Driving Forces for Bicarbonate Transport in the - Plant Physiology

Plant Physiol. (1 996) 1 1 2: 1573-1 584

Driving Forces for Bicarbonate Transport in the Cyanobacterium Synechococcus R-2 (PCC 7942)'

Raymond J. Ritchie*, Christiane Nadolny, and A.W.D. Larkum

Biology A-I 2, School of Biological Sciences, The University of Sydney, New South Wales 2006, Australia

Air-grown Synechococcus R-2 (PCC 7942) cultures grown in BC-11 medium are very alkaline (outside pH is 10.0) and use HC0,- as their inorganic carbon source. The cells showed a de- pendente on Na+ for photosynthesis, but low Na+ conditions (1 mo1 m-3) were sufficient to support saturating photosynthesis. The intracellular dissolved inorganic carbon in the light was greater than 20 mo1 m-3 in both low-Na+ conditions and in BC-11 medium containing the usual [Na+] (24 mo1 m-,, designated high-Na+ conditions). l h e electrochemical potential for HC0,- in the light was in excess of 25 kJ mol-', even in high-Na+ conditions. The Na+-motive force was greater than -12 kJ mol-' under both Na+ conditions. On thermodynamic grounds, an Na+-driven co-port process would need to have a stoichiometry of 2 or greater (22Na+i,,/HC03-'i,), but we show that Nat or K+ fluxes cannot be linked to HC0,- transport. Na+ and K+ fluxes were unaffected by the presence or absence of dissolved inorganic carbon. In low-Na+ conditions, Na+ fluxes are too low to support the observed net

C-carbon fixation rate. Active transport of HC0,- hyperpolarizes (not depolarizes) the membrane potential.

14

Cyanobacteria (Cyanophyta, the blue-green algae) gen- erally live in alkaline environments (Fogg, 1956) where HC0,- is the predominant form of DIC. Cyanobacteria show both a constitutive and an inducible inorganic C0,- concentrating mechanism, which enables the cells to accumulate DIC (Kaplan et al., 1980; Lucas and Berry, 1985; Badger and Price, 1992). Accumulation ratios of interna1 to external inorganic carbon concentrations of 1000 or more have been found (Badger and Gallagher, 1987; Kaplan et al., 1980; Salon et al., 1996a, 199613). Na+ is an essential element for cyanobacteria (Kratz and Meyers, 1955; Fogg, 1956; Batterton and Van Baalen, 1971). Synechococcus will not grow in culture in alkaline conditions (pH, 9.0 or higher) unless Nat is present (Miller et al., 1984). Nat plays a crucial role in control of pH, in cyanobacteria (Miller et al., 1984; Reinhold et al., 1984).

R.J.R. holds an Australian Research Council Postdoctoral Re- search Fellowship. This study was supported in part by a Univer- sity of Sydney/Institute of Advanced Studies (Australian National University, Canberra) Collaborative Research Grant awarded to R.J.R., A.W.D.L., M.R. Badger, and G.D. Price. C.N. gratefully acknowledges Rotary International for her graduate student schol- arship at The University of Sydney. Purchase of 42K+ was supported by a grant from the Australian Institute of Nuclear Science and Engineering.

* Corresponding author; e-mail [email protected]; fax 61- 2-9351-4771.

Cyanobacteria can use CO, and/or HC0,- to support photosynthesis (Volokita et al., 1984; Badger, 1987; Espie et al., 1988a; Miller et al., 1990a; Kaplan et al., 1991; Badger and Price, 1992; Badger et al., 1994), but in very alkaline conditions virtually all inorganic carbon exists in the form of HC0,- and CO$-. Cyanobacteria show an acclimation to different concentrations of external DIC during growth, so special attention has to be given to the growing conditions of the culture, as well as experimental conditions chosen so that HC0,- is the only carbon source (Badger and Andrews, 1982; Badger and Gallagher, 1987; Yu et al., 1994). The cells are capable of simultaneously taking up CO, and HC0,- if grown in ambient air conditions (Espie et al., 1988b; Yu et al., 1994; Salon et al., 1996~1, 199613). The conversion of HC0,- to CO, without carbonic anhydrase is very slow (Price and Badger, 1989). The use of CO, for DIC uptake can be negligible under sufficiently alkaline conditions (Miller and Colman, 1980b; Salon et al., 1996~1, 1996b).

Two different active transport mechanisms for HC0,-, which require different Nat concentrations, have been found. One uptake mechanism for HC0,-, which occurs only in cells grown under low CO, (equal to or lower than the concentration in ambient air) requires millimolar levels of Na+ (Miller et al., 1984; Miller and Canvin, 1985). The second HC0,- transport mechanism, which occurs in "standing culture" cells and in cells grown in CO, concentrations of 20 parts per million, seems to have no Na+ dependence (Espie and Canvin, 1987; Espie et al., 1991; Espie and Kandasamy, 1992).

The accumulation of HC0,- occurs against the ApHCO,-,,, and is therefore an energy-dependent process (Kaplan et al., 1980; Miller and Colman, 1980a, 1980b; Badger et al., 1985; Kaplan, 1985; Raven and Lucas, 1985). Little is known about the mechanisms underlying the en- ergization of the HC0,- uptake (Kaplan, 1985; Miller et al.,

Abbreviations: c (subscript), refers to the control-treated cells; CAI'S, 3-(cyclohexylamino)-~-propane-sulfonic acid; AG, Gibbs free energy; AG,,o, Gibbs free energy of transport process; AHC03-,,, electrochemical potentiallgradient for bicarbonate; ApH+,,, electrochemical potential/ gradient of protons; ApNati,,, electrochemical potential/gradíent of sodium; DIC, dissolved inorganic carbon (CO, + H,C03 + HC0,- + CO,2-); e (subscript), refers to the experimental treatment cells; in (subscript), inward; i (subscript), refers to the inside of the cells; K,,,, one-half maximum rate of photosynthesis; o (subscript), refers to the outside of the cells or bulk electrolyte.

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1574 Ritchie et al. Plant Physiol. Vol. 112, 1996

1990a, 1990b; Espie et al., 1991; Kaplan et al., 1991; Espie and Kandasamy, 1994).

In recent years it has become clear that some transport processes are linked to the ApNati,, (the Nai /HCO,- co-transport model) (Reinhold et al., 1984; Skulachev, 1988; Ritchie, 1991, 1992a). However, the possibility that an ion transport mechanism is a Na+-dependent process is often not suspected (Skulachev, 1988). The ApNa+,,, (or Na+mf,,,) seems a very good candidate for driving the active uptake of HC0,-, at least on thermodynamic grounds (Miller et al., 1984; Miller and Canvin, 1985; Espie et al., 1988a; Kaplan et al., 1991; Espie and Kandasamy, 1994), and would be consistent with the well-documented Na+ dependence of HC0,- assimilation.

For a Nat-driven mechanism to be adequate, both a thermodynamic and a kinetic condition need to be satis- fied: the ApNa+i,o must be large enough to support the observed accumulation of the transported ion, and the Na+ flux needs to be high enough to support the linked transport rate of the other ionic species (co-transport). For ex- ample, a stoichiometry for the putative co-transport mechanism that satisfies the thermodynamic condition (e.g. 2Na+,,/ HC03-i,) might not satisfy the kinetic condition. In Synechococcus, Na+ is the only ion with an electromotive driving force large enough to support the massive uphill accumulation of carbon into the cells (Ritchie 1991, 1992a).

