Osmotic mass transfer in the yeast Saccharomyces cerevisiae.

This paper reviews the passive mechanisms involved in the response of a yeast to changes in medium concentration and osmotic pressure. The results presented here were collected in our laboratory during the last decade and are experimentally based on the measurement of cell volume variations in response to changes in the medium composition. In the presence of isoosmotic concentration gradients of solutes between intracellular and extracellular media, mass transfers were found to be governed by the diffusion rate of the solutes through the cell membrane and were achieved within a few seconds. In the presence of osmotic gradients, mass transfers mainly consisting in a water flow were found to be rate limited by the mixing systems used to generate a change in the medium osmotic pressure. The use of ultra-rapid mixing systems allowed us to show that yeast cells respond to osmotic upshifts within a few milliseconds and to determine a very high hydraulic permeability for yeast membrane (Lp>6.10(-11) m x sec)-1) x Pa(-1)). This value suggested that yeast membrane may contain facilitators for water transfers between intra and extracellular media, i.e. aquaporins. Cell volume variation in response to osmotic gradients was only observed for osmotic gradients that exceeded the cell turgor pressure and the maximum cell volume decrease, observed during an hyperosmotic stress, corresponded to 60% of the initial yeast volume. These results showed that yeast membrane is highly permeable to water and that an important fraction of the intracellular content was rapidly transferred between intracellular and extracellular media in order to restore water balance after hyperosmotic stresses. Mechanisms implied in cell death resulting from these stresses are then discussed.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: Mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

Salt tolerance in the halophyte Suaeda maritima L. Dum.

Osmotic potentials and individual epidermal cell turgor pressures were measured in the leaves of seedlings of Suaeda maritima growing over a range of salinities. Leaf osmotic potentials were lower (more negative) the higher the salt concentration of the solution and were lowest in the youngest leaves and stem apices, producing a gradient of osmotic potential towards the apex of the plant. Epidermal cell turgor pressures were of the order of 0.25 to 0.3 MPa in the youngest leaves measured, decreasing to under 0.05 MPa for the oldest leaves. This pattern of turgor pressure was largely unaffected by external salinity. Calculation of leaf water potential indicated that the gradient between young leaves and the external medium was not altered by salinity, but with older leaves, however, this gradient diminished from being the same as that for young leaves in the absence of NaCl, to under 30% of this value at 400 mM NaCl. These results are discussed in relation to the growth response of S. maritima.     

The possible influence of osmotic poration on cell membrane water permeability

It has been hypothesized that pores in the plasma membrane form under conditions of rapid water efflux, allowing extracellular ice to grow into the cytoplasm under conditions of rapid freezing. When cells with intracellular ice are thawed slowly, the transmembrane ice crystal expands through recrystallization causing the cell to lyse. One of the implications of this hypothesis is that osmotic pores will provide an alternative route for water movement under conditions of osmotically induced flow. We show that the plasma membrane water permeability of a fibroblast cell changes as a function of the osmotic pressure gradient that is used to drive water movement. It is further shown that cell volume is more important than the magnitude of water flux in causing this departure from a uniform water permeability. We suggest that these data provide evidence of a transient route for water movement across cell membranes.     

渗透压突然改变对大肠埃希菌水通道蛋白GlpF基因表达的影响

目的 探讨水-甘油通道蛋白(glycerol protein ficilitator,GlpF)的生理功能及对细菌生长繁殖的影响.方法 将大肠埃希菌接种于等渗透压的液体培养基(1 IM)培养,18h后,突然改变培养液的渗透压,在30、60、120 min时间点检测细菌的A600nm值,RT-PCR分析其GlpF的表达.结果 突然改变渗透压后,大肠埃希菌数量均有不同程度的减少.在1/2 IM组、1/4 IM组细菌的A600nm值与其各自对照组相比,变化并不明显,但其细菌GlpF表达量明显降低.在高渗组,2 IM组的A600nm值与等渗组相比变化不大,而其GlpF表达量明显高于等渗组.结论 在环境渗透压突然改变时,细菌可以通过调控GlpF的表达来实现对细菌细胞内外水份的调节,以维持胞内环境稳定.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

Intracellular ethanol accumulation in Saccharomyces cerevisiae during fermentation.

