Rb+ uptake by isolated pea mesophyll protoplasts in light and darkness

Rb⁺ uptake into protoplasts isolated from the mesophyll of Pisum sativum L. cv. Dan has been followed at intervals of a few minutes in the light and in the dark. The progress curve for uptake in the dark decreased in slope after about 7 min; in the light, by contrast, the slope increased. This effect was more pronounced at pH 7 than at pH 5.5. The pH profile for uptake in the dark rose with increasing pH: in the light the profile flattened, or even fell somewhat, between pH 5.5 and pH 6.5, then rose again. In the dark the proton uncoupler carbonyl cyanide m-chlorphenylhydrazone (CCCP) had little or no effect, either at pH 5.5 or at pH 7.4; in the light CCCP was strongly inhibitory, particularly at pH 7.4. Increasing concentrations of CCCP produced progressively more and more severe inhibition in the light, but in the dark produced a slight rise in uptake. The ATPase inhibitors quercetin, rutin and diethyl-stilbestrol, as well as arsenate, all depressed uptake in the light, particularly at higher pH Dark uptake was sensitive only at pH 5.5, not at pH 7.4. In marked contrast to the case of methyl-3 glucose, where protoplasts which were switched from light to dark took up sugar at the accelerated light rate for the first 7 min in the dark, a switch to darkness produced a Rb⁺ uptake rate below that for protoplasts held continuously in the dark. It is inferred that the mechanism of Rb⁺ uptake does not involve proton cotransport. Information regarding the membrane potential was obtained by following the distribution of tetraphenyl phosphonium (TPP⁺) between protoplasts and medium. The potential was more negative in the light than in the dark. It was also more negative at pH 7 than at pH 5 both in the light and in the dark. Treatment with CCCP produced no appreciable depolarization within the first 20 min, indicating thet the CCCP inhibition of Rb⁺ uptake in the light cannot be ascribed to a reduction in potential. An ATP-fueled K⁺ porter, or K⁺-H⁺ antiporter, seems the most likely explanation. The maintenance of the rising pH profile in the dark, despite the presence of a CCCP concentration which drastically inhibits light uptake, suggests that the profile does not depend on the operation of the proton pump.     

Role of K+ in leaf growth: K+ uptake is required for light-stimulated H+ efflux but not solute accumulation

The stimulation of dicotyledonous leaf growth by light depends on increased H⁺ efflux, to acidify and loosen the cell walls, and is enhanced by K⁺ uptake. The role of K⁺ is generally considered to be osmotic for turgor maintenance. In coleoptiles, auxin‐induced cell elongation and wall acidification depend on K⁺ uptake through tetraethylammonium (TEA)‐sensitive channels (Claussen et al., Planta 201, 227–234, 1997), and auxin stimulates the expression of inward‐rectifying K⁺ channels ( Philippar et al. 1999) . The role of K⁺ in growing, leaf mesophyll cells has been investigated in the present study by measuring the consequences of blocking K⁺ uptake on several growth‐related processes, including solute accumulation, apoplast acidification, and membrane polarization. The results show that light‐stimulated growth and wall acidification of young tobacco leaves is dependent on K⁺ uptake. Light‐stimulated growth is enhanced three‐fold over dark levels with increasing external K⁺, and this effect is blocked by the K⁺ channel blockers, TEA, Ba⁺⁺ and Cs⁺. Incubation in 10 mm TEA reduced light‐stimulated growth and K⁺ uptake by 85%, and completely inhibited light‐stimulated wall acidification and membrane polarization. Although K⁺ uptake is significantly reduced in the presence of TEA, solute accumulation is increased. We suggest that the primary role of K⁺ in light‐stimulated leaf growth is to provide electrical counterbalance to H⁺ efflux, rather than to contribute to solute accumulation and turgor maintenance.     

