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PubMed1989

Protoplast volume:water potential relationship and bound water fraction in spinach leaves.

Methods used to estimate the (nonosmotic) bound water fraction (BWF) (i.e. apoplast water) of spinach (Spinacia oleracea L.) leaves were evaluated. Studies using three different methods of pressure/volume (P/V) curve construction all resulted in a similar calculation of BWF; approximately 40%. The theoretically derived BWF, and the water potential (Psi(w))/relative water content relationship established from P/V curves were used to establish the relationship between protoplast (i.e. symplast) volume and Psi(w). Another method of establishing the protoplast volume/Psi(w) relationship in spinach leaves was compared with the results from P/V curve experiments. This second technique involved the vacuum infiltration of solutions at a range of osmotic potentials into discs cut from spinach leaves. These solutions contained radioactively labeled H(2)O and sorbitol. This dual label infiltration technique allowed for simultaneous measurement of the total and apoplast volumes in leaf tissue; the difference yielded the protoplast volume. The dual label infiltration experiments and the P/V curve constructions both showed that below -1 megapascals, protoplast volume decreases sharply with decreasing water potential; with 50% reduction in protoplast volume occurring at -1.8 megapascals leaf water potential.

Journal Article↗
PubMed1990

Correlation between the Maintenance of Photosynthesis and in Situ Protoplast Volume at Low Water Potentials in Droughted Wheat.

Studies were undertaken to examine the relationship between water deficit effects on photosynthesis and the extent of protoplast volume reduction which occurs in leaves at low water potential (Psi(w)). This relationship was monitored in two cultivars (;Condor' and ;Capelle Desprez') of cultivated wheat (Triticum aestivum) that differed in sensitivity to drought, and in a wild relative of cultivated wheat (Triticum kotschyi) that has been previously found to be ;drought resistant.' When subjected to periods of water stress, Condor and T. kotschyi plants underwent osmotic adjustment; Capelle plants did not. Photosynthetic capacity was maintained to different extents in the three genotypes as leaf Psi(w) declined during stress; Capelle plants were most severely affected. Calculations of internal leaf [CO(2)] and stomatal conductance from gas exchange measurements indicated that differences in photosynthetic inhibition at low Psi(w) among the genotypes were primarily due to nonstomatal effects. The extent of protoplast volume reduction that occurred in leaves at low Psi(w) was also found to be different in the three genotypes; maintenance of protoplast volume and photosynthetic capacity in stressed plants of the genotypes appeared to be correlated. When the extent of water stress-induced inhibition of photosynthesis was plotted as a function of declining protoplast volume, this relationship appeared identical for the three genotypes. It was concluded that there is a correlative association between protoplast volume and photosynthetic capacity in leaves of wheat plants subjected to periods of water stress.

Journal Article↗
PubMed1991

Localization of a protease in protoplast preparations in infected cells of French bean nodules.

Protoplasts from infected and uninfected cells were isolated from the central nitrogen fixing tissue of French bean (Phaseolus vulgaris L. cv Contender) root nodules. Successive filtrations allowed the separation of the infected cells, whereas the small uninfected cells were isolated on a discontinuous Percoll gradient. Higher yields of intact protoplasts were obtained from young (4-week-old) nodules whereas no protoplasts could be isolated from the oldest nodules. When proteolysis was determined in the cytosolic fraction of both infected and uninfected cells, at pH 5.0 and 8.0, with leghemoglobin or azocasein as substrate, activity was present only in infected cell protoplasts and increased with nodule age. A protease with an acidic pH optimum, mainly responsible for this increasing activity, was highly purified from senescing nodules by electro-elution after nondenaturing polyacrylamide gel electrophoresis and used to produce polyclonal antibodies. Western blots of nodule protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with purified anti-protease immunoglobulin G showed the molecular mass of the protease to be 58 kilodaltons. Blots also confirmed that protease protein was located in infected cell protoplasts only, regardless of nodule age.

Journal Article↗
PubMed1992

Effects of Light on the Membrane Potential of Protoplasts from Samanea saman Pulvini : Involvement of K Channels and the H -ATPase.

