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.     

Electrochemical Potential Gradients of H+, K+, Ca2+, and Cl- across the Tonoplast of the Green Alga Eremosphaera Viridis

Using ion-selective microelectrodes, we measured the activity of H+, K+, Ca2+, and Cl- and the electrical potential both in the vacuole and in the cytoplasm of the unicellular green alga Eremosphaera viridis to obtain comparable values of the named parameters from the same object under identical conditions. The cytosol had a pH of 7.3, and activities of the other ions were 130 mM K+, 160 nM Ca2+, and 2.2 mM Cl-. We observed only small and transient light-dependent changes of the cytosolic Ca2+ activity. The vacuolar K+ activity did not differ significantly from the cytosolic one. The Ca2+ activity inside the vacuole was approximately 200 [mu]M, the pH was 5.0, and the Cl- activity was 6.2 mM. The concentrations of K+, Ca2+, and Cl- in cell extracts were measured by induction-coupled plasma spectroscopy and anion chromatography. This confirmed the vacuolar activities for K+ and Cl- obtained with ion-selective microelectrodes and indicated that approximately 60% of the vacuolar Ca2+ was buffered. The tonoplast potential was vanishingly low ([less than or equal to][plus or minus]2 mV). There was no detectable electrochemical potential gradient for K+ across the tonoplast, but there was, however, an obvious electrochemical potential gradient for Cl- (-26 mV), indicating an active accumulation of Cl- inside the vacuole.     

Short communication. A steep Ca2[IMAGE]-dependence of a K[IMAGE] channel in a unicellular green alga

An increase of cytosolic Ca² in the unicellular green alga Eremosphaera viridis activities Ca²-dependent K channels causing a hyperpolarization of the plasma membrane. Data from parallel calcium, and potential measurements were combined with I/V relationships. This yielded a steep Ca²-dependence of K channels with a co-operativity of 4 and an affinity of 300 nM.Key words: Eremosphaera viridis, plasma membrane, Ca²-dependent K channel, co-operative binding.      

Effect of Ammonium on Dark-CO2 Fixation and on Cytosolic and Vacuolar pH Values in Eremosphaera viridis de Bary (Chlorococcales)

At constant external [CO₂], rates of dark-CO₂ fixation of theunicellular green alga Eremosphaera viridis were drasticallyincreased (up to 40-fold) by addition of ammonium (NH₃+ NH₄⁺)at external pH values (pH₀) between 6.0 and 8.0. The cytosolicpH was monitored under identical conditions by micro-pH-electrodemeasurements, and cytosolic and vacuolar pH by the ³¹P-NMR technique.Addition of ammonium (5.0 mol m^– pH₀ 7.0) caused a rapidand transient acidification of the cytosol during the first4 min. Thereafter, the cytosolic pH remained constant at itsoriginal value. A rather constant cytosolic pH was also confirmedby ³¹P-NMR measurements, which, in addition, indicated a slowalkalization of the vacuole (about 0.5 units within 30 min afteraddition of ammonium). Since the dramatic stimulation of dark-CO₂ fixation by ammoniumis not mediated by an alkalization of the cytosol, nor by directammonium effects on phosphoenolpyruvate carboxylase (PEPC, E.C.4.1.1.31 [EC] ), the role of vacuolar alkalization as a possible triggerfor the stimulation of PEP-carboxylase is discussed. Key words: Cytosolic pH, dark-CO₂ fixation, pH-regulation, vacuolar pH     

Neutral Red as a Redox Dye Induces K+ Efflux and Current-Voltage Changes in Eremosphaera,Lemna, and Guard Cells*

Membrane effects of the redox and pH indicator neutral red were studied with the chlorococcal alga Eremosphaera viridis, with Lemna gibba, and with “isolated” guard cells in epidermal peels of Valerianella locusta. Neutral red was extracellularly reduced and caused transmembrane current-voltage changes, an increase in membrane conductance by about 14 nS, an apparent K⁺ net efflux of up to 120 μmol g^?1 FW in 5 min, and an intracellular acidification by up to 0.7 pH units. Neutral red-triggered K⁺ net efflux was most pronounced at low pH, at an E_o more positive than ?200 mV, and without extracellular Ca²⁺. From the experimental data it is concluded that, due to the redox function of the phenazine molecule, extracellular neutral red triggers a trans-plasmalemma e^? transfer, leading to strong membrane depolarization and charge compensating K⁺ net efflux, in addition to some unspecific ion release. As a consequence the intracellular concentration of strong cations relative to strong anions (SID) decreases, resulting in intracellular acidification.     

