Behaviour of plasma membrane, cortical ER and plasmodesmata during plasmolysis of onion epidermal cells

During plasmolysis of onion epidermal cells, the contracting protoplast remains connected to the cell wall by an intricate, branched system of plasma membrane (PM) ‘Hechtian strands’ which stain strongly with the fluorescent probe DiOC₆. In addition, extensive regions of the cortical endoplasmic reticulum (ER) network remain anchored to the cell wall during plasmolysis and do not become incorporated into the contracting protoplast with the other cell organelles. These ER profiles become tightly encased by the PM as the latter contracts towards the centre of the cell. Thus, although the cortical ER is left outside the main protoplast body, it is nonetheless still bound by the PM of the cell. As well as being anchored to the wall, the cortical ER remains intimately linked with plasmodesmata and retains continuity between cells via the central desmotubules which become distended during plasmolysis. The PM also remains in close contact with the plasmodesmatal pore following plasmolysis. It is suggested that plasmodesmata, although sealed, may not be broken during plasmolysis, their substructure being preserved by continuity of both ER and PM through the plasmodesmatal pore. A structural model is presented which links the behaviour of PM, ER and plasmodesmata during plasmolysis.     

Characterization of theEgeria densa leaf symplast: Response to plasmolysis,deplasmolysis and to aromatic amino acids

Summary The fluorescent dyes 6-carboxyfluorescein and fluorescein glutamylglutamic acid, which move freely in theEgeria densa leaf symplast, fail to move from cells subjected to plasmolysis, demonstrating that plasmolysis disrupts symplastic continuity. Dye movements begins again within 10 minutes of removal of the osmoticum and becomes more extensive with increasing recovery time. The re-established symplastic links show a number of distinctive features compared to untreated leaves: dyes of up to 1678 dalton can pass, compared to the normal limit of 665 dalton; and Ca²⁺ ions, which completely inhibit dye movement in untreated cells, only reduce the extent of dye movement. Aromatic amino acids and their fluorescein conjugates prevent intercellular movement in untreated cells. In deplasmolysed cells the aromatic conjugates move freely. The increased symplast permeability persists for at least 20 hours. Thus, after plasmolysis followed by deplasmolysis, the symplast shows a marked increase in permeability associated with an increased molecular exclusion limit, indicating an increase in pore size, and symplast permeability becomes relatively insensitive to Ca²⁺ ions or to the aromatic conjugates.     

Effects of Antibiotics on Induction, Viability, and Reversion Potential of L-Forms of Haemophilus influenzae

The physical interactions between Serratia marcescens and solutions of NaCl, CaCl(2), CaI(2), NaI, and Na(2)HPO(4) plus NaH(2)PO(4) were examined. Dilute (0.017 n) salt solutions did not cause cells to lose water, as evidenced by the unchanged weight of centrifugally packed cells. The cells preferentially adsorbed the cations and repelled the anions of most salts in these solutions. Concentrated (1.71 n) salt solutions markedly reduced the weight and water content of centrifugally packed cells, although these cells took up considerable amounts of salts. More than 90% of the water in the packed-cell pellets was available for the solution of NaCl at 4.2 to 4.4% concentration. The observation that salts apparently penetrated the cells freely and yet caused extensive dehydration was not readily compatible with conventional concepts of solute-induced plasmolysis. Alternative hypotheses to explain the data included the following. First, the cells lost weight and water to concentrated salt solutions through a nonosmotic competitive dehydration, causing a shrinkage of the protoplasmic gel. The shrinkage of the cell wall was limited because of the rigidity of its mucopeptide layer; therefore, a space appeared between the cell wall and the cell membrane. Second, cells may have equilibrated their water activity with that of their environment by two mechanisms: (i) the loss of water by plasmolysis or competitive dehydration, and (ii) alterations in cell permeability that admitted previously excluded solutes to the cell interior. Possibly, the correct explanation of the observations reported here involves elements of all three hypotheses, plasmolysis, competitive dehydration, and permeability alterations.     