An intracellular accumulation of ethanol in Saccharomyces cerevisiae was observed during the early stages of fermentation (3 h). However, after 12 h of fermentation, the intracellular and extracellular ethanol concentrations were similar. Increasing the osmotic pressure of the medium caused an increase in the ratio of intracellular to extracellular ethanol concentrations at 3 h of fermentation. As in the previous case, the intracellular and extracellular ethanol concentrations were similar after 12 h of fermentation. Increasing the osmotic pressure also caused a decrease in yeast cell growth and fermentation activities. However, nutrient supplementation of the medium increased the extent of growth and fermentation, resulting in complete glucose utilization, even though intracellular ethanol concentrations were unaltered. These results suggest that nutrient limitation is a major factor responsible for the decreased growth and fermentation activities observed in yeast cells at higher osmotic pressures.     

渗压震扰法释放Aeromonas sp.2016菌株外周间质中几丁质酶的研究

气单胞菌Aeromonassp．2016菌株能产生多种几丁质酶,其中的胞外酶C可能聚集于细胞外周胞质。为了避免破碎菌体而产生过多的杂蛋白,探索了用渗压震扰法(osmoticshock)来释放这部分酶。主要步骤是:先将菌体悬浮在20％蔗糖-0．03mol／LTris-HCI(pH8．0)高渗透压的溶液中,再快速转移到纯水低渗透压溶液中,产生瞬间渗压震荡,释放细胞外周胞质中的酶。结果表明,通过渗压震扰法释放出的酶纯度最高,比活力达到142．79U／g,比培养液上清液的54．46U／g和菌体破碎样品的14．66U／g分别高1．6倍和8．7倍,可用于纯化目的蛋白。     

Vesicle formation in the membrane of onion cells (Allium cepa) during rapid osmotic dehydration

Background and Aims

Optimization of osmotic dehydration in different plant cells has been investigated through the variation of parameters such as the nature of the sugar used, the concentration of osmotic solutions and the processing time. In micro-organisms such as the yeast, Saccharomyces cerevisiae, the exposure of a cell to a slow increase in osmotic pressure preserves cell viability after rehydration, while sudden dehydration involves a lower rate of cell viability, which could be due to membrane vesiculation. The aim of this work is to study cytoplasmic vesicle formation in onion epidermal cells (Allium cepa) as a function of the kinetics of osmotic pressure variation in the external medium.

Methods

Onion epidermal cells were submitted either to an osmotic shock or to a progressive osmotic shift from an osmotic pressure of 2 to 24 MPa to induce plasmolysis. After 30 min in the treatment solution, deplasmolysis was carried out. Cells were observed by microscopy during the whole cycle of dehydration–rehydration.

Key Results

The application of an osmotic shock to onion cells, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for <1 s, led to the formation of numerous exocytotic and osmocytic vesicles visualized through light and confocal microscopy. In contrast, after application of a progressive osmotic shift, from an initial osmotic pressure of 2 MPa to a final one of 24 MPa for 30 min, no vesicles were observed. Additionally, the absence of Hechtian strand connections led to the bursting of vesicles in the case of the osmotic shock.

Conclusions

It is concluded that the kinetics of osmotic dehydration strongly influence vesicle formation in onion cells, and that Hechtian strand connections between protoplasts and exocytotic vesicles are a prerequisite for successful deplasmolysis. These results suggest that a decrease in the area-to-volume ratio of a cell could cause cell death following an osmotic shock.     