To critically test the validity of the Na+/HCO,- co- transport model, it is necessary to compare the characteristics of Na+ transport with the characteristics of HC0,- transport under alkaline conditions, in which HC0,- is the actively transported carbon species. Simultaneous transport of both CO, and HC0,- occurs (Espie et al., 1988b; Salon et al., 1996a, 1996b) under mildly alkaline conditions (below pH, 9.0). Although Kaplan et al. (1989) studied proton fluxes and Na+ accumulation in Synechococcus, the only Na+ flux data available on a plasmalemma surface area basis are for cells at pH 7.5 grown on 5% CO, (Ritchie, 1992a), and most estimates of intracellular carbon pool sizes and HC0,- fluxes are not in sufficiently alkaline conditions to eliminate complications arising from CO, transport (Espie et al., 1988b; Badger et al., 1992; Espie and Kandasamy, 1994; Salon et al., 1996a, 1996b). Therefore, the critical set of data to test the Na+/HCO,- co-transport model is not yet available.

The well-documented Na+ dependence of inorganic carbon transport in cyanobacteria does not necessarily imply a direct link to the ApNa+i,o and the flux of Na' across the plasmalemma (Reinhold et al., 1984; Volokita et al., 1984; Kaplan et al., 1989). Na+ could act as an activator of the transporter (as suggested by Reinhtld et al., 1984) and might or might not be transported across the membrane during DIC uptake. It has been shown that C1- transport in Synechococcus is a Na+-stimulated but not a Na+-motive- force-driven mechanism (Ritchie, 1992b, 1992~). The observed effects of Na+ on DIC uptake could reflect its role in pHi regulation and not in HC0,- transport (Reinhold et al., 1984; Volokita et al., 1984).

Transitory hyperpolarization based on the use of rather unreliable lipophilic cation probes (Ritchie, 1984, 1991) has

been reported when Anabaena or Synechococcus cells were fed DIC (Kaplan et al., 1982, 1991; Miller et al., 1984; Zenvirth et al., 1984). Kaplan et al. (1982) observed that the supposed hyperpolarization effect of added DIC disap- peared in the presence of DCMU. Chlorophyll fluorescence changes (Miller and Canvin, 1987; Miller et al., 1988) in Synechococcus cells supplied with DIC could be interpreted as reflecting the hyperpolarization effects caused by an electrogenic pump.

At an experimental pH, of 10.0, as used in the present study, dissolved CO, is present in only trace concentrations and seems unlikely as an inorganic carbon source. We show that HC0,- is actively accumulated by Syneckococcus under these very alkaline conditions but that the uptake mechanism is not a proton-driven or a Na+-driven secondary active transport mechanism.

MATERIALS A N D METHODS

Chemicals and Radiochemicals

"Rb+, "Na+, and [14C]NaHC0, were from DuPont- New England Nuclear and [14C]Suc was from Amersham. 42K+ was from the Australian Nuclear Technology Orga- nization (Lucas Heights, Sydney, Australia). CAI'S, DCMU (recrystallized), Fluorinert FC-77, Hepes, and valinomycin were from Sigma-Aldrich.

General

Synechococcus R-2 (Synechococcus leopoliensis, Anacystis nidulans) (PCC 7942) was grown from axenic stock cultures in BG-11 medium (Allen, 1968) modified as described by Ritchie and Gibson (1987) and Ritchie (1996). The cells were grown in air (30°C, pH, 10.0-10.5) either semicontinuously (approximately one-quarter dilution each day) or in a continuous culture device in continuous light of about 150 pmol (quanta) m-' s-l PAR, using cool-white fluorescent lights. Similar light conditions were used in the experiments but at a temperature of 25°C. The major ions in BG-11 medium were (in mo1 m-,): Kt, 0.350; Na+, 18.036; Mg2+, 0.304; Ca2+, 0.245; C1-, 0.508; NO,-, 17.652; SO:-, 0.304; PO, (a11 forms), 0.175; and CO, (all forms), 0.189. Na'-free medium was prepared by eliminating NaNO, and replacing Na,CO, with MgCO,. Nitrate was added as 0.5 mo1 mP3 Ca(NO,),, a concentration sufficient to satu- rate NO,- uptake. Nominally Na+-free BG-11 had a [Na+] of approximately 6 mmol mP3 because of contamination.

The cells were harvested by centrifugation (25008) in a preparative centrifuge and washed three times in the experimental medium. Later, a more convenient filtration method was used. Cells were harvested on a polycarbonate filter (0.40 pm; Nuclepore, Pleasanton, CA), washed with the appropriate experimental medium, and then resus- pended in the experimental medium.

Intracellular volumes and cellular surface areas were calculated from cell numbers estimated by measuring the light scattering at 750 nm (Cary 1 spectrophotometer, Var- ian, Melbourne, Australia) as described by Ritchie and Gibson (1987). For comparative purposes, the material

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Active Bicarbonate Transport in Synechococcus R-2 (PCC 7942) 1575

used in the present study had 1.73 ? 0.14 (n = 48) mg Chl a m-,.

Buffer Solutions

The ionic composition of BG-11 medium was changed as little as possible: buffers were adjusted to the appropriate pH, using NaOH or Ca(OH),. CaCO, in the Ca(OH), was removed by filtration or sedimentation for preparing medium with limited inorganic carbon. For nominally Na+- free BG-11 and 1 mo1 m-' Na+ BG-11, Ca(OH), was used as the titrant and the buffer concentration was reduced to 5 mo1 m-'. Saturated MgCO, was used as the added inorganic carbon when NaHCO, was not appropriate. The following buffers were used at either 5 or 10 mo1 m-': Hepes/ Ca(OH),, pH 7.5; CAPSINaOH or CAI'S/ Ca(OH),, pH 10.0; CAPS/NaOH, pH 11.0.

Dark Techniques

Dark experiments were run in completely dark conditions (Ritchie, 1991). A dim, green safelight was only used for taking samples (about 10 s, 0.1 pmol [quanta] mP2 s-l PAR).

Membrane Filtration Techniques

Uptakes of 86Rb+, 42K+, and ,,Na+ were measured using filtration techniques as previously described (Ritchie and Gibson, 1987; Ritchie, 1991). The usual specific activity was approximately 10 to 500 GBq mol-'. Photosynthetic fixation of [14C]DIC could be easily followed using membrane filtration methods (Ritchie, 1996) but could not be used to estimate the size of the intracellular carbon pool because unfixed carbon was easily washed out of the cells.

The [Na+], was measured by incubating the cells in '*Na+ for 5 min, then washing three times with isotonic Ca(N0,)2 on filters, and counting. The exchange rate for ,,Na+ was estimated using efflux methods and an expo- nential efflux model (Ritchie, 1992a). Cells were left to stand in nominally DIC-free medium in the light for 30 to 60 min in sets of sealed Eppendorf tubes. A bead of ,'Na+ label was placed in the cap and labeling commenced by shaking. After 5 min, the equilibrium accumulation of ,,Na+ was measured as described above. The rate of loss of ,Na+ was estimated by placing ,,Na+-equilibrated cells on a filter and washing them for a measured time (about 6 s) with unlabeled medium before washing in isotonic Ca(NO,),. In DIC-treated cells, saturated Mg(HCO,), was added to the cap to give a final DIC concentration of 189 mmol m-', as well as the ,'Na+. The Na+ flux was estimated as for the control cells.

