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. 1998 Jan;116(1):193–201.

Uptake of HCO3− and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta1

Gabi Amoroso 1, Dieter Sültemeyer 1,*, Christoph Thyssen 1, Heinrich P Fock 1
PMCID: PMC35158

Abstract

Mass-spectrometric disequilibrium analysis was applied to investigate CO2 uptake and HCO3− transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO2 (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO2 and HCO3−, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO3− was the dominant inorganic C species taken up, whereas HCO3− and CO2 were used at similar rates by C. reinhardtii. The apparent affinities of HCO3− transport and CO2 uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO2 and HCO3−. For chloroplasts from C. reinhardtii, the concentrations of HCO3− and CO2 required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO3− and CO2 transporters at the chloroplast envelope membrane.

It is well documented that a number of green algae and cyanobacteria possess an inducible CCM that elevates the intracellular CO2 concentration around the primary CO2-fixing enzyme, Rubisco. As a result, the apparent photosynthetic affinity for CO2 increases, photorespiration and sensitivity to O2 decrease, and the CO2 compensation point is lowered (Badger and Price, 1992, 1994; Sültemeyer et al., 1993).

Several biochemical and biophysical components have been identified in photosynthetically active microorganisms possessing a CCM. First, to sustain an elevated CO2 concentration within the cells, it is necessary to minimize CO2 leakage. Although little work has been done on the leak-rate control mechanism, it seems that the plasma membrane is not a barrier for CO2 diffusion, suggesting that a CO2 concentration gradient is not built up along the plasmalemma (Sültemeyer and Rinast, 1996). Second, CA seems to be an absolute requirement for the operation of the CCM. Extracellular CA exists in high- and low-Ci cells of Chlamydomonas reinhardtii and Dunaliella tertiolecta, as well as in a number of eukaryotic micro- and macroorganisms, and the activity is substantially increased upon transfer of cells to low CO2 concentrations (Sültemeyer et al., 1993; Badger and Price, 1994; Sültemeyer, 1997). In addition, physiological and biochemical evidence indicates the presence of several intracellular CA activities (Sültemeyer et al., 1993, 1995a; Badger and Price, 1994; Katzman et al., 1994; Karlsson et al., 1995; Amoroso et al., 1996; Eriksson et al., 1996; Funke et al., 1997). Third, an energy-dependent transport system for Ci is responsible for its accumulation. Active transport of CO2 and HCO3− have been postulated for eukaryotic algae, with CO2 being the preferred Ci species taken up by the cells (Williams and Turpin, 1987; Goyal and Tolbert, 1989; Sültemeyer et al., 1989, 1991; Palmqvist et al., 1994; Matsuda and Colman, 1995).

Ci transport systems may be located solely at the plasma membrane (Rotatore and Colman, 1991) or at both the plasma membrane and the chloroplast envelope, as has been suggested for green algae (Goyal and Tolbert, 1989; Sültemeyer et al., 1989, 1991; Ramazanov and Cardenas, 1992; Badger and Price, 1994, Palmqvist et al., 1994). Some investigators have postulated that the chloroplast envelope is the primary site for Ci uptake, and that HCO3− may serve as its substrate (Moroney et al., 1987; Moroney and Mason, 1991). Several lines of evidence indicate that chloroplasts from C. reinhardtii and D. tertiolecta do play an essential role in a functional CCM: (a) plastids from low-Ci cells had an enhanced ability to accumulate Ci internally and considerably higher affinities for Ci (Moroney et al., 1987; Sültemeyer et al., 1988; Goyal and Tolbert, 1989; Moroney and Mason, 1991; Ramazanov and Cardenas, 1992); (b) chloroplastic CA activities increased up to 10-fold upon transfer of the cells from high to low CO2 concentrations (Ramazanov and Cardenas, 1992; Sültemeyer et al., 1993, 1995a; Badger and Price, 1994; Amoroso et al., 1996); (c) several low-CO2-inducible polypeptides seem to be specifically associated with the chloroplast, possibly with the inner envelope membrane (Thielmann et al., 1992; Ramazanov et al., 1993, 1995); (d) a correlation between the phosphorylation state of thylakoid proteins and acclimation to low CO2 concentrations has been reported (Marcus et al., 1986); (e) the intracellular position of the chloroplast depends on the CO2 provided during growth because it moves close to the plasma membrane on low CO2, whereas it remains centralized under high CO2 concentrations (Tsuzuki et al., 1986); and (f) a starch sheath is developed around the pyrenoid under limiting CO2 concentrations (Kuchitsu et al., 1988; Ramazanov et al., 1994), but its importance for a functional CCM is controversial (Villarejo et al., 1996).

