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PubMed1984

Surface galactolipids of wheat protoplasts as receptors for soybean agglutinin and their possible relevance to host-parasite interaction.

Soybean agglutinin, a lectin specific for N-acetyl-d-galactosamine and d-galactose, was previously shown to agglutinate wheat leaf protoplasts (Larkin 1978 Plant Physiol 61: 626-629). We investigated the receptors for soybean agglutinin on the plasma membrane of these protoplasts. After treatment of the protoplasts with galactose oxidase, they were no longer agglutinated by the lectin, whereas upon reduction of the galactose oxidase-treated protoplasts with sodium borohydride the susceptibility to agglutination was restored. Analysis of the glycolipids of protoplasts surface labeled by the galactose oxidase-borotritide method, revealed that the radioactivity was mainly present in monogalactosyldiglyceride and digalactosyldiglyceride. The same galactolipids were identified as the only receptors for soybean agglutinin by direct binding of the (125)I-labeled lectin to a thin layer chromatogram of the glycolipids of wheat leaf protoplasts.

Journal Article↗
PubMed1986

Improved Cytoplasmic Delivery to Plant Protoplasts via pH-Sensitive Liposomes.

We demonstrated that the liposomes composed of dioleolylphosphatidylethanolamine/cholesterol/oleic acid (4:4:2) dramatically release their contents at a pH of less than or equal to 6.0 and are capable of delivering their contents into the cytoplasm of higher plant protoplasts. This is shown by using a soluble fluorescent dye, calcein, as a liposome-entrapped marker. We found that calcein fluorescence was evenly distributed in the cytoplasm of wild carrot protoplasts after the incubation of protoplasts with liposomes in the presence of polyethylene glycol 6000. At 0.45 micro mole phospholipid per 6 x 10(5) protoplast, for example, the percentage of protoplasts which took up liposomes was 89% which was much higher than that achieved by conventional pH-insensitive liposomes. In this study, liposomes were prepared by a detergent dialysis method which avoided sonication and organic solvents. Thus macromolecules such as proteins and nucleic acids could be entrapped in the liposomes and delivered to the cytoplasm of the protoplasts.

Journal Article↗
PubMed1987

Kinetics of l-Valine Uptake in Suspension-Cultured Cells and Protoplast-Derived Cells of Tobacco: Comparison of Wild-Type and the Val-2 Mutant.

A kinetic analysis was made of l-valine uptake in protoplast-derived cells (mesophyll protoplasts cultured for 6 days) and in suspension-cultured cells of tobacco (Nicotiana tabacum L., cv Xanthi). Cells from wild-type and Val(r)-2 mutant plants were compared. A low-K(m) component was found in protoplast-derived cells (K(m) = 45 +/- 5 micromolar) as well as in suspension-cultured cells (K(m) = 84 +/- 21 micromolar). In the mutant cells the V(max) of this component was 12- to 14-fold less than in wild-type cells. A second component (K(m) = 2.4 +/- 0.7 millimolar) was found in suspension-cultured cells but not in protoplast-derived cells; its V(max) was the same in wild-type and mutant cells. A third component was apparently unsaturable (linear component). It was present in protoplast-derived cells but not in suspension-cultured cells, and had the same magnitude in wild-type and mutant cells. The results are discussed with reference to the uptake of l-valine in leaf tissue, in which the three kinetic components have been found simultaneously. The reduced V(max) of the low-K(m) component in the Val(r)-2 mutant, and the differential expression of the other two components in suspension-cultured cells and protoplast-derived cells indicate that the kinetically distinguishable components represent physically distinct transport systems.

Journal Article↗
PubMed1988

Heat Inducible Expression of a Chimeric Maize hsp70CAT Gene in Maize Protoplasts.

The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26 degrees C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with (35)S-methionine. Incubation at 40 degrees and 42 degrees C resulted in the synthesis of polypeptides not detectable at 26 degrees C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40 degrees C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress.

Journal Article↗
PubMed1990

Aluminum Ions Induce Oat Protoplasts to Produce an Extracellular (1-->3)beta-d-Glucan.