Light-induced cytoplasmic pH changes and their interrelation to the activity of the electrogenic proton pump in Riccia fluitans

In green thallus cells of the aquatic liverwort Riccia fluitans light-induced pH changes have been measured, using a turgor-resistant pH-sensitive microelectrode. (1) Light-off/-on causes oscillations of the cytoplasmic pH (pH_c), as well as of the membrane potential difference across the plasmalemma (ψ). Beside the well-known ψ_m changes, the first detectable pH_c change following light-off is a transient acidification of about 0.3 pH units, whereas light-on causes a transient alkalinization of roughly 0.4 pH units. (2) 1 μM DCMU eliminates these transients. (3) In the presence of 0.2 mM procaine, which alkalizes the cytoplasm to over pH 8, the light-induced ψ_m transients are enhanced, but are almost absent, if pH_c is acidified to 6.9 by 1 mM acetate. It is suggested that the transient light-induced changes in pH_c are caused by light-dependent proton translocation across the thylakoid membranes, and it is concluded that the subsequent changes in ψ_m are essentially the result of altered activities of the electrogenic proton pump in the plasmalemma, due to the observed fluctuations of its substrate, the proton.     

Spectral sensitivity of light induced respiratory activity of photoreceptor mitochondria in the intact fly

Summary FlyCalliphora erythrocephala (white eyed) photoreceptors were investigated in intact, living animals by microspectrofluorometry in vivo. The fluorescence of mitochondrial flavoproteins (Tinbergen and Stavenga 1986) was used to monitor transient changes in oxidative metabolism, which were induced by a test light following a stimulus of variable intensity.Two stimulus types were applied, a brief, activating illumination and a prolonged, adapting illumination, respectively. The intensity ranges of activation and adaptation appear to be separated by ca. 3 log units.Action spectra for inducing a criterion activation or adaptation of the light-dependent mitochondrial system are virtually indistinguishable and closely resemble the spectral sensitivity measured electrophysiologically, thus reinforcing the hypothesis (Hamdorf and Langer 1966; Stavenga and Tinbergen 1983) that the light-induced changes in oxidative metabolism in fly photoreceptors are closely linked to the phototransduction process.On the basis of the literature we conclude that a light-induced rise in cytosolic calcium concentration is the likely cause for enhancing mitochondrial activity.     

Secondary Carotenoids of Eremosphaera viridis De Bary (Chlorophyceae) Under Nitrogen Deficiency*

Under nitrogen deficiency the unicellular chlorococcalean green alga, Eremosphaera viridis De Bary, was able to synthesize secondary carotenoids (SC). Nine SC were identified as six astaxanthin esters, echinenone, canthaxanthin and a lutein ester, previously not described in green algae under nitrogen deficiency. These SC, jS-carotene and the main part of lutein were located in lipid bodies outside the chloroplasts in the cytosol. The synthesis of SC could be inhibited by the herbicides norflurazon and nicotine. This result supported the idea that SC in cells ot Eremosphaera viridis were synthesized de novo rather than derived from primary carotenoids.     

Transient Cytoplasmic pH Changes in Correlation with Opening of Potassium Channels in Eremosphaera

