Compartments and Fluxes of K, NA, and CL in Avena Coleoptile Cells

By the compartmental analysis method of MacRobbie and Dainty, and Pitman, estimates of K⁺, Na⁺, and Cl⁻ concentrations and fluxes were obtained for the cytoplasm and vacuole of coleoptile cells of oat, Avena sativa L. cv. Victory. Double labeling was used in experiments with ⁴²K plus ²²Na and with ⁴²K plus ³⁶Cl in a complete nutrient solution. At the plasmalemma, according to the Ussing-Teorell flux ratio equation, Na⁺ is pumped out and Cl⁻ is actively transported inward. The results with K⁺ are less conclusive, but it is probably pumped in. At the tonoplast there is an active inward transport of Na⁺ and probably of K⁺, but the status of Cl⁻ is uncertain, depending upon whether there is an electrical potential difference between the cytoplasm and vacuole. The results suggest that ion selectivity resides mostly in the plasmalemma. Possible errors in the estimates and interpretations are discussed.     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

The ionic relations of Porphyra purpurea(Roth) C.Ag. (Rhodophyta, Bangiales)

Abstract Radioisotope equilibration techniques have been used to determine the intracellular concentration of K⁺, Na⁺ and Cl_?, together with the unidirectional ion fluxes across the plasmalemma of Porphyra purpurea. Influx and efflux of ⁴²K⁺, ²⁴Na⁺ and ³⁶C1^? are biphasic, the rapid, initial uptake and loss of tracer from individual thalli being attributable to desorption from extracellular regions. Cellular fluxes are slower and monophasic, cells discriminating in favour of K⁺ and Cl^? and against Na⁺. A comparison between the equilibrium potential of individual ion species and the measured membrane potential demonstrates that there is an active component of K⁺ and Cl^? influx and Na⁺ efflux. ‘Active’ uptake and ‘passive’ loss of K⁺ and Cl^? are reduced when plants are kept in darkness, suggesting that a fraction of the transport of K⁺ and Cl^? may be due to ‘exchange diffusion’ (K⁺/K⁺ and Cl^?/Cl^?antiport).     

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: II. Selectivity and Kinetics

Protoplasts were enzymically isolated from suspension cultured cells of Nicotiana glutinosa L. and aspects of transport selectivity and kinetics were studied. In the presence of Ca²⁺, transport was selective for K⁺ (⁸⁶Rb) over Na⁺. ³⁶Cl⁻ transport was inhibited by Br⁻ or I⁻ but not by H₂PO₄⁻. The kinetic data for short term (30 minutes) K⁺ influx over the range of 0.05 to 100 millimolar KCl were complex but similar to those observed in other plant tissues. In contrast, the kinetic data for Cl⁻ and H₂³²PO₄⁻ over the same concentration range were different from those observed for K⁺, and could be accounted for by a single isotherm in the range of 0.05 to 4 millimolar and by an almost linear increase in influx rate above 4 millimolar. The kinetic data for Cl⁻ transport into intact cultured cells were identical in character to those observed for isolated protoplasts. The results support the view that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast.     

Na/H and k/h antiport in root membrane vesicles isolated from the halophyte atriplex and the glycophyte cotton

Proton fluxes have been followed into and out of membrane vesicles isolated from the roots of the halophyte Atriplex nummularia and the glycophyte Gossypium hirsutum, with the aid of the ΔpH probe [¹⁴C]methylamine. Evidence is presented for the operation of Na⁺/H⁺ and K⁺/H⁺ antiporters in the membranes of both plants. Cation supply after a pH gradient has been set up across the vesicle membrane (either as a result of providing ATP to the H⁺-ATPase or by imposing an artificial pH gradient) brings about dissipation of the ΔpH, but does not depolarize the membrane potential as observed in similar experiments, but in the absence of Cl⁻, using the ΔΨ probe SCN⁻. Cation/H⁺ exchange is thus indicated. This exchange is not due to nonspecific electric coupling, nor to competition for anionic adsorption sites on the membrane, nor to inhibition of the H⁺-ATPase; coupling of the opposed cation and H⁺ fluxes by a membrane component is the most likely explanation. Saturation kinetics have been observed for both Na⁺/H⁺ and K⁺/H⁺ antiport in Atriplex. Moreover, additive effects are obtained when Na⁺ is supplied together with saturating concentrations of K⁺, and vice versa, suggesting that separate antiporters for Na⁺ and for K⁺ may be operating. In the case of both Atriplex and Gossypium evidence was obtained suggesting the presence of antiporters in both plasmalemma and tonoplast.     

