelagic coupling through suspension feeders and their detrital pathways is integral to carbon transport in oceans. In food-poor ecosystems however, a novel mechanism of carbon recycling has been proposed that involves direct uptake of dissolved carbon by suspension feeders followed by shedding of cells as particulate carbon. We studied cell replacement rates in a range of cold-water sponge species to determine how universal this mechanism might be. We show that cell replacement rates of feeding epithelia in explants vary from 30 hours up to 7 days, and change during different seasons and life-history stages. We also found that feeding epithelia are not replaced through direct replication but instead arise from a population of stem cells that differentiate and integrate into epithelial tissues. Our results reveal a surprising amount of complexity in the control of cell processes in sponges, with cell turnover depending on environmental conditions and using stem cells as rate-limiting mechanisms. Our results also suggest that for species in cold water with high particulate organic matter, cell turnover is not the mechanism delivering carbon flux to surrounding communities. PMID:28018632

  7. Cell Wall Heterogeneity in Root Development of Arabidopsis

    Science.gov (United States)

    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. Influence of particle size and concentration on the diffuse backscattering of polarized light from tissue phantoms and biological cell suspensions

    Energy Technology Data Exchange (ETDEWEB)

    Hielscher, A.H.; Mourant, J.R.; Bigio, I.J. [Los Alamos National Laboratory, Bioscience and Biotechnology, CST-4, MS E535, Los Alamos, New Mexico 87545 (United States)

    1997-01-01

    We present experimental results that show the spatial variations of the diffuse-backscattered intensity when linearly polarized light is incident upon highly scattering media. Experiments on polystyrene-sphere and Intralipid suspensions demonstrate that the radial and azimuthal variations of the observed pattern depend on the concentration, and anisotropy factor {ital g} of the particles that constitute the scattering medium. Measurements performed on biological-cell suspensions show the potential of this method for cell characterization. {copyright} 1997 Optical Society of America

  9. Establishment and optimization of cell growth in suspension culture of Papaver bracteatum: a biotechnology approach for thebaine production

    OpenAIRE

    FARJAMINEZHAD, Reza; Nasser ZARE; ASGHARI-ZAKARIA, Rasool; Farjaminezhad, Manoochehr

    2013-01-01

    Iranian poppy (Papaver bracteatum) is an important medicinal plant that is the main source of the opium alkaloids codeine, morphine, and thebaine. To establish an efficient protocol for cell suspension culture and growth, the effects of different plant growth regulators (2,4-D, NAA, BAP, and kinetin) on callus induction and cell suspension culture of Iranian poppy were evaluated. The maximum percentage of callus induction (86.67%) and fresh weight of callus were obtained in MS medium suppleme...

  10. Cell Size Clues for the Allee Effect in Vegetative Amoeba Suspension Culture

    Science.gov (United States)

    Franck, Carl; Rappazzo, Brendan; Wang, Xiaoning; Segota, Igor

    That cells proliferate at higher rates with increasing density helps us appreciate and understand the development of multicellular behavior through the study of dilute cell systems. However, arduous cell counting with a microscope reveals that in the model eukaryote, Dictyostelium discoideum this transition is difficult to ascertain and thereby further explore despite our earlier progress (Phys. Rev. E 77, 041905, (2008)). Here we report preliminary evidence that the slow proliferation phase is well characterized by reduced cell size compared to the wide distribution of cell sizes in the familiar exponential proliferation phase of moderate densities. This observation is enabled by a new system for characterizing cells in stirred suspension cultures. Our technique relies on quickly acquiring magnitude distributions of detected flashes of laser light scattered in situ by cell targets.

  11. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    Science.gov (United States)

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A.; Ko, Minoru S.H.; Motohashi, Tsutomu; Kunisada, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the suspension culture, and their undifferentiated state and pluripotency were experimentally verified. DNA microarray analyses showed a close relationship between the elevated expression of genes related to cell adhesions. We suggest that this suspension culture condition provides a better alternative to the conventional attached cell culture condition, especially for possible therapeutic use, by limiting the exposure of ES cells to feeder cells and animal products. PMID:18624284

  12. Nitric oxide suppresses stomatal opening by inhibiting inward-rectifying Kin channels in Arabidopsis guard cells

    Institute of Scientific and Technical Information of China (English)

    XUE ShaoWu; YANG Pin; HE YiKun

    2008-01-01

    We explore nitric oxide (NO) effect on K+in channels in Arabidopsis guard cells. We observed NO inhib-ited K+in currents when Ca2+ chelator EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'tetraacetic acid) was not added in the pipette solution; K+in currents were not sensitive to NO when cytosolic Ca2+ was chelated by EGTA. NO inhibited the Arabidopsis stomatal opening, but when EGTA was added in the bath solution, inhibition effect of NO on stomatal opening vanished. Thus, it implies that NO ele-vates cytosolic Ca2+ by activating plasma membrane Ca2+ channels firstly, then inactivates K+in chan-nels, resulting in stomatal opening suppressed subsequently.

  13. System for the exposure of cell suspensions to power-frequency electric fields.

    Science.gov (United States)

    Kaune, W T; Frazier, M E; King, A J; Samuel, J E; Hungate, F P; Causey, S C

    1984-01-01

    A system is described that uses an oscillating magnetic field to produce power-frequency electric fields with strengths in excess of those produced in an animal or human standing under a high-voltage electric-power transmission line. In contrast to other types of exposure systems capable of generating fields of this size, no electrodes are placed in the conducting growth media: the possibility of electrode contamination of the exposed suspension is thereby eliminated. Electric fields in the range 0.02-3.5 V/m can be produced in a cell culture with total harmonic distortions less than 1.5%. The magnetic field used to produce electric fields for exposure is largely confined within a closed ferromagnetic circuit, and experimental and control cells are exposed to leakage magnetic flux densities less than 5 microT . The temperatures of the experimental and control cell suspensions are held fixed within +/- 0.1 degrees C by a water bath. Special chambers were developed to hold cell cultures during exposure and sham exposure. Chinese hamster ovary (CHO) cells incubated in these chambers grew for at least 48 h and had population doubling times of 16-17 h, approximately the same as for CHO cells grown under standard cell-culture conditions.

  14. Biochemical precursor effects on the fatty acid production in cell suspension cultures of Theobroma cacao L.

    Science.gov (United States)

    Parra, O; Gallego, A M; Urrea, A; Rojas, L F; Correa, C; Atehortúa, L

    2017-02-01

    Cocoa butter (CB) is composed of 96% palmitic, stearic, oleic, linoleic and linolenic fatty acids that are responsible for the hardness, texture and fusion properties of chocolate. Through in vitro plant cell culture it is possible to modify CB lipid profiles and to study the fatty acid biosynthesis pathway on a subcellular level, evaluating fundamental aspects to enhance in vitro fatty acid production in a specific and controlled way. In this research, culture media was supplemented with acetate, biotin, pyruvate, bicarbonate and glycerol at three different concentrations and the effects on the biomass production (g/L), cell viability, and fatty acids profile and production was evaluated in in vitro cell suspensions culture. It was found that biotin stimulated fatty acid synthesis without altering cell viability and cell growth. It was also evident a change in the lipid profile of cell suspensions, increasing middle and long chain fatty acids proportion, which are unusual to those reported in seeds; thus implying that it is possible to modify lipid profiles according to the treatment used. According to the results of sucrose gradients and enzyme assays performed, it is proposed that cacao cells probably use the pentose phosphate pathway, mitochondria being the key organelle in the carbon flux for the synthesis of reductant power and fatty acid precursors.

  15. Evaluation of limonoid production in suspension cell culture of Citrus sinensis

    Directory of Open Access Journals (Sweden)

    Elisângela Fumagali Gerolino

    2015-10-01

    Full Text Available ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L. Osbeck, Rutaceae and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds, callus cultures (originating from hypocotyls, cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight. Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.

  16. [Research on ursolic acid production of Eriobotrya japonica cell suspension culture in WAVE bioreactor].

    Science.gov (United States)

    Li, Hui-hua; Yao, De-heng; Xu, Jian; Wang, Wei; Chang, Qiang; Su, Ming-hua

    2015-05-01

    Through scale-up cultivation of Eriobotrya japonica suspension cells using WAVE bioreactor, the cell growth and ursolic acid (UA) accumulation were studied. The comparison test was carried out in the flask and the reactor with cell dry weight (DW) and UA content as evaluation indexes. The culture medium, DW and UA content were compared in 1 L and 5 L working volumes of bioreactor. The orthogonal test with main actors of inoculation amount, speed and angle of rotation was developed to find the optimal combination, in 1 L working volume of bioreactor. DW of the cell growth and the UA content in bioreactor were higher than those of the shaker by 105.5% and 27.65% respectively. In bioreactor, the dynamic changes of elements in the fluid culture, the dry weight of the cell growth and the UA content in 1 L and 5 L working volumes were similar. Inoculation of 80 g, rotational speed of 26 r · min(-1), and angle of 6 ° was the optimal combination, and the cell biomass of 19.01 g · L(-1) and the UA content of 27.750 mg · g(-1) were achieved after 100 h cultivation in 1 L working volume of bioreactor. WAVE Bioreactor is more suitable than flasks for the E. japonica cell suspension culture, and culture parameters can be achieved from 1 L to 5 L amplification.

  17. Ghost Cell Suspensions as Blood Analogue Fluid for Macroscopic Particle Image Velocimetry Measurements.

    Science.gov (United States)

    Jansen, Sebastian V; Müller, Indra; Nachtsheim, Max; Schmitz-Rode, Thomas; Steinseifer, Ulrich

    2016-02-01

    Spatially resolved measurement of blood flow is of great interest in the development of artificial blood-carrying devices such as blood pumps, heart valve prostheses, and oxygenators. Particle image velocimetry (PIV) is able to measure instantaneous velocity fields in a plane with high accuracy and is being used more frequently for the development of such devices. However, as this measurement technique is based on optical access, blood flow at physiological hematocrit values is difficult to measure due to its low transparency and multiscattering properties. So far, only very small dimensions (in the range of 400 μm) can be measured using PIV. A suspension of ghost cells (GCs) offers a higher optical transparency than blood while having a similar rheological behavior. In this study, a procedure for the production of GC suspensions containing a very low intracellular hemoglobin concentration is presented. With the help of multiple rounds of controlled cell lysis, the intracellular hemoglobin concentration could be decreased to a point where a standard macroscopic PIV measurement was possible. A velocity profile of a 44% GC suspension in a circular channel with a diameter of 9.5 mm was measured with high spatial resolution. Meanwhile, the rheological behavior was found to be comparable with blood.

  18. Where the linearized Poisson-Boltzmann cell model fails: Spurious phase separation in charged colloidal suspensions

    Science.gov (United States)

    Tamashiro, M. N.; Schiessel, H.

    2003-07-01

    The Poisson-Boltzmann (PB) spherical Wigner-Seitz cell model—introduced to theoretically describe suspensions of spherical charged colloidal particles—is investigated at the nonlinear and linearized levels. The linearization of the mean-field PB functional yields linearized Debye-Hückel-type equations agreeing asymptotically with the nonlinear PB results in the weak-coupling (high-temperature) limit. Both the canonical (fixed number of microions) as well as the semigrand-canonical (in contact with an infinite salt reservoir) cases are considered and discussed in a unified linearized framework. In disagreement with the exact nonlinear PB solution inside a Wigner-Seitz cell, the linearized theory predicts the occurrence of a thermodynamical instability with an associated phase separation of the homogeneous suspension into dilute (gas) and dense (liquid) phases, being thus a spurious result of the linearization. We show that these artifacts, although thermodynamically consistent with quadratic expansions of the nonlinear functional and osmotic pressure, may be traced back to the nonfulfillment of the underlying assumptions of the linearization. This raises questions about the reliability of the prediction of gas/liquid-like phase separation in deionized aqueous suspensions of charged colloids mediated by monovalent counterions obtained by linearized theories.

  19. Embryonic stem cells remain highly pluripotent following long term expansion as aggregates in suspension bioreactors.

    Science.gov (United States)

    zur Nieden, Nicole I; Cormier, Jaymi T; Rancourt, Derrick E; Kallos, Michael S

    2007-05-01

    Increasing attention has been drawn towards pluripotent embryonic stem cells (ESCs) and their potential use as the primary material in various tissue engineering applications. Successful clinical implementation of this technology would require a quality controlled reproducible culture system for the expansion of the cells to be used in the generation of functional tissues. Recently, we showed that suspension bioreactors could be used in the regulated large-scale expansion of highly pluripotent murine ESCs. The current study illustrates that these bioreactor protocols can be adapted for long term culture and that murine ESC cultures remain highly undifferentiated, when serially passaged in suspension bioreactors for extended periods. Flow cytometry analysis and gene expression profiles of several pluripotency markers, in addition to colony and embryoid body (EB) formation tests were conducted at the start and end of the experiment and all showed that the ESC cultures remained highly undifferentiated over extended culture time in suspension. In vivo teratoma formation and in vitro differentiation into neural, cardiomyocyte, osteoblast and chondrocyte lineages, performed at the end of the long term culture, further supported the presence of functional and undifferentiated ESCs in the expanded population. Overall, this system enables the controlled expansion of highly pluripotent murine ESC populations.

  20. Immunoprofiling reveals unique cell-specific patterns of wall epitopes in the expanding Arabidopsis stem.

    Science.gov (United States)

    Hall, Hardy C; Cheung, Jingling; Ellis, Brian E

    2013-04-01

    The Arabidopsis inflorescence stem undergoes rapid directional growth, requiring massive axial cell-wall extension in all its tissues, but, at maturity, these tissues are composed of cell types that exhibit markedly different cell-wall structures. It is not clear whether the cell-wall compositions of these cell types diverge rapidly following axial growth cessation, or whether compositional divergence occurs at earlier stages in differentiation, despite the common requirement for cell-wall extensibility. To examine this question, seven cell types were assayed for the abundance and distribution of 18 major cell-wall glycan classes at three developmental stages along the developing inflorescence stem, using a high-throughput immunolabelling strategy. These stages represent a phase of juvenile growth, a phase displaying the maximum rate of stem extension, and a phase in which extension growth is ceasing. The immunolabelling patterns detected demonstrate that the cell-wall composition of most stem tissues undergoes pronounced changes both during and after rapid extension growth. Hierarchical clustering of the immunolabelling signals identified cell-specific binding patterns for some antibodies, including a sub-group of arabinogalactan side chain-directed antibodies whose epitope targets are specifically associated with the inter-fascicular fibre region during the rapid cell expansion phase. The data reveal dynamic, cell type-specific changes in cell-wall chemistry across diverse cell types during cell-wall expansion and maturation in the Arabidopsis inflorescence stem, and highlight the paradox between this structural diversity and the uniform anisotropic cell expansion taking place across all tissues during stem growth.

  1. Effect of Magnetic Nanoparticles on Tobacco BY-2 Cell Suspension Culture

    Directory of Open Access Journals (Sweden)

    Rene Kizek

    2012-12-01

    Full Text Available Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles, electronics (high-resolution imaging, logical circuits on the molecular level and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases or imaging (contrast agents. Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs and modified magnetic nanoparticles (MNPs on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis — total protein content, thiols — reduced (GSH and oxidized (GSSG glutathione, phytochelatins PC2-5, glutathione S-transferase (GST activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension

  2. Effect of magnetic nanoparticles on tobacco BY-2 cell suspension culture.

    Science.gov (United States)

    Krystofova, Olga; Sochor, Jiri; Zitka, Ondrej; Babula, Petr; Kudrle, Vit; Adam, Vojtech; Kizek, Rene

    2012-12-20

    Nanomaterials are structures whose exceptionality is based on their large surface, which is closely connected with reactivity and modification possibilities. Due to these properties nanomaterials are used in textile industry (antibacterial textiles with silver nanoparticles), electronics (high-resolution imaging, logical circuits on the molecular level) and medicine. Medicine represents one of the most important fields of application of nanomaterials. They are investigated in connection with targeted therapy (infectious diseases, malignant diseases) or imaging (contrast agents). Nanomaterials including nanoparticles have a great application potential in the targeted transport of pharmaceuticals. However, there are some negative properties of nanoparticles, which must be carefully solved, as hydrophobic properties leading to instability in aqueous environment, and especially their possible toxicity. Data about toxicity of nanomaterials are still scarce. Due to this fact, in this work we focused on studying of the effect of magnetic nanoparticles (NPs) and modified magnetic nanoparticles (MNPs) on tobacco BY-2 plant cell suspension culture. We aimed at examining the effect of NPs and MNPs on growth, proteosynthesis - total protein content, thiols - reduced (GSH) and oxidized (GSSG) glutathione, phytochelatins PC2-5, glutathione S-transferase (GST) activity and antioxidant activity of BY-2 cells. Whereas the effect of NPs and MNPs on growth of cell suspension culture was only moderate, significant changes were detected in all other biochemical parameters. Significant changes in protein content, phytochelatins levels and GST activity were observed in BY-2 cells treated with MNPs nanoparticles treatment. Changes were also clearly evident in the case of application of NPs. Our results demonstrate the ability of MNPs to negatively affect metabolism and induce biosynthesis of protective compounds in a plant cell model represented by BY-2 cell suspension culture. The

  3. Regulation of Cytoplasmic and Vacuolar Volumes by Plant Cells in Suspension Culture

    DEFF Research Database (Denmark)

    Owens, Trevor; Poole, Ronald J

    1979-01-01

    Quantitative microscopical measurements have been made of the proportion of cell volume occupied by cytoplasm in a cell suspension culture derived from cotyledons of bush bean (cv. Contender). On a 7-day culture cycle, the content of cytoplasm varies from 25% at the time of transfer to 45...... by cytoplasm is roughly correlated with protein content, but shows no correlation with cell size or with intracellular concentrations of K or Na. The most striking observation is that the growth of cytoplasmic volume for the culture as a whole appears to be constant throughout the culture cycle, despite......% at the start of the phase of rapid cell division. If the culture is continued beyond 7 days, the vacuole volume reaches 90% of cell volume by day 12.Attempts to measure relative cytoplasmic volumes by compartmental analysis of nonelectrolyte efflux were unsuccessful. The proportion of cell volume occupied...

  4. Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens.

    Science.gov (United States)

    Baldes, R; Moos, M; Geider, K

    1987-03-01

    Cell wall regenerating protoplasts from soybean cells kept in suspension culture were cocultivated with bacteria which were derived from the nopaline strain C58 of Agrobacterium tumefaciens. When the bacteria carried an oncogenic Ti-plasmid, about 5% of the surviving protoplasts were able to form calli on hormone-free agar in contrast to controls, where bacteria without Ti-plasmid were applied, and where no calli were formed. After isolation of DNA from hormone-independently growing cells further evidence for transformation was obtained by hybridization to Ti-plasmid specific RNA and by rescue of a segment with a bacterial resistance gene which had been inserted before into the T-DNA. Transfer of T-DNA harboring a neomycin-resistance gene activated by the nos-promoter resulted in calli growing on kanamycin. Verification of segments located at the left and the right part of the T-DNA indicated the presence of its entire length in transformed soybean cells. Expression of T-DNA genes was measured by the assay of nopaline-synthase. Cells cultured on agar had a much higher level of nopaline-synthase than fast growing cells in suspension culture. Transferring them to agar or treatment with azacytidine strongly increased synthesis of nopaline-synthase indicating a reversible repression presumably via a methylation mechanism.

  5. Induction of phytic acid synthesis by abscisic acid in suspension-cultured cells of rice.

    Science.gov (United States)

    Matsuno, Koya; Fujimura, Tatsuhito

    2014-03-01

    A pathway of phytic acid (PA) synthesis in plants has been revealed via investigations of low phytic acid mutants. However, the regulation of this pathway is not well understood because it is difficult to control the environments of cells in the seeds, where PA is mainly synthesized. We modified a rice suspension culture system in order to study the regulation of PA synthesis. Rice cells cultured with abscisic acid (ABA) accumulate PA at higher levels than cells cultured without ABA, and PA accumulation levels increase with ABA concentration. On the other hand, higher concentrations of sucrose or inorganic phosphorus do not affect PA accumulation. Mutations in the genes RINO1, OsMIK, OsIPK1 and OsLPA1 have each been reported to confer low phytic acid phenotypes in seeds. Each of these genes is upregulated in cells cultured with ABA. OsITPK4 and OsITPK6 are upregulated in cells cultured with ABA and in developing seeds. These results suggest that the regulation of PA synthesis is similar between developing seeds and cells in this suspension culture system. This system will be a powerful tool for elucidating the regulation of PA synthesis.

  6. APOPTOSIS AND TAXOL PRODUCTION IN SUSPENSION CULTURES OF Taxus spp.CELLS

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    l.lntroductionSuspension cultures of Taxal chinensis var.mairei frequently accumulate Taxol (paclitaxel),which is clinically effective amineoplanic agent.TSXol is known to act by enhancing thePOlymeriZation of tubulin in the initiation andextension of microtubules, ac has been shown toinduce apoptosis in human and animal cellslll.Apoptosis, also known as programmed cell death, isthe active process of cell death which occurs duringdevelopment and in resPOnse tO enviboental cues ofa multicellular organism. In...

  7. Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells.

    Science.gov (United States)

    Paves, H; Truve, E

    2007-01-01

    Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation.

  8. Embryonic control of epidermal cell patterning in the root and hypocotyl of Arabidopsis.

    Science.gov (United States)

    Lin, Y; Schiefelbein, J

    2001-10-01

    A position-dependent pattern of epidermal cell types is produced during the development of the Arabidopsis seedling root and hypocotyl. To understand the origin and regulation of this patterning mechanism, we have examined the embryonic expression of the GLABRA2 (GL2) gene, which encodes a cell-type-specific transcription factor. Using in situ RNA hybridization and a sensitive GL2::GFP reporter, we discovered that a position-dependent pattern of GL2 expression is established within protodermal cells at the heart stage and is maintained throughout the remainder of embryogenesis. In addition, we show that an exceptional GL2 expression character and epidermal cell pattern arises during development of the root-hypocotyl junction, which represents an anatomical transition zone. Furthermore, we find that two of the genes regulating seedling epidermal patterning, TRANSPARENT TESTA GLABRA (TTG) and WEREWOLF (WER), also control the embryonic GL2 pattern, whereas the CAPRICE (CPC) and GL2 genes are not required to establish this pattern. These results indicate that position-dependent patterning of epidermal cell types begins at an early stage of embryogenesis, before formation of the apical meristems and shortly after the cellular anatomy of the protoderm and outer ground tissue layer is established. Thus, epidermal cell specification in the Arabidopsis seedling relies on the embryonic establishment of a patterning mechanism that is perpetuated postembryonically.

  9. Stimulation of Cell Elongation by Tetraploidy in Hypocotyls of Dark-Grown Arabidopsis Seedlings.

    Science.gov (United States)

    Narukawa, Hideki; Yokoyama, Ryusuke; Komaki, Shinichiro; Sugimoto, Keiko; Nishitani, Kazuhiko

    2015-01-01

    Plant size is largely determined by the size of individual cells. A number of studies showed a link between ploidy and cell size in land plants, but this link remains controversial. In this study, post-germination growth, which occurs entirely by cell elongation, was examined in diploid and autotetraploid hypocotyls of Arabidopsis thaliana (L.) Heynh. Final hypocotyl length was longer in tetraploid plants than in diploid plants, particularly when seedlings were grown in the dark. The longer hypocotyl in the tetraploid seedlings developed as a result of enhanced cell elongation rather than by an increase in cell number. DNA microarray analysis showed that genes involved in the transport of cuticle precursors were downregulated in a defined region of the tetraploid hypocotyl when compared to the diploid hypocotyl. Cuticle permeability, as assessed by toluidine-blue staining, and cuticular structure, as visualized by electron microscopy, were altered in tetraploid plants. Taken together, these data indicate that promotion of cell elongation is responsible for ploidy-dependent size determination in the Arabidopsis hypocotyl, and that this process is directly or indirectly related to cuticular function.

  10. Arabidopsis and Tobacco superman regulate hormone signalling and mediate cell proliferation and differentiation.

    Science.gov (United States)

    Nibau, Candida; Di Stilio, Verónica S; Wu, Hen-Ming; Cheung, Alice Y

    2011-01-01

    Arabidopsis thaliana superman (SUP) plays an important role during flower development by maintaining the boundary between stamens and carpels in the inner two whorls. It was proposed that SUP maintains this boundary by regulating cell proliferation in both whorls, as loss-of-function superman mutants produce more stamens at the expense of carpels. However, the cellular mechanism that underlies SUP function remains unknown. Here Arabidopsis or tobacco (Nicotiana tabacum) SUP was overexpressed in tobacco plants to substantiate SUP's role as a regulator of cell proliferation and boundary definition and provide evidence that its biological role may be mediated via hormonal changes. It was found that moderate levels of SUP stimulated cell growth and proliferation, whereas high levels were inhibitory. SUP stimulated auxin- and cytokinin-regulated processes, and cells overexpressing SUP displayed reduced hormone dependency for proliferation and regeneration into plants. SUP also induced proliferation of female traits in the second and third flower whorls and promoted differentiation of petaloid properties in sepals, further supporting a role for SUP as a boundary regulator. Moreover, cytokinin suppressed stamen development and promoted differentiation of carpeloid tissues, suggesting that SUP may regulate male and female development via its effect on cytokinin signalling. Taken together, these observations suggest a model whereby the effect of SUP on cell growth and proliferation involves the modulation of auxin- and cytokinin-regulated processes. Furthermore, differential SUP expression or different sensitivities of different cell types to SUP may determine whether SUP stimulates or suppresses their proliferation.

  11. A quantitative analysis of stem cell homeostasis in the Arabidopsis columella root cap.

    Science.gov (United States)

    Hong, Jing Han; Chu, Huangwei; Zhang, Chen; Ghosh, Dipanjana; Gong, Ximing; Xu, Jian

    2015-01-01

    The Lugol's staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of Arabidopsis roots since the late 1990s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labeling dividing stem cells with 5-ethynyl-2'-deoxyuridine (EdU), the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol's staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol's staining, provides a novel quantitative method to study the dynamics of stem cell behavior that govern homeostasis in the Arabidopsis columella root cap.

  12. A Quantitative Analysis of Stem Cell Homeostasis in the Arabidopsis Columella Root Cap

    Directory of Open Access Journals (Sweden)

    Jing Han eHong

    2015-03-01

    Full Text Available The Lugol’s staining method has been widely used to detect changes in the maintenance of stem cell fate in the columella root cap of Arabidopsis roots since the late ‘90s. However, various limitations of this method demand for additional or complementary new approaches. For instance, it is unable to reveal the division rate of columella root cap stem cells. Here we report that, by labelling dividing stem cells with 5-ethynyl-2´-deoxyuridine (EdU, the number and distribution of their labeled progeny can be studied so that the division rate of stem cells can be measured quantitatively and in addition, that the progression of stem cell progeny differentiation can be assessed in combination with Lugol’s staining. EdU staining takes few hours and visualization of the stain characteristics of columella root cap can be performed easily under confocal microscopes. This simple technology, when used together with Lugol’s staining, provides a novel quantitative method to study the dynamics of stem cell behaviour that govern homeostasis in the Arabidopsis columella root cap.

  13. Role of chromatin factors in Arabidopsis root stem cell maintenance

    NARCIS (Netherlands)

    Kornet, N.G.

    2008-01-01

    Stem cells replenish the cells present in an organism throughout its lifetime and sustain growth. They have unique characteristics: the capability to self-renew and the potential to differentiate into several cell types. Recently, it has become clear that chromatin factors support these unique featu

  14. Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis.

    Science.gov (United States)

    Goubet, Florence; Barton, Christopher J; Mortimer, Jennifer C; Yu, Xiaolan; Zhang, Zhinong; Miles, Godfrey P; Richens, Jenny; Liepman, Aaron H; Seffen, Keith; Dupree, Paul

    2009-11-01

    Mannans are hemicellulosic polysaccharides that have previously been implicated as structural constituents of cell walls and as storage reserves but which may serve other functions during plant growth and development. Several members of the Arabidopsis cellulose synthase-like A (CSLA) family have previously been shown to synthesise mannan polysaccharides in vitro when heterologously expressed. It has also been found that CSLA7 is essential for embryogenesis, suggesting a role for the CSLA7 product in development. To determine whether the CSLA proteins are responsible for glucomannan synthesis in vivo, we characterised insertion mutants in each of the nine Arabidopsis CSLA genes and several double and triple mutant combinations. csla9 mutants showed substantially reduced glucomannan, and triple csla2csla3csla9 mutants lacked detectable glucomannan in stems. Nevertheless, these mutants showed no alteration in stem development or strength. Overexpression of CSLA2, CSLA7 and CSLA9 increased the glucomannan content in stems. Increased glucomannan synthesis also caused defective embryogenesis, leading to delayed development and occasional embryo death. The embryo lethality of csla7 was complemented by overexpression of CSLA9, suggesting that the glucomannan products are similar. We conclude that CSLA2, CSLA3 and CSLA9 are responsible for the synthesis of all detectable glucomannan in Arabidopsis stems, and that CSLA7 synthesises glucomannan in embryos. These results are inconsistent with a substantial role for glucomannan in wall strength in Arabidopsis stems, but indicate that glucomannan levels affect embryogenesis. Together with earlier heterologous expression studies, the glucomannan deficiency observed in csla mutant plants demonstrates that the CSLA family encodes glucomannan synthases.

  15. Single-cell telomere-length quantification couples telomere length to meristem activity and stem cell development in Arabidopsis.

    Science.gov (United States)

    González-García, Mary-Paz; Pavelescu, Irina; Canela, Andrés; Sevillano, Xavier; Leehy, Katherine A; Nelson, Andrew D L; Ibañes, Marta; Shippen, Dorothy E; Blasco, Maria A; Caño-Delgado, Ana I

    2015-05-12

    Telomeres are specialized nucleoprotein caps that protect chromosome ends assuring cell division. Single-cell telomere quantification in animals established a critical role for telomerase in stem cells, yet, in plants, telomere-length quantification has been reported only at the organ level. Here, a quantitative analysis of telomere length of single cells in Arabidopsis root apex uncovered a heterogeneous telomere-length distribution of different cell lineages showing the longest telomeres at the stem cells. The defects in meristem and stem cell renewal observed in tert mutants demonstrate that telomere lengthening by TERT sets a replicative limit in the root meristem. Conversely, the long telomeres of the columella cells and the premature stem cell differentiation plt1,2 mutants suggest that differentiation can prevent telomere erosion. Overall, our results indicate that telomere dynamics are coupled to meristem activity and continuous growth, disclosing a critical association between telomere length, stem cell function, and the extended lifespan of plants.

  16. An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis.

    Science.gov (United States)

    Ge, Xiaochun; Dietrich, Charles; Matsuno, Michiyo; Li, Guojing; Berg, Howard; Xia, Yiji

    2005-03-01

    The components and pathways that regulate and execute developmental cell death programmes in plants remain largely unknown. We have found that the PROMOTION OF CELL SURVIVAL 1 (PCS1) gene in Arabidopsis, which encodes an aspartic protease, has an important role in determining the fate of cells in embryonic development and in reproduction processes. The loss-of-function mutation of PCS1 causes degeneration of both male and female gametophytes and excessive cell death of developing embryos. Conversely, ectopic expression of PCS1 causes the septum and stomium cells that normally die in the anther wall to survive instead, leading to a failure in anther dehiscence and male sterility. PCS1 provides a new avenue for understanding the mechanisms of the programmed cell death processes that are associated with developmental pathways in plants and makes available a useful tool for engineering the male sterility trait for hybrid seed production.

  17. Simple suspension culture system of human iPS cells maintaining their pluripotency for cardiac cell sheet engineering.

    Science.gov (United States)

    Haraguchi, Yuji; Matsuura, Katsuhisa; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

    2015-12-01

    In this study, a simple three-dimensional (3D) suspension culture method for the expansion and cardiac differentiation of human induced pluripotent stem cells (hiPSCs) is reported. The culture methods were easily adapted from two-dimensional (2D) to 3D culture without any additional manipulations. When hiPSCs were directly applied to 3D culture from 2D in a single-cell suspension, only a few aggregated cells were observed. However, after 3 days, culture of the small hiPSC aggregates in a spinner flask at the optimal agitation rate created aggregates which were capable of cell passages from the single-cell suspension. Cell numbers increased to approximately 10-fold after 12 days of culture. The undifferentiated state of expanded hiPSCs was confirmed by flow cytometry, immunocytochemistry and quantitative RT-PCR, and the hiPSCs differentiated into three germ layers. When the hiPSCs were subsequently cultured in a flask using cardiac differentiation medium, expression of cardiac cell-specific genes and beating cardiomyocytes were observed. Furthermore, the culture of hiPSCs on Matrigel-coated dishes with serum-free medium containing activin A, BMP4 and FGF-2 enabled it to generate robust spontaneous beating cardiomyocytes and these cells expressed several cardiac cell-related genes, including HCN4, MLC-2a and MLC-2v. This suggests that the expanded hiPSCs might maintain the potential to differentiate into several types of cardiomyocytes, including pacemakers. Moreover, when cardiac cell sheets were fabricated using differentiated cardiomyocytes, they beat spontaneously and synchronously, indicating electrically communicative tissue. This simple culture system might enable the generation of sufficient amounts of beating cardiomyocytes for use in cardiac regenerative medicine and tissue engineering.

  18. Gene Mining for Proline Based Signaling Proteins in Cell Wall of Arabidopsis thaliana

    Science.gov (United States)

    Ihsan, Muhammad Z.; Ahmad, Samina J. N.; Shah, Zahid Hussain; Rehman, Hafiz M.; Aslam, Zubair; Ahuja, Ishita; Bones, Atle M.; Ahmad, Jam N.

    2017-01-01

    The cell wall (CW) as a first line of defense against biotic and abiotic stresses is of primary importance in plant biology. The proteins associated with cell walls play a significant role in determining a plant's sustainability to adverse environmental conditions. In this work, the genes encoding cell wall proteins (CWPs) in Arabidopsis were identified and functionally classified using geneMANIA and GENEVESTIGATOR with published microarrays data. This yielded 1605 genes, out of which 58 genes encoded proline-rich proteins (PRPs) and glycine-rich proteins (GRPs). Here, we have focused on the cellular compartmentalization, biological processes, and molecular functioning of proline-rich CWPs along with their expression at different plant developmental stages. The mined genes were categorized into five classes on the basis of the type of PRPs encoded in the cell wall of Arabidopsis thaliana. We review the domain structure and function of each class of protein, many with respect to the developmental stages of the plant. We have then used networks, hierarchical clustering and correlations to analyze co-expression, co-localization, genetic, and physical interactions and shared protein domains of these PRPs. This has given us further insight into these functionally important CWPs and identified a number of potentially new cell-wall related proteins in A. thaliana. PMID:28289422

  19. Deciphering the responses of root border-like cells of Arabidopsis and flax to pathogen-derived elicitors.

    Science.gov (United States)

    Plancot, Barbara; Santaella, Catherine; Jaber, Rim; Kiefer-Meyer, Marie Christine; Follet-Gueye, Marie-Laure; Leprince, Jérôme; Gattin, Isabelle; Souc, Céline; Driouich, Azeddine; Vicré-Gibouin, Maïté

    2013-12-01

    Plant pathogens including fungi and bacteria cause many of the most serious crop diseases. The plant innate immune response is triggered upon recognition of microbe-associated molecular patterns (MAMPs) such as flagellin22 and peptidoglycan. To date, very little is known of MAMP-mediated responses in roots. Root border cells are cells that originate from root caps and are released individually into the rhizosphere. Root tips of Arabidopsis (Arabidopsis thaliana) and flax (Linum usitatissimum) release cells known as "border-like cells." Whereas root border cells of pea (Pisum sativum) are clearly involved in defense against fungal pathogens, the function of border-like cells remains to be established. In this study, we have investigated the responses of root border-like cells of Arabidopsis and flax to flagellin22 and peptidoglycan. We found that both MAMPs triggered a rapid oxidative burst in root border-like cells of both species. The production of reactive oxygen species was accompanied by modifications in the cell wall distribution of extensin epitopes. Extensins are hydroxyproline-rich glycoproteins that can be cross linked by hydrogen peroxide to enhance the mechanical strength of the cell wall. In addition, both MAMPs also caused deposition of callose, a well-known marker of MAMP-elicited defense. Furthermore, flagellin22 induced the overexpression of genes involved in the plant immune response in root border-like cells of Arabidopsis. Our findings demonstrate that root border-like cells of flax and Arabidopsis are able to perceive an elicitation and activate defense responses. We also show that cell wall extensin is involved in the innate immunity response of root border-like cells.

  20. A reliable method for spectrophotometric determination of glycine betaine in cell suspension and other systems.

    Science.gov (United States)

    Valadez-Bustos, Ma Guadalupe; Aguado-Santacruz, Gerardo Armando; Tiessen-Favier, Axel; Robledo-Paz, Alejandrina; Muñoz-Orozco, Abel; Rascón-Cruz, Quintin; Santacruz-Varela, Amalio

    2016-04-01

    Glycine betaine is a quaternary ammonium compound that accumulates in a large variety of species in response to different types of stress. Glycine betaine counteracts adverse effects caused by abiotic factors, preventing the denaturation and inactivation of proteins. Thus, its determination is important, particularly for scientists focused on relating structural, biochemical, physiological, and/or molecular responses to plant water status. In the current work, we optimized the periodide technique for the determination of glycine betaine levels. This modification permitted large numbers of samples taken from a chlorophyllic cell line of the grass Bouteloua gracilis to be analyzed. Growth kinetics were assessed using the chlorophyllic suspension to determine glycine betaine levels in control (no stress) cells and cells osmotically stressed with 14 or 21% polyethylene glycol 8000. After glycine extraction, different wavelengths and reading times were evaluated in a spectrophotometer to determine the optimal quantification conditions for this osmolyte. Optimal results were obtained when readings were taken at a wavelength of 290 nm at 48 h after dissolving glycine betaine crystals in dichloroethane. We expect this modification to provide a simple, rapid, reliable, and cheap method for glycine betaine determination in plant samples and cell suspension cultures.

  1. Optimizing the transient transfection process of HEK-293 suspension cells for protein production by nucleotide ratio monitoring

    DEFF Research Database (Denmark)

    de Los Milagros Bassani Molinas, Maria; Beer, Christiane; Hesse, F

    2014-01-01

    Large scale, transient gene expression (TGE) is highly dependent of the physiological status of a cell line. Therefore, intracellular nucleotide pools and ratios were used for identifying and monitoring the optimal status of a suspension cell line used for TGE. The transfection efficiency upon...... polyethyleneimine (PEI)-mediated transient gene delivery into HEK-293 cells cultured in suspension was investigated to understand the effect of different culture and transfection conditions as well as the significance of the culture age and the quality of the cell line used. Based on two different bicistronic model...

  2. Establishment of suspension cell culture of Gymnema sylvestre R.Br.- A threatened anti-diabetic plant

    Directory of Open Access Journals (Sweden)

    Karthic Raju

    2012-04-01

    Full Text Available A cell suspension culture was established from leaf explants of wild Gymnema sylvestre plants collected from Muniyankudisai,Tamilnadu, India. Murashige and Skoog medium supplemented with 9.0 μM l-1 of 2, 4- Dichlorophenoxy acetic acid and 2.1 μM l-1Benzyl adenine produced yellow friable callus with green patches.The cells were subcultured conscientiously twelve times to getconsistent growth of the cells in suspension. From the 10thsubculture onwards callus cells acclimatized to grow in suspensionwith aggregation and reached 168.6 g l-1 fw and 5.16 g l-1 dw of cellbiomass.

  3. Monitoring changes in proteome during stepwise adaptation of a MDCK cell line from adherence to growth in suspension.

    Science.gov (United States)

    Kluge, Sabine; Benndorf, Dirk; Genzel, Yvonne; Scharfenberg, Klaus; Rapp, Erdmann; Reichl, Udo

    2015-08-20

    Adaptation of continuous cell lines to growth in suspension in a chemically defined medium has significant advantages for design and optimization in manufacturing of biologicals. In this work, changes in the protein expression level during a step-wise adaptation of an adherent Madin Darby canine kidney (MDCK) cell line to suspension growth were analyzed. Therefore, three cell line adaptations were performed independently. Two adaptations were monitored closely to characterize short term changes in protein expression levels after serum deprivation. In addition, initial stages of suspension growth were analyzed for both adaptations. The third adaptation involved MDCK suspension cells (MDCKSUS2) grown over an extended time period to achieve robust growth characteristics. Here, cells of the final stage of adaptation were compared with their parental cell line (MDCKADH). A combination of two dimensional differential gel electrophoresis for relative protein quantification and tandem mass spectrometry for protein identification enabled insights into cellular physiology. The two closely monitored cell line adaptations followed different routes regarding specific changes in protein expression but resulted in similar proteome profiles at the initial stages of suspension growth analyzed. Compared to the MDCKADH cells more than 90% of all changes in the protein expression level were identified after serum deprivation and were related to cytoskeletal structure, genetic information processing and cellular metabolism. Myosin proteins, involved in cellular detachment by actin-myosin contractile mechanisms were also differentially expressed. Interestingly, for both of the two adaptations, proteins linked for tumorigenicity, like lactoylglutathione lyase and sulfotransferase 1A1 were differentially expressed. In contrast, none of these proteins were differentially expressed for the MDCKSUS2 cell line. Overall, proteomic monitoring allowed identification of key proteins involved in

  4. Conditioning of Parsley (Petroselinum crispum L.) Suspension Cells Increases Elicitor-Induced Incorporation of Cell Wall Phenolics.

    Science.gov (United States)

    Kauss, H.; Franke, R.; Krause, K.; Conrath, U.; Jeblick, W.; Grimmig, B.; Matern, U.

    1993-06-01

    The elicitor-induced incorporation of phenylpropanoid derivatives into the cell wall and the secretion of soluble coumarin derivatives (phytoalexins) by parsley (Petroselinum crispum L.) suspension cultures can be potentiated by pretreatment of the cultures with 2,6-dichloroisonicotinic acid or derivatives of salicylic acid. To investigate this phenomenon further, the cell walls and an extracellular soluble polymer were isolated from control cells or cells treated with an elicitor from Phytophthora megasperma f. sp. glycinea. After alkaline hydrolysis, both fractions from elicited cells showed a greatly increased content of 4-coumaric, ferulic, and 4-hydroxybenzoic acid, as well as 4-hydroxybenzaldehyde and vanillin. Two minor peaks were identified as tyrosol and methoxytyrosol. The pretreatment effect is most pronounced at a low elicitor concentration. Its specificity was elaborated for coumarin secretion. When the parsley suspension cultures were preincubated for 1 d with 2,6-dichloroisonicotinic, 4- or 5-chlorosalicylic, or 3,5- dichlorosalicylic acid, the cells exhibited a greatly increased elicitor response. Pretreatment with isonicotinic, salicylic, acetylsalicylic, or 2,6-dihydroxybenzoic acid was less efficient in enhancing the response, and some other isomers were inactive. This increase in elicitor response was also observed for the above-mentioned monomeric phenolics, which were liberated from cell walls upon alkaline hydrolysis and for "lignin-like" cell wall polymers determined by the thioglycolic acid method. It was shown for 5-chlorosalicylic acid that conditioning most likely improves the signal transduction leading to the activation of genes encoding phenylalanine ammonia lyase and 4-coumarate: coenzyme A ligase. The conditioning thus sensitizes the parsley suspension cells to respond to lower elicitor concentrations. If a similar mechanism were to apply to whole plants treated with 2,6-dichloroisonicotinic acid, a known inducer of systemic

  5. Live Cell Imaging Reveals Structural Associations between the Actin and Microtubule Cytoskeleton in Arabidopsis [W] [OA

    Science.gov (United States)

    Sampathkumar, Arun; Lindeboom, Jelmer J.; Debolt, Seth; Gutierrez, Ryan; Ehrhardt, David W.; Ketelaar, Tijs; Persson, Staffan

    2011-01-01

    In eukaryotic cells, the actin and microtubule (MT) cytoskeletal networks are dynamic structures that organize intracellular processes and facilitate their rapid reorganization. In plant cells, actin filaments (AFs) and MTs are essential for cell growth and morphogenesis. However, dynamic interactions between these two essential components in live cells have not been explored. Here, we use spinning-disc confocal microscopy to dissect interaction and cooperation between cortical AFs and MTs in Arabidopsis thaliana, utilizing fluorescent reporter constructs for both components. Quantitative analyses revealed altered AF dynamics associated with the positions and orientations of cortical MTs. Reorganization and reassembly of the AF array was dependent on the MTs following drug-induced depolymerization, whereby short AFs initially appeared colocalized with MTs, and displayed motility along MTs. We also observed that light-induced reorganization of MTs occurred in concert with changes in AF behavior. Our results indicate dynamic interaction between the cortical actin and MT cytoskeletons in interphase plant cells. PMID:21693695

  6. Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures.

    Science.gov (United States)

    Xu, Jianfeng; Ge, Xumeng; Dolan, Maureen C

    2011-01-01

    "Molecular farming" in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative "factory". However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.

  7. Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

    Science.gov (United States)

    Heidrich, Katharina; Wirthmueller, Lennart; Tasset, Céline; Pouzet, Cécile; Deslandes, Laurent; Parker, Jane E

    2011-12-01

    Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

  8. Expression of Arabidopsis hexokinase in citrus guard cells controls stomatal aperture and reduces transpiration

    Directory of Open Access Journals (Sweden)

    Nitsan eLugassi

    2015-12-01

    Full Text Available Hexokinase (HXK is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1 under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

  9. Molecular and Genetic Analysis of Hormone-Regulated Differential Cell Elongation in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Ecker, Joseph R.

    2005-09-15

    We have utilized the response of Arabidopsis seedlings to the plant hormone ethylene to identify new genes involved in the regulation of ethylene biosynthesis, perception, signal transduction and differential cell growth. In building a genetic framework for the action of these genes, we have developed a molecular model that has facilitated our understanding of the molecular requirements of ethylene for cell elongation processes. The ethylene response pathway in Arabidopsis appears to be primarily linear and is defined by the genes: ETR1, ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, EIN3, EIN5, EIN6, and EIN. Downstream branches identified by the HLS1, EIR1, and AUX1 genes involve interactions with other hormonal (auxin) signals in the process of differential cell elongation in the hypocotyl hook. Cloning and characterization of HLS1 (and three HLL genes) and ETO1 (and ETOL genes) in my laboratory has been supported under this award. HLS1 is required for differential elongation of cells in the hypocotyl and may act in the establishment of hormone gradients. Also during the previous period, we have identified and characterized a gene that genetically acts upstream of the ethylene receptors. ETO1 encodes negative regulators of ethylene biosynthesis.

