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Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability

2011; Wiley; Volume: 190; Issue: 3 Linguagem: Inglês

10.1111/j.1469-8137.2010.03633.x

1469-8137

K. Suffrian, Kai G. Schulz, Magdalena A. Gutowska, Ulf Riebesell, Markus Bleich,

Photosynthetic Processes and Mechanisms

New PhytologistVolume 190, Issue 3 p. 595-608 Full paperFree Access Cellular pH measurements in Emiliania huxleyi reveal pronounced membrane proton permeability K. Suffrian, K. Suffrian Physiologisches Institut, CAU Kiel, Olshausenstraße 40, D-24098 Kiel, GermanySearch for more papers by this authorK. G. Schulz, K. G. Schulz Leibniz Institute of Marine Sciences (IFM-GEOMAR), Düsternbrooker Weg 20, D-24105 Kiel, GermanySearch for more papers by this authorM. A. Gutowska, M. A. Gutowska Physiologisches Institut, CAU Kiel, Olshausenstraße 40, D-24098 Kiel, GermanySearch for more papers by this authorU. Riebesell, U. Riebesell Leibniz Institute of Marine Sciences (IFM-GEOMAR), Düsternbrooker Weg 20, D-24105 Kiel, GermanySearch for more papers by this authorM. Bleich, M. Bleich Physiologisches Institut, CAU Kiel, Olshausenstraße 40, D-24098 Kiel, GermanySearch for more papers by this author K. Suffrian, K. Suffrian Physiologisches Institut, CAU Kiel, Olshausenstraße 40, D-24098 Kiel, GermanySearch for more papers by this authorK. G. Schulz, K. G. Schulz Leibniz Institute of Marine Sciences (IFM-GEOMAR), Düsternbrooker Weg 20, D-24105 Kiel, GermanySearch for more papers by this authorM. A. Gutowska, M. A. Gutowska Physiologisches Institut, CAU Kiel, Olshausenstraße 40, D-24098 Kiel, GermanySearch for more papers by this authorU. Riebesell, U. Riebesell Leibniz Institute of Marine Sciences (IFM-GEOMAR), Düsternbrooker Weg 20, D-24105 Kiel, GermanySearch for more papers by this authorM. Bleich, M. Bleich Physiologisches Institut, CAU Kiel, Olshausenstraße 40, D-24098 Kiel, GermanySearch for more papers by this author First published: 07 February 2011 https://doi.org/10.1111/j.1469-8137.2010.03633.xCitations: 86 Author for correspondence:Markus BleichTel: +49 (0)431 880 2961Email: m.bleich@physiologie.uni-kiel.de AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Summary • To understand the influence of changing surface ocean pH and carbonate chemistry on the coccolithophore Emiliania huxleyi, it is necessary to characterize mechanisms involved in pH homeostasis and ion transport. • Here, we measured effects of changes in seawater carbonate chemistry on the fluorescence emission ratio of BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) as a measure of intracellular pH (pHi). Out of equilibrium solutions were used to differentiate between membrane permeation pathways for H+, CO2 and HCO3−. • Changes in fluorescence ratio were calibrated in single cells, resulting in a ratio change of 0.78 per pHi unit. pHi acutely followed the pH of seawater (pHe) in a linear fashion between pHe values of 6.5 and 9 with a slope of 0.44 per pHe unit. pHi was nearly insensitive to changes in seawater CO2 at constant pHe and HCO3−. An increase in extracellular HCO3− resulted in a slight intracellular acidification. In the presence of DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), a broad-spectrum inhibitor of anion exchangers, E. huxleyi acidified irreversibly. DIDS slightly reduced the effect of pHe on pHi. • The data for the first time show the occurrence of a proton permeation pathway in E. huxleyi plasma membrane. pHi homeostasis involves a DIDS-sensitive mechanism. Introduction Emiliania huxleyi is the most abundant and cosmopolitan calcifying phytoplankton (Paasche, 2002). This coccolithophore with a diameter of 4–5 μm thrives in the euphotic zone of cold temperate to tropical regions (Westbroek et al., 1993). It forms extensive blooms covering up to 250 000 km2 (Holligan et al., 1983; Balch et al., 2010) and is considered to be responsible for the production of up to 50% of calcite on Earth (Westbroek et al., 1989; Broecker & Clark, 2009). Calcification at the cellular level is related to photosynthesis and acid–base metabolism. Emiliania huxleyi produces calcite