INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

In many species of cyanobacteria and microalgae, the uptake of CO₂ for photosynthesis is mediated by an energy-dependent CO₂-concentrating mechanism (CCM). Several key components of the system have been identified in cyanobacteria and include metabolic influx pumps that actively transport and accumulate inorganic carbon (CO₂ + HCO₃ = Ci), carbonic anhydrase (CA), which catalyzes the conversion of accumulated HCO₃ to CO₂ near the site of Rubisco, and structurally intact carboxysomes, which house the majority of the cellular complement of Rubisco and CA (Kaplan and Reinhold, 1999; So et al., 2002). Physiological and biochemical studies indicate that there are multiple transport systems for Ci that recognize and use HCO₃ and CO₂ as substrates (Miller et al., 1990; Espie et al., 1991). It is widely accepted that Ci transport is light dependent and that photosynthetic electron transport provides the energy required for the active transport of both HCO₃ and CO₂ at the plasma membrane (Kaplan and Reinhold, 1999). In the cyanobacterium Synechococcus sp., there appears to be a division of labor within the photosystems in that HCO₃ transport is supported by linear electron flow, whereas cyclic electron transport supplies the energy for CO₂ uptake (Li and Canvin, 1998).

The CCM is more complex in eukaryotic algae because of the increased number of metabolic compartments. Species diversity in terms of the required components for the CCM and its specific mode of operation in microalgae have been extensively documented (Badger et al., 1998; Badger and Spalding, 2000). Diversity in the functional elements of the CCM include the participation (or absence thereof) of various forms of perplasmic CA (Spalding et al., 1983b; Fujiwara et al., 1990), one or more plasma membrane-localized and/or chloroplast envelope-localized, Ci transport systems (Badger and Spalding, 2000), pyrenoid-localized Rubisco (Lacoste-Royal and Gibbs, 1987), and intracellular CA localized within the pyrenoid and thylakoid membranes (Karlsson et al., 1998) and mitochondria (Eriksson et al., 1998). Early experiments using Chlamydomonas reinhardtii indicated that photosynthetic processes provided the energy for the operation of the CCM (Spalding et al., 1983a; Sultemeyer et al., 1993). The details of energy supply are yet to be fully established, however, inhibition of light-dependent Ci accumulation in isolated chloroplasts of C. reinhardtii by 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) strongly suggested a role for linear electron transport in Ci transport in this organelle (Spalding, 1998; Badger and Spalding, 2000).

In marine microalgae, studies of the CCM have concentrated largely on examining the Ci species transported during photosynthesis and on the role of CA (Raven, 1997). Recent studies with the marine cyanobacterium Synechococcus sp. WH7803 and the eustigmatophyte alga Nannochloropsis spp. have, however, identified a new phenomenon associated with the operation of the CCM (Sukenik et al., 1997; Tchernov et al., 1997; Huertas et al., 2000). In these species, illumination resulted in a significant and sustained rise in the external CO₂ concentration as photosynthesis proceeded, rather than the expected draw-down of the external CO₂ due to active Ci transport and photosynthesis (Miller et al., 1990; Colman et al., 2002). In other words, the cells acted as point-source CO₂ generators substantially elevating both the internal and external CO₂ concentration above their chemical equilibrium, in what might be considered a shot-gun approach to circumventing CO₂ limitation. CO₂ generation required two elements in Nannochloropsis spp.: an active HCO₃ transport system to accumulate intracellular Ci and an intracellular CA to convert HCO₃ to CO₂. It appeared that a substantial portion of the internal CO₂ subsequently leaked to the surroundings, and this leakage accounted for the unexpected rise in the external concentration. Inhibition of HCO₃ transport by 4,4'-diisothiocyanatolstilbene-2,2'-disulfonic acid (DIDS) or the inhibition of intracellular CA activity by ethoxyzolamide (EZ) both prevented the rise in CO₂ concentration in the surroundings and reduced the rate of photosynthesis (Huertas et al., 2000). As a consequence, the CCM of Nannochloropsis spp. may be described minimally as HCO₃ pump-CO₂ leak activity.