Effect of diethylstilbestrol on ion fluxes in oat roots

Effects of diethylstilbestrol (DES) on ion fluxes in oat roots (Avena sativa L.) were investigated by measuring K⁺ and Cl⁻ absorption and K⁺ efflux. DES rapidly decreased the absorption of K⁺ (⁸⁶Rb) and ³⁶Cl⁻ by excised roots; 10⁻⁴ molar DES inhibited Cl⁻ absorption in 1 minute and K⁺ absorption in 1 to 2 minutes. With a 10-minute incubation period, K⁺ and Cl⁻ absorption were inhibited 50% by 1.1×10⁻⁵ molar and 8.4×10⁻⁶ molar DES, respectively. Treatment for 3 minutes with 10⁻⁴ molar DES caused irreversible inhibition of K⁺ absorption. Increasing concentrations of KCl in the absorption media decreased the DES inhibition. Experiments with the DES analogs, DES dipropionate, dienestrol and hexestrol, showed that the steric configuration and the hydroxyl group of the DES molecule are important in determining the inhibitory capacity of the compound.     

Fluxes and compartmentation of potassium and chloride in the green alga Mougeotia

Summary Some ionic relations of the filamentous green alga Mougeotia sp. have been analyzed under different light conditions. Data from influx and efflux measurements using ⁸⁶Rb⁺ and ³⁶Cl^- fit the model of three cellular compartments (cell wall, cytoplasm, vacuole) in series. This result is remarkable, since in a Mougeotia cell at least two thirds of the cytoplasmic compartment are occupied by the cell-filling, flat and nearly rectangular chloroplast which is axially oriented. The chloroplast is concluded to be part of the cytoplasmic flux compartment.Photosynthetically saturating irradiances of continuous white light enhance the active and passive fluxes of K⁺ and Cl^- at the plasmalemma by a factor of 3. Photosystem II is responsible for the light-dependent increase of the uptake of Cl^- (³⁶Cl^-) whereas the uptake of K⁺ (⁸⁶Rb⁺) depends additionally on energy from photosystem I.Ion flux measurements performed after irradiations with red and far-red, respectively, show that the fluxes of K⁺ and Cl^- across the plasmalemma are not affected by the state of phytochrome.     

Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics

An investigation was conducted into the feasibility of using enzymically isolated protoplasts from suspension-cultured cells of Nicotiana glutinosa L. to study ion transport. Transport of K⁺ (⁸⁶Rb), ³⁶Cl⁻, H₂³²PO₄⁻ and ⁴⁵Ca²⁺ from 1 millimolar salt solutions was determined after separation of intact protoplasts from nonabsorbed tracers by centrifugation through a Ficoll step gradient. Influx of K⁺, Cl⁻, and H₂PO₄⁻ measured over a 30-minute period was reduced (up to 99%) by respiratory inhibitors such as 5 micrograms per milliliter oligomycin, 0.1 millimolar dinitrophenol, 0.1 millimolar cyanide, or N₂ gas. In contrast, Ca²⁺ influx was not tightly coupled to respiratory energy production. The influx of K⁺ was highest between pH 6.5 and 7.5 whereas the influx of H₂PO₄⁻ and Cl⁻ was greatest between pH 4.5 and 5.5. Influx of K⁺ and Cl⁻ was maximal at 35 and 45 C, respectively, and was almost completely inhibited below 10 C. Fusicoccin (0.01 millimolar) stimulated K⁺ influx by more than 200% but had no effect on the influx of either Cl⁻ or H₂PO₄⁻. Apparent H⁺ efflux, as measured by decrease in solution pH, was enhanced by K⁺, stimulated further by 0.01 millimolar fusicoccin, and inhibited by 0.1 millimolar dinitrophenol or 5 micrograms per milliliter oligomycin. The measured ionic fluxes into protoplasts were similar to those obtained with intact cultured cells. The results indicate that enzymic removal of the cell wall produced no significant alteration in the transport properties of the protoplast, and that it is feasible to use isolated protoplasts for studies on ion transport.     

