A plasmolytic cycle: The fate of cytoskeletal elements

Summary In most plant cells, transfer to hypertonic solutions causes osmotic loss of water from the vacuole and detachment of the living protoplast from the cell wall (plasmolysis). This process is reversible and after removal of the plasmolytic solution, protoplasts can re-expand to their original size (deplasmolysis). We have investigated this phenomenon with special reference to cytoskeletal elements in onion inner epidermal cells. The main processes of plasmolysis seem to be membrane dependent because destabilization of cytoskeletal elements had only minor effects on plasmolysis speed and form. In most cells, the array of cortical microtubules is similar to that found in nonplasmolyzed states except that longitudinal patterns seen in some control cells were never observed in plasmolyzed protoplasts of onion inner epidermis. As soon as deplasmolysis starts, cortical microtubules become disrupted and only slowly regenerate to form an oblique array, similar to most nontreated cells. Actin microfilaments responded rapidly to the plasmolysis-induced deformation of the protoplast and adapted to its new form without marked changes in organization and structure. Both actin microfilaments and microtubules can be present in Hechtian strands, which, in plasmolyzed cells, connect the cell wall to the protoplast. Anticytoskeletal drugs did not affect the formation of Hechtian strands.Abbreviations DIC differential interference contrast - DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Plasmolysis bays in Escherichia coli: are they related to development and positioning of division sites?

Plasmolysis bays, induced in Escherichia coli by hypertonic treatment, are flanked by zones of adhesion between the plasma membrane and the cell wall. To test the proposition of Cook et al. (W. R. Cook, F. Joseleau-Petit, T. J. MacAlister, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 84:7144-7148, 1987) that these zones, called periseptal annuli, play a role in determining the division site, we analyzed the positions of these zones by phase-contrast and electron microscopy. In situ treatment of cells grown in agar showed that the youngest cell pole was the most susceptible to plasmolysis, whereas the constriction site was resistant. Lateral bays occurred only at some distance from a polar bay or a resistant constriction site. Orienting cells with their most prominently plasmolyzed polar bay in one direction showed that the lateral bays were always displaced away from the polar bay at about half the distance to the other cell pole. If no poles were plasmolyzed, lateral bays occurred either in the centers of nonconstricting cells or at the 1/4 or 3/4 position of cell length in constricting cells. The asymmetric positions of lateral plasmolysis bays, caused by their abrupt displacement in the presence of polar bays or constriction sites, does not confirm the periseptal annulus model (Cook et al.), which predicts a gradual and symmetric change in the position of lateral bays with increasing cell length. Our analysis indicates that plasmolysis bays have no relation to the development and positioning of the future division site.     

Plasmolysis and recovery of different cell types in cryoprotected shoot tips of Mentha × piperita

Summary. Successful cryopreservation of plant shoot tips is dependent upon effective desiccation through osmotic or physical processes. Microscopy techniques were used to determine the extent of cellular damage and plasmolysis that occurs in peppermint (Mentha × piperita) shoot tips during the process of cryopreservation, using the cryoprotectant plant vitrification solution 2 (PVS2) (30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol, 0.4 M sucrose) prior to liquid-nitrogen exposure. The meristem cells were the smallest and least plasmolyzed cell type of the shoot tips, while the large, older leaf and lower cortex cells were the most damaged. When treated with cryoprotectant solutions, meristem cells exhibited concave plasmolysis, suggesting that this cell type has a highly viscous protoplasm, and protoplasts have many cell wall attachment sites. Shoot tip cells were most severely plasmolyzed after PVS2 treatment, liquid-nitrogen exposure, and warming in 1.2 M sucrose. Successful recovery may be dependent upon surviving the plasmolytic conditions induced by warming and diluting treated shoot tips in 1.2 M sucrose solutions. In peppermint shoot tips, clumps of young meristem or young leaf cells survive the cryopreservation process and regenerate plants containing many shoots. Cryoprotective treatments that favor survival of small, meristematic cells and young leaf cells are most likely to produce high survival rates after liquid-nitrogen exposure. Correspondence and reprints: National Center for Genetic Resources Preservation, U.S. Department of Agriculture, 1111 S. Mason Street, Fort Collins, CO 80521, U.S.A.     