  10. A rapid chemical method for lysing Arabidopsis cells for protein analysis

    Directory of Open Access Journals (Sweden)

    Takano Tetsuo

    2011-07-01

    Full Text Available Abstract Background Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once. Results We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them. Conclusions Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.

  11. Molecular and Genetic Analysis of Hormone-Regulated Differential Cell Elongation in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Ecker, Joseph R.

    2002-12-03

    The authors have utilized the response of Arabidopsis seedlings to the plant hormone ethylene to identify new genes involved in the regulation of ethylene biosynthesis, perception, signal transduction and differential cell growth. In building a genetic framework for the action of these genes, they developed a molecular model that has facilitated the understanding of the molecular requirements of ethylene for cell elongation processes. The ethylene response pathway in Arabidopsis appears to be primarily linear and is defined by the genes: ETR1, ETR2, ERS1, ERS2, EIN4, CTR1, EIN2, EIN3, EIN5 EIN6, and EIN. Downstream branches identified by the HLS1, EIR1, and AUX1 genes involve interactions with other hormonal (auxin) signals in the process of differential cell elongation in the hypocotyl hook. Cloning and characterization of HLS1 and three HLS1-LIKE genes in the laboratory has been supported under this award. HLS1 is required for differential elongation of cells in the hypocotyl and may act in the establishment of hormone gradients. Also during the award period, they have identified and begun preliminary characterization of two genes that genetically act upstream of the ethylene receptors. ETO1 and RAN1 encode negative regulators of ethylene biosynthesis and signaling respectively. Progress on the analysis of these genes along with HOOKLESS1 is described.

  12. Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A Cooperate in Cell Cycle–Associated Replication of Peroxisomes[W

    Science.gov (United States)

    Lingard, Matthew J.; Gidda, Satinder K.; Bingham, Scott; Rothstein, Steven J.; Mullen, Robert T.; Trelease, Richard N.

    2008-01-01

    Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle–associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ∼40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells. PMID:18539750

  13. Arabidopsis PEROXIN11c-e, FISSION1b, and DYNAMIN-RELATED PROTEIN3A cooperate in cell cycle-associated replication of peroxisomes.

    Science.gov (United States)

    Lingard, Matthew J; Gidda, Satinder K; Bingham, Scott; Rothstein, Steven J; Mullen, Robert T; Trelease, Richard N

    2008-06-01

    Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle-associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in approximately 40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.

  14. Germination of arabidopsis seed in space and in simulated microgravity: alterations in root cell growth and proliferation

    NARCIS (Netherlands)

    Manzano, A.I.; Matia, I.; Gonzalez-Camacho, F.; Carnero-Diaz, E.; van Loon, J.J.W.A.; Dijkstra, C.; Larkin, O.; Anthony, P.; Davey, M.R.; Marco, R.; Medina, F.J.

    2009-01-01

    Changes have been reported in the pattern of gene expression in Arabidopsis on exposure to microgravity. Plant cell growth and proliferation are functions that are potentially affected by such changes in gene expression. In the present investigation, the cell proliferation rate, the regulation of ce

  15. Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase I protein is present in sporophytic and gametophytic cells and undergoes endocytosis

    NARCIS (Netherlands)

    Kwaaitaal, M.A.C.J.; Vries, de S.C.; Russinova, E.T.

    2005-01-01

    Arabidopsis thaliana plants expressing AtSERK1 fused to yellow-fluorescent protein were generated. Fluorescence was detected predominantly at the cell periphery, most likely the plasma membrane, of cells in ovules, embryo sacs, anthers, and embryos and in seedlings. The AtSERK1 protein was detected

  16. Arabidopsis Heterotrimeric G-protein Regulates Cell Wall Defense and Resistance to Necrotrophic Fungi

    Institute of Scientific and Technical Information of China (English)

    Magdalena Delcado-Cerezo; Paul Schulze-Lefert; Shauna Somerville; José Manuel Estevez; Staffan Persson; Antonio Molina; Clara Sánchez-Rodríguez; Viviana Escudero; Eva Miedes; Paula Virginia Fernández; Lucía Jordá; Camilo Hernández-Blanco; Andrea Sánchez-Vallet; Pawel Bednarek

    2012-01-01

    The Arabidopsis heterotrimeric G-protein controls defense responses to necrotrophic and vascular fungi.The agb1 mutant impaired in the Gβ subunit displays enhanced susceptibility to these pathogens.Gβ/AGB1 forms an obligate dimer with either one of the Arabidopsis Gγ subunits (γ1/AGG1 and γ2/AGG2).Accordingly,we now demonstrate that the agg1 agg2 double mutant is as susceptible as agb1 plants to the necrotrophic fungus Plectosphaerella cucumerina.To elucidate the molecular basis of heterotrimeric G-protein-mediated resistance,we performed a comparative transcriptomic analysis of agb1-1 mutant and wild-type plants upon inoculation with P cucumerina.This analysis,together with metabolomic studies,demonstrated that G-protein-mediated resistance was independent of defensive pathways required for resistance to necrotrophic fungi,such as the salicylic acid,jasmonic acid,ethylene,abscisic acid,and tryptophan-derived metabolites signaling,as these pathways were not impaired in agb1 and agg1 agg2 mutants.Notably,many mis-regulated genes in agb1 plants were related with cell wall functions,which was also the case in agg1 agg2 mutant.Biochemical analyses and Fourier Transform InfraRed (FTIR) spectroscopy of cell walls from G-protein mutants revealed that the xylose content was lower in agb1 and agg1 agg2 mutants than in wild-type plants,and that mutant walls had similar FTIR spectratypes,which differed from that of wild-type plants.The data presented here suggest a canonical functionality of the Gβ and Gγ1/γ2 subunits in the control of Arabidopsis immune responses and the regulation of cell wall composition.

  17. Modulated differential photoacoustic cell to study the gelatinization in a starch-water suspension

    Directory of Open Access Journals (Sweden)

    J. A. Villada

    2014-06-01

    Full Text Available In this paper the design and implementation of a novel Differential Photoacoustic Cell (DPC system is presented. The system was used to study the thermo optic transition in water-starch suspension called gelatinization. The melting temperature of Gallium was used to calibrate the temperature of the system. Both temperature values for starch gelatinization and gallium melting were agreed with those obtained using differential scanning calorimetry (DSC. The results show that this system is suitable to study other thermal processes in food or any thermal transition at low temperature.

  18. Investigation of roles for LRR-RLKs PNL1 and PNL2 in asymmetric cell division in Arabidopsis thaliana

    OpenAIRE

    Rodriguez, Maiti Celina

    2008-01-01

    Asymmetric cell division is a vital component of plant development. It enables cell differentiation and cell diversity. A key component of asymmetric cell division is cell signaling. Signals are believed to control polarization and orientation of asymmetric divisions during stomatal development. The findings of this report suggest that PNL1 and PNL2, two LRR-RLKs found in Arabidopsis and closely related to maize PAN1 LRR-RLK, are possibly involved in the signaling events occurring during the ...

  19. Steroids are required for epidermal cell fate establishment in Arabidopsis roots.

    Science.gov (United States)

    Kuppusamy, Kavitha T; Chen, Andrew Y; Nemhauser, Jennifer L

    2009-05-12

    The simple structure of Arabidopsis roots provides an excellent model system to study epidermal cell fate specification. Epidermal cells in contact with 2 underlying cortical cells differentiate into hair cells (H cells; trichoblasts), whereas cells that contact only a single cortical cell differentiate into mature hairless cells (N cells; atrichoblasts). This position-dependent patterning, in combination with the constrained orientation of cell divisions, results in hair and nonhair cell files running longitudinally along the root epidermis. Here, we present strong evidence that steroid hormones called brassinosteroids (BRs) are required to maintain position-dependent fate specification in roots. We show that BRs are required for normal expression levels and patterns of WEREWOLF (WER) and GLABRA2 (GL2), master regulators of epidermal patterning. Loss of BR signaling results in loss of hair cells in H positions, likely as a consequence of reduced expression of CAPRICE (CPC), a direct downstream target of WER. Our observations demonstrate that in addition to their well-known role in cell expansion, BRs play an essential role in directing cell fate.

  20. Solid oxide fuel cell electrolytes produced via very low pressure suspension plasma spray and electrophoretic deposition

    Science.gov (United States)

    Fleetwood, James D.

    Solid oxide fuel cells (SOFCs) are a promising element of comprehensive energy policies due to their direct mechanism for converting the oxidization of fuel, such as hydrogen, into electrical energy. Both very low pressure plasma spray and electrophoretic deposition allow working with high melting temperature SOFC suspension based feedstock on complex surfaces, such as in non-planar SOFC designs. Dense, thin electrolytes of ideal composition for SOFCs can be fabricated with each of these processes, while compositional control is achieved with dissolved dopant compounds that are incorporated into the coating during deposition. In the work reported, sub-micron 8 mole % Y2O3-ZrO2 (YSZ) and gadolinia-doped ceria (GDC), powders, including those in suspension with scandium-nitrate dopants, were deposited on NiO-YSZ anodes, via very low pressure suspension plasma spray (VLPSPS) at Sandia National Laboratories' Thermal Spray Research Laboratory and electrophoretic deposition (EPD) at Purdue University. Plasma spray was carried out in a chamber held at 320 - 1300 Pa, with the plasma composed of argon, hydrogen, and helium. EPD was characterized utilizing constant current deposition at 10 mm electrode separation, with deposits sintered from 1300 -- 1500 °C for 2 hours. The role of suspension constituents in EPD was analyzed based on a parametric study of powder loading, powder specific surface area, polyvinyl butyral (PVB) content, polyethyleneimine (PEI) content, and acetic acid content. Increasing PVB content and reduction of particle specific surface area were found to eliminate the formation of cracks when drying. PEI and acetic acid content were used to control suspension stability and the adhesion of deposits. Additionally, EPD was used to fabricate YSZ/GDC bilayer electrolyte systems. The resultant YSZ electrolytes were 2-27 microns thick and up to 97% dense. Electrolyte performance as part of a SOFC system with screen printed LSCF cathodes was evaluated with peak

  1. The organization pattern of root border-like cells of Arabidopsis is dependent on cell wall homogalacturonan.

    Science.gov (United States)

    Durand, Caroline; Vicré-Gibouin, Maïté; Follet-Gueye, Marie Laure; Duponchel, Ludovic; Moreau, Myriam; Lerouge, Patrice; Driouich, Azeddine

    2009-07-01

    Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.

  2. Effects of medium nutrition on cell growth and isocamptothecin A and B production by suspension cell culture of Camptotheca acuminata

    Institute of Scientific and Technical Information of China (English)

    Zhang Dongyan; Yu Fang; Bai Fengwu; An Lijia

    2006-01-01

    The effects of initial sucrose concentration, nitrate to ammonium ratio, total N concentration and phosphate concentration in medium on cell growth and isocamptothecin A and B synthesis by suspension cell culture of Camptotheca acuminata were investigated in 250 mL shake flasks. 30 g L-1 sucrose concentration was beneficial to secondary metabolites synthesis. The cell growth and metabolites synthesis were also affected by the ratio of NO-3/NH+4, and nitrate was favourable for cell growth. The maximum dry weight was achieved when nitrate was used as the sole N source. The effect of total initial N on the cell cultures was also investigated with NO-3/NH+4 ratio of 1∶2. The final dry cell weight was similar throughout culture period and 50 mM initial N was favourable for secondary metabolite synthesis. 50 mM initial phosphate concentration facilitated both cell growth and secondary metabolites synthesis.

  3. Identification and characterization of inward K ~+-channels in plasma membranes of Arabidopsis root cortex cells

    Institute of Scientific and Technical Information of China (English)

    于川江; 武维华

    1999-01-01

    Patch clamping whole-cell reeording techniques were apphed to study the inward K+ channels in Arabidopsis root cortex cells. The inward K+-channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective tor K+ ions over Na+ ions. The channel activity was significantly inbibited by the external TEA(?) or Ba(?) The changes in cytoplasmic Ca2+ concentrations did not affect the whole-cell inward K+-currents. The possible asso(?)ation betw(?)en the channel selectivity to K+ and Na(?) ions and plant salt-tolerance was also discussed.

  4. Histology of embryoid development in oil palm (Elaeis guineensis Jacq. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Songrat Tinnongjig

    2001-11-01

    Full Text Available Embryos of oil palm (Elaeis guineensis Jacq. variety tenera were cultured on Eeuwens or Y3 (1976; 1978 medium supplemented with 2 mg/l 2,4-D. Calluses were initiated from these embryos. The eight-weekold calluses derived from embryos were transferred to modified Y3 liquid medium devoid of 2,4-D and supplemented with NAA, BA and coconut water to establish cell suspension culture. After a period of culture,these cells were then subcultured to the same medium without plant growth regulators to induce embryoid formation. The calluses and embryoids were harvested at various times, fixed, sectioned, stained and examined microscopically. Histological study revealed that embryoid occurred from meristematic cells with dense cytoplasm along the callus clumps.

  5. Autophagic components contribute to hypersensitive cell death in Arabidopsis

    DEFF Research Database (Denmark)

    Hofius, Daniel; Schultz-Larsen, Torsten; Joensen, Jan;

    2009-01-01

    Autophagy has been implicated as a prosurvival mechanism to restrict programmed cell death (PCD) associated with the pathogen-triggered hypersensitive response (HR) during plant innate immunity. This model is based on the observation that HR lesions spread in plants with reduced autophagy gene ex...... contributes to HR PCD and can function in parallel with other prodeath pathways....

  6. JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a non-cell-autonomous mechanism.

    Science.gov (United States)

    Hassan, Hala; Scheres, Ben; Blilou, Ikram

    2010-05-01

    In Arabidopsis, specification of the hair and non-hair epidermal cell types is position dependent, in that hair cells arise over clefts in the underlying cortical cell layer. Epidermal patterning is determined by a network of transcriptional regulators that respond to an as yet unknown cue from underlying tissues. Previously, we showed that JACKDAW (JKD), a zinc finger protein, localizes in the quiescent centre and the ground tissue, and regulates tissue boundaries and asymmetric cell division by delimiting SHORT-ROOT movement. Here, we provide evidence that JKD controls position-dependent signals that regulate epidermal-cell-type patterning. JKD is required for appropriately patterned expression of the epidermal cell fate regulators GLABRA2, CAPRICE and WEREWOLF. Genetic interaction studies indicate that JKD operates upstream of the epidermal patterning network in a SCRAMBLED (SCM)-dependent fashion after embryogenesis, but acts independent of SCM in embryogenesis. Tissue-specific induction experiments indicate non-cell-autonomous action of JKD from the underlying cortex cell layer to specify epidermal cell fate. Our findings are consistent with a model where JKD induces a signal in every cortex cell that is more abundant in the hair cell position owing to the larger surface contact of cells located over a cleft.

  7. Analysis of chlorophyll fluorescence reveals stage specific patterns of chloroplast-containing cells during Arabidopsis embryogenesis.

    Science.gov (United States)

    Tejos, Ricardo I; Mercado, Ana V; Meisel, Lee A

    2010-01-01

    The basic body plan of a plant is established early in embryogenesis when cells differentiate, giving rise to the apical and basal regions of the embryo. Using chlorophyll fluorescence as a marker for chloroplasts, we have detected specific patterns of chloroplast-containing cells at specific stages of embryogenesis. Non-randomly distributed chloroplast-containing cells are seen as early as the globular stage of embryogenesis in Arabidopsis. In the heart stage of embryogenesis, chloroplast containing cells are detected in epidermal cells as well as a central region of the heart stage embryo, forming a triangular septum of chloroplast-containing cells that divides the embryo into three equal sectors. Torpedo stage embryos have chloroplast-containing epidermal cells and a central band of chloroplast-containing cells in the cortex layer, just below the shoot apical meristem. In the walking-stick stage of embryogenesis, chloroplasts are present in the epidermal, cortex and endodermal cells. The chloroplasts appear reduced or absent from the provascular and columella cells of walking-stick stage embryos. These results suggest that there is a tight regulation of plastid differentiation during embryogenesis that generates specific patterns of chloroplast-containing cells in specific cell layers at specific stages of embryogenesis.

  8. Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

    Energy Technology Data Exchange (ETDEWEB)

    Zhongchi Liu

    2004-10-01

    Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to the molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam age

  9. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract.

    Science.gov (United States)

    Zhou, Liangyun; Yang, Jian; Yang, Guang; Kang, Chuanzhi; Xiao, Wenjuan; Lv, Chaogeng; Wang, Sheng; Tang, Jinfu; Guo, Lanping

    2016-09-14

    Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE) treatment. An ultra-performance liquid chromatography (UPLC) method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS) LC-MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2'-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 μg·g(-1)) at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-β-d-glucopyranoside. The content of 2'-Hydroxyaucuparin reached its highest (422.75 μg·g(-1)) at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

  10. Biphenyl Phytoalexin in Sorbus pohuashanensis Suspension Cell Induced by Yeast Extract

    Directory of Open Access Journals (Sweden)

    Liangyun Zhou

    2016-09-01

    Full Text Available Biphenyls are unique phytoalexins de novo synthesized in plants in response to pathogen attack. These compounds are found in Maloideae, a subfamily of the Rosaceae. The anti-microbial activities of biphenyls have been reported in a number of studies and they appear to represent an important defense strategy against pathogens common in the Maloideae, such as species in Malus, Pyrus, Sorbus, and Chaenomeles. Here, cell suspension cultures of Sorbus pohuashanensis were established to study biphenyl phytoalexins formation after yeast extract (YE treatment. An ultra-performance liquid chromatography (UPLC method coupled with quadrupole time of flight mass spectrometry (Q-TOF-MS LC−MS/MS was applied to determine the time course of these biphenyl biomarkers accumulation in YE-treated S. pohuashanensis suspension cells. The results of quantitative analyses show the content of Noraucuparin, 2′-Hydroxyaucuparin, and their glycosides initially increased, then decreased over time. The Noraucuparin content reached its highest (225.76 μg·g−1 at 18 h after treatment, 6 hours earlier than that of Noraucuparin 5-O-β-d-glucopyranoside. The content of 2′-Hydroxyaucuparin reached its highest (422.75 μg·g−1 at 30 h after treatment, also earlier than that of its glycoside. The understanding of phytoalexin metabolism in this study may provide a basis for improving Maloideae resistance to pathogens.

  11. Production of Gymnemic Acid from Cell Suspension Cultures of Gymnema sylvestre.

    Science.gov (United States)

    Nagella, Praveen; Dandin, Vijayalaxmi S; Murthy, Hosakatte Niranjana

    2016-01-01

    Gymnema sylvestre R. Br. is a popular herbal medicine. It has been used in ayurvedic system of medicine for thousands of years. It is popularly called as "Gur-mar" for its distinctive property of temporarily destroying the taste of sweetness and is used in the treatment of diabetes. The leaves of gymnema possess antidiabetic, antimicrobial, anti-hypercholesterolemic, anti-sweetener, anti-inflammatory, and hepatoprotective properties and have traditional uses in the treatment of asthma, eye complaints, and snake bite. The leaves contain triterpene saponins such as gymnemic acid which is an active ingredient of Gymnema. Since the cultivation of G. sylvestre is a very slow process and the content of gymnemic acid depends on the environmental factors, cell suspension culture is sought as an alternative means for the production of Gymnema biomass and to enhance the gymnemic acid content. In this chapter, the methods employed for the induction of callus and subsequent establishment of cell suspension cultures for the production of biomass and analysis of gymnemic acid using high performance liquid chromatography are described.

  12. An Efficient Rice Mutagenesis System Based on Suspension-Cultured Cells

    Institute of Scientific and Technical Information of China (English)

    Yuan-Ling Chen; Hui-Lin Liang; Xing-Liang Ma; Su-Lin Lou; Yong-Yao Xie; Zhen-Lan Liu; Le-Tian Chen; Yao-Guang Liu

    2013-01-01

    Plant mutants are important bio-resources for crop breeding and gene functional studies.Conventional methods for generating mutant libraries by mutagenesis of seeds with physical or chemical agents are of low efficiency.Here,we developed a highly-efficient ethyl methanesulfonate (EMS) mutagenesis system based on suspension-cultured cells,with rice (Oryza sativa L.) as an example.We show that treatment of suspension-cultured tiny cell clusters with 0.4% EMS for 18-22 h followed by differentiation and regeneration produced as high as 29.4% independent mutant lines with visible phenotypic variations,including a number of important agronomic traits such as grain size,panicle size,grain or panicle shape,tiller number and angle,heading date,male sterility,and disease sensitivity.No mosaic mutant was observed in the mutant lines tested.In this mutant library,we obtained a mutant with an abnormally elongated uppermost internode.Sequencing and functional analysis revealed that this is a new allelic mutant of eui (elongated uppermost internode) caused by two point mutations in the first exon of the EUI gene,representing a successful example of this mutagenesis system.

  13. Suspension-cultured plant cells as a tool to analyze the extracellular proteome.

    Science.gov (United States)

    Sabater-Jara, Ana B; Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Esteso, María J; Youssef, Sabry M; Casado-Vela, Juan; Vera-Urbina, Juan C; Sellés-Marchart, Susana; Bru-Martínez, Roque; Pedreño, María A

    2014-01-01

    Suspension-cultured cells (SCC) are generally considered the most suitable cell systems to carry out scientific studies, including the extracellular proteome (secretome). SCC are initiated by transferring friable callus fragments into flasks containing liquid culture medium for cell biomass growth, and they are maintained in an orbital shaker to supply the sufficient oxygen that allows cell growth. SCC increase rapidly during the exponential phase and after 10-20 days (depending on the cell culture nature), the growth rate starts to decrease due to limitation of nutrients, and to maintain for decades these kinds of cell cultures is needed to transfer a portion of these SCC into a fresh culture medium. Despite the central role played by extracellular proteins in most processes that control growth and development, the secretome has been less well characterized than other subcellular compartments, meaning that our understanding of the cell wall physiology is still very limited. Useful proteomic tools have emerged in recent years to unravel metabolic network that occurs in cell walls. With the recent progress made in mass spectrometry technology, it has become feasible to identify proteins from a given organ, tissue, cells, or even a subcellular compartment. Compared with other methods used to isolate cell wall proteins, the spent medium of SCC provides a convenient, continuous, and reliable and unique source of extracellular proteins. Therefore, this biological system could be used as a large-scale cell culture from which these proteins can be secreted, easily separated from cells without cell disruption, and so, without any cytosolic contamination, easily recovered from the extracellular medium. This nondestructive cell wall proteome approach discloses a set of proteins that are specifically expressed in the remodelling of the cell wall architecture and stress defense.

  14. Tissue-wide Mechanical Forces Influence the Polarity of Stomatal Stem Cells in Arabidopsis.

    Science.gov (United States)

    Bringmann, Martin; Bergmann, Dominique C

    2017-03-20

    Mechanical information is an important contributor to cell polarity in uni- and multicellular systems [1-3]. In planar tissues like the Drosophila wing, cell polarity reorients during growth as cells divide and reorganize [4]. In another planar tissue, the Arabidopsis leaf epidermis [5], polarized, asymmetric divisions of stomatal stem cells (meristemoid mother cells [MMCs]) are fundamental for the generation and patterning of multiple cell types, including stomata. The activity of key transcription factors, polarizing factors [6], and peptide signals [7] explains some local stomatal patterns emerging from the behavior of a few lineally related cells [6, 8-11]. Here we demonstrate that, in addition to locally acting signals, tissue-wide mechanical forces can act as organizing cues, and that they do so by influencing the polarity of individual MMCs. If the mechanical stress environment in the tissue is altered through stretching or cell ablations, cellular polarity changes in response. In turn, polarity predicts the orientation of cellular and tissue outgrowth, leading to increased mechanical conflicts between neighboring cells. This interplay among growth, oriented divisions, and cell specification could contribute to the characteristic patterning of stomatal guard cells in the context of a growing leaf.

  15. Gymnema sylvestre R. Br. suspension cell extract show antidiabetic potential in Alloxan induced diabetic albino male rats

    Institute of Scientific and Technical Information of China (English)

    R Karthic; S Nagaraj; P Arulmurugan; S Seshadri; R Rengasamy; K Kathiravan

    2012-01-01

    Objective: To study the antidiabetic effects of suspension cell extract of Gymnema sylvestre (G.sylvestre in vitro grown suspension cells of G. sylvestre along with field grown and wild plant leaves of G.sylvestre was tested on alloxan induced diabetic rats. Results: While oral administration of the extracts reduced the glucose content in blood and urine, sugar and lipids in serum significantly (P≤0.05), it also increased the body weight, total haemoglobin and plasma protein content.Conclusions:It can be concluded that G. sylvestre suspension cell extract show excellent) along with field grown and wild plants. Methods: The effect of ethanolic extracts of the antidiabetic potential against alloxan induced diabetic albino male rats therefore be considered as potent antidiabetic drug.

  16. Suspension Culture Alters Insulin Secretion in Induced Human Umbilical Cord Matrix-Derived Mesenchymal Cells

    Directory of Open Access Journals (Sweden)

    Fatemeh Seyedi

    2016-04-01

    Full Text Available Objective: Worldwide, diabetes mellitus (DM is an ever-increasing metabolic disorder. A promising approach to the treatment of DM is the implantation of insulin producing cells (IPC that have been derived from various stem cells. Culture conditions play a pivotal role in the quality and quantity of the differentiated cells. In this experimental study, we have applied various culture conditions to differentiate human umbilical cord matrix-derived mesenchymal cells (hUCMs into IPCs and measured insulin production. Materials and Methods: In this experimental study, we exposed hUCMs cells to pancreatic medium and differentiated them into IPCs in monolayer and suspension cultures. Pancreatic medium consisted of serum-free Dulbecco’s modified eagle’s medium Nutrient mixture F12 (DMEM/F12 medium with 17.5 mM glucose supplemented by 10 mM nicotinamide, 10 nM exendin-4, 10 nM pentagastrin, 100 pM hepatocyte growth factor, and B-27 serum-free supplement. After differentiation, insulin content was analyzed by gene expression, immunocytochemistry (IHC and the chemiluminesence immunoassay (CLIA. Results: Reverse transcription-polymerase chain reaction (RT-PCR showed efficient expressions of NKX2.2, PDX1 and INSULIN genes in both groups. IHC analysis showed higher expression of insulin protein in the hanging drop group, and CLIA revealed a significant higher insulin production in hanging drops compared with the monolayer group following the glucose challenge test. Conclusion: We showed by this novel, simple technique that the suspension culture played an important role in differentiation of hUCMs into IPC. This culture was more efficient than the conventional culture method commonly used in IPC differentiation and cultivation.

  17. Cell pattern in the Arabidopsis root epidermis determined by lateral inhibition with feedback.

    Science.gov (United States)

    Lee, Myeong Min; Schiefelbein, John

    2002-03-01

    In the root epidermis of Arabidopsis, hair and nonhair cell types are specified in a distinct position-dependent pattern. Here, we show that transcriptional feedback loops between the WEREWOLF (WER), CAPRICE (CPC), and GLABRA2 (GL2) genes help to establish this pattern. Positional cues bias the expression of the WER MYB gene, leading to the induction of CPC and GL2 in cells located in a particular position (N) and adoption of the nonhair fate. The truncated MYB encoded by CPC mediates a lateral inhibition mechanism to negatively regulate WER, GL2, and its own gene in the alternative position (H) to induce the hair fate. These results provide a molecular genetic framework for understanding the determination of a cell-type pattern in plants.

  18. In Silico Identification of Co-transcribed Core Cell Cycle Regulators and Transcription Factors in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Regulatory networks involving transcription factors and core cell cycle regulators are expected to play crucial roles in plant growth and development. In this report, we describe the identification of two groups of co-transcribed core cell cycle regulators and transcription factors via a two-step in silico screening. The core cell cycle regulators include TARDY ASYNCHRONOUS MEIOSIS (CYCA1;2), CYCB1;1, CYCB2;1, CDKB1;2, and CDKB2;2 while the transcription factors include CURLY LEAF, AINTEGUMENTA, a MYB protein, two Forkhead-associated domain proteins, and a SCARECROW family protein. Promoter analysis revealed a potential web of cross- and self-regulations among the identified proteins. Because one criterion for screening for these genes is that they are predominantly transcribed in young organs but not in mature organs, these genes are likely to be particularly involved in Arabidopsis organ growth.

  19. Cell identity regulators link development and stress responses in the Arabidopsis root.

    Science.gov (United States)

    Iyer-Pascuzzi, Anjali S; Jackson, Terry; Cui, Hongchang; Petricka, Jalean J; Busch, Wolfgang; Tsukagoshi, Hironaka; Benfey, Philip N

    2011-10-18

    Stress responses in plants are tightly coordinated with developmental processes, but interaction of these pathways is poorly understood. We used genome-wide assays at high spatiotemporal resolution to understand the processes that link development and stress in the Arabidopsis root. Our meta-analysis finds little evidence for a universal stress response. However, common stress responses appear to exist with many showing cell type specificity. Common stress responses may be mediated by cell identity regulators because mutations in these genes resulted in altered responses to stress. Evidence for a direct role for cell identity regulators came from genome-wide binding profiling of the key regulator SCARECROW, which showed binding to regulatory regions of stress-responsive genes. Coexpression in response to stress was used to identify genes involved in specific developmental processes. These results reveal surprising linkages between stress and development at cellular resolution, and show the power of multiple genome-wide data sets to elucidate biological processes.

  20. Cell Proliferation Analysis Using EdU Labeling in Whole Plant and Histological Samples of Arabidopsis.

    Science.gov (United States)

    Kazda, Anita; Akimcheva, Svetlana; Watson, J Matthew; Riha, Karel

    2016-01-01

    The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis.

  1. AtGRIP protein locates to the secretory vesicles of trans Golgi-network in Arabidopsis root cap cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Ying; ZHANG Wei; ZHAO Lei; LI Yan

    2008-01-01

    GRIP domain proteins, locating to the trans-Golgi network, are thought to play an essential role in Golgi apparatus trafficking in yeast and animal cells. In the present study, AtGRIP cDNA was amplified by reverse transcriptase PCR from RNA isolated from Arabidopsis seedling. The GST fusion protein of AtGRIP was affinity-purified and its rabbit polyclonal antibody was obtained. Immuno-blotting with the purified anti-AtGRIP polyclonal antibody demonstrated that the molecular mass of AtGRIP protein is about 92 kD, and its expression is not tissue-specific in Arabidopsis. Immunoflourescent labeling and confocal microscopy revealed that the AtGRIP protein was co-localized with Golgi stacks in Arabidop-sis root cells. Immuno-gold labeling and electron microscopy observation showed that AtGRIP protein was mainly located to the membrane of the secretory vesicles of trans-Golgi network in Arabidopsis root cap cells. Taken together, these results indicate that the localization of GRIP domain proteins be-tween plants and animal cells are conserved. These results also suggest that the AtGRIP may be in-volved in regulating the formation or sorting of Golgi-associated vesicles in plant cells.

  2. Hydrogen Peroxide-induced Cell Death in Arabidopsis : Transcriptional and Mutant Analysis Reveals a Role of an Oxoglutarate-dependent Dioxygenase Gene in the Cell Death Process

    NARCIS (Netherlands)

    Gechev, Tsanko S.; Minkov, Ivan N.; Hille, Jacques

    2005-01-01

    Hydrogen peroxide is a major regulator of plant programmed cell death (PCD) but little is known about the downstream genes from the H2O2-signaling network that mediate the cell death. To address this question, a novel system for studying H2O2-induced programmed cell death in Arabidopsis thaliana was

  3. Microtubules Are Essential for Guard-Cell Function in Vicia and Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    William Eisinger; David Ehrhardt; Winslow Briggs

    2012-01-01

    Radially arranged cortical microtubules are a prominent feature of guard cells.Guard cells expressing GFPtubulin showed consistent changes in the appearance of microtubules when stomata opened or closed.Guard cells showed fewer microtubule structures as stomata closed,whether induced by transfer to darkness,ABA,hydrogen peroxide,or sodium hydrogen carbonate.Guard cells kept in the dark (closed stomata) showed increases in microtubule structures and stomatal aperture on light treatment.GFP-EB1,marking microtubule growing plus ends,showed no change in number of plus ends or velocity of assembly on stomatal closure.Since the number of growing plus ends and the rate of plus-end growth did not change when microtubule structure numbers declined,microtubule instability and/or rearrangement must be responsible for the apparent loss of microtubules.Guard cells with closed stomata showed more cytosolic GFP-fluorescence than those with open stomata as cortical microtubules became disassembled,although with a large net loss in total fluorescence.Microtubule-targeted drugs blocked guard-cell function in Vicia and Arabidopsis.Oryzalin disrupted guard-cell microtubules and prevented stomatal opening and taxol stabilized guard-cell microtubules and delayed stomatal closure.Gas exchange measurements indicated that the transgenes for fluorescent-labeled proteins did not disrupt normal stomatal function.These dynamic changes in guard-cell microtubules combined with our inhibitor studies provide evidence for an active role of microtubules in guard-cell function.

  4. A feedback mechanism controlling SCRAMBLED receptor accumulation and cell-type pattern in Arabidopsis.

    Science.gov (United States)

    Kwak, Su-Hwan; Schiefelbein, John

    2008-12-23

    Cellular pattern formation in the root epidermis of Arabidopsis occurs in a position-dependent manner, generating root-hair (H) cells contacting two underlying cortical cells and nonhair (N) cells contacting one cortical cell. SCRAMBLED (SCM), a leucine-rich repeat receptor-like kinase (LRR-RLK), mediates this process through its effect on a downstream transcription factor regulatory network. After perception of a positional cue, the SCM signaling pathway is proposed to preferentially repress WEREWOLF (WER) transcription factor expression in H cells and thereby bias the outcome of mutual lateral inhibition acting between H and N cells. However, the molecular mechanism responsible for this preferential SCM signaling is unknown. Here, we analyze the distribution of the SCM receptor and the biological effect of altering its accumulation pattern. We find that SCM expression and accumulation in the epidermal cell layer is necessary and sufficient to direct the cell-type pattern. Further, SCM preferentially accumulates in H cells, and this accumulation pattern is dependent on the downstream transcription factors. Thus, SCM participates in an autoregulatory feedback loop, enabling cells engaged in SCM signaling to maintain high levels of SCM receptor, which provides a simple mechanism for reinforcing a bias in receptor-mediated signaling to ensure robust pattern formation.

  5. Trueness-to-type and agronomic characteristics of Coffea arabica trees micropropagated by the embryogenic cell suspension technique.

    Science.gov (United States)

    Etienne, H; Bertrand, B

    2001-09-01

    Trueness-to-type and agronomic characteristics of trees of four coffee (Coffea arabica L.) F(1) hybrid clones derived from embryogenic cell suspensions were compared with those of trees produced from in vitro microcuttings. Three types of variants were observed among the 644 trees derived from embryogenic suspensions. Total frequency of the variants was 2.1% for trees originating from embryogenic cell suspensions, whereas no variant was found among the trees produced from microcuttings. The variant known as "thick leaf" had thick leaves, many abnormally starry flowers and low yields of large fruit. The "dwarf" variant was characterized by slow growth and small fruit. The "dwarf peaberry" variant had abnormal seeds in a single cavity, in addition to the "thick leaf" and "dwarf" characteristics. Compared with normal trees, the variants differed in leaf density and number of chloroplasts per guard cell. The variants aside, there were no differences in the main agronomic characteristics between trees produced from embryogenic suspensions and those produced from microcuttings. For all four clones, the trees had vegetative characteristics, productivity, fertility, and bean biochemical, mineral and organoleptic characteristics that were identical to those of the controls. We conclude that it is possible to generate coffee trees commercially with normal agronomic performance from embryogenic suspensions, because the frequency with which somaclonal variants occur is limited.

  6. Maintenance of undifferentiated mouse embryonic stem cells in suspension by the serum- and feeder-free defined culture condition

    OpenAIRE

    Tsuji, Yukiiko; Yoshimura, Naoko; Aoki, Hitomi; Sharov, Alexei A; Minoru S.H. Ko; Motohashi, Tsutomu; KUNISADA, Takahiro

    2008-01-01

    The proven pluripotency of ES cells is expected to allow their therapeutic use for regenerative medicine. We present here a novel suspension culture method that facilitates the proliferation of pluripotent ES cells without feeder cells. The culture medium contains polyvinyl alcohol (PVA), free of either animal-derived or synthetic serum, and contains very low amounts of peptidic or proteinaceous materials, which are favorable for therapeutic use. ES cells showed sustained proliferation in the...

  7. A lipochito-oligosaccharide, Nod factor, induces transient calcium influx in soybean suspension-cultured cells.

    Science.gov (United States)

    Yokoyama, T; Kobayashi, N; Kouchi, H; Minamisawa, K; Kaku, H; Tsuchiya, K

    2000-04-01

    Lipochito-oligosaccharides (Nod factors) produced by Rhizobium or Bradyrhizobium are the key signal molecules for eliciting nodulation in their corresponding host legumes. To elucidate the signal transduction events mediated by Nod factors, we investigated the effects of Nod factors on the cytosolic [Ca2+] of protoplasts prepared from roots and suspension-cultured cells of soybean (Glycine max and G. soja) using a fluorescent Ca2+ indicator, Fura-PE3. NodBj-V (C18:1, MeFuc), which is a major component of Nod factors produced by Bradyrhizobium japonicum, induces transient elevation of cytosolic [Ca2+] in the cells of soybean within a few minutes. This effect is specific to soybean cells and was not observed in the tobacco BY-2 cells. Furthermore, NodBj-V without MeFuc did not induce any cytosolic [Ca2+] elevation in soybean cells. Exclusion of Ca2+ from the medium, as well as pre-treatment of the cells with an external Ca2+ chelator or with a plasma membrane voltage-dependent Ca2+ channel inhibitor, suppressed the Nod factor-dependent cytosolic [Ca2+] elevation. These results indicate that transient Ca2+ influx from extracellular fluid is one of the earliest responses of soybean cells to NodBj-V (C18:1, MeFuc) in a host-specific manner.

  8. Influences of Plant Growth Regulators,Basal Media and Carbohydrate Levels on Cell Suspension Culture of Panax ginseng

    Institute of Scientific and Technical Information of China (English)

    TangWei; WuJiongyuan; 等

    1995-01-01

    A cell suspension culture of Panax ginseng which may be continuously subcultured has been established.Embryogenic callus derived from clutured young leaves was used to initiate the culture,Plant growth regulators,basal medium formula and carbohydrate levels were examined to determine their various effects on suspension culture cell growth and development ,The best selection of plant growth regulator,basal medium and carbohydrate level is 2mg/L 2,4-D:0.5mg/L KT,MS and 3% sucrose respectively.

  9. Suspension cell culture as a tool for the characterization of class III peroxidases in sugarcane.

    Science.gov (United States)

    Cesarino, Igor; Araújo, Pedro; Paes Leme, Adriana Franco; Creste, Silvana; Mazzafera, Paulo

    2013-01-01

    Secreted class III peroxidases (EC 1.11.1.7) are implicated in a broad range of physiological processes throughout the plant life cycle. However, the unambiguous determination of the precise biological role of an individual class III peroxidase isoenzyme is still a difficult task due to genetic redundancy and broad substrate specificity in vitro. In addition, many difficulties are encountered during extraction and analysis of cell wall proteins. Since class III peroxidases are also secreted into the apoplast, the use of suspension cell cultures can facilitate isolation and functional characterization of individual isoforms. Here, we report on the characterization of class III peroxidases secreted in the spent medium of sugarcane suspension cell cultures. After treatment with specific inducers of cell wall lignification, peroxidases were isolated and activities assayed with guaiacol, syringaldazine and coniferyl alcohol. Enzymatic activity was not significantly different after treatments, regardless of the substrate, with the exception of methyl-jasmonate treatment, which led to a decreased guaiacol peroxidase activity. Remarkably, peroxidases isolated from the medium were capable of oxidizing syringaldazine, an analog to sinapyl alcohol, suggesting that sugarcane cultures can produce peroxidases putatively correlated to lignification. A proteomic approach using activity staining of 2-DE gels revealed a complex isoperoxidase profile, composed predominantly of cationic isoforms. Individual spots were excised and analyzed by LC-ESI-Q-TOF and homology-based search against the Sugarcane EST Database resulted in the identification of several proteins. Spatio-temporal expression pattern of selected genes was determined for validation of identified class III peroxidases that were preferentially expressed during sugarcane stem development.

  10. Involvement of sphingoid bases in mediating reactive oxygen intermediate production and programmed cell death in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Lihua Shi; Yusuf A Hannun; Jianru Zuo; Jacek Bielawski; Jinye Mu; Haili Dong; Chong Teng; Jian Zhang; Xiaohui Yang; Nario Tomishige; Kentaro Hanada

    2007-01-01

    Sphingolipids have been suggested to act as second messengers for an array of cellular signaling activities in plant cells, including stress responses and programmed cell death (PCD). However, the mechanisms underpinning these processes are not well understood. Here, we report that an Arabidopsis mutant, fumonisin Bl resistant11-1 (fbr11-1), which fails to generate reactive oxygen intermediates (ROIs), is incapable of initiating PCD when the mutant is challenged by fumonisin B1 (FB1), a specific inhibitor of ceramide synthase. Molecular analysis indicated that FBR11 encodes a long-chain basel (LCB1) subunit of serine palmitoyltransferase (SPT), which catalyzes the first rate-limiting step of de novo sphingolipid synthesis. Mass spectrometric analysis of the sphingolipid concentrations revealed that whereas the fbrll-1 mutation did not affect basal levels of sphingoid bases, the mutant showed attenuated formation of sphingoid bases in response to FB1 By a direct feeding experiment, we show that the free sphingoid bases dihydrosphingosine, phytosphingosine and sphingosine efficiently induce ROI generation followed by cell death. Conversely, ROI generation and cell death induced by dihydrosphingosine were specifically blocked by its phosphorylated form dihydrosphingosine-1 -phosphate in a dose-dependent manner, suggesting that the maintenance of homeostasis between a free sphingoid base and its phosphorylated derivative is critical to determining the cell fate. Because alterations of the sphingolipid level occur prior to the ROI production, we propose that the free sphingoid bases are involved in the control of PCD in Arabidopsis, presumably through the regulation of the ROI level upon receiving different developmental or environmental cues.

  11. Dual Role of Hydrogen Peroxide in Arabidopsis Guard Cells in Response to Sulfur Dioxide

    Directory of Open Access Journals (Sweden)

    Huilan Yi

    2014-01-01

    Full Text Available Sulfur dioxide (SO2 is a major air pollutant and has significant impacts on plant physiology. Plant can adapt to SO2 stress by controlling stomatal movement, gene expression, and metabolic changes. Here we show clear evidences that SO2-triggered hydrogen peroxide (H2O2 production mediated stomatal closure and cell death in Arabidopsis leaves. High levels of SO2 caused irreversible stomatal closure and decline in guard cell viability, but low levels of SO2 caused reversible stomatal closure. Exogenous antioxidants ascorbic acid (AsA and catalase (CAT or Ca2+ antagonists EGTA and LaCl3 blocked SO2-induced stomatal closure and decline in viability. AsA and CAT also blocked SO2-induced H2O2 and [Ca2+]cyt elevation. However, EGTA and LaCl3 inhibited SO2-induced [Ca2+]cyt increase but did not suppress SO2-induced H2O2 elevation. These results indicate that H2O2 elevation triggered stomatal closure and cell death via [Ca2+]cyt signaling in SO2-stimulated Arabidopsis guard cells. NADPH oxidase inhibitor DPI blocked SO2-induced cell death but not the stomatal closure triggered by low levels of SO2, indicating that NADPH oxidase-dependent H2O2 production plays critical role in SO2 toxicity but is not necessary for SO2-induced stomatal closure. Our results suggest that H2O2 production and accumulation in SO2-stimulated plants trigger plant adaptation and toxicity via reactive oxygen species mediating Ca2+ signaling.

  12. Transcriptional characteristics and differences in Arabidopsis stigmatic papilla cells pre- and post-pollination.

    Science.gov (United States)

    Matsuda, Tomoki; Matsushima, Mai; Nabemoto, Moe; Osaka, Masaaki; Sakazono, Satomi; Masuko-Suzuki, Hiromi; Takahashi, Hirokazu; Nakazono, Mikio; Iwano, Megumi; Takayama, Seiji; Shimizu, Kentaro K; Okumura, Katsuzumi; Suzuki, Go; Watanabe, Masao; Suwabe, Keita

    2015-04-01

    Pollination is an important early step in sexual plant reproduction. In Arabidopsis thaliana, sequential pollination events, from pollen adhesion onto the stigma surface to pollen tube germination and elongation, occur on the stigmatic papilla cells. Following successful completion of these events, the pollen tube penetrates the stigma and finally fertilizes a female gametophyte. The pollination events are thought to be initiated and regulated by interactions between papilla cells and pollen. Here, we report the characterization of gene expression profiles of unpollinated (UP), compatible pollinated (CP) and incompatible pollinated (IP) papilla cells in A. thaliana. Based on cell type-specific transcriptome analysis from a combination of laser microdissection and RNA sequencing, 15,475, 17,360 and 16,918 genes were identified as expressed in UP, CP and IP papilla cells, respectively, and, of these, 14,392 genes were present in all three data sets. Differentially expressed gene (DEG) analyses identified 147 and 71 genes up-regulated in CP and IP papilla cells, respectively, and 115 and 46 genes down-regulated. Gene Ontology and metabolic pathway analyses revealed that papilla cells play an active role as the female reproductive component in pollination, particularly in information exchange, signal transduction, internal physiological changes and external morphological modification. This study provides fundamental information on the molecular mechanisms involved in pollination in papilla cells, furthering our understanding of the reproductive role of papilla cells.