A model for proton and potassium co-transport during the uptake of glutamine and sucrose by tomato internode disks

Internode disks of tomato (Lycopersicon esculentum cv. Moneymaker) were shaken in glutamine and sucrose solutions. At low external pH (<±5.5), the uptake of these substances was accompanied with K⁺ efflux, at high pH (>±5.5) with K⁺ influx. Low concentrations of external K⁺ (2 mmol l^-1) stimulated the uptake of glutamine, which was strongly inhibited by the supply of high K⁺ concentrations (20 mmol l^-1). The effect of K⁺ was particularly pronounced at high pH-values. Addition of CCCP in light reduced the uptake of glutamine to the same level as in the dark, and stopped the K⁺ fluxes which coincided with the uptake. A model is presented wherein the movements of K⁺ across the membrane are related to co-transport, depending on the membrane potential and the Nernst potential of K⁺.Abbreviation CCCP carbonylcyanide-m-chlorophenylhydrazone     

Cation regulation in Anacystis nidulans

Summary Anacystis nidulans accumulates K⁺ in preference to Na⁺. The majority of the internal K⁺ exchanges with ⁴²K by a first order process at rates of about 1.3 pequiv·cm^-2·sec^-1 in the light and 0.26 pequiv·cm^-2·sec^-1 in the dark. Although the K⁺/K⁺ exchange was stimulated by light and inhibited by 10^-4 M CCCP and 10^-5 M DCMU there are several indications that this cation is passively distributed in Anacystis. Inhibition of the exchange by CCCP and DCMU occurred at concentrations greater than those required to inhibit photosynthesis and the K⁺ fluxes were stimulated by low temperatures. Moreover, although valinomycin stimulated the exchange this compound did not induce a net K⁺ leak. Assuming K⁺ is passively distributed and in free solution within the cytoplasm, as indicated by osmotic studies, would imply that there is an active Na⁺ extrusion pump operating in this organism. As yet there are no firm conclusions about the nature of the energy source for this efflux pump.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Uptake of mercury by Chlorella and its effect on potassium regulation

Summary Addition of mercuric chloride at concentrations which resulted in an overall binding level of about 8 mmoles Hg/l packed cells and above caused a breakdown in the permeability of the cell membrane as indicated by a net efflux of internal K⁺. Below this level in region of 2 mmoles Hg/l packed cells the rate of K⁺ transfer across the cell surface was stimulated without affecting the internal K⁺ level. Maintainence of the stimulation was dependent both on time and dose. Enhancement of the rate of K⁺ turnover was associated with a fast component of the inorganic mercury uptake which could be removed by washing with cysteine. The mercury stimulated K⁺/K⁺ exchange was inhibited by low temperature, by the uncoupler CCCP and the energy transfer inhibitor DCCD. Overall binding concentrations of inorganic mercury below 0.5 mmoles/l packed cells had no effect on the K⁺ transport system. In contrast to mercuric chloride, methyl mercuric chloride over similar concentration ranges did not seem to induce a breakdown in the permeability barrier or directly interact with the K⁺/K⁺ exchange but more likely influenced the latter by inhibiting intracellular processes.     

Inhibition of nitrate uptake by ammonia in a blue-green alga,Anabaena cylindrica

Ammonia at concentrations above 1×10^-5 M inhibits uptake of nitrate in the nitrogen-fixing blue-green alga, Anabaena cylindrica. This inhibition takes place both in the light and in the dark. The rate of nitrate uptake is stimulated by light. Addition of relatively high concentrations of nitrate (1–10 mM) reversibly inhibits ammonia uptake. FCCP, an uncoupler of phosphorylation, inhibits both nitrate and ammonia uptake. Ammonia may inhibit nitrate uptake by reducing the supply of energy (ATP) for active nitrate transport.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxy-phenylhydrazone - CCCP carbonyl cyanide m-chlorophenyl-hydrazone     

The effect of CO2 on potassium transport by Chlorella fusca

Abstract. The effect of CO₂ on net K⁺ uptake by Chlorella fusca grown on high CO₂ levels was examined by passing 1.5% CO₂ through algal suspensions gassed previously with air or CO₂-free air Addition of CO₂ in the light caused a large net uptake of K⁺ (initial velocity 4.2–9.2 mmol s^?1 m^?3 cells) which decreased the concentration of K⁺ in the supernatant from 0.1–0.2 mol m^?3 to 3–10 mmol m^?3. In the dark and in the presence of 30 mmol m^?3 DCMU, no effects were found. Measurement or the unidirectional K⁺ fluxes by using ⁸⁶Rb⁺ as a label showed that in the presence of 1.5% CO₂, influx of K⁺ was increased by a factor of 2–4 while efflux was inhibited completely. CO₂ hyperpolarized the membrane potential (determined through TPP⁺ uptake) from –120mV to –130 mV which could not explain the more than 15,000-fold K⁺ accumulations. In the light, CO₂ lowered the intracellular pH (determined with DMO) by 0.5 units. In the dark and in the presence of DCMU only, a small acidification of 0.1 units was found. During the first 15 min after addition of CO₂ the malate content of the cells increased from 0.7 to 1.5 mol m^?3 packed cells. On the basis of these and earlier results, CO₂-induced net K⁺ uptake is interpreted as a stimulation of an electroneutral ATP-dependent K⁺/H⁺ exchange at the plasmalemma. This exchange acts as a ‘pHstat’ by reducing the intracellular acidification caused by production of acidic assimilation products.     

