Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protoplasts”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources
PubMed2001

Detection of infectious viral particles in plant protoplasts inoculated with transcripts of full-length shallot virus X cDNA.

Flexible filamentous shallot virus X (ShVX) particles were detected in extracts of Beta vulgaris protoplasts inoculated with transcripts from a full-length ShVX cDNA. Extracts from ShVX-infected protoplast were infectious for ShVX-healthy shallot seedlings. Western blot analysis of inoculated plants revealed the accumulation of the ShVX coat protein, while electron microscopy confirmed the presence of ShVX virions. The results suggest that the in vitro RNA transcripts from full-length ShVX cDNA give rise to infectious viral particles.

Allium↗
PubMed2006

Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel.

Several advances have been made in the use of microfluidic devices for insect and mammalian cell cultures, but no reports of their use for plant cell cultures have been published. We, therefore, conducted a plant cell culture in a microfluidic device using polydimethylsiloxane. Nicotiana tabacum protoplasts were cultured in a variously shaped polydimethylsiloxane channel containing Nitsch medium supplemented with 0.5 g of NLN-13 vitamin mixture, 2.0 mg of alpha-naphthaleneacetic acid, and 0.5 mg of 6-benzyladenine per liter and 9% mannitol. Protoplasts in the polydimethylsiloxane channel showed cell division and microcolony formation within 4 weeks. The use of a microfluidic channel is a novel technique in the field of plant cell culture. The results of this study will encourage the utilization of polydimethylsiloxane-based microfluidic devices in plant cell engineering and cell analysis.

Cell Culture Techniques↗
PubMed1984

Quantitation of the delivery of liposome contents into plant protoplasts.

A procedure for the quantitation of the delivery of liposome contents into Catharanthus roseus protoplasts has been developed. The method is based on the uptake of liposome encapsulated methylumbelliferyl beta-D-glucoside and its enzymatic hydrolysis to yield fluorescent methylumbelliferone. Since the free glucoside is not taken up by the protoplasts to a significant extent, the delivery of material in the nanomole range can be measured with ease.

Biological Transport↗
PubMed1984

A batch assay using Calcofluor fluorescence to characterize cell wall regeneration in plant protoplasts.

A batch assay to study and measure the regeneration of cell walls during the early days of culture of primary protoplasts is presented. The assay involves the measurement of Calcofluor White fluorescence on a scanning fluorometer when the Calcofluor is adsorbed to the cellulosic component of the newly synthesized cell walls. The Calcofluor fluorescence, when standardized with microcrystalline cellulose, provided a measure of cell wall cellulose. The assay was used to study cell wall regeneration in Hyoscyamus muticus L. protoplasts during 8 days of culture.

Benzenesulfonates↗
PubMed1980

Metabolite levels during induction in the chloroplast and extrachloroplast compartments of spinach protoplasts.

The light activation of photosynthesis has been investigated in spinach palisade cell protoplasts. (1) After a short induction period, maximal rates of photosynthesis are achieved. (2) [14C]Bicarbonate initially labels anionic compounds in the chloroplast and then in the extrachloroplast compartments. These pools saturate within 2-4 min and radioactivity accumulates mainly in sucrose in the extrachloroplast compartment, in starch and in cationic compounds. (3) Enzymic determinations were made of metabolite levels during the induction period in the chloroplast and extrachloroplast compartments. There is no general build-up of intermediates. Perturbations of individual intermediates occurred, consistent with the activation of specific enzymes. (4) It is suggested that fructose-1,6-bisphosphatase and ribulose-1,5-bisphosphate carboxylase may limit flux in the Calvin cycle during induction. (5) The onset of sucrose synthesis is not accompanied by accumulation of intermediates in the cytosol. It is suggested that sucrose phosphate synthase or sucrose phosphate phosphatase is activated. (6) Measurements of metabolites in whole leaves during a 24 h illumination cycle confirmed that substrates are not depleted during the dark period, and that the onset of photosynthesis is not accompanied by a rise in intermediate levels. (7) It is concluded that the causes of the induction lag in protoplasts can differ from those in isolated chloroplasts.