Aluminum chloride induced mesophyll protoplasts of oat (Avena sativa) to produce an extracellular polysaccharide (EPS). EPS induced by AlCl(3) appeared identical to that produced in response to the phytotoxin victorin (JD Walton, ED Earle [1985] Planta 165: 407-415). Al ions at 1 millimolar were toxic to protoplasts, but maximum EPS production occurred at a sublethal concentration of 200 micromolar, assayed at pH 6.0. As measured by incorporation of [(14)C]glucose, AlCl(3) stimulated EPS production 10- to 15- fold. Pretreatment of protoplasts with cycloheximide prevented EPS production but not cell death in response to AlCl(3), indicating that protein synthesis was necessary for EPS production but not for the phytotoxicity of Al ions. The trivalent salts of Y, Yb, Gd, and In also induced EPS production but those of Sc, Fe, Ga, Cr, and La did not. Mesophyll protoplasts from an acid-soil tolerant oat cultivar, Coker 83-23, produced less EPS in response to AlCl(3) than the acid-soil sensitive cultivar Fla 501. EPS was also produced by wheat (Triticum aestivum) and barley (Hordeum vulgare) protoplasts in response to AlCl(3). An Al-tolerant cultivar of wheat, Atlas, produced less EPS than an Al-sensitive cultivar, Scout, but an Al-tolerant cultivar of barley, Dayton, produced more than the Al-sensitive cultivar Kearney. Therefore, production of EPS by protoplasts in response to Al ions did not appear to be related to Al ion tolerance at the level of whole plants. EPS fluoresced in the presence of Calcofluor and Sirofluor and was degraded by purified laminarinase [(1-->3)beta-d-glucanase] but not pectinase (polygalacturonase). EPS was composed solely of glucose in 1-->3 linkages; hence it is a (1-->3)beta-d-glucan (callose).

Journal Article↗
PubMed1982

Pullulan Elaboration by Aureobasidium pullulans Protoplasts.

Protoplasts of Aureobasidium pullulans are capable of producing pullulan. Biosynthesis of the polymer pullulan required induction with kinetics similar to those of whole cells. The protoplasts also produced a heteropolysaccharide component containing mannose, glucose, and galactose. The relative proportions of the pullulan and heteropolysaccharide fractions were a function of glucose concentration, with the pullulan content of the total polysaccharide rising from 20% at 2.5 mM glucose to 45% at 20 mM glucose. Elaboration of pullulan by both cells and protoplasts was sensitive to 0.6 M KCl, which was present as the osmotic stabilizer in protoplast experiments. The presence of KCl resulted in a shift in the pH optimum to a more acidic value. The molecular weight of the protoplast-derived pullulan was sharply reduced from the molecular weight of the whole-cell-derived product. Exposure of the protoplasts to proteolytic enzymes had no effect on polysaccharide elaboration.

Journal Article↗
PubMed1984

Transformation of Heat-Treated Clostridium acetobutylicum Protoplasts with pUB110 Plasmid DNA.

Heat treatment of Clostridium acetobutylicum SA-1 protoplasts at 55 degrees C for 15 min before transformation resulted in expression in this microorganism of the kanamycin resistance determinant associated with plasmid pUB110. No heat treatment, or heat treatment at 65 or 44 degrees C for various time intervals, resulted in no kanamycin resistance transformants being recovered on selective kanamycin-containing regeneration medium. DNase plate assay indicated that treatment at 55 degrees C for 15 min completely inactivated the DNase activity associated with SA-1 protoplasts. Treatment of protoplasts at 65 or 55 degrees C for various periods under simulated transformation conditions had an inhibitory effect, although prolonged treatment at 55 or 44 degrees C appeared to stimulate DNase activity. Inactivation of protoplast-associated DNase activity by heat treatment at 55 degrees C for 15 min correlated with successful expression of kanamycin resistance and suggests that an extremely active, heatsensitive, protoplast-associated DNase may be a factor in the polyethylene glycol-induced transformation of C. acetobutylicum SA-1 protoplasts. Plasmid pUB110 DNA was isolated from C. acetobutylicum SA-1 kanamycin-resistant (Km) transformant cultures by a modification of the procedure used for C. perfringens plasmids. Detection of pUB110 DNA was possible only when diethyl pyrocarbonate was incorporated into isolation protocols to inactivate DNase activity. Restriction studies further verified the presence of pUB110 DNA in C. acetobutylicum SA-1 Km transformants.

Journal Article↗
PubMed1984

Production and Regeneration of Lactobacillus casei Protoplasts.