Hyperosmotic stress-induced actin filament reorganization in leaf cells of Chlorophyton comosum

Actin filament (AF) organization was studied during the plasmolytic cycle in leaf cells of Chlorophyton comosum Thunb. In most cells the hyperosmotic treatment induced convex or concave plasmolysis and intense reorganization of the AF cytoskeleton. Thin cortical AFs disappeared and numerous cortical, subcortical and endoplasmic AFs arranged in thick and well-organized bundles were formed. Plasmolysed cells displayed a significant increase in the overall AF content compared with the control cells. Cortical AF bundles were preferentially localized in the shrunken protoplast areas, lining the detached plasmalemma regions. The endoplasmic AF bundles were mainly found in the perinuclear cytoplasm and on the tonoplast surface. AFs also traversed some of the Hechtian strands. AF disorganization after cytochalasin B (CB) treatment induced dramatic changes in the pattern of plasmolysis, which lasted for a longer time and led to a greater decrease of the protoplast volume compared to the untreated cells. In many of the above cells the protoplasts assumed an 'amoeboid' form and were often subdivided into sub-protoplasts. Soon after the removal of the plasmolytic solution both CB-treated and untreated cells were deplasmolysed, while the AF cytoskeleton gradually reassumed the organization observed in the control cells. The findings of this study revealed for the first time in angiosperm cells that plasmolysis triggers an extensive reorganization of the AF cytoskeleton, which is involved in the regulation of protoplast shape and volume. The probable mechanism(s) leading to AF reorganization as well as the function(s) of the atypical AF arrays in plasmolysed cells are discussed.     

Staphylococcus aureus giant protein Ebh is involved in tolerance to transient hyperosmotic pressure

Staphylococcus aureus is well known to colonize on human skin where the physiological condition is characterized by hypervariable water activity, i.e., repeated dehydration or rehydration. To determine the facilitating factors for the colonization under hypervariable water activity, we studied the giant protein Ebh (extracellular matrix (ECM)-binding protein homologue). The ebh mutant RAM8 showed invaginated vacuoles along the septum, similar to that found in partial plasmolysis, and the cells burst under osmotic upshift. RAM8 was also relatively susceptible to abrupt hyperosmotic upshift, teicoplanin, and Triton X-100. By using the green fluorescent protein (GFP) as a reporter, Ebh was localized over the entire cell surface. This suggests that Ebh might contribute to structural homeostasis by forming a bridge between the cell-wall and cytoplasmic membrane to avoid plasmolysis under hyperosmotic condition.     

Abnormal Stomatal Behaviour and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: EFFECT OF ABSCISIC ACID AND AUXIN ON STOMATAL BEHAVIOUR AND PEROXIDASE ACTIVITY

The wilty tomato mutant flacca and the normal variety RheinlandsRuhm were compared in terms of: (1) potassium transport intoand out of the guard cells, (2) cell wall properties which includeprotein, hydroxyproline and peroxidase activity, and (3) activityof indol-3yl-acetic acid oxidase. Also studied were the effectsof auxin on stomatal behaviour and peroxidase activity whenapplied to normal plants during development, and the short-termeffect of abscisic acid on the resistance of flacca stomatato closure under plasmolysis. Potassium transport, wall protein and hydroxyproline all seemedto be equal in mutant and normal plants. Peroxidase activitywas higher in the soluble and wall fractions of the mutant,and decreased toward normal in the mutant treated with abscisicacid. More stomata were open and peroxidase activity was higherin normal plants treated with auxin during development. Thepercentage of open stomata under plasmolysis was lower and theiraperture size was smaller in the epidermal strips taken fromabscisic-acid-treated mutant plants than from control mutantplants.     

Compartmentalization of the periplasmic space at division sites in gram-negative bacteria.

Phase-contrast and serial-section electron microscopy were used to study the patterns of localized plasmolysis that occur when cells of Salmonella typhimurium and Escherichia coli are exposed to hypertonic solutions of sucrose. In dividing cells the nascent septum was flanked by localized regions of periseptal plasmolysis. In randomly growing populations, plasmolysis bays that were not associated with septal ingrowth were clustered at the midpoint of the cell and at 1/4 and 3/4 cell lengths. The localized regions of plasmolysis were limited by continuous zones of adhesion that resembled the periseptal annular adhesion zones described previously in lkyD mutants of S. typhimurium (T. J. MacAlister, B. MacDonald, and L. I. Rothfield, Proc. Natl. Acad. Sci. USA 80:1372-1376, 1983). When cell division was blocked by growing divC(Ts) cells at elevated temperatures, the localized regions of plasmolysis were clustered along the aseptate filaments at positions that corresponded to sites where septum formation occurred when cell division was permitted to resume by a shift back to the permissive temperature. Taken together the results are consistent with a model in which extended zones of adhesion define localized compartments within the periplasmic space, predominantly located at future sites of cell division.     

