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At least 865 records · Page 48Linked to original sources
PubMed1988

Light-affected ca fluxes in protoplasts from vallisneria mesophyll cells.

In Vallisneria gigantea Graebner mesophyll cells, red light irradiation induces cytoplasmic streaming by decreasing the Ca(2+) concentration in the cytoplasm, while far-red light irradiation inhibits it by increasing the concentration (S Takagi, R Nagai 1985 Plant Cell Physiol 26: 941-951). To examine the effects of light irradiation on Ca(2+) fluxes across the cell membrane, protoplasts are isolated from the mesophyll cells. Changes in Ca(2+) concentration in a solution bathing the protoplasts are monitored by spectrophotometry, using the Ca(2+) -sensitive dye murexide. Red light irradiation induces an increase in Ca(2+) concentration, which means an efflux of Ca(2+) from the protoplasts. Subsequent far-red light irradiation produces a rapid decrease in Ca(2+) concentration down to the dark control level; however, this is not observed in the presence of the Ca(2+) -channel blocker nifedipine. Vanadate inhibits both the streaming and the Ca(2+) efflux induced by red light irradiation. The results suggest that red light and far-red light control Ca(2+) movements across the cell membrane, which in turn regulate the streaming.

Journal Article↗
PubMed1988

A comparative spin-label study of isolated plasma membranes and plasma membranes of whole cells and protoplasts from cold-hardened and nonhardened winter rye.

Lipid-lipid and lipid-protein interactions in the plasma membranes of whole cells and protoplasts and an isolated plasma membrane fraction from winter rye (Secale cereale L. cv Puma) have been studied by spin labeling. Spectra were recorded between -40 degrees C and 40 degrees C using the freely diffusing spin-label, 16-doxyl stearic acid, as a midbilayer membrane probe. The probe was reduced by the whole cells and protoplasts and reoxidized by external potassium ferricyanide. The reoxidized probe was assumed to be localized in the plasma membrane. The spectra consisted of the superposition of a narrow and a broad component indicating that both fluid and immobilized lipids were present in the plasma membrane. The two components were separated by digital subtraction of the immobilized component. Temperature profiles of the membranes were developed using the percentage of immobilized lipid present at each temperature and the separation between the outermost hyperfine lines for the fluid lipid component. Lipid immobilization was attributed to lipid-protein interactions, lipid-cell wall interactions, and temperature-induced lipid phase transitions to the gel-state. Temperature profiles were compared for both cold-hardened and nonhardened protoplasts, plasma membranes, and plasma membrane lipids, respectively. Although cold-hardening extended the range of lipid fluidity by 5 degrees C, it had no effect on lipid-protein interactions or activation energies of lipid mobility. Differences were found, however, between the temperature profiles for the different samples, suggesting that alterations in the plasma membrane occurred as a consequence of the isolation methods used.

Journal Article↗
PubMed1990

Tobacco Mesophyll Protoplasts Synthesize 1,3-beta-Glucanase, Chitinases, and "Osmotins" during in Vitro Culture.

Tobacco (Nicotiana tabacum) mesophyll protoplasts synthesize six basic proteins (a, a', a(1), b, b', and c) which are undetectable in the leaf and whose synthesis is reduced by auxin (Y Meyer, L Aspart, Y Chartier [1984] Plant Physiol 75: 1027-1033). Polypeptides a, a', and a(1) were shown to have similar mobilities on two-dimensional electrophoresis as one 1,3-beta-glucanase and two chitinases from tobacco mosaic virus-infected leaves. In immunoblotting experiments, polypeptide a was recognized by specific antibodies raised against the 1,3-beta-glucanase and a' and a(1) reacted with anti-chitinase antibodies. Similarly, b and b' comigrated with osmotin and its neutral counterpart, two proteins characteristic of salt-adapted tobacco cells, and reacted with anti-osmotin antibodies. In addition it has been shown that 1,3-beta-glucanase and chitinase activities increased at the same time as a, a', and a(1) accumulated in cultivated protoplasts. Finally, polypeptide c was also detected in tobacco mosaic virus-infected leaves but could not be identified as any of the pathogenesis-related proteins characterized so far in tobacco. Thus, cultivated tobacco protoplasts synthesize and accumulate typical stress proteins.