Bacterial plasmolysis as a physical indicator of viability.

Bacterial plasmolytic response to osmotic stress was evaluated as a physical indicator of membrane integrity and hence cellular viability. Digital image analysis and either low-magnification dark-field, high-magnification phase-contrast, or confocal laser microscopy, in conjunction with pulse application of a 1.5 M NaCl solution, were used as a rapid, growth-independent method for quantifying the viability of attached biofilm bacteria. Bacteria were considered viable if they were capable of plasmolysis, as quantified by changes in cell area or light scattering. When viable Salmonella enteritidis biofilm cells were exposed to 1.5 M NaCl, an approximately 50% reduction in cell protoplast area (as determined by high-magnification phase-contrast microscopy) was observed. In contrast, heat- and formalin-killed S. enteritidis cells were unresponsive to NaCl treatment. Furthermore, the mean dark-field cell area of a viable, sessile population of Pseudomonas fluorescens cells (approximately 1,100 cells) increased by 50% as a result of salt stress, from 1,035 +/- 162 to 1,588 +/- 284 microns2, because of increased light scattering of the condensed, plasmolyzed cell protoplast. Light scattering of ethanol-killed control biofilm cells underwent little change following salt stress. When the results obtained with scanning confocal laser microscopy and a fluorescent viability probe were compared with the accuracy of plasmolysis as a viability indicator, it was found that the two methods were in close agreement. Used alone or in conjunction with fluorochemical probes, physical indicators of membrane integrity provided a rapid, direct, growth-independent method for determining the viability of biofilm bacteria known to undergo plasmolysis, and this method may have value during efficacy testing of biocides and other antimicrobial agents when nondestructive time course analyses are required.     

Vectorial and nonvectorial transphosphorylation catalyzed by enzymes II of the bacterial phosphotransferase system

The plasmolytic response of Bacillus licheniformis 749/C cells to the increasing osmolarity of the surrounding medium was quantitated with stereological techniques. Plasmolysis was defined as the area (in square micrometers) of the inside surface of the bacterial wall not in association with bacterial membrane per unit volume (in cubic micrometers) of bacteria. This plasmolyzed surface area was zero when the cells were suspended in a concentration of sucrose solution lower than 0.5 M, but increased linearly when the sucrose molarity rose above 0.5 M, reaching a plateau value of 3.61 micrometers2/micrometers3 in 2 M sucrose. In contrast, when the bacterial cells were treated with lysozyme plasmolysis increased abruptly from 0.06 micrometers2/micrometers3 in 0.75 M sucrose to 4.09 micrometers2/micrometers3 in 1 M sucrose. When the time of exposure was prolonged, the degree of plasmolysis increased gradually for the duration of the experiment (30 min) after exposure to 1 M sucrose without lysozyme, whereas with lysozyme plasmolysis reached a maximum (4.09 micrograms2/micrometers3) in 2 to 5 min. The examination of ultrastructure showed that the protoplast bodies of lysozyme-treated cells in 1 M sucrose and untreated cells in 2 M sucrose are maximally retracted from the intact wall of the bacteria; hardly any retraction of protoplasts could be seen for untreated cells in 1 M sucrose. The data suggest that the B. licheniformis cells are isoosmotic to 800 to 1,100 mosM solutions, but are able to withstand much greater osmotic pressure with no signs of plasmolysis because the cell wall and the plasma membrane are held in close association, perhaps by a covalent bond. It is likely that lysozyme weakens this bond by degradation of the peptidoglycan layer. Cellular autolysis also weakens this wall-membrane association.     

Osmotic Pressure in Escherichia coli as Rendered Detectable by Lysozyme Attack

The enhanced susceptibility of plasmolyzed Escherichia coli to lysozyme attack was used to estimate the internal osmotic pressure of these cells under various conditions. Differences were detected between strains, culture media, stages in the growth cycle, and the osmotically active material used to produce plasmolysis. Lysozyme also was found to attack unplasmolyzed cells at 0 C and between 50 and 70 C.     

