Compartment analysis of plant cells by means of turgor pressure relaxation: II. Experimental results onChara corallina

Summary The three-compartment model (paper I) described turgor pressure relaxations as a sum of two exponential functions. The predicted shape of the curves could be confirmed inChara corallina by improving the recording and processing of data. An evaluation on the basis of the three-compartment model provided values for the hydraulic conductivity of the plasmalemma ofLp _p=2×10^–5 to 4×10^–5 cm sec^–1 bar^–1 and ofLp _i=3×10^–5 to 1×10^–4 cm sec^–1 bar^–1 for the tonoplast (assuming the area to be 90% of the plasmalemma area). The mean proportion of the total volume occupied by the cytoplasm was calculated to be 9%. This value is consistent with the concept of a highly vacuolated cell. Other explanations for the biphasic relaxation curves are discussed.     

Separate Determination of the Electrical Properties of the Tonoplast and the Plasmalemma of the Giant-Celled Alga Valonia utricularis: Vacuolar Perfusion of Turgescent Cells with Nystatin and Other Agents

In the giant-celled marine algae Valonia utricularis the turgor-sensing mechanism of the plasmalemma and the role of the tonoplast in turgor regulation is unknown because of the lack of solid data about the individual electrical properties of the plasmalemma and the vacuolar membrane. For this reason, a vacuolar perfusion technique was developed that allowed controlled manipulation of the vacuolar sap under turgescent conditions (up to about 0.3 MPa). Charge-pulse relaxation studies on vacuolarly perfused cells at different turgor pressure values showed that the area-specific resistance of the total membrane barrier (tonoplast and plasmalemma) exhibited a similar dependence on turgor pressure as reported in the literature for nonperfused cells: the resistance assumed a minimum value at the physiological turgor pressure of about 0.1 MPa. The agreement of the data suggested that the perfusion process did not alter the transport properties of the membrane barrier. Addition of 16 μm of the H⁺-carrier FCCP (carbonylcyanide p-trifluoromethoxyphenyhydrazone) to the perfusion solution resulted in a drop of the total membrane potential from +4 mV to −22 mV and in an increase of the area-specific membrane resistance from 6.8 × 10⁻² to 40.6 × 10⁻²Ωm². The time constants of the two exponentials of the charge pulse relaxation spectrum increased significantly. These results are inconsistent with the assumption of a high-conductance state of the tonoplast (R. Lainson and C.P. Field, J. Membrane Biol. 29:81–94, 1976). Depending on the site of addition, the pore-forming antibiotics nystatin and amphotericin B affected either the time constant of the fast or of the slow relaxation (provided that the composition of the perfusion solution and the artificial sea water were replaced by a cytoplasma-analogous medium). When 50 μm of the antibiotics were added externally, the fast relaxation process disappeared. Contrastingly, the slow relaxation process disappeared upon vacuolar addition. The antibiotics cannot penetrate biomembranes rapidly, and therefore, the findings suggested that the fast and slow relaxations originated exclusively from the electrical properties of the plasmalemma and the tonoplast respectively. This interpretation implies that the area-specific resistance of the tonoplast is significantly larger than that of the plasmalemma (consistent with the FCCP data) and that the area-specific capacitance of the tonoplast is unusually high (6.21 × 10⁻² Fm⁻² compared to 0.77 × 10⁻² Fm⁻² of the plasmalemma). Thus, we have to assume that the vacuolar membrane of V. utricularis is highly folded (by a factor of about 9 in relation to the geometric area) and/or contains a fairly high concentration of mobile charges of an unknown electrogenic ion carrier system. Received: 22 October 1996/Revised: 16 January 1997     