The increased level of Ci accumulation and the higher apparent affinities of photosynthesis in low-Ci chloroplasts compared with those from high-Ci cells indicate that the former are able to transport Ci actively. In this context, the central question is which Ci species, CO2 or HCO3−, is taken up. The aim of this work was to investigate whether CO2 or HCO3− is taken up by photosynthetically active chloroplasts isolated from C. reinhardtii and D. tertiolecta. We applied a recently developed MS disequilibrium technique (Badger et al., 1994) that allows the distinction between CO2 and HCO3− uptake during steady-state photosynthesis. The results provide evidence that chloroplasts from both algal species are able to transport CO2 and HCO3− simultaneously, regardless of the CO2 concentration provided during growth.

MATERIALS AND METHODS

Growth of Algae

Chlamydomonas reinhardtii (Dangeard) strain 11/32b and Dunaliella tertiolecta (Butcher) were obtained from the Sammlung für Algenkulturen (Göttingen, Germany). Cells of both species were grown photoautotrophically under nonsynchronized (for isolation of protoplasts and cells) or synchronized (for isolation of chloroplasts) conditions (Sültemeyer et al., 1988, 1995a). For C. reinhardtii, culture conditions were pH 8.0 and 30°C, whereas D. tertiolecta was grown at 1 m NaCl, pH 8.0, and 25°C (Amoroso et al., 1996; Sültemeyer, 1997). Either ambient air (0.035% [v/v] CO2; low-Ci cells) or air enriched with 5% (v/v) CO2 (high-Ci cells) was continuously passed through the cultures.

Preparation of Cells and Protoplasts for Ci Measurements

For each measurement with nonsynchronized cells of D. tertiolecta an aliquot of high- and low-Ci algae containing about 50 to 100 μg of total Chl was harvested by centrifugation (2000g, 5 min) and washed three times in 1 mL of CO2-free buffer (25 mm BTP-HCl, pH 8.0, containing 1 m NaCl). The final pellet was resuspended in a small volume (<100 μL) and used for the gas-exchange measurements.

To prepare protoplasts from cells of C. reinhardtii, an aliquot containing 100 μg of Chl was removed from the culture vessel and centrifuged. The pellet was washed once in 15 mm Hepes-KOH, pH 7.8, and incubated in 30 mL of autolysin (Sültemeyer et al., 1988). Protoplast formation was usually complete within 10 min at room temperature. Protoplasts were separated from autolysin and washed six times in 1 mL of BTP-HCl buffer, pH 8.0, to ensure complete removal of external CA (Sültemeyer et al., 1989, 1990).

Isolation of Chloroplasts

The methods of plastid isolation from the two algal species differ mainly in the disruption procedure. Cells of D. tertiolecta were mechanically disrupted by slowly releasing the cell suspension into a glass tube after equilibration with 90 kPa N2 for 2 to 3 min in a Yeda press (Goyal and Tolbert, 1989; Amoroso et al., 1996), whereas protoplasts from cells of C. reinhardtii were disrupted chemically with digitonin. The buffers used for cell lysis and for further purification of chloroplasts have been described previously for D. tertiolecta (Amoroso et al., 1996) and C. reinhardtii (Sültemeyer et al., 1988, 1995a). Contamination of the chloroplast preparations with cytosol and mitochondria was routinely analyzed according to the method of Amoroso et al. (1996) and was always found to be less than 4%.

MS Measurements of CO2 Uptake and HCO3− Transport

Gas-exchange measurements were performed in a reaction chamber connected to a mass spectrometer via a semipermeable membrane inlet system (Fock and Sültemeyer, 1989). Protoplasts from high- and low-Ci cells of C. reinhardtii were measured in 25 mm BTP-HCl, pH 8.0, at 30°C, and the assay contained 5 to 10 μg of Chl mL−1. High- and low-Ci cells of D. tertiolecta were analyzed at 25°C in 25 mm BTP-HCl, pH 8.0, supplemented with 1 m NaCl in the presence of 5 to 10 μg of Chl mL−1. Photosynthetically active chloroplasts from both species were tested at 25°C in an assay medium containing 150 mm mannitol, 25 mm KCl, 1 mm MgCl2, 0.2 mm K2HPO4, 25 mm BTP-HCl, pH 8.0, and 15 to 25 μg of Chl mL−1. Rates of O2 evolution, CO2 uptake, and HCO3− transport during steady-state photosynthesis were determined using the MS disequilibrium technique, which was recently developed by Badger et al. (1994).