Journal Article↗
PubMed1990

Flow Cytometric Fluorescence Anisotropy of Lipophilic Probes in Epidermal and Mesophyll Protoplasts from Water-Stressed Lupinus albus L.

The blue emission anisotropy, r, of two lipophilic probes, diphenylhexatriene (DPH) and its trimethyl-ammonium derivative (TMA-DPH), has been measured in foliar Lupinus albus L. protoplasts for the first time by flow cytometry. Distinctive values have been obtained for protoplasts of epidermal and mesophyll origin, identified by their intensities of chlorophyll fluorescence. Fluorescence microscopy confirmed that TMA-DPH remained in the plasma membrane while DPH penetrated into intracellular lipid domains. Typical emission anisotropy values at 22 degrees C for mesophyll and epidermal protoplasts, respectively, were 0.225 and 0.312 with TMA-DPH, and 0.083 and 0.104 with DPH. This indicates that epidermal cells-and notably their plasma membranes (TMA-DPH)-have higher lipid microviscosity and/or more ordered lipid structure. Two lupin genotypes characterized as resistant or susceptible to drought were analyzed with or without 9 days of water stress shown to increase ion leakage from foliar discs. Water stress greatly increased the apparent fluidity, and more so in the susceptible genotype; the effect was more pronounced in the chlorophyll-containing mesophyll cells than in the epidermal cells.

Journal Article↗
PubMed1991

Isoelectric focusing of cassava protoplasts.

Cassava (Manihot esculenta Crantz) protoplast was analyzed by using isoelectric focusing techniques. Two populations, representing 68 and 32% of the total sample, with mean isoelectric points of 4.48 and 4.60, were obtained using mesophyll protoplasts. The use of this technique allows demonstration of a discontinuous distribution of protoplast isoelectric point from one species according to their surface potential.

Journal Article↗
PubMed1991

Regulation of a chitinase gene promoter by ethylene and elicitors in bean protoplasts.

Chitinase gene expression has been shown to be transcriptionally regulated by a number of inducers, including ethylene, elicitors, and pathogen attack. To investigate the mechanism(s) responsible for induction of chitinase gene expression in response to various stimuli, we have developed a transient gene expression system in bean (Phaseolus vulgaris) protoplasts that is responsive to ethylene and elicitor treatment. This system was used to study the expression of a chimeric gene composed of the 5' flanking sequences of a bean endochitinase gene fused to the reporter gene beta-glucuronidase linked to a 3' fragment from nopaline synthase. Addition of 1-aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene, or elicitors such as chitin oligosaccharides or cell wall fragments derived from Colletotrichum lagenarium, to transformed protoplasts resulted in a rapid and marked increase in the expression of the chimeric gene. The kinetics and dose response for these treatments were similar to those observed for the native gene in vivo. Analyses of 5' deletion mutants in the protoplast system indicated that DNA sequences located between -305 and -236 are important for both ethylene and elicitor induction of the reporter gene.

Journal Article↗
PubMed1992

Regeneration of Transgenic Soybean (Glycine max) Plants from Electroporated Protoplasts.