Mobile charges in the cell membranes ofHalicystis parvula

Charge-pulse experiments were performed on cells of the giant marine algaHalicystis parvula. At normal pH (8.2), the voltage decay following a charge-pulse of 500 ns duration fed to the vacuole could be described by summing two exponential relaxations. The amplitudes and time constants of these relaxations were widely separated. The parameters of the two relaxation processes were found to be pH-dependent. Reduction of the external pH value from pH 8.2 to 5 resulted in a complete change of the two relaxation processes within a few minutes. Only one relaxation process could be observed at pH 5, within the time resolution of our instrumentation. The experimental data could not be explained by a two-membrane model with reasonable values for the specific capacitances of tonoplast and plasmalemma. The results of the charge-pulse relaxations were found to be consistent with the assumption that both membranes have very similar electrical properties and that both contain mobile charges with a total surface concentration of about 30 nmol·m^-2 and a translocation-rate constant of about 500·s^-1. The mobile charges became neutralized at pH 5 hhich led to a decrease of the apparent specific capacitance of the algal cells. They are presumably either part of a transport system for cations or connected with the chloride pump ofHalicystis parvula.Abbreviation RC (R)esistance·(C)apacitance     

Studies on the tonoplast action potential ofNitella flexilis

Summary The origins of the two peaks of the action potential inNitella flexilis were analyzed by inserting two microelectrodes. one into the vacuole and the other into the cytoplasm. It was unequivocally demonstrated that the rapid first peak was generated at the plasmalemma and the slow second peak at the tonoplast. MnCl₂ applied in the external medium abolished the second, tonoplast, peak but not the first, plasmalemma, peak, MnCl₂ also inhibited the cessation of the cytoplasmic streaming accompanying the action potential. CaCl₂ added in MnCl₂-containing medium recovered generation of the tonoplast action potential and the streaming cessation. Since it has been established that the cessation of cytoplasmic streaming on membrane excitation is caused by an increase in cytoplasmic free Ca^2– (Williamson, R.E., Ashley, C.C., 1982.Nature (London) 296:647–651: Tominaga, Y., Shimmen, T., Tazawa, M., 1983,Protoplasma 116:75–77), it is suggested that the tonoplast action potential is also induced by an increase in cytoplasmic Ca²⁺ resulting from the plasmalemma excitation. When vacuolar Cl was replaced with SO ₄ ² by vacuolar perfusion, the polarity of the second, slow peak was reversed from vacuolar positive to vacuolar negative with respect to the cytoplasm, supporting the previous report that the tonoplast action potential is caused by increase in Cl permeability (Kikuyama, M., Tazawa, M., 1976.J. Membrane Biol.29:95–110).     

Turgor pressure changes trigger characteristic changes in the electrical conductance of the tonoplast and the plasmalemma of the marine alga Valonia utricularis

The giant marine alga Valonia utricularis is capable of regulating its turgor pressure in response to changes in the osmotic pressure of the sea water. The turgor pressure response comprises two phases, a fast, exponential phase arising exclusively from water shifting between the vacuole and the external medium (time constant about 10 min) and a second very slow, almost exponential phase adjusting (but not always) the turgor pressure near to the original value by release or uptake of KCl (time constant about 5 h). The changes in the vacuolar membrane potential as well as in the individual conductances of the tonoplast and plasmalemma accompanying turgor pressure regulation were measured by using the vacuolar perfusion assembly (with integrated microelectrodes, pressure transducers and pressure‐regulating valves) as described by Wang et al. (J. Membrane Biology 157, 311–321, 1997). Measurements on pressure‐clamped cells gave strong evidence that the turgor pressure, but not effects related to water flow (i.e. electro‐osmosis or streaming potential) or changes in the internal osmotic pressure and in the osmotic gradients, triggers the cascade of osmotic and electrical events recorded after disturbance of the osmotic equilibrium. The findings definitely exclude the existence of osmosensors as postulated for other plant cells and bacteria. There was also evidence that turgor pressure signals were primarily sensed by ion transporters in the vacuolar membrane because conductance changes were first recorded in the many‐folded tonoplast and then significantly delayed in the plasmalemma independent of the direction of the osmotic challenge. Consistently, turgor pressure up‐regulation (but not down‐regulation) could be inhibited reversibly by external addition of the K⁺ transport inhibitor Ba²⁺ and/or by the Cl^– transport inhibitor 4,4′‐diisothiocyanatostilbene‐2,2′‐disulfonic acid (DIDS). Extensive studies under iso‐, hyper‐ and hypo‐osmotic conditions revealed that K⁺ and Cl^– contribute predominantly to the plasmalemma conductance. Addition of 0.3 mm NaCN showed further that part of the K⁺ and Cl^– transporters depended on ATP. These transporters are apparently up‐regulated upon hyper‐osmotic, but not hypo‐osmotic challenge. These findings explain the strong increase of the K⁺ influx upon lowering turgor pressure and the less pronounced pressure‐dependence of the Cl^– influx of V. utricularis reported in the literature. The data derived from the blockage experiments under hypo‐osmotic conditions were also equally consistent with the experimental findings that the K⁺ efflux is solely passive and progressively increases with increasing turgor pressure due to an increase of the volumetric elastic modulus of the cell wall. However, despite unravelling some of the sequences and other components involved in turgor pressure regulation of V. utricularis the co‐ordination between the ion transporters in the tonoplast and plasmalemma remains unresolved because of the failure to block the tonoplast transporters by addition of Ba²⁺ and DIDS from the vacuolar side.     