Application of this method is possible only if extracellular or extrachloroplastic CA activity is absent, because it relies on the spontaneous hydration and dehydration of CO2 and HCO3−. Therefore, 5 μm AZA was added to protoplasts, cells, and chloroplasts. Independent measurements have shown that this concentration of the CA inhibitor has no effect on intracellular or intrachloroplastic CA activity (data not shown). From the changes in the CO2 (m/z = 44) and O2 concentrations (m/z = 32), the fluxes of CO2 and HCO3− uptake were estimated using previously published formulas (Badger et al., 1994). The ratio of HCO3− to CO2 and the pseudo-first-order rate constant K2(HCO3−:CO2) were measured for each experimental condition and found to be 45 and 0.069 min−1 (25 mm BTP-HCl, pH 8.0, 30°C), 110 and 0.018 min−1 (25 mm BTP-HCl, pH 8.0, 1 m NaCl, 25°C), and 85 and 0.024 min−1 (assay buffer for chloroplasts, 25°C), respectively.

Other Parameters

Contamination of the chloroplast fractions by mitochondria and cytosol was examined by means of the marker enzymes succinate dehydrogenase and PEP carboxylase, respectively, as described by Amoroso et al. (1996). Total Chl was determined according to the method of Porra et al. (1989).

RESULTS

CO2 and HCO3− Uptake with Intact Cells

MS measurements of CO2 and O2 gas exchange provide a useful tool for determining the rates of CO2 and HCO3− uptake during steady-state photosynthesis (Badger et al., 1994; Palmqvist et al., 1994; Sültemeyer et al., 1995b). A typical time course of changes in the CO2 and O2 concentrations obtained with protoplasts from low-Ci cells of C. reinhardtii is shown in Figure 1. MS measurements of changes in the CO2 concentration by illuminated cells of C. reinhardtii and D. tertiolecta are complicated by the presence of external CA, which increases the rate of interconversion of CO2 and HCO3− over the uncatalyzed value of the reaction medium. With C. reinhardtii, this activity could be abolished by using washed protoplasts that showed no measurable external CA activity (Sültemeyer et al., 1989, 1990). To inhibit any additional CA activity possibly released by the protoplasts into the external medium during the experiment, AZA (5 μm) was added to the suspension before the onset of illumination.

Figure 1.

Figure 1

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Typical time course of changes in the CO2 and O2 concentrations in the dark and during illumination of protoplasts from low-Ci cells of C. reinhardtii measured by MS. The experiment was performed at 30°C and in 25 mm BTP-HCl, pH 8.0. Light (350 μmol photons m−2 s−1) was switched on and off as indicated. The Chl concentration was 6 μg mL−1. The concentrations of Ci and O2 at the beginning of the experiment were 200 and 220 μm, respectively. To ensure that no CA activity was released by the protoplasts during the course of the experiment, 5 μm AZA was included.

In vivo measurements of intracellular CA activity using 18O2 exchange from dual-labeled CO2 (13C18O2) (Sülte-meyer et al., 1995a; Amoroso et al., 1996) indicated that internal CA activity remains unaffected by this treatment (data not shown). With the beginning of the light period an initial rapid decrease in the extracellular CO2 concentration was observed, which declined rapidly and reached a steady-state rate after 1 to 2 min, almost at the same time that O2 evolution reached its maximum steady-state rate. When the light was switched off, there was a rapid rise in the CO2 concentration caused by reequilibration between external CO2 and HCO3−, CO2 release from the Ci pool, and respiratory CO2 evolution (Sültemeyer et al., 1989; 1991; Badger et al., 1994; Palmqvist et al., 1994). This rapid increase in the CO2 concentration declined to the final slow rate of dark respiratory CO2 release.

Measurements of CO2 and O2 gas exchange similar to those shown in Figure 1 were used to calculate net O2 evolution, HCO3− uptake, and CO2 uptake during steady-state photosynthesis by C. reinhardtii and D. tertiolecta in relation to their substrates (Figs. 2 and 3). It is apparent that C. reinhardtii and D. tertiolecta were able to use both CO2 and HCO3− simultaneously for photosynthesis and that this ability was independent of the Ci concentration provided during growth. For protoplasts from high- and low-Ci cells of C. reinhardtii, photosynthesis is supported by CO2 and HCO3− utilization almost by the same percentage over the entire Ci concentration range tested, which is in agreement with earlier results (Badger et al., 1994; Palmqvist et al., 1994) (Fig. 2). For high- and low-Ci cells of D. tertiolecta, HCO3− seems to be the more dominant substrate, with CO2 and HCO3− contributing to about 20 and 80% of C uptake, respectively (Fig. 3).

Figure 2.

Figure 2

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A, Photosynthesis and Ci uptake in protoplasts from high-Ci (open symbols) and low-Ci (closed symbols) cells of C. reinhardtii. Shown are the rates of net O2 evolution (▪, □) and net HCO3− transport (•, ○) during steady-state photosynthesis in relation to the external HCO3− concentration. B, Rates of CO2 transport (▴, ▵) during steady-state photosynthesis in relation to the external CO2 content. Data were calculated from experiments similar to the one shown in Figure 1. The assay conditions were the same as in Figure 1 except that the initial Ci concentration varied from 0.01 to 4 mm. The Chl content was 6.2 μg mL−1.