We have recently discovered that the CO₂ generating system in Nannochloropsis gaditana continues to function for up to 20 min in the dark. Like its counterpart in the light, the CO₂ generating system was sensitive to DIDS and EZ, suggesting that the same components involved in the HCO₃ pump-CO₂ leak activity of the CCM participated in CO₂ generation in the dark. This observation seriously challenges the notion that the CCM is exclusively energized by the light-dependent photosynthetic electron transport chain. In the present work, we use mass spectrometry and stable isotope techniques to measure fluxes of ¹²CO₂ and ¹³CO₂ in cell suspensions of the marine microalga N. gaditana to distinguish between fluxes arising from respiratory metabolism and the HCO₃ pump-CO₂ leak activity of the CCM in the dark and light. Flux measurements were made in the absence or presence of inhibitors of mitochondrial respiration and chloroplast linear electron transport to investigate the role of these organelles in providing energy for HCO₃ transport. Our results demonstrate that HCO₃ uptake in the dark and light is driven by mitochondrial respiration and, thus, identify a novel component of the CCM in this alga.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

N. gaditana Generates CO₂ in the Dark and Light

Illuminated cells of N. gaditana were allowed to reach the CO₂ compensation point, and the light was switched off (Fig. 1a). The ¹²CO₂ concentration in the medium rose to a very high level over the initial 5 min of the experiment and then gradually declined over the next 10 min. Addition of bovine CA to the reaction vessel (Fig. 1b) during any part of the time course resulted in a rapid diminution of the CO₂ signal, indicating that CO₂ was present in the medium at levels well above its chemical equilibrium value with HCO₃. The creation and maintenance of this chemical disequilibrium is indicative of the involvement of an energy-dependent step in this process. Such a large rise in CO₂ after darkening has been observed thus far only in N. gaditana (Huertas et al., 2000). The subsequent decline in CO₂ concentration would not be anticipated, however, because respiratory metabolism should contribute to a continuous, though slow, increase in the external CO₂ concentration.

View larger version (12K): [in this window] [in a new window]   Figure 1.   Measurement of 12CO2 () and 13CO2 (  ) fluxes in the dark and light. a, An illuminated (1 mmol m2 s1, photosynthetically active radiation) cell suspension of N. gaditana was allowed to reach the CO2 compensation point, and the light was turned off. Changes in 12CO2 concentration were followed with time, and the light was then turned on. The asterisk indicates the transition from the slow to the fast phase of 12CO2 decline. b, As in a except that bovine CA (40 µg mL1) was added to the darkened cell suspension during the rise in 12CO2. c, K213CO3 (100 µM) was added to reaction buffer (-cells) to determine the equilibrium 13CO2 concentration at pH 8.0 and 25°C. d, As in a except that 100 µM K213CO3 was added 2 min after darkening and both 12CO2 and 13CO2 concentrations were measured over time. The time courses are superimposed for comparison.

When the light was turned on (Fig. 1a), a new transient rise in CO₂ concentration was observed that was then followed by a persistent decline as photosynthesis proceeded. Addition of bovine CA during the light phase also resulted in a diminution of the CO₂ signal, indicating that the CO₂ concentration was above its chemical equilibrium level (data not shown; Huertas et al., 2000). A similar sustained evolution of CO₂ in the light during CO₂ fixation has been previously reported for N. gaditana and the marine cyanobacterium Synechococcus sp. WH7803(Sukenik et al., 1997; Tchernov et al., 1997) and Synechococcus spp. (Badger and Andrews, 1982). In contrast to these results, other eukaryotic algae and cyanobacteria draw-down the CO₂ concentration after illumination. Clearly, CO₂ fluxes in N. gaditana are complex and cannot be accounted for simply by considering photosynthetic CO₂ consumption in the light and respiratory CO₂ production in the dark, because in several instances the net flux was opposite in direction to the major metabolic flux of CO₂.