42K+ uptake fluxes were measured by labeling cells for O, 2, 4, or 6 min in the presence and absence of added inorganic carbon, and the uptake flux was calculated by linear regression.

Silicone Oil Cradients

Intracellular DIC and some of the pH, measurements were made using silicone oil gradient methods developed

previously (Ritchie and Gibson, 1987). A microfuge (model B, Beckman) with a vertical head (6300g) and 400-pL poly- ethylene tubes (no. 223-9502, Bio-Rad) were used.

Fluorinert FC-77 (density 1.78) was used as a high- density seat (10 pL) in each tube; a 1:3 mixture of the silicone oils AR20 and AR200 (Wackerchemie, Germany) with a density of 1.01 was used as the separating oil, and 200-pL samples were centrifuged through the oils into a killing solution. This killing solution (10 pL of 2 M KOH with 10% methanol) prevented metabolism of inorganic carbon in the centrifuged cell pellet. The extracellular volume of the cells after centrifugation through silicone oil gradients was measured using [14C]Suc as the extracellular label (Ritchie and Gibson, 1987).

O, Evolution

Photosynthesis was measured using a Clark-type electrode (Hansatech, Kings Lynn, Norfork, UK) at 25°C (Walker, 1990). The O, solubility algorithms of Carpenter (1966) and Colt (1984) were used to calculate the O, concentration in air-saturated medium. The chamber was illu- minated with saturating red light (peak 660 nm) provided by a light-emitting diode array (175 pmol mP2 s-' PAR).

Cells were harvested (301 [? 241 X 10" cells me3) onto polycarbonate membrane filters and then washed three times in experimental medium. The cells were then resus- pended in the appropriate experimental medium by shaking in an Eppendorf tube and preincubated in the light. Samples were purged with water-saturated N, gas before 1 mL of cell suspension was placed in the O, electrode chamber.

For the photosynthetic response of cells to external DIC, nominally carbon-free BG-11 buffered with 10 mo1 m-' CAPS/NaOH (pH 10.0) was used. After the cells had consumed the residual DIC of the medium (approximately 30 min), known amounts of KHCO, were added to the sample and net photosynthesis was measured. A new aliquot of cells was taken for each photosynthesis determination.

For the response of cells to external Na+, nominally Na+-free and DIC-free BG-11 buffered with 5 mo1 m-' CAPS/Ca(OH), was used. When net O, evolution of the cell suspension was zero, known amounts of NaNO, and 0.189 mo1 m-, KHCO, were added and O, evolution was measured. The Na+ concentration required to yield K,,, at a defined DIC concentration was determined as described below.

The O, electrode was used to routinely check that batches of cells used in experiments exhibited Na'- dependent photosynthesis and that DIC-depleted cells were used in the 14C-labeling experiments.

PHi The pH, was measured using the accumulation of the

weak base ['4C]methylamine (14CH,NH,) based on methods developed previously for [14C]ethylamine (Ritchie and Gibson, 1987; Ritchie, 1991). The pH, was calculated from the pH, and the accumulation ratio of the cationic species



1576 Ritchie et al. Plant Physiol. Vol. 11 2, 1996

using the Henderson-Hasselbalch equation (Ritchie and Gibson, 1987).

To use methylamine as a satisfactory pH, probe, it is necessary to use short labeling times and to add only low concentrations (100 mmol mP3) of methylamine. Ammonia (100 mmol m-’) was added to discourage methylamine metabolism. No significant binding of [14CH3NH3+] to the surface of the cells was observed based on labeling of cells with [‘4C]methylamine at pH, 5.0.

To measure the pHi, the pH of a buffered cell suspension was first measured. The suspension (1.2 mL in 1.5-mL Eppendorf tubes) was then labeled with [14C]methylamine for a short time (60-120 s) before a 0.5-mL sample was taken for counting to estimate the total [I4C]methylamine present in the cell suspension. The remaining volume of labeled cell suspension was centrifuged (Beckman microfuge) and 0.5 mL of the supernatant was counted. Equal volumes of unlabeled cell suspension were added to the clear supernatant samples to correct for quenching. Total intracellular methylamine was then calculated by subtraction. Using the known pH, and the pK, of methylamine at 25°C (10.65; Segel, 1976), the concentrations of l4CH,NH, and 14CH,NH3+ were calculated for the bulk electrolyte. The [l4CH,NH,+Ii was then calculated, making an allow- ance for the concentration of uncharged methylamine inside the cells.

Membrane Potential

The membrane potential was measured using the 86Rbf/ valinomycin technique described and discussed by Ritchie (1991) but using filtration and washing to measure valinomycin-mediated uptake of 86Rb+ by the cells. Valinomycin-mediated equilibration of “Rb+ occurred in about 0.5 h in the light and in the dark.

lntracellular Carbon

Kinetic experiments showed that the [DIC], can be very high, e.g. about 30 mo1 mP3 with a half-time of much less than 1 min (from the Y intercept of 14C uptake compared with time experiments), Most of the 14C-label taken up after the first few minutes of an uptake experiment repre- sented carbon metabolized into organic compounds. As a result, the intracellular [14C]DIC was measured using silicone oil gradient separation of cells from the medium, followed by volatilization by acid; controls were treated with alkali.

Cells were first incubated in nominally inorganic carbon- free medium in the light for 30 min and were then labeled with a known concentration of [14C]DIC for a known time. Duplicate cell suspension samples were centrifuged through silicone oil gradients into a killing solution of KOH. One pellet was heated in 0.5 mL of acidified alcohol in a fume hood (90% ethano1/100 mo1 m-’ HC1 final concentration) in scintillation vials, vortexed, and heated in a hot block at 80°C to dryness. A second lot of HCl/ethanol was added and distilled off; 0.5 mL of 100 mo1 m-’ NaOH was added and dried off, and then 0.5 mL of water was added before adding the scintillant. The other duplicate

pellet was placed into a scintillation via1 in 0.5 mL of 100 mo1 m-’ NaOH and heated to dryness; after drying, 0.5 mL of water was added before the scintillant was added. The [14C]DIC content of the cells (20-min counting period) was calculated by subtraction of the alkali versus the acid- treated cells. To ensure uniform quenching effects, two sets of standards (four) were made up. The standards contained the same number of unlabeled cells as the experimental treatments: one set was treated with alkali and dried down, and water and a known volume of 14C was added; the other set was treated with acid / ethanol and then alkali before the I4C was added. The two sets of standards gave counts that were not significantly different.