Coupling of ion transport in green cells ofAtriplex spongiosa leaves to energy sources in the light and in the dark

Summary The coupling of ion transport to energy sources in the light and in the dark in green cells ofAtriplex spongiosa leaves was investigated using light of different qualities, an inhibitor of electron transport (dichlorophenyl dimethyl urea), and an uncoupler (p-CF₃O-carbonyl cyanide phenylhydrazone). Two different mechanisms of ion uptake were, distinguished. (1) A light-dependent Cl^– pump which is linked to light-dependent K⁺ uptake. The energy for this pump is probably derived from photosynthetic electron transport or from nicotinamide adenine dinucleotide phosphate, reduced form. This mechanism is dichlorophenyl dimethyl urea-sensitive and enhanced by uncouplers. (2) A mechanism independent of light, which operates at the same rate in the light and in the dark. This mechanism is sensitive to uncouplers. It is probably aK–Na exchange mechanism since K⁺ and Cl^– uptake and a small net uptake of H⁺ are balanced by Na⁺ loss.     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

Early Effects of Penconazole on H+ and K+ Transport,Electrolyte Leakage and Transmembrane Electrical Potential in Egeria densa Leaves

The early effects of penconazole (PCZ) at relatively high concentration (10^?4 to 5 × 10^?4 M) on changes in pH and in titratable acidity of the medium, transmembrane electrical potential difference (Em), electrolyte leakage and cell morphology were investigated in Egeria densa leaves. At the lowest (10^?4 M) concentration and in the presence of a very low (10 μM) K⁺ concentration, triazole induced an early, moderate hyperpolarization of Em, associated with a decrease of net K⁺ uptake, suggesting some increase in the passive permeability to K⁺. This Em hyperpolarization was no longer detectable at high (2 mM) K⁺_out concentration. At high PCZ concentrations (3 × 10^?4 M and 5 × 10^?4 M) the early hyperpolarization detectable in the presence of a low K⁺_out concentration became transient, and was followed by a marked depolarization. PCZ, at these concentrations, suppressed acidification of the medium, stimulated electrolyte leakage and, in the mesophyll cells, induced some shrinking of the cytoplasm and its disconnection from the cell walls. These results are interpreted as due to an early effect of this triazole leading to the disorganization of the plasma membrane.     

Amine transport at the plasmalemma of Riccia fluitans

Green thallus cells of the aquatic liverwort, Riccia fluitans, are rapidly depolarized in the presence of 1–20 μM NH₄Cl and 5–100 μM CH₃NH₃Cl, respectively. Simultaneously, the membrane conductance is increased from 0.41 to 1.2 S · m^?2. Uptake of [¹⁴C]methylamine is stimulated by increasing [K⁺]_o and inhibited by increasing [Na⁺]_o or [H⁺]_o, is highly voltage sensitive, and saturates at low amine concentrations.Double-reciprocal plots of (a) maximal membrane depolarization and (b) methylamine uptake vs. external amine concentration give apparent K_m values of 2 ± 1 μM ammonia and 25–50 μM methylamine; K_m values for changes in conductance and membrane current are greater and voltage dependent. Whereas the amine transport into the cell is strongly inhibited by CN^?, the amine efflux is stimulated.The current-voltage characteristics of the ammonia transport are represented by a sigmoid curve with an equilibrium potential of ?60 mV, and this is understood as a typical carrier curve with a saturation current of about 70 mA · m^?2. It is further concluded that the evidently carrier-mediated transport is competitive for the two amines tested, and that ammonia and methylamine are transported in the protonated form as NH₄⁺ and CH₃NH₃⁺ into the cytoplasm.     