Light-Enhanced Dark Respiration (LEDR) and HCO₃ Pump-CO₂ Leak Activity Are Required for Dark CO₂ Generation

The initial rise in CO₂ concentration in the dark can be attributed to a number of different sources including the release of an internal Ci pool, a photorespiratory postillumination CO₂ burst, and mitochondrial respiration. By definition, the Ci pool at the compensation point is small, and in other algal species, it was released to the medium within 1 to 2 min after darkening, resulting in only a minimal rise in the extracellular CO₂ concentration. Similarly, the postillumination CO₂ burst would contribute only a small portion of the CO₂ because the CCM of N. gaditana suppressed the formation of photorespiratory substrates required to initiate the burst (Sukenik et al., 1997; Huertas et al., 2000). As a consequence, respiratory processes would be expected to be a major source of the CO₂ appearing in the medium. N. gaditana is one of a number of plant and algal species that display a LEDR (Xue et al., 1996; Hoefnagel et al., 1998), where the respiration rate immediately after a period of photosynthesis is substantially higher than the steady-state rate. Under our conditions, LEDR measured immediately after darkening (O₂ uptake) was on average 2.7-fold higher than the steady-state respiration rate measured 20 min after darkening (e.g. Fig. 2). The rate of LEDR gradually declined to the steady-state over a period of 5 to 6 min. Thus, the period of LEDR (O₂ uptake) was clearly associated with the postillumination period of maximum CO₂ release (Fig. 2), indicating that they are linked, probably through mitochondrial respiration. We have recently discovered that the HCO₃ transport system in N. gaditana remained active in the dark (Huertas et al., 2000), and it may, therefore, also contribute to the rise in CO₂ through its HCO₃ pump-CO₂ leak activity.

View larger version (17K): [in this window] [in a new window]   Figure 2.   Measurement of 12CO2 () and 16O2 (. . . . .) fluxes in a cell suspension of N. gaditana in the light (L) and dark (D) and in the absence and presence of the HCO3 transport inhibitor DIDS (500 µM). The experimental procedure was essentially the same as that for Figure 1. The asterisk indicates the transition from the slow to the fast phase of 12CO2 decline.

To follow CO₂ fluxes between the medium and the cells independent of respiratory ¹²CO₂ production, we added 100 µM ¹³Ci to the cell suspension 2 min after turning the light off (Fig. 1, c and d). In the absence of cells, the ¹³CO₂ concentration in the medium rose to the expected equilibrium level and then remained constant (Fig. 1c). In the presence of cells, the ¹³CO₂ concentration also increased with time (Fig. 1d) but to a level 5.6-fold the equilibrium value and then declined gradually over time in parallel with the ¹²CO₂ signal. Thus, the ¹³CO₂ signal displayed the same dynamics as the ¹²CO₂ signal, but in this case, the rise in ¹³CO₂ cannot be attributed to LEDR or steady-state respiration because mitochondrial substrates were not enriched with ¹³C. Participation of H¹²CO₃ in this energy-driven pump-leak activity would also be expected to occur because it is the natural substrate for the transporter. The rise in CO₂ concentration may also be due to a cellular acidification of the medium. However, measurement of extracellular pH indicated that this parameter remained constant during the experiments (data not shown). As a consequence, the unusually large rise in ¹²CO₂ concentration in the dark (Fig. 1a) can be attributed to two superimposed processes. First, LEDR-generated CO₂, which was rapidly released to the medium, and some of it was hydrated to form HCO₃. Second, this HCO₃ then served as substrate for the HCO₃ pump-CO₂ leak activity in the dark resulting in a further increase in CO₂ efflux. The combined processes of CO₂ formation occurred at a rate faster than the uncatalyzed conversion of CO₂ to HCO₃ in the medium as evidenced by the rapid diminution in CO₂ concentration when CA was added (Fig. 1b). In the light, similar processes occurred that accounted for the initial rise in CO₂ upon illumination. The rise was considerably smaller, however, because Rubisco-mediated fixation consumed a portion of the CO₂. Both the ¹²CO₂ and ¹³CO₂ signals ultimately declined to the CO₂ compensation concentration because the Ci was consumed in photosynthesis (Fig. 1d).