Electrochemical Potentials

The ApNa+,,, was estimated from the accumulation ratio of Na+ and the appropriate observed membrane potential, as described previously (Ritchie, 1991, 1992a), using the Nernst equation (Nobel, 1983). Activities were calculated from concentrations using the Bjerrum version of the Debye-Huckel equation (Hamer, 1968). The cell wall of Synechococcus also has a small but significant anion- exchange capacity (Ritchie and Gibson, 1987; Ritchie, 1992b, 1992c) that needs to be taken into account when estimating intracellular DIC. The equations of Vollenwei- der (1974), Ostrom (1975), and Golterman et al. (1978) were used to calculate intra- and extracellular concentrations of CO,, HC0,-, and C032p from the DIC measurements and pH values. Since HC0,- was the form of inorganic carbon taken up by the cells and was by far the dominant species present inside the cells at equilibrium, even if the inorganic carbon system were not quite in equilibrium (Price and Badger, 1989), the [HCO,-], would be very close to the calculated values. Activities of HC0,- inside and outside the cells and the membrane potential were then inserted into the Nernst equation to calculate the ApHC03-i,,.

Transitory Polarization of the Membrane Potential

Transitory electrogenic effects of added substrates on the membrane potential of Synechococcus (Ritchie, 1992a) were calculated from measurements of the uptake flux of 86Rbf in the presence of valinomycin under control conditions with a transported substrate added. Effects on membrane potentials lasting less than a couple of minutes cannot be detected because of difficulties in measuring differences in the valinomycin-mediated 86Rbt fluxes on such short time scales. Valinomycin was added to the prepared batch of cells before dividing them into the control and experimental groups so that both treatments experienced the same valinomycin concentration.

For modest changes in potential and where the control membrane potential is about -120 mV or greater, the membrane potential of the experimentally treated cells can be calculated as:




where A$= is the instantaneous membrane potential of cells given the experimental treatment at t = O; A$= is the membrane potential of the control cells (30-min incubation, assumed to be constant); +e is the valinomycin-mediated uptake flux of “Rb+ of the experimentally treated cells; and +e is the valinomycin-mediated flux of “Rb+ of the control cells.

The polarizing effect ( A I / J ~ , ~ ) was calculated as the difference between the control membrane potential (A$=) and the potential found in the experimental treatment in Eq. 1). The polarizing effects of added DIC were measured in DIC- depleted cells. The valinomycin-mediated uptake flux of 86Rb+ was measured at O, 1,2, and 3 or 4 min. In control cells only “Rb’ was added to the caps before incubation. In most experimentally treated cells the caps had “Rb+ plus a saturated solution of Mg(HCO,),, to give a final [DIC], of 189 mmol m-’. In the case of the cells pre-exposed to DIC for 15 min, the DIC was added and mixed without disturbing the “Rb+ in the cap. The 86Rb+ fluxes were measured by shaking the tubes and removing a cell sample for filtration after the appropriate labeling time. Membrane potentials were calculated from the equilibrium accumulation ratio of 86Rbf after labeling for 30 min in both control and experimentally treated cells using the Nernst equation.

Kinetics

As in O, electrode experiments, Synechococcus cells were left in nominally inorganic carbon-free medium in the light for 30 to 60 min in sealed Eppendorf tubes to allow the cells to deplete [DIC],. Parallel O, electrode experiments were used to make sure that the cells had depleted the contam- inating inorganic carbon before commencing the I4C experiments. Batches of cells were not used if the Na+- independent net photosynthetic rate was more than 5 nmol m-2 -1

Net I4C-uptake fluxes were measured in [DIC], from 1 mmol m-3 and, routinely, to 300 mmol m-3. A 1-min uptake was used to estimate the net flux of I4C. Cells were collected on filters and rinsed twice with 10 mo1 m-’ Ca(NO,), (pH adjusted to 7.0-8.0 using Ca[OH],) and then counted. K l l , (apparent K,) and V,,, were determined using a nonlinear least squares fit to the Michaelis-Menten equation (Neame and Richards, 1972; Ritchie and Prvan, 1996). Asymptotic SES of K l l , and V,,, were calculated as ‘described by Cleland (1979) and Johnson and Faunt (1992) and then 295% confidence limits were calculated using n-2 degrees of freedom (Zar, 1974). Experimental [DIC], values were selected so that they were as evenly spaced as possible or logarithmically spaced (Cleland, 1979; Endrenyi, 1981; Ritchie and Prvan, 1996).

s in the O, electrode.

Counting Methods

14C label was counted using the standard 14C channel of a scintillation counter (model Tri-CARB 300, Packard, Me- riden, CT). “Rb+ , 42 K+, and ‘,Na+ were counted on the ’,P channel. Intracellular ’,Na+ labeling was very low, so 60- min counts were used. Equal volumes of cell suspension were added to standards corrected for quenching.

Statistics

Data are quoted as means ?z 95% confidence limits with the number of data points in brackets. Where two numbers appear in brackets the first is the number of separate experiments conducted and the second is the total number of data points. Student‘s t tests, linear regression analyses including the error limits of the slopes (m) and Y intercepts, and other statistics were calculated as described by Zar (1974).

RESULTS

Defining the Experimental Material

The cells used in the present study were grown in BG-11 medium aerated with air; the doubling time was approximately 12 h. In the chemostat-grown culture, the steady- state pH, was 9.5 to 10.5 (9.93 5 0.20 [ZO]), and the DIC was found to be 1.01 2 0.17 mo1 m-’ [20] by titration (Golter- man et al., 1978). The semicontinuous cultures (n = 20) had a pH, of 10.6 2 0.11 and a DIC of 734 2 80 mmol m-’ after 24 h of growth. The cultures absorbed large amounts of CO, from the air and were strongly alkaline. The cells had a mean net photosynthetic rate (as shown by an O, electrode) of 59 2 3 [5, 601 nmol mP2 s-’ in CAPS-buffered BG-11 at pH, 10.0 and 0.189 mo1 m-’ DIC. The cells had a K l l , for DIC of 64 +- 22 mmol m-’ [24] using 14C labeling; this is equivalent to 40 ? 14 mmol m-3 for HC0,- (Table V), which is comparable to that found for air-grown cells by Yu et al. (1994) but somewhat higher than those found by Salon et al. (1996a, 1996b). The cells showed a depen- dente on Na’ for photosynthesis, with a K l l z for Na+ of 314 5 94 (5, 60) mmol m-’ using 189 mmol m-’ [DIC],. Thus, cells in 1 mo1 m-’ Na’ BG-11 would not have been Na’-limited. The cells showed a Na+ dependence for photosynthesis (using both O, electrode and carbon fixation methods), so they were not standing culture cells, which seem to have no Na’ dependence (Espie et al., 1988a; Espie and Kandasamy, 1992).

[DIC], and pH, measurements were used to estimate extracellular CO,, HCO,-, and C0,’- using the equations of Vollenweider (1974), Ostrom (1975), and Golterman et al. (1978). At pH, 10.0 and a [DIC], of 189 mmol m-’, the CO, in the medium would have been only 34 x 10-6 mo1 m-’, but the HC0,- concentration would have been 117 mmol m-’. Furthermore, the rate of conversion of HC0,- to CO, could not have supplied CO, fast enough to support the observed net photosynthetic rate of 59 2 3.3 nmol m-’ s-’. This rate is equivalent to a rate of 165 (2 16) x 10-6 mo1 m-’ s-l for a cell suspension with 301 (t 24) x 10” cells m-’, but the rate of production of CO, is only 18 x 10-6 mo1 m-3 s-’ (using the equations of Miller and Col- man [1980b] and Salon et al. [1996a, 1996b1). Only a small fraction of this CO, production in the bulk electrolyte would be accessible to any one cell because of diffusion limitations. Thus, under the conditions used in the present experiments, the cells could not have been using CO, in the bulk electrolyte as the source of inorganic carbon and must have been using HCO,-.