Influence of membrane structure on ion transport through lipid bilayer membranes

Summary Charge-pulse relaxation studies with the positively charged PV-K⁺ complex (cyclo-(d-Val-l-Pro-l-Val-d-Pro)₃) and the negatively charged lipophilic ion dipicrylamine (DPA^–) have been performed in order to study the influence of structural properties on ion transport through lipid bilayer membranes. First, the thickness of monoolein membranes was varied over a wide range using differentn-alkanes and slovent-free membranes. The thickness (d) of the hydrocarbon core of these membranes varied between 4.9 and 2.5 nm. For both transport systems the partition coefficient was found to be rather insensitive to variations ind. The same was valid for the translocation rate constantk _MS of PV-K⁺, whereas a strong increase of the translocation rate constantk _i of DPA-with decreasingd was observed. In a further set of experimental conditions the structure of the lipids, such as number and position of the double bonds in the hydrocarbon chain and its chain length as well as the nature of the polar head group, was varied. The translocation constantk _MS of PV-K⁺ transport was found to be much more sensitive to these variations thank _i of DPA-.Much larger variations ink _i andk _MS were observed in membranes made from lipids with ether instead of ester linkages between glycerol backbone and hydrocarbon chain. The results are in qualitative agreement with the surface potentials of monolayers made from corresponding lipids. Increasing amounts of cholesterol in membranes of dioleoylphosphatidylcholine caused a strong decrease ofk _MS (PV-K⁺), whereask _i was found to be rather insensitive to this variation.In monoolein membranes cholesterol causes a decrease ofk _MS up to sixfold and a increase ofk _i up to eightfold. The partition coefficient of DPA^– was insensitive to cholesterol, whereas of PV-K⁺ was found to decrease about eightfold in these membranes. The influence of cholesterol onk _MS is discussed on the basis of viscosity changes in the membrane and the change ink _i of DPA^– and of PV-K⁺ on the basis of a possible change of the dipole potential of the membranes. The other sterols, epicholesterol and ergosterol cause no change in the kinetics of the two probes.The different influence of membrane properties like thickness, viscosity, and dipole potential on the two transport systems is discussed under the assumption that the adsorption planes of the two probes have different positions in a membrane. Possibly because of a larger hydrophobic interaction, the adsorption plane of PV-K⁺ is located more towards the hydrocarbon side and that of DPA^– more towards the aqueous side of the dipole layer.     