Transgenic soybean (Glycine max [L.] Merr.) plants were regenerated from calli derived from protoplasts electroporated with plasmid DNA-carrying genes for a selectable marker, neomycin phosphotransferase (NPTII), under the control of the cauliflower mosaic virus 35-Svedberg unit promoter, linked with a nonselectable mannityl opine synthesis marker. Following electroporation and culture, the protoplast-derived colonies were subjected to kanamycin selection (50 micrograms per milliliter) beginning on day 15 for 6 weeks. Approximately, 370 to 460 resistant colonies were recovered from 1 x 10(6) electroporated protoplasts, giving an absolute transformation frequency of 3.7 to 4.6 x 10(-4). More than 80% of the kanamycin-resistant colonies showed NPTII activity, and about 90% of these also synthesized opines. This indicates that the linked marker genes were co-introduced and co-expressed at a very high frequency. Plants were regenerated from the transformed cell lines. Southern blot analysis of the transformed callus and leaf DNA demonstrated the integration of both genes. Single-plant assays performed with different plant parts showed that both shoot and root tissues express NPTII activity and accumulate opines. Experiments with NPTII and mannityl opine synthesis marker genes on separate plasmids resulted in a co-expression rate of 66%. These results indicate that electroporation can be used to introduce both linked and unlinked genes into the soybean to produce transformed plants.

Journal Article↗
PubMed1993

A Potato Virus X Resistance Gene Mediates an Induced, Nonspecific Resistance in Protoplasts.

The Rx locus in potato controls extreme resistance to most isolates of potato virus X (PVX). The resistance is expressed in whole plants and in protoplasts. Rx-mediated resistance in protoplasts causes reduced accumulation of all PVX RNA species, including the (-) strand RNA after a lag of 8 hr postinoculation. In work reported elsewhere, we have shown that the Rx-breaking property of PVXHB was associated with the coat protein gene of PVXUK3 and PVXCP4. Here, we describe how a frameshift mutation in the coat protein gene had no effect on Rx resistance breaking but compromised the Rx-mediated resistance to PVXCP4. We also describe how in coinoculation experiments, the Rx-mediated resistance could be induced to affect PVXHB or cucumber mosaic virus (CMV). In these experiments, PVXHB or CMV was coinoculated to protoplasts (Rx genotype) together with an isolate of PVX, which is affected by Rx. We interpret this data to indicate that Rx-mediated resistance is induced when the PVX coat protein is produced in the infected cells and that the induced resistance mechanism is effective against viruses unrelated to PVX.

Journal Article↗
PubMed1983

Induction of L-phase variant from protoplast of Staphylococcus aureus.

Protoplasts of Staphylococcus aureus 209P and Cowan 1 were induced by treatment with lysostaphin. These protoplasts were sensitive to detergent, a low concentration of sodium chloride and low temperature. Almost all protoplast cells spread on CLYS agar medium (casein hydrolysate, yeast extract, Na-lactate, and NaCl) formed typical L-form colonies. Horse serum (0.25%) and Mg2+ (100 mM) are essential factors for formation of the L-form colonies of 209P. In the case of Cowan 1, Mg2+ was not required. The active factor(s) in horse serum was heat-resistant and protein in nature.

Culture Media↗
PubMed1988

Genetically transformed maize plants from protoplasts.

Genetically transformed maize plants were obtained from protoplasts treated with recombinant DNA. Protoplasts that were digested from embryogenic cell suspension cultures of maize inbred A188 were combined with plasmid DNA containing a gene coding for neomycin phosphotransferase (NPT II) next to the 35S promoter region of cauliflower mosaic virus. A high voltage electrical pulse was applied to the protoplasts, which were then grown on filters placed over feeder layers of maize suspension cells (Black Mexican Sweet) and selected for growth in the presence of kanamycin. Selected cell lines showed NPT II activity. Plants were regenerated from transformed cell lines and grown to maturity. Southern analysis of DNA extracted from callus and plants indicated the presence of the NPT II gene.

Cell Membrane Permeability↗
PubMed1983

Highly Efficient Mutagenesis of Claviceps purpurea by Using Protoplasts.