Cytoplasmic protein mobility in osmotically stressed Escherichia coli

Facile diffusion of globular proteins within a cytoplasm that is dense with biopolymers is essential to normal cellular biochemical activity and growth. Remarkably, Escherichia coli grows in minimal medium over a wide range of external osmolalities (0.03 to 1.8 osmol). The mean cytoplasmic biopolymer volume fraction ((phi)) for such adapted cells ranges from 0.16 at 0.10 osmol to 0.36 at 1.45 osmol. For cells grown at 0.28 osmol, a similar phi range is obtained by plasmolysis (sudden osmotic upshift) using NaCl or sucrose as the external osmolyte, after which the only available cellular response is passive loss of cytoplasmic water. Here we measure the effective axial diffusion coefficient of green fluorescent protein (D(GFP)) in the cytoplasm of E. coli cells as a function of (phi) for both plasmolyzed and adapted cells. For plasmolyzed cells, the median D(GFP) (D(GFP)(m)) decreases by a factor of 70 as (phi) increases from 0.16 to 0.33. In sharp contrast, for adapted cells, D(GFP)(m) decreases only by a factor of 2.1 as (phi) increases from 0.16 to 0.36. Clearly, GFP diffusion is not determined by (phi) alone. By comparison with quantitative models, we show that the data cannot be explained by crowding theory. We suggest possible underlying causes of this surprising effect and further experiments that will help choose among competing hypotheses. Recovery of the ability of proteins to diffuse in the cytoplasm after plasmolysis may well be a key determinant of the time scale of the recovery of growth.     

Plasmolytic behavior of the donor cell may affect protoplast response

Protoplast donor tissues (leaves of shoots in culture) from a herbaceous plant ( Solanum etuberosum ) and two woody species ( Populus alba × P. grandidentata cv. Crandon and Betula platyphylla szechuanica ) were compared during plasmolysis in a range of osmotic agents and potentials. Cells from both Solanum and Populus , species proven to be amenable to protoplast division and regeneration, plasmolyzed readily at higher osmotic potentials than cells from Betula , a species recalcitrant to prolonged culture after protoplast isolation. Betula leaf mesophyll cells exhibited persistent membrane-to-wall attachments and many failed to plasmolyze even under extreme osmolarity. Although their leaves exhibited similar photosynthetic rates, photosynthetic capacity was lost from Betula protoplasts upon isolation, and retained by Solanum protoplasts. Differential stress after isolation was not detectable through vital staining, but only Solanum and Populus gave both high protoplast yields and high plating efficiencies in continued culture.     

Determining Some Osmotic Characteristics of White Pine Leaf Cells

When leaf cells of white pine, Pinus strobus L., were plasmolyzed in appropriate concentrations of glucose (0.85-0.95 M) they could be fixed in a mixture of formalin, acetic acid and alcohol, embedded in polyethylene glycol, and measurements of the degree of plasmolysis made without development of artifacts. Fresh material was prepared by freehand sectioning only with difficulty and did not always give reliable results, whereas embedding and microtome sectioning provided satisfactory material for determining the osmotic characteristics of pine foliage tissue.     

Plasmolysis of Escherichia coli B/r with Sucrose

Escherichia coli B/r cells were plasmolyzed in sucrose solutions and observed under phase contrast. The prevalence of plasmolysis under various conditions was noted, and the degree of plasmolysis was categorized as slight, extensive, or severe. The presence of ions reduced the prevalence of plasmolysis. Survival curves showed that extensive plasmolysis was not lethal to colony-forming ability.     