Contribution of HCO₃ Transport

To estimate the relative contributions of respiration and the HCO₃ pump-CO₂ leak activity to the rise in CO₂ concentration, experiments were conducted in the absence and presence of the HCO₃ transport inhibitor DIDS (Fig. 2). The addition of 500 µM DIDS to illuminated cells at the CO₂ compensation point significantly reduced the level of CO₂ efflux once the cells were darkened. However, DIDS only had a small, negative effect (10%) on the rate of LEDR dark O₂ consumption, indicating that the major effect of DIDS was on the HCO₃ pump-CO₂ leak activity. At the DIDS concentration used, HCO₃ transport was inhibited by about 90% (Huertas et al., 2000). Using the difference in peak heights in the dark as an estimate of activity (e.g. Fig. 2), HCO₃ pump-CO₂ leak activity contributed approximately 40% to 50% to the total rise in CO₂. Illumination of the cell suspension resulted in a rapid rise in extracellular CO₂ concentration, which was greatly reduced in cells treated with the HCO₃ transport inhibitor (Fig. 2), indicating that DIDS-sensitive HCO₃ transport activity was essential for the rise in CO₂ in the light and in the dark. Thus, the rise in ¹³CO₂ concentration above the equilibrium level was diagnostic for HCO₃ transport activity in N. gaditana.

Oxygen Is Required for HCO₃ Pump-CO₂ Leak Activity in the Dark

The decline in CO₂ concentration in the dark often displayed two distinct phases, an initial slow phase followed by a second and more rapid rate of disappearance (e.g. Figs. 1a and 2, *). The accelerated phase of CO₂ disappearance was also observed in the ¹³CO₂ signal (Fig. 1d, *) and its onset corresponded with the approach to anaerobic conditions in the medium, brought about by respiratory O₂ consumption (Fig. 2). Because the ¹³CO₂ signal solely reflects HCO₃ pump-CO₂ leak activity, this observation suggests a dependence of a component of the pump-leak activity on O₂ availability. To test this hypothesis, we examined the effect of O₂ concentration on the magnitude of CO₂ efflux (Fig. 3). The O₂ concentration in the medium was initially set by gassing with a stream of N₂, or by the addition of dithionite to achieve zero O₂. In the absence of O₂, darkening of a cell suspension at the CO₂ compensation point resulted in a very small rise in ¹²CO₂, consistent with the suggestion that O₂-dependent LEDR was responsible for part of the large rise in CO₂ concentration in the dark. The addition of 100 µM ¹³Ci, 2 min after darkening, resulted in a rise in ¹³CO₂ concentration only to the expected equilibrium level (Fig. 3a). In either case, illumination did not result in a rise in the CO₂ signals, as observed in the control (Fig. 1d, 230 µM O₂). As the O₂ concentration was increased, the ¹³CO₂ concentration also increased progressively in the dark, although the absolute amount of ¹³Ci added was the same in each case (Figs. 3, b-d, and 1, c and d). These data support the concept that the pump-leak activity associated with HCO₃ transport required O₂. The ¹²CO₂ concentration also rose progressively due to combined mitochondrial respiration and HCO₃ pump-CO₂ leak activity. The rise in CO₂ concentration expected upon illumination was also restored with increasing O₂ concentration (Figs. 3, a-d, and 1c).

View larger version (23K): [in this window] [in a new window]   Figure 3.   Measurement of 12CO2 () and 13CO2 (  ) fluxes in the dark and light in the presence of 0 (a), 1.5 (b), 5.6 (c), and 130 (d) µM O2. The plots obtained at 230 µM O2 are shown in Figure 1d. The time courses are superimposed for comparison. The experimental procedure was essentially the same as that for Figure 1; off, light turned off; on, light turned on.