  13. MIRO1 influences the morphology and intracellular distribution of mitochondria during embryonic cell division in Arabidopsis.

    Science.gov (United States)

    Yamaoka, Shohei; Nakajima, Masaki; Fujimoto, Masaru; Tsutsumi, Nobuhiro

    2011-02-01

    Regulating the morphology and intracellular distribution of mitochondria is essential for embryo development in animals. However, the importance of such regulation is not clearly defined in plants. The evolutionarily conserved Miro proteins are known to be involved in the regulation of mitochondrial morphology and motility. We previously demonstrated that MIRO1, an Arabidopsis thaliana orthologue of the Miro protein, is required for embryogenesis. An insertional mutation in the MIRO1 gene causes arrest of embryonic cell division, leading to abortion of the embryo at an early stage. Here we investigated the role of MIRO1 in the regulation of mitochondrial behaviour in egg cells and early-stage embryos using GFP-labeled mitochondria. Two-photon laser scanning microscopy revealed that, in miro1 mutant egg cells, mitochondria are abnormally enlarged, although egg cell formation is nearly unaffected. After fertilization and subsequent zygotic cell division, the homozygous miro1 mutant two-celled embryo contained a significantly reduced number of mitochondria in its apical cell compared with the wild type, suggesting that the miro1 mutation inhibits proper intracellular distribution of mitochondria, leading to an arrest of embryonic cell division. Our findings suggest that proper mitochondrial morphology and intracellular distribution are maintained by MIRO1 and are vital for embryonic cell division.

  14. CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis.

    Science.gov (United States)

    Collins, Carl; Maruthi, N M; Jahn, Courtney E

    2015-08-01

    A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants.

  15. The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells.

    Science.gov (United States)

    Bauer, W D; Talmadge, K W; Keegstra, K; Albersheim, P

    1973-01-01

    The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 m urea, endoglucanase, or 0.5 n NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the sycamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The structure of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of beta-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.

  16. Biosynthesis of sterols and triterpenes in cell suspension cultures of Uncaria tomentosa.

    Science.gov (United States)

    Flores-Sánchez, Isvett J; Ortega-López, Jaime; del Carmen Montes-Horcasitas, María; Ramos-Valdivia, Ana C

    2002-12-01

    Pectin administered to Uncaria tomentosa cell suspension cultures, was found to increase the production of triterpene acids (ursolic and oleanolic acid), however, neither growth nor sterol accumulation were affected. Cell cultures showed that pectin treatment caused a rapid threefold increase in the activities of enzymes involved in the biosynthesis of C(5) and C(30 )isoprenoid, such as isopentenyl diphosphate isomerase and squalene synthase. The activity of a farnesyl diphosphatase, which could divert the flux of farnesyl diphosphate to farnesol, was two times lower in elicited than in control cells. Elicited cells also transformed more rapidly a higher percentage of [5-(3)H]mevalonic acid into triterpene acids. Interestingly, addition of terbinafine, an inhibitor of squalene epoxidase, to elicited cell cultures inhibited sterol accumulation while triterpene production was not inhibited. These results suggest that in U. tomentosa cells, both the previously mentioned enzymes and those involved in squalene 2,3-oxide formation play an important regulatory role in the biosynthesis of sterols and triterpenes.

  17. Assessment of the electrochemical effects of pulsed electric fields in a biological cell suspension.

    Science.gov (United States)

    Chafai, Djamel Eddine; Mehle, Andraž; Tilmatine, Amar; Maouche, Bachir; Miklavčič, Damijan

    2015-12-01

    Electroporation of cells is successfully used in biology, biotechnology and medicine. Practical problems still arise in the electroporation of cells in suspension. For example, the determination of cell electroporation is still a demanding and time-consuming task. Electric pulses also cause contamination of the solution by the metal released from the electrodes and create local enhancements of the electric field, leading to the occurrence of electrochemical reactions at the electrode/electrolyte interface. In our study, we investigated the possibility of assessing modifications to the cell environment caused by pulsed electric fields using electrochemical impedance spectroscopy. We designed an experimental protocol to elucidate the mechanism by which a pulsed electric field affects the electrode state in relation to different electrolyte conductivities at the interface. The results show that a pulsed electric field affects electrodes and its degree depends on the electrolyte conductivity. Evolution of the electrochemical reaction rate depends on the initial free charges and those generated by the pulsed electric field. In the presence of biological cells, the initial free charges in the medium are reduced. The electrical current path at low frequency is longer, i.e., conductivity is decreased, even in the presence of increased permeability of the cell membrane created by the pulsed electric field.

  18. Simultaneous reduction of nitrate and selenate by cell suspensions of selenium-respiring bacteria

    Science.gov (United States)

    Oremland, R.S.; Blum, J.S.; Bindi, A.B.; Dowdle, P.R.; Herbel, M.; Stolz, J.F.

    1999-01-01

    Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was ~11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate- grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high- affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with 75Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.

  19. Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis.

    Science.gov (United States)

    Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

    2014-06-23

    Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites.

  20. Expression of the Arabidopsis high-affinity hexose transporter STP13 correlates with programmed cell death.

    Science.gov (United States)

    Norholm, Morten H H; Nour-Eldin, Hussam H; Brodersen, Peter; Mundy, John; Halkier, Barbara A

    2006-04-17

    We report the biochemical characterization in Xenopus oocytes of the Arabidopsis thaliana membrane protein, STP13, as a high affinity, hexose-specific H(+)-symporter. Studies with kinase activators suggest that it is negatively regulated by phosphorylation. STP13 promoter GFP reporter lines show GFP expression only in the vascular tissue in emerging petals under non-stressed conditions. Quantitative PCR and the pSTP13-GFP plants show induction of STP13 in programmed cell death (PCD) obtained by treatments with the fungal toxin fumonisin B1 and the pathogen Pseudomonas syringae. A role for STP13 in PCD is supported by microarray data from e.g. plants undergoing senescence and a strong correlation between STP13 transcripts and the PCD phenotype in different accelerated cell death (acd11) mutants.

  1. Substitution of L-fucose by L-galactose in cell walls of arabidopsis mur1

    Energy Technology Data Exchange (ETDEWEB)

    Zablackis, E.; York, W.S.; Pauly, M. [Univ. of Georgia, Athens (United States)

    1996-06-21

    An Arabidopsis thaliana mutant (mur1) has less than 2 percent of the normal amounts of L-fucose in the primary cell walls of aerial portions of the plant. The survival of mur1 plants challenged the hypothesis that fucose is a required component of biologically active oligosaccharides derived from cell wall xyloglucan. However, the replacement of L-fucose (that is, 6-deoxyl-L-galactose) by L-galactose does not detectably alter the biological activity of the oligosaccharides derived from xyloglucan. Thus, essential structural and conformational features of xyloglucan and xyloglucan-derived oligosaccharides are retained when L-galactose replaces L-fucose. 29 refs., 2 figs., 2 tabs.

  2. Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells.

    Science.gov (United States)

    Stevenson, T T; McNeil, M; Darvill, A G; Albersheim, P

    1986-04-01

    The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified alpha-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.

  3. An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus.

    Science.gov (United States)

    Dutt, M; Grosser, J W

    2010-11-01

    A method for the genetic transformation of several citrus cultivars is described, including cultivars observed to be recalcitrant to conventional epicotyl-mediated transformation. Embryogenic cell suspension cultures, established from unfertilized ovules were used as target tissues for Agrobacterium-mediated transformation. Several modifications were made to the culture environment to investigate factors required for efficient transfer of the T-DNA and the subsequent regeneration of transgenic citrus plants. It was determined that co-cultivation of citrus cells and Agrobacterium in EME medium supplemented with maltose (EME-M) and 100 μM acetosyringone for 5 days at 25°C was optimum for transformation of each of the citrus cultivars. Efficient selection was obtained and escapes were prevented when the antibiotic hygromycin B was used as a selection antibiotic following transformation with an Agrobacterium strain containing hptII in the T-DNA region. Transgenic embryo regeneration and development was enhanced in medium that contained a liquid overlay consisting of a 1:2 mixture of 0.6 M BH3 and 0.15 M EME-M media. PCR and Southern blot analyses confirmed the presence of the T-DNA and the stable integration into the genome of regenerated plants, while RT-PCR demonstrated variable amounts of RNA being transcribed in different transgenic lines. This protocol can create an avenue for insertion of useful traits into any polyembryonic citrus cultivar that can be established as embryogenic cell suspension cultures, including popular specialty mandarins and seedless cultivars.

  4. Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation.

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    Full Text Available BACKGROUND: Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited. METHODOLOGY/PRINCIPAL FINDINGS: We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins. CONCLUSION/SIGNIFICANCE: These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.

  5. Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells.

    Science.gov (United States)

    Moore, P J; Darvill, A G; Albersheim, P; Staehelin, L A

    1986-11-01

    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.

  6. Involvement of C2H2 zinc finger proteins in the regulation of epidermal cell fate determination in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    An Yan; Minjie Wu; Yongqin Zhao; Aidong Zhang; Bohan Liu; John Schiefelbein; Yinbo Gan

    2014-01-01

    Cell fate determination is a basic developmental process during the growth of multicellular organisms. Trichomes and root hairs of Arabidopsis are both readily accessible structures originating from the epidermal cells of the aerial tissues and roots respectively, and they serve as excellent models for understanding the molecular mecha-nisms controlling cell fate determination and cell morphogen-esis. The regulation of trichome and root hair formation is a complex program that consists of the integration of hormonal signals with a large number of transcriptional factors, including MYB and bHLH transcriptional factors. Studies during recent years have uncovered an important role of C2H2 type zinc finger proteins in the regulation of epidermal cell fate determination. Here in this minireview we briefly summarize the involvement of C2H2 zinc finger proteins in the control of trichome and root hair formation in Arabidopsis.

  7. Optimization of lycopene extraction from tomato cell suspension culture by response surface methodology.

    Science.gov (United States)

    Lu, Chi-Hua; Engelmann, Nancy J; Lila, Mary Ann; Erdman, John W

    2008-09-10

    Radioisotope-labeled lycopene is an important tool for biomedical research but currently is not commercially available. A tomato cell suspension culture system for the production of radioisotope-labeled lycopene was previously developed in our laboratory. In the current study, the goal was to optimize the lycopene extraction efficiency from tomato cell cultures for preparatory high-performance liquid chromatography (HPLC) separation. We employed response surface methodology (RSM), which combines fractional factorial design and a second-degree polynomial model. Tomato cells were homogenized with ethanol, saponified by KOH, and extracted with hexane, and the lycopene content was analyzed by HPLC-PDA. We varied five factors at five levels: ethanol volume (1.33-4 mL/g); homogenization period (0-40 s/g); saturated KOH solution volume (0-0.67 mL/g); hexane volume (1.67-3 mL/g); and vortex period (5-25 s/g). Ridge analysis by SAS suggested that the optimal extraction procedure to extract 1 g of tomato cells was at 1.56 mL of ethanol, 28 s homogenization, 0.29 mL of KOH, 2.49 mL of hexane, and 17.5 s vortex. These optimal conditions predicted by RSM were confirmed to enhance lycopene yield from standardized tomato cell cultures by more than 3-fold.

  8. Serum-free spheroid suspension culture maintains high proliferation and differentiation potentials of mesenchymal stem cells

    Science.gov (United States)

    Alimperti, Stella; Wen, Yuan; Lei, Pedro; Tian, Jun; Campbell, Andrew; Andreadis, Stelios T.

    2016-01-01

    There have been many clinical trials recently using ex vivo-expanded human mesenchymal stem cells (MSCs) to treat several indications such as graft-versus-host disease, acute myocardial infarction, Crohn’s disease, and multiple sclerosis. However, the conventional 2-dimensional (2D) culture of MSCs is laborious and limited in scale potential. The large dosage requirement for many of the indications further exacerbates this manufacturing challenge. In contrast, spheroid MSC culture does not require a cell attachment surface and is amenable to large-scale suspension cell culture techniques, such as stirred-tank bioreactors. In this present study, we developed and optimized serum free media for culturing MSC spheroids. We used Design of Experiment (DoE)-based strategies to systematically evaluate media mixtures and a panel of different components. The optimization yielded two prototype media that could allow MSCs to form aggregates and proliferate in both static cultures and dynamic cultures. The expanded MSCs expressed the expected surface markers for mesenchymal cells (CD73, CD90 and CD105). In addition, the expanded cells demonstrated multipotency and differentiated to the osteocyte, chondrocyte and adipocyte lineages, which showed similar or enhanced differentiation levels compared with serum-containing adherent cultures. PMID:24616445

  9. Apoptosis Induced by High Concentrations of Nicotinamide in Tobacco Suspension Cells

    Institute of Scientific and Technical Information of China (English)

    张贵友; 朱瑞宇; 戴尧仁

    2004-01-01

    As an inhibitor of poly(ADP-ribose) polymerase (PARP), nicotinamide has a restraining effect on apoptosis at certain low concentrations. In our present study, apoptosis induced by high concentrations of nicotinamide was observed in tobacco suspension cells. When cells were preincubated with 250 mmol/L nicotinamide for 24 h, the hallmarks of apoptosis were detected, including DNA fragments increasing in size by multiples of 180-200 bp, the condensation and peripheral distribution of nuclear chromatin, and a positive reaction to the TUNEL assay. At the same time, the degradation of PARP and the reduction in the potential of the inner membrane of mitochondria appeared in apoptotic cells induced by high concentrations of nicotinamide. This result indicates that apoptosis induced by high concentrations of nicotinamide is associated with caspase-3-like activity and with the opening of mitochondrial permeability pores. These results partially support the hypothesis that high concentrations of PARP inhibitor could force cells to enter an apoptotic pathway by delay of DNA repair in replicating cells.

  10. Induction of linalool as a pharmaceutical and medicinal metabolite via cell suspension culture of cumin (Cuminum cyminum L.).

    Science.gov (United States)

    Kazemi, N; Kahrizi, D; Mansouri, M; Karim, H; Vaziri, S; Zargooshi, J; Khanahmadi, M; Shokrinia, M; Mohammadi, N

    2016-05-30

    Cumin is an important medicinal plant in Iran. Plant cell suspension culture is a method for the production of medicinal and secondary metabolites. The linalool is a plant secondary metabolite that has been recognized as a neuroprotective agent. The purpose of this study was to evaluate the effects of salicylic acid elicitor on induction of linalool in cell suspension culture of cumin. For this purpose, the cumin seeds were prepared, to obtain sterile seedling, were disinfected with sodium hypochlorite and alcohol, and were cultured on MS basal medium. This research was conducted in two separate experiments including callus induction and suspension cultures. Leaf explants were prepared from sterile seedlings and used to produce callus on MS medium supplemented with 1 mg/l NAA and 0.5 mg/l BAP. In order to establish suspension culture, the appropriate calli were transferred to liquid medium. Then cell cultures were treated with elicitors. The effects of elicitor on the production of linalool secondary metabolite and cell viability were assessed by GC-Mass and tetrazolium test respectively. For this purpose, the salicylic acid (at concentrations of 0, 1, 2, 4 and 8 mg/l) was used. The experimental design was a completely randomized design with five treatments and three replications. The results of cell culture and GC-Mass analysis showed that salicylic acid had significant effects on the linalool production (suspension culture experiments was lower than control. Increasing the elicitor concentrations lead to reduction in cell survival. In conclusion it is possible to produce linalool as a secondary metabolite and pharmaceutical agent in cell culture of cumin. It is necessary to determine the best combination of medium and elicitor.

  11. Variable-angle total internal reflection fluorescence microscopy of intact cells of Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Kim Myung K

    2011-09-01

    Full Text Available Abstract Background Total internal reflection fluorescence microscopy (TIRFM is a powerful tool for observing fluorescently labeled molecules on the plasma membrane surface of animal cells. However, the utility of TIRFM in plant cell studies has been limited by the fact that plants have cell walls, thick peripheral layers surrounding the plasma membrane. Recently, a new technique known as variable-angle epifluorescence microscopy (VAEM was developed to circumvent this problem. However, the lack of a detailed analysis of the optical principles underlying VAEM has limited its applications in plant-cell biology. Results Here, we present theoretical and experimental evidence supporting the use of variable-angle TIRFM in observations of intact plant cells. We show that when total internal reflection occurs at the cell wall/cytosol interface with an appropriate angle of incidence, an evanescent wave field of constant depth is produced inside the cytosol. Results of experimental TIRFM observations of the dynamic behaviors of phototropin 1 (a membrane receptor protein and clathrin light chain (a vesicle coat protein support our theoretical analysis. Conclusions These findings demonstrate that variable-angle TIRFM is appropriate for quantitative live imaging of cells in intact tissues of Arabidopsis thaliana.

  12. Rapamycin treatment inhibits CHO cell death in a serum-free suspension culture by autophagy induction.

    Science.gov (United States)

    Lee, Jae Seong; Lee, Gyun Min

    2012-12-01

    Rapamycin, a specific mTOR inhibitor, has been used as a chemical activator in autophagy research both in vitro and in vivo. Recently, autophagy has received attention as an anti-cell death engineering target in addition to apoptosis in the Chinese hamster ovary (CHO) cell engineering field. Here, the effect of rapamycin and the subsequent autophagy induction is investigated on two CHO cell lines, DG44 host and an antibody-producing recombinant CHO (rCHO), in a serum-free suspension culture. In both cell lines, the rapamycin treatment delayed the viability drop and apoptosis induction. In particular, the improved cell viability of the antibody-producing rCHO cell line resulting from the rapamycin treatment led to a 21% increase in the maximum antibody concentration. From observations that a rapamycin derivative, everolimus, demonstrated similar positive effects in both cell lines, but not FK-506, which forms the same complex as rapamycin, but does not inhibit mTOR, it was demonstrated that the positive effects of rapamycin appear to be mTOR-dependent. In addition, the cultivation with rapamycin and/or an autophagy inhibitor, bafilomycin A1, indicated that the autophagy induction is related to the positive effects of rapamycin. The genetic perturbation of the autophagy pathway through the regulation of the expression level of Beclin-1, an important autophagy regulator, resulted in a delayed autophagy induction and apoptosis inhibition in response to the rapamycin treatment in the DG44 host cell line. Taken together, the results obtained in this study imply a positive role for autophagy and predict the usefulness of pro-autophagy engineering in CHO cell cultures.

  13. In vitro production of azadirachtin from cell suspension cultures of Azadirachta indica

    Indian Academy of Sciences (India)

    S Sujanya; B Poornasri Devi; Isha Sai

    2008-03-01

    The present study aimed to elucidate the effect of nutritional alteration on biomass content and azadirachtin production in cell suspensions of the elite neem variety crida-8. Variations in total nitrogen availability in the medium in terms of different ratios of nitrate:ammonium showed that the ratio 4:1 revealed a profound effect, leading to a 1.5-fold increase in the total extracellular azadirachtin production (5.59 mg/l) over the standard MS medium. Reduction in sucrose (15 mg/l) in the medium exhibited a reduction in biomass and absence of azadirachtin, whereas total phosphate reduction raised intracellular azadirachtin production (6.98 mg/l). An altered medium with a nitrate:ammonium ratio of 4:1 coupled with complete elimination of phosphate enhanced biomass by 36% (59.36 g/l).

  14. The WEREWOLF MYB protein directly regulates CAPRICE transcription during cell fate specification in the Arabidopsis root epidermis.

    Science.gov (United States)

    Ryu, Kook Hui; Kang, Yeon Hee; Park, Young-hwan; Hwang, Ildoo; Schiefelbein, John; Lee, Myeong Min

    2005-11-01

    The Arabidopsis root epidermis is composed of two types of cells, hair cells and non-hair cells, and their fate is determined in a position-dependent manner. WEREWOLF (WER), a R2R3 MYB protein, has been shown genetically to function as a master regulator to control both of the epidermal cell fates. To directly test the proposed role of WER in this system, we examined its subcellular localization and defined its transcriptional activation properties. We show that a WER-GFP fusion protein is functional and accumulates in the nucleus of the N-position cells in the Arabidopsis root epidermis, as expected for a transcriptional regulator. We also find that a modified WER protein with a strong activation domain (WER-VP16) promotes the formation of both epidermal cell types, supporting the view that WER specifies both cell fates. In addition, we used the glucocorticoid receptor (GR) inducible system to show that CPC transcription is regulated directly by WER. Using EMSA, we found two WER-binding sites (WBSs; WBSI and WBSII) in the CPC promoter. WER-WBSI binding was confirmed in vivo using the yeast one-hybrid assay. Binding between the WER protein and both WBSs (WBSI and WBSII), and the importance of the two WBSs in CPC promoter activity were confirmed in Arabidopsis. These results provide experimental support for the proposed role of WER as an activator of gene transcription during the specification of both epidermal cell fates.

  15. Atkinesin-13A modulates cell-wall synthesis and cell expansion in Arabidopsis thaliana via the THESEUS1 pathway.

    Directory of Open Access Journals (Sweden)

    Ushio Fujikura

    2014-09-01

    Full Text Available Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion.

  16. Metabolic cycles in primary metabolism of cell suspensions of Daucus carota L. analysed by C-NMR

    NARCIS (Netherlands)

    Krook, J.

    1999-01-01

    In the work described in this thesis, uptake and conversion of sugar by cells of batch-grown suspensions of Daucus carota L. were studied. Invasive techniques (measurements of enzyme activities and sugar and starch levels) and non-invasive techniques ( 13C-NMR) were used to

  17. Oxygen transport and stem cell aggregation in stirred-suspension bioreactor cultures.

    Directory of Open Access Journals (Sweden)

    Jincheng Wu

    Full Text Available Stirred-suspension bioreactors are a promising modality for large-scale culture of 3D aggregates of pluripotent stem cells and their progeny. Yet, cells within these clusters experience limitations in the transfer of factors and particularly O2 which is characterized by low solubility in aqueous media. Cultured stem cells under different O2 levels may exhibit significantly different proliferation, viability and differentiation potential. Here, a transient diffusion-reaction model was built encompassing the size distribution and ultrastructural characteristics of embryonic stem cell (ESC aggregates. The model was coupled to experimental data from bioreactor and static cultures for extracting the effective diffusivity and kinetics of consumption of O2 within mouse (mESC and human ESC (hESC clusters. Under agitation, mESC aggregates exhibited a higher maximum consumption rate than hESC aggregates. Moreover, the reaction-diffusion model was integrated with a population balance equation (PBE for the temporal distribution of ESC clusters changing due to aggregation and cell proliferation. Hypoxia was found to be negligible for ESCs with a smaller radius than 100 µm but became appreciable for aggregates larger than 300 µm. The integrated model not only captured the O2 profile both in the bioreactor bulk and inside ESC aggregates but also led to the calculation of the duration that fractions of cells experience a certain range of O2 concentrations. The approach described in this study can be employed for gaining a deeper understanding of the effects of O2 on the physiology of stem cells organized in 3D structures. Such frameworks can be extended to encompass the spatial and temporal availability of nutrients and differentiation factors and facilitate the design and control of relevant bioprocesses for the production of stem cell therapeutics.

  18. Assessment of cultivation factors that affect biomass and geraniol production in transgenic tobacco cell suspension cultures.

    Directory of Open Access Journals (Sweden)

    Nikolay Vasilev

    Full Text Available A large-scale statistical experimental design was used to determine essential cultivation parameters that affect biomass accumulation and geraniol production in transgenic tobacco (Nicotiana tabacum cv. Samsun NN cell suspension cultures. The carbohydrate source played a major role in determining the geraniol yield and factors such as filling volume, inoculum size and light were less important. Sucrose, filling volume and inoculum size had a positive effect on geraniol yield by boosting growth of plant cell cultures whereas illumination of the cultures stimulated the geraniol biosynthesis. We also found that the carbohydrates sucrose and mannitol showed polarizing effects on biomass and geraniol accumulation. Factors such as shaking frequency, the presence of conditioned medium and solubilizers had minor influence on both plant cell growth and geraniol content. When cells were cultivated under the screened conditions for all the investigated factors, the cultures produced ∼ 5.2 mg/l geraniol after 12 days of cultivation in shaking flasks which is comparable to the yield obtained in microbial expression systems. Our data suggest that industrial experimental designs based on orthogonal arrays are suitable for the selection of initial cultivation parameters prior to the essential medium optimization steps. Such designs are particularly beneficial in the early optimization steps when many factors must be screened, increasing the statistical power of the experiments without increasing the demand on time and resources.

  19. A Study of Noncultured Extracted Hair Follicle Outer Root Sheath Cell Suspension for Transplantation in Vitiligo

    Science.gov (United States)

    Shah, Aarti N; Marfatia, Ritu K; Saikia, Siddhartha S

    2016-01-01

    Context: Vitiligo surgeries have come a long way from tissue grafts to cultured and non cultured cell transplantation. Extracted hair follicle outer root sheath cell transplantation (EHF ORS) suspension is more enriched with melanocyte. In a hair bulb, there is one melanocyte for every five keratinocytes which is much higher than the epidermal melanin unit. Aims: To analyse the effectiveness of cultured EHF ORS and to perform objective evaluation based on clinical improvement & photographic evidence. To observe any untoward events or side effects. Settings and Design: The study was open and uncontrolled. All the patients were screened at preliminary visit. Reviews were done every two weeks. The endpoint selected was six months post procedure. Materials and Methods: Twenty five patients of stable Vitiligo were included in the study and follicular unit were harvested by Follicular Unit Extraction method. Outer root sheath cells were extracted by trypsinization. The solution was transplanted over dermabraded recipient site. Pressure dressing was given. Patients were followed up regularly. Statistical Analysis Used: Descriptive Statistics, Chi-Square. Results: Mean ± SD repigmentation was 80.15% ± 22.9% with excellent repigmentation (90-100%) in 60% of patients. Conclusions: This method is safe, effective, and simpler than the other methods involving cell culturing and requiring a laboratory set-up but selection of patients is crucial for the success of the outcome. PMID:27601859

  20. Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters.

    Science.gov (United States)

    Chu, Mingyu; Quiñonero, Carmen; Akdemir, Hülya; Alburquerque, Nuria; Pedreño, María Ángeles; Burgos, Lorenzo

    2016-05-01

    Although some works have explored the transformation of differentiated, embryogenic suspension-cultured cells (SCC) to produce transgenic grapevine plants, to our knowledge this is one of the first reports on the efficient transformation of dedifferentiated Vitis vinifera cv Monastrell SCC. This protocol has been developed using the sonication-assisted Agrobacterium-mediated transformation (SAAT) method. A construct harboring the selectable nptII and the eyfp/IV2 marker genes was used in the study and transformation efficiencies reached over 50 independent transformed SCC per gram of infected cells. Best results were obtained when cells were infected at the exponential phase. A high density plating (500 mg/dish) gave significantly better results. As selective agent, kanamycin was inefficient for the selection of Monastrell transformed SCC since wild type cells were almost insensitive to this antibiotic whereas application of paromomycin resulted in very effective selection. Selected eyfp-expressing microcalli were grown until enough tissue was available to scale up a new transgenic SCC. These transgenic SCC lines were evaluated molecularly and phenotypically demonstrating the presence and integration of both transgenes, the absence of Agrobacterium contamination and the ability of the transformed SCC to grow in highly selective liquid medium. The methodology described here opens the possibility of improving the production of valuable metabolites. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:725-734, 2016.

  1. Guard cell chloroplasts are essential for blue light-dependent stomatal opening in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Noriyuki Suetsugu

    Full Text Available Blue light (BL induces stomatal opening through the activation of H+-ATPases with subsequent ion accumulation in guard cells. In most plant species, red light (RL enhances BL-dependent stomatal opening. This RL effect is attributable to the chloroplasts of guard cell, the only cells in the epidermis possessing this organelle. To clarify the role of chloroplasts in stomatal regulation, we investigated the effects of RL on BL-dependent stomatal opening in isolated epidermis, guard cell protoplasts, and intact leaves of Arabidopsis thaliana. In isolated epidermal tissues and intact leaves, weak BL superimposed on RL enhanced stomatal opening while BL alone was less effective. In guard cell protoplasts, RL enhanced BL-dependent H+-pumping and DCMU, a photosynthetic electron transport inhibitor, eliminated this effect. RL enhanced phosphorylation levels of the H+-ATPase in response to BL, but this RL effect was not suppressed by DCMU. Furthermore, DCMU inhibited both RL-induced and BL-dependent stomatal opening in intact leaves. The photosynthetic rate in leaves correlated positively with BL-dependent stomatal opening in the presence of DCMU. We conclude that guard cell chloroplasts provide ATP and/or reducing equivalents that fuel BL-dependent stomatal opening, and that they indirectly monitor photosynthetic CO2 fixation in mesophyll chloroplasts by absorbing PAR in the epidermis.

  2. Endocytosis restricts Arabidopsis KNOLLE syntaxin to the cell division plane during late cytokinesis.

    Science.gov (United States)

    Boutté, Yohann; Frescatada-Rosa, Márcia; Men, Shuzhen; Chow, Cheung-Ming; Ebine, Kazuo; Gustavsson, Anna; Johansson, Lenore; Ueda, Takashi; Moore, Ian; Jürgens, Gerd; Grebe, Markus

    2010-02-03

    Cytokinesis represents the final stage of eukaryotic cell division during which the cytoplasm becomes partitioned between daughter cells. The process differs to some extent between animal and plant cells, but proteins of the syntaxin family mediate membrane fusion in the plane of cell division in diverse organisms. How syntaxin localization is kept in check remains elusive. Here, we report that localization of the Arabidopsis KNOLLE syntaxin in the plane of cell division is maintained by sterol-dependent endocytosis involving a clathrin- and DYNAMIN-RELATED PROTEIN1A-dependent mechanism. On genetic or pharmacological interference with endocytosis, KNOLLE mis-localizes to lateral plasma membranes after cell-plate fusion. Fluorescence-loss-in-photo-bleaching and fluorescence-recovery-after-photo-bleaching experiments reveal lateral diffusion of GFP-KNOLLE from the plane of division to lateral membranes. In an endocytosis-defective sterol biosynthesis mutant displaying lateral KNOLLE diffusion, KNOLLE secretory trafficking remains unaffected. Thus, restriction of lateral diffusion by endocytosis may serve to maintain specificity of syntaxin localization during late cytokinesis.

  3. Cell division plane orientation based on tensile stress in Arabidopsis thaliana.

    Science.gov (United States)

    Louveaux, Marion; Julien, Jean-Daniel; Mirabet, Vincent; Boudaoud, Arezki; Hamant, Olivier

    2016-07-26

    Cell geometry has long been proposed to play a key role in the orientation of symmetric cell division planes. In particular, the recently proposed Besson-Dumais rule generalizes Errera's rule and predicts that cells divide along one of the local minima of plane area. However, this rule has been tested only on tissues with rather local spherical shape and homogeneous growth. Here, we tested the application of the Besson-Dumais rule to the divisions occurring in the Arabidopsis shoot apex, which contains domains with anisotropic curvature and differential growth. We found that the Besson-Dumais rule works well in the central part of the apex, but fails to account for cell division planes in the saddle-shaped boundary region. Because curvature anisotropy and differential growth prescribe directional tensile stress in that region, we tested the putative contribution of anisotropic stress fields to cell division plane orientation at the shoot apex. To do so, we compared two division rules: geometrical (new plane along the shortest path) and mechanical (new plane along maximal tension). The mechanical division rule reproduced the enrichment of long planes observed in the boundary region. Experimental perturbation of mechanical stress pattern further supported a contribution of anisotropic tensile stress in division plane orientation. Importantly, simulations of tissues growing in an isotropic stress field, and dividing along maximal tension, provided division plane distributions comparable to those obtained with the geometrical rule. We thus propose that division plane orientation by tensile stress offers a general rule for symmetric cell division in plants.

  4. Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis.

    Science.gov (United States)

    Zhao, Qiao; Zeng, Yining; Yin, Yanbin; Pu, Yunqiao; Jackson, Lisa A; Engle, Nancy L; Martin, Madhavi Z; Tschaplinski, Timothy J; Ding, Shi-You; Ragauskas, Arthur J; Dixon, Richard A

    2015-04-01

    Pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutant of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.

  5. Light-dependent intracellular positioning of mitochondria in Arabidopsis thaliana mesophyll cells.

    Science.gov (United States)

    Islam, Md Sayeedul; Niwa, Yasuo; Takagi, Shingo

    2009-06-01

    Mitochondria, the power house of the cell, are one of the most dynamic cell organelles. Although there are several reports on actin- or microtubule-dependent movement of mitochondria in plant cells, intracellular positioning and motility of mitochondria under different light conditions remain open questions. Mitochondria were visualized in living Arabidopsis thaliana leaf cells using green fluorescent protein fused to a mitochondrion-targeting signal. In darkness, mitochondria were distributed randomly in palisade cells. In contrast, mitochondria accumulated along the periclinal walls, similar to the accumulation response of chloroplasts, when treated with weak blue light (470 nm, 4 micromol m(-2) s(-1)). Under strong blue light (100 micromol m(-2) s(-1)), mitochondria occupied the anticlinal positions similar to the avoidance response of chloroplasts and nuclei. While strong red light (660 nm, 100 micromol m(-2) s(-1)) induced the accumulation of mitochondria along the inner periclinal walls, green light exhibited little effect on the distribution of mitochondria. In addition, the mode of movement of individual mitochondria along the outer periclinal walls under different light conditions was precisely analyzed by time-lapse fluorescence microscopy. A gradual increase in the number of static mitochondria located in the vicinity of chloroplasts with a time period of blue light illumination clearly demonstrated the accumulation response of mitochondria. Light-induced co-localization of mitochondria with chloroplasts strongly suggested their mutual metabolic interactions. This is the first characterization of the light-dependent redistribution of mitochondria in plant cells.

  6. Establishment of Suspension Cell Culture from Agrobacterium-transformed Hairy Root Cells of Psammosilene tunicoides, an Endangered and Rare Medicinal Plant of China

    Directory of Open Access Journals (Sweden)

    Zhang Zong-Shen

    2015-08-01

    Full Text Available Psammosilene tunicoides is an important medicinal plant endemic in China. Its annual yield is severely limited due to slow growth, poor seed germination and excessive collection. To satisfy the growing market demands, it’s necessary to seek alternatives to field cultivation and wild resources of this endangered plant. Using Agrobacterium -transformed hairy roots as initial explants, here, we reported the development of a suspension cell culture system for P. tunicoides. Results showed the Agrobacterium -transformed hairy roots-derived suspension cells are fast in growth and strong in capacity for accumulation of bioactive metabolites. We established that 1/2MS was a suitable medium for culturing the hairy root-derived suspension cells and the optimal combination of phytohormones is 1.5 mg/L 2, 4-D+0.5 mg/L 6-BA+0.25 mg/L NAA+0.1 mg/L KT. Under this condition, the maximal biomass was achieved at the 20th day of culture with an average growth rate of 0.72 g/L/d; and the intracellular saponine content reached 0.92%, comparable to that of mother hairy roots. Compared with the normal P. tunicoides suspension cells, the hairy roots-derived suspension cells exhibited features of fast growth, short culture period and high concentration of saponines, suggesting that the large scale culture of hairy root-derived cells could be a feasible alternative to the wild resources of P. tunicoides.

  7. Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.

  8. Suppression of Arabidopsis peroxidase 72 alters cell wall and phenylpropanoid metabolism.

    Science.gov (United States)

    Fernández-Pérez, Francisco; Pomar, Federico; Pedreño, María A; Novo-Uzal, Esther

    2015-10-01

    Class III peroxidases are glycoproteins with a major role in cell wall maturation such as lignin formation. Peroxidases are usually present in a high number of isoenzymes, which complicates to assign specific functions to individual peroxidase isoenzymes. Arabidopsis genome encodes for 73 peroxidases, among which AtPrx72 has been shown to participate in lignification. Here, we report by using knock out peroxidase mutants how the disruption of AtPrx72 causes thinner secondary walls in interfascicular fibres but not in the xylem of the stem. This effect is also age-dependent, and AtPrx72 function seems to be particularly important when lignification prevails over elongation processes. Finally, the suppression AtPrx72 leads to the down-regulation of lignin biosynthesis pathway, as well as genes and transcription factors involved in secondary wall thickening.

  9. Site of clomazone action in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    1990-10-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [(14)C]mevalonate ([(14)C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [(14)C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [(14)C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase.

  10. METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

    Directory of Open Access Journals (Sweden)

    Sacha Escamez

    2016-02-01

    Full Text Available We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs that undergo programmed cell death (PCD and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9 was reduced using RNAi (MC9-RNAi. Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2 was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells.

  11. A new picture of cell wall protein dynamics in elongating cells of Arabidopsis thaliana: Confirmed actors and newcomers

    Directory of Open Access Journals (Sweden)

    Jamet Elisabeth

    2008-09-01

    Full Text Available Abstract Background Cell elongation in plants requires addition and re-arrangements of cell wall components. Even if some protein families have been shown to play roles in these events, a global picture of proteins present in cell walls of elongating cells is still missing. A proteomic study was performed on etiolated hypocotyls of Arabidopsis used as model of cells undergoing elongation followed by growth arrest within a short time. Results Two developmental stages (active growth and after growth arrest were compared. A new strategy consisting of high performance cation exchange chromatography and mono-dimensional electrophoresis was established for separation of cell wall proteins. This work allowed identification of 137 predicted secreted proteins, among which 51 had not been identified previously. Apart from expected proteins known to be involved in cell wall extension such as xyloglucan endotransglucosylase-hydrolases, expansins, polygalacturonases, pectin methylesterases and peroxidases, new proteins were identified such as proteases, proteins related to lipid metabolism and proteins of unknown function. Conclusion This work highlights the CWP dynamics that takes place between the two developmental stages. The presence of proteins known to be related to cell wall extension after growth arrest showed that these proteins may play other roles in cell walls. Finally, putative regulatory mechanisms of protein biological activity are discussed from this global view of cell wall proteins.

  12. Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells.

    Science.gov (United States)

    Nikolay, Alexander; Castilho, Leda R; Reichl, Udo; Genzel, Yvonne

    2017-03-23

    The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging. Here we present a production platform based on BHK-21 suspension (BHK-21SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1-48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKV(PE)) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21SUS cells with ZIKV(PE) in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×10(3)PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×10(7)cells/mL, and virus titers of 3.9×10(7)PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards

  13. Whole organ, venation and epidermal cell morphological variations are correlated in the leaves of Arabidopsis mutants.

    Science.gov (United States)

    Pérez-Pérez, José Manuel; Rubio-Díaz, Silvia; Dhondt, Stijn; Hernández-Romero, Diana; Sánchez-Soriano, Joaquín; Beemster, Gerrit T S; Ponce, María Rosa; Micol, José Luis

    2011-12-01

    Despite the large number of genes known to affect leaf shape or size, we still have a relatively poor understanding of how leaf morphology is established. For example, little is known about how cell division and cell expansion are controlled and coordinated within a growing leaf to eventually develop into a laminar organ of a definite size. To obtain a global perspective of the cellular basis of variations in leaf morphology at the organ, tissue and cell levels, we studied a collection of 111 non-allelic mutants with abnormally shaped and/or sized leaves, which broadly represent the mutational variations in Arabidopsis thaliana leaf morphology not associated with lethality. We used image-processing techniques on these mutants to quantify morphological parameters running the gamut from the palisade mesophyll and epidermal cells to the venation, whole leaf and rosette levels. We found positive correlations between epidermal cell size and leaf area, which is consistent with long-standing Avery's hypothesis that the epidermis drives leaf growth. In addition, venation parameters were positively correlated with leaf area, suggesting that leaf growth and vein patterning share some genetic controls. Positional cloning of the genes affected by the studied mutations will eventually establish functional links between genotypes, molecular functions, cellular parameters and leaf phenotypes.

  14. Oryzalin-modified disruption of microtubular cytoskeleton in Arabidopsis thaliana root cells under clinorotation

    Science.gov (United States)

    Kalinina, Ia.; Shevchenko, G.; Kordyum, E.

    There are data on gravisensitivity of cells not specialized to perceive a gravity vector but the molecular processes by which gravity affects not graviperceptive cells are still unclear Spaceflight experiments show that the microtubule self-organization in vitro is gravity-dependent Confocal microscopic analysis of the microtubule spatial organization under altered gravity with combination of approach drugs that disrupt normal microtubule behavior should give us a better understanding of the possible role of microtubule cytoskeleton in gravisensing on cellular level With this aim we examined influence of horizontal clinorotation 2 rpm on the spatial organization of microtubules in the root cortical and epidermal cells by means of LSM 5 PASCAL Zeiss Germany Microtubules were visualized by using stably transformed line of transgenic Arabidopsis thaliana expressing a green fluorescent protein-MAP4 fusion protein We inhibited microtubule function applying 5 956 M L oryzalin microtubule inhibitor in control and clinorotated seedlings Preliminary investigations show that cortical microtubule arrays were dense and predominantly transverse to the root long axis in the meristem and distal elongation zone in control and they got oblique direction when rapid cell elongation is finishing In the differentiation zone microtubules reorient with respect to the longitudinal growth axis of cell Under clinorotation cortical microtubules have the same configuration in the meristem central elongation zone and differentiation zone but it is observed appearances of several

  15. MYB98 is required for pollen tube guidance and synergid cell differentiation in Arabidopsis.

    Science.gov (United States)

    Kasahara, Ryushiro D; Portereiko, Michael F; Sandaklie-Nikolova, Linda; Rabiger, David S; Drews, Gary N

    2005-11-01

    The synergid cells of the female gametophyte play a role in many steps of the angiosperm fertilization process, including guidance of pollen tube growth to the female gametophyte. However, the mechanisms by which the synergid cells become specified and develop their unique features during female gametophyte development are not understood. We identified MYB98 in a screen for Arabidopsis thaliana genes expressed in the female gametophyte. MYB98 is a member of the R2R3-MYB gene family, the members of which likely encode transcription factors. In the context of the ovule, MYB98 is expressed exclusively in the synergid cells, and mutations in this gene affect the female gametophyte specifically. myb98 female gametophytes are affected in two unique features of the synergid cell, pollen tube guidance and the filiform apparatus, but are otherwise normal. MYB98 also is expressed in trichomes and endosperm. Homozygous myb98 mutants exhibit no sporophytic defects, including trichome and endosperm defects. Together, these data suggest that MYB98 controls the development of specific features within the synergid cell during female gametophyte development.

  16. MYB98 Is Required for Pollen Tube Guidance and Synergid Cell Differentiation in ArabidopsisW⃞

    Science.gov (United States)

    Kasahara, Ryushiro D.; Portereiko, Michael F.; Sandaklie-Nikolova, Linda; Rabiger, David S.; Drews, Gary N.

    2005-01-01

    The synergid cells of the female gametophyte play a role in many steps of the angiosperm fertilization process, including guidance of pollen tube growth to the female gametophyte. However, the mechanisms by which the synergid cells become specified and develop their unique features during female gametophyte development are not understood. We identified MYB98 in a screen for Arabidopsis thaliana genes expressed in the female gametophyte. MYB98 is a member of the R2R3-MYB gene family, the members of which likely encode transcription factors. In the context of the ovule, MYB98 is expressed exclusively in the synergid cells, and mutations in this gene affect the female gametophyte specifically. myb98 female gametophytes are affected in two unique features of the synergid cell, pollen tube guidance and the filiform apparatus, but are otherwise normal. MYB98 also is expressed in trichomes and endosperm. Homozygous myb98 mutants exhibit no sporophytic defects, including trichome and endosperm defects. Together, these data suggest that MYB98 controls the development of specific features within the synergid cell during female gametophyte development. PMID:16214903

  17. Parabens enable suspension growth of MCF-10A immortalized, non-transformed human breast epithelial cells.

    Science.gov (United States)

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

    Parabens (alkyl esters of p-hydroxybenzoic acid) are used extensively as preservatives in consumer products, and intact esters have been measured in several human tissues. Concerns of a potential link between parabens and breast cancer have been raised, but mechanistic studies have centred on their oestrogenic activity and little attention has been paid to any carcinogenic properties. In the present study, we report that parabens can induce anchorage-independent growth of MCF-10A immortalized but non-transformed human breast epithelial cells, a property closely related to transformation and a predictor of tumour growth in vivo. In semi-solid methocel suspension culture, MCF-10A cells produced very few colonies and only of a small size but the addition of 5 × 10(-4) M methylparaben, 10(-5) M n-propylparaben or 10(-5) M n-butylparaben resulted in a greater number of colonies per dish (P paraben concentrations in human breast tissue samples from 40 mastectomies (Barr et al., 2012) showed that 22/40 of the patients had at least one of the parabens at the site of the primary tumour at or above these concentrations. To our knowledge, this is the first study to report that parabens can induce a transformed phenotype in human breast epithelial cells in vitro, and further investigation is now justified into a potential link between parabens and breast carcinogenesis.

  18. Development of a low capital investment reactor system: application for plant cell suspension culture

    Science.gov (United States)

    Hsiao; Bacani; Carvalho; Curtis

    1999-01-01

    Growth of plant cell cultures is demonstrated in an uncontrolled, simple, and inexpensive plastic-lined vessel. Sustained specific growth rates of 0.22 day-1 for Hyoscyamus muticus cell suspension cultures are achieved in a low-cost gas-sparged bioreactor configuration (6.5 L working volume, wv) which is comparable to an "optimized" 5 L wv mechanically agitated fermentor. In an effort to reduce bioreactor costs, the need for an autoclavable vessel was eliminated. Sterilization is achieved by separate autoclaving of the plastic liner and by gas-phase sterilization using ethylene oxide. The initial run sterilized with ethylene oxide displayed a long lag, apparently due to residual sterilant gas. Because ethylene oxide could eliminate costs associated with autoclave rated vessels, a quantitative basis for aeration time was developed by experimental measurements and modeling of diffusion in the polymer liner. Operational techniques to eliminate toxicity are implemented to grow 0.2 kg dry weight of plant cells in 13 days in a 40 L (28.5 L wv) air-lift bioreactor without autoclave sterilization. The biomass yields for all reactors were statistically indistinguishable from shake flask culture.

  19. Efficient procedure for grapevine embryogenic suspension establishment and plant regeneration: role of conditioned medium for cell proliferation.