Steigner, W. Khler, K., Simonis, W. and Urbach, W. 1988. Transientcytoplasmic pH changes in correlation with opening of potassiumchannels in Eremosphaera.—J. exp. Bot. 39: 23–36. The role of the cytoplasmic pH (pH_c) of Eremosphaera viridisin the signal transduction chain after light-off from the chloroplaststo the K⁺ channels in the plasmalemma of this unicellular algawas investigated. The temporary opening of K⁺ channels is indicatedby a transient hypcrpolarization (TP). To record rapid changesof pH_c, continuous measurements with pH sensitive micro-electrodeswere carried out. (i) Under normal conditions pH_c in the light(7·56 ±0·2) did not differ from pHc inthe dark (7·62 ±0·2). (ii) The vacuolepH ranged between 4·8 and 5·2. (iii) After light-offa rapid transient acidification of pHc O19±0·07occurred and a TP was released, (iv) In every case, the startof the transient acidification after light-off preceded thehyperpolarization by about 3s. (v) Light-on caused a rapid transientalkalinization but never a TP. (vi) Change to acid externalmedium (3.2) transiently acidified the cytoplasm and was ableto release a TP. (vii) After addition of NH₄Cl, pH_c again showeda rapid transient acidification and the release of a TP. The origin of the protons appearing in the cytoplasm after light-offis discussed critically with respect to the buffer capacity.Either direct or indirect translocation is a possible mechanismfor the movement of H⁺ from the chloroplasts into the cytoplasm.The intracellular acidification and its relation to the openingof potassium channels in the plasmalemma leads us to suggestthat a sudden change of pH_c is a potent internal signal factorin Eremosphaera viridis. Key words: Cytoplasmic pH, transient potential, K⁺–channels, Eremosphaera viridis     

Aluminium induces rapid changes in cytosolic pH and free calcium and potassium concentrations in root protoplasts of wheat (Triticum aestivum)

Addition of aluminium chloride (50 μM Al) caused different effects on the transmembrane electrical potential (PD) of root cells in Al-tolerant wheat (Triticum aestivum) cv. Kadett and Al-sensitive cv. WW 20299. As changes in PD of plant cells may depend on transient fluxes of protons, potassium and/or calcium through cell membranes, the effect of Al was investigated on the cytosolic concentrations of these ions in protoplasts isolated from root tips of the same cultivars. The tetra[acetoxymethyl] esters of the fluorescent dyes bis-carboxyethyl-carboxyfluorescein, BCECF, K⁺-binding benzofuran isophthalate, PBFI, and the stilbene chromophore Fura 2-AM were used to determine pH, K⁺ and Ca²⁺, respectively. Changes in fluorescence ratios, directly reflecting changes in [H⁺], [K⁺] and [Ca²⁺] in the cytosol, were determined by photometry fluorescence microscopy. Additions and removals of Al to and from both cultivars caused hyperpolarizations and depolarizations, respectively, but only in the sensitive cv. WW 20299 did the resting PD decrease gradually. Addition of Al to the protoplasts caused rapid changes in cytosolic pH, free [K⁺] and [Ca²⁺]. In both cultivars Al caused a transient oscillating increase in cytosolic [Ca²⁺] for 1 or 2 min and a rapid pH-dependent change in cytosolic [K⁺]. At pH 5 the presence of K⁺ in the medium diminished the Al-induced decrease in cytosolic [K⁺]. Aluminium (50 μM) induced a transient increase in cytosolic [H⁺] (pH decreased) in both cultivars, but the cytosolic pH returned to its initial value only in the Al-tolerant cv. Kadett. In the Alsensitive cv. WW 20299, repeated additions of Al caused a gradual decline in pH. Moreover, in the presence of 1 mM KCl, pH recovered completely in both cultivars. Since only the effect on pH differed in the two cultivars, the more toxic effect of Al on the cv. WW 20299 should be related to the change in pH.     

Light-induced cytosolic calcium transients in green plant cells. I. Methodological aspects of chlorotetracycline usage in algae and higher-plant cells

The fluorescent dye chlorotetracycline (CTC) has several disadvantages compared with ratio dyes like Fura-dextran. However, in many plant tissues the derivatives of Fura cannot be loaded. Thus, the pitfalls and possible precautions for the measurement of the light-induced changes in cytosolic free calcium concentration ([Ca²⁺]_c) were investigated in algae and higher plants. Eremosphaeraviridis de Bary and the flowing cytosol in whorl cells of Characorallina Klein ex Willd. were used as examples for possible pressure injection of Fura-dextran or bis-carboxyethyl-carboxy-fluorescein (BCECF) dextran, illustrating the better calibration in absolute terms provided by these dyes. However, here it is shown that CTC works better than Fura-dextran for monitoring the light-induced changes in [Ca²⁺]_c in the ectoplasm close to the plasma membrane in Chara. Protoplasts of Solanumnigrum L. and whole intact leaves of Viciafaba L. and Nicotianatabacum L. were used as examples of cells that were too fragile for pressure injection of Fura-dextran. The sensitivity of CTC to pH may cause artefacts when light-induced changes in [Ca²⁺]_c in intact leaves are to be measured. If some precautions are met, this problem and others (requirement of constant temperature, sensitivity to other ions, effect on plasma-membrane Ca²⁺ permeability) can be circumvented, thus making CTC a suitable dye for monitoring light-induced changes in [Ca²⁺]_c in a broad spectrum of different plant cells, tissues and species. Received: 3 May 1997 / Accepted: 19 May 1998     