Methods for the production and regeneration of Lactobacillus casei protoplasts are described. Protoplasts of L. casei strains were obtained by treatment with mutanolysin or with mutanolysin and lysozyme together in a protoplast formation buffer containing 0.02 M HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (pH 7.0), 1 mM MgCl(2), 0.5% gelatin, and 0.3 M raffinose. Cells were regenerated on a complex medium supplemented with bovine serum albumin, MgCl(2), CaCl(2), gelatin, and raffinose. Lengthy digestion with lytic enzymes inhibited the capacity of protoplasts to regenerate. The optimum conditions of protoplast formation varied from strain to strain. Using predetermined optimal conditions it was possible to prepare protoplasts of several L. casei strains and regenerate them with 10 to 40% efficiency. The methods were applicable to other species of lactobacilli as well.

Journal Article↗
PubMed1993

Formation and Regeneration of Methanococcus voltae Protoplasts.

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50% lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35 degrees C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.

Journal Article↗
PubMed1985

Protoplast dehydration correlated with heat resistance of bacterial spores.

Water content of the protoplast in situ within the fully hydrated dormant bacterial spore was quantified by use of a spore in which the complex of coat and outer (pericortex) membrane was genetically defective or chemically removed, as evidenced by susceptibility of the cortex to lysozyme and by permeability of the periprotoplast integument to glucose. Water content was determined by equilibrium permeability measurement with 3H-labeled water (confirmed by gravimetric measurement) for the entire spore, with 14C-labeled glucose for the integument outside the inner (pericytoplasm) membrane, and by the difference for the protoplast. The method was applied to lysozyme-sensitive spores of Bacillus stearothermophilus, B. subtilis, B. cereus, B. thuringiensis, and B. megaterium (four types). Comparable lysozyme-resistant spores, in which the outer membrane functioned as the primary permeability barrier to glucose, were employed as controls. Heat resistances were expressed as D100 values. Protoplast water content of the lysozyme-sensitive spore types correlated with heat resistance exponentially in two distinct clusters, with the four B. megaterium types in one alignment, and with the four other species types in another. Protoplast water contents of the B. megaterium spore types were sufficiently low (26 to 29%, based on wet protoplast weight) to account almost entirely for their lesser heat resistance. Corresponding values of the other species types were similar or higher (30 to 55%), indicating that these spores depended on factors additional to protoplast dehydration for their much greater heat resistance.

Cell Compartmentation↗
PubMed1966

Retention of episomes during protoplasting and during propagation in the L state.

Kawakami, Masaya (Georgetown University, Washington, D.C.), and Otto E. Landman. Retention of episomes during protoplasting and during propagation in the L state. J. Bacteriol. 92:398-404. 1966.-In earlier work, it was observed that the mesosomes of Bacillus magaterium and B. subtilis are expelled from the cell interior during protoplasting and that mesosome fragments are released into the supernatant fluid as the cell wall disintegrates. Since the resultant protoplasts remain intact and capable of reproduction, the expelled contents of the mesosome "bag" are presumably external to the protoplast membrane and nonessential to survival. Accordingly, if episomes (plasmids) were localized in the extramembrane mesosome "bag," it would be predicted that protoplasting would cure cells of their episomes. This prediction was tested in three different systems: B. subtilis W23 carrying phage SP-10, B. megaterium 216 carrying megacinogenic factors A and C, and B. megaterium C4M(-) carrying megacinogenic factor C. No curing due to protoplasting was observed. Even during propagation in the L form, when septation is not functioning and the distribution of deoxyribonucleic acid to daughter cells is severely disrupted, curing was not observed in any of the above systems nor in Salmonella L forms carrying F(0)lac. It is concluded that episomes are located at a position on the interior side of the cell membrane and that their distribution to daughter cells is coordinated with that of the chromosome.

Journal Article↗
PubMed1964

ELECTRON MICROSCOPE STUDY OF THE RELATIONSHIP BETWEEN MESOSOME LOSS AND THE STABLE L STATE (OR PROTOPLAST STATE) IN BACILLUS SUBTILIS.