Electrical tolerance (breakdown) of theChara corallina plasmalemma: II. Inductive property of membrane and effects of pH o and impermeable monovalent cations on breakdown phenomenon

Summary Changes in the chord conductanceG and the membrane electromotive forceE _m in the so-called breakdown region of large negative potential of theChara plasmalemma were analyzed in more detail. In addition to the increase inG, the voltage sensitivity of the change inG increased, which was the cause of marked inductive current in the breakdown region. The breakdown potential, defined as a critical potential at which both low and high slope conductances of theI–V _m relationship cross, almost coincided with the potential at which an inductive current began to appear. This breakdown potential level changed with pH _o in a range between 5 and 9. TheChara plasmalemma was electrically most tolerant around pH _o 7.In some cellsE _m shifted to a positive level as large as +50+70 mV during the breakdown phenomenon. Such a large positive shift ofE _m is caused mainly by the increase in conductance of Cl^– and partly Ca²⁺ and K⁺.     

Characterization of the mobile charges in the membrane ofValonia utricularis

Summary An attempt for the characterization of the mobile charges has been made by investigation of the voltage relaxations following a charge pulse at various temperatures between 2 and 40°C. The translocation ratek, the membrane conductivity 1/R _m and the total concentration of mobile chargesN _t within the membrane were calculated from recently developed theory (U. Zimmermann. K.-H. Büchner and R. Benz,J. Membrane Biol. 67:183–197, 1982). Data from 21 cells show that the concentration of mobile charges does not change significantly over a temperature range of 5 to 34°C, whereas both the translocation rate and the membrane conductivity reveal a strong but reversible temperature dependence. In the temperature range of 18 to 5°C,k decreases by a factor of 10 to 20, while between 18 and 34°C the increase ink is only two- or threefold with a maximum around 25°C. In principle, the same temperature dependence was observed for the membrane conductivity. Hysteresis effects occurring in the low temperature range as well as at high temperatures indicate that a protein component is involved in the mobile charge system. Furthermore, addition of cycloheximide leads to a marked decrease in both the translocation rate and the membrane conductivity, however, leaving the concentration of mobile charges almost unchanged. Therefore, it is assumed that the mobile charges are coupled to, but not part of a carrier protein.     

Comparison of a lipophilic cation and microelectrodes to measure membrane potentials of the giant-celled algae,Chara australis (Charophyta) andGriffithsia monilis (Rhodophyta)

Summary The vacuolar equilibrium potential of the lipophilic cation TPMP⁺ (triphenyl methyl phosphonium) in the giant algaeChara australis andGriffithsia monilis was directly measured. The TPMP⁺ equilibrium potential was approximately 100mV less negative than the measured vacuolar electrical potential. Thus TPMP⁺ does not act as a probe of the vacuolar electrical potential and appears to be extruded against an electrochemical gradient. Measurement of the plasmalemma equilibrium potential of TPMP⁺ showed that extrusion of TPMP⁺ apparently occurred at both the tonoplast and plasmalemma inChara and at the plasmalemma inGriffithsia. It is concluded that TPMP⁺ cannot be used as a membrane potential probe inChara orGriffithsia.     

Turgor regulation inValonia macrophysa following acute osmotic shock

Summary The marine algaValonia macrophysa an inhabitant of shallow subtropical waters, is subjected to sudden dilutions of external seawater during rain showers. This study describes the mechanisms involved in turgor pressure regulation following acute hyposmotic shock. Turgor regulation is 88% effective and complete within 4 hr following hyposmotic shocks of up to –10 bar. Loss of vacuolar K⁺, Na⁺ and Cl^– accounts for the decrease in vacuolar osmotic pressure associated with turgor regulation. A novel mechanism of turgor regulation is exhibited byValonia macrophysa given hyposmotic shocks greater than about –4 bar. Such an osmotic shock causes cell wall tension to increase above a critical value of about 6×10⁵ dyne/cm, whereupon the protoplasm ruptures and the cell wall stretches irreversibly at a localized site. The protoplasm rupture is suggested by (1) a large abrupt increase in K⁺ efflux (as measured by⁸⁶Rb⁺), (2) a rapid decrease in turgor pressure as measured with a pressure probe, and (3) sudden depolarization of the vacuole potential. Evidence for an increase in cell wall permeability includes efflux from the vacuole of dextran (mol wt 70,000), which normally has a very low cell wall permeability, and scanning electron micrographs which show a trabeculated scar area in the cell wall. This mechanism of turgor regulation is physiologically important because 98% of the cells regained normal growth rate and turgor following acute osmotic shock.     