Mitochondrial Respiration Energizes HCO₃ Transport

The occurrence of HCO₃ pump-CO₂ leak activity in the dark and its dependence on O₂ suggested that mitochondrial respiration may be involved in energizing a component of that system. The most likely candidate is HCO₃ transport because CA is a freely reversible enzyme, whereas the uptake of HCO₃ would be against its electrochemical potential. To assess mitochondrial involvement, CO₂ flux experiments were carried out in the presence of various inhibitors of respiration (Fig. 4; Table I). The addition of 250 µM KCN to cell suspensions during the dark CO₂ efflux phase mimicked the effect of anoxia and resulted in a rapid decline in both ¹²CO₂ and ¹³CO₂ concentrations (Fig. 4b). Upon illumination, the efflux of ¹³CO₂ was abolished and that of ¹²CO₂ was markedly reduced. Inclusion of KCN in the cell suspension at the CO₂ compensation point in the light resulted in a significant inhibition of ¹²CO₂ efflux after darkening (Fig. 4c) and a 82% decrease in O₂ consumption (data not shown), confirming inhibition of mitochondrial respiration. Inhibition of HCO₃ pump-CO₂ leak activity was also evident because the addition of 100 µM ¹³Ci resulted in a rise in ¹³CO₂ only to its equilibrium level. The efflux of both ¹²CO₂ and ¹³CO₂ was abolished during a subsequent dark-light transition. Concentrations of KCN as low as 25 µM were also effective in inhibiting CO₂ efflux in the dark and in the light (Table I), suggesting a requirement for mitochondrial complex IV in HCO₃ pump-CO₂ leak activity. Consistent with this hypothesis was the observation that NaN₃, another inhibitor of complex IV, inhibited ¹²CO₂ and ¹³CO₂ efflux in the dark and light (Table I) and could also mimic the effect of anoxia (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 4.   Measurement of 12CO2 () and 13CO2 (  ) fluxes in the dark and light in the absence (a; control) and presence (b and c) of 250 µM KCN. KCN was added during the slow decline in CO2 (b) or 2 min before darkening (c). The time courses are superimposed for comparison. The experimental procedure was essentially the same as that for Figure 1; off, lights turned off; on, lights turned on.

In the light, KCN may also inhibit photosynthetic electron transport directly at plastocyanin and lead to the inhibition of CO₂ fixation and O₂ evolution. At an external Ci concentration (0.1 mM) just sufficient to saturate photosynthesis (Huertas and Lubian, 1998), 250 µM KCN abolished O₂ evolution (Fig. 5). However, increasing the external Ci to 50 mM resulted in a restoration of the photosynthetic rate to 75% of the control rate. Although KCN has a clear effect on photosynthesis, these results also indicate that the ability of the photosynthetic electron transport system to supply energy to the Calvin cycle remained largely functional and that it was the availability of Ci that was the limiting factor, consistent with an impairment of the CCM not directly related to chloroplast energy supply.

View larger version (21K): [in this window] [in a new window]   Figure 5.   Effect of 250 µM KCN on the time course of photosynthetic O2 evolution in the presence of various levels of external Ci (). For comparison, O2 evolution in the absence of KCN at 0.1 mM Ci () is also shown.

The results obtained with KCN and NaN₃ were in marked contrast to those observed with DCMU, an inhibitor of chloroplast linear electron transport (Fig. 6). At 10 µM, the rise in ¹³CO₂ was unaffected by the inhibitor in the dark or the light, indicating that the HCO₃ pump-CO₂ leak activity could be sustained when the energy supply from photosynthetic linear electron transport was restricted. As anticipated, photosynthetic O₂ evolution was strongly inhibited (6.4-fold), indicating that DCMU effectively blocked linear electron transport in N. gaditana. At 5 times the DCMU concentration (Table I), ¹³CO₂ efflux in the dark occurred at 86% of the control level, but ¹³CO₂ efflux in the light was abolished, indicating that chloroplast energy supply may also contribute to some extent in the pump-leak activity (Table I).

View larger version (21K): [in this window] [in a new window]   Figure 6.   Measurement of 12CO2 (), 13CO2 (  ), and 16O2 (. . . . .) fluxes in the dark and light in the absence and presence of 10 µM DCMU. The time courses are superimposed for comparison. The experimental procedure was essentially the same as that for Figure 1.

DCMU reduced ¹²CO₂ efflux in the dark, but had no effect on ¹³CO₂ efflux (Fig. 6) or on the steady-state rate of O₂ uptake. The effect of DCMU on LEDR-dependent ¹²CO₂ efflux was likely indirect and the result of a decreased supply of oxidizable substrate to the mitochondria from the chloroplasts, leading to less CO₂ production and ultimately to reduced availability of HCO₃ for the pump-leak activity. This possibility seems reasonable because Xue et al. (1996) have shown that LEDR was substantially, but indirectly, reduced by DCMU in C. reinhardtii.