Ryter, Antoinette (Institut Pasteur, Paris, France), and Otto E. Landman. An electron microscope study of the relationship between mesosome loss and the stable L-state (or protoplast state) in Bacillus subtilis. J. Bacteriol. 88:457-467. 1964.-In a prior publication, it was postulated that inability of protoplasts to restart cell-wall synthesis and cell division and the inability of stable mass-conversion L forms to return to the bacillary state were both equivalent and both due to the interruption of a membrane-associated reaction sequence. It was further postulated that this reaction sequence might reside in the mesosome. In the present publication, it is shown by means of electron microscopy of thin sections that protoplasts and L forms do not contain mesosomes. The sequence of events leading to loss of the mesosomes during protoplasting is as follows. Soon after lysozyme addition, the mesosomes are extruded from the cell interior into the space between cell wall and cytoplasmic membrane. Mesosome fragments in the form of small vesicles gather at the poles of the cells and are released, along with intact protoplasts, when the wall fragments. (Sudden shift of bacilli to hypertonic environment also causes extrusion and fragmentation of mesosomes, but this damage is later repaired.) In intact bacilli, mesosomes are in contact with both the peripheral membrane and nuclear material. Upon extrusion of the mesosomes, a direct attachment between nuclear material and cytoplasmic membrane is observed. Deoxyribonucleic acid (DNA)-membrane attachment may play a role in the control of DNA replication. Bacillus subtilis L-colonies consist of irregularly shaped bodies of varying sizes, bounded only by a membrane. Many of the smaller bodies do not contain nuclear material, and many of the large ones appear inviable. Division is accomplished by a disorganized-appearing constriction process. There are no septa.

Bacillus↗
PubMed2006

Magnetic field exposure stiffens regenerating plant protoplast cell walls.

Single suspension-cultured plant cells (Catharanthus roseus) and their protoplasts were anchored to a glass plate and exposed to a magnetic field of 302 +/- 8 mT for several hours. Compression forces required to produce constant cell deformation were measured parallel to the magnetic field by means of a cantilever-type force sensor. Exposure of intact cells to the magnetic field did not result in any changes within experimental error, while exposure of regenerating protoplasts significantly increased the measured forces and stiffened regenerating protoplasts. The diameters of intact cells or regenerating protoplasts were not changed after exposure to the magnetic field. Measured forces for regenerating protoplasts with and without exposure to the magnetic field increased linearly with incubation time, with these forces being divided into components based on the elasticity of synthesized cell walls and cytoplasm. Cell wall synthesis was also measured using a cell wall-specific fluorescent dye, and no changes were noted after exposure to the magnetic field. Analysis suggested that exposure to the magnetic field roughly tripled the Young's modulus of the newly synthesized cell wall without any lag.

Catharanthus↗
PubMed1991

Determination of cytochrome P-450 in Cunninghamella elegans intact protoplasts and cell-free preparations capable of steroid hydroxylation.

Cytochrome P-450 was shown to be involved in 11 alpha-, and 11 beta-hydroxylation of Substance S in intact C. elegans protoplasts. The steroid transformation was inhibited by carbon monoxide, the inhibitory effect being dependent on CO concentration. The function of cyt P-450 in intact protoplasts was confirmed by the estimation of strong absorption at 450 nm in the CO difference spectrum. The presence of antimycin A was necessary to prevent the reduction of the cytochrome oxidase and its interference with the cyt P-450 in the spectrophotometric analysis. The intracellular content of cyt P-450 could be increased from 5.25 pM/mg protein to 26.88 pM/mg protein when the steroid inducer was present in the medium at each stage of protoplast preparation and during cyt P-450 determination. The enriched microsomal fraction obtained from the crude extract of ruptured protoplasts contained the steroid 11 alpha-hydroxylase system of C. elegans. The activity of 11 beta-hydroxylase could not be detected under the conditions of the experiment. The localization of steroid hydroxylases of C. elegans in microsomes was confirmed by cyt P-450 detection in the 9600 x g supernatant. Membranous fractions (pellets 1100 x g and 9600 x g) of the concanavaline A stabilized protoplasts, carrying the marker plasma-membrane-bound and mitochondrial ATPases, did not show maximum absorption at 450 nm in the CO difference spectrum.

Antimycin A↗
PubMed1976

Regeneration of yeast protoplasts. A freeze-etching study.