Plasmalemma- and tonoplast-ATPase activity in mesophyll protoplasts,vacuoles and microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana

Adenosine-triphosphatase activity on the plasmalemma and tonoplast of isolated mesophyll protoplasts, isolated vacuoles and tonoplast-derived microsomes of the Crassulacean-acid-metabolism plant Kalanchoe daigremontiana Hamet et Perr., was localized by a cytochemical procedure using lead citrate. Enzyme activity was detected on the cytoplasmic surfaces of the plasmalemma and tonoplast. The identity of the enzymes was confirmed by various treatments differentiating the enzymes by their sensitivity to inhibitors of plasmalemma and tonoplast H⁺-ATPase. Isolated vacuoles and microsomes prepared from isolated vacuoles clearly exhibited single-sided deposition on membrane surfaces.Abbveviations CAM Crassulacean acid metabolism - H⁺-ATPase proton-translocating ATPase     

Rectification characteristics ofNitella membranes in respect to water permeability

Summary One of the membrane characteristics of plant cells, rectification, or the direction dependence of water permeability, was investigated inCharaceae internodes using the procedures we developed (Tazawa andKiyosawa 1973) for determining the endosmotic (k _pen) and exosmotic (k _pex) water permeabilities of the membranes (plasmalemma and tonoplast) in the transcellular osmosis system. Bothk _pen andk _pex were dependent on the osmotic pressure ( _o ) of the mannitol solution, which is the driving force for the transcellular osmosis. Thus, k_pen increased andk _pex decreased with _o . The rectification parameter, or the polarity (_p), defined ask _pen/k _pex tended to unity when _o approached zero.InNitella flexilis the specific resistances of the membranes to endosmosis and exosmosis,k _pen ^–1 andk _pex ^–1 , were linearly dependent on ₀. When the cell was partitioned into two equal halves,k _pen ^–1 =4.2×10⁴–1.1×10³₀,k _pex ^–1 =4.2×10⁴+2.9×10³₀, where the specific resistances are represented in cm^–1 sec atm. When _o is 0.1, 0.2, 0.3, 0.4, and 0.5 M mannitol eq., the rectification parameter is calculated as 1.3, 1.6, 1.9, 2.4, and 2.9, respectively. Essentially the same results were also obtained withChara australis.Results were discussed on the basis of changes in the hydration of the cytoplasm. Assuming that the driving force across the protoplasmic layer can be divided into two forces; one driving water across the plasmalemma and the other driving water across the tonoplast, we deduced that the cytoplasm on the endosmosis side is hydrated, while the cytoplasm on the exosmosis side is dehydrated. Analysis showed that changes in hydration depend on the rate of flow.This work was supported partly by a Research Grant from the Ministry of Education of Japan.     

The relative electrical resistances of the plasmalemma and tonoplast in higher plants

Summary Measurements on 3–6 days old root hair cells of cucumber, oats and maize, in a medium of 1.0 mN KCl plus 1.0 mN CaCl₂, gave an average d.c. surface resistance value of 3,000 ohm·cm² for the plasmalemma and of 3,500 or 3.600 ohm·cm² for the plasmalemma and tonoplast in series. The average value for the combined surface resistance of the two membranes was about 1.2 times that of the plasmalemma alone, showing that in the above cells the latter membrane has a resistance appreciably more than that of the tonoplast.It is concluded that the plasmalemma offers a major barrier to the passive uptake of ions. This conclusion is supported by a consideration of transverse impedance measurements of young and old tissues.     