Synthetic Capabilities of Plasmolyzed Cells and Spheroplasts of Escherichia coli

Effects of plasmolysis and spheroplast formation on deoxyribonucleic acid (DNA), ribonucleic acid (RNA), protein, and phospholipid synthesis by Escherichia coli strain THU were studied. RNA and protein synthesis were severely diminished. DNA and phospholipid synthesis were inhibited, but less so; they could be partly restored. DNA synthesis could be restored by replacing thymine in the medium with thymidine, and phospholipid synthesis, by adding back small quantities of soluble cell extract. Plasmolysis effected marked reductions in rates of growth and macro-molecule synthesis, and temporarily reduced culture viability. Plasmolysis also caused an anomalous stimulation of phospholipid synthesis. Spheroplasts and plasmolyzed cells synthesized small amounts of ribosomal RNA that sedimented normally. However, this ribosomal RNA was very inefficiently packaged to ribosome subunits. Spheroplasts were unable to carry out induced synthesis of beta-galactosidase, and plasmolyzed cells were delayed in this function. Radioautographs examined in an electron microscope showed that DNA synthesis in plasmolyzed cells and spheroplasts was performed by a substantial fraction of the culture populations. That DNA and membrane were associated in the spheroplasts used in this study was suggested by formation of M-bands containing membrane and most of the cell's DNA. The results are discussed in terms of alterations of membrane structure and conformation attending plasmolysis and spheroplasting.     

Nucleoid release from Escherichia coli cells.

The time course of morphological changes during lysis of Escherichia coli cells was examined with respect to an undisturbed release of nucleoids. The addition of detergents to plasmolyzed, osmotic sensitive cells resulted in the immediate reversal of plasmolysis followed by the appearance of rod-shaped ghost cells without any detectable spheroplast formation. Electron microscopic examination of the rod-shaped ghost cells revealed a zonal gap in the cell envelope, allowing the free release of the nucleoid. Due to the high ionic strength, a suitable cell lysis was shown to require higher incubation temperatures. However, in the absence of an appropriate control this may result in the sphering and vesiculation of ghost cell envelopes and even the unfolding of released nucleoids. To avoid this unfavorable consequence of lysis at high temperatures, a microscopic examination on the course of rod-shaped ghost formation is suggested.     

Hyperosmotic stress induces formation of tubulin macrotubules in root-tip cells of Triticum turgidum: their probable involvement in protoplast volume control

Treatment of root-tip cells of Triticum turgidum with 1 M mannitol solution for 30 min induces microtubule (Mt) disintegration in the plasmolyzed protoplasts. Interphase plasmolyzed cells possess many cortical, perinuclear and endoplasmic macrotubules, 35 nm in mean diameter, forming prominent arrays. In dividing cells macrotubules assemble into aberrant mitotic and cytokinetic apparatuses resulting in the disturbance of cell division. Putative tubulin paracrystals were occasionally observed in plasmolyzed cells. The quantity of polymeric tubulin in plasmolyzed cells exceeds that in control cells. Root-tip cells exposed for 2-8 h to plasmolyticum recover partially, although the volume of the plasmolyzed protoplast does not change detectably. Among other events, the macrotubules are replaced by Mts, chromatin assumes its typical appearance and the cells undergo typical cell divisions. Additionally, polysaccharidic material is found in the periplasmic space. Oryzalin and colchicine treatment induced macrotubule disintegration and a significant reduction of protoplast volume in every plasmolyzed cell type examined, whereas cytochalasin B had only minor effects restricted to differentiated cells. These results suggest that Mt destruction by hyperosmotic stress, and their replacement by tubulin macrotubules and putative tubulin paracrystals is a common feature among angiosperms and that macrotubules are involved in the mechanism of protoplast volume regulation.     

Plasmolysis and Cell Shape Depend on Solute Outer-Membrane Permeability during Hyperosmotic Shock in E. coli

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.     