Chloroplasts↗
PubMed1979

Interactions between bacterial membranes and peptidolipids: lysis of micrococcus luteus protoplasts by derivatives of peptidolipidic antibiotics from bacillus subtilis.

The lysis of protoplasts of Micrococcus luteus has been tested with various derivatives of three peptidolipidic antibiotics: iturin A, mycosubtilin and bacillomycin L. The lytic activity is dependent to the nature of the substituting group and to the position of the substituted aminoacid residue. The acetylation of OH groups leads to a decrease of the lytic activity of the natural antibiotics. The methylation of aspartyl residues of bacillomycin L gives a strong lytic activity while natural bacillomycin L has no lytic activity. The methylation of the tyrosyl residue enhances the lytic activities of iturin A and of bacillomycin L-dimethyl ester and reduces that of mycosubtilin. Correlations between the structures of derivatives and their lytic action on M. luteus protoplasts are discussed.

Anti-Bacterial Agents↗
PubMed1984

Evaluation of the silica microbead method for isolation of red beet protoplast plasma membrane sheets.

The silica microbead procedure was utilized for the isolation of plasma membrane sheets from protoplasts of a higher plant, the red beet (Beta vulgaris L.). Membrane yields, as determined by recovery of an exogenous membrane marker were approx. 75%. The plasma membrane fraction contained the enzyme marker, pH 6.5, vanadate-sensitive, K+-stimulated, Mg2+-ATPase and small amounts of mitochondria, endoplasmic reticulum, and possibly tonoplast. The silica microbead procedure was also used for the isolation of intact vacuoles from microbead-coated protoplasts.

Cell Fractionation↗
PubMed1987

Intracellular pH topography of Penicillium cyclopium protoplasts. Maintenance of delta pH by both passive and active mechanisms.

The intracellular pH distribution in protoplasts of Penicillium cyclopium has been studied using the recently developed fluorescent probe microscopic technique. The technique gives detailed pH maps of the interior of the protoplasts with the exception of vacuoles (no fluorescence signal from vacuoles was observed). In the cytoplasm two separate layers were distinguished: a thinner outer layer with acidic pH (around 5) and the larger core region with near neutral pH. The pH of the core region is decreased by the addition of uncouplers, inhibitors of respiration and during the uptake of L-phenylalanine. These compounds do not change the pH of the surface layer, which is, however, acidified by addition of vanadate, an inhibitor of the proton pump of the plasmalemma. We suggest that the pH of the surface layer is maintained by the combined effects of a Donnan distribution of protons (bound to postulated anion binding proteins) and the proton extrusion via the plasmalemma proton pump. This mechanism explains the protection of the cytoplasmic core of acidophilic eukaryotes from the influence of the usually acidic environment.

Hydrogen-Ion Concentration↗
PubMed1977

Kinetic analysis of fraction I protein biosynthesis in young protoplasts of tobacco leaves.

At maximum inhibition chloramphenicol reduced [35S] methionine incorporation into acid-insoluble materials of sterile protoplasts from young tobacco leaves 5-7 cm in length by 30% compared to 70% by cycloheximide, indicating that 30% of the [35S] methionine became incorporated into chloroplast proteins and 70% into cytoplasmic proteins. [35S] Methionine became incorporated into both the large and small subunits of Fraction I protein, the major soluble protein of chloroplasts. Rifampicin and streptolydigin inhibited [3H] uridine incorporation into the 23 and 16 S rRNAs of chloroplasts to a much greater extent than into the 25 and 18 S cytoplasmic rRNAs. Rifampicin inhibited [35S] metionine incorporation into Fraction I protein after the third hour of incubation; streptolydigin after 2 h, the former evidently preventing initiation of mRNA for the large subunit of Fraction I protein and the latter its elongation. About 2.5 h was required between initiation of the large subunit mRNA synthesis, and appearance of the protein. It was estimated that 45 min is required for transcription of the mRNA which has a half-life of 2 h and that 105 min is required for its translation into approximately 350 amino acids constituting the large subunit monomeric polypeptide. The effect of chloramphenicol, cycloheximide and 2-(4-methyl-2,6-dinitroanaline)-N-methyl propionamide, the latter an inhibitor of protein initiation by 80 S ribosomes, on kinetics of Fraction I protein synthesis indicate that protoplasts contain a pool of small subunit polypeptides and that 30 min is required to polymerize the approximately 100 amino acids constituting the primary structure.