Table 1. pH membrane potential, and [Na], in Synechococcus i n alkaline media

High-Na+ BG-11 usually contained CAPS/NaOH buffer, so the [Na+] was 23.7 mo1 m-3 at pH, 10, but in one pHi experiment, light and dark determinations were made using 10 mo1 m-’ glycine/NaOH. All high-Na+ BG-11 experiments run at pH, 11 .O used CAPS/NaOH buffer, giving a total [Na+] of 34 mo1 m-3. Low-Na+ BG-11 medium contained 1 mo1 m-3 [Na+] and 5 mo1 m-’ CAPS/Ca(OH), buffer. Cells were preincubated in the experimental conditions. DCMU-treated cells were incubated in 1 O mmol m-’ DCMU. = membrane potential; E&+ ,,o

= Nerust equilibrium potential of Na+.

Conditions

High-Na+ BG-11 buffered to pH, 10.0 Light Dark Light, DCMU

Light Dark

Light Dark Light, DCMU

High-Na+ BG-11 buffered to pH, 11 .O

Low-Na+ BG-1 1 buffered to pH, 10.0

PHi

7.22 t 0.0714 [6, 361 7.36 t 0.106 [4, 241 7.40 C 0.025 [4, 241

7.28 C 0.018 I4, 241 7.96 t 0.034 [4, 241

7.27 2 0.01 7 [4, 181 7.14 t 0.040 [4, 181 7.27 t 0.035 14, 241

A k O

mV

-131 t 5 [5, 201 -133 C 5 [3, 121 - 124 2 0.8 181

-133 2 3 [3, 121 -134 2 1.4 [3, 121

-153 t 2 [4, 161 -150 t 2 [2, 81 -150 C 9 (4, 181

9.57 t 1.1 o 32.0 t 4.76

a -

23.1 2 2.29 92.0 2 13.0

3.04 C 0.45 11.8C 1.5

-

E N a +

mV

+23 C 2.9 [3, 121 -8 t 3.8 [3, 121

-

+10 t 2.5 [3, 121 -27 C 3.6 [3, 121

+25 t 3.8 [4, 201 -60 C 3.2 [4, 161

-

a -, Not measured.

pH, and [Na+]

Table I shows the pH, membrane potential, and [Na+], for Synechococcus cells in the light, in the dark, and in the light with 10 mmol m-’ DCMU. Care was taken to add enough DIC so that cells did not run out of inorganic carbon before the [‘*C]methylamine, “Rb+ / valinomycin, or ”Na+ incubations were complete.

In normal BG-11 medium with CAPSINaOH buffer, the [Na+], was 23.7 mo1 m-’ at pH, 10.0 and 34 mo1 m-’ at pH, 11.0: these will be defined as high-Na+ conditions. At pH, 10.0, the pH, was s ign i f i can t ly d i f fe ren t at P < 2% leve1 but there was a much greater difference in pH, in the light and dark at pH, 11.0. DCMU had little if any effect on pH, or membrane potential.

Table I also shows the pH,, membrane potential, and [Na+], of Synechococcus cells in alkaline media in the light, in the light in DCMU, and in the dark for cells in 1 mo1 m-’ Na+ BG-11 medium with 5 mo1 m-’ CAPS/Ca(OH), buffer, defined here as low-Naf conditions. The pH, values found in the light, light plus DCMU, and dark at pH, 10.0,

but with a low [Na+],, are similar to those found in cells in the more usual BG-11 medium. The membrane potential values are somewhat higher and the [Na+], lower than in the high-Na+ medium.

Proton-Motive Force and Na+-Motive Force

Table I1 shows the APH+~,, (or proton-motive force,,,) and ApNa+,,, for cells in both low- and high-Na+ BG-11 medium. For protons, the electrochemical potential was positive in the light, light plus DCMU, and in the dark at pH, 10.0 and even more so at pH, 11.0, indicating that protons are actively taken up by the cells. Na+ ions are actively extruded by the cells (about -13 kJ mol-’) at pH, 10.0 in the light and dark and in the light at pH, 11.0. ”Uphill” or active extrusion of Nat is less pronounced in the dark at pH, 11.0. There is a large Na+-motive force across the plasmalemma of the cells, but the proton-motive force is very small.

Table II. ApHC,,, and ApNa+i,, across plasmalemma of Synechococcus

AfiHti and AfiNa’; were calculated from the data in Table I .

A N + i , o APNa+,,, Conditions

mV kl mol-’ mV kl mol-’

High-Na+ BG-11 medium, pH, 10.0 Light +33 2 6.5 +3.18 C 0.6 -154 t 5.8 -14.9 2 0.6 Dark +23 t 8.0 +2.22 t 0.8 -125 t 6.3 -12.1 2 0.6 Light, DCMU +30 t 2 +2.9 t 0.2 - -

Light +87 C 3.2 +8.39 2 0.3 -143 C 3.9 -1 3.8 2 0.4 Dark +46 t 2.5 +4.43 t 0.2 -107 2 3.9 -10.3 C 0.4

a

High-Na+ BG-11 medium, pH, 11 .O

Low-Na+ BG-1 1 medium, pHo 10.0 Light +9 t 2.2 +0.8 -t 0.3 -128 C 4.3 -1 2.4 C 0.4 Dark +19 2 3.1 +1.9 C 0.3 -91 rt 3.8 -8.7 2 0.4 Light, DCMU f 1 2 C 9 +1.1 2 0 . 9 - -

a -, Not measured.




Table III. fffect of Na+ on the pH, in Synechococcus CAPS/Ca(OH), buffer (5 mo1 m-,) at pH, 10.0 was used. The

nominally Na+-free BC-11 would have been contaminated with about 6 mmol m-3 Na+. The Na+-treated cells were exposed to 18 mo1 m-3 Nat for about 1 O min before determination of the pHi. The pHi measurements were made as described in the text less than 1 h after the cells had been prepared in Na+-free BC-11.

Conditions Na+-free BC-11 High-Na+ BC-11

Light Dark

7.60 i 0.1 7 [4, 241 7.87 i 0.24 [4, 241

7.35 t 0.16 [4, 241 7.62 t 0.16 14, 241

pH, Control and Na+ Availability

Table 111 shows a comparison of pH, of Synechococcus cells incubated in nominally Na+-free BG-11 to cells given a normal leve1 of Na+ after first collecting the cells in Na+-free medium. The cells in 18 mo1 m-’ Na+ had a pH, similar to control cells after incubating in the light (Table I). It was notable that cells fed Na+ in the dark for only 10 min and not exposed to the light before the pH, measurement had a significantly more alkaline pH, than control cells in the dark. Preparation of cells in Na+-free medium at pH, 10.0 had some deleterious longer-term effects on the cells. Cells in Na+-free BG-11 had abnormally alkaline pH,. O, electrode studies found that the cells could not tolerate Na+-free medium in the dark for more than about 1 h.