Zones of membrane adhesion in the cryofixed envelope of Escherichia coli

The envelopes of Escherichia coli B and E. coli K29 were examined using cryofixation and freeze substitution. Emphasis was directed toward the question whether membrane adhesion zones (which connect inner membrane (IM) and outer membrane (OM) after plasmolysis in 10-20% sucrose) can be visualized with the use of cryotechniques. Plasmolysis in 10-20% sucrose was observed to have no effect on cell viability. We found that simple plunge-freezing methods preserve adhesion sites, whereas these sites were not observed after impact-freezing. Also, plasmolysis "bays," visible in light microscopic preparations of living cells, were seen to be maintained intact after plunge-freezing. Employment of photocrosslinking with UV-flashes before or after plasmolysis showed a significant increase in the number of adhesion areas compared to noncrosslinked specimens. To control the contact speed of the specimen during immersion into the cryogen, a hollow rotor was constructed in which the cryogenic liquid is moving at desired high speeds. Adhesion sites presented themselves in the plasmolyzed cell as sites of close contact of the outer and inner membrane, an arrangement that would leave very limited space for peptidoglycan layers at the contact site of the two membranes. Adhesion sites may occur either as single, isolated sites or within stretches of IM/OM apposition where they appear to function as "spot welds" between the two membranes. Exposure of cells to sucrose concentrations of 35% caused rupture of adhesions with cytoplasmic fragments remaining attached to the envelope. The cryofixation procedures described here do not presently yield the number of membrane adhesions obtainable with conventional aldehyde fixation. However, since the combination of millisecond photocrosslinking and cryofixation of plasmolyzed cells resulted in a higher membrane stabilization and in an increase of the number of adhesion sites, this combination appears to be a useful tool for the analysis of sensitive membrane structures.     

Sensitivity of Escherichia coli to cephaloridine at different growth rates.

Steady-state populations of Escherichia coli B/r were treated with cephaloridine at minimal inhibitory concentrations. The antibiotic sensitivity of the cells and the localization of spheroplast emergence along the cell surface were examined as a function of cell length and growth rate. In fast-growing populations (greater than 1 division per h) the sites of cephaloridine interaction occurred preferentially at the cell pole in the smaller cells and at the cell center in dividing cells. At decreasing growth rates the cells became more resistant to cephaloridine, and a gradual shift from the cell pole toward the cell center was observed for the sphere position. A similar growth rate-dependent change in localization was found for sucrose-induced plasmolysis vacuoles.     

Cellular osmotic phenomena during stomatal movements ofCommelina communis

Summary The accuracy of most of the published values for guard cell osmotic pressures is disputed and it is considered that many values are grossly in error. Since most of the values were obtained from incipient plasmolysis experiments limitations of the technique were investigated. It was concluded that it is not possible to use the incipient plasmolysis method for accurately determining guard cell osmotic pressures since all concentrations of plasmolytica (concentrations down to 0.1 M sucrose or calcium nitrate were used) bring about incipient plasmolysis depending on the period of time the tissue is immersed in the plasmolytica. In other words, the concentration of a plasmolyticum at which incipient plasmolysis occurs continues to decrease as the plasmolysing time increases. Furthermore, the time taken for incipient plasmolysis to occur varies according to the solutes in the plasmolyticum and the extent of stomatal aperture.A reason for the changing values of guard cell osmotic pressures was the loss of K⁺, and to a lesser extent, Cl^–, Ca²⁺ and Na⁺, and sugars and organic acids from the tissue during exposure to graded concentrations of plasmolytica (sucrose and calcium nitrate). A good correlation between loss of solutes from the epidermal tissue and decrease in guard cell osmotic pressure was not observed, however.Histochemical tests for K⁺ support the view that leakage of K⁺ from the guard cells occurs while the tissue is immersed in the plasmolytica except when high concentrations of sucrose (2.0 M) and calcium nitrate (greater than 1.0 M) were used and then leakage was minimal. However, these high concentrations of plasmolytica caused cell damage.The osmotic relationships of the various cell types within the epidermis ofCommelina communis were investigated during stomatal movements. Although absolute values for the osmotic pressures of the various cell types could not be evaluated it was apparent from the rates of changes of the osmotic pressures that when stomata closed guard cell osmotic pressures decreased while epidermal and subsidiary cell osmotic pressures increased to almost the same values as the guard cells.     