Rhythmic light-sensitive movements of the leaflets of Samanea saman depend upon ion fluxes across the plasma membrane of extensor and flexor cells in opposing regions of the leaf-movement organ (pulvinus). We have isolated protoplasts from the extensor and flexor regions of S. saman pulvini and have examined the effects of brief 30-second exposures to white, blue, or red light on the relative membrane potential using the fluorescent dye, 3,3'-dipropylthiadicarbocyanine iodide. White and blue light induced transient membrane hyperpolarization of both extensor and flexor protoplasts; red light had no effect. Following white or blue light-induced hyperpolarization, the addition of 200 millimolar K(+) resulted in a rapid depolarization of extensor, but not of flexor protoplasts. In contrast, addition of K(+) following red light or in darkness resulted in a rapid depolarization of flexor, but not of extensor protoplasts. In both flexor and extensor protoplasts, depolarization was completely inhibited by tetraethylammonium, implicating channel-mediated movement of K(+) ions. These results suggest that K(+) channels are closed in extensor plasma membranes and open in flexor plasma membranes in darkness and that white and blue light, but not red light, close the channels in flexor plasma membranes and open them in extensor plasma membranes. Vanadate treatment inhibited hyperpolarization in response to blue or white light, but did not affect K(+) -induced depolarization. This suggests that white or blue light-induced hyperpolarization results from activation of the H(+) -ATPase, but this hyperpolarization is not the sole factor controlling the opening of K(+) channels.

Journal Article↗
PubMed1998

Ca2+, annexins, and GTP modulate exocytosis from maize root cap protoplasts

Protoplasts isolated from root cap cells of maize were shown to secrete fucose-rich polysaccharides and were used in a patch-clamp study to monitor changes in whole-cell capacitance. Ca2+ was required for exocytosis, which was measured as an increase in cell capacitance during intracellular dialysis with Ca2+ buffers via the patch pipette. Exocytosis was stimulated significantly by small increases above normal resting [Ca2+]. In the absence of Ca2+, protoplasts decreased in size. In situ hybridization showed significant expression of the maize annexin p35 in root cap cells, differ-entiating vascular tissue, and elongating cells. Dialysis of protoplasts with maize annexins stimulated exocytosis at physiological [Ca2+], and this could be blocked by dialysis with antibodies specific to maize annexins. Dialysis with milli-molar concentrations of GTP strongly inhibited exocytosis, causing protoplasts to decrease in size. GTPgammaS and GDPbetaS both caused only a slight inhibition of exocytosis at physiological Ca2+. Protoplasts were shown to internalize plasma membrane actively. The results are discussed in relation to the regulation of exocytosis in what is usually considered to be a constitutively secreting system; they provide direct evidence for a role of annexins in exocytosis in plant cells.

Journal Article↗
PubMed1982

Optimal Cultural and Physiological Conditions for Handling Streptomyces rimosus Protoplasts.

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no alpha, epsilon-ll-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.

Journal Article↗
PubMed1994

Natural and Electroporation-Mediated Transformation of Methanococcus voltae Protoplasts.

The lack of high-efficiency transformation systems has severely impeded genetic research on methanogenic members of the kingdom Archaeobacteria. By using protoplasts of Methanococcus voltae and an integration vector, Mip1, previously shown to impart puromycin resistance, we obtained natural transformation frequencies that were about 80-fold higher (705 transformants per mug of transforming DNA) than that reported with whole cells. Electroporation-mediated transformation of M. voltae protoplasts with covalently closed circular Mip1 DNA was possible, but at lower frequencies of ca. 177 transformants per mug of vector DNA. However, a 380-fold improvement (3,417 transformants per mug of DNA) over the frequency of natural transformation with whole cells was achieved by electroporation of protoplasts with linearized DNA. This general approach, of using protoplasts, should allow the transformation of other methanogens, especially those that may be gently converted to protoplasts as a result of their tendency to lyse in hypotonic solutions.

Journal Article↗
PubMed1971

Growth of streptococcal protoplasts and L-colonies on membrane filters.

Membrane filters (Millipore Corp.; pore sizes 1.2 to 0.22 mum) were placed on the surface of L-phase growth medium solidified with agar. The filter and the surrounding medium were inoculated with either protoplasts or stable broth-grown L-phase variants obtained from Streptococcus faecium strain F24. The L-phase inoculum gave rise to viable L-colonies on the filters and on the medium, whereas protoplasts gave colony formation only on the medium. However, when the Millipore filters were covered by a layer of solid L-phase medium, 75 mum or greater in depth, before inoculation with protoplasts, colony formation resulted but with atypical morphology. In contrast, inoculation of protoplasts on Nuclepore and Sartorius membrane filters did give rise to L-colonies on the surface and underneath the filters after 2 days of incubation at 37 C. Submicroscopic, viable L-phase elements produced during colony formation were capable of passing through membrane filters with pore channels as small as 0.22 mum; these elements required transfer from underneath the filters to fresh agar medium in order to develop into L-phase colonies. Membrane filters were also placed on the surface of L-phase growth medium solidified with gelatin. Inoculation of the filters and surrounding medium with a lysozyme-prepared protoplast suspension gave rise to streptococci on the surface of the filters and on the medium. However, inoculation with the stable broth-grown L-phase variants gave rise to atypical colonies on the medium and only small patches of abortive growth on the filters.