platelets, so-called coccoliths, in a specialized intracellular compartment, the coccolith vesicle (Paasche, 2002), from where they are exocytosed upon completion. For calcification E. huxleyi depends on the supply of dissolved inorganic carbon (DIC). The same applies for photosynthesis. As in other algae, primary carbon fixation is CO2-dependent, as it is mediated by the enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) in the Calvin–Benson cycle, producing C3 compounds. However, recent studies have shown a C4 anaplerotic β-carboxylation reaction, producing C4 compounds concomitantly operating in E. huxleyi (Tsuji et al., 2009). This pathway could supplement the cells as a temporal 'CO2 storage' in DIC uptake. Both calcification and photosynthesis have the potential to interfere with cytosolic pH homeostasis. It is therefore of special interest by which mechanisms the cell provides and regulates cell membrane permeability to CO2, HCO3− and H+. Membrane permeability for a substrate depends on the lipid composition in addition to the functional expression of membrane proteins such as ion channels, transporters, and pumps. Unfortunately, composition of membrane lipids, membrane proteins, and electrical properties are not well characterized in E. huxleyi. CO2 permeability and a HCO3− transport pathway have been suggested (Paasche, 1968, 2002; Nimer et al., 1996; Herfort et al., 2002; Brownlee & Taylor, 2004). Herfort et al. (2002) found the HCO3− pathway to be sensitive to DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), a relatively unspecific blocker of a set of anion transporters. In Coccolithus pelagicus, a larger coccolithophore, a voltage-dependent and DIDS-inhibitable Cl− current was shown (Taylor & Brownlee, 2003). More information is available on pHi in coccolithophores. Presently there are three datasets on pH measurements in E. huxleyi, reporting a whole-cell pH of between 6.77 ± 0.31 for a low calcifying strain and 7.29 ± 0.11 for a high calcifying strain (Dixon et al., 1989; Nimer et al., 1994a). Cytosol pH was reported to be c. 7.0 (Dixon et al., 1989; Anning et al., 1996), pH of the chloroplast 8.0 (Anning et al., 1996), and pH inside the coccolith vesicle was measured to be 7.1 ± 0.3 (Anning et al., 1996). At a seawater pH of c. 8.1, the H+ gradient across the plasma membrane is about one order of magnitude (Dixon et al., 1989; Anning et al., 1996). This might reflect either high cytosolic H+ production at a limited export capacity, or H+ uptake mechanisms driven by ion gradients or membrane voltage, or both processes at the same time. In the present study we monitored BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) fluorescence in E. huxleyi as a measure of cytosolic pH. The use of the pH-sensitive dye BCECF is a well-established method to measure pHi in different organisms since the early 1980s (Rink et al., 1982). It is introduced into the cells as an uncharged acetoxymethyl ester form (BCECF-AM), and only emits fluorescence, after cleavage to the free acid by (cellular) esterases. This hydrolysis also traps the dye inside the cells, as the BCECF molecule is now charged (see also Pörtner et al., 2010). The BCECF signal can be calibrated after permeabilization of the plasma membrane by the H+/K+ exchanger nigericin and elimination of the transmembrane K+ gradient. Calibration has to be performed at the end of every single experiment to obtain individual pHi values, whereas ratio changes as a measure of pHi are fairly constant between experiments. With this method we got a first semiquantitative insight into the membrane permeability properties with respect to CO2, HCO3−, and H+. The challenge in these experiments was to differentiate between isolated effects of CO2, HCO3−, and H+ on physiological processes. Under steady-state conditions it is impossible to alter one of the three carbon species while keeping the other two constant because they are in a dynamic equilibrium (Eqn 1). (Eqn 1) Different approaches have been applied to overcome this problem, taking advantage of the slow reaction rates between CO2 and HCO3−. The isotopic disequilibrium technique (Rost et al., 