Cell Wall Regeneration In Protoplasts Research Articles

Caffeine inhibits cell-wall regeneration in protoplasts isolated from tobacco BY-2 cells

Caffeine inhibits cell-wall regeneration in protoplasts isolated from tobacco BY-2 cells

The Cell Wall Regeneration of Tobacco Protoplasts Based on Microfluidic System

The cell wall, serving as the exoskeleton of plants, is naturally a barrier to resist external stresses. Protoplasts can be obtained by dissolving the cell walls of plant cells without damaging the cell membrane, and are widely used in the rapid propagation, transgenic breeding, and somatic hybridization of plants. However, to regenerate the cell wall is a precondition for cell division. Therefore, to study the culture condition and influencing factors during the cell wall regeneration of protoplasts is vital. Traditionally, culture medium is used to cultivate protoplasts, but it has some disadvantages. Herein, a microfluidic system with crossed channels was constructed to isolate and cultivate the protoplasts of tobacco. Then, the cell wall regeneration of the tobacco protoplasts was also studied based on this microfluidic system. It was found that, compared with the control, benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) could accelerate the regeneration of the cell wall, while Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) could inhibit the regeneration of the cell wall within 24 h. To conclude, this study demonstrated that a crossed microfluidic chip could be an effective tool to study cell wall regeneration or other behavior of plant cells in situ with high resolution. In addition, this study revealed the rate of cell wall regeneration under BTH and Pst DC3000 treatment.

Dynamics of structural polysaccharides deposition on the plasma-membrane surface of plant protoplasts during cell wall regeneration

In this study, dynamic changes in structural polysaccharide deposition on the plasma membrane and cortical microtubules (CMTs) behavior were monitored in protoplasts isolated from white birch callus using confocal laser scanning microscopy and atomic force microscopy. We focused on the influence of an environmental stimulus on cell wall regeneration in protoplasts by employing an acidic culture medium containing a high concentration of Ca2+ (the stress condition). Under the non-stress condition, cellulose microfibrils and callose were initially synthesized, and thereafter deposited on the plasma membrane as “primary cell wall material”. Under the stress condition, callose micro-sized fibers were secreted without cell wall regeneration. Behavior of CMTs labeled with mammalian microtubule-associated protein 4 with green fluorescent protein in transgenic protoplasts was monitored by time-lapse video analysis. Under the non-stress condition, CMTs behavior showed a linear arrangement at a fixed position, whereas unfixed manner of CMTs behavior was observed under the stress condition. These findings indicate that excessive Ca2+ affects cellulose synthesis and CMTs dynamics in plant protoplasts. Current study first demonstrated dynamics of cell wall regeneration and CMTs in woody protoplast, which provides novel insight to aid in understanding early stages of primary cell wall formation in plants.

Studies on cell wall regeneration in protoplast culture of legumes - the effect of organic medium additives on cell wall components

The cell wall regeneration in mesophyll protoplasts of yellow lupin and grass pea was studied. The occurrence of cell wall components: cellulose, callose and arabinogalactan proteins was analysed during 15 days of culture. Protoplasts were cultured in different media to test the effect of culture environment on the cell wall regeneration. Medium supplementation with 2 mg/l chitosan resulted in prolonged viability, more balanced cellulose resynthesis, increased callose formation and induction of mitotic divisions in protoplast-derived cells of both examined legumes. In chitosan-enriched medium arabinogalactan proteins were detected in cell plates of divided cells. Medium rich in additional organic compounds, i.e. free amino acids, organic acids and monosaccharides, was inferior to media of simpler composition. In both species the relatively quick cellulose resynthesis negatively affected the viability of protoplast-derived cells. In grass pea cellulose appeared during 24 h of culture. In yellow lupin the process started significantly later and after 10 days the frequency of walled cells did not exceed 50%. Callose was detected in cultures of both species and its pattern suggested that the synthesis was unlikely to be a result of protoplast wounding. Arabinogalactan proteins were localized in cell walls of different types of cells: dividing, elongating, but predominantly in degenerating ones. Occurrence and organization of the cell wall components studied were discussed in relation to recalcitrance of grass pea and yellow lupin protoplasts.

