Plant Physiol. (1998) 116: 193-201

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

Gabi Amoroso, Dieter Sültemeyer^*, Christoph Thyssen, and Heinrich P. Fock

Fachbereich Biologie der Universität, Postfach 3049, D-67653 Kaiserslautern, Germany

	ABSTRACT

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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.

	INTRODUCTION

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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

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Growth of Algae

Preparation of Cells and Protoplasts for Ci 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

MS Measurements of CO₂ Uptake and HCO₃ Transport

1

1

1

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 min¹ (25 mm BTP-HCl, pH 8.0, 30°C), 110 and 0.018 min¹ (25 mm BTP-HCl, pH 8.0, 1 m NaCl, 25°C), and 85 and 0.024 min¹ (assay buffer for chloroplasts, 25°C), respectively.

Other Parameters

	RESULTS

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CO₂ and HCO₃ Uptake with Intact Cells

View larger version (12K): [in this window] [in a new window]   Figure 1. 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 m2 s1) was switched on and off as indicated. The Chl concentration was 6 µg mL1. 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).

View larger version (22K): [in this window] [in a new window]   Figure 2. 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 mL1.

View larger version (22K): [in this window] [in a new window]   Figure 3. 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 mL1.

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 K_1/2(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 K_1/2(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 K_1/2(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).

View this table: [in this window] [in a new window]   Table I. Apparent affinities of CO2 and HCO3 uptake in intact cells 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

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 mg¹ Chl h¹ 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).

View larger version (13K): [in this window] [in a new window]   Figure 4. 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 m2 s1). 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 m2 s1) 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 mL1 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 K_1/2(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.

View larger version (21K): [in this window] [in a new window]   Figure 5. 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 mL1.

View larger version (22K): [in this window] [in a new window]   Figure 6. 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 mL1.

View this table: [in this window] [in a new window]   Table II. Apparent affinities of CO2 and HCO3 uptake in intact chloroplasts 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 K_1/2(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 K_1/2(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

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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 mg¹ Chl h¹ 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 V_max 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 K_1/2(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 K_1/2(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 K_1/2(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 (K_1/2[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.

	FOOTNOTES

   Received June 16, 1997; accepted September 23, 1997.

	ABBREVIATIONS

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. K_1/2(CO₂) and K_1/2(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).

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