    Science.gov (United States)

    Ben Amar, A; Cobanov, P; Boonrod, K; Krczal, G; Bouzid, S; Ghorbel, A; Reustle, G M

    2007-09-01

    An efficient system for the establishment and multiplication of highly prolific embryogenic cell cultures of grapevine (Vitis sp.) was developed. Using anther-derived pro-embryogenic masses as starting material, cell suspensions of different grapevine cultivars (Tempranillo, Cabernet-Sauvignon) and rootstocks (Kober 125 AA, Kober 5 BB, 110 Richter) were initiated in liquid medium containing NOA (1.0 mg l(-1)) and BAP (0.25 mg l(-1)) as growth regulators. Conditioned medium was recovered and utilised for establishing new, highly totipotent cell cultures. The suspensions obtained, showed embryogenic competence resulting in somatic embryo induction and subsequent plant regeneration. In this study, a simplified establishment procedure for grapevine embryogenic cell suspension allowing the fast multiplication of embryogenic material is described. Evidence for the promoting effect of the protein fraction derived from conditioned medium, on cell proliferation was found. In bioassays, addition of ss-D: -GlcY affect cell proliferation suggesting that arabinogalactan proteins are required for growth processes in grapevine cell cultures.

  20. Nitric oxide consumption through lipid peroxidation in brain cell suspensions and homogenates.

    Science.gov (United States)

    Keynes, Robert G; Griffiths, Charmaine H; Hall, Catherine; Garthwaite, John

    2005-05-01

    Mechanisms which inactivate NO (nitric oxide) are probably important in governing the physiological and pathological effects of this ubiquitous signalling molecule. Cells isolated from the cerebellum, a brain region rich in the NO signalling pathway, consume NO avidly. This property was preserved in brain homogenates and required both particulate and supernatant fractions. A purified fraction of the particulate component was rich in phospholipids, and NO consumption was inhibited by procedures that inhibited lipid peroxidation, namely a transition metal chelator, the vitamin E analogue Trolox and ascorbate oxidase. The requirement for the supernatant was accounted for by its content of ascorbate which catalyses metal-dependent lipid peroxidation. The NO-degrading activity of the homogenate was mimicked by a representative mixture of brain lipids together with ascorbate and, under these conditions, the lipids underwent peroxidation. In a suspension of cerebellar cells, there was a continuous low level of lipid peroxidation, and consumption of NO by the cells was decreased by approx. 50% by lipid-peroxidation inhibitors. Lipid peroxidation was also abolished when NO was supplied at a continuously low rate (approximately 100 nM/min), which explains why NO consumption by this process is saturable. Part of the activity remaining after the inhibition of lipid peroxidation was accounted for by contaminating red blood cells, but there was also another component whose activity was greatly enhanced when the cells were maintained under air-equilibrated conditions. A similar NO-consuming process was present in cerebellar glial cells grown in tissue culture but not in blood platelets or leucocytes, suggesting a specialized mechanism.

  1. Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures.

    Science.gov (United States)

    Luna-Palencia, Gabriela R; Cerda-García-Rojas, Carlos M; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2005-01-01

    Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.

  2. Dynamics of defense responses and cell fate change during Arabidopsis-Pseudomonas syringae interactions.

    Directory of Open Access Journals (Sweden)

    Safae Hamdoun

    Full Text Available Plant-pathogen interactions involve sophisticated action and counteraction strategies from both parties. Plants can recognize pathogen derived molecules, such as conserved pathogen associated molecular patterns (PAMPs and effector proteins, and subsequently activate PAMP-triggered immunity (PTI and effector-triggered immunity (ETI, respectively. However, pathogens can evade such recognitions and suppress host immunity with effectors, causing effector-triggered susceptibility (ETS. The differences among PTI, ETS, and ETI have not been completely understood. Toward a better understanding of PTI, ETS, and ETI, we systematically examined various defense-related phenotypes of Arabidopsis infected with different Pseudomonas syringae pv. maculicola ES4326 strains, using the virulence strain DG3 to induce ETS, the avirulence strain DG34 that expresses avrRpm1 (recognized by the resistance protein RPM1 to induce ETI, and HrcC(- that lacks the type three secretion system to activate PTI. We found that plants infected with different strains displayed dynamic differences in the accumulation of the defense signaling molecule salicylic acid, expression of the defense marker gene PR1, cell death formation, and accumulation/localization of the reactive oxygen species, H2O2. The differences between PTI, ETS, and ETI are dependent on the doses of the strains used. These data support the quantitative nature of PTI, ETS, and ETI and they also reveal qualitative differences between PTI, ETS, and ETI. Interestingly, we observed the induction of large cells in the infected leaves, most obviously with HrcC(- at later infection stages. The enlarged cells have increased DNA content, suggesting a possible activation of endoreplication. Consistent with strong induction of abnormal cell growth by HrcC(-, we found that the PTI elicitor flg22 also activates abnormal cell growth, depending on a functional flg22-receptor FLS2. Thus, our study has revealed a comprehensive

  3. UV-B-induced signaling events leading to enhanced-production of catharanthine in Catharanthus roseus cell suspension cultures

    Directory of Open Access Journals (Sweden)

    Chelliah Jayabaskaran

    2007-11-01

    Full Text Available Abstract Background Elicitations are considered to be an important strategy towards improved in vitro production of secondary metabolites. In cell cultures, biotic and abiotic elicitors have effectively stimulated the production of plant secondary metabolites. However, molecular basis of elicitor-signaling cascades leading to increased production of secondary metabolites of plant cell is largely unknown. Exposure of Catharanthus roseus cell suspension culture to low dose of UV-B irradiation was found to increase the amount of catharanthine and transcription of genes encoding tryptophan decarboxylase (Tdc and strictosidine synthase (Str. In the present study, the signaling pathway mediating UV-B-induced catharanthine accumulation in C. roseus suspension cultures were investigated. Results Here, we investigate whether cell surface receptors, medium alkalinization, Ca2+ influx, H2O2, CDPK and MAPK play required roles in UV-B signaling leading to enhanced production of catharanthine in C. roseus cell suspension cultures. C. roseus cells were pretreated with various agonists and inhibitors of known signaling components and their effects on the accumulation of Tdc and Str transcripts as well as amount of catharanthine production were investigated by various molecular biology techniques. It has been found that the catharanthine accumulation and transcription of Tdc and Str were inhibited by 3–4 fold upon pretreatment of various inhibitors like suramin, N-acetyl cysteine, inhibitors of calcium fluxes, staurosporine etc. Conclusion Our results demonstrate that cell surface receptor(s, Ca2+ influx, medium alkalinization, CDPK, H2O2 and MAPK play significant roles in UV-B signaling leading to stimulation of Tdc and Str genes and the accumulation of catharanthine in C. roseus cell suspension cultures. Based on these findings, a model for signal transduction cascade has been proposed.

  4. Three-dimensional patterns of cell division and expansion throughout the development of Arabidopsis thaliana leaves.

    Science.gov (United States)

    Kalve, Shweta; Fotschki, Joanna; Beeckman, Tom; Vissenberg, Kris; Beemster, Gerrit T S

    2014-12-01

    Variations in size and shape of multicellular organs depend on spatio-temporal regulation of cell division and expansion. Here, cell division and expansion rates were quantified relative to the three spatial axes in the first leaf pair of Arabidopsis thaliana. The results show striking differences in expansion rates: the expansion rate in the petiole is higher than in the leaf blade; expansion rates in the lateral direction are higher than longitudinal rates between 5 and 10 days after stratification, but become equal at later stages of leaf blade development; and anticlinal expansion co-occurs with, but is an order of magnitude slower than periclinal expansion. Anticlinal expansion rates also differed greatly between tissues: the highest rates occurred in the spongy mesophyll and the lowest in the epidermis. Cell division rates were higher and continued for longer in the epidermis compared with the palisade mesophyll, causing a larger increase of palisade than epidermal cell area over the course of leaf development. The cellular dynamics underlying the effect of shading on petiole length and leaf thickness were then investigated. Low light reduced leaf expansion rates, which was partly compensated by increased duration of the growth phase. Inversely, shading enhanced expansion rates in the petiole, so that the blade to petiole ratio was reduced by 50%. Low light reduced leaf thickness by inhibiting anticlinal cell expansion rates. This effect on cell expansion was preceded by an effect on cell division, leading to one less layer of palisade cells. The two effects could be uncoupled by shifting plants to contrasting light conditions immediately after germination. This extended kinematic analysis maps the spatial and temporal heterogeneity of cell division and expansion, providing a framework for further research to understand the molecular regulatory mechanisms involved.

  5. Proteomic identification of S-nitrosylated proteins in Arabidopsis

    DEFF Research Database (Denmark)

    Lindermayr, C.; Saalbach, G.; Durner, J.

    2005-01-01

    purified and analyzed using nano liquid chromatography in combination with mass spectrometry. We identified 63 proteins from cell cultures and 52 proteins from leaves that represent candidates for S-nitrosylation, including stress-related, redox-related, signaling/regulating, cytoskeleton, and metabolic......Although nitric oxide (NO) has grown into a key signaling molecule in plants during the last few years, less is known about how NO regulates different events in plants. Analyses of NO-dependent processes in animal systems have demonstrated protein S-nitrosylation of cysteine (Cys) residues...... to be one of the dominant regulation mechanisms for many animal proteins. For plants, the principle of S-nitrosylation remained to be elucidated. We generated S-nitrosothiols by treating extracts from Arabidopsis (Arabidopsis thaliana) cell suspension cultures with the NO-donor S...

  6. Detection of Changes in the Medicago sativa Retinoblastoma-Related Protein (MsRBR1) Phosphorylation During Cell Cycle Progression in Synchronized Cell Suspension Culture.

    Science.gov (United States)

    Ayaydin, Ferhan; Kotogány, Edit; Ábrahám, Edit; Horváth, Gábor V

    2017-01-01

    Deepening our knowledge on the regulation of the plant cell division cycle depends on techniques that allow for the enrichment of cell populations in defined cell cycle phases. Synchronization of cell division can be achieved using different plant tissues; however, well-established cell suspension cultures provide large amount of biological sample for further analyses. Here, we describe the methodology of the establishment, propagation, and analysis of a Medicago sativa suspension culture that can be used for efficient synchronization of the cell division. A novel 5-ethynyl-2'-deoxyuridine (EdU)-based method is used for the estimation of cell fraction that enters DNA synthesis phase of the cell cycle and we also demonstrate the changes in the phosphorylation level of Medicago sativa retinoblastoma-related protein (MsRBR1) during cell cycle progression.

  7. Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco.

    Science.gov (United States)

    Kater, M M; Franken, J; van Aelst, A; Angenent, G C

    2000-08-01

    Molecular and genetic analyses have shown that the Arabidopsis thaliana gene SUPERMAN (SUP) has at least two functions in Arabidopsis flower development. SUP is necessary to control the correct distribution of cells with either a stamen or carpel fate, and is essential for proper outgrowth of the ovule outer integument. Both these functions indicate a role for SUP in cell proliferation. To study the function of the Arabidopsis SUP gene in more detail, we over-expressed the SUP gene in petunia and tobacco in a tissue-specific manner. The petunia FLORAL BINDING PROTEIN 1 (FBP1) gene promoter was used to restrict the expression of SUP to petals and stamens. The development of petals and stamens was severely affected in both petunia and tobacco plants over-expressing SUP. Petals remained small and did not unfold, resulting in closed flowers. Stamen filaments were thin and very short. Detailed analysis of these floral organs from the petunia transformants showed that cell expansion was dramatically reduced without affecting cell division. These results reveal a novel activity for SUP as a regulator of cell expansion.

  8. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  9. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    Science.gov (United States)

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  10. A theoretical model for ROP localisation by auxin in Arabidopsis root hair cells.

    Directory of Open Access Journals (Sweden)

    Robert J H Payne

    Full Text Available Local activation of Rho GTPases is important for many functions including cell polarity, morphology, movement, and growth. Although a number of molecules affecting Rho-of-Plants small GTPase (ROP signalling are known, it remains unclear how ROP activity becomes spatially organised. Arabidopsis root hair cells produce patches of ROP at consistent and predictable subcellular locations, where root hair growth subsequently occurs.We present a mathematical model to show how interaction of the plant hormone auxin with ROPs could spontaneously lead to localised patches of active ROP via a Turing or Turing-like mechanism. Our results suggest that correct positioning of the ROP patch depends on the cell length, low diffusion of active ROP, a gradient in auxin concentration, and ROP levels. Our theory provides a unique explanation linking the molecular biology to the root hair phenotypes of multiple mutants and transgenic lines, including OX-ROP, CA-rop, aux1, axr3, tip1, eto1, etr1, and the triple mutant aux1 ein2 gnom(eb.We show how interactions between Rho GTPases (in this case ROPs and regulatory molecules (in this case auxin could produce characteristic subcellular patterning that subsequently affects cell shape. This has important implications for research on the morphogenesis of plants and other eukaryotes. Our results also illustrate how gradient-regulated Turing systems provide a particularly robust and flexible mechanism for pattern formation.

  11. Immune suppression of human lymphoid tissues and cells in rotating suspension culture and onboard the International Space Station.

    Science.gov (United States)

    Fitzgerald, Wendy; Chen, Silvia; Walz, Carl; Zimmerberg, Joshua; Margolis, Leonid; Grivel, Jean-Charles

    2009-12-01

    The immune responses of human lymphoid tissue explants or cells isolated from this tissue were studied quantitatively under normal gravity and microgravity. Microgravity was either modeled by solid body suspension in a rotating, oxygenated culture vessel or was actually achieved on the International Space Station (ISS). Our experiments demonstrate that tissues or cells challenged by recall antigen or by polyclonal activator in modeled microgravity lose all their ability to produce antibodies and cytokines and to increase their metabolic activity. In contrast, if the cells were challenged before being exposed to modeled microgravity suspension culture, they maintained their responses. Similarly, in microgravity in the ISS, lymphoid cells did not respond to antigenic or polyclonal challenge, whereas cells challenged prior to the space flight maintained their antibody and cytokine responses in space. Thus, immune activation of cells of lymphoid tissue is severely blunted both in modeled and true microgravity. This suggests that suspension culture via solid body rotation is sufficient to induce the changes in cellular physiology seen in true microgravity. This phenomenon may reflect immune dysfunction observed in astronauts during space flights. If so, the ex vivo system described above can be used to understand cellular and molecular mechanisms of this dysfunction.

  12. Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

    Science.gov (United States)

    Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

    2014-01-01

    The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

  13. A Dynamic Gene Regulatory Network Model That Recovers the Cyclic Behavior of Arabidopsis thaliana Cell Cycle

    Science.gov (United States)

    Ortiz-Gutiérrez, Elizabeth; García-Cruz, Karla; Azpeitia, Eugenio; Castillo, Aaron; Sánchez, María de la Paz; Álvarez-Buylla, Elena R.

    2015-01-01

    Cell cycle control is fundamental in eukaryotic development. Several modeling efforts have been used to integrate the complex network of interacting molecular components involved in cell cycle dynamics. In this paper, we aimed at recovering the regulatory logic upstream of previously known components of cell cycle control, with the aim of understanding the mechanisms underlying the emergence of the cyclic behavior of such components. We focus on Arabidopsis thaliana, but given that many components of cell cycle regulation are conserved among eukaryotes, when experimental data for this system was not available, we considered experimental results from yeast and animal systems. We are proposing a Boolean gene regulatory network (GRN) that converges into only one robust limit cycle attractor that closely resembles the cyclic behavior of the key cell-cycle molecular components and other regulators considered here. We validate the model by comparing our in silico configurations with data from loss- and gain-of-function mutants, where the endocyclic behavior also was recovered. Additionally, we approximate a continuous model and recovered the temporal periodic expression profiles of the cell-cycle molecular components involved, thus suggesting that the single limit cycle attractor recovered with the Boolean model is not an artifact of its discrete and synchronous nature, but rather an emergent consequence of the inherent characteristics of the regulatory logic proposed here. This dynamical model, hence provides a novel theoretical framework to address cell cycle regulation in plants, and it can also be used to propose novel predictions regarding cell cycle regulation in other eukaryotes. PMID:26340681

  14. Design of serum-free medium for suspension culture of CHO cells on the basis of general commercial media.

    Science.gov (United States)

    Miki, Hideo; Takagi, Mutsumi

    2015-08-01

    The design of serum-free media for suspension culture of genetically engineered Chinese hamster ovary (CHO) cells using general commercial media as a basis was investigated. Subcultivation using a commercial serum-free medium containing insulin-like growth factor (IGF)-1 with or without FCS necessitated additives other than IGF-1 to compensate for the lack of FCS and improve cell growth. Suspension culture with media containing several combinations of growth factors suggested the effectiveness of addition of both IGF-1 and the lipid signaling molecule lysophosphatidic acid (LPA) for promoting cell growth. Subcultivation of CHO cells in suspension culture using the commercial serum-free medium EX-CELL™302, which contained an IGF-1 analog, supplemented with LPA resulted in gradually increasing specific growth rate comparable to the serum-containing medium and in almost the same high antibody production regardless of the number of generations. The culture with EX-CELL™302 supplemented with LPA in a jar fermentor with pH control at 6.9 showed an apparently higher cell growth rate than the cultures without pH control and with pH control at 6.8. The cell growth in the medium supplemented with aurintricarboxylic acid (ATA), which was much cheaper than IGF-1, in combination with LPA was synergistically promoted similarly to that in the medium supplemented with IGF-1 and LPA. In conclusion, the serum-free medium designed on the basis of general commercial media could support the growth of CHO cells and antibody production comparable to serum-containing medium in suspension culture. Moreover, the possibility of cost reduction by the substitution of IGF-1 with ATA was also shown.

  15. Conjugation of the mycotoxins alternariol and alternariol monomethyl ether in tobacco suspension cells.

    Science.gov (United States)

    Hildebrand, Andreas A; Kohn, Beate N; Pfeiffer, Erika; Wefers, Daniel; Metzler, Manfred; Bunzel, Mirko

    2015-05-20

    The mycotoxins alternariol (AOH) and alternariol-9-O-methyl ether (AME) carry three and two phenolic hydroxyl groups, respectively, which makes them candidates for the formation of conjugated metabolites in plants. Such conjugates may escape routine methods of analysis and have therefore been termed masked or, more recently, modified mycotoxins. We report now that AOH and AME are extensively conjugated in suspension cultures of tobacco BY-2 cells. Five conjugates of AOH were identified by MS and NMR spectroscopy as β-D-glucopyranosides (attached in AOH 3- or 9-position) as well as their 6'-malonyl derivatives, and as a gentiobiose conjugate. For AME, conjugation resulted in the d-glucopyranoside (mostly attached in the AME 3-position) and its 6'- and 4'-malonyl derivatives. Pronounced differences were noted for the quantitative pattern of AOH and AME conjugates as well as for their phytotoxicity. Our in vitro study demonstrates for the first time that masked mycotoxins of AOH and AME can be formed in plant cells.

  16. Controlling Expansion and Cardiomyogenic Differentiation of Human Pluripotent Stem Cells in Scalable Suspension Culture

    Directory of Open Access Journals (Sweden)

    Henning Kempf

    2014-12-01

    Full Text Available To harness the potential of human pluripotent stem cells (hPSCs, an abundant supply of their progenies is required. Here, hPSC expansion as matrix-independent aggregates in suspension culture was combined with cardiomyogenic differentiation using chemical Wnt pathway modulators. A multiwell screen was scaled up to stirred Erlenmeyer flasks and subsequently to tank bioreactors, applying controlled feeding strategies (batch and cyclic perfusion. Cardiomyogenesis was sensitive to the GSK3 inhibitor CHIR99021 concentration, whereas the aggregate size was no prevailing factor across culture platforms. However, in bioreactors, the pattern of aggregate formation in the expansion phase dominated subsequent differentiation. Global profiling revealed a culture-dependent expression of BMP agonists/antagonists, suggesting their decisive role in cell-fate determination. Furthermore, metallothionein was discovered as a potentially stress-related marker in hPSCs. In 100 ml bioreactors, the production of 40 million predominantly ventricular-like cardiomyocytes (up to 85% purity was enabled that were directly applicable to bioartificial cardiac tissue formation.

  17. Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures.

    Science.gov (United States)

    Chappell, J; Von Lanken, C; Vögeli, U; Bhatt, P

    1989-05-01

    The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [(14)C]acetate and [(3)H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.

  18. High lipid order of Arabidopsis cell-plate membranes mediated by sterol and DYNAMIN-RELATED PROTEIN1A function.

    Science.gov (United States)

    Frescatada-Rosa, Márcia; Stanislas, Thomas; Backues, Steven K; Reichardt, Ilka; Men, Shuzhen; Boutté, Yohann; Jürgens, Gerd; Moritz, Thomas; Bednarek, Sebastian Y; Grebe, Markus

    2014-12-01

    Membranes of eukaryotic cells contain high lipid-order sterol-rich domains that are thought to mediate temporal and spatial organization of cellular processes. Sterols are crucial for execution of cytokinesis, the last stage of cell division, in diverse eukaryotes. The cell plate of higher-plant cells is the membrane structure that separates daughter cells during somatic cytokinesis. Cell-plate formation in Arabidopsis relies on sterol- and DYNAMIN-RELATED PROTEIN1A (DRP1A)-dependent endocytosis. However, functional relationships between lipid membrane order or lipid packing and endocytic machinery components during eukaryotic cytokinesis have not been elucidated. Using ratiometric live imaging of lipid order-sensitive fluorescent probes, we show that the cell plate of Arabidopsis thaliana represents a dynamic, high lipid-order membrane domain. The cell-plate lipid order was found to be sensitive to pharmacological and genetic alterations of sterol composition. Sterols co-localize with DRP1A at the cell plate, and DRP1A accumulates in detergent-resistant membrane fractions. Modifications of sterol concentration or composition reduce cell-plate membrane order and affect DRP1A localization. Strikingly, DRP1A function itself is essential for high lipid order at the cell plate. Our findings provide evidence that the cell plate represents a high lipid-order domain, and pave the way to explore potential feedback between lipid order and function of dynamin-related proteins during cytokinesis.

  19. The histidine kinase AHK5 integrates endogenous and environmental signals in Arabidopsis guard cells.

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    Radhika Desikan

    Full Text Available BACKGROUND: Stomatal guard cells monitor and respond to environmental and endogenous signals such that the stomatal aperture is continually optimised for water use efficiency. A key signalling molecule produced in guard cells in response to plant hormones, light, carbon dioxide and pathogen-derived signals is hydrogen peroxide (H(2O(2. The mechanisms by which H(2O(2 integrates multiple signals via specific signalling pathways leading to stomatal closure is not known. PRINCIPAL FINDINGS: Here, we identify a pathway by which H(2O(2, derived from endogenous and environmental stimuli, is sensed and transduced to effect stomatal closure. Histidine kinases (HK are part of two-component signal transduction systems that act to integrate environmental stimuli into a cellular response via a phosphotransfer relay mechanism. There is little known about the function of the HK AHK5 in Arabidopsis thaliana. Here we report that in addition to the predicted cytoplasmic localisation of this protein, AHK5 also appears to co-localise to the plasma membrane. Although AHK5 is expressed at low levels in guard cells, we identify a unique role for AHK5 in stomatal signalling. Arabidopsis mutants lacking AHK5 show reduced stomatal closure in response to H(2O(2, which is reversed by complementation with the wild type gene. Over-expression of AHK5 results in constitutively less stomatal closure. Abiotic stimuli that generate endogenous H(2O(2, such as darkness, nitric oxide and the phytohormone ethylene, also show reduced stomatal closure in the ahk5 mutants. However, ABA caused closure, dark adaptation induced H(2O(2 production and H(2O(2 induced NO synthesis in mutants. Treatment with the bacterial pathogen associated molecular pattern (PAMP flagellin, but not elf peptide, also exhibited reduced stomatal closure and H(2O(2 generation in ahk5 mutants. SIGNIFICANCE: Our findings identify an integral signalling function for AHK5 that acts to integrate multiple signals via H

  20. Heterotrimeric G-protein is involved in phytochrome A-mediated cell death of Arabidopsis hypocotyls

    Institute of Scientific and Technical Information of China (English)

    Qing Wei; Wenbin Zhou; Guangzhen Hu; Jiamian Wei; Hongquan Yang; Jirong Huang

    2008-01-01

    The heterotrimeric guanine nucleotide-binding protein (G-protein) has been demonstrated to mediate various signaling pathways in plants. However,its role in phytochrome A (phyA) signaling remains elusive. In this study,we discover a new phyA-mediated phenotype designated far-red irradiation (FR) preconditioned cell death,which occurs only in the hypocotyls of FR-grown seedlings following exposure to white light (WL). The cell death is mitigated in the Ga mutant gpal but aggravated in the Gβ mutant agbl in comparison with the wild type (WT),indicative of antagonistic roles of GPAI and AGB1 in the phyA-mediated cell-death pathway. Further investigation indicates that FR-induced accumulation of nonphotoconvertible protochlorophyllide (Pchlide633),which generates reactive oxygen species (ROS)on exposure to WL,is required for FR-preconditioned cell death. Moreover,ROS is mainly detected in chloroplasts using the fluorescent probe. Interestingly,the application of H2O2 to dark-grown seedlings results in a phenotype similar to FR-preconditioned cell death. This reveals that ROS is a critical mediator for the cell death. In addition,we observe that agbl is more sensitive to H2O2 than WT seedlings,indicating that the G-protein may also modify the sensitivity of the seedlings to ROS stress. Taking these results together,we infer that the G-protein may be involved in the phyA signaling pathway to regulate FR-preconditioned cell death of Arabidopsis hypocotyls.Apossible mechanism underlying the involvement of the G-protein in phyA signaling is discussed in this study.

  1. Live imaging of companion cells and sieve elements in Arabidopsis leaves.

    Directory of Open Access Journals (Sweden)

    Thibaud Cayla

    Full Text Available The phloem is a complex tissue composed of highly specialized cells with unique subcellular structures and a compact organization that is challenging to study in vivo at cellular resolution. We used confocal scanning laser microscopy and subcellular fluorescent markers in companion cells and sieve elements, for live imaging of the phloem in Arabidopsis leaves. This approach provided a simple framework for identifying phloem cell types unambiguously. It highlighted the compactness of the meshed network of organelles within companion cells. By contrast, within the sieve elements, unknown bodies were observed in association with the PP2-A1:GFP, GFP:RTM1 and RTM2:GFP markers at the cell periphery. The phloem lectin PP2-A1:GFP marker was found in the parietal ground matrix. Its location differed from that of the P-protein filaments, which were visualized with SEOR1:GFP and SEOR2:GFP. PP2-A1:GFP surrounded two types of bodies, one of which was identified as mitochondria. This location suggested that it was embedded within the sieve element clamps, specific structures that may fix the organelles to each another or to the plasma membrane in the sieve tubes. GFP:RTM1 was associated with a class of larger bodies, potentially corresponding to plastids. PP2-A1:GFP was soluble in the cytosol of immature sieve elements. The changes in its subcellular localization during differentiation provide an in vivo blueprint for monitoring this process. The subcellular features obtained with these companion cell and sieve element markers can be used as landmarks for exploring the organization and dynamics of phloem cells in vivo.

  2. Involvement of Arabidopsis Hexokinase1 in Cell Death Mediated by Myo -Inositol Accumulation

    KAUST Repository

    Bruggeman, Quentin

    2015-06-05

    Programmed cell death (PCD) is essential for several aspects of plant life, including development and stress responses. We recently identified the mips1 mutant of Arabidopsis thaliana, which is deficient for the enzyme catalyzing the limiting step of myo-inositol (MI) synthesis. One of the most striking features of mips1 is the light-dependent formation of lesions on leaves due to salicylic acid (SA)-dependent PCD. Here, we identified a suppressor of PCD by screening for mutations that abolish the mips1 cell death phenotype. Our screen identified the hxk1 mutant, mutated in the gene encoding the hexokinase1 (HXK1) enzyme that catalyzes sugar phosphorylation and acts as a genuine glucose sensor. We show that HXK1 is required for lesion formation in mips1 due to alterations in MI content, via SA-dependant signaling. Using two catalytically inactive HXK1 mutants, we also show that hexokinase catalytic activity is necessary for the establishment of lesions in mips1. Gas chromatography-mass spectrometry analyses revealed a restoration of the MI content in mips1 hxk1 that it is due to the activity of the MIPS2 isoform, while MIPS3 is not involved. Our work defines a pathway of HXK1-mediated cell death in plants and demonstrates that two MIPS enzymes act cooperatively under a particular metabolic status, highlighting a novel checkpoint of MI homeostasis in plants. © 2015 American Society of Plant Biologists. All rights reserved.

  3. Overproduction of stomatal lineage cells in Arabidopsis mutants defective in active DNA demethylation.

    Science.gov (United States)

    Yamamuro, Chizuko; Miki, Daisuke; Zheng, Zhimin; Ma, Jun; Wang, Jing; Yang, Zhenbiao; Dong, Juan; Zhu, Jian-Kang

    2014-06-05

    DNA methylation is a reversible epigenetic mark regulating genome stability and function in many eukaryotes. In Arabidopsis, active DNA demethylation depends on the function of the ROS1 subfamily of genes that encode 5-methylcytosine DNA glycosylases/lyases. ROS1-mediated DNA demethylation plays a critical role in the regulation of transgenes, transposable elements and some endogenous genes; however, there have been no reports of clear developmental phenotypes in ros1 mutant plants. Here we report that, in the ros1 mutant, the promoter region of the peptide ligand gene EPF2 is hypermethylated, which greatly reduces EPF2 expression and thereby leads to a phenotype of overproduction of stomatal lineage cells. EPF2 gene expression in ros1 is restored and the defective epidermal cell patterning is suppressed by mutations in genes in the RNA-directed DNA methylation pathway. Our results show that active DNA demethylation combats the activity of RNA-directed DNA methylation to influence the initiation of stomatal lineage cells.

  4. Endogenous TasiRNAs mediate non-cell autonomous effects on gene regulation in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Rebecca Schwab

    Full Text Available BACKGROUND: Different classes of small RNAs (sRNAs refine the expression of numerous genes in higher eukaryotes by directing protein partners to complementary nucleic acids, where they mediate gene silencing. Plants encode a unique class of sRNAs, called trans-acting small interfering RNAs (tasiRNAs, which post-transcriptionally regulate protein-coding transcripts, as do microRNAs (miRNAs, and both sRNA classes control development through their targets. TasiRNA biogenesis requires multiple components of the siRNA pathway and also miRNAs. But while 21mer siRNAs originating from transgenes can mediate silencing across several cell layers, miRNA action seems spatially restricted to the producing or closely surrounding cells. PRINCIPAL FINDINGS: We have previously described the isolation of a genetrap reporter line for TAS3a, the major locus producing AUXIN RESPONS FACTOR (ARF-regulating tasiRNAs in the Arabidopsis shoot. Its activity is limited to the adaxial (upper side of leaf primordia, thus spatially isolated from ARF-activities, which are located in the abaxial (lower side. We show here by in situ hybridization and reporter fusions that the silencing activities of ARF-regulating tasiRNAs are indeed manifested non-cell autonomously to spatially control ARF activities. CONCLUSIONS/SIGNIFICANCE: Endogenous tasiRNAs are thus mediators of a mobile developmental signal and might provide effective gene silencing at a distance beyond the reach of most miRNAs.

  5. Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

    Institute of Scientific and Technical Information of China (English)

    李景川; 马忠海; 元英进; 孙安慈; 胡昌序

    2001-01-01

    The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of “partition” and “bifurcation” were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

  6. Incorporation and Degradation of 14C and 3H-labeled Thymidine by Sugarcane Cells in Suspension Culture 12

    Science.gov (United States)

    Lesley, Stanley M.; Maretzki, Andrew; Nickell, Louis G.

    1980-01-01

    Sugarcane cells growing in suspension culture degrade exogenous thymidine, releasing thymine. Thymine is not utilized for DNA synthesis. Thymine is rapidly catabolized to β-aminoisobutyric acid which is found within the cell. Thymidine in the medium is used for DNA synthesis. The label of [2-14C]thymidine is lost as 14CO2, but the label of [3H]methylthymidine is found in the cell as [3H]β-aminoisobutyric acid, some of which is used for the synthesis of other cell components. The degradation of thymidine can be partially inhibited by addition of certain substituted pyrimidines. PMID:16661365

  7. Laminin-adherent versus suspension-non-adherent cell culture conditions for the isolation of cancer stem cells in the DAOY medulloblastoma cell line.

    Science.gov (United States)

    de la Rosa, Javier; Sáenz Antoñanzas, Ander; Shahi, Mehdi H; Meléndez, Bárbara; Rey, Juan A; Castresana, Javier S

    2016-09-01

    Medulloblastoma (MB) is a highly malignant tumor of childhood. MB seems to be initiated and maintained by a small group of cells, known as cancer stem cells (CSCs). The CSC hypothesis suggests that a subset of tumor cells is able to proliferate, sustain the tumor, and develop chemoresistance, all of which make of CSC an interesting target for new anticancer therapies. The MB cell line DAOY was cultured in suspension by a medullosphere traditional culturing method and in adherent conditions by laminin-pre-coated flasks and serum-free medium enriched with specific growth factors. An increase in the stem features was shown when cells were successively cultured in hypoxia conditions. By contrast, a reduction in these properties was appreciated when cells were exposed to differentiation conditions. In addition, the CD133+ and CD133- subpopulations were isolated from cells grown in laminin-pre-coated flasks, and in vitro experiments showed that the CD133+ fraction represented the stem population and it could have CSC with a higher probability than the CD133- fraction. We can conclude that the laminin culture method in adherent conditions and the medullosphere traditional culturing method in suspension are similarly good for obtaining stem-like cells in the DAOY cell line.

  8. A gene regulatory network for root epidermis cell differentiation in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Angela Bruex

    2012-01-01

    Full Text Available The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 "core" root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

  9. Blue light-dependent nuclear positioning in Arabidopsis thaliana leaf cells.

    Science.gov (United States)

    Iwabuchi, Kosei; Sakai, Tatsuya; Takagi, Shingo

    2007-09-01

    The plant nucleus changes its intracellular position not only upon cell division and cell growth but also in response to environmental stimuli such as light. We found that the nucleus takes different intracellular positions depending on blue light in Arabidopsis thaliana leaf cells. Under dark conditions, nuclei in mesophyll cells were positioned at the center of the bottom of cells (dark position). Under blue light at 100 mumol m(-2) s(-1), in contrast, nuclei were located along the anticlinal walls (light position). The nuclear positioning from the dark position to the light position was fully induced within a few hours of blue light illumination, and it was a reversible response. The response was also observed in epidermal cells, which have no chloroplasts, suggesting that the nucleus has the potential actively to change its position without chloroplasts. Light-dependent nuclear positioning was induced specifically by blue light at >50 mumol m(-2) s(-1). Furthermore, the response to blue light was induced in phot1 but not in phot2 and phot1phot2 mutants. Unexpectedly, we also found that nuclei as well as chloroplasts in phot2 and phot1phot2 mutants took unusual intracellular positions under both dark and light conditions. The lack of the response and the unusual positioning of nuclei and chloroplasts in the phot2 mutant were recovered by externally introducing the PHOT2 gene into the mutant. These results indicate that phot2 mediates the blue light-dependent nuclear positioning and the proper positioning of nuclei and chloroplasts. This is the first characterization of light-dependent nuclear positioning in spermatophytes.

  10. Single-Beam Acoustic Trapping of Red Blood Cells and Polystyrene Microspheres in Flowing Red Blood Cell Saline and Plasma Suspensions.

    Science.gov (United States)

    Liu, Hsiao-Chuan; Li, Ying; Chen, Ruimin; Jung, Hayong; Shung, K Kirk

    2017-02-21

    Single-beam acoustic tweezers (SBATs) represent a new technology for particle and cell trapping. The advantages of SBATs are their deep penetration into tissues, reduction of tissue damage and ease of application to in vivo studies. The use of these tools for applications in drug delivery in vivo must meet the following conditions: large penetration depth, strong trapping force and tissue safety. A reasonable penetration depth for SBATs in the development of in vivo applications was established in a previous study conducted in water with zero velocity. However, capturing objects in flowing fluid can provide more meaningful results. In this study, we investigated the capability of SBATs to trap red blood cells (RBCs) and polystyrene microspheres in flowing RBC suspensions. Two different types of RBC suspension were prepared in this work: an RBC phosphate-buffered saline (PBS) suspension and an RBC plasma suspension. The results indicated that SBATs successfully trapped RBCs and polystyrene microspheres in a flowing RBC PBS suspension with an average steady velocity of 1.6 cm/s in a 2-mm-diameter polyimide. Furthermore, SBATs were found able to trap RBCs in a flowing RBC PBS suspension at speeds as high as 7.9 cm/s in a polyimide tube, which is higher than the velocity in capillaries (0.03 cm/s) and approaches the velocity in arterioles and venules. Moreover, the results also indicated that polystyrene microspheres can be trapped in an RBC plasma suspension, where aggregation is observed. This work represents a step forward in using this tool in actual in vivo experimentation.

  11. Cellulose binding protein from the parasitic nematode Heterodera schachtii interacts with Arabidopsis pectin methylesterase: cooperative cell wall modification during parasitism.

    Science.gov (United States)

    Hewezi, Tarek; Howe, Peter; Maier, Tom R; Hussey, Richard S; Mitchum, Melissa Goellner; Davis, Eric L; Baum, Thomas J

    2008-11-01

    Plant-parasitic cyst nematodes secrete a complex of cell wall-digesting enzymes, which aid in root penetration and migration. The soybean cyst nematode Heterodera glycines also produces a cellulose binding protein (Hg CBP) secretory protein. To determine the function of CBP, an orthologous cDNA clone (Hs CBP) was isolated from the sugar beet cyst nematode Heterodera schachtii, which is able to infect Arabidopsis thaliana. CBP is expressed only in the early phases of feeding cell formation and not during the migratory phase. Transgenic Arabidopsis expressing Hs CBP developed longer roots and exhibited enhanced susceptibility to H. schachtii. A yeast two-hybrid screen identified Arabidopsis pectin methylesterase protein 3 (PME3) as strongly and specifically interacting with Hs CBP. Transgenic plants overexpressing PME3 also produced longer roots and exhibited increased susceptibility to H. schachtii, while a pme3 knockout mutant showed opposite phenotypes. Moreover, CBP overexpression increases PME3 activity in planta. Localization studies support the mode of action of PME3 as a cell wall-modifying enzyme. Expression of CBP in the pme3 knockout mutant revealed that PME3 is required but not the sole mechanism for CBP overexpression phenotype. These data indicate that CBP directly interacts with PME3 thereby activating and potentially targeting this enzyme to aid cyst nematode parasitism.

  12. Antioxidant metabolism of coffee cell suspension cultures in response to cadmium.

    Science.gov (United States)

    Gomes-Junior, Rui A; Moldes, Carlos A; Delite, Fabricio S; Pompeu, Georgia B; Gratão, Priscila L; Mazzafera, Paulo; Lea, Peter J; Azevedo, Ricardo A

    2006-11-01

    The antioxidant responses of coffee (Coffea arabica L.) cell suspension cultures to cadmium (Cd) were investigated. Cd accumulated very rapidly in the cells and this accumulation was directly correlated with an increase in applied CdCl(2) concentration in the external medium. At 0.05mM CdCl(2), growth was stimulated, but at 0.5mM CdCl(2), the growth rate was reduced. An alteration in activated oxygen metabolism was detected by visual analysis as well as by an increase in lipid peroxidation at the higher CdCl(2) concentration. Catalase (CAT; EC 1.11.1.6), glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (SOD; EC 1.15.1.1) activity increased, particularly at the higher concentration of CdCl(2). Ascorbate peroxidase (APX; EC 1.11.1.11) activity was increased at the lower CdCl(2) concentration used, but could not be detected in cells growing in the higher CdCl(2) concentration after 24h of growth, whilst guaiacol peroxidase (GOPX; EC 1.11.1.7) did not show a clear response to Cd treatment. An analysis by non-denaturing PAGE followed by staining for enzyme activity, revealed one CAT isoenzyme, nine SOD isoenzymes and four GR isoenzymes. The SOD isoenzymes were differently affected by CdCl(2) treatment and one GR isoenzyme was shown to specifically respond to CdCl(2). The results suggest that the higher concentrations of CdCl(2) may lead to oxidative stress. The main response appears to be via the induction of SOD and CAT activities for the removal of reactive oxygen species (ROS), and by the induction of GR to ensure the availability of reduced glutathione for the synthesis of Cd-binding peptides, which may also be related to the inhibition of APX activity probably due to glutathione and ascorbate depletion.

  13. Acaricidal activities of whole cell suspension, cell-free supernatant,and crude cell extract of Xenorhabdus stokiae against mushroom mite (Luciaphorus sp.)

    Institute of Scientific and Technical Information of China (English)

    Prapassom BUSSAMAN; Chirayu SA-UTH; Paweena RATTANAS ENA; Angsumarn CHANDRAPATYA

    2012-01-01

    Xenorhabdus bacterium has been used as a biological control agent against Luciaphorus sp.,a mushroom mite endemic in Thailand.To develop an effective formulation of Xenorhabdus stokiae,treatments using different parts of X.stokiae isolate PB09 culture,including whole cell suspension,cell-free supernatant,and crude cell extract,were performed.The results show that different parts ofX.stokiae isolate PB09 culture could induce variable effects on mite mortality and fecundity.Application with cell-free supernatant of X.stokiae culture resulted in both the highest mite mortality rate [(89.00+3.60)%] and the lowest mite fecundity [(41.33+23.69) eggs/gravid female].Whole cell suspension of X.stokiae isolate PB09 culture was found to be slightly less effective than its cell-free supernatant,suggesting that X.stokiae was more likely to release its metabolites with acaricidal activities to the surrounding culture media.Crude cell extract of X.stokiae was not effective against mites.Cell-free supernatant of X.stokiae isolate PB09 was the most effective biological control agent and it could be conveniently used in future formulations instead of live bacteria.

  14. Evidence for 'silicon' within the cell walls of suspension-cultured rice cells.

    Science.gov (United States)

    He, Congwu; Wang, Lijun; Liu, Jian; Liu, Xin; Li, Xiuli; Ma, Jie; Lin, Yongjun; Xu, Fangsen

    2013-11-01

    Despite the ubiquity and beneficial role of silicon (Si) in plant biology, structural and chemical mechanisms operating at the single-cell level have not been extensively studied. To obtain insights regarding the effect of Si on individual cells, we cultivated suspended rice (Oryza sativa) cells in the absence and presence of Si and analyzed single cells using a combination of physical techniques including atomic force microscopy (AFM). Si is naturally present as a constituent of the cell walls, where it is firmly bound to the cell wall matrix rather than occurring within intra- or extracellular silica deposition, as determined by using inductively coupled plasma mass spectrometry (ICP-MS) and X-ray photoelectron spectroscopy (XPS). This species of Si, linked with the cell wall matrix, improves the structural stability of cell walls during their expansion and subsequent cell division. Maintaining cell shape is thereby enhanced, which may be crucial for the function and survival of cells. This study provides further evidence that organosilicon is present in plant cell walls, which broadens our understanding of the chemical nature of 'anomalous Si' in plant biology.

  15. Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture

    Directory of Open Access Journals (Sweden)

    Thu V. Vuong

    2014-06-01

    Full Text Available Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA, salicylic acid, β-glucan (GLU, and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 μM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert.

  16. The reduction of starch accumulation in transgenic sugarcane cell suspension culture lines.

    Science.gov (United States)

    Ferreira, Stephanus J; Kossmann, Jens; Lloyd, James R; Groenewald, Jan-Hendrik

    2008-11-01

    Starch only occurs in small amounts in sugarcane, but is, nevertheless an unwanted product because it reduces the amount of sucrose that can be crystallized from molasses. In an attempt to reduce the starch content of sugarcane, the activities of ADP-glucose pyrophosphorylase (AGPase) and beta-amylase were manipulated using transgenic approaches. Transformation vectors to reduce AGPase activity and to increase plastidial beta-amylase activity were constructed and used for the transformation of sugarcane calli. The results of the manipulations were analyzed in suspension cultures. AGPase activity was reduced down to between 14 and 54% of the wild-type control. This led to a reduction in starch concentration down to 38% of the levels of the wild-type control. beta-Amylase activity was increased in the transgenic lines by 1.5-2 times that of the wild-type control. This increase in activity led to a reduction in starch amounts by 90% compared to wild-type control cells. In both experiments, the changes in starch concentrations could be correlated with the change in enzyme activity. There were no significant effects on sucrose concentrations in either experiment, indicating that these approaches might be useful to engineer regenerated sugarcane for optimized sucrose production.

  17. Fabrication and electrochemical performance of solid oxide fuel cell components by atmospheric and suspension plasma spray

    Institute of Scientific and Technical Information of China (English)

    XIA Wei-sheng; YANG Yun-zhen; ZHANG Hai-ou; WANG Gui-lan

    2009-01-01

    The theory of functionally graded material (FGM) was applied in the fabrication process of PEN (Positive- Electrolyte-Negative),the core component of solid oxide fuel cell (SOFC).To enhance its electrochemical performance,the functionally graded PEN of planar SOFC was prepared by atmospheric plasma spray (APS).The cross-sectional SEM micrograph and element energy spectrum of the resultant PEN were analyzed.Its interface resistance was also compared with that without the graded layers to investigate the electrochemical performance enhanced by the functionally graded layers.Moreover,a new process,suspension plasma spray (SPS) was applied to preparing the SOFC electrolyte.Higher densification of the coating by SPS,1.61%,is observed,which is helpful to effectively improve its electrical conductivity.The grain size of the electrolyte coating fabricated by SPS is also smaller than that by APS,which is more favourable to obtain the dense electrolyte coatings.To sum up,all mentioned above can prove that the hybrid process of APS and SPS could be a better approach to fabricate the PEN of SOFC stacks,in which APS is for porous electrodes and SPS for dense electrolyte.

  18. PATHOGEN IMPACT ON THE ACTIVITY DYNAMICS OF POTATO SUSPENSION CELLS EXTRA-CELLULAR PEROXIDASE

    Directory of Open Access Journals (Sweden)

    Graskova I.A.