2002; Endeward et al., 2006) combines radioactive or stable isotopes of the CO2 and HCO3− pools with mass spectrometric measurements of the resulting metabolites. The out-of-equilibrium (OOE) approach (Zhao et al., 1995) allows the impact of any of the three species on pHi to be monitored online and at a single cell level. We used the OOE method to investigate membrane permeability properties for H+, CO2 and HCO3− and used DIDS as a pharmacological tool. Materials and Methods Cell culture Emiliania huxleyi cells used in this study were isolated in 2005 during the PeECE III mesocosm study in the Raune Fjord (Norway) by M. N. Müller (Riebesell et al., 2007). The cultures were grown in artificial seawater (ASW) modified from Kester et al.(1967), with an initial pH of 8.05 ± 0.05 (Table 1) and enriched with nutrients according to f/20-Si, modified after Guillard (1975). Cells were exposed to daylight (Osram Lumilux L 18W/950 Color Proof Daylight G13; Osram, Munich, Germany) in a simulated diurnal cycle with a maximum photosynthetically active radiation (PAR, 400–700 nm; QSL-2101, Biospherical Instruments, San Diego, CA, USA) of 170 μmol photons m−2 s−1 for 6 h, framed by a 6 h increment and a 6 h decrement in three steps, respectively. The mean PAR during the illumination phase was 100 μmol photons m−2 s−1. Cells were kept at 17°C in a growth chamber (KBWF 240, Binder GmbH, Tuttlingen, Germany) in 50 ml polystyrene culture flasks (Sarstedt, Nümbrecht, Germany). Table 1. Artificial seawater (ASW) solutions (data are mmol kg−1) Seawater 1 2 3 4 5 ASWculture ASWc ASWstrip 0 HCO3− ASWnig Na+ 498 504 504 447 488 437 K+ 9.9 9.9 9.9 10.0 10.0 97.4 Mg2+ 53 53 53 50 53 52 Ca2+ 10.4 10.3 10.3 0 10.0 9.7 Sr2+ 0.09 0.09 0 0 0 0 Cl− 546 544 545 555 569 532 SO42− 28 28 28 0 28 27 Br– 0.84 0.82 0 0 0 0 F– 0.07 0.07 0 0 0 0 H3BO3 0.42 0.42 0 0 0 0 HCO3− 1.98 2.35 2.35 2.00 0 0 HEPES 0 0 0 0 5 5 Gluconate 0 37.6 37.6 0 0 71 EGTA 0 0 0 25 0 0 Calculated values (CO2SYS) HCO3− 2.14 1.80 CO32− 0.19 0.19 CO2 0.02 0.01 pCO2 [μatm] 592.40 363.20 pH 8.08 ± 0.03 (as ind.) (as ind.) Osmolality 1070 ± 10 Salinity 35 ± 1 ASWc, control ASW; ASWculture, ASW used for culture; ASWnig, calibration solution containing 100 mmol kg−1 K+ and 10 μmol kg−1 nigericin; ASWstrip, EGTA-containing solution; as ind., as indicated in the respective results. ASW solutions were designed according to values of pH, osmolality and salinity measured in North Sea water. Standard seawater composition was modified after Zeebe & Wolf-Gladrow (2001). All solutions were allowed to equilibrate and, if necessary, adjusted to the exact pH at 20°C (NaOH or HCl). ±, indicator of the accuracy achieved and allowed in generation of the respective solution. For the experiments, cells were grown to high densities (4 × 105–1.8 × 106 cells ml−1, achieved during days 7–10) under nutrient limitation (from day 3–4 onwards) to get a highly calcified cell population (Shiraiwa, 2003), which was found to be best suitable for the experiments as a result of high cell numbers, good loading properties and increased adhesiveness to the bath chamber. Decalcification and protoplast isolation A quantity of 10–15 ml of cell culture was centrifuged at 1882 g for 5 min and the supernatant was discarded. The cell pellet was resuspended in an ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid-containing solution (EGTA, solution 3, ASWstrip) by gentle mixing with a plastic transfer pipette and then incubated for 15 min to detach and dissolve the coccoliths (adapted from Taylor & Brownlee, 2003). Cells were centrifuged again and incubated in ASWstrip for another 10 min. Thereafter, cells were mechanically agitated by a series of rapid aspirations and expulsions through polyethylene tubing (inner diameter 350 μm) attached to a 1 ml syringe to remove remnants of coccospheres. After another centrifugation step, the cells were transferred to solution 2 (ASWc, pH 8.05 ± 0.05) where they were kept for 2 h to allow recovery. Apart from centrifugation, all steps were performed under illumination to allow photosynthesis during the long stripping process. Dye loading (BCECF) Stock solutions of BCECF-AM (10 mmol l−1 in dimethyl sulfoxide, Invitrogen), and Pluronic F-127 (10% in H2O, Invitrogen) were stored in aliquots at −20°C until use. Cells were incubated at 17°C with a final concentration of 50 μmol l−1 BCECF-AM and 0.5% Pluronic for 120 min in ASW to allow sufficient uptake and cleaving of the esterified dye. Cells were centrifuged and the supernatant was discarded. After resuspension in ASW, cells were transferred into the bath chamber and allowed to settle and adhere to the poly-D-lysine-coated bottom cover slip (Sigma-Aldrich) for at least 30 min. Thereafter a sufficient number of cells firmly adhered and allowed a rapidly flowing bath solution. With this dye loading procedure we achieved a signal-to-noise relation for the emission signal > 10 throughout a 1 h experimental period in most cells. Cells with weak dye loading below this threshold or showing signs of overloading were excluded. Viability tests To ensure that the cells were viable after the isolation and incubation procedure, we added trypan blue (458 μmol l−1) to test samples and monitored dye uptake. Exclusion of the dye was expected for intact protoplasts and observed in > 90% of the tested cells. In a second approach we tested the ability of cell samples to recalcify under normal cell culture conditions. Virtually all of the observed cells were able to recalcify within the observation period of 2 d and the culture did not show any significant change in growth rate compared with the control. Microfluorimetry Fluorescence was monitored with an imaging system (Visitron) using a charge coupled device (CCD) camera (CoolSNAP HQ2; Photometrics, Tucson, AZ, USA) mounted on an inverted microscope (Zeiss Axiovert 35 M). The microscope was equipped with an A-Plan 100×/1.25 Oil objective (×100, Zeiss, Jena, Germany). At a rate of 0.2 Hz the dye was alternatively excited at 486 and 440 nm (± 10 nm bandwidth) for 24 and 60 ms, respectively. Emission was recorded at 525 nm (emission filter ET525/50 nm; Chroma Technology Corp, Bellows Falls, VT, USA) and the integrated ratio of the emission intensities at the two excitation wavelengths over the whole cell was calculated after subtraction of system immanent camera offset and background signal (MetaFluor Meta Series Software 7.6.1; Meta Imaging System, Molecular Devices, Inc., Sunnyvale, CA, USA). From each experiment five to 20 cells were selected for analysis, and each cell was analysed individually for fluorescence ratio, representing pHi. Our study focussed on changes in pHi as a result of changes in ambient CO2, HCO3− and H+ concentrations and we decided not to perform an individual calibration for each cell. However, in order to gain a magnitude for pHi changes we performed a calibration of the ratio change in a separate experimental series. The cells were checked for autofluorescence after excitation at the experimental wavelengths 486 and 440 nm, known to induce chlorophyll autofluorescence. No autofluorescence was detected at the emission wavelength of 525 ± 25 nm. Calibration of ΔpHi with nigericin Nigericin was used (Pressman, 1976) to calibrate relative changes in pHi of living cells. It is an ionophore and acts as a K+/H+ exchanger. To obtain absolute pHi measurements, [K+]e in calibration solutions has to be adjusted to equal [K+]i in order to remove the driving force for K+ and to depolarize the cell. Under these conditions, internal and external [H+] can equilibrate. Values for [K+]i in E. huxleyi have been reported over a wide range from 100 to 260 mmol l−1 (Sikes & Wilbur, 1982; Ho et al., 2003). Hence, we limited our calibration to relative changes in pHi as this only requires high [K+]e.We did not calculate absolute pHi values in this study. A stock solution of nigericin (Nigericin sodium salt, 72445, Sigma Aldrich, 10 mmol l−1 in ethanol) was prepared and stored in aliquots at −20°C until use. Emiliania huxleyi cells were exposed to 10 μmol l−1 nigericin in the presence of 100 mmol l−1 K+ (solution 5) at varying pH values. Ratio changes were monitored (Fig. 4a,b). The calibration curve (Fig. 4c) was linear, allowing an estimate to be made of the relationship between the detected