Key-word system of indexing

The effects of uncouplers of photophosphorylation on the P-S₁ transient of the fluorescence induction in darkadapted intact chloroplasts of Bryopsis maxima were studied to examine the mechanism of light-dependent regulatory changes in electron transport. (1) Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and nigericin slowed down the fluorescence quenching from P to S₁, whereas the transient was significantly accelerated by the addition of NH₄Cl and methylamine. (2) The P-S₁ decline was slowed down at low pH of the suspending medium, suggesting sensitivity of the transient to the stroma pH. The inhibitory effect of nigericin was markedly enhanced at low pH and at low KCl concentrations, whereas the ionophore stimulated the transient at high pH and at high KCl concentrations. Similar results were obtained on the combined addition of CCCP and valinomycin. (3) Nigericin and the CCCP-valinomycin couple altered the internal pH of intact chloroplast through the H⁺-K⁺ exchange across the outer limiting membrane. The fluorescence decline was rapid at alkaline internal pH but was suppressed with lowering internal pH below 8.0 (4) A similar internal pH dependence of the transient was obtained when the internal pH was changed by the addition of NH₄Cl and acetate. (5) It is proposed that the photoactivation of electron transport is regulated by the stroma pH. The progress of the photoactivation is slow at acidic or neutral pH but is significantly accelerated by light-induced alkalinization near the light-regulation site of electron transport located on the outer surface of the thylakoid membrane.     

Different Xanthines Cause Membrane Potential Oscillations in a Unicellular Green Alga Pointing to a Ryanodine/cADPR Receptor Ca2+ Channel

In the unicellular green alga Eremosphaera viridis caffeineinduces oscillations of cytosolic Ca²⁺ which cause membranepotential oscillations. Application of the caffeine analoguesisocaffeine, theophylline, and isotheophylline resulted in membranepotential oscillations with a structure-activity relationshipcomparable to isolated ryanodine/cyclic ADPribose (cADPR) receptorCa²⁺ channels from animal cells. (Received November 24, 1998; Accepted February 1, 1999)     

Comparative characterization of the two primary pumps, H+-ATPase and Na+-ATPase, in the plasma membrane of the marine alga Tetraselmis viridis

The transport characteristics of the plasma membrane H⁺‐ATPase (PMHA) and Na⁺‐ATPase (PMNA) from marine unicellular green alga Tetraselmis viridis Rouch. were studied using sealed plasma membrane vesicles isolated from this species. The activities of the ATPases were investigated by monitoring the ATP‐dependent pH changes in the vesicle lumen. PMHA operation led to acidification of the vesicle lumen, whereas Na⁺ translocation into plasma membrane vesicles catalysed by PMNA was accompanied by H⁺ efflux, namely the alkalization of the vesicle lumen (Balnokin et al., FEBS Lett 462: 402–406, 1999). The intravesicular acidification and alkalization were detected with the ΔpH probe acridine orange and the pH probe pyranine, respectively. PMHA and PMNA were found to operate in distinct pH regions, maximal activity of PMHA being observed at pH 6.5 and that of PMNA at pH 7.8. Kinetic studies revealed that the ATPases have similar affinities to their primary substrate, MgATP complex (an apparent K_m = 34 ± 6.2 µM for PMHA and 73 ± 8.7 µM for PMNA). At the same time, the ATPases were differently affected by free Mg²⁺ and ATP. Free Mg²⁺ appeared to be a mixed‐type inhibitor for PMNA (K_i′ = 210 µM) but it did not suppress PMHA. Conversely, free ATP markedly suppressed PMHA being a mixed‐type inhibitor (K_i′ = 330 µM), but PMNA was affected by free ATP only slightly. Furthermore, the ATPases substantially differed in their sensitivities to the inhibitors of membrane ATPases, such as orthovanadate, N‐ethylmaleimide and N,N′‐dicyclohexylcarbodiimide. The differences found in the properties of the PMHA and PMNA are discussed in terms of regulation of their activities and their capacity to be involved in cytosolic ion homeostasis in T. viridis cells.     