    2005-08-01

    Full Text Available Changes in the activity of extracellular peroxidases were measured in cell suspension cultures of potato infected by Clavibacter michiganensis subsp. sepedonicus (Spieck. et Kotth. Skapt et Burkh. The total extracellular peroxidases activity of the resistant potato variety was higher than that of the sensitive variety both before and after infection. The enzyme of the resistant variety had a рН optimum of 6.2, while that of the sensitive variety was 5.4. Extracellular peroxidases of the sensitive potato variety were activated 10 minutes after infection, and displayed highest activity 1.5-2 hours later. In the resistant variety, peroxidase activity rose sharply in the first minutes of infection, and second peak of activity occurred 1.5-2 hours later. The increase of extracellular peroxidases activity of the sensitive potato variety under pathogenesis is connected with the change of genome expression and synthesis of proteins. The increase of enzyme activity of resistant potato variety in the first moments of infection is not related to proteins synthesis and is apparently conditioned by the change of kinetic parameters.

  19. Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures.

    Science.gov (United States)

    Hanley, K; Chappell, J

    1992-01-01

    Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-beta-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a K(m) for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH.

  20. Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

    Directory of Open Access Journals (Sweden)

    Yoichiro Fukao

    2016-01-01

    Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

  1. The use of the CELLection kit in the isolation of carcinoma cells from mononuclear cell suspensions

    DEFF Research Database (Denmark)

    Werther, K; Normark, M; Hansen, B F;

    2000-01-01

    A study was performed to evaluate in vitro the sensitivity, specificity and variability of a new immunomagnetic microbead isolation technique which provides subsequent immunological staining of captured carcinoma cells. In a mixture of peripheral blood mononuclear cells (PBMCs) and human carcinoma...... cells the epithelial cancer cells were isolated with the Dynal((R)) RAM IgG1 CELLection Kit using Dynabeads M-280 coated with a rat monoclonal antibody (Mab) against mouse IgG1. The rat Mab was biotinylated and attached to Dynabeads via streptavidin and a DNA linker. The anti-epithelial monoclonal mouse...... an average recovery of approximately 60% of a human colon carcinoma cell line HCC-2998 seeded in 5.10(6) PBMCs was obtained, and the recovered cells could subsequently be immunologically stained for the surface antigen CD87 (urokinase plasminogen activator receptor). No positive stained cells were found...

  2. Analysis of a transcription factor using transient assay in Arabidopsis protoplasts.

    Science.gov (United States)

    Iwata, Yuji; Lee, Mi-Hyun; Koizumi, Nozomu

    2011-01-01

    Regulation of gene expression by transcription factors is a fundamental mechanism in essentially all aspects of cellular processes. Transient expression assay of a reporter plasmid containing a reporter gene driven by a promoter of interest and an effector plasmid expressing a transcription factor has been a powerful tool for analyzing transcription factors. Here we present a protocol for polyethylene glycol (PEG)-mediated transformation of Arabidopsis protoplasts. It details preparation of protoplasts from Arabidopsis suspension cultured cells or leaves of soil-grown Arabidopsis plants and subsequent PEG-mediated transformation with reporter and effector plasmids. This protocol can be completed within 24 h from protoplast preparation to reporter assay. As an example, analysis of the membrane-bound transcription factor AtbZIP60 and its target BiP3 promoter is shown.

  3. Single-cell and coupled GRN models of cell patterning in the Arabidopsis thaliana root stem cell niche

    Directory of Open Access Journals (Sweden)

    Alvarez-Buylla Elena R

    2010-10-01

    Full Text Available Abstract Background Recent experimental work has uncovered some of the genetic components required to maintain the Arabidopsis thaliana root stem cell niche (SCN and its structure. Two main pathways are involved. One pathway depends on the genes SHORTROOT and SCARECROW and the other depends on the PLETHORA genes, which have been proposed to constitute the auxin readouts. Recent evidence suggests that a regulatory circuit, composed of WOX5 and CLE40, also contributes to the SCN maintenance. Yet, we still do not understand how the niche is dynamically maintained and patterned or if the uncovered molecular components are sufficient to recover the observed gene expression configurations that characterize the cell types within the root SCN. Mathematical and computational tools have proven useful in understanding the dynamics of cell differentiation. Hence, to further explore root SCN patterning, we integrated available experimental data into dynamic Gene Regulatory Network (GRN models and addressed if these are sufficient to attain observed gene expression configurations in the root SCN in a robust and autonomous manner. Results We found that an SCN GRN model based only on experimental data did not reproduce the configurations observed within the root SCN. We developed several alternative GRN models that recover these expected stable gene configurations. Such models incorporate a few additional components and interactions in addition to those that have been uncovered. The recovered configurations are stable to perturbations, and the models are able to recover the observed gene expression profiles of almost all the mutants described so far. However, the robustness of the postulated GRNs is not as high as that of other previously studied networks. Conclusions These models are the first published approximations for a dynamic mechanism of the A. thaliana root SCN cellular pattering. Our model is useful to formally show that the data now available are not

  4. Possible Involvement of NADPH Oxidase in Lanthanide Cation-Induced Superoxide Anion Generation in BY-2 Tobacco Cell Suspension Culture

    Institute of Scientific and Technical Information of China (English)

    Yang Shengchang

    2006-01-01

    A rapid and concentration-dependent generation of superoxide anion (·O-2), measured with a superoxide-specific Cypridina luciferin-derived chemiluminescent reagent, was observed when two lanthanide salts (LaCl3 and GdCl3) were added to tobacco (Nicotiana tabacum) cell suspension culture.Addition of superoxide dismutase (480 U·ml-1) and Tiron (5 μmol·L-1) to cell culture suspension decreases the level of lanthanide cation-induced ·O-2 generation, suggesting that ·O-2 generation is extra-cellular.Pretreatment of the cell culture suspension with diphenyleneiodonium (10 and 50 μmol·L-1), quinacrine (1 and 5 mmol·L-1) and imidazol (10 mmol·L-1), inhibitors of NADPH oxidase, notably inhibits the generation of superoxide induced by lanthanide cation, implying the possible involvement of activation of NADPH oxidase.In addition, addition of SHAM (1 and 5 mmol·L-1), azide (0.2 and 1 mmol·L-1), inhibitor of peroxidase, has no influence on ·O-2 generation.

  5. The MYB23 gene provides a positive feedback loop for cell fate specification in the Arabidopsis root epidermis.

    Science.gov (United States)

    Kang, Yeon Hee; Kirik, Victor; Hulskamp, Martin; Nam, Kyoung Hee; Hagely, Katherine; Lee, Myeong Min; Schiefelbein, John

    2009-04-01

    The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis.

  6. Phytosulfokine-α controls hypocotyl length and cell expansion in Arabidopsis thaliana through phytosulfokine receptor 1.

    Directory of Open Access Journals (Sweden)

    Nils Stührwohldt

    Full Text Available The disulfated peptide growth factor phytosulfokine-α (PSK-α is perceived by LRR receptor kinases. In this study, a role for PSK signaling through PSK receptor PSKR1 in Arabidopsis thaliana hypocotyl cell elongation is established. Hypocotyls of etiolated pskr1-2 and pskr1-3 seedlings, but not of pskr2-1 seedlings were shorter than wt due to reduced cell elongation. Treatment with PSK-α did not promote hypocotyl growth indicating that PSK levels were saturating. Tyrosylprotein sulfotransferase (TPST is responsible for sulfation and hence activation of the PSK precursor. The tpst-1 mutant displayed shorter hypocotyls with shorter cells than wt. Treatment of tpst-1 seedlings with PSK-α partially restored elongation growth in a dose-dependent manner. Hypocotyl elongation was significantly enhanced in tpst-1 seedlings at nanomolar PSK-α concentrations. Cell expansion was studied in hypocotyl protoplasts. WT and pskr2-1 protoplasts expanded in the presence of PSK-α in a dose-dependent manner. By contrast, pskr1-2 and pskr1-3 protoplasts were unresponsive to PSK-α. Protoplast swelling in response to PSK-α was unaffected by ortho-vanadate, which inhibits the plasma membrane H(+-ATPase. In maize (Zea mays L., coleoptile protoplast expansion was similarly induced by PSK-α in a dose-dependent manner and was dependent on the presence of K(+ in the media. In conclusion, PSK-α signaling of hypocotyl elongation and protoplast expansion occurs through PSKR1 and likely involves K(+ uptake, but does not require extracellular acidification by the plasma membrane H(+-ATPase.

  7. Erwinia amylovora type three-secreted proteins trigger cell death and defense responses in Arabidopsis thaliana.

    Science.gov (United States)

    Degrave, A; Fagard, M; Perino, C; Brisset, M N; Gaubert, S; Laroche, S; Patrit, O; Barny, M-A

    2008-08-01

    Erwinia amylovora is the bacterium responsible for fire blight, a necrotic disease affecting plants of the rosaceous family. E. amylovora pathogenicity requires a functional type three secretion system (T3SS). We show here that E. amylovora triggers a T3SS-dependent cell death on Arabidopsis thaliana. The plants respond by inducing T3SS-dependent defense responses, including salicylic acid (SA)-independent callose deposition, activation of the SA defense pathway, reactive oxygen species (ROS) accumulation, and part of the jasmonic acid/ethylene defense pathway. Several of these reactions are similar to what is observed in host plants. We show that the cell death triggered by E. amylovora on A. thaliana could not be simply explained by the recognition of AvrRpt2 ea by the resistance gene product RPS2. We then analyzed the role of type three-secreted proteins (T3SPs) DspA/E, HrpN, and HrpW in the induction of cell death and defense reactions in A. thaliana following infection with the corresponding E. amylovora mutant strains. HrpN and DspA/E were found to play an important role in the induction of cell death, activation of defense pathways, and ROS accumulation. None of the T3SPs tested played a major role in the induction of SA-independent callose deposition. The relative importance of T3SPs in A. thaliana is correlated with their relative importance in the disease process on host plants, indicating that A. thaliana can be used as a model to study their role.

  8. The U-Box/ARM E3 ligase PUB13 regulates cell death, defense, and flowering time in Arabidopsis.

    Science.gov (United States)

    Li, Wei; Ahn, Il-Pyung; Ning, Yuese; Park, Chan-Ho; Zeng, Lirong; Whitehill, Justin G A; Lu, Haibin; Zhao, Qingzhen; Ding, Bo; Xie, Qi; Zhou, Jian-Min; Dai, Liangying; Wang, Guo-Liang

    2012-05-01

    The components in plant signal transduction pathways are intertwined and affect each other to coordinate plant growth, development, and defenses to stresses. The role of ubiquitination in connecting these pathways, particularly plant innate immunity and flowering, is largely unknown. Here, we report the dual roles for the Arabidopsis (Arabidopsis thaliana) Plant U-box protein13 (PUB13) in defense and flowering time control. In vitro ubiquitination assays indicated that PUB13 is an active E3 ubiquitin ligase and that the intact U-box domain is required for the E3 ligase activity. Disruption of the PUB13 gene by T-DNA insertion results in spontaneous cell death, the accumulation of hydrogen peroxide and salicylic acid (SA), and elevated resistance to biotrophic pathogens but increased susceptibility to necrotrophic pathogens. The cell death, hydrogen peroxide accumulation, and resistance to necrotrophic pathogens in pub13 are enhanced when plants are pretreated with high humidity. Importantly, pub13 also shows early flowering under middle- and long-day conditions, in which the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 and FLOWERING LOCUS T is induced while FLOWERING LOCUS C expression is suppressed. Finally, we found that two components involved in the SA-mediated signaling pathway, SID2 and PAD4, are required for the defense and flowering-time phenotypes caused by the loss of function of PUB13. Taken together, our data demonstrate that PUB13 acts as an important node connecting SA-dependent defense signaling and flowering time regulation in Arabidopsis.

  9. Over-expression of Arabidopsis CAP causes decreased cell expansion leading to organ size reduction in transgenic tobacco plants.

    Science.gov (United States)

    Barrero, Roberto A; Umeda, Masaaki; Yamamura, Saburo; Uchimiya, Hirofumi

    2003-04-01

    Cyclase-associated proteins (CAP) are multifunctional proteins involved in Ras-cAMP signalling and regulation of the actin cytoskeleton. It has recently been demonstrated that over-expression of AtCAP1 in transgenic arabidopsis plants causes severe morphological defects owing to loss of actin filaments. To test the generality of the function of AtCAP1 in plants, transgenic tobacco plants over-expressing an arabidopsis CAP (AtCAP1) under the regulation of a glucocorticoid-inducible promoter were produced. Over-expression of AtCAP1 in transgenic tobacco plants led to growth abnormalities, in particular a reduction in the size of leaves. Morphological alterations in leaves were the result of reduced elongation of epidermal and mesophyll cells.

  10. AMIODARONE INDUCES THE SYNTHESIS OF HSPS IN SACCHAROMYCES CEREVISIAE AND ARABIDOPSIS THALIANA CELLS

    Directory of Open Access Journals (Sweden)

    Pyatrikas D.V.

    2012-08-01

    Full Text Available Many biotic and abiotic stresses cause an increase of cytosolic Ca2+ level in cells. Calcium is one of the most important second messengers, regulating many various activities in the cell and was known to affect expression of stress activated genes. Mild heat shock induces the expression of heat shock proteins (Hsps which protect cell from drastic heat shock exposure. There are some literature data permitting to suggest that transient elevation of cytosolic Ca2+ level in plant cells is important for activation of Hsps expression. On the other hand mitochondria are known to regulate the intracellular calcium and reactive oxygen species signaling. It has been shown recently that mild heat shock induces hyperpolarization of inner mitochondrial membrane in plant and yeast cells and this event is critically important for activation of Hsps expression. To reveal the relationship between mitochondrial activity, intracellular calcium homeostasis and Hsps expression an antiarrhythmic drug amiodarone (AMD have been used. AMD is known to cause transient increase of cytosolic Ca2+ level in Saccharomyces cerevisiae. Obtained results have showed that AMD treatment induced the synthesis of Hsp104p in S. cerevisiae cells and Hsp101p in A. thaliana cell culture. Induction of Hsp104p synthesis leads to enhanced yeast capability to survive lethal heat shock exposure. Development of S. cerevisiae thermotolerance depended significantly on the presence of Hsp104p. Elevation of Hsp104p level in the result of AMD treatment was shown to be governed by activity of Msn2p and Msn4p transcription factors. Deletion of the MSN2 and MSN4 genes abrogated the AMD ability to induce Hsp104p synthesis. Mild heat shock and AMD treatment induced the hyperpolarization of the inner mitochondrial membrane in yeast and Arabidopsis cells which accompanied by HSP synthesis and development of thermotolerance. It was suggested that increase of cytosolic Ca2+ level after AMD treatment

  11. Immunocytochemical characterization of the cell walls of bean cell suspensions during habituation and dehabituation to dichlobenil

    DEFF Research Database (Denmark)

    Garcia-Angulo, P.; Willats, W. G. T.; Encina, A. E.

    2006-01-01

    analysed showed calcofluor-stained appositions. However, in habituated and dehabituated cells, appositions were not recognized by an anticallose antibody. This finding suggested the accumulation of an extracellular polysaccharide different to callose, probably a 1,4-ß-glucan in these cell lines...

  12. LC/MS profiling of flavonoid glycoconjugates isolated from hairy roots, suspension root cell cultures and seedling roots of Medicago truncatula

    OpenAIRE

    Staszków, Anna; Swarcewicz, Barbara; Banasiak, Joanna; Muth, Dorota; Jasiński, Michał; Stobiecki, Maciej

    2011-01-01

    Hairy roots and suspension cell cultures are commonly used in deciphering different problems related to the biochemistry and physiology of plant secondary metabolites. Here, we address about the issue of possible differences in the profiles of flavonoid compounds and their glycoconjugates derived from various plant materials grown in a standard culture media. We compared profiles of flavonoids isolated from seedling roots, hairy roots, and suspension root cell cultures of a model legume plant...

  13. The mycorrhizal fungus Amanita muscaria induces chitinase activity in roots and in suspension-cultured cells of its host Picea abies.

    Science.gov (United States)

    Sauter, M; Hager, A

    1989-08-01

    A cell-wall fraction of the mycorrhizal fungus Amanita muscaria increased the chitinase activity in suspension-cultured cells of spruce (Picea abies (L.) Karst.) which is a frequent host of Amanita muscaria in nature. Chitinase activity was also increased in roots of spruce trees upon incubation with the fungal elicitor. Non-induced levels of chitinase activity in spruce were higher in suspension cells than in roots whereas the elicitorinduced increase of chitinase activity was higher in roots. Treatment of cells with hormones (auxins and cytokinin) resulted in a severalfold depression of enzyme activity. However, the chitinase activity of hormone-treated as well as hormone-free cells showed an elicitor-induced increase. Suspension cells of spruce secreted a large amount of enzyme into the medium. It is postulated that chitinases released from the host cells in an ectomycorrhizal system partly degrade the fungal cell walls, thus possibly facilitating the exchange of metabolites between the symbionts.

  14. A lower content of de-methylesterified homogalacturonan improves enzymatic cell separation and isolation of mesophyll protoplasts in Arabidopsis.

    Science.gov (United States)

    Lionetti, Vincenzo; Cervone, Felice; De Lorenzo, Giulia

    2015-04-01

    Cell adhesion occurs primarily at the level of middle lamella which is mainly composed by pectin polysaccharides. These can be degraded by cell wall degrading enzymes (CWDEs) during developmental processes to allow a controlled separation of plant cells. Extensive cell wall degradation by CWDEs with consequent cell separation is performed when protoplasts are isolated from plant tissues by using mixtures of CWDEs. We have evaluated whether modification of pectin affects cell separation and protoplast isolation. Arabidopsis plants overexpressing the pectin methylesterase inhibitors AtPMEI-1 or AtPMEI-2, and Arabidopsis pme3 plants, mutated in the gene encoding pectin methylesterase 3, showed an increased efficiency of isolation of viable mesophyll protoplasts as compared with Wild Type Columbia-0 plants. The release of protoplasts was correlated with the reduced level of long stretches of de-methylesterified homogalacturonan (HGA) present in these plants. Response to elicitation, cell wall regeneration and efficiency of transfection in protoplasts from transgenic plants was comparable to those of wild type protoplasts.

  15. TCS1, a Microtubule-Binding Protein, Interacts with KCBP/ZWICHEL to Regulate Trichome Cell Shape in Arabidopsis thaliana.

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    Liangliang Chen

    2016-10-01

    Full Text Available How cell shape is controlled is a fundamental question in developmental biology, but the genetic and molecular mechanisms that determine cell shape are largely unknown. Arabidopsis trichomes have been used as a good model system to investigate cell shape at the single-cell level. Here we describe the trichome cell shape 1 (tcs1 mutants with the reduced trichome branch number in Arabidopsis. TCS1 encodes a coiled-coil domain-containing protein. Pharmacological analyses and observations of microtubule dynamics show that TCS1 influences the stability of microtubules. Biochemical analyses and live-cell imaging indicate that TCS1 binds to microtubules and promotes the assembly of microtubules. Further results reveal that TCS1 physically associates with KCBP/ZWICHEL, a microtubule motor involved in the regulation of trichome branch number. Genetic analyses indicate that kcbp/zwi is epistatic to tcs1 with respect to trichome branch number. Thus, our findings define a novel genetic and molecular mechanism by which TCS1 interacts with KCBP to regulate trichome cell shape by influencing the stability of microtubules.

  16. The durative use of suspension cells and callus for volatile oil by comparative with seeds and fruits in Capparis spinosa L.

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    Yongtai Yin

    Full Text Available Capparis spinosa is one of the most important eremophytes among the medicinal plants, and continued destruction of these plants poses a major threat to species survival. The development of methods to extract compounds, especially those of medicinal value, without harvesting the whole plant is an issue of considerable socioeconomic importance. On the basis of an established system for culture of suspension cells and callus in vitro, Gas Chromatograph-Mass Spectrometer (GC-MS was used for the volatile oil composition analyzing in seed, fruit, suspension cells and callus. Fatty acids were the major component, and the highest content of alkanes was detected in seed, with <1.0% in suspension cells and callus. Esters, olefins and heterocyclic compounds were significantly higher in fruit than in the other materials. The content of acid esters in the suspension cells and callus was significantly higher than in seed and fruit. This indicated that the suspension cells and callus could be helpful for increasing the value of volatile oil and replacing seeds and fruit partially as a source of some compounds of the volatile oil and may also produce some new medical compounds. The above results give valuable information for sustainable use of C. spinosa and provide a foundation for use of the C. spinosa suspension cells and callus as an ongoing medical resource.

  17. The Arabidopsis EDR1 Protein Kinase Negatively Regulates the ATL1 E3 Ubiquitin Ligase to Suppress Cell Death[W

    Science.gov (United States)

    Serrano, Irene; Gu, Yangnan; Qi, Dong; Dubiella, Ullrich

    2014-01-01

    Loss-of-function mutations in the Arabidopsis thaliana ENHANCED DISEASE RESISTANCE1 (EDR1) gene confer enhanced programmed cell death under a variety of abiotic and biotic stress conditions. All edr1 mutant phenotypes can be suppressed by missense mutations in the KEEP ON GOING gene, which encodes a trans-Golgi network/early endosome (TGN/EE)-localized E3 ubiquitin ligase. Here, we report that EDR1 interacts with a second E3 ubiquitin ligase, ARABIDOPSIS TOXICOS EN LEVADURA1 (ATL1), and negatively regulates its activity. Overexpression of ATL1 in transgenic Arabidopsis induced severe growth inhibition and patches of cell death, while transient overexpression in Nicotiana benthamiana leaves induced cell death and tissue collapse. The E3 ligase activity of ATL1 was required for both of these processes. Importantly, we found that ATL1 interacts with EDR1 on TGN/EE vesicles and that EDR1 suppresses ATL1-mediated cell death in N. benthamiana and Arabidopsis. Lastly, knockdown of ATL1 expression suppressed cell death phenotypes associated with the edr1 mutant and made Arabidopsis hypersusceptible to powdery mildew infection. Taken together, our data indicate that ATL1 is a positive regulator of programmed cell death and EDR1 negatively regulates ATL1 activity at the TGN/EE and thus controls stress responses initiated by ATL1-mediated ubiquitination events. PMID:25398498

  18. Enhanced arsenic accumulation by engineered yeast cells expressing Arabidopsis thaliana phytochelatin synthase.

    Science.gov (United States)

    Singh, Shailendra; Lee, Wonkyu; Dasilva, Nancy A; Mulchandani, Ashok; Chen, Wilfred

    2008-02-01

    Phytochelatins (PCs) are naturally occurring peptides with high-binding capabilities for a wide range of heavy metals including arsenic (As). PCs are enzymatically synthesized by phytochelatin synthases and contain a (gamma-Glu-Cys)(n) moiety terminated by a Gly residue that makes them relatively proteolysis resistant. In this study, PCs were introduced by expressing Arabidopsis thaliana Phytochelatin Synthase (AtPCS) in the yeast Saccharomyces cerevisiae for enhanced As accumulation and removal. PCs production in yeast resulted in six times higher As accumulation as compared to the control strain under a wide range of As concentrations. For the high-arsenic concentration, PCs production led to a substantial decrease in levels of PC precursors such as glutathione (GSH) and gamma-glutamyl cysteine (gamma-EC). The levels of As(III) accumulation were found to be similar between AtPCS-expressing wild type strain and AtPCS-expressing acr3Delta strain lacking the arsenic efflux system, suggesting that the arsenic uptake may become limiting. This is further supported by the roughly 1:3 stoichiometric ratio between arsenic and PC2 (n = 2) level (comparing with a theoretical value of 1:2), indicating an excess availability of PCs inside the cells. However, at lower As(III) concentration, PC production became limiting and an additive effect on arsenic accumulation was observed for strain lacking the efflux system. More importantly, even resting cells expressing AtPCS pre-cultured in Zn(2+) enriched media showed PCs production and two times higher arsenic removal than the control strain. These results open up the possibility of using cells expressing AtPCS as an inexpensive sorbent for the removal of toxic arsenic.

  19. Allocation of Heme is Differentially Regulated by Ferrochelatase Isoforms in Arabidopsis Cells

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    Nino Asuela Espinas

    2016-08-01

    Full Text Available Heme is involved in various biological processes as a cofactor of hemoproteins located in various organelles. In plant cells, heme is synthesized by two isoforms of plastid-localized ferrochelatase, FC1 and FC2. In this study, by characterizing Arabidopsis T-DNA insertional mutants, we showed that the allocation of heme is differentially regulated by ferrochelatase isoforms in plant cells. Analyses of weak (fc1-1 and null (fc1-2 mutants suggest that FC1-producing heme is required for initial growth of seedling development. In contrast, weak (fc2-1 and null (fc2-2 mutants of FC2 showed pale green leaves and retarded growth, indicating that FC2-producing heme is necessary for chloroplast development. During the initial growth stage, FC2 deficiency caused reduction of plastid cytochromes. In addition, although FC2 deficiency marginally affected the assembly of photosynthetic reaction center complexes, it caused relatively larger but insufficient light-harvesting antenna to reaction centers, resulting in lower efficiency of photosynthesis. In the later vegetative growth, however, fc2-2 recovered photosynthetic growth, showing that FC1-producing heme may complement the FC2 deficiency. On the other hand, reduced level of cytochromes in microsomal fraction was discovered in fc1-1, suggesting that FC1-producing heme is mainly allocated to extraplastidic organelles. Furthermore, the expression of FC1 is induced by the treatment of an elicitor flg22 while that of FC2 was reduced, and fc1-1 abolished the flg22-dependent induction of FC1 expression and peroxidase activity. Consequently, our results clarified that FC2 produces heme for the photosynthetic machinery in the chloroplast, while FC1 is the housekeeping enzyme providing heme cofactor to the entire cell. In addition, FC1 can partly complement FC2 deficiency and is also involved in defense against stressful conditions.

  20. Crosstalks between myo-inositol metabolism, programmed cell death and basal immunity in Arabidopsis.

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    Ping Hong Meng

    Full Text Available BACKGROUND: Although it is a crucial cellular process required for both normal development and to face stress conditions, the control of programmed cell death in plants is not fully understood. We previously reported the isolation of ATXR5 and ATXR6, two PCNA-binding proteins that could be involved in the regulation of cell cycle or cell death. A yeast two-hybrid screen using ATXR5 as bait captured AtIPS1, an enzyme which catalyses the committed step of myo-inositol (MI biosynthesis. atips1 mutants form spontaneous lesions on leaves, raising the possibility that MI metabolism may play a role in the control of PCD in plants. In this work, we have characterised atips1 mutants to gain insight regarding the role of MI in PCD regulation. METHODOLOGY/PRINCIPAL FINDINGS: - lesion formation in atips1 mutants depends of light intensity, is due to PCD as evidenced by TUNEL labelling of nuclei, and is regulated by phytohormones such as salicylic acid - MI and galactinol are the only metabolites whose accumulation is significantly reduced in the mutant, and supplementation of the mutant with these compounds is sufficient to prevent PCD - the transcriptome profile of the mutant is extremely similar to that of lesion mimic mutants such as cpr5, or wild-type plants infected with pathogens. CONCLUSION/SIGNIFICANCE: Taken together, our results provide strong evidence for the role of MI or MI derivatives in the regulation of PCD. Interestingly, there are three isoforms of IPS in Arabidopsis, but AtIPS1 is the only one harbouring a nuclear localisation sequence, suggesting that nuclear pools of MI may play a specific role in PCD regulation and opening new research prospects regarding the role of MI in the prevention of tumorigenesis. Nevertheless, the significance of the interaction between AtIPS1 and ATXR5 remains to be established.

  1. Effect of pH on acid production from sorbitol in washed cell suspensions of oral bacteria.

    Science.gov (United States)

    Kalfas, S; Maki, Y; Birkhed, D; Edwardsson, S

    1990-01-01

    The acid production from sorbitol and glucose was studied under anaerobic conditions in resting cell suspensions of bacteria from the predominant sorbitol-fermenting human dental plaque flora, belonging to the genera Streptococcus, Lactobacillus and Actinomyces. The acid production activity of the bacterial cells was followed by titration with alkali, at environmental pH 7.0, 6.0 and 5.0 after addition of carbohydrate solution. The metabolic end products formed in the suspensions were analyzed thereafter by isotachophoretic and enzymatic methods. The results showed that sorbitol was fermented at a slower rate than glucose. Lowering the environmental pH decreased the acid production activity from the two carbohydrates. Compared with glucose, the catabolism of sorbitol was affected to greater extent by the pH conditions. The total amount of acids formed from sorbitol was considerably less than from glucose. Lactic acid, which was the major end product in glucose-challenged suspensions, was produced only in low concentrations from sorbitol by all strains tested. The ratio strong (formic + lactic)/weak acids was moreover lower for sorbitol than for glucose. The present results further illustrate some of the mechanisms behind the low cariogenic potential of this sugar substitute.

  2. Effects of mercury (II) species on cell suspension cultures of catharanthus roseus

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, L. (Hangzhou Univ. (China)); Cullen, W.R. (Univ. of British Columbia, Vancouver, British Columbia (Canada))

    1994-11-01

    Mercury has received considerable attention because of its high toxicity. Widespread contamination with mercury poses severe environmental problems despite our extensive knowledge of its toxicity in living systems. It is generally accepted that the toxicity of mercury is related to its oxidation states and species, the organic forms being more toxic than the inorganic forms. In the aquatic environment, the toxicity of mercury depends on the aqueous speciation of the mercuric ion (Hg[sup 2+]). Because of the complex coordination chemistry of mercury in aqueous systems, the nature of the Hg[sup 2+] species present in aquatic environments is influenced greatly by water chemistry (e. g, pH, inorganic ion composition, and dissolved organics). Consequently, the influence of environmental factors on the aqueous speciation of mercury has been the focus of much attention. However, there is very little information available regarding the effects of the species and speciation on Hg (II) toxicity in plant-tissue cultures. Catharanthus roseus (C. roseus), commonly called the Madagascar Periwinkle, is a member of the alkaloid rich family Apocynaceae. The present investigation was concerned with the toxicity of mercury on the growth of C. roseus cell suspension cultures as influenced by mercury (II) species and speciation. The specific objectives of the study were to (a) study the effects of mercury species on the growth of C. roseus cultures from the point of view of environmental biology and toxicology; (b) evaluate the effects of selenate, selenite and selected ligands such as chloride, 1-cysteine in the media on the acute toxicity of mercuric oxide; (c) determine the impact of the initial pH of the culture media on the toxicities of mercuric compounds; (d) discuss the dependence of the toxicity on the chemical species and speciation of Hg (II). 11 refs., 7 figs., 2 tabs.

  3. Structure of Plant Cell Walls: XI. GLUCURONOARABINOXYLAN, A SECOND HEMICELLULOSE IN THE PRIMARY CELL WALLS OF SUSPENSION-CULTURED SYCAMORE CELLS.

    Science.gov (United States)

    Darvill, J E; McNeil, M; Darvill, A G; Albersheim, P

    1980-12-01

    The isolation, purification, and partial characterization of a glucuronoarabinoxylan, a previously unobserved component of the primary cell walls of dicotyledonous plants, are described. The glucuronoarabinoxylan constitutes approximately 5% of the primary walls of suspension-cultured sycamore cells. This glucuronoarabinoxylan possesses many of the structural characteristics of analogous polysaccharides that have been isolated from the primary and secondary cell walls of monocots as well as from the secondary cell walls of dicots. The glucuronoarabinoxylan of primary dicot cell walls has a linear beta-1,4-linked d-xylopyranosyl backbone with both neutral and acidic sidechains attached at intervals along its length. The acidic sidechains are terminated with glucuronosyl or 4-O-methyl glucuronosyl residues, whereas the neutral sidechains are composed of arabinosyl and/or xylosyl residues.

  4. 3D Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae Using Focused Ion Beam–Scanning Electron Microscopy

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    Bhawana

    2014-06-01

    Full Text Available Premise of the study: Focused ion beam–scanning electron microscopy (FIB-SEM combines the ability to sequentially mill the sample surface and obtain SEM images that can be used to create 3D renderings with micron-level resolution. We have applied FIB-SEM to study Arabidopsis cell architecture. The goal was to determine the efficacy of this technique in plant tissue and cellular studies and to demonstrate its usefulness in studying cell and organelle architecture and distribution. Methods: Seed aleurone, leaf mesophyll, stem cortex, root cortex, and petal lamina from Arabidopsis were fixed and embedded for electron microscopy using protocols developed for animal tissues and modified for use with plant cells. Each sample was sectioned using the FIB and imaged with SEM. These serial images were assembled to produce 3D renderings of each cell type. Results: Organelles such as nuclei and chloroplasts were easily identifiable, and other structures such as endoplasmic reticula, lipid bodies, and starch grains were distinguishable in each tissue. Discussion: The application of FIB-SEM produced 3D renderings of five plant cell types and offered unique views of their shapes and internal content. These results demonstrate the usefulness of FIB-SEM for organelle distribution and cell architecture studies.

  5. Constitutive activation of AtMEK5, a MAPK kinase, induces salicylic acid-independent cell death in Arabidopsis thaliana

    Institute of Scientific and Technical Information of China (English)

    LIU Hongxia; WANG Ying; ZHOU Tianhong; SUN Yujing; LIU Guoqin; REN Dongtao

    2004-01-01

    AtMEK5DD is an active mutant of AtMEK5, a MAP kinase kinase in Arabidopsis. Induction of AtMEK5DD expression in transgenic plants leads to activation of 44 and 48 kD MAPKs and causes a rapid cell death. To compare the cell death induced by the expression of AtMEK5DD with the HR-cell death induced by avirulence pathogen infection, we analyzed the activation of downstream MAP Kinase and induction of PR genes expression in permanent transgenic Arabidopsis plants. In-gel kinase activity assay revealed that the infection of Pseudomonas syringae DC3000 harboring Avr Rpt2 gene also lead to activation of 44 and 48 kD MAPKs. PAL, PR1 and PR5 were strongly induced in plants undergoing HR-cell death caused by the infection of P. Syringae DC3000, while only the expression of PR5 was strongly induced in transgenic plants expressing AtMEK5DD protein. NahG protein in AtMEK5DD×NahG plants cannot suppress the cell death induced by AtMEK5DD. And AtMEK5DD protein expressed AtMEK5DD×NahG plants showed no significant change in salicylic acid (SA)level.All these suggest that the cell death induced by the activation of AtMEK5 is salicylic acid-independent.

  6. Fasciclin-like arabinogalactan proteins: specialization for stem biomechanics and cell wall architecture in Arabidopsis and Eucalyptus.

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    MacMillan, Colleen P; Mansfield, Shawn D; Stachurski, Zbigniew H; Evans, Rob; Southerton, Simon G

    2010-05-01

    The ancient cell adhesion fasciclin (FAS) domain is found in bacteria, fungi, algae, insects and animals, and occurs in a large family of fasciclin-like arabinogalactan proteins (FLAs) in higher plants. Functional roles for FAS-containing proteins have been determined for insects, algae and vertebrates; however, the biological functions of the various higher-plant FLAs are not clear. Expression of some FLAs has been correlated with the onset of secondary-wall cellulose synthesis in Arabidopsis stems, and also with wood formation in the stems and branches of trees, suggesting a biological role in plant stems. We examined whether FLAs contribute to plant stem biomechanics. Using phylogenetic, transcript abundance and promoter-GUS fusion analyses, we identified a conserved subset of single FAS domain FLAs (group A FLAs) in Eucalyptus and Arabidopsis that have specific and high transcript abundance in stems, particularly in stem cells undergoing secondary-wall deposition, and that the phylogenetic conservation appears to extend to other dicots and monocots. Gene-function analyses revealed that Arabidopsis T-DNA knockout double mutant stems had altered stem biomechanics with reduced tensile strength and a reduced tensile modulus of elasticity, as well as altered cell-wall architecture and composition, with increased cellulose microfibril angle and reduced arabinose, galactose and cellulose content. Using materials engineering concepts, we relate the effects of these FLAs on cell-wall composition with stem biomechanics. Our results suggest that a subset of single FAS domain FLAs contributes to plant stem strength by affecting cellulose deposition, and to the stem modulus of elasticity by affecting the integrity of the cell-wall matrix.

  7. Arabidopsis brassinosteroid biosynthetic mutant dwarf7-1 exhibits slower rates of cell division and shoot induction

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    Schulz Burkhard

    2010-12-01

    Full Text Available Abstract Background Plant growth depends on both cell division and cell expansion. Plant hormones, including brassinosteroids (BRs, are central to the control of these two cellular processes. Despite clear evidence that BRs regulate cell elongation, their roles in cell division have remained elusive. Results Here, we report results emphasizing the importance of BRs in cell division. An Arabidopsis BR biosynthetic mutant, dwarf7-1, displayed various characteristics attributable to slower cell division rates. We found that the DWARF4 gene which encodes for an enzyme catalyzing a rate-determining step in the BR biosynthetic pathways, is highly expressed in the actively dividing callus, suggesting that BR biosynthesis is necessary for dividing cells. Furthermore, dwf7-1 showed noticeably slower rates of callus growth and shoot induction relative to wild-type control. Flow cytometric analyses of the nuclei derived from either calli or intact roots revealed that the cell division index, which was represented as the ratio of cells at the G2/M vs. G1 phases, was smaller in dwf7-1 plants. Finally, we found that the expression levels of the genes involved in cell division and shoot induction, such as PROLIFERATING CELL NUCLEAR ANTIGEN2 (PCNA2 and ENHANCER OF SHOOT REGENERATION2 (ESR2, were also lower in dwf7-1 as compared with wild type. Conclusions Taken together, results of callus induction, shoot regeneration, flow cytometry, and semi-quantitative RT-PCR analysis suggest that BRs play important roles in both cell division and cell differentiation in Arabidopsis.

  8. Evaluation of cell wall preparations for proteomics: a new procedure for purifying cell walls from Arabidopsis hypocotyls

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    Canut Hervé

    2006-05-01

    Full Text Available Abstract Background The ultimate goal of proteomic analysis of a cell compartment should be the exhaustive identification of resident proteins; excluding proteins from other cell compartments. Reaching such a goal closely depends on the reliability of the isolation procedure for the cell compartment of interest. Plant cell walls possess specific difficulties: (i the lack of a surrounding membrane may result in the loss of cell wall proteins (CWP during the isolation procedure, (ii polysaccharide networks of cellulose, hemicelluloses and pectins form potential traps for contaminants such as intracellular proteins. Several reported procedures to isolate cell walls for proteomic analyses led to the isolation of a high proportion (more than 50% of predicted intracellular proteins. Since isolated cell walls should hold secreted proteins, one can imagine alternative procedures to prepare cell walls containing a lower proportion of contaminant proteins. Results The rationales of several published procedures to isolate cell walls for proteomics were analyzed, with regard to the bioinformatic-predicted subcellular localization of the identified proteins. Critical steps were revealed: (i homogenization in low ionic strength acid buffer to retain CWP, (ii purification through increasing density cushions, (iii extensive washes with a low ionic strength acid buffer to retain CWP while removing as many cytosolic proteins as possible, and (iv absence of detergents. A new procedure was developed to prepare cell walls from etiolated hypocotyls of Arabidopsis thaliana. After salt extraction, a high proportion of proteins predicted to be secreted was released (73%, belonging to the same functional classes as proteins identified using previously described protocols. Finally, removal of intracellular proteins was obtained using detergents, but their amount represented less than 3% in mass of the total protein extract, based on protein quantification. Conclusion The

  9. Systematic secretome analyses of rice leaf and seed callus suspension-cultured cells: workflow development and establishment of high-density two-dimensional gel reference maps.

    Science.gov (United States)

    Jung, Young-Ho; Jeong, Seung-Hee; Kim, So Hee; Singh, Raksha; Lee, Jae-Eun; Cho, Yoon-Seong; Agrawal, Ganesh Kumar; Rakwal, Randeep; Jwa, Nam-Soo

    2008-12-01

    Secreted proteins control a multitude of biological and physiological processes in multicellular organisms such as plants. Identification of secreted proteins in reference plants like Arabidopsis and rice under normal growth conditions and adverse environmental conditions will help better understand the secretory pathways. Here, we have performed a systematic in planta and in vitro analyses of proteins secreted by rice leaves (in planta) and seed callus suspension-cultured cells (SCCs; in vitro), respectively, using a combination of biochemical and two-dimensional gel electrophoresis (2-DGE) coupled with liquid chromatography mass spectrometry analyses. Secreted proteins prepared from either leaves or SCCs medium were essentially free from contamination of intracellular proteins as judged by biochemical and Western blot analyses. 2-DGE analyses of secreted proteins collectively identified 222 protein spots with only 6 protein spots common to both in planta and in vitro derived data sets. Data were used to establish high-resolution and high-density 2-D gel reference maps for both in planta and in vitro secreted proteins. Identified proteins belonged to 11 (in planta) and 6 (in vitro) functional classes. Proteins involved in carbon metabolism (33%) and cell wall metabolism having plant defense mechanism (18%) were highly represented in the in planta secreted proteins accounting for 51% of total identified proteins, whereas proteins of cell wall metabolism having plant defense mechanism (64%) were predominant in the in vitro secreted proteins. Interestingly, secreted proteins possessing signal peptides were significantly lower in an in planta (27%) prepared secreted protein population than in vitro (76%) as predicted by SignalP prediction tool, implying the notion that plant might possess yet unidentified secretory pathway(s) in addition to the classical endoplasmic reticulum/Golgi pathway. Taken together, this systematic study provides evidence for (i) significant

  10. Novel Nuclear Protein ALC-INTERACTING PROTEIN1 is Expressed in Vascular and Mesocarp Cells in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    Fang Wang; Dong-Qiao Shi; Jie Liu; Wei-Cai Yang

    2008-01-01

    Pod shattering is an agronomical trait that is a result of the coordinated action of cell differentiation and separation. In Arabidopsis, pod shattering is controlled by a complex genetic network in which ALCATRAZ (ALC), a member of the basic helix-loop-helix family, is critical for cell separation during fruit dehiscence. Herein, we report the identification of ALC-INTERACTiNG PROTEIN1 (ACI1) via the yeast two-hybrid screen. ACI1 encodes a nuclear protein with a lysine-rich domain and a C-terminal serine-rich domain. ACI1 is mainly expressed in the vascular system throughout the plant and mesocarp of the valve in siliques. Our data showed that ACI1 interacts strongly with the N-terminal portion of ALC in yeast cells and in plant cells in the nucleus as demonstrated by bimolecular fluorescence complementation assay. Both ACl1 and ALC share an overlapping expression pattern, suggesting that they likely function together in planta. However, no detectable phenotype was found in plants with reduced ACI1 expression by RNA interference technology, suggesting that ACI1 may be redundant. Taken together, these data indicate that ALC may interact with ACll and its homologs to control cell separation during fruit dehiscence in Arabidopsis.

  11. The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules.

    Science.gov (United States)

    Bemer, Marian; Wolters-Arts, Mieke; Grossniklaus, Ueli; Angenent, Gerco C

    2008-08-01

    MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein-beta-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.

  12. EBE, an AP2/ERF transcription factor highly expressed in proliferating cells, affects shoot architecture in Arabidopsis.

    Science.gov (United States)

    Mehrnia, Mohammad; Balazadeh, Salma; Zanor, María-Inés; Mueller-Roeber, Bernd

    2013-06-01

    We report about ERF BUD ENHANCER (EBE; At5g61890), a transcription factor that affects cell proliferation as well as axillary bud outgrowth and shoot branching in Arabidopsis (Arabidopsis thaliana). EBE encodes a member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor superfamily; the gene is strongly expressed in proliferating cells and is rapidly and transiently up-regulated in axillary meristems upon main stem decapitation. Overexpression of EBE promotes cell proliferation in growing calli, while the opposite is observed in EBE-RNAi lines. EBE overexpression also stimulates axillary bud formation and outgrowth, while repressing it results in inhibition of bud growth. Global transcriptome analysis of estradiol-inducible EBE overexpression lines revealed 48 EBE early-responsive genes, of which 14 were up-regulated and 34 were down-regulated. EBE activates several genes involved in cell cycle regulation and dormancy breaking, including D-type cyclin CYCD3;3, transcription regulator DPa, and BRCA1-ASSOCIATED RING DOMAIN1. Among the down-regulated genes were DORMANCY-ASSOCIATED PROTEIN1 (AtDRM1), AtDRM1 homolog, MEDIATOR OF ABA-REGULATED DORMANCY1, and ZINC FINGER HOMEODOMAIN5. Our data indicate that the effect of EBE on shoot branching likely results from an activation of genes involved in cell cycle regulation and dormancy breaking.

  13. AtPGL3 is an Arabidopsis BURP domain protein that is localized to the cell wall and promotes cell enlargement

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    Jiyoung ePark

    2015-06-01

    Full Text Available The BURP domain is a plant-specific protein domain that has been identified in secretory proteins, and some of these are involved in cell wall remodeling. Among Arabidopsis BURP domain proteins, three proteins exhibit strong amino acid similarities with the tomato polygalacturonase 1 beta (PG1β protein that interacts with a pectin-digesting enzyme. To investigate biological roles of the Arabidopsis PG1β-like proteins (AtPGLs, we generated Arabidopsis lines in which expression of AtPGLs is altered. Among the three AtPGLs, AtPGL3 exhibited highest transcriptional activity throughout all developmental stages. When tissue-specific expression pattern of AtPGL3 was examined, the gene was observed to be active in epidermal cell layers of rosette leaves and in the trichomes. AtPGL triple mutant plants were smaller than wild type plants because cells were smaller in the mutant plants. Interestingly, when we overexpressed AtPGL3 using a 35S promoter, cells in transgenic plants grew larger than those of the wild type, suggesting that AtPGL3 plays a role in cell expansion. A C-terminal GFP fusion protein of AtPGL3 complemented phenotypes of the triple mutant plants and localized to the cell wall. A truncated AtPGL3-GFP fusion protein that lacks the BURP domain failed to rescue the mutant phenotypes even though the GFP protein was targeted to the cell wall, indicating that the BURP domain is required for its effect on cell expansion. Quantitative RT-PCR and immunoblot analyses indicated that 2 α-expansin genes are down-regulated and up-regulated in the triple mutant and overexpressor lines, respectively. Taken together, AtPGL3 is a cell wall protein required for normal cell expansion and the coexpression results suggest that AtPGLs regulate cell wall loosening, in conjunction with α-expansins, to promote cell growth.