Figure 3.

Figure 3

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A, Photosynthesis and Ci uptake in high-Ci (open symbols) and low-Ci cells (closed symbols) of D. tertiolecta. Shown are rates of net O2 evolution (▪, □) and net HCO3− transport (•, ○) during steady-state photosynthesis in relation to the external HCO3− concentration. B, Rates of CO2 transport (▴, ▵) during steady-state photosynthesis in relation to the external CO2 content. The reaction buffer contained 25 mm BTP-HCl, pH 8.0, 1 m NaCl, and 5 μm AZA. The temperature was 25°C and the Chl content was 8 μg mL−1.

The apparent affinity of net O2 evolution and net HCO3− uptake for external HCO3− substantially increased after acclimation to a low CO2 concentration in both algal species (Table I). The K1/2(HCO3−) values for net O2 evolution and net HCO3− uptake in protoplasts from high-Ci-grown cells of C. reinhardtii were 436 and 316 μm, respectively, whereas the corresponding values for protoplasts from low-Ci-grown cells were 101 and 78 μm (Fig. 2; Table I). This indicates a 4- to 5-fold increase in the apparent affinity for HCO3− for net O2 evolution and HCO3− uptake. In high-Ci cells of D. tertiolecta, the K1/2(HCO3−) values for net O2 evolution and HCO3− uptake were 541 and 362 μm, respectively, and the corresponding values for low-Ci cells were 195 and 143 μm HCO3− (Fig. 3; Table I). Similar to HCO3− transport, the efficiency of CO2 uptake was found to be dependent on the CO2 supply during growth. In C. reinhardtii the K1/2(CO2) for CO2 uptake in protoplasts decreased from 8.3 μm in high-Ci cells to 0.9 μm in low-Ci cells (Fig. 2; Table I). The corresponding values in D. tertiolecta were 7-fold lower in low-Ci cells than in high-Ci cells, 1.7 versus 13.9 μm CO2 (Fig. 3; Table I).

Table I.

Apparent affinities of CO2 and HCO3− uptake in intact cells

Type of Cells K1/2 Values
Ci HCO3− CO2
μm
C. reinhardtii
 High Ci 436  ± 80 316  ± 35 8.3  ± 0.9
 Low Ci 101  ± 9 78  ± 7 0.9  ± 0.1
D. tertiolecta
 High Ci 541  ± 55 362  ± 33 13.9  ± 2
 Low Ci 195  ± 32 143  ± 17.4 1.7  ± 0.2
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Summary of the K1/2 values of net O2 evolution, net HCO3− transport, and gross CO2 transport for their respective substrates during steady-state photosynthesis by high- and low-Ci cells of C. reinhardtii and D. tertiolecta. The data represent mean values ± sd from three to four independent experiments similar to those shown in Figures 2 and 3.

Experiments with Intact Chloroplasts

The examination of Ci uptake properties of C. reinhardtii and D. tertiolecta chloroplasts during steady-state photosynthesis requires the isolation and characterization of intact chloroplasts. The intactness of the plastids isolated from both algal species was checked by the ferricyanide reduction assay (McLilley et al., 1975) and was found to be greater than 90% (data not shown). Cytosolic and mitochondrial contamination of the plastid fraction, determined separately by marker-enzyme measurements, was less than 4% (Amoroso et al., 1996), regardless of the Ci concentration provided during growth. The high purity of the plastids is comparable to that of other chloroplast preparations from D. tertiolecta and C. reinhardtii (Goyal et al., 1988; Sültemeyer et al., 1988; Ramazanov and Cardenas, 1994).

Figure 4A shows a typical time course of O2 gas exchange in the dark and during illumination in the presence of 1 mm Ci in chloroplasts isolated from low-Ci cells of C. reinhardtii. In contrast to intact cells (Fig. 1), no O2 uptake before illumination could be detected in isolated chloroplasts under our experimental conditions, which indicates the absence of respiration. After the light was turned on, chloroplasts from both species took an unusually long period (up to 20 min) before a linear rate of O2 evolution between 20 and 40 μmol mg−1 Chl h−1 could be observed. It should be noted, however, that this was true only for the first light period after the isolation procedure. Subsequent dark/light cycles revealed a normal lag phase for net O2 evolution of between 2 and 3 min (Fig. 4B). Typically, photosynthetic O2 evolution by the chloroplasts could be completely inhibited by the addition of 10 mm Pi (Fig. 4A), whereas whole cells were not affected by this Pi concentration (data not shown). Similar results concerning the inhibition of photosynthetic O2 evolution by chloroplasts from D. tertiolecta by 10 mm Pi have been reported (Goyal et al., 1988).