[DICli

[DIC], in the light was approximately 25 mo1 m-’ in both high- and low-Na+ conditions (Table IV), which is comparable to recent estimates by Salon et al. (1996~1, 1996b). [DIC], was very low in the dark: approximately 1 mo1 m-’ in both high- and low-Na+ concentrations (each using n = 15 data points). Cells in DCMU in the light accumulated large amounts of inorganic carbon, even

though no significant photosynthetic carbon fixation occurred. [DIC], in the light in DCMU was about 8.5 mo1 m-’ in low-Na+ conditions and 11 mo1 m-’ in high-Na+ conditions.

Table IV shows the calculated [CO,],, [HCO,-],, and [C0,2-], (assuming the inorganic carbon system has had time to equilibrate and using pH, estimates from Table I). The equilibrium [CO,], was mueh greater than the [CO,], and so CO, was not being taken up by simple diffusion (Nobel, 1983).

HC0,- was not in electrochemical equilibrium in the light, in the light in the presence of DCMU, or in the dark, as observed using membrane potentials (Table I) and the Nernst criteria. In the light, the ApHC0,- exceeded 20 kJ mol-I and approached 30 kJ mol-’ in the light in 1 mo1 mP3 Na+ BG-11. DCMU-treated cells also actively accumulated HC0,- (>20 kJ mel-,) in the light. The intracellular carbon species inside such cells would be expected to be in equilibrium because there was no net flux of carbon through the system. The observed inorganic pool size in DCMU-treated cells is consistent with recent estimates of CO, permeability (Salon et al., 1996a, 1996b) and the oper- ation of a HC0,- uptake pump at a rate comparable to the observed carbon fixation rate in control cells in the light (about 30-60 nmol m-’ s-’).

For a Na+-driven chemiosmotic mechanism to work in the desired direction, the ApNati,, must provide the necessary energy to drive HC0,- against its electrochemical gradient into the cell with an overall negative AGi,, (Ritchie, 1991, 1992a). The ApNa+,,, found in the conditions used in the present study was -12 kJ mol-’ (low Na+) and -15 kJ mol-’ (high Na+) (Table 11). The ApHCO,-,,, observed in the light was 25 to 28 kJ mol-’ (Table IV). At least two (more likely three) Na+ ions would need to be transported to provide enough energy for the inward transport of one HC0,- anion; otherwise the over- a11 AGi,, would not be negative.

Table IV. [DlC], at a pH, of 10.0 Low-Na+ BG-11 contained 1 mo1 m-3 Na+ and 5 mo1 m-3 CAPS/Ca(OH), buffer; high-Na+ BG-11 contained 23.7 mo1 m-, Na+ and 10 mo1

m-3 CAPS/NaOH buffer. The pH, values from Table I were used to calculate [CO,],, [HCO,],, and [CO,’-],. The [HCO,-l, values were then used to calculate ApHC03-,,, in kJ mol-’ using membrane potential data from Table I.

[DICI, DIC IDICI,

Dark Light LighVDCMU mo/ m-3 mo/ m-’ mo/ m-3 mo/ m-’

Cells in High-Na+ BC-11 (23.7 mo1 m-’ Na+) 11.3 i 2.35 IDICI 0.1 89 1.20 i 0.1 2 24 i 4.0

lHCO,-l 0.117 1.09 t 0.15 21 2 4 10.37 t 4.4 34 X 10-6 0.101 i 0.014 2.7 i 0.40 0.876 i 0.136

0.072 0.005 2 0.002 0.067 i 0.01 1 0.049 i 0,013

IDlCl 0.1 89 1.65 i 0.26 3 6 % 13 8.48 i 0.078

[HCO,-I 0.1 17 1.43 t 0.24 32 t 1 1 7.59 i 1.30 [co3’-l 0.072 0.004 2 0.001 0.1 1 4 i 0.040 0.0267 i 0.007

[CO,I

Ico,2-1 Cells in Low-Na+ BG-11 (1 mo1 m-3 [Na+])

. [CO,I 34 X 10-6 0.219 2 0.015 3.7 2 1.3 0.865 i 0.0078

kJ mo/- ’ kJ mo/- kJ mo/-

Thermodynamic analysis of HC0,- electrochemical gradient High-Na+ BC-11 18.0 i 0.59 25 2 0.7 22.7 i 1.05

24.4 i 0.97 Low-Na+ BG-11 20.3 i 0.46 28 i 0.9




Table V. Comparison o f Na+ and K+ fluxes of cells using HC0,- as the carbon source (pH, 10.0) Buffers and Na+ concentrations of media were as for Table IV.

Low-Na+ BC-11 High-Na+ BC-11

Na+ fluxes (nmol m-2 s-’, 22Na+ efflux labeling) DIC supply DIC-free medium + 189 mmol m-3 DIC

DIC supply DIC-free medium

102 t 28 [3, 241 25.7 ? 5.12 14, 321 22.4 ? 3.3 [4, 321 80 t- 14.5 [4, 321

K+ fluxes (nmol m-2 s-’, 42K+ uptake labeling)

3.26 t 0.58 [2, 321 3.68 L 0.31 [2, 321 + 189 mmol m-3 DIC 3.95 t 0.51 [2, 321 4.16 L 0.64 [2, 321

Net Photosynthesis Rate and Na+ and K+ Fluxes

Table V shows measurements of Na+- and K+-exchange fluxes, which will now be compared with measurements of

that the net photosynthetic rates are too high to be supported by a 2Nafin/ HCO,-,, co-transport mechanism, particularly in low-Na+ conditions.

the net photosynthetic rate, which can be taken as a mini- mal estimate of the net HC0,- uptake rate (Salon et al., 1996a, 1996b). ,’Na+ fluxes were estimated in cells incubated in DIC-free BG-11 at pH, 10.0 and in cells fed inorganic carbon at the same time that the ,’Na+ flux determinations were made. The Na+ fluxes in the presence and absence of DIC were not significantly different ( t test; Zar, 1974) and so the Na’ flux in low-Na+ cells was 24 +- 3 nmol m-’s-’ (n = 64) and 89 C 14 nmol mP2 sK1 (n = 56) for cells in high-Na+ BG-11.

Table V also shows estimates of 42K+ fluxes in cells incubated in DIC-free BG-11 at pH, 10.0 and cells fed inorganic carbon at the same time that the 42K+ flux determinations were made. The K+ fluxes in the presence and absence of DIC were not significantly different (Zar, 1974) in either low- or high-Na+ concentrations and there was no significant effect of Na+ on the K+ flux. The K+ flux (about 4 nmol m-’ s-’) was too low for it to play a role in HC0,- transport.