Direct turgor pressure measurements in individual leaf cells of Tradescantia virginiana

The water relations of leaves of Tradescantia virginiana were studied using the miniaturized pressure probe (Hüsken, E. Steudle, Zimmermann, 1978 Plant Physiol. 61, 158–163). Under well-watered conditions cell turgor pressures, P _o, ranged from 2 to 8 bar in epidermal cells. In subsidiary cells P _o was about 1.5 to 4.5 bar and in mesophyll cells about 2 to 3.5 bar. From the turgor pressure, relaxation induced in individual cells by changing the turgor pressure directly by means of the pressure probe, the half-time of water exchange was measured to be between 3 and 100 s for the epidermal, subsidiary, and mesophyll cells. The volumetric elastic modulus, , of individual cells was determined by changing the cell volume by a defined amount and simultaneously measuring the corresponding change in cell turgor pressure. The values for the elastic modulus for epidermal, subsidiary, and mesophyll cells are in the range of 40 to 240 bar, 30 to 200 bar, and 6 to 14 bar, respectively. Using these values, the hydraulic conductivity, L _p, for the epidermal, subsidiary, and mesophyll cells is calculated from the turgor pressure relaxation process (on the basis of the thermodynamics of irreversible processes) to be between 1 and 55·10^-7 cm s^-1 bar^-1. The data for the volumetric elastic modulus of epidermal and subsidiary cells indicate that the corresponding elastic modulus for the guard cells should be considerably lower due to the large volume changes of these cells during opening or closing. Recalculation of experimental data obtained by K. Raschke (1979, Encycl. Plant Physiol. N.S., vol. 7, pp 383–441) on epidermal strips of Vicia faba indicates that the elastic modulus of guard cells of V. faba is in the order of 40–80 bar for closed stomata. However, with increasing stomatal opening, i.e., increasing guard cell volume, decreases. Therefore, in our opinion Raschke's results would indicate a relationship between guard cell volume and which would be inverse to that for plant cells known in the literature. assumes values between 20–40 bar when the guard cell colume is soubled.     

Tonoplast action potential inNitella in relation to vacuolar chloride concentration

Summary The action potential ofNitella internode was studied in relation to K⁺ and Cl^– concentrations in the vacuole. When the vacuole ofNitella pulchella was filled with an artificial solution with extremely low Cl^– concentration, a diphasic action potential (DAP) was observed. The first phase consists of a rapid depolarization followed by a relatively rapid repolarization, and the second one consists of a strong hyperpolarization followed by a gradual return to the resting potential.When the cell was stimulated immediately after the generation ofDAP, a monophasic action potential which resembles an action potential of the natural cell was observed, indicating that theDAP consists of two components with different refractory periods. The refractory period of the component responsible for the depolarizing phase is shorter than that of a component responsible for the hyperpolarizing phase. Measuring the plasmalemma potential and vacuolar potential separately, it was demonstrated that the hyperpolarizing component ofDAP originates from the tonoplast.The action potential of the tonoplast, in contrast with that of the plasmalemma, could be generated independently of concentration of K⁺ in the vacuole. Since the maximum amplitude of hyperpolarization decreased significantly by increasing Cl^– concentration of the vacuole, it is concluded that the tonoplast is very sensitive to Cl^– during excitation.     

Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, , determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5–2·10^–6 cm s^–1 bar^–1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.     

灵武长枣果实质膜和液泡膜H~+-ATPase和H~+-PPase活性与果实糖分积累

以不同发育时期灵武长枣(Ziziphus jujuba cv.Lingwuchangzao)的果实为材料,通过测定与分析果肉组织中细胞质膜、液泡膜H+-ATPase和H+-PPase活性、果实糖分含量变化,研究了灵武长枣果实质膜、液泡膜H+-ATPase和H+-PPase活性与糖积累特性的关系。结果表明:(1)果实第二次快速生长期之前主要积累葡萄糖和果糖,之后果实迅速积累蔗糖,葡萄糖和果糖含量则逐渐下降,成熟期果实主要积累蔗糖。(2)在果实发育的缓慢生长期S1,质膜H+-ATPase活性最低;第一次快速生长期,质膜H+-ATPase活性最高;缓慢生长期S2,其活性降低;第二次快速生长期,质膜H+-ATPase活性升至次高;完熟期,质膜H+-ATPase活性下降幅度较大。(3)在果实发育过程中,液泡膜H+-ATPase和H+-PPase活性的变化趋势相似。缓慢生长期S1,液泡膜H+-ATPase和H+-PPase活性较低;从缓慢生长期S1至第一次快速生长期缓慢下降至最低;从第一次快速生长期开始,液泡膜H+-ATPase和H+-PPase活性呈现为逐渐增高的变化趋势;除第二次快速生长期以外,液泡膜H+-PPase活性始终高于H+-ATPase。由此推测,质膜H+-ATPase和液泡膜H+-ATPase、H+-PPase对灵武长枣果实糖分的跨膜次级转运起到重要的调控作用。     