Auxin causes oscillations of cytosolic free calcium and pH inZea mays coleoptiles

In epidermal cells of maize (Zea mays L.) coleoptiles, cytosolic pH (pH_c), cytosolic free calcium, membrane potential and changes thereof were monitored continuously and simultaneously (pH_c/, _m, Ca²⁺/ _m) using double-barrelled ion-sensitive microelectrodes. In the resting cells the cytosolic pH was 7.3–7.5 and the concentration of free calcium was 119±24 nM. One-micromolar indole-3-acetic acid (IAA), added to the external medium at pH 6.0 triggered oscillations in _m, pH_c and free calcium with a period of 20 to 30 min. Acidification of the cytosolic pH increased the cytosolic free calcium. The _m oscillations are attributed to changes in activity of the H⁺-extrusion pump at the plasmalemma, triggered off by pH and controlled by pH regulation (pH oscillation). The origin of the pH_c and Ca²⁺ changes remains unclear, but is possibly caused by auxin-receptor-induced lipid breakdown and subsequent second-messenger formation. It is suggested that the observed cytosolic pH and Ca²⁺ changes are intrinsically interrelated, and it is concluded that this onset of regulatory processes through the phytohormone IAA is indicative of calcium and protons mediating early auxin action in maize coleoptiles. It is further concluded that the double-barrelled ion-sensitive microelectrode is an invaluable tool for investigating in-vivo hormone action in plant tissues.Abbreviations and symbols FC fusicoccin - IAA indole-3-acetic acid - Mes 2-(N-morpholino)ethanesulfonic acid - pH_c cytosolic pH - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - _m membrane potential difference (mV)     

Ca2+-requirement for a blue-light-induced chloroplast translocation inEremosphaera viridis

Summary The light-mediated translocation of chloroplasts inEremosphaera viridis is dependent on blue light near 450 nm, while wavelengths longer than 500 nm are inactive. The plastid translocation results in an aggregation of the organelles close to the nucleus in the center of the cell. After cessation of irradiation, the cells begin to redistribute their plastids in the cytoplasm immediately. Treatments that alter the Ca²⁺ concentration in the cytoplasm ofEremosphaera suggest that the translocation is regulated by calcium. Ultrastructural investigation ofEremosphaera reveals a very characteristic, multilayered and highly-ordered cell wall.     

Review article. Light-dependent signal transduction and transient changes in cytosolic Ca2+ in a unicellular green alga

The physiological function and the molecular mechanisms of Ca²⁺-mediated signal transduction processes were studied in the unicellular green alga Eremosphaera viridis by different electrophysiological and microfluorimetric techniques. A sudden blockage of photosynthetic electron transport by darkening or inhibitors causes a transient hyperpolarization of the plasma membrane. For the alga this transient hyperpolarization seems to be an important mechanism to release monovalent ions and to drive the uptake of divalent cations. The transient hyperpolarization is due to the opening of K⁺ channels and is caused by a rapid transient elevation of the cytosolic free Ca²⁺ concentration ([Ca²⁺]cy spike). Different agonists like caffeine or InsP3 which are known to release Ca²⁺ from internal stores in animal cells, also cause a transient hyperpolarization and a [Ca²⁺]cy spike, similar to darkening. In Eremosphaera the transient hyperpolarization can be used as an indicator for [Ca²⁺]cy spikes. The InsP3 gated and the ryanodine/cADPR gated Ca²⁺ channels which obviously both mediate Ca²⁺ release from internal stores in Eremosphaera do not seem to be involved in the dark-induced [Ca²⁺]cy spikes. Besides single [Ca²⁺]cy spikes, the addition of Sr²⁺ (or caffeine in the absence of divalent cations) causes repetitive [Ca²⁺]cy spikes which may last hours and resemble [Ca²⁺]cy oscillations observed in excitable animal cells. These observations suggest that some principal molecular mechanisms causing single or repetitive [Ca²⁺]cy spikes are conserved from animal to plant cells.     