Membrane Organization of the Desiccation-Tolerant Moss Tortula ruralis in Dehydrated States

Membrane organization of the desiccation tolerant moss Tortula ruralis was studied in several intensely dehydrated states (75% relative humidity [RH], 90% RH, plasmolysis in molar salt, freezing to −20°C) by ³¹P nuclear magnetic resonance and ultrastructural analyses. Both methods revealed that even at 75% RH (−400 bars), the moss cellular membranes retained extended phospholipid bilayers. Ultrastructural analyses of the fully hydrated moss showed an extensive proliferation of membrane vesicles in the endoplasmic reticulum. During dehydration, these vesicles form layers of membrane under the plasmalemma and in some cases appear to fuse with the surface membrane. This suggests that these vesicles may serve as a reservoir of membranes to accommodate for membrane surface area changes during desiccation and subsequent rehydration.     

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.     

Physiological and Morphological Changes Induced by Nutrient Limitation of Pseudomonas fluorescens 378 in Continuous Culture

Pseudomonas fluorescens 378 was studied in continuous culture at a dilution rate of 0.05 or 0.15 h⁻¹ and under a limitation of carbon/energy, nitrogen, phosphorus, iron(III), or oxygen. Cultures were examined for nutritional consumption, production of biosurfactant AP-6 and lipase, and electron microscopy morphology. Morphological features were lysis and plasmolysis of the cells, vacuoles in the cells, granules in cell nuclei, and DNA coagulation during transmission electron microscopy preparation. Biosurfactant and lipase production were lost after 8 to 15 retention times, but under iron limitation and at low dilution rate they were maintained for more than 30 retention times. Consumption of nutrients varied between different cultures. Between 2.4 and 6.0 g of succinic acid per g (dry weight) was consumed; the highest value was obtained under phosphorus limitation. The uptake of nitrogen was mostly about 0.16 g/g (dry weight), and that of phosphorus varied between 13 and 58 mg/g (dry weight). Phosphorus-limited cells reduced their phosphorus consumption by at least 50% compared with other limitations. Cell morphology varied among different cultures. Up to 25% cell lysis occurred at the higher dilution rate. The frequencies of plasmolysis varied between 0 and 85%. Granules in nuclei were found in 65 to 100% of the cells. Vacuoles appeared mostly in low numbers, but at the lower dilution rate under phosphorus or iron limitation the frequencies increased to between 25 and 85%. At high dilution rate, the DNA coagulated in 30 to 70% of the cells. Multivariate data analysis demonstrated a general difference between the two tested dilution rates; i.e., both nutritional and morphological features differed more between the two tested dilution rates than between the different limitations. Cultures at the lower dilution rate changed more with time; this was especially pronounced for phosphorus or iron limitation. The data analysis also showed a correlation between plasmolysis or vacuoles in the cells and an increased carbon uptake under phosphorus limitation.     

The outer membrane of Pseudomonas aeruginosa is a barrier against the penetration of disaccharides

The outer membrane of Pseudomonas aeruginosa acted as a barrier against the penetration of di- (Mr, 342), tri- (Mr, 504) and tetrasaccharides (Mr, 666), whereas the membrane allowed the penetration of pentose (Mr, 150) and methylhexoses (Mr, 194) into the periplasm. When the intact cells of P. aeruginosa were treated with 600 mosM saccharides of various sizes and observed under an electron microscope, saccharides of Mr larger than 342 caused the extensive shrinking of the outer membrane. Whereas the cells treated with the saccharides of Mr less than 194 or with sucrose in the presence of EDTA showed plasmolysis. Determination of the extent of saccharide penetration into the periplasm of the cells treated with 600 mosM sodium chloride or with 600 mosM saccharides of various sizes showed that only pentose and hexoses, so far examined, were penetrable but di-, tri- and tetrasaccharides were impenetrable.     

Actin filaments regulate the adhesion between the plasma membrane and the cell wall of tobacco guard cells

During the opening and closing of stomata, guard cells undergo rapid and reversible changes in their volume and shape, which affects the adhesion of the plasma membrane (PM) to the cell wall (CW). The dynamics of actin filaments in guard cells are involved in stomatal movement by regulating structural changes and intracellular signaling. However, it is unclear whether actin dynamics regulate the adhesion of the PM to the CW. In this study, we investigated the relationship between actin dynamics and PM–CW adhesion by the hyperosmotic-induced plasmolysis of tobacco guard cells. We found that actin filaments in guard cells were depolymerized during mannitol-induced plasmolysis. The inhibition of actin dynamics by treatment with latrunculin B or jasplakinolide and the disruption of the adhesion between the PM and the CW by treatment with RGDS peptide (Arg-Gly-Asp-Ser) enhanced guard cell plasmolysis. However, treatment with latrunculin B alleviated the RGDS peptide-induced plasmolysis and endocytosis. Our results reveal that the actin depolymerization is involved in the regulation of the PW–CW adhesion during hyperosmotic-induced plasmolysis in tobacco guard cells.     