Figure 4.

Figure 4

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A, Effect of Pi on photosynthetic O2 evolution by chloroplasts from low-Ci cells of C. reinhardtii measured in an O2 electrode. Evolution of O2 was initiated by turning the light on (250 μmol photons m−2 s−1). From a 1 m KPi stock solution, pH 8.0, Pi was added to a final concentration of 10 mm where indicated. The concentrations of Ci and O2 at the beginning of the experiment were 1 mm and 230 μm, respectively. Note the rather long lag period after the onset of illumination. B, Changes in the CO2 and O2 concentration in the dark and during illumination of chloroplasts from high-Ci cells of C. reinhardtii measured by MS. Light (250 μmol photons m−2 s−1) was switched on and off as indicated. To fully inhibit extrachloroplastic CA activity in the chloroplast suspension, 5 μm AZA was included. At the beginning of the light period the Ci concentration was 1 mm and the initial O2 concentration was 225 μm. Both experiments were performed at 25°C in assay medium containing 150 mm mannitol, 25 mm KCl, 1 mm MgCl2, 0.2 mm K2HPO4, and 25 mm BTP-HCl, pH 8.0. The Chl concentration was adjusted to 16 μg mL−1 in both cases.

A typical time course for determination of CO2 and HCO3− flux rates during steady-state photosynthesis obtained with chloroplasts from high-Ci cells of C. reinhardtii is depicted in Figure 4B. The process of measuring Ci fluxes with chloroplasts was the same as that described for whole cells. It was also necessary to add 5 μm AZA to inhibit CA activity likely released into the external medium by broken chloroplasts. After the onset of illumination, a rapid decline in the CO2 concentration was observed, which reached a steady-state rate after about 2 min, whereas the O2 evolution reached a maximum steady-state rate. The rapid rise in the CO2 concentration in the dark is caused by reequilibration between the external Ci species and by CO2 released from the Ci pool. As shown in Figure 4A, dark respiration was almost zero in chloroplast preparations and, therefore, CO2 evolution was negligible.

MS measurements as shown in Figure 4B were used to calculate the rates of net O2 evolution, HCO3− uptake, and CO2 uptake during steady-state photosynthesis by chloroplasts isolated from high- and low-Ci cells of C. reinhardtii and D. tertiolecta (Figs. 5 and 6). It is obvious that plastids isolated from both algal species had the capacity to transport both CO2 and HCO3−. This ability is independent of the Ci concentration provided during growth, with CO2 and HCO3− contributing about 50% of the total C uptake over the concentration range tested. Values for K1/2(HCO3−) for net O2 evolution and HCO3− uptake by chloroplasts from high-Ci-grown cells of C. reinhardtii were 585 and 446 μm, respectively, and the corresponding values for chloroplasts from low-Ci-grown cells were 45 and 33 μm (Table II). Thus, the apparent affinities of net O2 evolution and HCO3− transport were more than 10-fold higher in low-Ci cells than in high-Ci cells.

Figure 5.

Figure 5

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A, Rates of net O2 evolution (▪, □) and net HCO3− transport (•, ○) during steady-state photosynthesis in relation to the external HCO3− concentration. B, Rates of CO2 transport (▴, ▵) during steady-state photosynthesis in relation to the external CO2 content. Data were calculated from MS experiments with chloroplasts from high-Ci (open symbols) and low-Ci cells (closed symbols) of C. reinhardtii. The experimental conditions were the same as given for Figure 4B. The Chl content ranged from 15 to 25 μg mL−1.

Figure 6.

Figure 6

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A, Rates of net O2 evolution (▪, □) and net HCO3− transport (•, ○) during steady-state photosynthesis in relation to the external HCO3− concentration. B, Rates of CO2 transport (▴, ▵) during steady-state photosynthesis in relation to the external CO2 content. Data were obtained from MS experiments with chloroplasts from high-Ci (open symbols) and low-Ci cells (closed symbols) of D. tertiolecta. The experimental conditions were 25°C in assay buffer containing 150 mm mannitol, 25 mm KCl, 1 mm MgCl2, 0.2 mm K2HPO4, 25 mm BTP-HCl, pH 8.0, and 5 μm AZA. The Chl content ranged from 15 to 25 μg mL−1.

Table II.

Apparent affinities of CO2 and HCO3− uptake in intact chloroplasts

Type of Cells K1/2 Values
Ci HCO3− CO2
μm
C. reinhardtii
 High Ci 585  ± 59 446  ± 45 6.8  ± 0.8
 Low Ci 45  ± 9 33  ± 6 0.6  ± 0.1
D. tertiolecta
 High Ci 318  ± 38 203  ± 12 5.8  ± 0.6
 Low Ci 89  ± 12 58  ± 5 0.5  ± 0.08
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Summary of the K1/2 values of net O2 evolution, net HCO3− transport, and gross CO2 transport for their respective substrates during steady-state photosynthesis by chloroplasts from high- and low-Ci cells of C. reinhardtii and D. tertiolecta. The data represent mean values ± sd from two to four independent experiments similar to those shown in Figures 5 and 6.