The submicroscopical structure of yeast protoplasts regenerating the new cell wall or merely its fibrillar component was studied by freeze-etching. No relation was found between the number and distribution of plasma membrane particles at various stages of regeneration. Hexagonal arrangement of the particles was found only solitarily even in protoplasts synthesizing intensely glucan microfibrils in liquid media. The fibrillar network on protoplasts grown in liquid medium or fibrillar groundwork of the cell wall on protoplasts grown in gelatine medium were exposed only after etching on etched faces. The microfibrils did not penetrate the outer leaflet of the unit membrane, which consequently indicates that no structural relation could exist between the fibrils and the plasma membrane particles. During conversion of cells to protoplasts, plasma membrane invaginations were arranged end-to-end to form prolonged furrows which persisted until cell wall regeneration had been completed. Then the long furrows broke into short units. Thus plasma membrane invaginations appear to be loca, rigid differentiations of the plasma membrane which may migrate laterally. Neither the plasma membrane nor the adjacent cytoplasm showed signs of reverse pinocytosis. The endoplasmic reticulum, which was hypertrophic during regeneration, consisted of extensive membranes, often parallel in arrangement. The cytoplasm frequently contained groups of small globular particles without characteristic localization.

Cell Membrane↗
PubMed1981

Microbiological implications of electric field effects. III. Stimulation of yeast protoplast fusion by electric field pulses.

Prototrophic colonies could be selected on minimal medium after mixing of protoplasts from diauxotrophic mutants of the yeasts Saccharomycopsis lipolytica and/or Lodderomyces elongisporus and treatment with polyethylene glycol (PEG) in the presence of calcium chloride. This is the result of protoplast fusion and complementation of auxotrophic deficiencies. Under identical conditions an electric field pulse in the mus-range applied via an electric discharge to the protoplast-PEG mixture resulted in a drastic enhancement of the protoplast fusion rate. The presence of polyethylene glycol was demonstrated to be a prerequisite for fusion in this case, too. The frequency of hybrid formation detected a prototrophic colonies could be increased in the case of intraspecific fusion at initial electric field strengths between 2.5 and 5 kV . cm-1. The application of an electric field pulse of proper strength and duration to a yeast protoplast suspension turned out to be a more effective tool in production of fusion products that conventional methods. Large numbers of parasexual hybrids for different selection programmes in yeast genetics and for industrial purpose may be delivered by this technique.

Electricity↗
PubMed1983

[Protoplast liberation and regeneration in the ascomycete Hypomyces ochraceus (Pers.) Tul].

A rapid and convenient method for producing protoplasts from 3 d old mycelium of the ascomycete Hypomyces ochraceus is described. The procedure involves a Helix pomatia enzyme preparation and sucrose (20%) for stabilization. Pretreatment with disulfide bond reducing agents reduced the amount of viable protoplasts. Formation of protoplasts and different stages of regeneration were observed by phase contrast microscopy. There was only one type of true regeneration from protoplasts to hyphae in 15-30% gelatine medium by direct forming a germ tube from the original protoplast. Cytological events and physiological conditions are discussed.

Animals↗
PubMed1996

In vitro transcripts from cloned cDNAs of the lettuce infectious yellows closterovirus bipartite genomic RNAs are competent for replication in Nicotiana benthamiana protoplasts.

Full-length cloned cDNAs of lettuce infectious yellows closterovirus (LIYV) RNAs 1 and 2 were constructed and fused to the bacteriophage T3 RNA polymerase promoter. To assess RNA replication, Nicotiana benthamiana protoplasts were inoculated with LIYV virion RNAs and LIYV cDNA-derived in vitro transcripts. Analysis of protoplasts inoculated with LIYV virion RNAs or capped (m7GpppG) in vitro transcripts from LIYV RNA 1 and 2 cDNAs showed accumulation of LIYV genomic and putative subgenomic RNAs (sgRNAs), synthesis of LIYV coat protein, and formation of LIYV virions. Furthermore, protoplasts inoculated with only capped in vitro transcripts from LIYV RNA 1 cDNA showed accumulation of LIYV RNA 1 and its putative sgRNA, indicating that LIYV RNA 1 can replicate in the absence of LIYV RNA 2. Conversely, accumulation of LIYV RNA 2 was not detectable in protoplasts inoculated with only LIYV RNA 2 cDNA-derived capped in vitro transcripts. These data demonstrate that LIYV genomic RNAs are competent for replication in mesophyll protoplasts and that infectious in vitro transcripts can be derived from the cloned cDNAs of a closterovirus genome.

Capsid↗