Agar↗
PubMed1962

STABILIZATION OF PROTOPLASTS AND SPHEROPLASTS BY SPERMINE AND OTHER POLYAMINES.

Tabor, Celia W. (National Institute of Arthritis and Metabolic Diseases, Bethesda, Md.). Stabilization of protoplasts and spheroplasts by spermine and other polyamines. J. Bacteriol. 83:1101-1111. 1962.-Spermine (10(-3)m) or spermidine prevents lysis of lysozyme-produced protoplasts of Escherichia coli W, E. coli B, and Micrococcus lysodeikticus in hypotonic media. Spheroplasts prepared by the action of penicillin are also stabilized by these concentrations of spermine and spermidine, but the protection is not as complete. Streptomycin, polylysine, and Ca(++) are also effective or partially effective stabilizers, but 1,4-diaminobutane, 1,5-diaminopentane, ornithine, Mg(++), and monovalent cations have no protective action at 10(-3)m concentration, and only a slight effect at higher concentrations. The osmotic stability conferred on protoplasts by spermine is irreversible. However, the protective effect of polyamines against lysis is not accompanied by restoration of viability to lysozyme protoplasts. There is a marked reduction in the loss of ultra-violet-absorbing material from the protoplasts to the medium when 10(-3)m spermine is present.

Journal Article↗
PubMed1966

Genetic Recombination of Transforming Deoxyribonucleic Acid Molecules with the Recipient Genome and Among Themselves in Protoplasts of Bacillus subtilis.

Hirokawa, Hideo (Southwest Center for Advanced Studies, Dallas, Tex.), and Yonosuke Ikeda. Genetic recombination of transforming deoxyribonucleic acid molecules with the recipient genome and among themselves in protoplasts of Bacillus subtilis. J. Bacteriol. 92:455-463. 1966.-Re-extraction of transforming deoxyribonucleic acid (DNA) from protoplasts of Bacillus subtilis is much more efficient than from intact competent cells. This facilitated the detection of physical recombination between donor and recipient DNA molecules, as indicated by a high cotransfer index of ind(+) and his(+) markers which were originally located in exogenous and endogenous DNA molecules, respectively. This recombinant DNA was extracted after 30 min of incubation of ind his(+) protoplasts with ind(+)his DNA, previously extracted from a corresponding mutant strain of B. subtilis. The intracellular formation of recombinant molecules (ind(+)his(+)) bearing markers from two different exogenous DNA species was also detected 15 min after exposure of ind his recipient protoplasts to a mixture of ind(+)his and ind his(+) donor DNA molecules. The unity of the recombinant molecule was ascertained by dilution experiments and by its being resistant to ribonuclease and trypsin treatment (but being sensitive to deoxyribonuclease). The formation of recombinant molecules showed an inverse kinetics to that of the intracellularly induced loss of linkage between the corresponding markers in the wild-type DNA, thus suggesting a breakage and reunion process which is also favored by the absence of DNA synthesis in the protoplasts and the effect of some specific inhibitors.

Journal Article↗
PubMed1965

ELECTRON MICROSCOPE OBSERVATIONS ON INTACT CELLS, PROTOPLASTS, AND THE CYTOPLASMIC MEMBRANE OF BACILLUS STEAROTHERMOPHILUS.

Abram, Dinah (Purdue University, Lafayette, Ind.). Electron microscope observations on intact cells, protoplasts, and the cytoplasmic membrane of Bacillus stearothermophilus. J. Bacteriol. 89:855-873. 1965.-Negatively stained preparations of protoplasts and fragments of cytoplasmic membranes from cells of Bacillus stearothermophilus ruptured by treatment with sonic oscillation, partial lysis with lysozyme, autolysis, or phage infection were examined electron microscopically. Specimens of intact cells also were examined by the same technique. The following structural details were revealed. Intact or nearly intact, partially swollen, elongated protoplasts and their ghosts have a characteristic differentiated surface texture and can easily be distinguished from the cell wall. Infoldings of the cytoplasmic membrane can be observed in these protoplasts, to which flagella are attached; the latter originate via hooks from "basal structures" that are in close association with the cytoplasmic membrane or part of it. Abundant intracytoplasmic membranous elements, which appear to be tubular or vesicular, can be seen in whole cells of three of the strains studied. The fine structure of the cytoplasmic membrane and probably that of its intracytoplasmic infoldings was observed on flattened and folded membrane fragments, one layer thick. Structural units, roughly spherical, 65 to 85 A in diameter, were present on one side of the cytoplasmic membrane, facing the cytoplasm. They were attached loosely to the membrane by fine stalks, 40 to 60 A long, and were easily detached, probably leaving the stalks behind them on the membrane. While the greater stability of membranes from thermophiles made this study of the fine structure possible, the structural units described were demonstrated also on cytoplasmic membranes from mesophiles.