Early stages in development of the Escherichia coli cell-division site

Development of the Escherichia coli cell division site was studied in wild-type cells and in non-septate filaments of ftsZ null and ftsZTs mutant cells. Localized regions of plasmolysis were used as markers for the positions of annular structures that are thought to be related to the periseptal annuli that flank the ingrowing septum during cytokinesis. The results show that these structures are localized at potential division sites in non-septate filaments of FtsZ^- cells, contrary to previous reports. The positions of the structures along the long axis of the cells in both wild-type cells and FtsZ^- filaments were unaffected by the presence of plasmolysis bays at the cell poles. These results do not agree with a previous suggestion that the apparent association of plasmolysis bays with future division sites was artefactual. They support the view that division sites begin to differentiate before the initiation of septal ingrowth and that plasmolysis bays and the annular attachments that define them, mark the locations of these early events in the division process.     

暖温带森林生态系统主要树种若干水分参数的季节变化

本文通过PV技术对暖温带落叶阔叶林主要树种辽东栎(Quercus liaotungensis)、棘桦(Betula dahurica)、五角枫(Acer mono)、大叶白蜡(Fraxinus rhychophylla)及其林下灌木六道木(Abelia biflora)、山地针叶林的主要树种油松(Pinus tabulaeformis)以及次生灌丛主要树种山杏(Prunus armeniaca var.ansu)、山桃(Prunus davidiana)、大果榆(Ulmus macrocarpa)、北京丁香(Syringa pekinensis)的日最低叶水势Ψmin、饱和含水量时的最大渗透势Ψs sat、初始质壁分离时渗透势Ψs tlp、初始质壁分离的渗透水的相对含量(ROWCtlp)、相对含水量(RWCtlp)、质外体水的相对含量(AWC)6种水分生理指标的季节变化进行了研究。结果表明：1)所研究的10种树种叶水势Ψw min的季节进程基本与土壤湿度的变化一致。乔木Ψw min季节均值由低到高的顺序为油松<辽东栎<大叶白蜡<棘皮桦<五角枫。次生灌丛4种树种Ψw min季节均值由低到高依次为：山杏<山桃<北京丁香<大果榆。在生长季的不同时期,同为灌木、生长于落叶阔叶林下的六道木Ψw min均高于灌丛样地4类树种的同期值,且季节变幅较小。2)各树种饱和含水量时的最大渗透势Ψs sat和初始质壁分离时渗透势Ψs tlp,表现出随季节进程而不断降低的趋势;Ψs tlp和Ψs sat的季节变化并未表现出与土壤水分状况变化的一致关系,而与树木自身生长发育的物候节律有关。从Ψs tlp和Ψs sat指标所反映的树木耐旱能力的季节变化来看,表现为随季节进程不断上升的趋势。一般在展叶和随后的生长旺盛期,上述树种的耐旱能力较弱。3)初始质壁分离的渗透水的相对含量(ROWCtlp)和相对含水量 (RWCtlp)的季节变化在各树种间呈现大体相同的趋势,5月下旬的生长高峰期,达到生长季中的最高值。而后随林木当年生叶的成熟和嫩枝的木质化,ROWCtlp和RWCtlp呈下降趋势。4)各树种质外体水的相对含量(AWC)表现为随季节进程而上升的趋势。     

Shrinkage of growing Escherichia coli cells by osmotic challenge.

The immediate response of growing Escherichia coli to changing external osmotic pressure was studied with stopped-flow turbidimetric measurements with a narrow-beam spectrophotometer. It is shown theoretically that in such a photometer rod-shaped bacteria have an apparent absorbance which is proportional to the inverse of the surface area. The apparent optical density, corrected for effects of alteration of the index of refraction of the medium, increased continuously as the external osmotic pressure was raised. Because of the short time scale of the measurements, the turbidity increases could result either from shrinkage of the cells or from plasmolysis, or both, but not from growth or metabolic adaptation. With low concentrations of pentose such that the external osmotic pressure was not greater than that inside the cells, plasmolysis would not occur and, consequently, only shrinkage of the previously stretched sacculus remains to account for the observed optical effects. Taking the osmotic pressure of the growing cells as 5 atmospheres (506 kPa), the turbidity changes correspond to the murein fabric having been stretched 20% beyond its unstressed equilibrium area during growth under the conditions used.     

