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.     

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.     

The Selective Uptake of Alkali Cations by Red Beet Root Tissue

The selective uptake of alkali cations by red beet root tissuefrom solutions of chlorides has been investigated. It is shownthat when disks are transferred from distilled water to a solutionof salts, there is a rapid initial uptake of cations which isneither particularly selective, nor directly related to metabolism.On the other hand, the prolonged active accumulation of cationsexhibits strong selectivity, Na being preferred to other ions. Evidence is presented to show that the alkali cations competewith one another for the same absorption mechanism. In thisrespect the material apparently differs from that investigatedby Epstein and Hagen, in which the operation of distinct mechanismsfor some of these ions was visualized. The validity of Epsteinand Hagen's conclusion is discussed in relation to the resultspresented here.     

The conduction of sap

Summary Experiments are described in which the uptake of water by leaves exposed to severe water stress (80–95% R. W. C.) was found to deviate markedly from those of leaves with smaller water deficits. In its most extreme form the deviation appears as a curious increase in rate of water absorption some time after uptake has commenced. It could not be detected during the absorption of water by leaf discs suffering comparable severe water deficits.It is suggested that the differences are caused by cavitation in the xylem conducting channels during wilting. On restoring a water supply the reverse process takes place causing unusual patterns of water uptake.Since restoration takes place at 1° C it appears that metabolism is not involved; the process is physical in character.     

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.     

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.     

The osmotic property and fluorescent tracer movement of developing orchid embryos of Phaius tankervilliae (Aiton) Bl

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.     

The Exchangeability of Potassium and Bromide Ions in Cells of Red Beetroot Tissue

The exchangeability of potassium and bromide ions accumulatedby cells of red beetroot tissue has been examined under variousexperimental conditions by means of radioactive tracers. It is established that the cells contain a certain amount ofeasily exchanged ions which is largely independent of the saltcontent of the tissue. The remainder of the salt does not exchangeappreciably within 24 hours either during absorption or whenaccumulation is stopped by low temperature, potassium cyanide,high internal salt content, or by an insufficient preliminarywashing of the material. An hypothesis is proposed that the easily exchanged ions aredistributed throughout the intercellular spaces, cell walls,and in parts of the protoplasm, whilst those which do not readilyexchange may be situated in the cell vacuoles, or else stronglyassociated with protoplasmic constituents. The results suggestthat a considerable barrier to the free diffusion and exchangeof ions is located in the region of the tonoplast of a plantcell, and the metabolic transport of ions across this membraneis discussed. An examination of the changes which occur in the amounts ofreadily exchanged potassium during the washing of freshly cutbeet disks in aerated distilled water indicates that the protoplasmprobably acquires a capacity to fix ions more strongly as aresult of this treatment. This supports the view that an effectof washing on the capacity of cells to absorb salts metabolicallyis to increase the number of ‘absorption centres’involved.     

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.     

The Role of the Epidermal Cells in the Stomatal Movements

The water deficit of the leaves, the osmotic values of the stomatal cells and epidermal cells at incipiment plasmolysis, as well as the width of the stomatal apparatus and pore opening, were measured every hour from 6-17 o'clock under natural environmental conditions. During the noon hours, the intensity of light in clear weather ranged from 40,000-55,000 lux in the open position, and from 15,000-20,000 lux in the shade. The temperature was usually 15–20°C. The experimental object was Vicia Faba growing in a field, both plants freely rooted and plants in pots buried in the soil. The experiments resulted in the following observations and conclusions: 1. When leaves are exposed to strong light, the osmotic value at incipient plasmolysis changes not only in the guard cells, but also in the epidermal cells. If the epidermal cells' osmotic value rises, water is sucked from the guard cells and their uptake of water by suction is decreased, which promotes closure and counteracts opening, respectively. If the value falls, the effect is the reverse. The guard cells react passively to these epidermal changes. The passive stomatal movement eliciteed in this way has therefore been denoted as “osmopassive”, in contrast to the long known passive movement caused by a change in turgor of the epidermal cells, and which has therefore been denoted as “turgorpasslve”. The osmopassive component of stomatal closure has an earlier and more rapid onset than the hydroactive closing reaction, which consists of a decrease in the guard cells' osmotic value. Stomatat closure often starts with the osmopassive rapid process, and is completed and stabilized by the hydroactive process. It has not been possible to determine whether the osmopassive closing reaction is identical with the rapid reaction previously described, and interpreted as of adenoid nature, and tlius belonging to the active group. 2. The osmotic potential of the guard cells - i.e., the difference between the osmotic value of guard cells and epidermal cells at incipient plasmolysis - is, therefore, formed not only by a cbange in the osmotic value of the former cells, but also by a cbange in that of the latter. 3. Although the pore width runs largely parallel to the osmotic value of the guard cells, there is greater agreement between pore width and osmotic potential. When the water deficit of the leaf exceeds a certain threshold value, potential and stomatal width start to decrease. Closure is completed when the fall in potential approaches the zero value. If the water deficit subsequently continues to increase, the potential becomes negative and the stomata remain closed. 4. The stomatal movements are regulated by physiological processes which form two kinds of equilibrium between increase and decrease of the osmotic potential of the guard cells, i.e. the osmopassive increase - osmopassive decrease and the photoactive increase - hydroactive decrease. These equilibria complement each other in rate and stability. The osmopassive processes start rapidly and as soon as the deficit cbanges; hydroactive closure and sometimes also photoactive opening, are, on the contrary, time-consuming. When the water deficit is suboptimal, turgorpassive opening and closing are superadded, but only in those cases in which the osmotic potential of the guard cetls is positive.     