Tracheary element differentiation is correlated with inhibition of cell expansion in xylogenic mesophyll suspension cultures.

To test the hypothesis that xylogenesis is coupled to cell growth suppression, cell expansion in Zinnia elegans L. var. Envy mesophyll suspension cultures was manipulated by varying the extracellular osmolarity and the effect on xylogenesis was examined. Cell expansion and tracheary element differentiation were inversely related along a gradient of extracellular osmolarity ranging from 200 to 400 mOsm, supporting the hypothesis that tracheary element differentiation is coupled to cessation of cell expansion. Above 300 mOsm, reduction in the number of cells that differentiated into tracheary elements coincided with an increase in the number of plasmolyzed cells as extracellular osmolarity was increased, indicating that plasmolysis inhibits tracheary element differentiation, although not specifically. Using the plasmolysis method we showed that cellular osmolarity within populations of isolated Zinnia mesophyll cells ranges from 250 to 600 mOsm with a mean of 425 mOsm. The broad range in cellular osmolarity within Zinnia mesophyll cell populations, coupled with inhibition of differentiation in the low range due to cell expansion and in the high range due to plasmolysis, may help explain why tracheary element differentiation in Zinnia suspension cultures is never complete nor perfectly synchronous and enable further optimization of this culture system.     

Transmembrane Protein (Perfringolysin O) Association with Ordered Membrane Domains (Rafts) Depends Upon the Raft-Associating Properties of Protein-Bound Sterol

The concentration of chemicals inside the bacterial cytoplasm generates an osmotic pressure, termed turgor, which inflates the cell and is necessary for cell growth and survival. In Escherichia coli, a sudden increase in external concentration causes a pressure drop across the cell envelope that drives changes in cell shape, such as plasmolysis, where the inner and outer membranes separate. Here, we use fluorescence imaging of single cells during hyperosmotic shock with a time resolution on the order of seconds to examine the response of cells to a range of different conditions. We show that shock using an outer-membrane impermeable solute results in total cell volume reduction with no plasmolysis, whereas a shock caused by outer-membrane permeable ions causes plasmolysis immediately upon shock. Slowly permeable solutes, such as sucrose, which cross the membrane in minutes, cause plasmolysis to occur gradually as the chemical potential equilibrates. In addition, we quantify the detailed morphological changes to cell shape during osmotic shock. Nonplasmolyzed cells shrink in length with an additional lateral size reduction as the magnitude of the shock increases. Quickly plasmolyzing cells shrink largely at the poles, whereas gradually plasmolyzing cells invaginate along the cell cylinder. Our results give a comprehensive picture of the initial response of E. coli to hyperosmotic shock and offer explanations for seemingly opposing results that have been reported previously.     

Salt-induced Contraction of Bacterial Cell Walls

Intact Bacillus megaterium cells were found to contract as much as 26% in terms of dextran-impermeable volume when transferred from water to unbuffered, non-plasmolyzing NaCl solutions. This shrinkage appeared to be primarily due to electrostatic wall contraction rather than to any osmotic response of the cells. A variety of salts (but not sucrose) added to water suspensions of isolated cell walls caused protons to be released from the walls with resultant lowering of suspension pH and contraction of the structures. In effect, B. megaterium walls behaved as flexible, amphoteric polyelectrolytes, and their compactness in aqueous suspensions was affected by changes in environmental ionic strength and pH. Isolated walls were most compact in low ionic strength media with a pH of about 4, a value close to the apparent isoelectric pH of wall peptidoglycan. Electrostatic attractions appeared to play a major role in determining the compactness of highly contracted walls, and the walls responded to increased environmental ionic strength by expanding. In contrast, electrostatic repulsions were dominant in highly expanded walls, and increased environmental ionic strength induced wall contraction. Walls of whole bacteria also shrank when the cells were plasmolyzed. This second type of contraction seemed to result from relief of wall tension during plasmolysis, and it could be induced with nonionic solutes. Thus, cell wall tone in B. megaterium appeared to be set both by mechanical tension and by electrostatic interactions among wall ions.