Membrane deletion during plasmolysis in hardened and non-hardened plant cells

Abstract Video recordings of interference phase contrast microscopy were used to study plasmalemma deletion during plasmolysis in hardened and non-hardened suspension cultured cells of Brassica napus, alfalfa, and cells isolated from rye seedlings. Although different hardening regimes and different cells were used, the responses to plasmolysis were consistent. Hardened cells uncoupled the volume to surface area ratio during plasmolysis both by forming a large number of strands between the cell wall and protoplast and by leaving rivulet-like networks of membranes on the cell wall surface. Tonoplast membrane was deleted as sac-like intrusions into the vacuole. Non-hardened cells produced few strands during plasmolysis. They also deleted plasmalemma and tonoplast into the vacuole as endocytotic vesicles. During deplasmolysis of hardened cells both the individual membrane strands and the rivulets of membrane material vesiculated into strings of vesicles. The vesicles were osmotically active and were re-incorporated into the expanding protoplast. Conversely, deplasmolysis in non-hardened cells resulted in few osmotically active vesicles and many broken strands. The vacuolar sac-like intrusions in hardened cells were re-incorporated into the vacuole whereas the endocytotic vesicles in non-hardened cells were not re-incorporated. Therefore, the non-hardened cells underwent expansion-induced lysis.     

The Absorption of Potassium Ions by Plasmolysed Cells

The absorption of potassium ions by cells of red beetroot tissueplasmolysed in various media has been examined and comparedwith that of unplasmolysed cells under similar experimentalconditions. It is established that although the plasmolysing agents in themselvestend to promote the absorption of ions at the concentrationsemployed, the effect of plasmolysis is to inhibit the rate ofpotassium uptake. Evidence is provided that this is due to anincrease in the rate of leakage of ions from plasmolysed cells,and to a reduction of gross uptake. These results are discussed in terms of the structural and physiologicalchanges which are associated with plasmolysis. It is concludedthat alterations in the surface area, and thickness or densityof the protoplasts, modifications of the vacuolar concentrationof ions, and respiratory influences are all involved. Anotherfactor, the nature of which has not been elucidated, also appearsto be involved.     

Tissue Tensions during Cell Elongation in Wheat Roots and a Comparison with Contractile Roots

A study has been made of tissue tensions in growing Triticum roots. After the initial phase of elongation with both a lateral and an exponential longitudinal growth, the tissues are under strong longitudinal stress. Plasmolysis in situ or splitting of the root in the pericyclic region causes an increase in length of the perivascular part. Freed from the stele the perivascular part contracts on plasmolysis. During the following only longitudinal growth, the stress decreases under decreased root diameter. When elongation has ceased, the root in situ contracts on plasmolysis. It is suggested that during radial growth longitudinal stresses are built up, which are released when the stele catches up with the growth of the cortex. The similarity to conditions in contractile roots of Arum (Lamant and Heller 1967) is pointed out. A difference is found in the polarity of growth and thus in the mode of release of the stresses. The cause of the tensions and methodical difficulties are discussed.     

Effect of different sugars on stomatal behaviour inMerremia aegyptia (L.) Urban andM. dissecta Hallier F.

The effect of mannitol, glucose and sucrose on the stomatal behaviour of two desert species,Merremia aegyptia andM. dissecta has been studied. Stomatal opening did not uniformly depend on the decrease in turgor of the epidermal and subsidiary cells caused by the different osmotic potential of the sugars. Sucrose caused plasmolysis of the subsidiary cells only but this was not accompanied by the opening of the stomatal pore. InM. aegyptia, no plasmolysis was seen either in epidermal or subsidiary cells, even the stomata opened; inM. dissecta, on the other hand, plasmolysis occurred in these cells without any stomatal opening, after incubation in glucose or mannitol. Mannitol is least absorbed, glucose slightly more and sucrose is absorbed to a very large extent in the guard cells when the materials were inoubated in the respective sugar solutions. However, the absorption of these three sugars was almost always larger in isolated epidermal strips than in discs; in detached intact leaves it was still more reduced.     

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.