Turgor regulation in a brackish water charophyte,Lamprothamnium succinctum

Internodal cells of a brackish water charophyte,Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic pressures. During turgor regulation upon hypotonic treatment, net effluxes of K⁺ and Cl⁻ from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase in concentration of cytoplasmic Ca²⁺ are induced. Activation of the plasmalemma Ca²⁺ channels and the Ca²⁺-controlled passive effluxes of K⁺ and Cl⁻ through the plasmalemma ion channels are postulated.     

Osmotic Water Permeability of Vacuolar and Plasma Membranes Isolated from Maize Roots

The method of stopped flow was used to follow the changes in light scattering by the vesicles of plasmalemma and tonoplast isolated from maize (Zea maysL.) roots and treated by osmotic pressure. In both membrane preparations, the rate of the process depended on the osmotic gradient and was described with the simple exponential function. The rate constants derived from these functions were the following: the coefficient of water permeability in the tonoplast (P= 165 ± 7 m/s) exceeded by an order of magnitude the corresponding index for plasmalemma (11 ± 2 m/s). The presence of HgCl₂(1.6 nmol/g membrane protein) decreased the tonoplast water permeability by 80%. Microviscosity studies of the hydrocarbon zone in the isolated membranes by using a fluorescent diphenylhexatriene probe demonstrated that the two membranes do not differ in the phase state of their lipid bilayer. The authors conclude that the observed difference in water permeability does not depend on the state of the lipid phase and probably reflects the dissimilar functional activity of plasmalemma and tonoplast aquaporins.     

Elucidation of the Mechanisms Underlying Hypo-osmotically Induced Turgor Pressure Regulation in the Marine Alga Valonia utricularis

Exposure of the giant marine alga Valonia utricularis to acute hypo-osmotic shocks induces a transient increase in turgor pressure and subsequent back-regulation. Separate recording of the electrical properties of tonoplast and plasmalemma together with turgor pressure was performed by using a vacuolar perfusion assembly. Hypo-osmotic turgor pressure regulation was inhibited by external addition of 300 microM of the membrane-permeable ion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). In the presence of 100 microM NPPB, regulation could only be inhibited by simultaneous external addition of 200 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), a membrane-impermeable inhibitor of Cl(-) transport. At concentrations of about 100 microM, NPPB seems to selectively inhibit Cl(-) transporters in the tonoplast and K(+) transporters in the plasmalemma, whereas 300 microM NPPB inhibits K(+) and Cl(-) transporters in both membranes. Evidence was achieved by measuring the tonoplast and plasmalemma conductances (G(t) and G(p)) in low-Cl(-) and K(+)-free artificial seawater. Inhibition of turgor pressure regulation by 300 microM NPPB was accompanied by about 85% reduction of G(t) and G(p). Vacuolar addition of sulfate, an inhibitor of tonoplast Cl(-) transporters, together with external addition of DIDS and Ba(2+) (an inhibitor of K(+) transporters) also strongly reduced G(p) and G(t) but did not affect hypo-osmotic turgor pressure regulation. These and many other findings suggest that KCl efflux partly occurs via electrically silent transport systems. Candidates are vacuolar entities that are disconnected from the huge and many-folded central vacuole or that become disconnected upon disproportionate swelling of originally interconnected vacuolar entities upon acute hypo-osmotic challenge.