change in emission ratio of BCECF and the respective change in pHi. Experimental procedure General For all experiments the bath chamber (350 μl volume) was mounted on the stage of an inverted microscope and perfused by gravity at a rate of 6–8 ml min−1 at 17°C, ensuring rapid bath exchange rates. In this background of bath perfusion, OOE solutions were directly applied to the investigated cells by a micromanipulated superfusion pipette system, enabling the mixing time to be kept < 10 s. During application of the OOE solutions, the bath was continuously rinsed by solution 2 (ASWc), securing permanent removal of the OOE solution. The sequence of bath solution exchanges is described in detail for each series in the respective Results section. OOE mixing unit Dual-syringe pumps (50 ml, Perfusor Secura, B. Braun, Messungen, Germany) were used to drive solution pairs a and b (Table 2) at a constant rate of 7–10 ml h−1 to a Teflon mixing unit (Supporting Information, Fig. S1) with six inlets, each secured with a unidirectional restrictor valve, for a consecutive application of up to three OOE solutions. Stainless steel inlet pairs for the 1 : 1 combination of a and b solutions were situated opposite to ensure optimal mixing. In addition, mixing was improved and dead space minimized by a mesh (60 μm pore size nylon filter, Millipore NY60) mounted inside the mixing unit in front of the outflow cannula. The opening of this cannula was positioned directly before and above the cells (c. 30° lateral) under investigation to ensure laminar superfusion and to prevent any mixing of solutions around the cells. The total dead space volume of the system after mixing was c. 20 μl. In consequence, the time between mixing and supply of OOE solutions to the cells was 7–10 s, and thus well within the time required to prevent any significant equilibration (see Fig. 1). Table 2. Out-of-equilibrium (OOE) solutions (data are mmol kg−1) 6a 6b 6 7a 7b 7 High CO2 (a + b) OOE High HCO3− (a + b) OOE Na+ 484 415 450 563 361 462 K+ 10 10 10 10 10 10 Mg2+ 52 52 52 0 103 52 Ca2+ 10 10 10 0 19 10 Cl− 563 494 528 530 508 519 SO42− 27 27 27 0 55 27 HEPES 0 63 31.7 0 63 31.7 HCO3− 0 0 0 43 0 21 CO2 (%) 5 0 2.4 0 0 0 Calculated values HCO3− 0.1 0 0.05 10.6 0 20 CO32− 0 0 0 33.4 0 2 CO2 1.72 0 0.86 0 0 0 (%) 5.0 0 2.5 0 0 0 pH 4.90 8.07 8.08 9.24 7.69 8.07 Osmolality 1070 ± 10 Salinity 35 ± 1 OOE solutions a and b were adjusted or aerated to the measured pH value at 20°C. Solutions were generated shortly before application in the experiment. ±, indicator of the accuracy achieved and allowed in generation of the respective solution. Figure 1Open in figure viewerPowerPoint Reaction kinetics upon mixing of out-of-equilibrium (OOE) solutions. Changes in concentrations against time upon mixing of two solutions (see Table 2) with different carbonate chemistry on a logarithmic scale (10−10–105 s) (a–f) and on a linear scale (1–200 s) (g–l). Shown are pH on the free scale, pHF (a, g); concentrations of the unprotonated form of HEPES, [A−] (b, h); the protonated form of HEPES, [AH] (c, i); carbon dioxide [CO2] (d, j); bicarbonate, [HCO3−] (e, k); carbonate, [CO32−] (f, l). Solid lines illustrate evolution of carbonate chemistry speciation in solution 6 (high CO2). Dashed lines show carbonate chemistry kinetics in solution 7 (high HCO3−). Calculations of carbonate chemistry speciation were done at a salinity of 35 and at 20°C. The vertical line represents the experimental time range of 7–10 s after mixing. Light, intermediate and dark gray shaded areas mark the time ranging from 10−10 to 10−5, 10−5 to 10−1 and 10−1 to 1000 s, respectively. Note that the left ordinates in (j–l) give the values for the solid lines (high CO2), while the additional right ordinate in (j–l) gives the values for the dashed lines (high HCO3−). pH measurements in experimental solutions pH was measured with a pH-sensitive single-rod measuring cell (Blueline 16 pH; Schott Instruments, Mainz, Germany) with a microprobe. This enabled us to measure the pH of seawater (pHe) directly at the outlet of the OOE mixing unit to check for target pH. pH values are presented on the NBS (National Bureau of Standards, USA) scale and a pH of 8.05 ± 0.05 was defined as the control pH. Values in Fig. 1, however, are given on free scale. Solutions ASW solutions Artificial seawater solutions were designed after Zeebe & Wolf-Gladrow (2001). At a sea surface temperature of 17°C, a pH of 8.2 on the NBS scale and a salinity of 35, the carbonate system consists of 1900–2000 μmol kg−1 HCO3−, 200 μmol kg−1 CO3− and 15 μmol kg−1 CO2. Osmolality and salinity (35 ± 1) were chosen according to measurements of natural seawater (NSW). All experimental solutions (Table 1) were adjusted to an osmolality of 1070 ± 10 mosm kg−1 at the expense of NaCl or by addition of Na-gluconate. pH, if not indicated otherwise, was 8.08 ± 0.05. Calculations for the carbonate system were performed using CO2SYS (Lewis & Wallace, 1998), based on measurements of pH and total inorganic carbon concentration. Dissociation constants (K1, K2) for carbonic acid were taken from Roy et al. (1993), KSO4− from Dickson (1990). pH was measured and calculations are given on the NBS scale. All chemicals were purchased at highest grade of purity from Merck and Sigma, Germany. DIDS was dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 0.1 mol l−1 and added at a final concentration of 0.1 mmol l−1 to the respective experimental solutions, unless indicated otherwise. DMSO did not exceed a concentration of 0.1%. DIDS autofluorescence did not interfere with BCECF fluorescence at the selected wavelengths. OOE solutions Out-of-equilibrium solutions were designed to have either a comparatively high [CO2] and low [HCO3−], or a high [HCO3−] and a very low [CO2], at a typical surface ocean pH of 8.05. The enzymatically catalyzed equilibration of experimental solutions directly at the extracellular surface of the cells was neglected since, in E. huxleyi, only very low external carbonic anhydrase (CA) activities have been observed under various conditions (Rost et al., 2003, 2006). Although there is strain variance and there might be strains with higher CA expression and activity, dependent on nutrient concentration (Nimer et al., 1994b), no CA inhibitors were added as they might have dampened cytosolic reactions in pH homeostasis. A chemical model of the carbonate system, including all important reactions in seawater together with HEPES buffer kinetics, was implemented according to Schulz et al. (2006). The resulting seven differential equations were integrated numerically with the matlab 'ode15s' solver for 'stiff' problems (Shampine & Reichelt, 1997), and used to calculate the reaction kinetics in carbonate chemistry speciation upon mixing of two different OOE solutions. The model also allows the degree of disequilibrium at any given point in time to be estimated and the actual concentrations of, for instance, [H+], [CO2] and [HCO3−] to be derived. Calculations and statistics Each cell was analyzed individually for changes in emission ratio as a measure of pHi and the resulting changes are shown as means ± SEM. Data were pooled from multiple cells in different experiments where (n, m) indicate the number of cells (n) from m experiments. Paired Student's t-test was applied, and P < 0.01 was accepted for statistical significance. Statistics and calculations were performed using Excel 2003 (Microsoft) or OriginPro 7.5G (OriginLab Corporation, Northampton, MA, USA). Absolute and relative changes were calculated vs values derived under control conditions unless indicated otherwise. Results OOE solutions The present experiments with OOE solutions were performed in the time range 7–10 s after mixing of the respective solution pairs a and b. We calculated the reaction kinetics for the carbonate chemistry according to Schulz et al. (2006). The results nicely validate the fact that the cells under investigation were exposed to solutions that were still out of equilibrium (Fig. 1). We generated two OOE solutions with either high [CO2] or high [HCO3−]. The two solutions are depicted as a solid line (high [CO2]) and a dashed line (high [HCO3−]). The development of the solutions can be identified in Fig. 1(d) and (e), which show [CO2] and [HCO3−] vs time. The