In D. tertiolecta, the differences in the K1/2(HCO3−) for net O2 evolution and net HCO3− uptake between high- and low-Ci plastids were less obvious, with the apparent affinities for HCO3− being three to four times higher in low-Ci chloroplasts. In addition, the kinetic characteristics of CO2 uptake by chloroplasts from both species were also changed during acclimation to low Ci. The K1/2(CO2) was 6.8 and 5.8 μm, respectively, for plastids from high-Ci cells of C. reinhardtii and D. tertiolecta, but decreased to 0.6 and 0.5 μm in chloroplasts from the respective low-Ci cells (Table II).

DISCUSSION

Using a recently developed MS disequilibrium technique (Badger et al., 1994), we were able to distinguish between CO2 and HCO3− utilization in cells and chloroplasts from D. tertiolecta and C. reinhardtii and to measure the Ci fluxes in relation to the external CO2 and HCO3− concentrations in the medium. Low-Ci cells from both species showed the ability to take up CO2 and HCO3− during steady-state photosynthesis (Figs. 2 and 3; Table I), confirming previous reports of the presence of a HCO3−-transport mechanism in intact cells of eukaryotic green alga (Williams and Turpin, 1987; Sültemeyer et al., 1989, 1991; Ramazanov and Cardenas, 1992; Badger et al., 1994; Palmqvist et al., 1994; Matsuda and Colman, 1995).

The fact that even high-Ci cells from C. reinhardtii and D. tertiolecta are able to transport HCO3−, although with a lower apparent affinity (Table I), is consistent with other data (Palmqvist et al., 1994), and contradicts previous views (Moroney et al., 1985) that high-Ci cells depend largely on CO2 uptake. Apparently, CO2 and HCO3− transport in C. reinhardtii and D. tertiolecta can be distinguished into low- and high-affinity transport systems, present in high- and low-Ci cells, respectively. The differences between the high- and low-affinity transport systems for CO2 and HCO3− in the two algal species appear to be mainly in substrate affinities and not in maximal activities (Figs. 2 and 3; Table I), indicating that the increase in Ci-transport efficiency after acclimation to low CO2 concentrations is caused by a modification(s) of the transport mechanism rather than by a quantitative increase in the number of transport components. Other authors have reached similar conclusions with Scenedesmus obliquus (Palmqvist et al., 1994) and two cyanobacteria, Synechococcus sp. strain PCC7942 (Yu et al., 1994) and Synechococcus sp. strain PCC7002 (Sültemeyer et al., 1995b).

In addition to different photosynthetic activities, another important difference between the two algal species appears to be in the use of the Ci species for photosynthesis. Whereas in D. tertiolecta HCO3− is the dominant Ci species taken up by the cells regardless of the CO2 concentration during growth, in high- and low-Ci cells of C. reinhardtii HCO3− uptake contributes only about 50% to O2 evolution (Figs. 2 and 3). In fact, during acclimation to a low-Ci concentration, transport of CO2 in cells of C. reinhardtii becomes even more important. The preference for different Ci species by the two algae could be explained by the growth conditions. Cells of D. tertiolecta were grown at pH 8.0 under a high salinity of 1 m NaCl and a HCO3−:CO2 ratio of about 115 in the external medium (Yokota and Kitaoka, 1985).

In contrast, the ratio of HCO3− to CO2 in the growth medium for C. reinhardtii is only around 20, so that HCO3− is by far the most dominant Ci species in the marine environment. Moreover, periplasmic CA activity is, irrespective of the Ci concentration provided during growth, one degree of magnitude lower in cells of D. tertiolecta than in cells of C. reinhardtii (Sültemeyer et al., 1993; Badger and Price, 1994; Amoroso et al., 1996; Sültemeyer, 1997). The facilitated conversion of HCO3− to CO2 by periplasmic CA contributes to the dominant role of CO2 transport, especially in low-Ci cells of C. reinhardtii, and the lower external CA activity in D. tertiolecta leads to the development of a predominant HCO3− transport system, which seems to be typical for marine organisms, inasmuch as similar results were obtained with the marine cyanobacterium Synechococcus sp. strain PCC7002 (Sültemeyer et al., 1995b).