Claviceps purpurea ATCC 20102, which is aconidial under laboratory conditions, was grown in submerged culture in the presence of mutagens and various nutritional additives. Protoplasts from such cultures were prepared and regenerated on solid medium to obtain colonies from single cell units. Frequencies of auxotrophs and high alkaloid producers were on the order of 1 to 2%. Some of the auxotrophic mutants derived from strain ATCC 20102 were constantly segregating prototrophs. High-alkaloid-producing derivatives showed sclerotia-like morphology and violet-brown pigmentation, in contrast to the parent strain; some of them also showed segregation sectors when grown as giant colonies. Mutagenesis of strain 1029, isolated during this study and having an increased level of alkaloid synthesis and sclerotia-like cell morphology, was done in the same fashion as with the original parent strain, ATCC 20102. Mutants obtained from this strain were all stable with respect to their genotypes. However, a large proportion of colonies derived from regenerated protoplasts, even in the mutagen-free controls, showed a lowered level of alkaloid production and were morphologically more similar to the original wild type, ATCC 20102. The influence of protoplast preparation or regeneration or both on the stability of genes involved in differentiation is discussed.

Journal Article↗
PubMed1985

Improvement of protoplast regeneration by growing yeast cells hypertonically.

The high osmotic pressure provided for in vitro transformation processes to support naked protoplasts of Saccharomyces cerevisiae was found to hurt regeneration of the protoplasts to walled cells. Preadaptation of the cells by growth under the same hypertonic conditions as those used for transformation doubled the rate of protoplast regeneration and in turn resulted in a higher yield of transformants.

Journal Article↗
PubMed1988

Escherichia coli Spheroplast-Mediated Transfer of pBR322 Carrying the Cloned Ruminococcus albus Cellulase Gene into Anaerobic Mutant Strain FEM29 by Protoplast Fusion.

Intergeneric protoplast fusion between Escherichia coli HB101 with pBR322 carrying the cloned o-(carboxymethyl)cellulase (CMCase) gene of Ruminococcus albus (Pro Leu Ap Km) and an anaerobic mutant strain, FEM29 (Trp His Ap Km), with dehydrodivanillin-degrading activity was performed in the presence of 40% polyvinyl alcohol 300 under aerobic and anaerobic conditions to transfer the cloned cellulase gene into the mutant. The mutant FEM29 had a unique property. When it was incubated in liquid medium with 1% glucose and sucrose, protoplasts could be produced autogenously and regenerated on the agar slant. E. coli spheroplasts formed from a plasmid-amplified overnight culture after 10 min of treatment with lysozyme (20 mug/ml) in a hypertonic solution (0.01 M Tris hydrochloride [pH 7.5], 0.4 M mannitol). Protoplast regeneration rates of FEM29 and HB101 were 30 and 83%, respectively, on the agar-yeast extract medium. Ap Km fusants were obtained at high frequency: 1.7 x 10 anaerobically and 8.2 x 10 aerobically. These fusants showed 23 to 57% of CMCase and dehydrodivanillin-degrading activities, respectively, as compared with parental strains. All the fusants isolated were gram-negative rods with main phenotypes such as urease and catalase activities as in HB101 and esterase and chymotrypsin activities as in FEM29. Southern hybridization experiments suggested that pBR322 with the cloned CMCase gene existed autonomously in the fusant cells. This is the first report describing transfer of pBR322 with a cloned cellulase gene into an anaerobic mutant by polyvinyl alcohol-mediated fusion with an E. coli spheroplast.

Journal Article↗
PubMed1981

Mutanolysin-induced spheroplasts of Streptococcus mutants are true protoplasts.

A method is described for the preparation of protoplasts of Streptococcus mutans BHT. The muralytic enzyme mutanolysin was prepared free of contaminating proteinases and shown to completely dissolve cell walls of this strain. Whole cells were converted to stabilizable protoplasts by using the enzyme in an isotonic medium containing 40% raffinose. Experiments using [3H]thymidine and [14C]leucine as cytoplasmic pool markers revealed only minimal (10%) leakage during a 1-h incubation. Examination by electron microscopy revealed the apparent absence of structural cell wall on the enlarged spherical bodies. Quantitative chemical analyses of membranes prepared by lysing protoplasts demonstrated only very small amounts of rhamnose and trace amounts of galactose. These sugars are the principal components of the BHT cell wall polysaccharide. Also, there were only small amounts of peptidoglycan components (e.g., N-acetylglucosamine) in the purified membranes obtained by this method.