Effect of osmotic pressure,Na+/K+ ratio and medium concentration on the enzyme activity and growth of L cells in suspension culture

Summary The effect of changes in osmotic pressure and in the Na⁺/K⁺ ratio on the doubling time, maximum cell population, enzyme activity, and isoenzyme distribution pattern in suspension cultures of L cells was determined. The growth of viable cells is relatively flat over a rather wide range of osmotic pressures (220 to 440 mOsm per kg). The presence of extra salt or sucrose beyond that needed to reach the minimum osmotic pressure (220) is detrimental to cell growth as reflected by a delay in the onset of logarithmic growth, a slower growth rate, a decreased maximum population, and accelerated death phase. Excessive K⁺ ion is toxic, but the level at which it is toxic interacts with osmotic pressure of the medium. Enzyme activity and isoenzyme distribution patterns for those enzymes studied did not vary as a function of osmotic pressure, ionic ratios, or medium concentration.     

Determination of free space, growth, solute concentrations and parameters of water relations of suspension-cultured tobacco cells

Abstract Methods were developed for measuring water content of the free space of suspension-cultured tobacco cells using ³H- or ¹⁴C-sorbitol. Sorbitol was not taken up by cells in significant quantities over the 3 min taken to label free space. Free space accounted for 50–60% of the water content of cell pellets irrespective of whether ³H- or ¹⁴-C-sorbitol was used. ¹⁴C-inulin labelled 13.5% less of the water in cell pellets than ³H-sorbitol, probably due to inadequate penetration by inulin into the free space in the cell wall matrix and within clumps of cells. Measurement of free space is necessary for measuring growth on a fresh or dry weight basis, solute concentrations and parameters of water relations of cells. Techniques for making these measurements on tobacco cells were also developed in this study. Solutes were measured after extraction from cells by expressing sap or by boiling cells in ethanol. Similar solute concentrations were found using both methods of extraction. By expressing sap from cells grown in culture medium with an osmotic pressure of 0.24 MPa, the cells were found to have an internal osmotic pressure of 0.70 MPa. Glucose, fructose, sucrose, amino acids and K⁺ accounted for 60% of this osmotic pressure. Elastic moduli were estimated using the Boyle-Van't Hoff relationship after suspending cells in solutions with different osmotic pressures and assessing their water content or internal osmotic pressure. For two different lines of tobacco cells, elastic modulus varied between 1 MPa and 5.4 MPa at turgor pressures of 0.15–0.52 MPa (line 1) and between 0.2 MPa and 4.2 MPa at turgor pressures of 0.04–0.26 MPa (line 2).     

Permeability properties of erythrocyte ghosts

1. Erythrocyte ghosts from human blood were produced by gentle water hemolysis. The ghost-containing hemolysate (about 20 mN) was added to media of different composition (KCl, NaCl, glucose, sucrose, etc.) and varying concentration ranging from 8 to 840 mN. The volume changes of the ghost cells were followed by a light absorption method. The potassium and sodium concentrations were also analyzed in some representative cases. 2. The ghosts shrank, or swelled, in two stages. An initial phase with a momentary expulsion, or uptake, of water leading to an osmotic equilibrium, was followed by a second phase in which a slow swelling or shrinking proceeded toward a final constant volume. 3. The ghosts were semipermeable in the sense that water always passed rapidly in either direction so as to maintain isotonicity with the external medium. The relation between ghost cell volumes (V) and the total concentration (C(e)) of the suspension medium can be expressed by a modified van't Hoff-Mariotte law: (C(e) + a)(V - b) = constant. Here a is a term correcting for an internal pressure and b is the non-solvent volume of the ghost cells. This means that the ghosts behave as perfect osmometers. 4. On the other hand appreciable concentration differences of the K and Na ions could be maintained across the intact ghost cell membranes for long periods. Whether this phenomenon is due simply to very low cation permeability or to active transport processes cannot be decided, although the first assumption appears more probable. 5. When the ghosts were treated with small concentrations of a lytic substance like Na oleate, the alkali ion transfer was greatly increased. This seems to be a simple exchange diffusion process with simultaneous, continued maintenance of osmotic equilibrium (= the second phase). A simplified theory is also given for the kinetics of the volume variations and ion exchange during the second phase (cf. the Appendix). 6. Miscellaneous observations on the effects of pH, and of some other substances are discussed. Some shape transformations of the ghost cells are also described.     