Our data on CO2 and HCO3− uptake with intact cells do not permit exact determination of the location of either of these transport processes. We therefore decided to isolate photosynthetically active chloroplasts from C. reinhardtii and D. tertiolecta. In general, maximum rates of photosynthetic O2 evolution were found to be around 30 and 40 μmol O2 mg−1 Chl h−1 for chloroplasts from C. reinhardtii and D. tertiolecta, respectively, regardless of the CO2 concentration provided during growth (Figs. 5 and 6). Similar maximum photosynthetic rates have been reported for chloroplasts from eukaryotic algae (Moroney et al., 1987; Goyal et al., 1988; Goyal and Tolbert, 1989; Moroney and Mason, 1991; Ramazanov and Cardenas, 1992).

To further interpret the Ci uptake measurements obtained with isolated plastids, one must be certain that contamination by whole cells does not contribute significantly to the CO2 and HCO3− uptake measurements with the chloroplast preparation (Moroney and Mason, 1991). Several lines of evidence indicate that this was indeed the case in our experiments: (a) comparative measurements of marker enzyme activities, such as PEP carboxylase and succinate dehydrogenase, with lysates from cells and chloroplasts show less than 4% contamination with cytosol and mitochondria, respectively (Amoroso et al., 1996) (this value is consistent with the approximately 1% contamination by intact cells generally observed by phase-contrast microscopy when an aliquot from the chloroplast preparation is treated with water, which causes lysis of the plastids but not of intact cells [Sültemeyer et al., 1988]; (b) isolated chloroplasts showed almost no O2 consumption in the dark (Fig. 4), which is indicative of a low degree of contamination by mitochondria and the absence of viable cells because respiration of intact cells is usually higher in 150 mm mannitol than in standard growth medium (D. Sültemeyer, unpublished data); and (c) photosynthetic O2 evolution was completely abolished by 10 mm Pi (Fig. 4A), which is typical for isolated intact chloroplasts (Goyal et al., 1988; Rotatore and Colman, 1990, 1991; Moroney and Mason, 1991).

Pi at this concentration does not affect photosynthesis by intact cells at all and, in fact, the Pi concentration in the growth medium is around 20 mm. In addition, photosynthetic O2 evolution by the chloroplast preparations became less sensitive to Pi inhibition in the presence of 1 mm 3-P-glycerate, and O2 evolution was stimulated 10 to 40% by the latter in the absence of high Pi concentrations (Rolland et al., 1997). This effect of 3-P-glycerate on O2 evolution is consistent with the presence of a plastid triose-P translocator and indicates that the chloroplast inner envelope membrane is intact (Moroney and Mason, 1991). It also demonstrates that the chloroplasts are not retained in resealed plasma membranes, because 3-P-glycerate is not taken up by protoplasts and has no stimulatory effect on O2 evolution by cells and protoplasts (data not shown; Moroney et al., 1991). In addition, because the chloroplasts from D. tertiolecta, which were isolated with a pressure-disruption method, had Ci uptake characteristics similar to those of chloroplasts from C. reinhardtii, which were isolated with a digitonin method (Figs. 5 and 6; Table II), it is likely that neither breakage method interferes with the Ci uptake systems at the chloroplast envelope membrane.

The measurements of Ci fluxes during steady-state photosynthesis by isolated chloroplasts (Figs. 5 and 6) show that plastids from high- and low-Ci cells of C. reinhardtii and D. tertiolecta are able to use CO2 and HCO3− for photosynthesis. The major change in the transport characteristics, which occurs when the cells are adapted to low Ci concentrations, is an increase in the apparent affinity for the uptake of both Ci species (Table II) rather than any dramatic change in the Vmax values. Therefore, the Ci transporters can be separated into high- and low-affinity uptake systems in a manner similar to intact cells. The observation that chloroplasts from high-Ci cells of both species possess the ability to transport HCO3− is new and deserves some comment.

Previously published data have indicated that a chloroplastic HCO3− uptake system is present only when the cells are adapted to low Ci concentrations, whereas photosynthesis by plastids from high-Ci algae was assumed to rely solely on CO2 diffusion without additional HCO3− uptake (Moroney et al., 1987; Goyal and Tolbert, 1989; Rotatore and Colman, 1990, 1991; Moroney and Mason, 1991; Ramazanov and Cardenas, 1992; Katzmann et al., 1994). However, in these earlier studies a detailed kinetic analysis of HCO3− uptake was not performed with chloroplasts from high-Ci cells. In fact, in these plastids Ci uptake was examined at rather low external HCO3− concentrations, well below the K1/2(HCO3−) value reported in this work (Table II). Therefore, a simple explanation for the above-mentioned discrepancies could be the presence of the low-affinity HCO3− transporter that was not detected by the previous methods. In this context it is noteworthy that even chloroplasts from cells grown on acetate show evidence of a low-affinity HCO3− transport system similar to that of high-Ci cells (data not shown).