Pga13 in Candida albicans is localized in the cell wall and influences cell surface properties, morphogenesis and virulence

The fungal cell wall is an essential organelle required for maintaining cell integrity and also plays an important role in the primary interactions between pathogenic fungi and their hosts. PGA13 encodes a GPI protein in the human pathogen Candida albicans, which is highly up-regulated during cell wall regeneration in protoplasts. The Pga13 protein contains a unique tandem repeat, which is present five times and is characterized by conserved spacing between the four cysteine residues. Furthermore, the mature protein contains 38% serine and threonine residues, and therefore probably is a highly glycosylated cell wall protein. Consistent with this, a chimeric Pga13-V5 protein could be localized to the cell wall, but only after deglycosylation was performed. Disruption of PGA13 led to increased sensitivity to Congo red, Calcofluor white, and zymolyase, and to a diminished ability of protoplasts to recover their cell wall. In addition, pga13Δ mutants exhibited delayed filamentation, a higher surface hydrophobicity, and increased adherence and flocculation (cell–cell interactions). Furthermore, transcript profiling showed that expression of four members of the ALS family (adhesin-encoding genes) is up-regulated in the pga13Δ null mutant. Altogether, these results indicate that Pga13 is a wall-localized protein that contributes to cell wall synthesis and is important for acquiring normal surface properties. The contribution of Pga13 to surface hydrophilicity may be important for cell dispersal during development of invasive infections, and possibly for morphological development. This is consistent with the observed reduced virulence of pga13Δ mutants in a mouse model of disseminated candidiasis.

Transglutaminase-like activity participates in cell wall biogenesis in Saccharomyces cerevisiae

Transglutaminases (TGases) catalyze the cross-linking between protein molecules by formation of an amide bond between γ-carboxyamide group of glutamine and the e-amine group of lysine under deamination of glutamine. We have demonstrated the participation of transglutaminase-like activity in the isolated cell walls and in the process of cell wall regeneration in protoplasts of the yeast Saccharomyces cerevisiae. A radioactive TGase substrate [3H]putrescine was incorporated into the isolated cell walls and into the TCA-insoluble fraction in regenerating protoplasts. The incorporation was increased by adding exogenous artificial substrate of TGase N,N’-dimethylcasein and was inhibited by TGase inhibitor cystamine and/or EDTA. These results suggest the existence of a TGase-type reaction involved in the formation of covalent cross-links between glycoprotein molecules during cell wall construction in S. cerevisiae.

A Proteomic Approach to Apoplastic Proteins Involved in Cell Wall Regeneration in Protoplasts of Arabidopsis Suspension-cultured Cells

To clarify the mechanisms of cell wall construction, we used a proteomic approach to investigate the proteins secreted into cell wall spaces during cell wall regeneration from the protoplasts of Arabidopsis suspension-cultured cells. We focused on cell wall proteins loosely bound to the cell wall architecture and extractable with 1 M KCl solutions from: (i) native suspension cultured cells; (ii) protoplasts that had been allowed to regenerate their cell walls for 1 h; and (iii) protoplasts allowed to regenerate their cell walls for 3 h. We adopted a non-destructive extraction procedure without disrupting cellular integrity, thereby avoiding contamination from cytoplasmic proteins. Using two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), we separated, mapped and identified 71 proteins derived from the native cell wall, and 175 and 212 proteins derived from the 1 and 3 h regenerated protoplasts, respectively. Quite different sets of proteins with differing status of their post-translational modifications, including phosphorylation and glycosylation, were identified in the three protein fractions. This indicated dynamic in muro changes in the cell wall proteins during cell wall regeneration in the protoplasts. The analysis revealed a set of enzymes specifically involved in cell wall expansion and construction in suspension-cultured cells. This approach has also determined a set of cell wall proteins that had not been predicted to be localized in cell wall spaces.

Preparation of Protoplasts from Chlorella vulgaris K-73122 and Cell Wall Regeneration of Protoplasts from C. vulgaris K-73122 and C-27

Preparation of Protoplasts from Chlorella vulgaris K-73122 and Cell Wall Regeneration of Protoplasts from C. vulgaris K-73122 and C-27

The Effects of Extracellular Calmodulin on Cell Wall Regeneration of Protoplasts and Cell Division

The Effects of Extracellular Calmodulin on Cell Wall Regeneration of Protoplasts and Cell Division

Interaction between Pseudomonas syringae pv. Pisi and Isolated Pea Mesophyll Protoplasts

AbstractThe interaction between five races of Pseudomonas syringae pv. pisi (PSP) and isolated mesophyll protoplasts obtained from five pea cultivars was studied. There was no trend in the attachment of bacterial cells to surfaces of compatible and incompatible host protoplasts. The viability of protoplasts from compatible and incompatible host‐pathogen interactions did not differ significantly; however, changes in the viability of cultivar Kelvedon Wonder protoplasts, compatible with all five races, was relatively more stable following inoculation with bacteria than those of cultivar Fortune, incompatible with all of the five races. Protoplast cell wall regeneration did not take place until 24–48 h after isolation. It is concluded that the use of pea protoplasts as a model system for studying the pea‐PSP interaction appears to have considerable potential for the future, but more basic research is required.

Isolation and Cell Wall Regeneration of Protoplasts from Botrytis cinerea Pers.