An artificial osmotic cell: a model system for simulating osmotic processes and for studying phenomena of negative pressure in plants

Abstract An artificial osmotic cell has been constructed using reverse osmosis membranes. The cell consisted of a thin film of an osmotic solution (thickness: 100 to 200 μm) containing a non-permeating solute and was bounded between the membrane and the front plate of a pressure transducer which continuously recorded cell turgor. The membrane was supported by metal grids to withstand positive and negative pressures (P). At maximum, negative pressures of up to –0.7 MPa (absolute) could be created within the film on short-term and pressures of up to –0.3 MPa could be maintained without cavitation for several hours. As with living plant cells, the application of osmotic solutions of a non-permeating solute resulted in monophasic relaxations of turgor pressure from which the hydraulic conductivity of the membrane (Lp) and the elastic modulus of the cell (?) could be estimated. The application of solutions with permeating solutes resulted in biphasic pressure relaxation curves (as for living cells) from which the permeability (P_s) and reflection (σ_s) coefficients could be evaluated for the given membrane. Lp, P_s, and σ_s were independent of P and did not change upon transition from the positive to the negative range of pressure. It is concluded that the artificial cell could be used to simulate certain transport properties of living cells and to study phenomena of negative pressure as they occur in the xylem and, perhaps, also in living cells of higher plants.     

Sequence of occurring damages in yeast plasma membrane during dehydration and rehydration: mechanisms of cell death

Yeasts are often exposed to variations in osmotic pressure in their natural environments or in their substrates when used in fermentation industries. Such changes may lead to cell death or activity loss. Although the involvement of the plasma membrane is strongly suspected, the mechanism remains unclear. Here, the integrity and functionality of the yeast plasma membrane at different levels of dehydration and rehydration during an osmotic treatment were assessed using various fluorescent dyes. Flow cytometry and confocal microscopy of cells stained with oxonol, propidium iodide, and lucifer yellow were used to study changes in membrane polarization, permeabilization, and endocytosis, respectively. Cell volume contraction, reversible depolarization, permeabilization, and endovesicle formation were successively observed with increasing levels of osmotic pressure during dehydration. The maximum survival rate was also detected at a specific rehydration level, of 20 MPa, above which cells were strongly permeabilized. Thus, we show that the two steps of an osmotic treatment, dehydration and rehydration, are both involved in the induction of cell death. Permeabilization of the plasma membranes is the critical event related to cell death. It may result from lipidic phase transitions in the membrane and from variations in the area-to-volume ratio during the osmotic treatment.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Water activity: a fundamental parameter of aroma production by microorganisms

Summary The water activity of the medium, which is analogous to osmotic pressure in liquid medium, is a fundamental parameter for the mass transfer of water and solutes across the cell membrane. The control of this parameter could be used to modify the metabolic production or excretion of a microorganism, as demonstrated in this work for aroma production by a fungus and a yeast. Offprint requests to: P. Gervais     

Osmotic pressure effects and intracellular accumulation of ethanol in yeast during fermentation

Summary The intracellular accumulation of ethanol in yeast and its potential effects on growth and fermentation have been topics of controversy for the past several years. The determination of intracellular ethanol based on the exclusion of [¹⁴C]sorbitol to estimate aqueous cell volume was used to examine the question of intracellular ethanol accumulation. An intracellular accumulation of ethanol inSaccharomyces cerevisiae was observed during the early stages of fermentation. However, as fermentation continued, the intracellular and extracellular concentrations of ethanol became similar. Increasing the osmotic pressure of the medium with glucose or sorbitol was observed to cause an increase in the intracellular ethanol concentration. Associated with this was a decrease in yeast growth and fermentation rates. In addition, increasing the osmotic pressure of the medium was observed to cause an increase in glycerol production. Supplementation of the media with excess peptone, yeast extract, magnesium sulfate and potassium phosphate was found to relieve the detrimental effects of high osmotic pressure. Under these conditions, though, no effect on the intracellular and extracellular ethanol distribution was observed. These results indicate that nutrient limitation, and not necessarily intracellular ethanol accumulation, plays a key role during yeast fermentations in media of high osmolarity.     