  14. Putrescine Alleviates Iron Deficiency via NO-Dependent Reutilization of Root Cell-Wall Fe in Arabidopsis.

    Science.gov (United States)

    Zhu, Xiao Fang; Wang, Bin; Song, Wen Feng; Zheng, Shao Jian; Shen, Ren Fang

    2016-01-01

    Plants challenged with abiotic stress show enhanced polyamines levels. Here, we show that the polyamine putrescine (Put) plays an important role to alleviate Fe deficiency. The adc2-1 mutant, which is defective in Put biosynthesis, was hypersensitive to Fe deficiency compared with wild type (Col-1 of Arabidopsis [Arabidopsis thaliana]). Exogenous Put decreased the Fe bound to root cell wall, especially to hemicellulose, and increased root and shoot soluble Fe content, thus alleviating the Fe deficiency-induced chlorosis. Intriguingly, exogenous Put induced the accumulation of nitric oxide (NO) under both Fe-sufficient (+Fe) and Fe-deficient (-Fe) conditions, although the ferric-chelate reductase (FCR) activity and the expression of genes related to Fe uptake were induced only under -Fe treatment. The alleviation of Fe deficiency by Put was diminished in the hemicellulose-level decreased mutant-xth31 and in the noa1 and nia1nia2 mutants, in which the endogenous NO levels are reduced, indicating that both NO and hemicellulose are involved in Put-mediated alleviation of Fe deficiency. However, the FCR activity and the expression of genes related to Fe uptake were still up-regulated under -Fe+Put treatment compared with -Fe treatment in xth31, and Put-induced cell wall Fe remobilization was abolished in noa1 and nia1nia2, indicating that Put-regulated cell wall Fe reutilization is dependent on NO. From our results, we conclude that Put is involved in the remobilization of Fe from root cell wall hemicellulose in a process dependent on NO accumulation under Fe-deficient condition in Arabidopsis.

  15. A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal.

    Science.gov (United States)

    Sivanandhan, Ganeshan; Kapil Dev, Gnanajothi; Jeyaraj, Murugaraj; Rajesh, Manoharan; Muthuselvam, Manickam; Selvaraj, Natesan; Manickavasagam, Markandan; Ganapathi, Andy

    2013-08-01

    Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.

  16. WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning.

    Science.gov (United States)

    Lee, M M; Schiefelbein, J

    1999-11-24

    The formation of the root epidermis of Arabidopsis provides a simple and elegant model for the analysis of cell patterning. A novel gene, WEREWOLF (WER), is described here that is required for position-dependent patterning of the epidermal cell types. The WER gene encodes a MYB-type protein and is preferentially expressed within cells destined to adopt the non-hair fate. Furthermore, WER is shown to regulate the position-dependent expression of the GLABRA2 homeobox gene, to interact with a bHLH protein, and to act in opposition to the CAPRICE MYB. These results suggest a simple model to explain the specification of the two root epidermal cell types, and they provide insight into the molecular mechanisms used to control cell patterning.

  17. Identifying new components participating in the secondary cell wall formation of vessel elements in zinnia and Arabidopsis.

    Science.gov (United States)

    Endo, Satoshi; Pesquet, Edouard; Yamaguchi, Masatoshi; Tashiro, Gen; Sato, Mayuko; Toyooka, Kiminori; Nishikubo, Nobuyuki; Udagawa-Motose, Makiko; Kubo, Minoru; Fukuda, Hiroo; Demura, Taku

    2009-04-01

    Xylem vessel elements are hollow cellular units that assemble end-to-end to form a continuous vessel throughout the plant body; the xylem vessel is strengthened by the xylem elements' reinforced secondary cell walls (SCWs). This work aims to unravel the contribution of unknown actors in xylem vessel differentiation using the model in vitro cell culture system of Zinnia elegans differentiating cell cultures and the model in vivo system of Arabidopsis thaliana plants. Tracheary Element Differentiation-Related6 (TED6) and TED7 were selected based on an RNA interference (RNAi) screen in the Zinnia system. RNAi reduction of TED6 and 7 delayed tracheary element (TE) differentiation and co-overexpression of TED6 and 7 increased TE differentiation in cultured Zinnia cells. Arabidopsis TED6 and 7 were expressed preferentially in differentiating vessel elements in seedlings. Aberrant SCW formation of root vessel elements was induced by transient RNAi of At TED7 alone and enhanced by inhibition of both TED6 and 7. Protein-protein interactions were demonstrated between TED6 and a subunit of the SCW-related cellulose synthase complex. Our strategy has succeeded in finding two novel components in SCW formation and has opened the door for in-depth analysis of their molecular functions.

  18. Diuretics Prime Plant Immunity in Arabidopsis thaliana

    Science.gov (United States)

    Noutoshi, Yoshiteru; Ikeda, Mika; Shirasu, Ken

    2012-01-01

    Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application. PMID:23144763

  19. Diuretics prime plant immunity in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Yoshiteru Noutoshi

    Full Text Available Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application.

  20. Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

    Science.gov (United States)

    Dores, C; Rancourt, D; Dobrinski, I

    2015-05-01

    To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling.

  1. Host-Pathogen Interactions : XXIV. Fragments Isolated from Suspension-Cultured Sycamore Cell Walls Inhibit the Ability of the Cells to Incorporate [C]Leucine into Proteins.

    Science.gov (United States)

    Yamazaki, N; Fry, S C; Darvill, A G; Albersheim, P

    1983-07-01

    A bioassay to measure the incorporation of [(14)C]leucine into acid-precipitable polymers of suspension-cultured sycamore (Acer pseudoplatanus L.) cells is described. Using this assay, cell wall fragments solubilized from sycamore cell walls by partial acid hydrolysis are shown to contain components that inhibit the incorporation of [(14)C]leucine into the acid-precipitable polymers. This inhibition was not attributable to a suppression of [(14)C]leucine uptake. The effectiveness of the wall fragments in inhibiting [(14)C]leucine incorporation was substantially relieved by plasmolysis of the cells. Fragments released from starch and citrus pectin are shown not to possess such inhibitory activities.

  2. Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.

    Science.gov (United States)

    Cavez, Damien; Hachez, Charles; Chaumont, François

    2009-09-01

    Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

  3. Molecular cell biology of male meiotic chromosomes and isolation of male meiocytes in Arabidopsis thaliana.

    Science.gov (United States)

    Wang, Yingxiang; Cheng, Zhihao; Lu, Pingli; Timofejeva, Ljudmilla; Ma, Hong

    2014-01-01

    Plants typically produce numerous flowers whose meiotic chromosomes are relatively easy to observe, making them excellent structures for studying the cellular processes underlying meiosis. In recent years, breakthroughs in light and electron microscopic technologies for small chromosomes, combined with molecular genetic methods, have resulted in major advances in the understanding of meiosis in the model plant Arabidopsis thaliana. In this chapter, we summarize protocols for basic cytology, fluorescence in situ hybridization, immunofluorescence, electron microscopy, and isolation of male meiocytes for the analysis of Arabidopsis meiosis.

  4. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    Directory of Open Access Journals (Sweden)

    Takemoto Daigo

    2008-06-01

    Full Text Available Abstract Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the

  5. Bio-inactivation of human malignant cells through highly responsive diluted colloidal suspension of functionalized magnetic iron oxide nanoparticles

    Science.gov (United States)

    Ferreira, Roberta V.; Silva-Caldeira, Priscila P.; Pereira-Maia, Elene C.; Fabris, José D.; Cavalcante, Luis Carlos D.; Ardisson, José D.; Domingues, Rosana Z.

    2016-04-01

    Magnetic fluids, more specifically aqueous colloidal suspensions containing certain magnetic nanoparticles (MNPs), have recently been gaining special interest due to their potential use in clinical treatments of cancerous formations in mammalians. The technological application arises mainly from their hyperthermic behavior, which means that the nanoparticles dissipate heat upon being exposed to an alternating magnetic field (AMF). If the temperature is raised to slightly above 43 °C, cancer cells are functionally inactivated or killed; however, normal cells tend to survive under those same conditions, entirely maintaining their bioactivity. Recent in vitro studies have revealed that under simultaneous exposure to an AMF and magnetic nanoparticles, certain lines of cancer cells are bio-inactivated even without experiencing a significant temperature increase. This non-thermal effect is cell specific, indicating that MNPs, under alternating magnetic fields, may effectively kill cancer cells under conditions that were previously thought to be implausible, considering that the temperature does not increase more than 5 °C, which is also true in cases for which the concentration of MNPs is too low. To experimentally test for this effect, this study focused on the feasibility of inducing K562 cell death using an AMF and aqueous suspensions containing very low concentrations of MNPs. The assay was designed for a ferrofluid containing magnetite nanoparticles, which were obtained through the co-precipitation method and were functionalized with citric acid; the particles had an average diameter of 10 ± 2 nm and a mean hydrodynamic diameter of approximately 40 nm. Experiments were first performed to test for the ability of the ferrofluid to release heat under an AMF. The results show that for concentrations ranging from 2.5 to 1.0 × 103 mg L-1, the maximum temperature increase was actually less than 2 °C. However, the in vitro test results from K562 cells and suspensions

  6. Inducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture.

    Science.gov (United States)

    Broussau, Sophie; Jabbour, Nadine; Lachapelle, Guillaume; Durocher, Yves; Tom, Rosanne; Transfiguracion, Julia; Gilbert, Rénald; Massie, Bernard

    2008-03-01

    We have developed new packaging cell lines (293SF-PacLV) that can produce lentiviral vectors (LVs) in serum-free suspension cultures. A cell line derived from 293SF cells, expressing the repressor (CymR) of the cumate switch and the reverse transactivator (rtTA2(S)-M2) of the tetracycline (Tet) switch, was established first. We next generated clones stably expressing the Gag/Pol and Rev genes of human immunodeficiency virus-1, and the glycoprotein of vesicular stomatitis virus (VSV-G). Expression of Rev and VSV-G was tightly regulated by the cumate and Tet switches. Our best packaging cells produced up to 2.6 x 10(7) transducing units (TU)/ml after transfection with the transfer vector. Up to 3.4 x 10(7) TU/ml were obtained using stable producers generated by transducing the packaging cells with conditional-SIN-LV. The 293SF-PacLV was stable, as shown by the fact that some producers maintained high-level LV production for 18 weeks without selective pressure. The utility of the 293SF-PacLV for scaling up production in serum-free medium was demonstrated in suspension cultures and in a 3.5-L bioreactor. In shake flasks, the best packaging cells produced between 3.0 and 8.0 x 10(6) TU/ml/day for 3 days, and the best producer cells, between 1.0 and 3.4 x 10(7) TU/ml/day for 5 days. In the bioreactor, 2.8 liters containing 2.0 x 10(6) TU/ml was obtained after 3 days of batch culture following the transfection of packaging cells. In summary, the 293SF-PacLV possesses all the attributes necessary to become a valuable tool for scaling up LV production for preclinical and clinical applications.

  7. Comparison of mesencephalic free-floating tissue culture grafts and cell suspension grafts in the 6-hydroxydopamine-lesioned rat

    DEFF Research Database (Denmark)

    Meyer, Morten; Widmer, H R; Wagner, B;

    1998-01-01

    . The amphetamine-induced rotational behavior of all 6-OHDA-lesioned animals was monitored at various time points from 18 days before transplantation and up to 80 days after transplantation. Tyrosine hydroxylase (TH) immunostaining of the histologically processed brains served to assess the long-term survival...... improvements in terms of significant reductions in amphetamine-induced rotations were observed in rats grafted with FFRT cultures (127%) and rats grafted with cell suspensions (122%), while control animals showed no normalization of rotational behavior. At 84 days after transplantation, there were similar...

  8. A conserved function for Arabidopsis SUPERMAN in regulating floral-whorl cell proliferation in rice, a monocotyledonous plant.

    Science.gov (United States)

    Nandi, A K; Kushalappa, K; Prasad, K; Vijayraghavan, U

    2000-02-24

    Studies of floral organ development in two dicotyledonous plants, Arabidopsis thaliana and Antirrhinum majus, have shown that three sets of genes (A, B and C) can pattern sepals, petals, stamens and carpels [1] [2]. Mechanisms that define boundaries between these floral whorls are unclear, however. The Arabidopsis gene SUPERMAN (SUP), which encodes a putative transcription factor, maintains the boundary between stamens and carpels [3] [4] [5], possibly by regulating cell proliferation. By overexpressing SUP cDNA in rice, we examined whether its effects on whorl boundaries are conserved in a divergent monocotyledonous species. High-level ectopic SUP expression in transgenic rice resulted in juvenile death or dwarf plants with decreased axillary growth. Plants with lower levels of SUP RNA were vegetatively normal, but the flowers showed ubiquitous ventral carpel expansion. This was often coupled with reduced stamen number, or occurrence of third-whorl stamen-carpel mosaic organs. Additionally, proliferation of second-whorl ventral cells produced adventitious lodicules, and flowers lost the asymmetry that is normally inherent to this whorl. We predict that SUP is a conserved regulator of floral whorl boundaries and that it affects cell proliferation.

  9. Glycyrrhiza glabra (Linn.) and Lavandula officinalis (L.) cell suspension cultures-based biotransformation of β-artemether.

    Science.gov (United States)

    Patel, Suman; Gaur, Rashmi; Upadhyaya, Mohita; Mathur, Archana; Mathur, Ajay K; Bhakuni, Rajendra S

    2011-07-01

    The biotransformation of β-artemether (1) by cell suspension cultures of Glycyrrhiza glabra and Lavandula officinalis is reported here for the first time. The major biotransformed product appeared as a grayish-blue color spot on thin-layer chromatography (TLC) with transparent crystal-like texture. Based on its infrared (IR) and (1)H nuclear magnetic resonance (NMR) spectra, the product was characterized as a tetrahydrofuran (THF)-acetate derivative (2). The highest conversion efficiencies of 57 and 60% were obtained when 8-9-day-old cell suspensions of G. glabra and L. officinalis were respectively fed with 4-7 mg of compound 1 in 40 ml of medium per culture and the cells were harvested after 2-5 days of incubation. The addition of compound 1 at the beginning of the culture cycle caused severe growth depression in a dose-dependent manner, resulting in poor bioconversion efficiency of ~25% at 2-5 mg/culture dose only.

  10. Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia.

    Science.gov (United States)

    dos Santos, André Luis Wendt; El Gueddari, Nour Eddine; Trombotto, Stéphane; Moerschbacher, Bruno Maria

    2008-12-01

    Suspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia , using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1-69%), chitin oligomers of different degrees of polymerization (DP 3-6), and chitosan oligomer of different DA (0-91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24-72 h in the presence of 1-10 microg mL(-1) chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia , the induction of an oxidative burst is not directly coupled to the induction of other defense reactions.

  11. Suspension model for blood flow through a catheterized arterial stenosis with peripheral layer of plasma free from cells

    Science.gov (United States)

    Ponalagusamy, R.

    2016-06-01

    The present article describes the blood flow in a catheterized artery with radially symmetric and axially asymmetric stenosis. To understand the effects of red cell concentration, plasma layer thickness and catheter size simultaneously, blood is considered by a two-layered model comprising a core region of suspension of all the erythrocytes (particles) supposed to be a particle-fluid mixture and a peripheral zone of cell-free plasma. The analytical expressions for flow features, such as fluid phase and particle phase velocities, flow rate, wall shear stress and resistive force are obtained. It is witnessed that the presence of the catheter causes a substantial increase in the frictional forces on the walls of arterial stenosis and catheter, shear stress and flow resistance, in addition to that, have occurred due to the presence of red cells concentration (volume fraction density of the particles) and the absence of peripheral plasma layer near the wall of the stenosed artery. The introduction of an axially asymmetric nature of stenosis and plasma layer thickness causes significant reduction in flow resistance. One can notice that the two-phase fluid (suspension model) is more profound to the thickness of peripheral plasma layer and catheter than the single-phase fluid.

  12. Xylogalacturonan exists in cell walls from various tissues of Arabidopsis thaliana

    NARCIS (Netherlands)

    Zandleven, J.S.; Sorensen, S.; Harbolt, J.; Beldman, G.; Schols, H.A.; Scheller, H.V.; Voragen, A.G.J.

    2007-01-01

    Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH)

  13. Brownian Dynamics of a Suspension of Particles with Constrained Voronoi Cell Volumes

    KAUST Repository

    Singh, John P.

    2015-06-23

    © 2015 American Chemical Society. Solvent-free polymer-grafted nanoparticle fluids consist of inorganic core particles fluidized by polymers tethered to their surfaces. The attachment of the suspending fluid to the particle surface creates a strong penalty for local variations in the fluid volume surrounding the particles. As a model of such a suspension we perform Brownian dynamics of an equilibrium system consisting of hard spheres which experience a many-particle potential proportional to the variance of the Voronoi volumes surrounding each particle (E = α(Vi-V0)2). The coefficient of proportionality α can be varied such that pure hard sphere dynamics is recovered as α → 0, while an incompressible array of hairy particles is obtained as α →. As α is increased the distribution of Voronoi volumes becomes narrower, the mean coordination number of the particle increases and the variance in the number of nearest neighbors decreases. The nearest neighbor peaks in the pair distribution function are suppressed and shifted to larger radial separations as the constraint acts to maintain relatively uniform interstitial regions. The structure factor of the model suspension satisfies S(k=0) → 0 as α → in accordance with expectation for a single component (particle plus tethered fluid) incompressible system. The tracer diffusivity of the particles is reduced by the volume constraint and goes to zero at φ 0.52, indicating an earlier glass transition than has been observed in hard sphere suspensions. The total pressure of the suspension grows in proportion to (αkBT)1/2 as the strength of the volume-constraint potential grows. This stress arises primarily from the interparticle potential forces, while the hard-sphere collisional contribution to the stress is suppressed by the volume constraint.

  14. Engineering of red cells of Arabidopsis thaliana and comparative genome-wide gene expression analysis of red cells versus wild-type cells.

    Science.gov (United States)

    Shi, Ming-Zhu; Xie, De-Yu

    2011-04-01

    We report metabolic engineering of Arabidopsis red cells and genome-wide gene expression analysis associated with anthocyanin biosynthesis and other metabolic pathways between red cells and wild-type (WT) cells. Red cells of A. thaliana were engineered for the first time from the leaves of production of anthocyanin pigment 1-Dominant (pap1-D). These red cells produced seven anthocyanin molecules including a new one that was characterized by LC-MS analysis. Wild-type cells established as a control did not produce anthocyanins. A genome-wide microarray analysis revealed that nearly 66 and 65% of genes in the genome were expressed in the red cells and wild-type cells, respectively. In comparison with the WT cells, 3.2% of expressed genes in the red cells were differentially expressed. The expression levels of 14 genes involved in the biosynthetic pathway of anthocyanin were significantly higher in the red cells than in the WT cells. Microarray and RT-PCR analyses demonstrated that the TTG1-GL3/TT8-PAP1 complex regulated the biosynthesis of anthocyanins. Furthermore, most of the genes with significant differential expression levels in the red cells versus the WT cells were characterized with diverse biochemical functions, many of which were mapped to different metabolic pathways (e.g., ribosomal protein biosynthesis, photosynthesis, glycolysis, glyoxylate metabolism, and plant secondary metabolisms) or organelles (e.g., chloroplast). We suggest that the difference in gene expression profiles between the two cell lines likely results from cell types, the overexpression of PAP1, and the high metabolic flux toward anthocyanins.

  15. Salicylic acid antagonism of EDS1-driven cell death is important for immune and oxidative stress responses in Arabidopsis.

    Science.gov (United States)

    Straus, Marco R; Rietz, Steffen; Ver Loren van Themaat, Emiel; Bartsch, Michael; Parker, Jane E

    2010-05-01

    Reactive oxygen species (ROS) have emerged as signals in the responses of plants to stress. Arabidopsis Enhanced Disease Susceptibility1 (EDS1) regulates defense and cell death against biotrophic pathogens and controls cell death propagation in response to chloroplast-derived ROS. Arabidopsis Nudix hydrolase7 (nudt7) mutants are sensitized to photo-oxidative stress and display EDS1-dependent enhanced resistance, salicylic acid (SA) accumulation and initiation of cell death. Here we explored the relationship between EDS1, EDS1-regulated SA and ROS by examining gene expression profiles, photo-oxidative stress and resistance phenotypes of nudt7 mutants in combination with eds1 and the SA-biosynthetic mutant, sid2. We establish that EDS1 controls steps downstream of chloroplast-derived O(2)(*-) that lead to SA-assisted H(2)O(2) accumulation as part of a mechanism limiting cell death. A combination of EDS1-regulated SA-antagonized and SA-promoted processes is necessary for resistance to host-adapted pathogens and for a balanced response to photo-oxidative stress. In contrast to SA, the apoplastic ROS-producing enzyme NADPH oxidase RbohD promotes initiation of cell death during photo-oxidative stress. Thus, chloroplastic O(2)(*-) signals are processed by EDS1 to produce counter-balancing activities of SA and RbohD in the control of cell death. Our data strengthen the idea that EDS1 responds to the status of O(2)(*-) or O(2)(*-)-generated molecules to coordinate cell death and defense outputs. This activity may enable the plant to respond flexibly to different biotic and abiotic stresses in the environment.

  16. Reprogramming of enteroendocrine K cells to pancreatic β-cells through the combined expression of Nkx6.1 and Neurogenin3, and reaggregation in suspension culture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Esder; Ryu, Gyeong Ryul; Moon, Sung-Dae; Ko, Seung-Hyun; Ahn, Yu-Bae; Song, Ki-Ho, E-mail: kihos@catholic.ac.kr

    2014-01-17

    Highlights: •K cells were selected from STC-1 cells, a heterogeneous enteroendocrine cell line. •K cells did not express Nkx6.1 and Neurogenin3. •Combined expression of Nkx6.1 and Neurogenin3 reprogrammed K cells to β-cells. •Reprogramming of K cells to β-cells was not complete. -- Abstract: Recent studies have demonstrated that adult cells such as pancreatic exocrine cells can be converted to pancreatic β-cells in a process called cell reprogramming. Enteroendocrine cells and β-cells share similar pathways of differentiation during embryonic development. Notably, enteroendocrine K cells express many of the key proteins found in β-cells. Thus, K cells could be reprogrammed to β-cells under certain conditions. However, there is no clear evidence on whether these cells convert to β-cells. K cells were selected from STC-1 cells, an enteroendocrine cell line expressing multiple hormones. K cells were found to express many genes of transcription factors crucial for islet development and differentiation except for Nkx6.1 and Neurogenin3. A K cell clone stably expressing Nkx6.1 (Nkx6.1{sup +}-K cells) was established. Induction of Neurogenin3 expression in Nkx6.1{sup +}-K cells, by either treatment with a γ-secretase inhibitor or infection with a recombinant adenovirus expressing Neurogenin3, led to a significant increase in Insulin1 mRNA expression. After infection with the adenovirus expressing Neurogenin3 and reaggregation in suspension culture, about 50% of Nkx6.1{sup +}-K cells expressed insulin as determined by immunostaining. The intracellular insulin content was increased markedly. Electron microscopy revealed the presence of insulin granules. However, glucose-stimulated insulin secretion was defective, and there was no glucose lowering effect after transplantation of these cells in diabetic mice. In conclusion, we demonstrated that K cells could be reprogrammed partially to β-cells through the combined expression of Nkx6.1 and Neurogenin3, and

  17. Increased sesquiterpenoid biosynthesis and an apparent decrease in sterol biosynthesis in elicitor-treated tobacco cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Voegeli, U.; Bhatt, P.N.; Chappell, J.

    1987-04-01

    Addition of fungel elicitor prepared from Phytophthora parasitica to tobacco cell suspension cultures leads to an increased production of the phytoalexin capsidiol. Capsidiol is a sesquiterpenoid which is most likely synthesized from farnesylpyrophosphat (FPP) by a bicyclic cyclase reaction. Because FPP is also a substrate for squalene synthetase and therefore a precursor of sterol biosynthesis, the question arises whether or not the accumulation of capsidiol in elicitor-treated cells occurs at the expense of sterol biosynthesis. (/sup 14/C)-acetate was given to elicitor-treated and control (no treatment) cell cultures and incorporation into sterols and capsidiol determined. No labeled capsidiol was detected in control cells. In elicitor-treated cells about 12-15% of the radioactivity taken up by the cells was incorporated into capsidiol. In contrast, control cells incorporated 4 times more radioactivity into sterols than elicitor-treated cells. Similar results were obtained using (/sup 3/H)-mevalonate as a precursor of capsidiol and sterol biosynthesis. Likely explanations for the apparently decline in sterol biosynthesis in elicitor-treated cells include: (1) inhibition of squalene synthetase; (2) induction of capsidiol synthesizing enzymes; and (3) metabolic channeling of FPP into capsidiol versus sterols. These possibilities will be discussed further together with other results.

  18. Conserved CDC20 cell cycle functions are carried out by two of the five isoforms in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Zoltán Kevei

    Full Text Available BACKGROUND: The CDC20 and Cdh1/CCS52 proteins are substrate determinants and activators of the Anaphase Promoting Complex/Cyclosome (APC/C E3 ubiquitin ligase and as such they control the mitotic cell cycle by targeting the degradation of various cell cycle regulators. In yeasts and animals the main CDC20 function is the destruction of securin and mitotic cyclins. Plants have multiple CDC20 gene copies whose functions have not been explored yet. In Arabidopsis thaliana there are five CDC20 isoforms and here we aimed at defining their contribution to cell cycle regulation, substrate selectivity and plant development. METHODOLOGY/PRINCIPAL FINDINGS: Studying the gene structure and phylogeny of plant CDC20s, the expression of the five AtCDC20 gene copies and their interactions with the APC/C subunit APC10, the CCS52 proteins, components of the mitotic checkpoint complex (MCC and mitotic cyclin substrates, conserved CDC20 functions could be assigned for AtCDC20.1 and AtCDC20.2. The other three intron-less genes were silent and specific for Arabidopsis. We show that AtCDC20.1 and AtCDC20.2 are components of the MCC and interact with mitotic cyclins with unexpected specificity. AtCDC20.1 and AtCDC20.2 are expressed in meristems, organ primordia and AtCDC20.1 also in pollen grains and developing seeds. Knocking down both genes simultaneously by RNAi resulted in severe delay in plant development and male sterility. In these lines, the meristem size was reduced while the cell size and ploidy levels were unaffected indicating that the lower cell number and likely slowdown of the cell cycle are the cause of reduced plant growth. CONCLUSIONS/SIGNIFICANCE: The intron-containing CDC20 gene copies provide conserved and redundant functions for cell cycle progression in plants and are required for meristem maintenance, plant growth and male gametophyte formation. The Arabidopsis-specific intron-less genes are possibly "retrogenes" and have hitherto undefined

  19. The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan12[C][W

    Science.gov (United States)

    Durand, Caroline; Vicré-Gibouin, Maïté; Follet-Gueye, Marie Laure; Duponchel, Ludovic; Moreau, Myriam; Lerouge, Patrice; Driouich, Azeddine

    2009-01-01

    Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip. PMID:19448034

  20. Cell Wall Targeted in planta Iron Accumulation Enhances Biomass Conversion and Seed Iron Concentration in Arabidopsis and Rice

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Haibing; Wei, Hui; Ma, Guojie; Antunes, Mauricio S.; Vogt, Stefan; Cox, Joseph; Zhang, Xiao; Liu, Xiping; Bu, Lintao; Gleber, S. Charlotte; Carpita, Nicholas C.; Makowski, Lee; Himmel, Michael E.; Tucker, Melvin P.; McCann, Maureen C.; Murphy, Angus S.; Peer, Wendy A.

    2016-10-01

    Conversion of nongrain biomass into liquid fuel is a sustainable approach to energy demands as global population increases. Previously, we showed that iron can act as a catalyst to enhance the degradation of lignocellulosic biomass for biofuel production. However, direct addition of iron catalysts to biomass pretreatment is diffusion-limited, would increase the cost and complexity of biorefinery unit operations and may have deleterious environmental impacts. Here, we show a new strategy for in planta accumulation of iron throughout the volume of the cell wall where iron acts as a catalyst in the deconstruction of lignocellulosic biomass. We engineered CBM-IBP fusion polypeptides composed of a carbohydrate-binding module family 11 (CBM11) and an iron-binding peptide (IBP) for secretion into Arabidopsis and rice cell walls. CBM-IBP transformed Arabidopsis and rice plants show significant increases in iron accumulation and biomass conversion compared to respective controls. Further, CBM-IBP rice shows a 35% increase in seed iron concentration and a 40% increase in seed yield in greenhouse experiments. CBM-IBP rice potentially could be used to address iron deficiency, the most common and widespread nutritional disorder according to the World Health Organization.

  1. The companion cell-specific Arabidopsis disaccharide carrier AtSUC2 is expressed in nematode-induced syncytia.

    Science.gov (United States)

    Juergensen, Katja; Scholz-Starke, Joachim; Sauer, Norbert; Hess, Paul; van Bel, Aart J E; Grundler, Florian M W

    2003-01-01

    Cyst nematodes induce a metabolically highly active syncytial cell complex in host roots. The syncytia are symplastically isolated. Because they form a strong sink, assimilates must be imported via the apoplast, thus suggesting that specific membrane-bound sugar transport proteins are expressed and activated. To identify possible candidate genes, transgenic Arabidopsis plants expressing different reporter genes under the control of different promoters from Arabidopsis sugar transporter genes were infected with the beet cyst nematode (Heterodera schachtii). With polymerase chain reaction, 13 additional sugar transporters were tested for their presence in the syncytia through the use of a syncytium-specific cDNA library. Analysis of the infected roots showed that the promoter of the sucrose (Suc) transporter AtSUC2 gene that codes for a companion cell-specific Suc transporter in noninfected plants was found to be expressed in syncytia. Its expression patterns in beta-glucuronidase and green fluorescent protein plants were monitored. Syncytium-specific gene expression was confirmed by reverse transcriptase-polymerase chain reaction. Results support the idea that AtSUC2 mediates the transmembrane transfer of Suc. AtSUC2 is the first disaccharide carrier described to be activated by pathogens.

  2. Transmission Fourier transform infrared microspectroscopy allows simultaneous assessment of cutin and cell-wall polysaccharides of Arabidopsis petals.

    Science.gov (United States)

    Mazurek, Sylwester; Mucciolo, Antonio; Humbel, Bruno M; Nawrath, Christiane

    2013-06-01

    A procedure for the simultaneous analysis of cell-wall polysaccharides, amides and aliphatic polyesters by transmission Fourier transform infrared microspectroscopy (FTIR) has been established for Arabidopsis petals. The combination of FTIR imaging with spectra derivatization revealed that petals, in contrast to other organs, have a characteristic chemical zoning with high amount of aliphatic compounds and esters in the lamina and of polysaccharides in the stalk of the petal. The hinge region of petals was particular rich in amides as well as in vibrations potentially associated with hemicellulose. In addition, a number of other distribution patterns have been identified. Analyses of mutants in cutin deposition confirmed that vibrations of aliphatic compounds and esters present in the lamina were largely associated with the cuticular polyester. Calculation of spectrotypes, including the standard deviation of intensities, allowed detailed comparison of the spectral features of various mutants. The spectrotypes not only revealed differences in the amount of polyesters in cutin mutants, but also changes in other compound classes. For example, in addition to the expected strong deficiencies in polyester content, the long-chain acyl CoA synthase 2 mutant showed increased intensities of vibrations in a wavelength range that is typical for polysaccharides. Identical spectral features were observed in quasimodo2, a cell-wall mutant of Arabidopsis with a defect in pectin formation that exhibits increased cellulose synthase activity. FTIR thus proved to be a convenient method for the identification and characterization of mutants affected in the deposition of cutin in petals.

  3. Hydrodynamic interaction between two red blood cells in simple shear flow: its impact on the rheology of a semi-dilute suspension

    Science.gov (United States)

    Omori, Toshihiro; Ishikawa, Takuji; Imai, Yohsuke; Yamaguchi, Takami

    2014-10-01

    Blood is a suspension of red blood cells (RBCs) and its rheology is important when discussing the physiology of the cardiovascular system. In this study, we performed a numerical investigation of the rheological properties of an RBC suspension from the dilute to semi-dilute regime. RBCs were modelled as a capsule with a two-dimensional hyperelastic membrane. Large deformation of the thin membrane was calculated by a finite element method. Due to the small size of the RBC, fluid motion around the RBC was assumed to follow Stokes flow and was solved by a boundary element method. In the dilute limit, cell-cell interactions were omitted and the bulk stress of the suspension was calculated by the stresslet generated on a single RBC. Interestingly, the effective shear viscosity of the dilute suspension decreased with increasing viscosity of the internal liquid. In the semi-dilute regime, cells can be considered as showing pairwise interactions. The effective shear viscosity of the semi-dilute suspension shows a quadratic increase with respect to the volume fraction. These findings are important for understanding the complex phenomena of blood rheology.

  4. Development, characterization, and optimization of a new suspension chicken-induced pluripotent stem cell line for the production of Newcastle disease vaccine

    Science.gov (United States)

    Traditionally, substrates for production of vaccines have been embryonated eggs or adherent cell culture. The daunting challenge of scaling up these technologies in the face of an outbreak has been a limitation for industrial applicability. Suspension cell lines are better suited in many ways to e...

  5. Blue light-dependent changes in loosely bound calcium in Arabidopsis mesophyll cells: an X-ray microanalysis study.

    Science.gov (United States)

    Łabuz, Justyna; Samardakiewicz, Sławomir; Hermanowicz, Paweł; Wyroba, Elżbieta; Pilarska, Maria; Gabryś, Halina

    2016-06-01

    Calcium is involved in the signal transduction pathway from phototropins, the blue light photoreceptor kinases which mediate chloroplast movements. The chloroplast accumulation response in low light is controlled by both phot1 and phot2, while only phot2 is involved in avoidance movement induced by strong light. Phototropins elevate cytosolic Ca(2+) after activation by blue light. In higher plants, both types of chloroplast responses depend on Ca(2+), and internal calcium stores seem to be crucial for these processes. Yet, the calcium signatures generated after the perception of blue light by phototropins are not well understood. To characterize the localization of calcium in Arabidopsis mesophyll cells, loosely bound (exchangeable) Ca(2+) was precipitated with potassium pyroantimonate and analyzed by transmission electron microscopy followed by energy-dispersive X-ray microanalysis. In dark-adapted wild-type Arabidopsis leaves, calcium precipitates were observed at the cell wall, where they formed spherical structures. After strong blue light irradiation, calcium at the apoplast prevailed, and bigger, multilayer precipitates were found. Spherical calcium precipitates were also detected at the tonoplast. After red light treatment as a control, the precipitates at the cell wall were smaller and less numerous. In the phot2 and phot1phot2 mutants, calcium patterns were different from those of wild-type plants. In both mutants, no elevation of calcium after blue light treatment was observed at the cell periphery (including the cell wall and a fragment of cytoplasm). This result confirms the involvement of phototropin2 in the regulation of Ca(2+) homeostasis in mesophyll cells.

  6. A novel xylogenic suspension culture model for exploring lignification in Phyllostachys bamboo

    Directory of Open Access Journals (Sweden)

    Ogita Shinjiro

    2012-09-01

    Full Text Available Abstract Background Some prominent cultured plant cell lines, such as the BY-2 cell line of tobacco (Nicotiana tabacum cv. ‘Bright Yellow 2’ and the T87 cell line of Arabidopsis (Arabidopsis thaliana L. Heynh., ecotype Columbia are used as model plant cells. These suspension cell culture systems are highly applicable for investigating various aspects of plant cell biology. However, no such prominent cultured cell lines exist in bamboo species. Results We standardized a novel xylogenic suspension culture model in order to unveil the process of lignification in living bamboo cells. Initial signs of lignin deposition were able to be observed by a positive phloroglucinol-HCl reaction at day 3 to 5 under lignification conditions (LG, i.e., modified half-strength Murashige and Skoog medium (m1/2MS containing 10 μM 6-benzyladenine (BA and 3% sucrose. Two types of xylogenic differentiation, both fiber-like elements (FLEs with cell wall thickening and tracheary elements (TEs with formation of perforations in the cell wall, were observed under these conditions. The suspension cells rapidly formed secondary cell wall components that were highly lignified, making up approximately 25% of the cells on a dry weight basis within 2 weeks. Detailed features involved in cell growth, differentiation and death during lignification were characterized by laser scanning microscopic imaging. Changes in transcript levels of xylogenesis-related genes were assessed by RT-PCR, which showed that the transcription of key genes like PAL1, C4H, CCoAOMT, and CCR was induced at day 4 under LG conditions. Furthermore, interunit linkage of lignins was compared between mature bamboo culms and xylogenic suspension cells by heteronuclear single quantum coherence (HSQC NMR spectroscopy. The presence of the most common interunit linkages, including β-aryl ether (β-O-4, phenylcoumaran (β-5 and resinol (β-β structures was identified in the bamboo cultured cell lignin (BCCL

  7. Single cell dual adherent-suspension co-culture micro-environment for studying tumor-stromal interactions with functionally selected cancer stem-like cells.

    Science.gov (United States)

    Chen, Yu-Chih; Zhang, Zhixiong; Fouladdel, Shamileh; Deol, Yadwinder; Ingram, Patrick N; McDermott, Sean P; Azizi, Ebrahim; Wicha, Max S; Yoon, Euisik

    2016-08-07

    Considerable evidence suggests that cancer stem-like cells (CSCs) are critical in tumor pathogenesis, but their rarity and transience has led to much controversy about their exact nature. Although CSCs can be functionally identified using dish-based tumorsphere assays, it is difficult to handle and monitor single cells in dish-based approaches; single cell-based microfluidic approaches offer better control and reliable single cell derived sphere formation. However, like normal stem cells, CSCs are heavily regulated by their microenvironment, requiring tumor-stromal interactions for tumorigenic and proliferative behaviors. To enable single cell derived tumorsphere formation within a stromal microenvironment, we present a dual adherent/suspension co-culture device, which combines a suspension environment for single-cell tumorsphere assays and an adherent environment for co-culturing stromal cells in close proximity by selectively patterning polyHEMA in indented microwells. By minimizing dead volume and improving cell capture efficiency, the presented platform allows for the use of small numbers of cells (cells). As a proof of concept, we co-cultured single T47D (breast cancer) cells and primary cancer associated fibroblasts (CAF) on-chip for 14 days to monitor sphere formation and growth. Compared to mono-culture, co-cultured T47D have higher tumorigenic potential (sphere formation rate) and proliferation rates (larger sphere size). Furthermore, 96-multiplexed single-cell transcriptome analyses were performed to compare the gene expression of co-cultured and mono-cultured T47D cells. Phenotypic changes observed in co-culture correlated with expression changes in genes associated with proliferation, apoptotic suppression, tumorigenicity and even epithelial-to-mesechymal transition. Combining the presented platform with single cell transcriptome analysis, we successfully identified functional CSCs and investigated the phenotypic and transcriptome effects induced by

  8. Signal interaction between nitric oxide and hydrogen peroxide in heat shock-induced hypericin production of Hypericum perforatum suspension cells

    Institute of Scientific and Technical Information of China (English)

    XU MaoJun; DONG JuFang; ZHANG XinBo

    2008-01-01

    Heat shock (HS, 40℃, 10 min) induces hypericin production, nitric oxide (NO) generation, and hydrogen peroxide (H2O2) accumulation of Hypericum perforatum suspension cells. Catalase (CAT) and NO spe-cific scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) suppress not only the HS-induced H2O2 generation and NO burst, but also the HS-triggered hypericin produc-tion. Hypericin contents of the cells treated with both NO and H2O2 are significantly higher than those of the cells treated with NO alone, although H2O2 per se has no effects on hypericin production of the cells, which suggests the synergistic action between H2O2 and NO on hypericin production. NO treatmentenhances H2O2 levels of H. perforatum cells, while external application of H2O2 induces NO generation of cells. Thus, the results reveal a mutually amplifying action between H2O2 and NO in H. perforatum cells. CAT treatment inhibits both HS-induced H2O2 accumulation and NO generation, while cPTIO can also suppress H2O2 levels of the heat shocked cells. The results imply that H2O2 and NO may enhance each other's levels by their mutually amplifying action in the heat shocked cells. Membrane NAD(P)H oxidase inhibitor diphenylene iodonium (DPI) and nitric oxide synthase (NOS) inhibitor S,S'-1,3-phenylene-bis(1,2-ethanediyl)-bis-isothiourea (PBITU) not only inhibit the mutually amplifying action between H2O2 and NO but also abolish the synergistic effects of H2O2 and NO on hypericin production, showing that the synergism of H2O2 and NO on secondary metsbolite biosynthesis might be dependent on their mutual amplification. Taken together, data of the present work demonstrate that both H2O2 and NO are essential for HS-induced hypericin production of H. perforatum suspension cells. Furthermore, the results reveal a special interaction between the two signal molecules in mediating HS-triggered secondary metabolite biosynthesis of the cells.

  9. Distinct isoforms of ADPglucose pyrophosphatase and ADPglucose pyrophosphorylase occur in the suspension-cultured cells of sycamore (Acer pseudoplatanus L.).

    Science.gov (United States)

    Baroja-Fernández, E; Zandueta-Criado, A; Rodríguez-López, M; Akazawa, T; Pozueta-Romero, J

    2000-09-01

    The intracellular localizations of ADPglucose pyrophosphatase (AGPPase) and ADPglucose pyrophosphorylase (AGPase) have been studied using protoplasts prepared from suspension-cultured cells of sycamore (Acer pseudoplatanus L.). Subcellular fractionation studies revealed that all the AGPPase present in the protoplasts is associated with amyloplasts, whereas more than 60% of AGPase is in the extraplastidial compartment. Immunoblots of amyloplast- and extraplastid-enriched extracts further confirmed that AGPase is located mainly outside the amyloplast. Experiments carried out to identify possible different isoforms of AGPPase in the amyloplast revealed the presence of soluble and starch granule-bound isoforms. We thus propose that ADPglucose levels linked to starch biosynthesis in sycamore cells are controlled by enzymatic reactions catalyzing the synthesis and breakdown of ADPglucose, which take place both inside and outside the amyloplast.

  10. Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures.

    Science.gov (United States)

    Robinson, S A; Stewart, G R; Phillips, R

    1992-03-01

    Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.

  11. Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b6/f Complex

    Institute of Scientific and Technical Information of China (English)

    张贵友; 李萍; 朱瑞宇; 田瑞华; 戴尧仁

    2004-01-01

    An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco suspension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei,evidenced by DNA electrophoresis and fluorescence microscopy observations.Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still exhibited the apoptosis-inducing activity,but the supernatant had no effect,which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble.The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO,the specific inhibitor of Caspase-3,implying involvement of a Caspase-3-like protease during the process.Furthermore,extensive apoptosis in nuclei was induced by cytochrome b6/f on the thylakoid membrane,indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.

  12. Im"plant"ing of Mammalian Glycosyltransferase Gene into Plant Suspension-Cultured Cells Using Agrobacterium-Mediated Transformation.

    Science.gov (United States)

    Kajiura, Hiroyuki; Fujiyama, Kazuhito

    2015-01-01

    Enzymatic activity assay of exogenous glycosyltransferase (GT) and glycosylhydrolase (GH) expressed in plants is an important analysis for determination of the expression of the gene of interest. However, generations and establishment of in planta transgenic lines are time-consuming. Furthermore, the expression levels and the activities of the exogenous GTs and GHs are quite low and weak, the radiolabeled donor substrate had to be used to analyze the enzymatic activity. Here, we describe a protocol for the generation of transgenic plants using suspension-cultured cells and a high sensitive assay for GT, especially β1,4-galactosyltransferase, using microsomal fraction from plant cells and fluorescent-labeled sugar chains as an acceptor substrate. This method enables less-time-consuming preparation of stable transgenic plants, non-radiolabeled, high-throughput detail analysis which includes mass spectrometric analysis and exo-glycosidase digestions.

  13. A two-stage process with temperature-shift for enhanced anthocyanin production in strawberry cell suspension cultures

    Institute of Scientific and Technical Information of China (English)

    张卫; Shintaro; Furusaki; Chris; Franco

    1999-01-01

    A two-stage process with temperature-shift has been developed to enhance the anthocyanin yield in suspension cultures of strawberry cells. The effect of the temperature-shift interval and the shift-time point was investigated for the optimization of this strategy. In this process, strawberry cells were grown at 30℃ (the optimum temperature for cell growth) for a certain period as the first stage, with the temperature shifted to a lower temperature for the second stage. In response to the temperature shift-down, anthoeyanin synthesis was stimulated and a higher content could be achieved than that at both boundary temperatures but cell growth was suppressed. When the lower boundary temperature was deereased, cell growth was lowered and a delayed, sustained maximum anthocyanin content was achieved. Anthocyanin synthesis was strongly influeneed by the shift-time point but cell growth was not. Consequently, the maximum anthocyanin content of 2.7 mg(?)g-fresh cell-1 was obtained on day 9 by a temperature-

  14. Elicitation of gymnemic acid production in cell suspension cultures of Gymnema sylvestre R.Br. through endophytic fungi.

    Science.gov (United States)

    Netala, Vasudeva Reddy; Kotakadi, Venkata Subbaiah; Gaddam, Susmila Aparna; Ghosh, Sukhendu Bikash; Tartte, Vijaya

    2016-12-01

    The enhancement of plant secondary metabolite production in cell suspension cultures through biotic or abiotic elicitation has become a potential biotechnological approach for commercialization or large-scale production of bioactive compounds. Gymnema sylvestre R.Br. is an important medicinal plant, rich in a group of oleanane triterpenoid saponins called gymnemic acid, well known for its anti-diabetic activity. Two endophytic fungal strains were isolated from the leaves of G. sylvestre and identified as Polyancora globosa and Xylaria sp. based on the PCR amplification and internal transcribed spacer (ITS 1-5.8S-ITS 2) sequencing of 18S rRNA gene. The process of elicitation of cell suspension cultures of G. sylvestre with dried powder of fungal mycelia (DPFM) and extracellular culture filtrate (ECF) of endophytic fungi consistently enhanced the accumulation of gymnemic acid and the DPFM was proved to be an effective elicitor when compared to the ECF. The DPFM elicited the gymnemic acid content in the range of 2.57-10.45-fold, while the ECF elicited the gymnemic acid content in the range of 2.39-7.8-fold. P. globosa, a novel and a rare endophytic fungal strain, has shown a great influence on the production of gymnemic acid. Cell suspension cultures elicited with DPFM of P. globosa produced higher amount of gymnemic acid content (124.23 mg/g dried cell weight) compared to the cultures elicited with DPFM of Xylaria sp. (102.24 mg/g DCW). But the cultures treated with consortium of DPFM of both fungi showed great influence on the production of gymnemic acid (139.98 mg/g DCW) than the cultures treated with DPFM alone. Similarly, cultures treated with consortium of ECF of both fungi produced more gymnemic acid content (94.86 mg/g DCW) compared with cultures treated with ECF of Xylaria sp. (77.93 mg/g DCW) and ECF of P. globosa (78.65 mg/g DCW) alone.