Bacillus↗
PubMed1965

LYSIS OF BACTERIAL PROTOPLASTS AND SPHEROPLASTS BY STAPHYLOCOCCAL ALPHA-TOXIN AND STREPTOLYSIN S.

Bernheimer, Alan W. (New York University School of Medicine, New York, N.Y.), and Lois L. Schwartz. Lysis of bacterial protoplasts and spheroplasts by staphylococcal alpha-toxin and streptolysin S. J. Bacteriol. 89:1387-1392. 1965.-Protoplasts of Bacillus megaterium, Sarcina lutea, and Streptococcus pyogenes, and spheroplasts of Escherichia coli were lysed by staphylococcal alpha-toxin, whereas spheroplasts of Vibrio metschnikovii and V. comma were not. In the spectrum of its lytic action, streptolysin S qualitatively resembled staphylococcal alpha-toxin except for failure to lyse S. pyogenes protoplasts. In contrast to the two foregoing agents, streptolysin O did not lyse protoplasts and spheroplasts. The observations are interpreted in relation to similarities and differences in lipid composition of bacterial and mammalian cell membranes.

Animals↗
PubMed1996

Flow cytometric analysis of cytosolic pH of mesophyll cell protoplasts from the crabgrass Digitaria sanguinalis.

Phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) is a key enzyme of photosynthesis in C4 plants; it is specifically localized in the cytosol of mesophyll cells and is regulated by a phosphorylation/dephosphorylation process. The light-dependent phosphorylation of PEPC is triggered by an increase in the cytosolic pH (pHc) of mesophyll cell protoplasts. An epifluorescence and confocal microscopy analysis showed that the specific pH probe 2',7'-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein, acetoxymethyl ester (BCECF-AM), when used at low concentration, was essentially localized in the protoplast cytosol. By the nigericin null-point method and flow cytometry, the pHc of freshly isolated protoplasts was estimated to be 6.4. To observe the full activity of PEPC kinase and maximal phosphorylation of PEPC in vivo, such protoplast suspensions must first be treated with a permeant weak base. The present report shows that 20 mM NH4Cl raised the final pHc to 7.4. This method can be useful for estimating rapid changes of pHc in plant cells.

Ammonium Chloride↗
PubMed1993

Conditional inhibition of beta-glucuronidase expression by antisense gene fragments in petunia protoplasts.

Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of beta-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60-70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.

Base Sequence↗
PubMed1991

Barley aleurone layer cell protoplasts as a transient expression system.

Protoplasts were prepared from barley aleurone layers using 'Onozuka' cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of alpha-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley alpha-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormone-responsive elements in hydrolase genes.

Abscisic Acid↗
PubMed1992

Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses.

Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris. Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed. cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized. The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5-mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids. The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation. The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.

Amino Acid Sequence↗
PubMed1989

Transient gene expression in aleurone protoplasts isolated from developing caryopses of barley and wheat.

Methods have been developed for the isolation of aleurone protoplasts from developing caryopses of Hordeum vulgare and Triticum aestivum in order to study transient expression of introduced genes. Chimaeric gene constructs were introduced into aleurone protoplasts by polyethylene glycol (PEG). Transient expression directed by the 35S promoter from cauliflower mosaic virus (CaMV) of the reporter gene encoding chloramphenicol acetyl transferase (CAT) was detected in aleurone protoplasts from developing barley and wheat grains. Using a similar construct, CAT activity increased when the alcohol dehydrogenase intron 1 fragment from maize was ligated between the 35S promoter and the CAT coding region. The demonstration of transient expression in protoplasts from developing aleurone layers indicates that they may be useful for investigating tissue and developmental control of genes coding for cereal seed proteins.

Brassica↗
PubMed1994

Patch clamp analysis of the dominant plasma membrane K+ channel in root cell protoplasts of Plantago media L. Its significance for the P and K state.

Ion channels in the plasma membrane of root cell protoplasts of Plantago media L. were studied with the patch clamp technique in the cell-attached patch and outside-out patch configuration. An outward rectifying potassium channel was dominantly present in the plasma membrane. It appears responsible for the diffusional part, dominated by the K+ diffusion potential, of the cell membrane potential, in vivo. This channel is activated at potentials near to and more positive than the K+ diffusion potential. The dependence of this ion channel on K+ activity and voltage has been characterized. The current-voltage relationships of the open channel at various K+ concentrations are described by a four-state model. The membrane potential of intact protoplasts appears either dominated by the K+ diffusion potential, the protoplast is then said to be in the K state, or by the pump potential generated by the plasma membrane-bound proton pump/H+ ATPase, the P state. An experimental procedure is described to determine in cell-attached patch mode the state of the protoplast, either K or P state.

Cell Membrane↗