Dynamics of stomatal water relations following leaf excision

We examined the stomatal response to leaf excision in an evergreen woody shrub, Photinia x fraseri, using a novel combination of gas exchange, traditional water relations and modelling. Plants were kept outdoors in mild winter conditions (average daily temperature range: -1 to 12 degrees C) before being transferred to a glasshouse (temperature range: 20-30 degrees C) and allowed to acclimate for different periods before experiments. 'Glasshouse plants' were acclimated for at least 9 d, and 'outdoor plants' were acclimated for fewer than 3 d before laboratory gas exchange experiments. The transient stomatal opening response to leaf excision was roughly twice as long in outdoor plants as in glasshouse plants. To elucidate the reason for this difference, we inferred variables of stomatal water relations (epidermal and guard cell turgor pressures and guard cell osmotic pressure: Pe, Pg and pi g, respectively) from stomatal conductance (gs) and bulk leaf water potential (psi l), using a hydromechanical model of gs. psi l was calculated from cumulative post-excision transpirational water loss using empirical relationships between psi l and relative water content obtained on similar leaves. Inferred Pg and Pe both declined immediately after leaf excision. Inferred pi g also declined after a lag period. The kinetics of pi g adjustment after the lag were similar in outdoors and glasshouse plants, but the lag period was much longer in outdoor plants. This suggests that the longer transient opening response in outdoor plants resulted from slower induction, not slower execution, of guard cell osmoregulation. We discuss the implications of our results for the mechanism of short-term stomatal responses to hydraulic perturbations, for dynamic modelling of gs and for leaf water status regulation.     

THE EFFECT OF SALT CONCENTRATION OF THE MEDIUM ON THE RATE OF OSMOSIS OF WATER THROUGH THE MEMBRANE OF LIVING CELLS

1. Using the unfertilized egg of the sea urchin, Arbacia, as osmometer, it was found that the rate with which water enters or leaves the cell depends on the osmotic pressure of the medium: the velocity constant of the diffusion process is higher when the cell is in concentrated sea water, and lower when the sea water medium is diluted with distilled water. Differences of more than tenfold in the value of the velocity constant were obtained in this way. When velocity constants are plotted against concentration of medium, a sigmoid curve is obtained. 2. These results are believed to indicate that cells are more permeable to water when the osmotic pressure of the medium is high than when it is low. This relation would be accounted for if water should diffuse through pores in a partially hydrated gel, constituting the cell membrane. In a medium of high osmotic pressure, the gel is conceived to give up water, to shrink, and therefore to allow widening of its pores with more ready diffusion of water through them. Conversely, in solutions of lower osmotic pressure, the gel would take up water and its pores become narrow.     

Early changes of water diffusional transfer in maize roots under the influence of water stress

The time dependent response of the hydrodynamic root system to PEG-induced water stress was studied in intact maize Zea mays L. seedlings at intervals varying from several seconds to 3 h by detecting diffusional water transfer with the use of pulsed NMR. In order to establish the contribution of water transfer through aquaporins in response to water stress, the transmembrane water transport in control roots and roots treated with aquaporin blocker was detected. Changes in diffusional water transfer under stress were shown to depend on the duration of osmotic treatment, and include the series of heterogeneous processes. A transient pulsed jump in diffusional water transfer detected several seconds after beginning the osmotic treatment is associated with the spread of the wave of hydraulic pressure along the root. It is proposed that early responses of the hydrodynamic system of maize roots to PEG-induced water stress lies in the unequal change in water permeability of the plasmalemma and tonoplast resulting from the changes in aquaporin activity and perhaps in the escalation of water transfer along the cell vacuome.