  15. Induction of Systemic Resistance against Aphids by Endophytic Bacillus velezensis YC7010 via Expressing PHYTOALEXIN DEFICIENT4 in Arabidopsis

    Science.gov (United States)

    Rashid, Md. Harun-Or-; Khan, Ajmal; Hossain, Mohammad T.; Chung, Young R.

    2017-01-01

    Aphids are the most destructive insect pests. They suck the sap and transmit plant viruses, causing widespread yield loss of many crops. A multifunctional endophytic bacterial strain Bacillus velezensis YC7010 has been found to induce systemic resistance against bacterial and fungal pathogens of rice. However, its activity against insects attack and underlying cellular and molecular defense mechanisms are not elucidated yet. Here, we show that root drenching of Arabidopsis seedlings with B. velezensis YC7010 can induce systemic resistance against green peach aphid (GPA), Myzus persicae. Treatment of bacterial suspension of B. velezensis YC7010 at 2 × 107 CFU/ml to Arabidopsis rhizosphere induced higher accumulation of hydrogen peroxide, cell death, and callose deposition in leaves compared to untreated plants at 6 days after infestation of GPA. Salicylic acid, jasmonic acid, ethylene, and abscisic acid were not required to confer defense against GPA in Arabidopsis plants treated by B. velezensis YC7010. Bacterial treatment with B. velezensis YC7010 significantly reduced settling, feeding and reproduction of GPA on Arabidopsis leaves via strongly expressing senescence-promoting gene PHYTOALEXIN DEFICIENT4 (PAD4) while suppressing BOTRYTIS-INDUCED KINASE1 (BIK1). These results indicate that B. velezensis YC7010-induced systemic resistance to the GPA is a hypersensitive response mainly dependent on higher expression of PAD4 with suppression of BIK1, resulting in more accumulation of hydrogen peroxide, cell death, and callose deposition in Arabidopsis. PMID:28261260

  16. Induction of Systemic Resistance against Aphids by Endophytic Bacillus velezensis YC7010 via Expressing PHYTOALEXIN DEFICIENT4 in Arabidopsis.

    Science.gov (United States)

    Rashid, Md Harun-Or-; Khan, Ajmal; Hossain, Mohammad T; Chung, Young R

    2017-01-01

    Aphids are the most destructive insect pests. They suck the sap and transmit plant viruses, causing widespread yield loss of many crops. A multifunctional endophytic bacterial strain Bacillus velezensis YC7010 has been found to induce systemic resistance against bacterial and fungal pathogens of rice. However, its activity against insects attack and underlying cellular and molecular defense mechanisms are not elucidated yet. Here, we show that root drenching of Arabidopsis seedlings with B. velezensis YC7010 can induce systemic resistance against green peach aphid (GPA), Myzus persicae. Treatment of bacterial suspension of B. velezensis YC7010 at 2 × 10(7) CFU/ml to Arabidopsis rhizosphere induced higher accumulation of hydrogen peroxide, cell death, and callose deposition in leaves compared to untreated plants at 6 days after infestation of GPA. Salicylic acid, jasmonic acid, ethylene, and abscisic acid were not required to confer defense against GPA in Arabidopsis plants treated by B. velezensis YC7010. Bacterial treatment with B. velezensis YC7010 significantly reduced settling, feeding and reproduction of GPA on Arabidopsis leaves via strongly expressing senescence-promoting gene PHYTOALEXIN DEFICIENT4 (PAD4) while suppressing BOTRYTIS-INDUCED KINASE1 (BIK1). These results indicate that B. velezensis YC7010-induced systemic resistance to the GPA is a hypersensitive response mainly dependent on higher expression of PAD4 with suppression of BIK1, resulting in more accumulation of hydrogen peroxide, cell death, and callose deposition in Arabidopsis.

  17. Arabidopsis thaliana T-DNA Mutants Implicate GAUT Genes in the Biosynthesis of Pectin and Xylan in Cell Walls and Seed Testa

    Institute of Scientific and Technical Information of China (English)

    Kerry H. Caffall; Sivakumar Pattathil; Sarah E. Phillips; Michael G. Hahn; Debra Mohnen

    2009-01-01

    Galacturonosyltransferase 1 (GAUT1) is an α1,4-D-galacturonosyltransferase that transfers galacturonic acid from uridine 5'-diphosphogalacturonic acid onto the pectic polysaccharide homogalacturonan (Sterling et al., 2006). The 25-member Arabidopsis thaliana GAUT1-related gene family encodes 15 GAUT and 10 GAUT-like (GATL) proteins with, respectively, 56-84 and 42-53% amino acid sequence similarity to GAUT1. Previous phylogenetic analyses of AtGAUTs indicated three clades: A through C. A comparative phylogenetic analysis of the Arabidopsis, poplar and rice GAUT families has sub-classified the GAUTs into seven clades: clade A-1 (GAUTs 1 to 3); A-2 (GAUT4); A-3 (GAUTs 5 and 6); A-4 (GAUT7); B-1(GAUTs 8 and 9); B-2 (GAUTs 10 and 11); and clade C (GAUTs 12 to 15). The Arabidopsis GAUTs have a distribution com-parable to the poplar orthologs, with the exception of GAUT2, which is absent in poplar. Rice, however, has no orthologs of GAUTs 2 and 12 and has multiple apparent orthologs of GAUTs 1, 4, and 7 compared with eitherArabidopsis or poplar. The cell wall glycosyl residue compositions of 26 homozygous T-DNA insertion mutants for 13 of 15 Arabidopsis GAUTgenes reveal significantly and reproducibly different cell walls in specific tissues of gaut mutants 6, 8, 9, 10, 11, 12, 13, and 14 from that of wild-type Arabidopsis walls. Pectin and xylan polysaccharides are affected by the loss of GAUT function, as dem-onstrated by the altered galacturonic acid, xylose, rhamnose, galactose, and arabinose composition of distinct gaut mu-tant walls. The wall glycosyl residue compositional phenotypes observed among the gaut mutants suggest that at least six different biosynthetic linkages in pectins and/or xylans are affected by the lesions in these GAUTgenes. Evidence is also presented to support a role for GAUT11 in seed mucilage expansion and in seed wall and mucilage composition.

  18. Localization of the Arabidopsis Senescence- and Cell Death-Associated BFN1 Nuclease: From the ER to Fragmented Nuclei

    Institute of Scientific and Technical Information of China (English)

    Sarit Farage-Barhom; Shaul Burd; Lilian Sonego; Ana Mett; Eduard Belausov; David Gidoni; Amnon Lers

    2011-01-01

    Plant senescence- or PCD-associated nucleases share significant homology with nucleases from different organisms.However,knowledge of their function is limited.Intracellular localization of the Arabidopsis senescenceand PCD-associated nuclease BFN1 was investigated.Analysis of BFN1-GFP localization in transiently transformed tobacco protoplasts revealed initial localization in filamentous structures spread throughout the cytoplasm,which then clustered around the nuclei as the protoplasts senesced.These filamentous structures were identified as being of ER origin.In BFN1GFP-transgenic Arabidopsis plants,similar localization of BFN1-GFP was observed in young leaves,that is,in filamentous structures that reorganized around the nuclei only in senescing cells.In late senescence,BFN1-GFP was localized with fragmented nuclei in membrane-wrapped vesicles.BFN1's postulated function as a nucleic acid-degrading enzyme in senescence and PCD is supported by its localization pattern.Our results suggest the existence of a dedicated compartment mediating nucleic acid degradation in senescence and PCD processes.

  19. A general G1/S-phase cell-cycle control module in the flowering plant Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Xin'Ai Zhao

    Full Text Available The decision to replicate its DNA is of crucial importance for every cell and, in many organisms, is decisive for the progression through the entire cell cycle. A comparison of animals versus yeast has shown that, although most of the involved cell-cycle regulators are divergent in both clades, they fulfill a similar role and the overall network topology of G1/S regulation is highly conserved. Using germline development as a model system, we identified a regulatory cascade controlling entry into S phase in the flowering plant Arabidopsis thaliana, which, as a member of the Plantae supergroup, is phylogenetically only distantly related to Opisthokonts such as yeast and animals. This module comprises the Arabidopsis homologs of the animal transcription factor E2F, the plant homolog of the animal transcriptional repressor Retinoblastoma (Rb-related 1 (RBR1, the plant-specific F-box protein F-BOX-LIKE 17 (FBL17, the plant specific cyclin-dependent kinase (CDK inhibitors KRPs, as well as CDKA;1, the plant homolog of the yeast and animal Cdc2⁺/Cdk1 kinases. Our data show that the principle of a double negative wiring of Rb proteins is highly conserved, likely representing a universal mechanism in eukaryotic cell-cycle control. However, this negative feedback of Rb proteins is differently implemented in plants as it is brought about through a quadruple negative regulation centered around the F-box protein FBL17 that mediates the degradation of CDK inhibitors but is itself directly repressed by Rb. Biomathematical simulations and subsequent experimental confirmation of computational predictions revealed that this regulatory circuit can give rise to hysteresis highlighting the here identified dosage sensitivity of CDK inhibitors in this network.

  20. The location of aluminium in protoplasts and suspension cells taken from Coffea arabica L. with different tolerance of Al.

    Science.gov (United States)

    Ramírez-Benítez, J Efraín; Hernández-Sotomayor, S M Teresa; Muñoz-Sánchez, J Armando

    2009-11-01

    Biotechnological advances in coffee research (in vitro manipulation, multiplication, generation and development of transgenic coffee plants with specific traits like high yield and good quality) have contributed to description of the metabolic pathways involved in the response mechanisms to environmental factors like abiotic stress. Coffea arabica L. plants grow in acidic soils, and therefore aluminium (Al) toxicity is a major negative impact on crop productivity. To understand Al toxicity mechanisms in cells via the Al absorption kinetic, we isolated protoplasts from two C. arabica L. suspension cell lines: Al-sensitive (L2) and Al-tolerant (LAMt). Protoplasts of LAMt line exhibited lower Al absorption levels than protoplasts of the L2 line. Use of two fluorescent tracers (morin and calcofluor white) indicated that Al interacts with internal cell structures, such as the plasma membrane and nucleus, with differences in both cell lines. Al-tolerance in the LAMt is probably associated with the cell wall as well as intracellular structures. These data will help to better understand Al toxicity in C. arabica, and Al toxicity mechanisms in plant cells.

  1. The impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture

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    Maycock Christopher

    2010-10-01

    Full Text Available Abstract Background Nanotechnology has the potential to provide agriculture with new tools that may be used in the rapid detection and molecular treatment of diseases and enhancement of plant ability to absorb nutrients, among others. Data on nanoparticle toxicity in plants is largely heterogeneous with a diversity of physicochemical parameters reported, which difficult generalizations. Here a cell biology approach was used to evaluate the impact of Quantum Dots (QDs nanocrystals on plant cells, including their effect on cell growth, cell viability, oxidative stress and ROS accumulation, besides their cytomobility. Results A plant cell suspension culture of Medicago sativa was settled for the assessment of the impact of the addition of mercaptopropanoic acid coated CdSe/ZnS QDs. Cell growth was significantly reduced when 100 mM of mercaptopropanoic acid -QDs was added during the exponential growth phase, with less than 50% of the cells viable 72 hours after mercaptopropanoic acid -QDs addition. They were up taken by Medicago sativa cells and accumulated in the cytoplasm and nucleus as revealed by optical thin confocal imaging. As part of the cellular response to internalization, Medicago sativa cells were found to increase the production of Reactive Oxygen Species (ROS in a dose and time dependent manner. Using the fluorescent dye H2DCFDA it was observable that mercaptopropanoic acid-QDs concentrations between 5-180 nM led to a progressive and linear increase of ROS accumulation. Conclusions Our results showed that the extent of mercaptopropanoic acid coated CdSe/ZnS QDs cytotoxicity in plant cells is dependent upon a number of factors including QDs properties, dose and the environmental conditions of administration and that, for Medicago sativa cells, a safe range of 1-5 nM should not be exceeded for biological applications.

  2. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures

    Energy Technology Data Exchange (ETDEWEB)

    Norman, M.A.; Liebl, R.A.; Widholm, J.M. (Univ. of Illinois, Urbana (USA))

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max (L.) Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum (L.) cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I{sub 50} values for growth, chlorophyll (Chl), {beta}-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in ({sup 14}C)clomazone uptake cannot account for selectivity since there were significantly greater levels of domazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  3. Uptake and metabolism of clomazone in tolerant-soybean and susceptible-cotton photomixotrophic cell suspension cultures.

    Science.gov (United States)

    Norman, M A; Liebl, R A; Widholm, J M

    1990-03-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I(50) values for growth, chlorophyll (Chl), beta-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [(14)C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action.

  4. Uptake and Metabolism of Clomazone in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the uptake and metabolism of the pigment synthesis inhibiting herbicide clomazone in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) photomixotrophic cell suspensions. Soybean and cotton on a whole plant level are tolerant and susceptible to clomazone, respectively. Preliminary studies indicated that I50 values for growth, chlorophyll (Chl), β-carotene, and lutein were, respectively, >22, 14, 19, and 23 times greater for the soybean cell line (SB-M) 8 days after treatment (DAT) compared to the cotton cell line (COT-M) 16 DAT. Differences in [14C]clomazone uptake cannot account for selectivity since there were significantly greater levels of clomazone absorbed by the SB-M cells compared to the COT-M cells for each treatment. The percentage of absorbed clomazone converted to more polar metabolite(s) was significantly greater by the SB-M cells relative to COT-M cells at 6 and 24 hours after treatment, however, only small differences existed between the cell lines by 48 hours after treatment. Nearly identical levels of parental clomazone was recovered from both cell lines for all treatments. A pooled metabolite fraction isolated from SB-M cells had no effect on the leaf pigment content of susceptible velvetleaf (Abutilon theophrasti Medic.) or soybean seedlings. Conversely, a pooled metabolite fraction from COT-M cells reduced the leaf Chl content of velvetleaf. Soybean tolerance to clomazone appears to be due to differential metabolism (bioactivation) and/or differences at the site of action. PMID:16667349

  5. Expression of hemagglutinin protein from the avian influenza virus H5N1 in a baculovirus/insect cell system significantly enhanced by suspension culture

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    Spencer Lynn

    2006-02-01

    Full Text Available Abstract Background Prevention of a possible avian influenza pandemic necessitates the development of rapid diagnostic tests and the eventual production of a vaccine. Results For vaccine production, hemagglutinin (HA1 from avian influenza H5N1 was expressed from a recombinant baculovirus. Recombinant HA1 was expressed in monolayer or suspension culture insect cells by infection with the recombinant baculovirus. The yield of rHA1 from the suspension culture was 68 mg/l, compared to 6 mg/l from the monolayer culture. Immunization of guinea pigs with 50 μg of rHA1 yielded hemagglutinin inhibition and virus neutralization titers of 1:160 after two times vaccination with rHA1 protein. Conclusion Thus, the production of rHA1 using an insect suspension cell system provides a promising basis for economical production of a H5 antigen.

  6. ROS enhancement by silicon nanoparticles in X-ray irradiated aqueous suspensions and in glioma C6 cells

    Energy Technology Data Exchange (ETDEWEB)

    David Gara, Pedro M. [CITOMA, Fundacion Avanzar, Instituto de Terapia Radiante S.A., CIO La Plata (Argentina); Garabano, Natalia I. [University of Buenos Aires, Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, UBA (Argentina); Llansola Portoles, Manuel J. [UNLP, INIFTA, Departamento de Quimica, Facultad de Ciencias Exactas (Argentina); Moreno, M. Sergio [Centro Atomico Bariloche (Argentina); Dodat, Diego; Casas, Oscar R. [CITOMA, Fundacion Avanzar, Instituto de Terapia Radiante S.A., CIO La Plata (Argentina); Gonzalez, Monica C., E-mail: gonzalez@inifta.unlp.edu.ar [UNLP, INIFTA, Departamento de Quimica, Facultad de Ciencias Exactas (Argentina); Kotler, Monica L., E-mail: kotler@qb.fcen.uba.ar [University of Buenos Aires, Departamento de Quimica Biologica, Facultad de Ciencias Exactas y Naturales, UBA (Argentina)

    2012-03-15

    The capability of silicon nanoparticles to increase the yield of reactive species upon 4 MeV X-ray irradiation of aqueous suspensions and C6 glioma cell cultures was investigated. ROS generation was detected and quantified using several specific probes. The particles were characterized by FTIR, XPS, TEM, DLS, luminescence, and adsorption spectroscopy before and after irradiation to evaluate the effect of high energy radiation on their structure. The total concentration of O{sub 2}{sup Bullet -}/HO{sub 2}{sup Bullet}, HO{sup Bullet}, and H{sub 2}O{sub 2} generated upon 4-MeV X-ray irradiation of 6.4 {mu}M silicon nanoparticle aqueous suspensions were on the order of 10 {mu}M per Gy, ten times higher than that obtained in similar experiments but in the absence of particles. Cytotoxic {sup 1}O{sub 2} was generated only in irradiation experiments containing the particles. The particle surface became oxidized to SiO{sub 2} and the luminescence yield reduced with the irradiation dose. Changes in the surface morphology did not affect, within the experimental error, the yields of ROS generated per Gy. X-ray irradiation of glioma C6 cell cultures with incorporated silicon nanoparticles showed a marked production of ROS proportional to the radiation dose received. In the absence of nanoparticles, the cells showed no irradiation-enhanced ROS generation. The obtained results indicate that silicon nanoparticles of <5 nm size have the potential to be used as radiosensitizers for improving the outcomes of cancer radiotherapy. Their capability of producing {sup 1}O{sub 2} upon X-ray irradiation opens novel approaches in the design of therapy strategies.

  7. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Abolghasem Abbasi Kajani

    2012-10-01

    Full Text Available Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale.MethodsWe investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing.ResultsThe yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  8. Optimization of the basal medium for improving production and secretion of taxanes from suspension cell culture of Taxus baccata L

    Directory of Open Access Journals (Sweden)

    Kajani Abolghasem

    2012-10-01

    Full Text Available Abstract Background and purpose of the study Taxol is one of the most effective anticancer drugs that isolated from Taxus sp. due to the slow growth of Taxus trees and low concentration of Taxol in the tissues, the biotechnological approaches especially plant cell culture have been considered to produce Taxol in commercial scale. Methods We investigated the effects of basal medium type used in culture media on production of Taxol and other taxane compounds from cell suspension culture of T. baccata L. Briefly, five commonly basal media including Gamborg, Murashige and Skoog, Woody Plant, Schenk and Hildebrandt, and Driver and Kuniyuki medium were used for preparing separate suspension culture media. The intra- and extra-cellular yields of taxanes were analyzed by using HPLC after 21 days period of culturing. Results The yields of taxanes were significantly different for the cultures prepared by different basal media. Moreover, the effects of basal medium on the yield of products differed for varius taxane compounds. Maximum yields of Baccatin III (10.03 mgl-1 and 10-deacetyl baccatin III (4.2 mgl-1 were achieved from the DKW basal media, but the yield of Taxol was maximum (16.58 mgl-1 in the WPM basal media. Furthermore, the secretion of taxanes from the cells into medium was also considerably affected by the type of basal medium. The maximum extra-cellular yield of Taxol (7.81 mgl-1, Baccatin III (5.0 mgl-1, and 10-deacetyl baccatin III (1.45 mgl-1 were also obtained by using DKW basal medium that were significantly higher than those obtained from other culture media.

  9. Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides.

    Science.gov (United States)

    Ishii, T; Thomas, J; Darvill, A; Albersheim, P

    1989-02-01

    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-alpha-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-alpha-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na(2)CO(3) at 1 and 22 degrees C. These previously uncharacterized polysaccharides accounted for approximately 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 NaCO(3)-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 Na(2)CO(3) at 1 degrees 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.

  10. Comparison of the Production of Recombinant Protein in Suspension Culture of CHO Cells in Spinner Flask and Shake Flask System

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    S.N.Z Zainul Abidin

    2011-12-01

    Full Text Available Chinese hamster ovary (CHO cells have been most widely used as the production host for the commercial production of biopharmaceuticals product. They have been extensively studied and developed, and today provide a stable platform for producing monoclonal antibodies and recombinant proteins. This study was focusing on comparison of suspension culture system by using spinner flask and shake flask for the growth and production of recombinant protein in CHO cell line. The CHO cells were transfected with an expression of DNA plasmid containing lac Z gene which codes for β-galactosidase. The recombinant genes in these CHO cells and the β-galactosidase expressing cells were adapted to suspension culture. The agitation speed for both spinner and shake flask were adjusted accordingly. The experiments were carried out in duplicate and samples were taken for cell count, determination of glucose consumption, lactate production and protein level by using biochemical assay. The result showed that, the cell growth in spinner flask is more favorable then in shake flask. The cell concentration in spinner flask is 58% higher than in shake flask. On the other hand, specific activity of β-galactosidase is 25% higher in spinner flask compared to shake flask, at the same agitation speed.ABSTRAK: Sel ovari hamster China (Chinese hamster ovary (CHO digunakan secara meluas dalam hos pembiakan untuk tujuan komersil produk biofarmaseutikal. Ia telah dikaji dan dibangunkan secara ekstensif, dan kini ia menyediakan landasan yang stabil untuk penghasilan antibodi monoklon dan protein rekombinan. Kajian ini memfokuskan tentang penghasilan protein rekombinan menggunakan kultur ampaian sel CHO di dalam kelalang putar dan kelalang goncang. Sel CHO dimasukkan dengan plasmid DNA yang mengandungi gen lac Z yang juga memberikan kod untuk β-galaktosidase. Sel CHO β-galaktosidase-terungkap dimasukkan ke dalam kultur ampaian. Kelajuan agitasi untuk kedua-dua kelalang putar

  11. Identification of genes involved in the ACC-mediated control of root cell elongation in Arabidopsis thaliana

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    Markakis Marios

    2012-11-01

    Full Text Available Abstract Background Along the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone. Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified. Results Using a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone. Conclusions ACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream

  12. Cell wall accumulation of fluorescent proteins derived from a trans-Golgi cisternal membrane marker and paramural bodies in interdigitated Arabidopsis leaf epidermal cells.

    Science.gov (United States)

    Akita, Kae; Kobayashi, Megumi; Sato, Mayuko; Kutsuna, Natsumaro; Ueda, Takashi; Toyooka, Kiminori; Nagata, Noriko; Hasezawa, Seiichiro; Higaki, Takumi

    2017-01-01

    In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis.

  13. [Establishment of embryogenic cell suspension culture and plant regeneration of edible banana Musa acuminata cv. Mas (AA)].

    Science.gov (United States)

    Wei, Yue-Rong; Huang, Xue-Lin; Li, Jia; Huang, Xia; Li, Zhe; Li, Xiao-Ju

    2005-01-01

    Conventional breeding for dual resistance of disease and pest of Musa cultivars remains a difficult endeavor, as the plant is polyploidic and high in sterility. Biotechnological techniques, eg., genetic engineering, in vitro mutation breeding, or protoplast fusion, may overcome the difficulties and improve the germplasm. Establishment of a stable embryogenic cell suspension (ECS) is a prerequisite for any of the biotechnological breeding methods. In this study an embryogenic cell suspension was established from immature male flower of Musa acuminata cv. Mas (AA), a popular commercial variety of banana in the South-East Asian region. After culture for 5-6 months on callus induction media, which consisted of MS salts, different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 4.1 micromol/L biotin, 5.7 micromol/L indoleacetic acid (IAA), 5.4 micromol/L naphthaleneacetic acid (NAA), other vitamins, 87 mmol/L sucrose, and solidified with 7 g/L agarose, meristematic globules and yellow, friable embryogenic cultures were induced from the explants of 1-15th row young floral hands of immature male flowers. Of the four treatments of 2,4-D, 9 micromol/L was the most effective on the callus induction, it transformed 40.96% and 7.45% of the cultivated male floral hands into callus and embryogenic callus respectively. The explants to produce highest frequency of the embryogenic calli were floral hands of 6 to 12th rows, which generated 5.79% of the embryogenic calli. Suspension cultures were initiated from these embryogenic calli in liquid medium supplemented with 4.5 micromol/L 2, 4-D. After sieving selection of the cultures using a stainless steel metallic strainer with pore sizes of 154 microm at 15 day intervals for 3 months, homogeneous and yellow embryogenic cell suspensions, composed of single cells and small cell aggregates, were established. Based upon the growth quantity and growth rate of ECS, it was determined that the appropriate inoculum was 2.0 mL PCV

  14. SCARECROW, SCR-LIKE 23 and SHORT-ROOT control bundle sheath cell fate and function in Arabidopsis thaliana.

    Science.gov (United States)

    Cui, Hongchang; Kong, Danyu; Liu, Xiuwen; Hao, Yueling

    2014-04-01

    Bundle sheath (BS) cells form a single cell layer surrounding the vascular tissue in leaves. In C3 plants, photosynthesis occurs in both the BS and mesophyll cells, but the BS cells are the major sites of photosynthesis in C4 plants, whereas the mesophyll cells are only involved in CO2 fixation. Because C4 plants are more efficient photosynthetically, introduction of the C4 mechanism into C3 plants is considered a key strategy to improve crop yield. One prerequisite for such C3-to-C4 engineering is the ability to manipulate the number and physiology of the BS cells, but the molecular basis of BS cell-fate specification remains unclear. Here we report that mutations in three GRAS family transcription factors, SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE 23 (SCL23), affect BS cell fate in Arabidopsis thaliana. SCR and SCL23 are expressed specifically in the BS cells and act redundantly in BS cell-fate specification, but their expression pattern and function diverge at later stages of leaf development. Using ChIP-chip experiments and sugar assays, we show that SCR is primarily involved in sugar transport whereas SCL23 functions in mineral transport. SHR is also essential for BS cell-fate specification, but it is expressed in the central vascular tissue. However, the SHR protein moves into the BS cells, where it directly regulates SCR and SCL23 expression. SHR, SCR and SCL23 homologs are present in many plant species, suggesting that this developmental pathway for BS cell-fate specification is likely to be evolutionarily conserved.

  15. Anti-Cancer Activity of Resveratrol and Derivatives Produced by Grapevine Cell Suspensions in a 14 L Stirred Bioreactor

    Directory of Open Access Journals (Sweden)

    Laetitia Nivelle

    2017-03-01

    Full Text Available In the present study, resveratrol and various oligomeric derivatives were obtained from a 14 L bioreactor culture of elicited grapevine cell suspensions (Vitis labrusca L.. The crude ethyl acetate stilbene extract obtained from the culture medium was fractionated by centrifugal partition chromatography (CPC using a gradient elution method and the major stilbenes contained in the fractions were subsequently identified by using a 13C-NMR-based dereplication procedure and further 2D NMR analyses including HSQC, HMBC, and COSY. Beside δ-viniferin (2, leachianol F (4 and G (4′, four stilbenes (resveratrol (1, ε-viniferin (5, pallidol (3 and a newly characterized dimer (6 were recovered as pure compounds in sufficient amounts to allow assessment of their biological activity on the cell growth of three different cell lines, including two human skin malignant melanoma cancer cell lines (HT-144 and SKMEL-28 and a healthy human dermal fibroblast HDF line. Among the dimers obtained in this study, the newly characterized resveratrol dimer (6 has never been described in nature and its biological potential was evaluated here for the first time. ε-viniferin as well as dimer (6 showed IC50 values on the three tested cell lines lower than the ones exerted by resveratrol and pallidol. However, activities of the first two compounds were significantly decreased in the presence of fetal bovine serum although that of resveratrol and pallidol was not. The differential tumor activity exerted by resveratrol on healthy and cancer lines was also discussed.

  16. Ultrasonic manipulation of yeast cells in suspension for absorption spectroscopy with an immersible mid-infrared fiberoptic probe.

    Science.gov (United States)

    Koch, Cosima; Brandstetter, Markus; Lendl, Bernhard; Radel, Stefan

    2013-06-01

    Recent advances in combining ultrasonic particle manipulation with attenuated total reflection infrared spectroscopy of yeast suspensions are presented. Infrared spectroscopy provides highly specific molecular information about the sample. It has not been applicable to in-line monitoring of cells during fermentation, however, because positioning cells in the micron-thin measurement region of the attenuated total reflection probe was not possible. Ultrasonic radiation forces exerted on suspended particles by an ultrasonic standing wave can result in the buildup of agglomerates in the nodal planes, hence enabling the manipulation of suspended cells on the microscopic scale. When a chamber setup and a prototype in-line applicable probe were used, successful control over the position of the yeast cells relative to the attenuated total reflection sensor surface could be proven. Both rate of increase and maximum mid-infrared absorption of yeast-specific bands during application of a pushing frequency (chamber setup: 1.863 MHz, in-line probe: 1.990 MHz) were found to correlate with yeast cell concentration.

  17. Comparison of use of Vero cell line and suspension culture of murine macrophage to attenuation of virulence of Neospora caninum.

    Science.gov (United States)

    Khordadmehr, Monireh; Namavari, Mehdi; Khodakaram-Tafti, Azizollah; Mansourian, Maryam; Rahimian, Abdollah; Daneshbod, Yahya

    2013-10-01

    In this study the tachyzoite yields of Neospora caninum were compared in two cell lines: Vero (African Green Monkey Kidney) and suspension culture of murine macrophage (J774) cell lines. Then, N. caninum were continuously passaged in these cell lines for 3 months and the effect of host cells on virulence of tachyzoites was assessed by broiler chicken embryonated eggs. Inoculation was performed in the chorioallantoic (CA) liquid of the embryonated eggs with different dilutions (0.5 × 10(4), 1.0 × 10(4), 1.5 × 10(4)) of tachtzoites isolated from these cell cultures. The mortality pattern and pathological changes of the dead embryos and hatched chickens were noted. Tissue samples of brain, liver and heart were examined by histopathological and detection of DNA of parasite by polymerase chain reaction (PCR). Also, consecutive sections of the tissues examined histologically were used for immunohistochemical (IHC) examination. Embryos inoculated with tachyzoites derived from Vero cell line (group V) showed a higher mortality rate (100%) than the embryos that received tachyzoites derived from J774 cell line (group J) (10% mortality rate). The results of this study indicated that the culture of N. caninum in J774 cell led to a marked increase in the number of tachyzoite yields and rapid attenuation in comparison to Vero, so the results were confirmed by IHC and PCR. This study is the first report of the significant effect of host cell on the attenuation of virulence of N. caninum tachyzoites. These findings could potentially provide a practical approach in the mass production of N. caninum tachyzoites, and also in producing live attenuated vaccine.

  18. [Analysis of the mechanism of intensification of fermentation process using yeast cells in a suspension of high-dispersed oxides].

    Science.gov (United States)

    Bagatskaya, A N; Mazurenko, R V; Makhno, S N; Gorbik, P P

    2014-01-01

    The differential microcalorimetry was used to explore an influence of particles of silicon dioxide, and also other high-dispersed oxides (0.05% of masses.) in water suspension of yeast cells on intensification of the process of their fermentation in endogenous metabolic conditions. It was shown that intensification of the processes of the vital activity of yeast microorganisms was observed in the specified interval of the concentration of silicon dioxide hydrosol particles. Mechanisms of interaction between SiO2 particles and a surface of a cellular organism, as well as interaction between SiO2 particles and one of metabolism products--carbon dioxide were studied. It was found out, that Al2O3, TiO2 hydrosols also had a stimulating effect, but it is lower compared to that of SiO2.

  19. A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem.

    Science.gov (United States)

    Burian, Agata; Ludynia, Michal; Uyttewaal, Magalie; Traas, Jan; Boudaoud, Arezki; Hamant, Olivier; Kwiatkowska, Dorota

    2013-12-01

    Cortical microtubules (CMTs) are often aligned in a particular direction in individual cells or even in groups of cells and play a central role in the definition of growth anisotropy. How the CMTs themselves are aligned is not well known, but two hypotheses have been proposed. According to the first hypothesis, CMTs align perpendicular to the maximal growth direction, and, according to the second, CMTs align parallel to the maximal stress direction. Since both hypotheses were formulated on the basis of mainly qualitative assessments, the link between CMT organization, organ geometry, and cell growth is revisited using a quantitative approach. For this purpose, CMT orientation, local curvature, and growth parameters for each cell were measured in the growing shoot apical meristem (SAM) of Arabidopsis thaliana. Using this approach, it has been shown that stable CMTs tend to be perpendicular to the direction of maximal growth in cells at the SAM periphery, but parallel in the cells at the boundary domain. When examining the local curvature of the SAM surface, no strict correlation between curvature and CMT arrangement was found, which implies that SAM geometry, and presumed geometry-derived stress distribution, is not sufficient to prescribe the CMT orientation. However, a better match between stress and CMTs was found when mechanical stress derived from differential growth was also considered.

  20. Cell wall pectic arabinans influence the mechanical properties of Arabidopsis thaliana inflorescence stems and their response to mechanical stress.

    Science.gov (United States)

    Verhertbruggen, Yves; Marcus, Susan E; Chen, Jianshe; Knox, J Paul

    2013-08-01

    Little is known of the dynamics of plant cell wall matrix polysaccharides in response to the impact of mechanical stress on plant organs. The capacity of the imposition of a mechanical stress (periodic brushing) to reduce the height of the inflorescence stem of Arabidopsis thaliana seedlings has been used to study the role of pectic arabinans in the mechanical properties and stress responsiveness of a plant organ. The arabinan-deficient-1 (arad1) mutation that affects arabinan structures in epidermal cell walls of inflorescence stems is demonstrated to reduce the impact on inflorescence stem heights caused by mechanical stress. The arabinan-deficient-2 (arad2) mutation, that does not have detectable impact on arabinan structures, is also shown to reduce the impact on stem heights caused by mechanical stress. The LM13 linear arabinan epitope is specifically detected in epidermal cell walls of the younger, flexible regions of inflorescence stems and increases in abundance at the base of inflorescence stems in response to an imposed mechanical stress. The strain (percentage deformation) of stem epidermal cells in the double mutant arad1 × arad2 is lower in unbrushed plants than in wild-type plants, but rises to wild-type levels in response to brushing. The study demonstrates the complexity of arabinan structures within plant cell walls and also that their contribution to cell wall mechanical properties is a factor influencing responsiveness to mechanical stress.

  1. Arabidopsis Kinesins HINKEL and TETRASPORE Act Redundantly to Control Cell Plate Expansion during Cytokinesis in the Male Garnetophyte

    Institute of Scientific and Technical Information of China (English)

    Sung-Aeong Oh; Valérie Bourdon; Madhumita Das'Pal; Hugh Dickinson; David Twell

    2008-01-01

    Asymmetric cell division at pollen mitosis I(PMI)is required to specify the differentiaI fate of the daughter vegetative and generative cells.Cytokinesis at PMI displays specialized features,and it has been suggested that there might be distinct molecular pathways underpinning different modes of cytokinesis in plants.Activation of the NACKPQR MAP kinase signaling pathway,which is essentiaI for somatic cell cytokinesis in tobacco,depends upon the NACK1and NACK2 kinesin-related proteins.Their Arabidopsis orthologs.HINKEL(HIK)and TETRAsPORE(TES).were reported to be essential for cytokinesis in somatic cells and in microsporoctes.respectively.More recently,HIK and TES were shown to have a functionally redundant role in female gametophytic cvtokinesis.We report here that HIK and TES are co-expressed in microspores and developing pollen,and,through analysis of microspore and pollen development in double heterozygote mutants.the occurrence of cell plate expansion defects during cytokinesis at PMI.The data demonstrate a functionally redundant role for HIK and TES in cell plate expansion during male gametophytic cytokinesis.extending the concept that different modes of cytokinesis are executed by a common signaling pathway,but reinforcing the individuality of gametophytic cytokinesis in its requirement for either TES or HIK.

  2. Discrimination of intra- and extracellular 23Na + signals in yeast cell suspensions using longitudinal magnetic resonance relaxography

    Science.gov (United States)

    Zhang, Yajie; Poirer-Quinot, Marie; Springer, Charles S.; Balschi, James A.

    2010-07-01

    This study tested the ability of MR relaxography (MRR) to discriminate intra- (Nai+) and extracellular (Nae+)23Na + signals using their longitudinal relaxation time constant ( T1) values. Na +-loaded yeast cell ( Saccharomyces cerevisiae) suspensions were investigated. Two types of compartmental 23Na +T1 differences were examined: a selective Nae+T1 decrease induced by an extracellular relaxation reagent (RR e), GdDOTP 5-; and, an intrinsic T1 difference. Parallel studies using the established method of 23Na MRS with an extracellular shift reagent (SR e), TmDOTP 5-, were used to validate the MRR measurements. With 12.8 mM RR e, the 23Nae+T1 was 2.4 ms and the 23Nai+T1 was 9.5 ms (9.4T, 24 °C). The Na + amounts and spontaneous efflux rate constants were found to be identical within experimental error whether measured by MRR/RR e or by MRS/SR e. Without RR e, the Na +-loaded yeast cell suspension 23Na MR signal exhibited two T1 values, 9.1 (±0.3) ms and 32.7 (±2.3) ms, assigned to 23Nai+ and 23Nae+, respectively. The Nai+ content measured was lower, 0.88 (±0.06); while Nae+ was higher, 1.43 (±0.12) compared with MRS/SR e measures on the same samples. However, the measured efflux rate constant was identical. T1 MRR potentially may be used for Nai+ determination in vivo and Na + flux measurements; with RR e for animal studies and without RR e for humans.

  3. Gravitational field related changes in gene expression after short-term exposure of Arabidopsis thaliana cell cultures

    Science.gov (United States)

    Babbick, Maren; Cogoli-Greuter, Marianne; Lowe, Kenneth C.; Power, J. Brian; Anthony, Paul; Dijkstra, Camelia; Davey, Michael R.; Hampp, Rüdiger

    2005-08-01

    Cell cultures of Arabidopsis thaliana (cv. Columbia) were used to screen for early changes in gene expression in response to altered gravitatonal fields. Genes of interest (mainly components of signalling chains) were selected from a larger group, the expression of which was affected under hypergravity [Martzivanou M. and Hampp R., Physiol. Plant., 118, 221-231, 2003]. Transcriptional changes of these genes were studied within a period of up to 10 min of exposure to clinorotation (random positioning machine), magnetophoresis, and hypergravity (8 g). Microarrays identified a set of transcription factor genes which responded in a treatment-specific way. The respective transcripts were quantified by real time RT PCR. As most responses occurred within 10 min of treatment, such genes can be used for the investigation of microgravity-related alterations in gene expression under sounding rocket conditions (TEXUS, MAXUS).

  4. Cultivation of Thalictrum rugosum cell suspension in an improved airlift bioreactor: stimulatory effect of carbon dioxide and ethylene on alkaloid production.

    Science.gov (United States)

    Kim, D I; Pedersen, H; Chin, C K

    1991-08-05

    Airlift bioreactor operations have been studied for the growth-associated production of secondary metabolites from plant cell suspension cultures. The model system used in this work was Thalictrum rugosum producing berberine, an isoquinoline alkaloid. The airlift system was well suited for growth of Thalictrum cell suspension cultures unless the cell density was high. At high cell density, the airlift system with a draught tube was not adequate due to large aggregates clogging the recirculation paths. This was overcome by use of a cell scraper in the reactor. For berberine production, gas-stripping also played a significant role and it was discovered that CO(2) and ethylene were important for product formation. By supplying a mixture of CO(2) and ethylene into the airlift system, the specific berberine content was increased twofold. It is evident that continuous gas sparging was harmful for the production of berberine without supplementation with other gases.

  5. Enhanced accumulation of phytosterols and phenolic compounds in cyclodextrin-elicited cell suspension culture of Daucus carota.

    Science.gov (United States)

    Miras-Moreno, Begoña; Almagro, Lorena; Pedreño, M A; Sabater-Jara, Ana Belén

    2016-09-01

    In this work, suspension-cultured cells of Daucus carota were used to evaluate the effect of β-cyclodextrins on the production of isoprenoid and phenolic compounds. The results showed that the phytosterols and phenolic compounds were accumulated in the extracellular medium (15100μgL(-1) and 477.46μgL(-1), respectively) in the presence of cyclodextrins. Unlike the phytosterol and phenolic compound content, β-carotene (1138.03μgL(-1)), lutein (25949.54μgL(-1)) and α-tocopherol (8063.82μgL(-1)) chlorophyll a (1625.13μgL(-1)) and b (9.958 (9958.33μgL(-1)) were mainly accumulated inside the cells. Therefore, cyclodextrins were able to induce the cytosolic mevalonate pathway, increasing the biosynthesis of phytosterols and phenolic compounds, and accumulate them outside the cells. However, in the absence of these cyclic oligosaccharidic elicitors, carrot cells mainly accumulated carotenoids through the methylerythritol 4-phosphate pathway. Therefore, the use of cyclodextrins would allow the extracellular accumulation of both phytosterols and phenolic compounds by diverting the carbon flux towards the cytosolic mevalonate/phenylpropanoid pathway.

  6. Testing automated liquid-based cytology samples with a manual liquid-based cytology method using residual cell suspensions from 500 ThinPrep cases.

    Science.gov (United States)

    Maksem, John A; Dhanwada, Vijaya; Trueblood, Joy E; Weidmann, James; Kane, Bruce; Bolick, David R; Bedrossian, Carlos W M; Kurtycz, Daniel F I; Stewart, Jim

    2006-06-01

    We report a technical improvement upon a previously disclosed manual liquid-based cytology (MLBC) method; and, we use the improved method to prepare slides from residual ThinPrep specimens in order to see how often ThinPrep diagnoses correspond to diagnoses derived from exhaustive examination of their parent sample suspensions. Residual cell suspensions from 500 ThinPrep cases comprising (1) 20 low-grade squamous intraepithelial lesions (LSILs); (2) 200 high risk (HR) negatives and 20 ASC-US; and (3) 260 screening cytology specimens were studied. Institutional review committee guidelines allowed us to know diagnoses by groups of specimens, but did not allow us to know individual patient diagnoses, so we could not perform case-by-case matched outcome-comparisons. Cells were concentrated by conventional centrifugation and sedimented into a polymer gel that was then vortex-mixed and converted into a viscous cell-rich suspension. The cell suspension was smeared between two clean glass slides, which were air-dried and stained with the Papanicolaou stain. Two study-sets were created, comprising one slide from each case. Each of the two study sets was examined by two cytopathologists, and discordant diagnoses were adjudicated. Because of the ambiguity involved in the "atypical" (ASC-US, ASC-H, AGC) diagnosis categories, only outcomes at the level of LSIL or greater were recorded. All MLBC SILs were digitally imaged and abnormal slides plus digital images were sent to the laboratory that provided the residual automated liquid-based cytology (ALBC) suspensions. The final diagnoses were confirmed by the laboratory that provided the residual ALBC specimens. MLBC slides of the 20 LSIL cases afforded 2 high-grade squamous intraepithelial lesions (HSILs) and 18 LSILs. Those of the 200 HR-Negatives showed 3 HSILs and 30 LSILs; and those of the 20 HR-ASC-US showed 3 HSILs and 9 LSILs. MLBC slides of the 260 screening cytology specimens showed 1 Carcinoma, 3 HSILs and 20 LSILs

  7. Habituation to thaxtomin A in hybrid poplar cell suspensions provides enhanced and durable resistance to inhibitors of cellulose synthesis

    Directory of Open Access Journals (Sweden)

    Beaulieu Carole

    2010-12-01

    Full Text Available Abstract Background Thaxtomin A (TA, a phytotoxin produced by the phytopathogen Streptomyces scabies, is essential for the development of potato common scab disease. TA inhibits cellulose synthesis but its actual mode of action is unknown. Addition of TA to hybrid poplar (Populus trichocarpa x Populus deltoides cell suspensions can activate a cellular program leading to cell death. In contrast, it is possible to habituate hybrid poplar cell cultures to grow in the presence of TA levels that would normally induce cell death. The purpose of this study is to characterize TA-habituated cells and the mechanisms that may be involved in enhancing resistance to TA. Results Habituation to TA was performed by adding increasing levels of TA to cell cultures at the time of subculture over a period of 12 months. TA-habituated cells were then cultured in the absence of TA for more than three years. These cells displayed a reduced size and growth compared to control cells and had fragmented vacuoles filled with electron-dense material. Habituation to TA was associated with changes in the cell wall composition, with a reduction in cellulose and an increase in pectin levels. Remarkably, high level of resistance to TA was maintained in TA-habituated cells even after being cultured in the absence of TA. Moreover, these cells exhibited enhanced resistance to two other inhibitors of cellulose biosynthesis, dichlobenil and isoxaben. Analysis of gene expression in TA-habituated cells using an Affymetrix GeneChip Poplar Genome Array revealed that durable resistance to TA is associated with a major and complex reprogramming of gene expression implicating processes such as cell wall synthesis and modification, lignin and flavonoid synthesis, as well as DNA and chromatin modifications. Conclusions We have shown that habituation to TA induced durable resistance to the bacterial toxin in poplar cells. TA-habituation also enhanced resistance to two other structurally

  8. Methionine catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and leads to S-methylcysteine and isoleucine syntheses.