Glucose transport and metabolism in Gymnodinium breve

Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K_FD of 11 μM and a V_max of 1 × 10^?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (K_i = 40 μM), mannose (K_i = 12O μM), and 2-deoxy-d-glucose (K_i = 190 μM) and non-competitively inhibited by galactose (K_i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.     

Uncoupler and anaerobic resistant transport of phosphate in Escherichia coli

Active transport of inorganic phosphate into whole cells of a strain (AB3311) derived from $\text{Escherichia coli}$ K12 was found to be partially resistant to 50 μM carbonyl cyanide $\text{m}$-chlorophenyl hydrazone (CCCP), a powerful uncoupler of oxidative phosphorylation. The presence of 10 mM dithiothreitol (DTT) before the addition of CCCP completely prevented the inhibition of phosphate uptake caused by the uncoupler. The addition of DTT to the CCCP-inhibited system restored phosphate uptake to the control rate even when added 5 min after the phosphate transport assay was started. This uncoupler resistant transport is insensitive to anaerobiosis, or the addition of 10 mM KCN which reduces oxygen consumption to less than 1% that of aerobic controls. Additional studies of transport in a mutant (CBT302) deficient in membranebound Ca²⁺-, Mg²⁺-ATPase activity also demonstrated the retention of appreciable inorganic phosphate uptake under anaerobic conditions.     

Nature of the light-induced h efflux and na uptake in cyanobacteria

We investigated the nature of the light-induced, sodium-dependent acidification of the medium and the uptake of sodium by Synechococcus. The rate of acidification (net H⁺ efflux) was strongly and specifically stimulated by sodium. The rates of acidification and sodium uptake were strongly affected by the pH of the medium; the optimal pH for both processes being in the alkaline pH range. Net proton efflux was severely inhibited by inhibitors of adenosine triphosphatase activity, energy transfer, and photosynthetic electron transport, but was not affected by the presence of inorganic carbon (C_i). Light and C_i stimulated the uptake of sodium, but the stimulation by C_i was observed only when C_i was present at the time sodium was provided. Amiloride, a potent inhibitor of Na⁺/H⁺ antiport and Na⁺ channels, stimulated the rate of acidification but inhibited the rate of sodium uptake. It is suggested that acidification might stem from the activity of a light dependent proton excreting adenosine triphosphatase, while sodium transport seems to be mediated by both Na⁺/H⁺ antiport and Na⁺ uniport.     

Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores

(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg²⁺). I₅₀ values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min^?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg²⁺ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg²⁺ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg²⁺. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg²⁺ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg²⁺ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.     

Stimulation of growth and ion uptake in bean leaves by red and blue light

Red and blue light both stimulate growth and ion accumulation in bean (Phaseolus vulgaris L.) leaves, and previous studies showed that the growth response is mediated by phytochrome and a blue-light receptor. Results of this study confirm that there is an additional photosynthetic contribution from the growing cells that supports ion uptake and growth. Disc expansion in the light was enhanced by exogenous K⁺ and Rb⁺, but was not specific for anions. Light increased K⁺ accumulation and the rate of ⁸⁶Rb⁺ uptake by discs, over darkness, with no effect of light quality. The photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, inhibited light-driven ⁸⁶Rb⁺ uptake by 75%. Light quality caused differences in short-term kinetics of growth and acidification of the leaf surface. At comparable fluence rates (50 μmol m⁻² s⁻¹), continuous exposure to blue light increased the growth rate 3-fold after a 2-min lag, whereas red light caused a smaller growth response after a lag of 12 min. In contrast, the acidification of the leaf surface normally associated with growth was stimulated 3-fold by red light but only slightly (1.3-fold) by blue light. This result shows that, in addition to acidification caused by red light, a second mechanism specifically stimulated by blue light is normally functioning in light-driven leaf growth.     