Aminoglycosides↗
PubMed1987

Controlled cell lysis and protoplast formation by enhancement of inhibitors of alanine racemase by glycine.

beta-D-chloroalanine (200-800 micrograms/ml) addition to Bacillus subtilis SB 19 caused a slight inhibition of growth in MLN broth. Inhibitor plus 1% glycine initiated rapid lysis of cells (complete in 2 hours). D-alanine (500 micrograms/ml) prevented lysis. D-cycloserine behaved similarly. Inhibitors in 0.5 M sucrose and 1% glycine formed stable "primed protoplasts" which were converted to the bacillary form by addition of D-alanine. Characteristics of Dal+ cells with inhibitor are similar to Dal- mutants. Thus Dal+ cells in the presence of alanine racemase inhibitors + glycine can be used for controlled lysis and protoplast formation (suitable for PEG transformation with plasmid DNA). Inhibitors in 1% glycine exhibit a strong antibacterial action similar to potent antibiotics which inhibit cell wall formation.

Alanine↗
PubMed1987

Effects of abscisic acid on K+ channels in Vicia faba guard cell protoplasts.

Potassium channels were resolved in Vicia faba guard cell protoplasts by patch voltage-clamp. Whole-cell currents and single K+ channels had linear instantaneous current-voltage relations, reversing at the calculated Nernst potential for K+. Whole cell K+ currents activated exponentially during step depolarizations, with half-activation times of 400-450 msec at +80 mV and 90-110 msec at +150 mV. Single K+ channel conductance was 65 +/- 5 pS with a mean open time of 1.25 +/- 0.30 msec at 150 mV. Potassium channels were blocked by internal Cs+ and by external TEA+, but they were insensitive to external 4-aminopyridine. Application of 10 microM abscisic acid increased mean open time and caused long-lasting bursts of channel openings. Since internal and external composition can be controlled, patch-clamped protoplasts are ideal systems for studying the role of ion channels in plant physiology.

Abscisic Acid↗
PubMed1983

Destabilization of the plasma membrane of isolated plant protoplasts during a freeze-thaw cycle: the influence of cold acclimation.

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways--intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.

Acclimatization↗
PubMed1989

Quantitative analysis of the probability of intracellular ice formation during freezing of isolated protoplasts.

(i) A quantitative prediction of the temperature and cooling-rate dependence of IIF requires information on the probability distribution for IIF temperature; the super-cooling tolerance; and membrane permeability, initial cell size, Boyle-van't Hoff relation, and other parameters associated with the thermodynamic description of cell volumetric behavior. (ii) A probabilistic analysis of IIF was developed on the basis that IIF occurs if the underlying potential IIF temperature T* falls within the range of temperatures over which the cell is supercooled beyond its tolerance delta T*. The "location" of the transition range of cooling rates, in which the Pr(IIF) increases from 0 to 1, depends on the mean of T* and delta T*. The breadth of the transition range increases strongly with variability in T*. (iii) Cryomicroscopic studies of IIF in rye protoplasts isolated from cold acclimated and nonacclimated leaves showed that the range of IIF temperatures and the incidence of IIF are strongly dependent on the solute used to manipulate osmolality in the suspending medium. The ionic solutes used in this study (NaCl + CaCl2, KCl + CaCl2, KNO3 + CaCl2) generally yielded median IIF temperatures lower than those of the nonionic solutes (sucrose, sorbitol, proline). Cold acclimation reduced the median IIF temperature for all suspending media and reduced the incidence of IIF for KCl + CaCl2, sorbitol, and proline. A Weibull distribution was found to describe adequately the distribution of IIF temperatures at the highest cooling rate. (iv) Estimates of the supercooling tolerance were generally from 1.0 to 1.5 degrees C based on the location of the transition range of cooling rates and on the estimates of Lp and delta E from (6). The breadth of the transition range of cooling rates could mostly be attributed to random variability in the underlying potential IIF temperature. The rise in median IIF temperature with higher cooling rates was correctly predicted by the model when the supercooling tolerance was also assumed to be random. For rye protoplasts, both the model and the data showed that cooling-rate dependence in median IIF temperatures occurs within the transition range of cooling rates.