Potassium channels in Eremosphaera viridis

The dependence of the membrane potential of Eremosphaera viridis on different external concentrations of potassium, sodium, calcium, and protons was compared with the diffusion potential measured in the dark and in the presence of NaN₃. In contrast to some other algae, the membrane potential in the light as well as in the dark seemed to be predominantly determined by the calculated diffusion potential and less by an electrogenic pump which, however, seemed to be involved at potassium concentrations >1 mol·m^-3 and at higher pH_os (>pH 6). Furthermore, some characteristics of an action-potential-like response (CAP) triggered by light-off, and independent of the membrane-potential threshold value, were determined. The CAP had a delay period of 5.4 s and needed 4.5 s for polarization to a plateau. On average, the plateau held for 8.8 s and the CAP lasted 37.7 s. The peak amplitudes of CAP (P _AP) exactly followed the Nernst potential of potassium. Other cations like sodium, calcium and protons did not appreciably affect the peak amplitudes of CAP. From these and other results it can be assumed that the CAP is caused by a temporary opening of potassium channels in the plasma membrane of Eremosphaera (Köhler et al., 1983, Planta 159, 165–171). The release of a CAP by light-off has been partly explained by the participation of a transient increase of proton concentration in the cytoplasm. It was possible to trigger a CAP by external pH changes and by the addition of sodium acetate, thus supporting the hypothesis that a pH decrease in the cytoplasm may be one element of the signal transfer from the photosynthetic system to the potassium channels in the plasmalemma. Calcium also seemed to have an influence on triggering the CAP.Abbreviations and symbols CAP chemical-induced action-potential-like response - E _D calculated diffusion potential (mV) - E _D ^* measured diffusion potential (mV) - E _K potassium equilibrium potential (mV) - E _m membrane potential (mV) - P _AP peak of action potential (mV) Part II will appear in Planta, Vol. 167, No. 1, 1986     

Isolation of Protoplasts from the Coccal Green Alga Eremosphaera viridis De Bary for Patch-clamp Measurements

A method for enzymatic isolation of protoplasts from the unicellular green alga Eremosphaera viridis for patch-clamp measurements is described. Viable protoplasts with “patch-clean” plasma membranes could only be isolated when combining high enzyme concentrations and long incubation times. In whole-cell recordings the protoplasts exhibited electrical properties similar to those measured in intact cells. Taken together with the protoplasts' ability for rapid deplasmolysis after transfer into hypotonic solution, this indicates the viability of the isolated protoplasts.     

Suitability of Arabidopsis for studies on intracellular pH regulation: correlation between H+ pump activity, cytosolic pH and malate level in wild-type and in a mutant partially insensitive to fusicoccin

Data are presented on the suitability of Arabidopsis thaliana seedlings for studies on intracellular pH regulation. In this material, grown in the dark in liquid medium, the determination of weak acid distribution at equilibrium provides an adequate method for calculating cytosolic pH values, in spite of the failure of benzylamine as a vacuolar pH probe. The stimulation of the H⁺ pump by K⁺ or K⁺ and fusicoccin (FC) is associated with a marked alkalinization of both cytosol and cell sap, and with a strong increase in malate level, whereas its inhibition by erythrosin B (EB) leads to the opposite effects. A good quantitative correlation is evident between the changes in net H⁺ extrusion and those in intracellular pH and malate content, in particular, with FC+K⁺. Cell sap buffer capacity is strongly influenced by the different treatments, its changes being substantially accounted for by changes in malate level. A comparison between the values of intracellular pH and malate level in wt and in the 5-2 mutant shows that in the mutant the cytosolic pH is always more acidic, and the intracellular alkalinization induced by FC+K⁺ and also by K⁺ alone is significatively lower. These results support the view that the partial insensitivity of 5-2 to FC is due to a reduced functionality of the H⁺-extruding system on which FC acts, and that the depression of the H⁺ pump activity in the mutant does not depend on a possible regulation by constitutively higher cytosolic pH values.