Abstract A procedure for efficient isolation and cell wall regeneration of protoplasts from Botrytis cinerea is described. Protoplasts were obtained from mycelia using a lytic enzyme mixture containing β‐Glucuronidase, Cellulase R10 and Driselase with mannitol for osmotic support. The digestion of cell walls was checked by fluorescence microscopy. Protoplasts were purified from cell debris and lytic enzymes. Regeneration and reversion were performed by incubation on agar plates.

Modification of protoplast cell wall regeneration by membrane perturbation

Protoplasts derived from cells ofBoergesenia forbesii regenerated aberrant cell walls when treated with cholesteryl hemisuccinate (CHS). Protoplasts treated with CHS, for a short period during the initial stages of cell wall regeneration, developed a patchwork cell wall, possessing regions devoid of cell wall. This effect was reversible, and treated cells ultimately developed a normal, confluent cell wall when removed from the CHS. Freeze fracture studies revealed that for CHS-treated cells, regions without microfibril impressions did possess intramembranous particles (IMP's) but that these regions contained small domains free of IMP's suggestive of lateral phase separation. The data implies that the physical characteristics of the plasma membrane lipid are important to the deposition of cell wall microfibrils during cell wall regeneration. This effect may be attributed to altered lipid-protein interactions, modified membrane fusion characteristics, or altered membrane flow.

Protoplast fusion of Lactobacillus casei.

Lactobacillus casei ATCC 7469 was successfully converted to protoplasts by treatment with endo-N-acetyl muramidase in sucrose phosphate buffer. For full hydrolysis of cell walls, a high concentration of sucrose and a cold shock were necessary. Mg2+ ions enhanced the stability of protoplasting cells. The cell wall regeneration of protoplasts was more effective on gelatin-induced regeneration medium than with the soft overlay method. The optimal concentration of gelatin was 2.5%. The frequency of regeneration was found to be about 6% for the protoplast prepared by enzyme treatment for 20min. The mutants having streptomycin resistance and rifampicin resistance, as selection markers for the detection of fusion, were isolated by UV irradiation and NTG treatment. These mutants were stable for at least several transfers. Protoplast fusion was carried out using PEG (50% solution of polyethyleneglycol, M.W. 6, 000). The frequency of protoplast fusion was found to be about 10-5.

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.

Curing of plasmids during a protoplast-cell wall regeneration cycle in Bacillus subtilis strains

Curing of plasmids during a protoplast-cell wall regeneration cycle in Bacillus subtilis strains

Curing of plasmids during a protoplast-cell wall regeneration cycle inBacillus subtilisstrains

Curing of plasmids during a protoplast-cell wall regeneration cycle in<i>Bacillus subtilis</i>strains

Effect of isoprothiolane on Pyricularia oryzae Cavara (I). Cell wall regeneration of protoplasts.

Effects of isoprothiolane (IPT) on the regeneration process of protoplasts isolated from hyphae of Pyricularia oryzae Cav. were examined by electron, phase-contrast and fluorescence microscopies. Electron microscopy revealed that freshly-prepared protoplasts were completely devoid of cell wall. The plasmalemma became corrugated, and small, vesicle-like protrusions appeared from it before any sign of incipient cell wall regeneration, regardless of the presence or absence of IPT. Electron-dense, particle-like wall materials were deposited on the original protoplasts prior to budding within 2hr after incubation; they were loosely organized along the plasmalemma and developed to form cell walls of fibrillar structure in controls. The overall features and developmental process of cell walls formed in the presence of 20ppm IPT were similar to those in controls, whereas cell walls formed in the presence of 50ppm IPT were thinner and less compact than those of controls. Protoplasts incubated in the regenerating medium formed bud-like protrusions by 2hr after incubation. Regardless of the presence or absence of IPT they continued to repeat budding-like divisions until 8hr, resulting in chains of budded cells. Normal hyphae arose from either original protoplasts or budded cells in chains at 8hr. By 24hr after incubation in control solutions nearly 50% of incubated protoplasts had produced hyphae. However, hyphae arose from only 1% of protoplasts in 20ppm IPT and were never produced in 50ppm, even after 24hr. IPT evidently interfered with cell wall regeneration of protoplasts of P. oryzae, but whether the observed effect was the expression of the primary or secondary action of IPT remains uncertain.

Composition of the cell wall formed by protoplasts isolated from cell suspension cultures of Vinca rosea

The biochemistry of cell-wall regeneration in protoplasts obtained from Vinca rosea L. (Catharanthus roseus (L.) G. Don) cells grown in suspension culture by isolating the regenerated wall and the extracellular polysaccharides of protoplasts cultured for various periods, and investigating their composition. Gas-liquid chromatography and tracer studies with D-[U-(14)C]glucose showed that the sugar composition of the extracellular polysaccharides was similar to that of the original cell culture, consisting mainly of polyuronide and 3,6-linked arabinogalactan. the regenerated cell wall was composed of non-cellulosic glucans having 1,3- and 1,4-linkages, while its content in pectic and hemicellulosic components was very low.