Effects of ultraviolet radiation on the membranes ofChara corallina

Summary The effects of 253.7 nm ultraviolet (UV) radiation on the membrane properties ofChara corallina have been studied. UV irradiation caused depolarization of the membrane potential (p.d.) and a decrease in membrane resistance. These effects were largely reversible with steady values being obtained within 40 minutes after the UV was turned off. The effects on ionic fluxes of Na⁺, K⁺ and Cl^– have also been studied using radioactive tracer techniques. The influxes were unchanged by irradiation. The chloride efflux was increased sevenfold during the irradiation period but recovered to the pre-irradiation value within 30 minutes after the irradiation period. The potassium efflux was also increased and reached a maximum 10 minutes after irradiation. The resting potential and the average depolarized p.d. reached during irradiation were in good agreement with those calculated from permeability coefficients indicated by the observed passive fluxes, using the Goldman equation for p.d. However, the plasmalemma resistance and its change due to irradiation did not match the values calculated from the same permeability coefficients used to estimate p.d. This disagreement, and an apparent imbalance in the charge transferred across the resting or irradiated plasmalemma, suggest the participation of another ion species as well as K⁺, Na⁺ and Cl^–.     

Ion fluxes and phytochrome protons in mung bean hypocotyl segments: I. Fluxes of potassium

K⁺ [⁸⁶Rb⁺] uptake by Phaseolus aureus Roxb. hypocotyl segments cut immediately below the hook is inhibited by the active form of phytochrome (Pfr). Short load-short wash experiments indicate that the inhibition of uptake occurs across the plasmalemma. A maximal inhibition of short term uptake occurs in 10 to 50 millimolar KCI. Low temperature had only a small effect on influx and the inhibition of influx from 50 millimolar KCI. A consideration of the electrochemical gradient for K⁺ suggests that passive K⁺ fluxes may predominate under these conditions. Red light induces small depolarizations of membrane potential in subhook cells. Far red light antagonizes this effect. Pfr inhibits efflux of K⁺[⁸⁶Rb⁺] from subhook segments. This effect is also relatively insensitive to low temperature. This inhibition of efflux may reflect inhibition of a K⁺ -K⁺ exchange process, or reduced passive permeability of the plasmalemma to K⁺. In contrast, Pfr enhances short term uptake of K⁺[⁸⁶Rb⁺] in apical hypocotyl hook segments of Phaseolus aureus Roxb. Short load-short wash experiments indicate that fluxes across the plasmalemma are modified by Pfr. A maximal enhancement of short term influx occurs in 50 millimolar KCI. Influx and the red light enhancement of influx from 50 millimolar KCI are relatively insensitive to low temperature. Pfr also enhances efflux of K⁺[⁸⁶Rb⁺] from preloaded apical hook segments. This increased influx may reflect enhancement of a K⁺ -K⁺ exchange process or increased passive permeability of the plasmalemma to K⁺.     

Potassium Transport in Corn Roots : IV. Characterization of the Linear Component

A detailed examination was conducted on the linear, or first-order kinetic component for K⁺(⁸⁶Rb⁺) influx into root segments of both low- and high-salt grown corn seedlings (Zea mays [A632 × Oh 43]). In tissue from both low- and high-salt grown roots, replacement of Cl⁻ in the uptake solution by either SO₄²⁻, H₂PO₄⁻, or NO₃⁻ caused a significant (50-60%) and specific inhibition of the linear component of K⁺ influx. The anion transport inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid, was found to abolish saturable Cl⁻ influx in corn roots while causing a significant (50-60%) and specific inhibition of the linear K⁺ uptake system; this inhibition was identical to that observed when Cl⁻ was replaced by other anions in the K⁺ uptake solution. Additionally, the quaternary ammonium cation, tetraethylammonium, which has been shown to block K⁺ channels in nerve axons, also caused a dramatic (70%) and specific inhibition of the linear component of K⁺ influx, but this was obtained only in high-salt roots. The reasons for this difference are discussed with respect to the differing abilities of low- and high-salt roots to absorb tetraethylammonium.

Our present results indicate that the linear component of K⁺ influx may occur by a passive process involving transmembrane K⁺ channels. Fluxes through these K⁺ channels may be partly coupled to a saturating Cl⁻ influx mechanism.