The effects of two additional inhibitors of mitochondrial energy metabolism on ¹³CO₂ efflux were also examined (Table I). The respiratory electron transport inhibitor rotenone, which blocks complex I, reduced ¹³CO₂ efflux in the dark and the light by 40% and 80%, respectively. The transport inhibitor N-ethylmaleimide, which prevents ATP export from the mitochondria, reduced ¹³CO₂ efflux to 60% of the control level in the dark and abolished it in the light. With time (> 45 min), NEM ultimately reduced dark efflux to zero (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

N. gaditana generated CO₂ internally in the light and dark, which raised the extracellular CO₂ concentration to levels well above its equilibrium with HCO₃. CO₂ generation was associated, in part, with HCO₃ transport and intracellular CA activity (Sukenik et al., 1997; Tchernov et al., 1997; Huertas et al., 2000). This HCO₃ pump-CO₂ leak activity was resolved from respiratory ¹²CO₂ release by using ¹³Ci (H¹³CO₃) as the substrate for the HCO₃ transporter and monitoring the rise in ¹³CO₂ in the medium.¹³CO₂ concentrations above the measured equilibrium value were diagnostic for HCO₃ pump-CO₂ leak activity (Fig. 1).

Mitochondrial Energization of the CCM

The locations of the internal sites of CO₂ generation are not known other than that they coincide with sites where CA is located (Huertas et al., 2000). Net diffusion of CO₂ away from these sites would occur randomly, following the chemical potential between the cells and the medium. As a consequence, high levels of internal CO₂ would be distributed more or less evenly throughout the cells and would be dependent on HCO₃ transport. Minimally, the internal CO₂ concentration would be expected to be equal to the highest extracellular CO₂ concentration observed. In the light, HCO₃ pump-CO₂ leak activity would function as a rudimentary CCM increasing the CO₂ concentration in the chloroplast (and other compartments), thereby facilitating high rates of CO₂ fixation and suppressing photorespiration. N. gaditana lacks CO₂ transport capability, extracellular CA, and pyrenoids (Santos and Leedale, 1995; Huertas et al., 2000), features that in other algae play an important role in the efficient and refined operation of the CCM.(Spalding, 1998; Badger and Spalding, 2000). It is this unique set of circumstances in N. gaditana that allow us to detect HCO₃ pump-CO₂ leak activity in this organism by mass spectrometry.

HCO₃ pump-CO₂ leak activity in the light and dark was inhibited by DIDS (Fig. 2), by EZ (Huertas et al., 2000), and by anoxia (Fig. 3). The common effect of these very different treatments on reducing CO₂ generation indicated that the same biochemical components mediated HCO₃ pump-CO₂ leak activity in the light and dark. Thus, information obtained from experiments in the dark is relevant in interpreting the results of the experiment in the light and vice versa. The O₂ requirement of active HCO₃ transport (Fig. 3) and its occurrence in the dark was inconsistent with light being the sole or primary energy source to drive this process. Inhibitors of respiration such as KCN, NEM, and others (Table I) reduced or completely inhibited HCO₃ pump-CO₂ leak activity in the light and dark, consistent with a requirement for mitochondrial energy supply in the process. A primary role for light in energizing HCO₃ pump-CO₂ leak activity (CCM) was further challenged by the observation that low concentrations of DCMU had little effect on ¹³CO₂ efflux, but significantly reduced the photosynthetic rate. Energy supply from the mitochondrion, therefore, was critical to HCO₃ pump-CO₂ leak activity, whereas energy from chloroplast linear electron transport was not. We cannot, however, completely exclude a role for light in HCO₃ pump-CO₂ leak activity because high levels of DCMU blocked ¹³CO₂ efflux in the light, but not in the dark. Thus, complex interactions between chloroplasts and mitochondria may also be associated with the process.

In other algae and cyanobacteria, active Ci transport rapidly ceases after darkening, reflecting a requirement for photosynthetically derived energy to fuel Ci accumulation (Kaplan and Reinhold, 1999; Badger and Spalding, 2000). In N. gaditana, HCO₃ pump-CO₂ leak activity continues for at least 20 min in the dark before it stops. Thus, regulation of HCO₃ transport by chloroplast energy supply seems unlikely. Restoration of dark HCO₃ pump-CO₂ leak activity can be achieved by briefly (15-45 s) interrupting the dark phase with white light (Huertas et al., 2000). Thus, light may activate, but not energize, HCO₃ transport. The continuation of HCO₃ pump-CO₂ leak activity in the dark may, therefore, reflect a rather prolonged deactivation process that is more in tune with the gradual decrease in light during the natural day-night cycle than with the abrupt light-dark transition of our experiments.