Net photosynthetic rates were measured using 14C labeling and O, electrode methods. In low Na+, the carbon fixation rate was about 42 5 4 [4, 321 nmol m-’ s-l and 55 ? 6 [6,70] nmol m-’s-l in 18 mo1 m-3 Na+ BG-11. The 14C net fixation rate for cells in high Na+ compares well with estimates based on the O, electrode (59.1 ? 3.3 [5, 601 nmol m-’ s-l). Comparison with the Na+ flux data shows

Hyperpolarization Effects of lnorganic Carbon Uptake

Table VI shows the polarizing effects of added inorganic carbon on the membrane potentials of the cells. Apparent hyperpolarization of the membrane potential in the pres- ente of inorganic carbon occurred in the light at pH, 7.5 and 10.0 in cells in high-Na+ BG-11. No significant hyperpolarization occurred in cells exposed to DIC in Na+-free medium. The hyperpolarization in 1 mo1 m-’ Na+ BG-11 was small but significant, even though it was very much smaller than in cells in high-Na+ conditions. There was no significant hyperpolarization in the dark or in the presence of DCMU in the light. The observed effects of DCMU show that the hyperpolarization effect is associated with net carbon fixation, since these cells actively accumulate HC0,-. The hyperpolarization effect is transitory: after a 30-min incubation there is only a very small difference in the membrane potential of cells in DIC-free medium and in cells exposed to DIC. Most of the initial hyperpolarization observed in cells in 18 mo1 m-’ Na+ BG-11 had disap- peared after 15 min of exposure to DIC. Net uptake of HC0,- seems to involve a net uptake of negative charge.

K+ uptake fluxes were unaffected by the presence or absence of DIC (Table V). Thus, the observed changes in the valinomycin-mediated Rb+ uptake flux reflected a

Table VI. Polarization effects o f inorganic carbon

Low-Na+ BG-1 1 contained 1 mo1 m-’ Na+ and 5 mo1 m-’ CAPS/Ca(OH), buffer; high-Na+ BC-1 1 contained 23.7 mo1 m-3 Na+ and 1 O mo1 m-3 CAPS/NaOH buffer. The nominally Na+-free media contained about 6 mmol m-3 Nat due to contaminants in the laboratory chemicals; 5 mo1 m-3 CAPS/Ca(OH), buffer was used. Cells were preincubated in nominally DIC-free BG-1 1 medium in capped Eppendorf tubes for 30 min. Control and experimental cells were incubated as matched pairs.

A$,,, “1 Conditions PHO Polarization

Control 30 min DIC

High-Na+ BC-11, light 7.5 -104 t 4 -110 t 1.5 -66 t- 24 [2, 321 -157 t 14 [2,321 High-Na+ BG-11, light 10.0 - 1 1 7 t 2.9 -126 t 4

High-Na+ BG-11, exposed 10.0 -125 t 9 -130 t 8 -43 t 24 [2, 321

High-Na+ BG-1 1 , 10 10.0 -124 t 0.8 -126 t 0.9 +4.3 t 15 (2, 401

High-Naf BG-1 1 , dark 10.0 - 1 3 3 t 1.7 -129 t 2.9 +19 t 19 [2, 361 Low-Na+ BG-1 1, light 10.0 - 1 4 9 t 1.7 -1 56 t 0.94 -1 7 t 8.8 12, 401 Na+-free BC-l 1, light 10.0 -92 t 2.1 -94 t 2 -19 t 23 [ l , 161

to DIC 15 min, light

mmol m-3 DCMU, light




change in the membrane potential and not an activation of a K+ pump in the presence of inorganic carbon.

DISCUSSION

The present study has shown that Synechococcus R-2 cannot use CO, as its carbon source and must actively accumulate HC0,- in the light and dark in alkaline medium. To make an assessment of the most likely mechanisms for DIC accumulation (Reinhold et al., 1984; Zenvirth et al., 1984), a comprehensive set of ion transport data under the same experimental conditions was needed.

Thermodynamic Analysis

The values of pH, and membrane potential are similar to those reported previously for C0,-grown cells (pH, 7.5) (Ritchie, 1991, 1992a), meaning that the APH+,,~ is near O and of the wrong sign (active in) for it to be a driving force for secondary active transport mechanisms (Table 11). An H+ / HC0,- symport can be rejected on thermodynamic grounds (Nicholls, 1982; Nobel, 1983; Skulachev, 1988). Similarly, K+ is close to electrochemical equilibrium in Synechococcus and so its electrochemical potential is very unlikely to drive secondary active transport systems (Ritchie, 1991). Thus, ApNa+i,o is the only electromotive force large enough to be a candidate for driving HC0,- transport. Under the alkaline conditions of the present study, ApNa+i,o is in excess of -12 kJ mol-' in both high- and low-Na+ conditions (Table 11) and is similar to that found previously in C0,-grown cells (Ritchie, 1992a).

Na+-Driven Transport Systems

It is important to distinguish between Na+-dependent uptake of ions and uptake mechanisms actually driven by the ApNa+i,o. Involvement of Na+ in transport of inorganic carbon species could be direct (Na+,, / HCO,-,, co- transport), as proposed by Zenvirth et al. (1984), Reinhold et al. (1984), and Espie and Kandasamy (1994), or indirect (Miller et al., 1984; Reinhold et al., 1984; Espie et al., 198813) through effects on ATP supply, or through Na+ acting as a cofactor for transport proteins, or through the role of Na+ in pH, regulation.

The present study shows that Na+ is required for HC0,- uptake; however, HC0,- uptake rate is too high to be supported by the observed Naf fluxes (Table V), even though it would appear that there is a sufficient Nat- motive force available (Table 11). This conclusion seems to closely parallel the case of chloride transport in Synecho- coccus (Ritchie, 1992a, 1992b, 1992~). There is other evi- dente for a biochemical rather than a chemiosmotic role of Na+ in cyanobacteria (Ritchie, 1991, 1992a, 1992b, 1992~). Table I11 confirms the finding of Miller et al. (1984) that Na+ is required for the close regulation of pH,. Reinhold et al. (1984) raised the possibility that the Na+ dependence of HC0,- transport in Anabaena was primarily a result of its role in control of pH, (Table 111). This hypothesis is not inconsistent with the present results and could be tested experimentally (Ritchie and Gibson, 1987).

lnorganic Carbon Accumulation

Light-stimulated active uptake of inorganic carbon (25-30 kJ mol-', Table IV) occurs in the light on the basis of both the Fick's First Law criterion for CO, and the Nernst criterion for HC0,- movement (Nobel, 1983). DCMU only partially inhibited active uptake of HC03- (Table IV), even though carbon fixation was essentially completely inhibited by DCMU (<<1 nmol m-' s-I). This shows that cyclic photophosphorylation can at least partially drive HC0,- uptake in Synechococcus. Miller and Colman (1980a) observed similar effects of DCMU on the blue-green alga Coccochloris.

The [DIC], is also apparently not in equilibrium with the outside of the cells in the dark. This has important impli- cations. Using the recent permeability estimate of 30 ? 1 nm s-' for cells at pH, 8.0 (Salon et al., 1996a, 1996b), and the calculated [CO,], in the dark (Table IV), we estimate that the COz flux out of the cells would be about 3 to 10 nmol m-' s-'. This is very comparable to respiration rate estimates we have made using the O, electrode. Hence, the size of the intracellular carbon pool in the dark is due to a buildup of respiratory CO, inside the cells and rate- limiting diffusion of CO, out of the cells (Badger, 1985; Badger et al., 1985; Price and Badger, 1989; Salon et al., 1996a, 1996b). Additionally, the observed size of the intracellular carbon pool in DCMU-treated cells is consistent with DCMU inhibiting carbon fixation but not the HC0,- uptake pump.

Test of the Na+/HCO,- Co-Transport Hypothesis

with the data shown in Tables I to V we are in a position to critically test whether a Na' /HCO,- co-transport mechanism could account for the very strongly uphill, active uptake of HC0,- in the light (ApHC03-,,,, Table IV).