Cell Membrane↗
PubMed1984

Transformation of protoplasted yeast cells is directly associated with cell fusion.

The frequency of cell fusion during transformation of yeast protoplasts with various yeast plasmids with a chromosome replicon (YRp or YCp) or 2 mu DNA (YEp) was estimated by two methods. In one method, a mixture of protoplasts of two haploid strains with identical mating type and complementary auxotrophic nuclear markers with or without cytoplasmic markers was transformed. When the number of various phenotypic classes of transformants for the nuclear markers was analyzed by equations derived from binominal distribution theory, the frequency of nuclear fusion among the transformants was 42 to 100% in transformations with the YRp or YCp plasmids and 28 to 39% with the YEp plasmids. In another method, a haploid bearing the sir mutation, which allows a diploid (or polyploid) homozygous for the MAT (mating type) locus to sporulate by the expression of the silent mating-type loci HML and HMR, was transformed with the plasmids. Sporulation ability was found in 43 to 95% of the transformants with the YRp or YCp plasmids, and 26 to 31% of the YEp transformants. When cytoplasmic mixing was included with the nuclear fusion, 96 to 100% of the transformants were found to be cell fusants. Based upon these observations, we concluded that transformation of yeast protoplasts is directly associated with cell fusion.

Cell Fusion↗
PubMed1987

Visualization of surface antigens on yeast protoplasts by gold or ferritin markers.

Surface antigens of Saccharomyces cerevisiae protoplast were visualized immunocytochemically with protein A-gold or ferritin conjugated antibodies. Ferritin particles were densely distributed at the surface of yeast protoplast after treatment with rabbit anti-protoplast serum and ferritin conjugated antibody against rabbit IgM. Control experiments demonstrated that the binding of these ferritin markers was specific for surface antigens. The composition of these surface antigens, however, remain to be investigated.

Antigens, Fungal↗
PubMed1987

Physico-chemical interaction of mycobacillin with Aspergillus niger protoplast membrane, the site of its action.

Mycobacillin partially quenched the strong fluorescence when 1-anilino naphthalene 8-sulfonate (ANS) was added to protoplast or plasma membrane but is without any effect on weak fluorescence when added to cell-free extract. There are two classes of ANS binding sites on protoplast or plasma membrane of which one class is sensitive to mycobacillin, being competitively abolished by it. Mycobacillin also non-competitively inhibits the binding of pyrene, a lipid specific probe. Thus it follows from the inhibition by mycobacillin of ANS or pyrene binding to protoplast or plasma membrane that the site of action of the antibiotic is located in the plasma membrane. Interaction between mycobacillin and the plasma membrane is physico-chemical in nature.

Anilino Naphthalenesulfonates↗
PubMed1998

Optimization of parameters for isolation of protoplasts from Gracilaria verrucosa (Rhodophyta).

A systematic method was designed for the isolation of a large number of protoplasts from an agarophyte alga Gracilaria verrucosa using agarase from a marine bacterium Vibrio sp. PO-303 and commercial enzymes (Cellulase Onozuka RS and Macerozyme R-10). Pretreatment of the tissue with 5% papain at 22 degreesC for 30 min before digestion with polysaccharide-degrading enzymes increased the protoplast yield. Suitable pH and temperature for the polysaccharide-degrading enzyme reaction were 6.5 and 22 degreesC, respectively. Mannitol (0.7 M) was found to be an excellent osmotic stabilizer. When the tissue (1 g, fresh wt.) of G. verrucosa pretreated with 5% papain solution (20 mM MES buffer, pH 7.5, containing 0.7 M mannitol) was digested with an enzyme mixture consisting of 4 units of agarase, 4% Cellulase Onozuka, 2% Macerozyme, and 0.7 M mannitol in 20 mM MES buffer (pH 6.5) with gentle agitation for 150 min at 22 degreesC, 1.03 x 10(8) protoplasts were obtained.

Journal Article↗