     

Inhibition of anion transport in corn root protoplasts

The effects of several amino-reactive disulfonic stilbene derivatives and N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate on Cl⁻, SO₄²⁻, and inorganic phosphate (Pi) uptake in protoplasts isolated from corn root tissue were studied. 4-Acetamido-4′-isothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, 4,4′-diamino-2,2′-stilbenedisulfonic acid, and NAP-taurine inhibited Cl⁻ and SO₄²⁻ but not Pi and K⁺ uptake in corn root protoplasts; whereas mersalyl inhibited Pi but not Cl⁻ or SO₄²⁻ uptake. The rate of uptake of all anions decreased with increasing external pH. In addition, these reagents markedly inhibited plasmalemma ATPase activity isolated from corn root tissue. Excised root segments were less sensitive to Cl⁻ and SO₄²⁻ transport inhibitors.     

Effect of Salinity upon Cell Membrane Potential in the Marine Halophyte, Salicornia bigelovii Torr

The electrophysiology of root cells of the marine halophyte, Salicornia bigelovii Torr., has been investigated. Cellular concentrations of K⁺, Cl⁻, and Na⁺ and resulting cell membrane potentials were determined as functions of time and exposure to dilutions of artificial seawater. Treatment of these data by the Nernst criterion suggests that Cl⁻ is actively transported into these root cells, but that active transport need not be invoked to explain the accumulation of Na⁺ at all salinities investigated nor for K⁺ at moderate to high salinities. In low environmental salinity, the cell electropotential of Salicornia root cells was found to respond to inhibitors in a fashion similar to that observed in glycophytes; in high environmental salinity, root cell membrane potential appears to be insensitive to bathing salinity and m-chlorocarbonylcyanide phenylhydrazone induces membrane hyperpolarization, in contrast to the response of glycophytes to such treatments. The fact that measured membrane potentials exceed diffusion potentials for Na⁺, K⁺, and Cl⁻ and the observation of a rapid depolarization by CO in the dark suggests an electrogenic component in Salicornia root cell membrane potentials.     

Light-Dependent Changes of the Cytoplasmic H and Cl Activity in the Green Alga Eremosphaera viridis

Ion-sensitive microelectrodes were used to measure Cl⁻ and H⁺ activities in the cytoplasm of the unicellular green alga Eremosphaera viridis de Bary. In the light, cytoplasmic Cl⁻ activity was 2.2 millimolar at most and cytoplasmic H⁺ activity was about 5.4·10⁻⁸ molar (pH 7.3). Darkening resulted in a permanent increase of the Cl⁻ activity to 3.2 millimolar and in a transient acidification, which was compensated within 3 to 5 minutes. Switching light on again decreased the Cl⁻ activity to the light level (2.2 millimolar). Simultaneously, a transient alkalization of the cytoplasm was observed. The transient character of the light-dependent pH changes was probably caused by pH-stat mechanisms, whereas the light-dependent Cl⁻ activity changes were compensated to a much smaller degree. Studies with different inhibitors (3-(3,4-dichlorophenyl)-1, 1-dimethylurea, piretanide, venturicidin) indicated a direct relation between the light-driven H⁺ flow across the thylakoid membrane and the observed light-dependent Cl⁻ and H⁺ activity changes in the cytoplasm. It is suggested that light-driven H⁺ flux across the thylakoid membrane was in part electrically compensated by a parallel Cl⁻ flux. The resulting Cl⁻ and H⁺ activity changes in the stroma were compensated by Cl⁻ and H⁺ fluxes across the chloroplast envelope giving rise to the observed Cl⁻ and H⁺ activity changes in the cytoplasm.     

Electrodiffusion,Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl⁻ conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl⁻ conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K⁺ and Cl⁻ were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl⁻ effluxes, induced by valinomycin or gramicidin at varied [K⁺]_o, were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K⁺]_o. Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P_K/P_Cl is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P_Cl. Fitting the constant field current-voltage equation to the measured Cl⁻ currents yields P _Cl = 0.13 h⁻¹ with DIDS, compared to 0.49 h⁻¹ without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl⁻ current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. IV. Comparative ion flux characteristics of the turion and the vegetative frond and the effect of ABA during early turion development

Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca²⁺, K⁺ and Cl^- have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca²⁺ exchange across the tonoplast and low vacuolar Ca²⁺ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca²⁺ concentration in the cytoplasm. The concentration of K⁺ and Cl^- is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K⁺ plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca²⁺, K⁺ or Cl^- fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport.     