time axis is logarithmic and covers the whole period from initial mixing to equilibrium. The time range of the experiment is indicated by a vertical line. The high-CO2 solution (solid line) shows a virtually constant [CO2] until c. 1 s (Fig. 1d) and remains above c. 600 μmol kg−1 CO2 during the 7–10 s experimental period. The formation of HCO3− during this period is negligible (Fig. 1e). Only after minutes does the conversion of CO2 into HCO3− reach equilibrium. The target pH value in this solution is already met 10 μs after mixing (Fig. 1a). The high-HCO3− solution (dashed line) shows a substantial increase in [HCO3−] to the target value in the time range of ms (Fig. 1e) by the protonation of CO32− (Fig. 1f). This value stays virtually constant during the time of the experiment. CO2 formation in this solution does not exceed 50 μmol kg−1. In this solution the target pH value is reached within ms after mixing (Fig. 1a). The initial pH changes reflect buffering and protonation of CO32−. Fig. 1(b) and (c) show the respective changes in HEPES buffer components. For a higher time and concentration resolution of the equilibration phase refer to Fig. 1(g)–(l), plotted with linear time axis. The left ordinate gives the scale for the high-CO2 solution (solid line), the right ordinate for the high-HCO3− solution (dashed line). In essence, the slow conversion between CO2 and HCO3− allows the use of OOE solutions in the time-frame between 0.1 and 10 s after mixing without relevant equilibration. Even after 10 s the most obvious change in [CO2] in the high-CO2 solution (Fig. 1j) results in a [CO2] that is still threefold above [HCO3−] in the same solution. BCECF measurements After dye loading, E. huxleyi was allowed to equilibrate for a few minutes to control conditions, resulting in a stable ratio after initial rundown. Experiments were started c. 120 s thereafter. The resulting BCECF fluorescence ratio as a measure of pHi resembled a Gaussian distribution over all analysed experiments (Fig. 2a). This was also observed for batches of cells within one experiment, indicating different individual starting pHi values. Since our study focussed on the changes of pHi as a result of changes in ambient CO2, HCO3− and H+ concentrations, we decided not to perform an individual calibration for each cell and therefore we do not give absolute pHi values. The mean ratios under control conditions in the experimental series (Table 3) were between 2.49 and 2.54. In some experiments we observed a decline in ratio over time. Cells with a lower loading signal were monitored in the same experiments, and showed qualitatively the same results (data not shown); however, because of the low signal, the effect was dampened and the signal approached the detection limit already before completion of the experiment. Figure 2Open in figure viewerPowerPoint Frequency distribution of measured emission ratios of BCECF (2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein) fluorescence and confocal images showing dye distribution. (a) Analysis of the distribution of measured emission ratios under initial control conditions of n = 299 cells displayed as absolute number in clusters of 0.2. (b) Confocal false color image of Emiliania huxleyi cells loaded with BCECF-acetoxymethyl ester (BCECF-AM). (I) BCECF fluorescence intensity, green (excitation 488nm, bandpass 530–550 nm). (II) Chloroplast autofluorescence intensity, red (excitation 488 nm, emission long pass 600 nm). (III) Merged image indicates differential localization of fluorescence signals. No BCECF loading of chloroplast. Table 3. Effects of changes in CO2, HCO3− and H+ on intracellular pH (pHi) Solution (mol kg−1) n m Mean d abs d (%) P (a) 7 HCO3− 0.0220 85 5 2.31 ± 0.02 −0.12 ± 0.01 −5 ± 0.3 < 0.01 6 CO2 0.0017 148 8 2.42 ± 0.02 −0.01 ± 0.01 −1 ± 0.4 0.18 4 H+ 1 × 10–7 42 3 1.93 ± 0.03 −0.62 ± 0.04 −24 ± 1.3 < 0.01 4 H+DIDS 1 × 10–7 46 3 1.70 ± 0.04 −0.40 ± 0.02 −19 ± 1.0 < 0.01 Solution pHe H+ (mol kg−1) n m Mean d abs d (%) P (b) 4 9.0 1 × 10–9 31 6 2.62 ± 0.05 −0.13 ± 0.02 −5 ± 0.7 < 0.01 8.5 5 × 10–9 2.49 ± 0.05 −0.18 ± 0.03 −7 ± 1.3 < 0.01 8.0 1 × 10–8 2.31 ± 0.04 −0.28 ± 0.02 −7