NaCl-Induced Alterations in Both Cell Structure and Tissue-Specific Plasma Membrane H+ -ATPase Gene Expression

NaCl-induced plasma membrane H+-ATPase gene expression, which occurs in roots and fully expanded leaves of the halophyte Atriplex nummularia L. (X. Niu, M.L. Narasimhan, R.A. Salzman, R.A. Bressan, P.M. Hasegawa [1993] Plant Physiol 103: 713-718), has been differentially localized to specific tissues using in situ RNA hybridization techniques. Twenty-four-hour exposure of plants to 400 mM NaCl resulted in substantial accumulation of H+ pump message in the epidermis of the root tip and the endodermis of the root elongation/differentiation zone. In expanded leaves, NaCl induction of plasma membrane H+-ATPase message accumulation was localized to bundle-sheath cells. Ultrastructural analyses indicated that significant cytological adaptations in root cells included plasmolysis that is accompanied by plasma membrane invaginations, formation of Hechtian strands and vesiculation, and vacuolation. These results identify specific tissues that are involved in the regulation of Na+ and Cl- uptake into different organs of the halophyte A. nummularia and provide evidence of the intercellular and interorgan coordination that occurs in the mediation of NaCl adaptation.     

Functional identification of organic cation transporter 1 as an atenolol transporter sensitive to flavonoids

Atenolol, a β1-adrenergic receptor blocker, is administered orally and its intestinal absorption has recently been indicated to be mediated by carrier protein and reduced markedly by ingestion of some fruit juices, such as apple and orange juices. This could be postulated to be a problem arising from the interaction of some components of fruit juices with atenolol at a transporter involved in its intestinal uptake, but the responsible transporter and its interacting components have not been identified yet. In an attempt to examine that possibility, we could successfully find that human organic cation transporter 1 (OCT1/SLC22A1), which is suggested to be expressed at the brush border membrane of enterocytes, is highly capable of transporting atenolol. In this attempt, OCT1 was stably expressed in Madin-Darby canine kidney II cells and the specific uptake of atenolol by the transporter was found to be saturable, conforming to the Michaelis-Menten kinetics with the maximum transport rate (V_max) of 4.00 nmol/min/mg protein and the Michaelis constant (K_m) of 3.08 mM. Furthermore, the OCT1-specific uptake was found to be inhibited by various flavonoids, including those contained in fruit juices that have been suggested to interfere with intestinal atenolol absorption. Particularly, phloretin and quercetin, which are major components of apple juice, were potent in inhibiting OCT1-mediated atenolol transport with the inhibition constants of 38.0 and 48.0 µM, respectively. It is also notable that the inhibition by these flavonoids was of the noncompetitive type. These results indicate that OCT1 is an atenolol transporter that may be involved in intestinal atenolol uptake and sensitive to fruit juices, although its physiological and clinical relevance remains to be further examined.     

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.     

On the Nature of the Heat Stabilizing Effect of Inorganic Ions on Escherichia coli

The growth lag of Escherichia coli at 45°C was reduced by the addition of sodium, potassium, magnesium and calcium ions to the growth medium. A method to quantitatively determine the lag-reducing effect of these ions was developed. The results obtained showed that equivalent amounts of the ions produced the same reduction of the growth lag. According to the results of plasmolysis experiments cells of E. coli suspended in peptone broth were permeable to all four ions. The course of plasmolysis and subsequent deplasmolysis was registered as changes in the cells' ability to scatter light. The heat stability of catalase from E. coli was increased by addition of the four ions. This was observed in experiments with intact cells and with a crude cell-free preparation of catalase. The results of our experiments are most easily explained by assuming a stabilizing effect of the ions tested on the intracellular bacterial proteins.     