Symport of proton and sucrose in plasma membrane vesicles isolated from spinach leaves

The mechanism of sucrose transport was investigated in plasma membrane (PM) vesicles isolated from spinach (Spinacia oleracea L.) leaves. PM vesicles were isolated by aqueous two-phase partitioning and were equilibrated in pH 7.8 buffer containing K⁺. The vesicles rapidly accumulated sucrose in the presence of a transmembrane pH gradient (ΔpH) with external pH set at 5.8. The uptake rate was slow at pH 7.8. The K⁺-selective ionophore, valinomycin, stimulated uptake in the presence of a ΔpH, and the protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), greatly inhibited ΔpH-dependent sucrose uptake. Addition of sucrose to the vesicles resulted in immediate alkalization of the medium. Alkalization was stimulated by valinomycin, was abolished by CCCP, and was sucrose-specific. These results demonstrate the presence of a tightly coupled H⁺/sucrose symporter in PM vesicles isolated from spinach leaves.     

Effect of ouabain upon diuretic-sensitive K+ transport in cultured cells evidence for separate modes of operation of the transporter

(1) Unidirectional K⁺ (⁸⁶Rb) influx and efflux were measured in subconfluent layers of MDCK renal epithelial cells and HeLa carcinoma cells. (2) In both MDCK and HeLa cells, the furosemide-inhibitable and chloride-dependent component of K⁺ influx/efflux was stimulated 2-fold by a 30 min incubation in 1 · 10^?3 M ouabain. (3) Measurements of net K⁺ loss and Na⁺ gain in ouabain-treated cells at 1 h failed to show any diuretic sensitive component, confirming the exchange character of the diuretic-sensitive fluxes. (4) Prolonged incubations for 2.5 h in ouabain revealed a furosemide- and anion-dependent K⁺ (Cl^?) outward net flux uncoupled from net Na⁺ movement. Net K⁺ (Cl^?) outward flux was half-maximally inhibited by 2 μM furosemide. (5) After 2.5 h ouabain treatment, the anion and cation dependence of the diuretic-sensitive K⁺ influx/efflux were essentially unchanged when compared to untreated controls.     

CHLORIDE UPTAKE BY ANACYSTIS NIDULANS (CYANOPHYCEAE)1

Anacystis nidulans (Richt.) Drouet & Daily (UTEX 625), grown in batch culture with 0.5% CO₂ in air, was supplied with chloride labelled with ³⁶Cl in light and dark. Uptake in light was stimulated relative to uptake in darkness. A single transport system for Cl^? with an apparent K_m for Cl^? of 0.14 mM was identified. Chloride in the cells reached a maximum value after 30–50 min at 25 C. At this point the internal Cl^? concentration was calculated to be 60-fold the external (0.1 mM) in light and 37-fold in darkness. DCMU (3-[3,4-dichlorophenyl]–1, 1-dime-thylurea), at concentrations which abolished photosynthetic O₂ evolution did not inhibit Cl^? uptake in light. Carbonyl cyanide m-chlorophenyl hydrazone (CCCP), at uncoupling concentrations for photosynthesis and dark respiration, strongly inhibited Cl^? uptake in light and darkness. N,N'-dicyclohexyl carbodiimide (DCCD), an energy transfer inhibitor, inhibited light Cl^? uptake more slowly than photosynthesis but had no effect on dark Cl^? uptake. It is concluded that Cl^? uptake in A. nidulans was active in light and darkness, and that ATP was the probable energy source for transport.     

Light-dependent potassium uptake by Pisum sativum leaf fragments

A net K⁺ influx into chopped pea leaves bathed in 5 mM KCl,0.26 M sucrose and illuminated with 4000 lux amounted to about7.5 µmoles/g fresh weight-hr, while essentially no netflux occurred in the dark. This light-dependent K⁺ uptake waslinear with time for nearly 2 hr and continuously increasedas the light intensity was raised to 9000 lux. Over half ofthe K⁺ uptake was balanced by H⁺ release into the bathing solution,possibly by a mechanism in which bicarbonate was the anion accompanyingK⁺. The replacement of Cl^– by HCO₃^– increased thelight-dependent K⁺ uptake to 56 µmoles/g fresh weight-hr.About 23% of the light-dependent K⁺ uptake in 5 mM KCl was accompaniedby a Cl^– uptake. This net Cl^– influx was less sensitiveto the uncoupler tri-Fl-CCP and more sensitive to DCMU in thebathing solution than was the K⁺ uptake. The remaining net K⁺influx into pea leaf fragments was balanced by effluxes of sodium(accounting for 5%), magnesium (8%) and calcium (1%). (Received March 31, 1969; )