Edible Grain↗
PubMed1992

Effects of thionins on beta-glucuronidase in vitro and in plant protoplasts.

Thionins cause the irreversible inactivation of beta-glucuronidase (GUS) in vitro in a dose- and time-dependent manner. The enzyme is also sensitive to externally added thionins when expressed in the cytoplasmic compartment of tobacco protoplasts transformed with the Gus gene under the 35S promoter of the cauliflower mosaic virus. In protoplasts transformed with the Gus gene fused to a signal peptide, where GUS is translocated into the lumen of the endoplasmic reticulum, the activity is significantly increased both by externally-added and by transiently-expressed thionin, suggesting that it interferes with GUS secretion.

Antimicrobial Cationic Peptides↗
PubMed1993

Detection of GTP-binding proteins in barley aleurone protoplasts.

We report the existence of several families of GTP-binding proteins in barley aleurone protoplasts. Partial purified plasma membrane proteins were separated by SDS-PAGE, transferred to a nitrocellulose filter and incubated with either antisera raised against a highly conserved animal G protein alpha subunit peptide/or Ras protein, or with [alpha-32P]GTP. Two sets of proteins of M(r) = 32-36 kDa and 22-24 kDa were strongly recognized by the antisera. Binding of [alpha-32P]GTP was detected on Western blots with proteins of M(r) = 22-24 kDa and 16 kDa. Binding was inhibited by 10(-7)-10(-6) M GTP gamma S, GTP or GDP; binding was not affected by 10(-6)-10(-5) M ATP gamma S or ADP. The kinetics, specificity and the effects of phytohormones in a [35S]GTP gamma S binding assay were also studied in isolated plasma membranes of barley aleurone protoplasts.

Binding Sites↗
PubMed1994

Calcium channel antagonists induce direct inhibition of the outward rectifying potassium channel in tobacco protoplasts.

Verapamil, nifedipine and bepridil, three antagonists of L-type calcium channels in animal cells, were shown to induce direct inhibition of outward rectifying potassium current in Nicotiana tabacum cv. Xanthi protoplasts with IC50 of 5 microM, 5 microM and 1 microM, respectively. In the outside-out configuration, verapamil reduced the open probability of the ion channel responsible for the outward rectifying potassium conductance. Verapamil also blocked the outward rectifying potassium conductance in protoplasts from the N. tabacum cv. Bright Yellow cell line. Thus, studies using these molecules to demonstrate the involvement of calcium channels in plant physiological responses should be regarded with caution.

Bepridil↗
PubMed1996

Ionic transport in the plasma membrane of carrot protoplasts from embryogenic cell-suspension cultures.

Ionic transport properties of protoplasts obtained from embryogenic carrot suspension cells were studied by the patch-clamp technique. In the whole-cell configuration, carrot protoplasts presented macroscopic time-dependent outward currents, showing kinetics of activation which did not depend appreciably on the amplitude of the stimulus. Time- and voltage-dependent whole-cell inward rectifying currents as well as instantaneous non-selective currents were also observed. Both time-dependent inward and outward currents are carried by potassium ions. In a cell-attached configuration, two types of single-channel signals, displaying conductances of 10 and 17 pS, were observed; the instantaneous 10 pS channel was also present in outside-out excised patches.

Cell Membrane↗