Electric Conductivity and Internal Osmolality of Intact Bacterial Cells

Intact cells of Streptococcus faecalis and Micrococcus lysodeikticus were found to have high-frequency electric conductivities of 0.90 and 0.68 mho/m, respectively. These measured values, which reflect movements of ions both within the cytoplasm and within the cell wall space, were only about one-third of those calculated on the basis of determinations of the amounts and types of small ions within the cells. Concentrated suspensions of bacteria with damaged membranes showed similarly large disparities between measured and predicted conductivities, whereas the conductivities of diluted suspensions were about equal to predicted values. Thus, the low mobilities of intracellular ions appeared to be interpretable in terms of the physicochemical behavior of electrolytes in concentrated mixtures of small ions and cell polymers. In contrast to the low measured values for conductivity of intact bacteria, values for intracellular osmolality measured by means of a quantitative plasmolysis technique were higher than expected. For example, the plasmolysis threshold for S. faecalis cells indicated an internal osmolality of about 1.0 osmol/kg, compared with a value of only 0.81 osmol/liter of cell water calculated from a knowledge of the cell content and the distribution of small solutes. In all, our results indicate that most of the small ions within vegetative bacterial cells are free to move in an electric field and that they contribute to cytoplasmic osmolality.     

Regulation of H Excretion : EFFECTS OF OSMOTIC SHOCK

Osmotic shock, a 15-minute plasmolysis followed by a 15-minute rehydration in the cold, is a nondestructive technique which inhibits fusicoccin-stimulated H⁺ excretion from oat mesophyll cells (Avena sativa L.). Osmotic shock also causes a loss of intracellular solutes and stimulates H⁺ uptake, but osmoregulation can still occur, and enhanced H⁺ uptake is observed only at low external pH. It is concluded that osmotic shock interferes directly with the excretion of H⁺ rather than affecting only H⁺ or counter ion uptake.     

Excision repair of ultraviolet-irradiated deoxyribonucleic acid in plasmolyzed cells of Escherichia coli.

A system of cells made permeable by treatment with high concentrations of surcrose (plasmolysis) has been exploited to study the excision repair of ultraviolet-irradiated deoxyribonucleic acid in Escherichia coli. It is demonstrated that adenosine 5'-triphosphate is required for incision breaks to be made in the bacterial chromosome as well as in covalently closed bacteriophage lambda deoxyribonucleic acid. After plasmolysis, uvrC mutant strains appear as defective in the incision step as the uvrA-mutated strains. This is in contrast to the situation in intact cells where uvrC mutants accumulate single-strand breaks during postirradiation incubation. These observations have led to the proposal of a model for excision repair, in which the ultraviolet-specific endonuclease, coded for by the uvrA and uvrB genes, exists in a complex with the uvrC gene product. The complex is responsible for the incision and possibly also the excision steps of repair. The dark-repair inhibitors acriflavine and caffeine are both shown to interfere with the action of the adenosine 5'-triphosphate-dependent enzyme.     

Acquisition of dietary copper: a role for anion transporters in intestinal apical copper uptake

Copper is an essential micronutrient in humans and is required for a wide range of physiological processes, including neurotransmitter biosynthesis, oxidative metabolism, protection against reactive oxygen species, and angiogenesis. The first step in the acquisition of dietary copper is absorption from the intestinal lumen. The major human high-affinity copper uptake protein, human copper transporter hCTR1, was recently shown to be at the basolateral or blood side of both intestinal and renal epithelial cell lines and thus does not play a direct role in this initial step. We sought to functionally identify the major transport pathways available for the absorption of dietary copper across the apical intestinal membrane using Caco2 cells, a well-established model for human enterocytes. The initial rate of apical copper uptake into confluent monolayers of Caco2 cells is greatly elevated if amino acids and serum proteins are removed from the growth media. Uptake from buffered saline solutions at neutral pH (but not at lower pH) is inhibited by either d- or l-histidine, unaltered by the removal of sodium ions, and inhibited by ～90% when chloride ions are replaced by gluconate or sulfate. Chloride-dependent copper uptake occurs with Cu(II) or Cu(I), although Cu(I) uptake is not inhibited by histidine, nor by silver ions. A well-characterized inhibitor of anion exchange systems, DIDS, inhibited apical copper uptake by 60-70%, while the addition of Mn(II) or Fe(II), competitive substrates for the divalent metal transporter DMT1, had no effect on copper uptake. We propose that anion exchangers play an unexpected role in copper absorption, utilizing copper-chloride complexes as pseudo-substrates. This pathway is also observed in mouse embryonic fibroblasts, human embryonic kidney cells, and Cos-7 cells. The special environment of low pH, low concentration of protein, and protonation of amino acids in the early intestinal lumen make this pathway especially important in dietary copper acquisition.     

pH effect on cellular uptake of Sn(IV) chlorine e6 dichloride trisodium salt by cancer cells in vitro

The effects of pH value and presence of serum in an incubation medium on photosensitizer drug cellular uptake in MCF7 cancer cells have been investigated. The results showed that the presence of serum in an incubation medium reduced the drug cellular uptake at all pH values. It has been found that decreasing on pH values of the incubation medium increased the cellular uptake of the drug, demonstrating selective uptake of the sensitizer. The HepG2 liver cancer cells exhibited more drug cellular uptake than CCD-18CO normal colon cells, which assessed the selectivity uptake of photosensitizer on cancerous cells. The concentration of photosensitizer measured in 10⁶ cells showed a good correlation to the incubation time. Fluorescence and absorption spectroscopy been have used to examine the cells.     

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.