Potassium and Phosphate Uptake in Corn Roots: Further Evidence for an Electrogenic H/K Exchanger and an OH/Pi Antiporter

Evidence is presented that K⁺ uptake in corn root segments is coupled to an electrogenic H⁺/K⁺ -exchanging plasmalemma ATPase while phosphate uptake is coupled to an OH⁻/Pi antiporter. The plasmalemma ATPase inhibitor, diethylstilbestrol, or the stimulator, fusicoccin, altered K⁺ uptake directly and phosphate uptake indirectly. On the other hand, mersalyl, an OH⁻/Pi antiporter inhibitor, inhibited phosphate uptake instantly but only slightly affected K⁺ uptake. Collapse of the proton gradient across the membrane by (p-trifluoromethoxy) carbonyl cyanide phenylhydrazone resulted in immediate inhibition of K⁺ uptake but only later inhibited phosphate uptake. Changing the pH of the absorption solution had opposite effects on K⁺ and phosphate uptake. In addition, a 4-hour washing of corn root tissue induced a 5-fold increase in the rate of K⁺ uptake with little or no lag, but only a 2- to 3-fold increase in phosphate uptake with a 30- to 45-minute lag. Collectively these differences strongly support the coupling of an electrogenic H⁺/K⁺ -exchanging ATPase to an OH⁻/Pi antiporter in corn root tissue.     

Characteristics of sulfate transport across plasmalemma and tonoplast of carrot root cells

Compartmental analysis of ³⁵SO₄²⁻ exchange kinetics is used to obtain SO₄²⁻ fluxes and compartment contents in carrot (Daucus carota L.) storage root cells, where 2 to 5% of the SO₄²⁻ taken up is reduced to organic form. The necessary curve fitting is verified by (a) consistency between `content versus time' and `rate versus time' plots of washout data; (b) agreement between loading and washout kinetics; and (c) correct identification of the fastest exchange phase as being from extracellular spaces.

Sulfate is actively transported up an electrochemical potential gradient at both plasmalemma and tonoplast. The plasmalemma influx is from 2 to 10 times higher than the tonoplast influx, is much greater than the SO₄²⁻ reduction rate, and would not limit the rate of either. This is consistent with the finding that the plasmalemma influx is not regulated by internal SO₄²⁻ or cysteine (Cram 1982 Plant Sci Lett, in press).

Both SO₄²⁻ influxes rise with only limited saturation as the external SO₄²⁻ concentration increases up to 50 millimolarity. Both effluxes appear to be passive, with extensive recycling in the plasmalemma influx pump. SO₄²⁻ permeability is about 10⁻¹¹ meter per second at both membranes.

The high, nonlimiting fluxes of SO₄²⁻ at the plasmalemma relative to the tonoplast (found also in Lemna; Thoiron, Thoiron, Demarty, Thellier 1981 Biochim Biophys Acta 644: 24-35) contrasts with SO₄²⁻ fluxes in bacteria and with Cl⁻ fluxes in plant cells. Their implications for work on characteristics and regulation of SO₄²⁻ uptake in roots and tissue cultures are discussed.

     

Plasmalemma Transport of OH in Chara corallina: III. FURTHER STUDIES ON TRANSPORT SUBSTRATE AND DIRECTIONALITY

The identity of the plasmalemma-transported species that develops the alkaline bands of Chara corallina was investigated. The effect of fusicoccin on the rate of HCO₃⁻ assimilation, and on the time-dependent alkaline band pH buildup following low pH flushing, was found to be small, with no stimulatory effect. Computer simulation of the flushing experiments showed that in the experimental situation the alkaline band transport system was slowed down, rather than speeded up, by low pH flushing. A detailed theoretical examination of the maximum rate of proton production from water showed that measured alkaline band fluxes are too large to be explicable in terms of an H⁺ influx system. The experimental and theoretical results indicate that the plasmalemma transport of OH⁻ ions is responsible for the measured negative external electric potential and alkalinity flux which are associated with the alkaline band phenomenon. Consequently, HCO₃⁻ influx across the characean plasmalemma must be charge-balanced by the efflux of OH⁻ ions.