(Bush, 1995; Trewavas et al., 1996; Webb et al., 1996) and animal cells (Petersen et al., 1994; Bootman and Berridge, 1995 Tsien, 1990; Fewtrell, 1993; Petersen et al., 1994). For plant cells only a few reports about [Ca 2ϩ ] cy oscillations exist. Phytohormone-induced [Ca 2ϩ ] cy fluctuations reported earlier were strongly damped and ceased after a few repetitions (Felle, 1988; Schroeder and Hagiwara, 1990). Only recently stable [Ca 2ϩ ] cy oscillations were observed in plant cells (Johnson et al., 1995; McAinsh et al., 1995; Ehrhardt et al., 1996; Bauer et al., 1997). In some cases these [Ca 2ϩ ] cy oscillations display a baseline spiking pattern, which means that repetitive [Ca 2ϩ ] cy spikes are separated by a constant [Ca 2ϩ ] cy baseline (Ehrhardt et al., 1996; Bauer et al., 1997). There are indications for a physiological function of [Ca 2ϩ ] cy oscillations in plant cells (Johnson et al., 1995; McAinsh et al., 1995; Ehrhardt et al., 1996). The mechanisms generating [Ca 2ϩ ] cy oscillations in plant cells are unknown.The unicellular green alga Eremosphaera viridis responds to various stimuli with single or repetitive [Ca 2ϩ ] cy spikes (Bauer et al., 1997

Using ion-selective microelectrodes, we measured the activity of H+, K+, CaZ+, and CI-and the electrical potential both in the vacuole and in the cytoplasm of the unicellular green alga €remo-sphaera viridis to obtain comparable values of the named parameters from the same object under identical conditions. l h e cytosol had a pH of 7.3, and activities of the other ions were 130 mM K+, 160 nM Ca2+, and 2.2 mM CI-. W e observed only small and transient light-dependent changes of the cytosolic ca*+ activity. The vacuolar Kf activity did not differ significantly from the cytosolic one. l h e Ca2+ activity inside the vacuole was approximately 200 ~LM, the pH was 5.0, and the CI-activity was 6.2 mM. l h e concentrations of K+, Ca*+, and CI-in cell extracts were measured by induction-coupled plasma spectroscopy and anion chromatography. l h i s confirmed the vacuolar activities for K+ and CI-obtained with ion-selective microelectrodes and indicated that approximately 60% of the vacuolar Ca2+ was buffered. The tonoplast potential was vanishingly low ( 5 2 2 mV). There was no detectable electrochemical potential gradient for K+ across the tonoplast, but there was, however, an obvious electrochemical potential gradient for CI-(-26 mV), indicating an active accumulation of CI-inside the vacuole.

Summary• Short-term cytosolic pH regulation has three components: H + binding by buffering groups; H + transport out of the cytosol; and H + transport into the vacuole. To understand the large differences plants show in their tolerance to acidic environments, these three components were quantified in the acidophilic unicellular green alga Eremosphaera viridis.• Intracellular pH was recorded using ion-selective microelectrodes, whereas constant doses of weak acid were applied over different time intervals. A mathematical model was developed that describes the recorded cytosolic pH changes. Recordings of cytosolic K + and Na + activities, and application of anion channel inhibitors, revealed which ion fluxes electrically compensate H + transport.• The cytosolic buffer capacity was β = 30 mM. Acidification resulted in a substantial and constant H + efflux that was probably driven by the plasmalemma H + -ATPase, and a proportional pH regulation caused by H + pumped into the vacuole. Under severe cytosolic acidification (≥ 1 pH) more than 50% of the ATP synthesized was used for H + pumping. While H + influx into the vacuole was compensated by cation release, H + efflux out of the cell was compensated by anion efflux.• The data presented here give a complete and quantitative picture of the ion fluxes during acid loading in an acidophilic green plant cell.

Summary. Recently Plieth et al. [Protoplasma (1997) 198: 107-124; 199: 223] gave a quantitative picture of the Ca 2+ and H § buffers in green algae which we would like to comment. In that paper a mechanistic model was derived which describes the relationship between cytosolic Ca 2+ and H + assuming that Ca z+ and H + interact with the same binding site of a Ca>-H+-exchange buffer. But the increase of the cytosolic free Ca 2+ concentration observed upon acidification can alternatively be described by a co-operative (n = 2) protonation of a Ca2+/I-I+-binding buffer pointing to an allosteric mechanism of Ca 2+ liberation. Furthermore we present evidences that the cytosolic buffer capacities for H + (90 mM/pH) and Ca 2+ (20 mM/pCa) given for Eremosphaera viridis were overestimated by a factor of three and three orders of magnitude, respectively.

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