    Science.gov (United States)

    Rébeillé, Fabrice; Jabrin, Samuel; Bligny, Richard; Loizeau, Karen; Gambonnet, Bernadette; Van Wilder, Valérie; Douce, Roland; Ravanel, Stéphane

    2006-10-17

    Despite recent progress in elucidating the regulation of methionine (Met) synthesis, little is known about the catabolism of this amino acid in plants. In this article, we present several lines of evidence indicating that the cleavage of Met catalyzed by Met gamma-lyase is the first step in this process. First, we cloned an Arabidopsis cDNA coding a functional Met gamma-lyase (AtMGL), a cytosolic enzyme catalyzing the conversion of Met into methanethiol, alpha-ketobutyrate, and ammonia. AtMGL is present in all of the Arabidopsis organs and tissues analyzed, except in quiescent dry mature seeds, thus suggesting that AtMGL is involved in the regulation of Met homeostasis in various situations. Also, we demonstrated that the expression of AtMGL was induced in Arabidopsis cells in response to high Met levels, probably to bypass the elevated Km of the enzyme for Met. Second, [13C]-NMR profiling of Arabidopsis cells fed with [13C]Met allowed us to identify labeled S-adenosylmethionine, S-methylmethionine, S-methylcysteine (SMC), and isoleucine (Ile). The unexpected production of SMC and Ile was directly associated to the function of Met gamma-lyase. Indeed, we showed that part of the methanethiol produced during Met cleavage could react with an activated form of serine to produce SMC. The second product of Met cleavage, alpha-ketobutyrate, entered the pathway of Ile synthesis in plastids. Together, these data indicate that Met catabolism in Arabidopsis cells is initiated by a gamma-cleavage process and can result in the formation of the essential amino acid Ile and a potential storage form for sulfide or methyl groups, SMC.

  9. Microbial cell disruption for improving lipid recovery using pressurized CO2 : Role of CO2 solubility in cell suspension, sugar broth and spent media.

    Science.gov (United States)

    Howlader, Md Shamim; French, William Todd; Shields-Menard, Sara A; Amirsadeghi, Marta; Green, Magan; Rai, Neeraj

    2017-04-03

    The study of in situ gas explosion to lyse the triglyceride-rich cells involves the solubilization of gas (e.g. carbon dioxide, CO2 ) in lipid rich cells under pressure followed by a rapid decompression, which allows the gas inside the cell to rapidly expand and rupture the cell from inside out. The aim of this study was to perform the cell disruption using pressurized CO2 as well as to determine the solubility of CO2 in Rhodotorula glutinis cell suspension, sugar broth media, and spent media. Cell disruption of R. glutinis was performed at two pressures of 2000 and 3500 kPa, respectively at 295.2 K, and it was found from both scanning electron microscopy (SEM) and plate count that a substantial amount of R. glutinis was disrupted due to the pressurized CO2 . We also found a considerable portion of lipid present in the aqueous phase after the disruption at P = 3500 kPa compared to control (no pressure) and P = 2000 kPa, which implied that more intracellular lipid was released due to the pressurized CO2 . Solubility of CO2 in R. glutinis cell suspension was found to be higher than the solubility of CO2 in both sugar broth media and spent media. Experimental solubility was correlated using the extended Henry's law, which showed a good agreement with the experimental data. Enthalpy and entropy of dissolution of CO2 were found to -14.22 kJ.mol(-1) and 48.10 kJ.mol(-1) .K(-1) , 9.64 kJ.mol(-1) and 32.52 kJ.mol(-1) .K(-1) , and 7.50 kJ.mol(-1) and 25.22 kJ.mol(-1) .K(-1) in R. glutinis, spent media and sugar broth media, respectively. This article is protected by copyright. All rights reserved.

  10. Genome-wide Expression Profiling in Seedlings of the Arabidopsis Mutant uro that is Defective in the Secondary Cell Wall Formation

    Institute of Scientific and Technical Information of China (English)

    Zheng Yuan; Xuan Yao; Dabing Zhang; Yue Sun; Hai Huang

    2007-01-01

    Plant secondary growth is of tremendous importance, not only for plant growth and development but also for economic usefulness.Secondary tissues such as xylem and phloem are the conducting tissues in plant vascular systems, essentially for water and nutrient transport, respectively.On the other hand, products of plant secondary growth are important raw materials and renewable sources of energy.Although advances have been recently made towards describing molecular mechanisms that regulate secondary growth, the genetic control for this process is not yet fully understood.Secondary cell wall formation in plants shares some common mechanisms with other plant secondary growth processes.Thus, studies on the secondary cell wall formation using Arabidopsis may help to understand the regulatory mechanisms for plant secondary growth.We previously reported phenotypic characterizations of an Arabidopsis semi-dominant mutant,upright rosette (uro), which is defective in secondary cell wall growth and has an unusually soft stem.Here, we show that lignification in the secondary cell wall in uro is aberrant by analyzing hypocotyl and stem.We also show genome-wide expression profiles of uro seedlings, using the Affymetrix GeneChip that contains approximately 24 000 Arabidopsis genes.Genes identified with altered expression levels include those that function in plant hormone biosynthesis and signaling,cell division and plant secondary tissue growth.These results provide useful information for further characterizations of the regulatory network in plant secondary cell wall formation.

  11. Reference: 798 [Arabidopsis Phenome Database[Archive

    Lifescience Database Archive (English)

    Full Text Available iption factors, control the delicately tuned reorientation and timing of cell div...EZ and SOMBRERO control the orientation of cell division plane in Arabidopsis root stem cells. 6 913-22 1908

  12. Site of Clomazone Action in Tolerant-Soybean and Susceptible-Cotton Photomixotrophic Cell Suspension Cultures 1

    Science.gov (United States)

    Norman, Michael A.; Liebl, Rex A.; Widholm, Jack M.

    1990-01-01

    Studies were conducted to determine the herbicidal site of clomazone action in tolerant-soybean (Glycine max [L.] Merr. cv Corsoy) (SB-M) and susceptible-cotton (Gossypium hirsutum [L.] cv Stoneville 825) (COT-M) photomixotrophic cell suspension cultures. Although a 10 micromolar clomazone treatment did not significantly reduce the terpene or mixed terpenoid content (microgram per gram fresh weight) of the SB-M cell line, there was over a 70% reduction in the chlorophyll (Chl), carotenoid (CAR), and plastoquinone (PQ) content of the COT-M cell line. The tocopherol (TOC) content was reduced only 35.6%. Reductions in the levels of Chl, CAR, TOC, and PQ indicate that the site of clomazone action in COT-M cells is prior to geranylgeranyl pyrophosphate (GGPP). The clomazone treatment did not significantly reduce the flow of [14C]mevalonate ([14C]MEV) (nanocuries per gram fresh weight) into CAR and the three mixed terpenoid compounds of SB-M cells. Conversely, [14C]MEV incorporation into CAR and the terpene moieties of Chl, PQ, and TOC in COT-M cells was reduced at least 73%, indicating that the site of clomazone action must be after MEV. Sequestration of clomazone away from the chloroplast cannot account for soybean tolerance to clomazone since chloroplasts isolated from both cell lines incubated with [14C]clomazone contained a similar amount of radioactivity (disintegrations per minute per microgram of Chl). The possible site(s) of clomazone inhibition include mevalonate kinase, phosphomevalonate kinase, pyrophosphomevalonate decarboxylase, isopentenyl pyrophosphate isomerase, and/or a prenyl transferase. PMID:16667768

  13. Enhanced homologous recombination is induced by alpha-particle radiation in somatic cells of Arabidopsis thaliana

    Science.gov (United States)

    Bian, Po; Liu, Ping; Wu, Yuejin

    Almost 9 percent of cosmic rays which strike the earth's atmosphere are alpha particles. As one of the ionizing radiations (IR), its biological effects have been widely studied. However, the plant genomic instability induced by alpha-particle radiation was not largely known. In this research, the Arabidopsis thaliana transgenic for GUS recombination substrate was used to evaluate the genomic instability induced by alpha-particle radiation (3.3MeV). The pronounced effects of systemic exposure to alpha-particle radiation on the somatic homologous recombination frequency (HRF) were found at different doses. The 10Gy dose of radiation induced the maximal HRF which was 1.9-fold higher than the control. The local radiation of alpha-particle (10Gy) on root also resulted in a 2.5-fold increase of somatic HRF in non-radiated aerial plant, indicating that the signal(s) of genomic instability was transferred to non-radiated parts and initiated their genomic instability. Concurrent treatment of seedlings of Arabidopsis thaliana with alpha-particle and DMSO(ROS scavenger) both in systemic and local radiation signifi- cantly suppressed the somatic HR, indicating that the free radicals produced by alpha-particle radiation took part in the production of signal of genomic instability rather than the signal transfer. Key words: alpha-particle radiation, somatic homologous recombination, genomic instability

  14. Transient gibberellin application promotes Arabidopsis thaliana hypocotyl cell elongation without maintaining transverse orientation of microtubules on the outer tangential wall of epidermal cells

    KAUST Repository

    Sauret-Güeto, Susanna

    2011-11-25

    The phytohormone gibberellin (GA) promotes plant growth by stimulating cellular expansion. Whilst it is known that GA acts by opposing the growth-repressing effects of DELLA proteins, it is not known how these events promote cellular expansion. Here we present a time-lapse analysis of the effects of a single pulse of GA on the growth of Arabidopsis hypocotyls. Our analyses permit kinetic resolution of the transient growth effects of GA on expanding cells. We show that pulsed application of GA to the relatively slowly growing cells of the unexpanded light-grown Arabidopsis hypocotyl results in a transient burst of anisotropic cellular growth. This burst, and the subsequent restoration of initial cellular elongation rates, occurred respectively following the degradation and subsequent reappearance of a GFP-tagged DELLA (GFP-RGA). In addition, we used a GFP-tagged α-tubulin 6 (GFP-TUA6) to visualise the behaviour of microtubules (MTs) on the outer tangential wall (OTW) of epidermal cells. In contrast to some current hypotheses concerning the effect of GA on MTs, we show that the GA-induced boost of hypocotyl cell elongation rate is not dependent upon the maintenance of transverse orientation of the OTW MTs. This confirms that transverse alignment of outer face MTs is not necessary to maintain rapid elongation rates of light-grown hypocotyls. Together with future studies on MT dynamics in other faces of epidermal cells and in cells deeper within the hypocotyl, our observations advance understanding of the mechanisms by which GA promotes plant cell and organ growth. © 2011 Blackwell Publishing Ltd.

  15. AtHKT1;1 mediates nernstian sodium channel transport properties in Arabidopsis root stelar cells.

    Directory of Open Access Journals (Sweden)

    Shaowu Xue

    Full Text Available The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na⁺ transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na⁺ and K⁺ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K⁺ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na⁺ over K⁺ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na⁺ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na⁺ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na⁺ channel transport properties.

  16. Highly protein-resistant coatings and suspension cell culture thereon from amphiphilic block copolymers prepared by RAFT polymerization.

    Science.gov (United States)

    Haraguchi, Kazutoshi; Kubota, Kazuomi; Takada, Tetsuo; Mahara, Saori

    2014-06-09

    Novel amphiphilic block copolymers composed of hydrophobic (poly(2-methoxyethyl acrylate): M) and hydrophilic (poly(N,N-dimethylacrylamide): D) segments were synthesized by living radical polymerization: a reversible addition-fragmentation chain-transfer polymerization. Two types of amphiphilic block copolymers, triblock (MDM) and 4-arm block ((MD)4) copolymers with specific compositions (D/M = (750-1500)/250), were prepared by a versatile one-pot synthesis. These copolymers show good adhesion to various types of substrates (e.g., polystyrene, polycarbonate, polypropylene, Ti, and glass), and the surface coating showed high protein repellency and a low contact angle for water, regardless of the substrate. The two opposing characteristics of high protein repellency and good substrate adhesion were achieved by the combined effects of the molecular architecture of the block copolymers, the high molecular weight, and the characteristics of each segment, that is, low protein adsorption capability of both segments and low glass transition temperature of the hydrophobic segment. Further, a polystyrene dish coated with the MDM block copolymer could be sterilized by γ-ray irradiation and used as a good substrate for a suspension cell culture that exhibits low cell adhesion and good cell growth.

  17. Induction of two prenyltransferases for the accumulation of coumarin phytoalexins in elicitor-treated Ammi majus cell suspension cultures.

    Science.gov (United States)

    Hamerski, D; Schmitt, D; Matern, U

    1990-01-01

    Two dimethylallyl diphosphate:umbelliferone dimethylallyltransferase (prenyltransferase) activities, catalysing the 6-prenylation and the 7-O-prenylation, respectively, of umbelliferone in the course of phytoalexin synthesis, increased in Ammi majus cell suspension cultures in response to elicitor treatment. Both enzyme activities were dependent on Mg2+ or Mn2+ with significant preference for Mg2+ in the 6-prenylation reaction. Whereas dark-grown cells did not contain these activities, both prenyltransferase activities were induced rapidly by the addition of elicitor reaching a first maximum after 10-14 hr and a second maximum beyond 30 hr. Other coumarin specific, elicitor-induced enzyme activities of A. majus cells, in contrast, showed only one maximum of activity within the 50 hr experimental period, while the pattern of induction of phenylalanine ammonia-lyase activity resembled that of the prenyltransferases with maxima at ca 8 hr and 20-30 hr. Preliminary data suggest that the apparent biphasic induction of these enzyme activities is due to post-translational enzyme modifications.

  18. Hydrodynamic stress induces monoterpenoid oxindole alkaloid accumulation by Uncaria tomentosa (Willd) D. C. cell suspension cultures via oxidative burst.

    Science.gov (United States)

    Trejo-Tapia, Gabriela; Sepúlveda-Jiménez, Gabriela; Trejo-Espino, José Luis; Cerda-García-Rojas, Carlos M; de la Torre, Mayra; Rodríguez-Monroy, Mario; Ramos-Valdivia, Ana C

    2007-09-01

    Uncaria tomentosa cell suspension cultures were grown in a 2-L stirred tank bioreactor operating at a shear rate gamma(.)(avg)=86 s(-1). The cultures showed an early monophasic oxidative burst measured as H2O2 production (2.15 micromol H2O2 g(-1) dw). This response was followed by a transient production of monoterpenoid oxindole alkaloids (178 +/- 40 microg L(-1) at 24 h). At the stationary phase (144 h), the increase of the shear rate gamma(.)(avg) up to 150 s(-1) and/or oxygen tension up to 85% generated H2O2, restoring oxindole alkaloid production. U. tomentosa cells cultured in Erlenmeyer flasks also exhibited the monophasic oxidative burst but the H2O2 production was 16-fold lower and the alkaloids were not detected. These cells exposed to H2O2 generated in situ produced oxindole alkaloids reaching a maximum of 234 +/- 40 microg L(-1). A positive correlation was observed between the oxindole alkaloid production and the endogenous H2O2 level. On the other hand, addition of 1 microM diphenyleneiodonium (NAD(P)H oxidase inhibitor) or 10 microM sodium azide (peroxidases inhibitor) reduced both H2O2 production and oxindole alkaloids build up, suggesting that these enzymes might play a role in the oxidative burst induced by the hydrodynamic stress.

  19. [Enhanced production of taxuyunnanine c in cell suspension cultures of Taxus chinensis by methyl jasmonate elicitation and in situ absorption].

    Science.gov (United States)

    Gao, Mingbo; Zhang, Wei; Yu, Xingju

    2010-02-01

    A bioprocess intensification strategy that combines both elicitation and in situ absorption was developed to improve the production of taxuyunnanine c (Tc) in cell suspension cultures of Taxus chinensis. When 100 micromol/L methyl jasmonate was added as an elicitor on Day 7, the Tc content and yield increased 3.6 and 3.3 times respectively, however the cell growth was reduced by 10%-30%. Significant improvement in Tc yield was observed when an absorbent XAD-7 was added on different time of the culture period. The optimum Tc yield was achieved when 100 g/L XAD-7 was added simultaneously with 100 micromol/L methyl jasmonate on Day 7. The maximum Tc yield of 477.4 mg/L was obtained on Day 21 of the culture, being 6.3-fold of the control and 1.9-fold of the 100 micromol/L methyl jasmonate treatment alone. In the combined treatment, 94% of the Tc produced was secreted outside of the cells and absorbed on XAD-7 absorbents. The results demonstrated that the process strategy combining elicitation and in situ absorption was effective to intensify the Tc biosynthesis via elicitation with the removal of product feedback inhibition via absorption, presenting a great potential in commercial applications.

  20. Phosphorylation switch modulates the interdigitated pattern of PIN1 localization and cell expansion in Arabidopsis leaf epidermis

    Institute of Scientific and Technical Information of China (English)

    Hongjiang Li; Deshu Lin; Pankaj Dhonukshe; Shingo Nagawa; Dandan Chen; Ji(r)í Friml; Ben Scheres; Hongwei Guo; Zhenbiao Yang

    2011-01-01

    Within a multicellular tissue cells may coordinately form a singular or multiple polar axes,but it is unclear whether a common mechanism governs different types of polar axis formation. The phosphorylation status of PIN proteins,which is directly affected by the PINOID (PID) protein kinase and the PP2A protein phosphatase,is known to regulate the apical-basal polarity of PIN localization in bipolar cells of roots and shoot apices. Here,we provide evidence that the phosphorylation status-mediated PIN polarity switch is widely used to modulate cellular processes in Arabidopsis including multipolar pavement cells (PC) with interdigitated lobes and indentations. The degree of PC interdigitation was greatly reduced either when the FYPP1 gene,which encodes a PP2A called phytochromeassociated serine/threonine protein phosphatase,was knocked out or when the PID gene was overexpressed (35S::PID).These genetic modifications caused PIN1 localization to switch from lobe to indentation regions. The PP2A and PID mediated switching of PIN1 localization is strikingly similar to their regulation of the apical-basal polarity switch of PIN proteins in other cells. Our findings suggest a common mechanism for the regulation of PIN1 polarity formation,a fundamental cellular process that is crucial for pattern formation both at the tissue/organ and cellular levels.

  1. Amino acid substitution converts WEREWOLF function from an activator to a repressor of Arabidopsis non-hair cell development.

    Science.gov (United States)

    Tominaga-Wada, Rumi; Nukumizu, Yuka; Wada, Takuji

    2012-02-01

    Root hair cell or non-hair cell fate determination in Arabidopsis thaliana root epidermis is model system for plant cell development. Two types of MYB transcription factors, the R2R3-type MYB, WEREWOLF (WER), and an R3-type MYB, CAPRICE (CPC), are involved in this cell fate determination process. To study the molecular basis of this process, we analyzed the functional relationship of WER and CPC. WER-CPC chimeric constructs were made from WER where all or parts of the MYB R3 region were replaced with the corresponding regions from CPC R3, and the constructs were introduced into the cpc-2 mutant. Although, the WER gene did not rescue the cpc-2 mutant 'small number of root hairs' phenotype, the WER-CPC chimera with two amino acids substitution (WC6) completely rescued the cpc-2 mutant phenotype. Furthermore, the WER-CPC chimera with 37 amino acids substitution (WC5) excessively rescued the cpc-2 mutant and induced 2.5 times more root hairs than wild-type. Consistent with this phenotype, GL2 gene expression was strongly reduced in WC5 in a cpc-2 background. Our results suggest that swapping at least two amino acids is sufficient to convert WER to CPC function. Therefore, these key residues may have strongly contributed to the selection of these important functions over evolution.

  2. Control of patterns of symmetric cell division in the epidermal and cortical tissues of the Arabidopsis root.

    Science.gov (United States)

    Zhang, Yanwen; Iakovidis, Michail; Costa, Silvia

    2016-03-15

    Controlled cell division is central to the growth and development of all multicellular organisms. Within the proliferating zone of the Arabidopsis root, regular symmetric divisions give rise to patterns of parallel files of cells, the genetic basis of which remains unclear. We found that genotypes impaired in the TONNEAU1a (TON1a) gene display misoriented symmetric divisions in the epidermis and have no division defects in the underlying cortical tissue. The TON1a gene encodes a microtubule-associated protein. We show that in the ton1a mutant, epidermal and cortical cells do not form narrow, ring-like preprophase bands (PPBs), which are plant-specific, cytoskeletal structures that predict the position of the division plane before mitosis. The results indicate that in the cortex but not in the epidermis, division plane positioning and patterning can proceed correctly in the absence of both a functional TON1a and PPB formation. Differences between tissues in how they respond to the signals that guide symmetric division orientation during patterning might provide the basis for organised organ growth in the absence of cell movements.

  3. Transcriptome profiling in Arabidopsis inflorescence stems grown under hypergravity in terms of cell walls and plant hormones

    Science.gov (United States)

    Tamaoki, D.; Karahara, I.; Nishiuchi, T.; De Oliveira, S.; Schreiber, L.; Wakasugi, T.; Yamada, K.; Yamaguchi, K.; Kamisaka, S.

    2009-07-01

    Land plants rely on lignified secondary cell walls in supporting their body weight on the Earth. Although gravity influences the formation of the secondary cell walls, the regulatory mechanism of their formation by gravity is not yet understood. We carried out a comprehensive analysis of gene expression in inflorescence stems of Arabidopsis thaliana L. using microarray (22 K) to identify genes whose expression is modulated under hypergravity condition (300 g). Total RNA was isolated from the basal region of inflorescence stems of plants grown for 24 h at 300 g or 1 g. Microarray analysis showed that hypergravity up-regulated the expression of 403 genes to more than 2-fold. Hypergravity up-regulated the genes responsible for the biosynthesis or modification of cell wall components such as lignin, xyloglucan, pectin and structural proteins. In addition, hypergravity altered the expression of genes related to the biosynthesis of plant hormones such as auxin and ethylene and that of genes encoding hormone-responsive proteins. Our transcriptome profiling indicates that hypergravity influences the formation of secondary cell walls by modulating the pattern of gene expression, and that auxin and/or ethylene play an important role in signaling hypergravity stimulus.

  4. Arabidopsis SMALL ORGAN 4, a homolog of yeast NOP53, regulates cell proliferation rate during organ growth

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ran Zhang; Zhixiang Qin; Xiao Zhang; Yuxin Hu

    2015-01-01

    Cel proliferation is a fundamental event essential for plant organogenesis and contributes greatly to the final organ size. Although the control of cel proliferation in plants has been extensively studied, how the plant sets the cel number required for a single organ is largely elusive. Here, we describe the Arabidopsis SMALL ORGAN 4 (SMO4) that functions in the regulation of cell proliferation rate and thus final organ size. The smo4 mutant exhibits a reduced size of organs due to the decreased cell number, and further analysis reveals that such phenotype results from a retardation of the cell cycle progression during organ development. SMO4 encodes a homolog of NUCLEOLAR PROTEIN 53 (NOP53) in Saccharomy-ces cerevisiae and is expressed primarily in tissues undergoing cel proliferation. Nevertheless, further complementation tests show that SMO4 could not rescue the lethal defect of NOP53 mutant of S. cerevisiae. These results define SMO4 as an important regulator of cell proliferation during organ growth and suggest that SMO4 might have been evolutionarily divergent from NOP53.

  5. A multidirectional non-cell autonomous control and a genetic interaction restricting tobacco etch virus susceptibility in Arabidopsis.

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    Suresh Gopalan

    Full Text Available BACKGROUND: Viruses constitute a major class of pathogens that infect a variety of hosts. Understanding the intricacies of signaling during host-virus interactions should aid in designing disease prevention strategies and in understanding mechanistic aspects of host and pathogen signaling machinery. METHODOLOGY/PRINCIPAL FINDINGS: An Arabidopsis mutant, B149, impaired in susceptibility to Tobacco etch virus (TEV, a positive strand RNA virus of picoRNA family, was identified using a high-throughput genetic screen and a counterselection scheme. The defects include initiation of infection foci, rate of cell-to-cell movement and long distance movement. CONCLUSIONS/SIGNIFICANCE: The defect in infectivity is conferred by a recessive locus. Molecular genetic analysis and complementation analysis with three alleles of a previously published mutant lsp1 (loss of susceptibility to potyviruses indicate a genetic interaction conferring haploinsufficiency between the B149 locus and certain alleles of lsp1 resulting in impaired host susceptibility. The pattern of restriction of TEV foci on leaves at or near the boundaries of certain cell types and leaf boundaries suggest dysregulation of a multidirectional non-cell autonomous regulatory mechanism. Understanding the nature of this multidirectional signal and the molecular genetic mechanism conferring it should potentially reveal a novel arsenal in the cellular machinery.

  6. Manipulation of culture strategies to enhance capsaicin biosynthesis in suspension and immobilized cell cultures of Capsicum chinense Jacq. cv. Naga King Chili.

    Science.gov (United States)

    Kehie, Mechuselie; Kumaria, Suman; Tandon, Pramod

    2014-06-01

    Manipulation of culture strategies was adopted to study the influence of nutrient stress, pH stress and precursor feeding on the biosynthesis of capsaicin in suspension and immobilized cell cultures of C. chinense. Cells cultured in the absence of one of the four nutrients (ammonium and potassium nitrate for nitrate and potassium stress, potassium dihydrogen orthophosphate for phosphorus stress, and sucrose for sugar stress) influenced the accumulation of capsaicin. Among the stress factors studied, nitrate stress showed maximal capsaicin production on day 20 (505.9 ± 2.8 μg g(-1) f.wt) in immobilized cell, whereas in suspension cultures the maximum accumulation (345.5 ± 2.9 μg g(-1) f.wt) was obtained on day 10. Different pH affected capsaicin accumulation; enhanced accumulation of capsaicin (261.6 ± 3.4 μg g(-1) f.wt) was observed in suspension cultures at pH 6 on day 15, whereas in case of immobilized cultures the highest capsaicin content (433.3 ± 3.3 μg g(-1) f.wt) was obtained at pH 5 on day 10. Addition of capsaicin precursors and intermediates significantly enhanced the biosynthesis of capsaicin, incorporation of vanillin at 100 μM in both suspension and immobilized cell cultures resulted in maximum capsaicin content with 499.1 ± 5.5 μg g(-1) f.wt on day 20 and 1,315.3 ± 10 μg g(-1) f.wt on day 10, respectively. Among the different culture strategies adopted to enhance capsaicin biosynthesis in cell cultures of C. chinense, cells fed with vanillin resulted in the maximum capsaicin accumulation. The rate of capsaicin production was significantly higher in immobilized cells as compared to freely suspended cells.

  7. Phenotypic screening of Arabidopsis T-DNA insertion lines for cell wall mechanical properties revealed ANTHOCYANINLESS2, a cell wall-related gene.

    Science.gov (United States)

    Mabuchi, Atsushi; Soga, Kouichi; Wakabayashi, Kazuyuki; Hoson, Takayuki

    2016-02-01

    We performed a phenotypic screening of confirmed homozygous T-DNA insertion lines in Arabidopsis for cell wall extensibility, in an attempt to identify genes involved in the regulation of cell wall mechanical properties. Seedlings of each line were cultivated and the cell wall extensibility of their hypocotyls was measured with a tensile tester. Hypocotyls of lines with known cell wall-related genes showed higher or lower extensibility than those of the wild-type at high frequency, indicating that the protocol used was effective. In the first round of screening of randomly selected T-DNA insertion lines, we identified ANTHOCYANINLESS2 (ANL2), a gene involved in the regulation of cell wall mechanical properties. In the anl2 mutant, the cell wall extensibility of hypocotyls was significantly lower than that of the wild-type. Levels of cell wall polysaccharides per hypocotyl, particularly cellulose, increased in anl2. Microarray analysis showed that in anl2, expression levels of the major peroxidase genes also increased. Moreover, the activity of ionically wall-bound peroxidases clearly increased in anl2. The activation of peroxidases as well as the accumulation of cell wall polysaccharides may be involved in decreased cell wall extensibility. The approach employed in the present study could contribute to our understanding of the mechanisms underlying the regulation of cell wall mechanical properties.

  8. Effect of Plant Growth Regulators on Callus, Cell Suspension and Cell Line Selection for Flavonoid Production from Pegaga (centella asiatica L. urban

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    Suat H. Tan

    2010-01-01

    Full Text Available Problem statement: Considering pegaga medicinal properties and over-exploitation, the requirement for a tissue culture technique as an alternative production system was crucial. Approach: Investigation of cell suspension culture response to different plant growth regulators (PRGs for flavonoid production from elite cell line was carried out. Callus cultures were initiated from the leaf explants of Centella asiatica on Murashige and Skoog (MS medium containing B5 vitamins and 30 g L−1 sucrose supplemented with different concentrations (0.5-2.5 mg L−1 of 2,4-D, NAA, Dicamba, Picloram and IBA supplied singly and in combination with different concentrations (0.5-1.5 mg L−1 of kinetin, BAP and TDZ. Results: Callus induction was observed for all the PGRs tested. The highest callus induction frequency (86.67% was observed in MS medium containing 2.0 mg L−1 2,4-D while the combination of 2.0 mg L−1 2,4-D and 1 mg L−1 kinetin in MS medium gave the highest biomass yield (0.27 g dry weight culture−1. This combination was also found to be best for callus proliferation for all the accessions investigated. Among the four accessions tested, UPM03 was found to have the highest biomass yield (0.041 g DW culture−1 and hydrolysed flavonoid content (10.75 mg g−1 DW after the 12th day of culture. The flavonoids present in the four accessions were quercetin, kaempherol, luteolin and rutin based on high performance liquid chromatography (HPLC analysis. These results indicated that C. asiatica accession UPM03 was the potential elite cell line in mass production of flavonoid, especially luteolin. Coclusions/Recommendations: In the establishment of cell suspension culture, 2 mg L−1 2,4-D and 1 mg L−1 kinetin were the best PGRs in supporting the cell growth and flavonoid production. This is the first report on the use of PRGs on the establishment of cell suspension cultures in flavonoid production of C. asiatica.

  9. Identification and expression analysis of methyl jasmonate responsive ESTs in paclitaxel producing Taxus cuspidata suspension culture cells

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    Lenka Sangram K

    2012-04-01

    Full Text Available Abstract Background Taxol® (paclitaxel promotes microtubule assembly and stabilization and therefore is a potent chemotherapeutic agent against wide range of cancers. Methyl jasmonate (MJ elicited Taxus cell cultures provide a sustainable option to meet the growing market demand for paclitaxel. Despite its increasing pharmaceutical importance, the molecular genetics of paclitaxel biosynthesis is not fully elucidated. This study focuses on identification of MJ responsive transcripts in cultured Taxus cells using PCR-based suppression subtractive hybridization (SSH to identify genes involved in global pathway control. Results Six separate SSH cDNA libraries of paclitaxel-accumulating Taxus cuspidata P991 cell lines were constructed at three different post-elicitation time points (6h, 18h and 5 day to identify genes that are either induced or suppressed in response to MJ. Sequencing of 576 differentially screened clones from the SSH libraries resulted in 331 unigenes. Functional annotation and Gene Ontology (GO analysis of up-regulated EST libraries showed enrichment of several known paclitaxel biosynthetic genes and novel transcripts that may be involved in MJ-signaling, taxane transport, or taxane degradation. Macroarray analysis of these identified genes unravelled global regulatory expression of these transcripts. Semi-quantitative RT-PCR analysis of a set of 12 candidate genes further confirmed the MJ-induced gene expression in a high paclitaxel accumulating Taxus cuspidata P93AF cell line. Conclusions This study elucidates the global temporal expression kinetics of MJ responsive genes in Taxus suspension cell culture. Functional characterization of the novel genes identified in this study will further enhance the understanding of paclitaxel biosynthesis, taxane transport and degradation.

  10. INSECTICIDAL POTENTIALITY OF FLAVONOIDS FROM CELL SUSPENSION CULTURE OF MARCHANTIA LINEARIS LEHM. & LINDENB AGAINST SPODOPTERA LITURA F.

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    Remya Krishnan

    2015-05-01

    Full Text Available Bryophytes were diverse, primitive non vascular am phibious taxa distributed worldwide and form the second largest category of plants. Bryophytes synthesize an array of phytochemicals to combat against the unhospitable environmental conditions including predation, UV radiation, high temperature and pest and pathogens. The present investigation was undertaken to elucidate flavonoids from in vitro cell cultures of the liverwort Marchantia linearis Lehm. & Lindenb. its fractionation and analysis of insecticidal potentialities. Initially, callus culture was initiated from spores in MS/5 media containing gr owth regulators BAP and NAA at the concentration of 2 mg/L and 0.5 mg/L. Agitation of the friable callus at lowe r rpm bring about lower leve l of cell dispersion, on the contrary at higher rpm might have risk of cell collision that is why rpm was kept at moderate speed i.e., 110 rpm. Continuous sub culturing process substantially improves cell growth and biomass. In the second phase, the flavonoids were isolated from cell suspension cultures of M. linearis and were fractionated by TLC and HPLC PAD chromatogram, which revealed the presence of quer cetin, luteolin, apigenin , rutin and kaempferol. In vivo insecticidal analysis revealed significant antifeedan t, larvicidal and pupicidal activities at all the concentrations against 5 th instar larvae of Spodoptera litura . The extract also exhibited feeding deterrent activity with M. linearis. Similarly, the nutritional parameters were also affected i.e., reduced ECI (Efficiency of conversion of ingested food and ECD (Efficiency of conversion of digested food and increased AD (Approximate digestibility and metabolic cost for the larvae, when compared with the control. The consumption of the basal diet with the incorporation of flavonoids by S. litura larvae was not significantly different compared to the co nsumption of the control diet by the larvae. Faecal production reduced proportionally with

  11. Aluminium-induced phospholipid signal transduction pathway in Coffea arabica suspension cells and its amelioration by silicic acid.

    Science.gov (United States)

    Quintal-Tun, Fausto; Muñoz-Sánchez, J Armando; Ramos-Díaz, Ana; Escamilla-Bencomo, Armando; Martínez-Estévez, Manuel; Exley, Christopher; Hernández-Sotomayor, S M Teresa

    2007-02-01

    Coffee (Coffea arabica L.) is of economic importance worldwide. Its growth in organic-rich acidic soils is influenced by aluminium such that coffee yield may be impaired. Herein we have used the Al-sensitive C. arabica suspension cell line L2 to analyse the effect of two different Al species on the phosphoinositide signal transduction pathway. Our results have shown that the association of Al with coffee cells was affected by the pH and the form of Al in media. More Al was associated with cells at pH 4.3 than 5.8, whereas when Al was present as hydroxyaluminosilicates (HAS) the association was halved at pH 4.3 and unchanged at pH 5.8. Two signal transduction elements were also evaluated; phospholipase C (PLC) activity and phosphatidic acid (PA) formation. PLC was inhibited ( approximately 50%) when cells were incubated for 2 h in the presence of either AlCl(3) or Al in the form of HAS. PA formation was tested as a short-term response to Al. By way of contrast to what was found for PLC, incubation of cells for 15 min in the presence of AlCl(3) decreased the formation of PA whereas the same concentration of Al as HAS produced no effect upon its formation. These results suggest that Al is capable to exert its effects upon signal transduction as Al((aq))(3+) acting upon a mechanism linked to the phosphoinositide signal transduction pathway.

  12. Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis.

    Science.gov (United States)

    Wang, Shucai; Li, Eryang; Porth, Ilga; Chen, Jin-Gui; Mansfield, Shawn D; Douglas, Carl J

    2014-05-23

    Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

  13. Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Shucai [Northeast Normal Univ., Changchun (China); Univ. of British Columbia, Vancouver, BC (Canada); Li, Eryang [Univ. of British Columbia, Vancouver, BC (Canada); Porth, Ilga [Univ. of British Columbia, Vancouver, BC (Canada); Chen, Jin-Gui [Univ. of British Columbia, Vancouver, BC (Canada); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Mansfield, Shawn D. [Univ. of British Columbia, Vancouver, BC (Canada); Douglas, Carl [Univ. of British Columbia, Vancouver, BC (Canada)

    2014-05-23

    Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

  14. Multi-omics analysis identifies genes mediating the extension of cell walls in the Arabidopsis thaliana root elongation zone

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    Michael H Wilson

    2015-02-01

    Full Text Available Plant cell wall composition is important for regulating growth rates, especially in roots. However, neither analyses of cell wall composition nor transcriptomes on their own can comprehensively reveal which genes and processes are mediating growth and cell elongation rates. This study reveals the benefits of carrying out multiple analyses in combination. Sections of roots from five anatomically and functionally defined zones in Arabidopsis thaliana were prepared and divided into three biological replicates. We used glycan microarrays and antibodies to identify the major classes of glycans and glycoproteins present in the cell walls of these sections, and identified the expected decrease in pectin and increase in xylan from the meristematic zone (MS, through the rapid and late elongation zones (REZ, LEZ to the maturation zone and the rest of the root, including the emerging lateral roots. Other compositional changes included extensin and xyloglucan levels peaking in the REZ and increasing levels of arabinogalactan-proteins (AGP epitopes from the MS to the LEZ, which remained high through the subsequent mature zones. Immuno-staining using the same antibodies identified the tissue and (subcellular localization of many epitopes. Extensins were localized in epidermal and cortex cell walls, while AGP glycans were specific to different tissues from root-hair cells to the stele. The transcriptome analysis found several gene families peaking in the REZ. These included a large family of peroxidases (which produce the reactive oxygen species needed for cell expansion, and three xyloglucan endo-transglycosylase/hydrolase genes (XTH17, XTH18 and XTH19. The significance of the latter may be related to a role in breaking and re-joining xyloglucan cross-bridges between cellulose microfibrils, a process which is required for wall expansion. Knockdowns of these XTHs resulted in shorter root lengths, confirming a role of the corresponding proteins in root

  15. Effects of mutations in the Arabidopsis Cold Shock Domain Protein 3 (AtCSP3) gene on leaf cell expansion.

    Science.gov (United States)

    Yang, Yongil; Karlson, Dale

    2012-08-01

    The cold shock domain is among the most evolutionarily conserved nucleic acid binding domains from prokaryotes to higher eukaryotes, including plants. Although eukaryotic cold shock domain proteins have been extensively studied as transcriptional and post-transcriptional regulators during various developmental processes, their functional roles in plants remains poorly understood. In this study, AtCSP3 (At2g17870), which is one of four Arabidopsis thaliana c old s hock domain proteins (AtCSPs), was functionally characterized. Quantitative RT-PCR analysis confirmed high expression of AtCSP3 in reproductive and meristematic tissues. A homozygous atcsp3 loss-of-function mutant exhibits an overall reduced seedling size, stunted and orbicular rosette leaves, reduced petiole length, and curled leaf blades. Palisade mesophyll cells are smaller and more circular in atcsp3 leaves. Cell size analysis indicated that the reduced size of the circular mesophyll cells appears to be generated by a reduction of cell length along the leaf-length axis, resulting in an orbicular leaf shape. It was also determined that leaf cell expansion is impaired for lateral leaf development in the atcsp3 loss-of-function mutant, but leaf cell proliferation is not affected. AtCSP3 loss-of-function resulted in a dramatic reduction of LNG1 transcript, a gene that is involved in two-dimensional leaf polarity regulation. Transient subcellular localization of AtCSP3 in onion epidermal cells confirmed a nucleocytoplasmic localization pattern. Collectively, these data suggest that AtCSP3 is functionally linked to the regulation of leaf length by affecting LNG1 transcript accumulation during leaf development. A putative function of AtCSP3 as an RNA binding protein is also discussed in relation to leaf development.

  16. A role for katanin in plant cell division: microtubule organization in dividing root cells of fra2 and lue1Arabidopsis thaliana mutants.

    Science.gov (United States)

    Panteris, Emmanuel; Adamakis, Ioannis-Dimosthenis S; Voulgari, Georgia; Papadopoulou, Galini

    2011-07-01

    Severing of microtubules by katanin has proven to be crucial for cortical microtubule organization in elongating and differentiating plant cells. On the contrary, katanin is currently not considered essential during cell division in plants as it is in animals. However, defects in cell patterning have been observed in katanin mutants, implying a role for it in dividing plant cells. Therefore, microtubule organization was studied in detail by immunofluorescence in dividing root cells of fra2 and lue1 katanin mutants of Arabidopsis thaliana. In both, early preprophase bands consisted of poorly aligned microtubules, prophase spindles were multipolar, and the microtubules of expanding phragmoplasts were elongated, bended toward and connected to the surface of daughter nuclei. Accordingly, severing by katanin seems to be necessary for the proper organization of these microtubule arrays. In both fra2 and lue1, metaphase/anaphase spindles and initiating phragmoplasts exhibited typical organization. However, they were obliquely oriented more frequently than in the wild type. It is proposed that this oblique orientation may be due to prophase spindle multipolarity and results in a failure of the cell plate to follow the predetermined division plane, during cytokinesis, producing oblique cell walls in the roots of both mutants. It is therefore concluded that, like in animal cells, katanin is important for plant cell division, influencing the organization of several microtubule arrays. Moreover, failure in microtubule severing indirectly affects the orientation of the division plane.

  17. Sulfonamides identified as plant immune-priming compounds in high-throughput chemical screening increase disease resistance in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Yoshiteru eNoutoshi

    2012-10-01

    Full Text Available Plant activators are agrochemicals that protect crops from diseases by activating the plant immune system. To isolate lead compounds for use as practical plant activators, we screened 2 different chemical libraries composed of various bioactive substances by using an established screening procedure that can selectively identify immune-priming compounds. We identified and characterized a group of sulfonamide compounds—sulfameter, sulfamethoxypyridazine, sulfabenzamide, and sulfachloropyridazine—among the various isolated candidate molecules. These sulfonamide compounds enhanced the avirulent Pseudomonas-induced cell death of Arabidopsis suspension cell cultures and increased disease resistance in Arabidopsis plants against both avirulent and virulent strains of the bacterium. These compounds did not prevent the growth of pathogenic bacteria in minimal liquid media at 200 µM. They also did not induce the expression of defense-related genes in Arabidopsis seedlings, at least not at 24 and 48 h after treatment, suggesting that they do not act as salicylic acid analogs. In addition, although sulfonamides are known to be folate biosynthesis inhibitors, the application of folate did not restore the potentiation effects of the sulfonamides on pathogen-induced cell death. Our data suggest that sulfonamides potentiate Arabidopsis disease resistance by their novel chemical properties.

  18. Double Fertilization in Arabidopsis thaliana Involves a Polyspermy Block on the Egg but Not the Central Cell

    Institute of Scientific and Technical Information of China (English)

    Rod J.Scott; Susan J.Armstrong; James Doughty; Melissa Spielman

    2008-01-01

    In animal reproduction,thousands of sperm may compete to fertilize a single egg,but polyspermy blocks prevent multiple fertilization that would otherwise lead to death of the embryo.In flowering plants,successfuI seed development requires that only two sperm are delivered to the embryo sac,where each must fertilize a female gamete(egg or central cell)to produce the embryo and endosperm.Therefore,polyspermy must be avoided,not only to prevent abnormalities in offspring,but to ensure double fertilization.It is not understood how each sperm fertilizes only one female gamete,nor has the existence of polyspermy barriers been directly tested in vivo.Here,we sought evidence for poly-spermy blocks in angiosperms using the polyspermic tetraspore(tes)mutant of Arabidopsis,which allows in-vivo challenge of egg and central cell with multiple male gametes.We show that tes mutant pollen tubes can transmit more than one sperm pair to an embryo sac,and that sperm from more than one pair can participate in fertilization.We detected endosperms but not embryos with ploidies that could only result from multiple fertilization.Our results therefore dem-onstrate an in-vivo polyspermy block on the egg,but not the central cell of a flowering plant.

  19. Enhanced extracellular production of trans-resveratrol in Vitis vinifera suspension cultured cells by using cyclodextrins and coronatine.

    Science.gov (United States)

    Almagro, Lorena; Belchí-Navarro, Sarai; Martínez-Márquez, Ascensión; Bru, Roque; Pedreño, María A

    2015-12-01

    In the present work the effect of cyclodextrin and coronatine on both trans-resveratrol production and the expression of stilbene biosynthetic genes in Vitis vinifera L. cv Monastrell suspension cultured cells were evaluated. The results showed the maximum level of trans-resveratrol produced by cells and secreted to the culture medium with 50 mM cyclodextrins and 1 μM coronatine. Since the levels of trans-resveratrol produced in the combined treatment were higher than the sum of the individual treatments, a synergistic effect between both elicitors was assumed. In addition, all the analysed genes were induced by cyclodextrins and/or coronatine. The expression of the phenylalanine ammonia lyase and stilbene synthase genes was greatly enhanced by coronatine although an increase in the amount of trans-resveratrol in the spent medium was not detected. Therefore, despite the fact that trans-resveratrol production is related with the expression of genes involved in the biosynthetic process, other factors may be involved, such as post-transcriptional and post-traductional regulation. The expression maximal levels of cinnamate 4-hydroxylase and 4-coumarate-CoA ligase genes were found with cyclodextrins alone or in combination with coronatine suggesting that the activity of these enzymes could be not only important for the formation of intermediates of trans-R biosynthesis but also for those intermediates involved in the biosynthesis of lignins and/or flavonoids.

  20. Role of Changes in Cell Fatty Acids Composition in the Increasing of Frost Resistance of Winter Wheat Suspension Culture

    Directory of Open Access Journals (Sweden)

    I.V. Lyubushkina

    2013-11-01

    Full Text Available Influences of low temperatures (4 and 8 ° С on the frost tolerance and fatty acid compositions of cells in a winter wheat suspension culture have been studied. It has been found that treatment of the culture with 4 °C (7 days did not protect cells from subsequent freezing temperature action (-8 °С, 6 h and was not accompanied significant changes in the fatty acid composition. On the contrary, the treatment of the culture with the temperature 8 °C (7 days prevented the death caused by freezing temperature and the content of saturated fatty acids decreased: pentadecanoic acid (by 35,0%, palmitic acid (by 19,9% and stearic acid (by 65,4%, and the content of α-linolenic acid increased by 94%. That was the cause of the double bond index (DBI increase by 16%. The role of fatty acids composition changes in the process of increasing frost tolerance in plants are discussed.

  1. Confocal restricted-height imaging of suspension cells (CRISC) in a PDMS microdevice during apoptosis

    NARCIS (Netherlands)

    Munoz-Pinedo, Cristina; Green, Douglas R.; Berg, van den Albert

    2005-01-01

    We have monitored and imaged cell death induced in human leukemic U937 cells over time using three-color confocal imaging. Three different apoptotic inducers, anti-Fas, TNF- and Etoposide were used. Individual cascaded events such as loss of mitochondrial transmembrane potential, exposure of phospha

  2. Confocal restricted-height imaging