During our analysis of HCO3− uptake in high- and low-Ci chloroplasts from C. reinhardtii and D. tertiolecta, another interesting observation was made. Over the entire HCO3− range tested, HCO3− transport activity reached about 50% of the photosynthetic O2 evolution rate measured at the same time (Figs. 5 and 6), which was not enough to maintain photosynthesis. Consequently, another Ci species such as CO2 had to enter the chloroplast to satisfy C supply for CO2 fixation. This is exactly what was found, and it is apparent from Figures 5 and 6 that CO2 and HCO3− uptake contribute more or less equally to O2 evolution regardless of the CO2 concentration under which the parent cells were grown.

The nature of the CO2 uptake during steady-state photosynthesis is unknown but the low K1/2(CO2) values for chloroplasts from low-Ci cells (around 0.5 μm; Table II) clearly indicate that this process is active and is not simply diffusion that depends on CO2 fixation by Rubisco. If the CO2 consumption measured with chloroplasts during steady-state photosynthesis was attributed largely to the carboxylation reaction one would expect a similar K1/2(CO2) value as was found for Rubisco, which for green algae such as C. reinhardtii and D. tertiolecta is about 30 μm (Chen et al., 1988). This is clearly not the case and, in fact, it is not even true for chloroplasts from high-Ci algae (Table II). Therefore, we believe that even chloroplasts from high-Ci cells possess a functional CO2 pump but with a reduced efficiency compared with plastids from low-Ci cells.

This conclusion is supported by CO2 uptake measurements with a Ci-pump mutant (pmp-1-6-5K) that is unable to accumulate Ci internally (Spalding et al., 1983) and has a considerably lower apparent affinity for CO2 (K1/2[CO2] = 25 μm) than wild-type high-Ci grown cells (G. Amoroso, S. Haupt, D. Sültemeyer, H.P. Fock, unpublished data). In addition, similar kinetic characteristics of CO2 uptake were found with chloroplasts from a recently constructed mutant of C. reinhardtii in which the chloroplastic gene ycf10 (cemA) has been inactivated (Rolland et al., 1997).

The results presented in this paper indicate that the predominant location of Ci transporters, both for CO2 and HCO3−, is at the envelope of the chloroplasts from C. reinhardtii and D. tertiolecta. In this respect, both species differ from Chlorella ellipsoidea because photosynthetically active chloroplasts from this alga show no evidence of CO2 uptake and only a limited capacity to transport HCO3− (Rotatore and Colman, 1990, 1991). Our data with C. reinhardtii and D. tertiolecta, however, do not rule out the possibility that additional transporters exist at the plasma membrane. The fact that all cell types are able to take up HCO3− from the external medium (Figs. 2 and 3) indicates that the plasmalemma contains a mechanism for either passive or active HCO3− transport. In this context it is interesting to note that HCO3− transport with intact cells of D. tertiolecta, in particular, is distinct from that in chloroplasts (Figs. 3 and 6).

In high- and low-Ci cells the ratios of O2 evolution to HCO3− transport and HCO3− transport to CO2 uptake are about 1 and 4, respectively (Fig. 3). The same ratios reach values of around 0.5 and 1, respectively, in chloroplasts (Fig. 6). This shows that proportionally more HCO3− transport occurs with intact cells and indicates that a HCO3− transport component may exist at the plasma membrane, which supports Ci transport at the chloroplast level. On the other hand, active CO2 transport may occur only at the chloroplast envelope, thus creating a CO2 sink so that entry of CO2 into the cells may occur by passive diffusion.

Our experimental results support the same model for Ci transport that has been proposed for C. reinhardtii (Moroney et al., 1987; Moroney and Mason, 1991; Sültemeyer et al., 1991, 1993; Ramazanov and Cardenas, 1992; Badger and Price, 1994) and for D. tertiolecta (Goyal and Tolbert, 1989), which invokes the operation of active Ci transport systems across both the plasma membrane and the chloroplast envelope. However, some evidence indicates that only one predominant Ci-transport system functions in eukaryotic cells. The inability of the Ci-pump mutant (pmp-1-6-5K) to accumulate Ci internally is most likely caused by a single nuclear mutation (Spalding et al., 1983) and, therefore, one would expect a malfunction of a single primary transport process. This possibility is currently under investigation in our laboratory.

Abbreviations:

AZA

acetazolamide

BTP

1,3-bis[tris(hydroxymethyl)methylamino]propane

CA

carbonic anhydrase

CCM

CO2-concentrating mechanism

Chl

chlorophyll

Ci

inorganic carbon

high-Ci cells

cells grown in air enriched with 5% (v/v) CO2

K1/2(CO2) and K1/2(HCO3−)

concentration of CO2 or HCO3−, respectively, required for half-maximal activity

low-Ci cells

cells grown in ambient air (0.035% [v/v] CO2)

Footnotes

1

This research was supported by Deutsche Forschungsgemeinschaft grant no. Fo-72/16-1.

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