For a Na'-driven chemiosmotic mechanism to work in the desired direction, the ApNa+,,, must provide an overall negative AGi,o high enough to supply the energy necessary to drive HC0,- against its electrochemical gradient into the cell (Ritchie, 1991, 1992a). A Na+-driven co-transport would need to have a stoichiometry of 2 or greater (2 or 3Na+,,/HC03-,,) for the overall AG to be negative (Tables I1 and 1V).

Na+ fluxes must be high enough to account for the rate of the linked transport process. Table V shows that Na+ fluxes at pH, 10.0 are considerably lower than those found previously at pH, 7.5 (about 275 nmol m-, s-', Ritchie, 1992a). Na'-exchange fluxes were unaffected by the presence or absence of DIC in both high and low concentrations of Na+ (Table V): an implausible circum- stance if there were an obligate linkage between Naf and HC0,- transport. In high-Na+ concentrations the entire Na+ flux would need to be linked to active uptake of HC0,- in the light (Table V). In low-Na+ conditions the observed Na+ fluxes (24 nmol m-'s-') are far too low to support the observed net photosynthetic rates based on I4C fixation (42 nmol m-' s- ') . K+ fluxes are very low and seem unaffected by either low or high Na+ or the presence or absence of inorganic carbon (Table V). Thus, neither




Na+ nor K+ transport seems linked to uptake of inorganic carbon.

The above calculations take into account only the observed net fixation of carbon in the light. The actual transport of inorganic carbon into the cells would need to be higher because of leakage of inorganic carbon out of the cells as CO, and some back-flux of HC0,- (Badger, 1985; Raven and Lucas, 1985; Badger et al., 1985; Salon et al., 1996a, 1996b).

Electrogenicity of HC0,- Uptake

Significant hyperpolarization effects of added inorganic carbon were detected using comparisons of the valinomycin-mediated uptake flux of "Rb+ (Table VI), despite the inherent limitations of the method: polarization effects are not experimentally detectable if less than about 2 20 mV and it is not possible to measure very short-term effects on membrane potential (Ritchie, 1992a). The hyperpolarization effect appears to be real because the

K uptake flux is independent of the presence of HC0,-, and the K+ flux is very low. These depolarization effects are more clear-cut than in previously reported studies. The very substantial hyperpolarization effect of HC0,- uptake on the membrane potential shows that net uptake of HC0,- is negatively electrogenic (Table VI). This observa- tion argues against electrogenic 2Na+,, / HCO,-,, or 3Na+,, / HCO,-,, co-transport mechanisms, both of which should depolarize the membrane potential (the opposite of the effect observed).

42 +

Charge Balance

The accumulation rates of inorganic carbon found in the present study are comparable to or somewhat lower than those previously reported. Badger and Andrews (1982) reported HC0,- fluxes as high as 144 nmol m-'s-' and accumulation ratios for HC0,- of 3500:l (ApHCO,-,,i = 35 kJ mol-l, assuming a membrane potential of -120 mV). The inorganic carbon pool rapidly increases from about 1 mo1 m-' in the dark to about 25 mo1 m-' in the light (mostly in the form of HCO,-, Table IV). This net uptake of inorganic carbon is accompanied by a temporary hyperpolarization of the membrane potential (Table VI) that is absent in DCMU-treated cells, even though they do actively accumulate inorganic carbon (Table IV). Thus, the cells must maintain a charge balance to compensate for the uptake of HC0,- and fixation as CO, (Price and Badger, 1989). However, Tables I and V clearly show that the balancing ion cannot be either Na+ or K+: the fluxes of these ions are too low. Therefore, this study supports pre- vious evidence that either H+ or OH- is the balancing ion involved in HC0,- uptake (Kaplan et al., 1982,1989,1991; Zenvirth et al., 1984; Ogawa and Kaplan, 1987). The observed hyperpolarization indicates a lack of direct linkage of HC0,- and H+ / OH- charge compensation; otherwise, hyperpolarization effects would not be observed on a time scale of minutes.

Cost of HC0,- Transport

Kaplan (1985) suggested the possibility that there was a primary or ATP-driven HC0,- pump in cyanobacteria. An HCO,- / ATP pump with a stoichiometry of one HC0,- / ATP could actively accumulate HC0,- ion to the observed maximum ApHCO,-,,, (approximately +28 kJ mol-', Ta- ble IV). The pump would need to have a thermodynamic efficiency of about 6O%, assuming a AG of hydrolysis of ATP of -50 kJ mol-I (Reid and Walker, 1983; Muchl and Peschek, 1984).

It is possible to make an estimate of the energetic "cost" of active transport of HC0,- from the product of estimates of the electrochemical gradient of the ion and the net photosynthetic rate (Ritchie, 1985, 1991, 1992a). This is a minimum estimate of the power consumption of a membrane transport process. The power consumption of a primary ATP-driven pump would be the product of the free energy available from the hydrolysis of ATP (see above) and the flux of the transported ion.

W = +HC03-,,i X ApHC03-i,, (2)

W = +HC03-,,,i X AGATp (3)

where W is the power requirement of transport in mW m-'. It is possible to make minimum estimates of the power

consumption of HC0,- transport using the data from Ta- bles IV and V and compare it with the energy available from Glc formed in photosynthesis. The power requirement for net uptake of HC0,- in low- and high-Na+ conditions would be greater than 1 mW m-'. The free energy available from one CH20 fragment of Glc is 482 kJ mol-' (Ritchie, 1985) and so the power available from net photosynthesis is approximately 20 mW m-' in low-Na' medium and 27 mW m-' in standard BG-11 medium. Thus, Synechococcus would consume at least the equivalent of 5% of the power available from fixed carbon in active uptake of HC0,- by such a mechanism. Active uptake of H+ or extrusion of OH- needed to achieve charge balance would add to the power cost (1-8 kJ mol-l, Table 111).

A primary ATP-driven pump would be much more costly in terms of power since 50 kJ mol-I would be consumed in the transport of each HC0,- ion. If such a mechanism existed in Synechococcus, the power consumption of HC0,- transport would increase to 2 to 3 mW m-' or about 10% of the power available from the carbon after fixation. Such calculations take no account of the "leak of some inorganic carbon out of the cells (perhaps 30% of total uptake, Badger et al., 1985; Salon et al., 1996a, 1996b). The total uptake flux of HC0,- must be greater than the actual carbon fixation rate (Badger et al., 1985; Reinhold et al., 1989).

CONCLUSIONS

Measurement of Na+ and K+ fluxes, combined with data on the electrochemical potentials of Na+ and protons and HC0,-, have demonstrated that the Na+-dependent HC0,- uptake pump of Synechococcus is a negatively electrogenic pump. But it does not appear to be a second-




ary active transport system. Mechanisms linked t o the electrochemical potentials H+, K+, or Na+ have each been refuted on thermodynamic and / or kinetic grounds. Pro- tons or OH- appear t o be the balancing ions for HC0,-, b u t this raises important questions about how the cells a re able to regulate pH, within very nar row limits.

ACKNOWLEDCMENTS

The authors wish to thank Drs. G.D. Price and M. Badger for their help with this project.

Received March 21, 1996; accepted August 27, 1996. Copyright Clearance Center: 0032-0889/96/ 112/1573/12.

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