One hypothesis that can account for the experimental data is that mitochondria-derived ATP is exported to the cytoplasm where it is used to drive, either directly on indirectly, a plasma membrane-localized active HCO₃ transport system in the dark and light. The accumulated HCO₃ is converted by a thermodynamically reversible CA to CO₂ resulting in a high level within the cell and a rise in the external CO₂ concentration. In the light, the effect is to concentrate CO₂ in the chloroplast and to provide saturating levels of substrate for Rubisco. In the dark, LEDR releases CO₂ to the medium, some of which is converted nonenzymatically to HCO₃. This HCO₃ serves as the substrate for the HCO₃ pump, and the resulting leakage of CO₂ is superimposed upon the respiratory efflux. As LEDR diminishes with time, the rate of CO₂ production declines, but HCO₃ pump-CO₂ leak activity and the uncatalyzed conversion of CO₂ to HCO₃ continue with the net result being a decline in CO₂ in the medium. This decline corresponds to the slow decline phase seen in the time course experiments (e.g. Figs. 1a and 2). When respiration depletes the available O₂, oxidative ATP and CO₂ production stop, causing a cessation in CO₂ release to the medium and in HCO₃ pump-CO₂ leak activity. But conversion of CO₂ to HCO₃ in the medium continues, causing a further decrease in CO₂ concentration that corresponds to the second, more-rapid phase in the CO₂ decline curve.

At present, we cannot rule out involvement of a chloroplast-localized HCO₃ transport system powered by mitochondria-derived ATP. However, either the ATP-binding site of the transporter would need to be oriented toward the cytosol or an import of mitochondria-derived ATP to the chloroplast would have to occur (Neuhaus and Wagner, 2000).

In the light, it is also possible that a complex shuttle of reductant from the chloroplast to the mitochondria may also contribute to ATP generation in the mitochondrion, which is used to transport HCO₃. This situation could explain the inhibition by high concentrations of DCMU of ¹³CO₂ efflux in the light.

The association of active HCO₃ transport with mitochondrial energy supply and the operation of the CCM has not been observed before. However, a role for mitochondria in the acclimation of the green algal C. reinhardtii to CO₂-limiting growth conditions has been proposed, based on the observations that specific mitochondrial CAs were induced upon transfer to a low-CO₂ environment and the relocation of mitochondria from central region of the cells to the periphery (Geraghty and Spalding, 1996; Eriksson et al., 1998). Participation of mitochondria in the CCM in other species may be masked by the presence of external CA, which would prevent the rise in external CO₂ concentration, or by active CO₂ transport, which would rapidly recycle CO₂ that leaked from the cells (Espie et al., 1991). New experimental approaches will be needed to detect mitochondrial involvement in the CCM in these species and to determine whether this phenomenon is widespread. The dependence of HCO₃ uptake on mitochondria energy supply may well be a primitive characteristic of algae and confined to a few species. The genus Nannochloropsis is a member of the class Eustigmatophyceae within the division Heterkonta and is considered to be one of its most primitive members (Whatley, 1995), based on the lack several photosynthetic pigments found in other members of the division (Hoek et al., 1995). Intracellular structures of eustigmatophycean algae indicate that they may have arisen by a secondary endosymbiosis where a eukaryotic alga was engulfed by a phagotrophic oomycete. This may explain the features of Nannochloropsis spp. in that the oomycete host may have had a constitutive HCO₃ transporter, but is unlikely to have had either an external CA or an active transport of CO₂ to facilitate Ci acquisition.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Organism and Growth

The unicellular marine microalga Nannochloropsis gaditana was grown at 20°C in artificial seawater (Sigma, St. Louis) supplemented with F/2 medium (Guillard and Ryther, 1962) and bubbled with air (0.035% [v/v] CO₂) at a rate of 60 mL min¹ (Huertas et al., 2000). Cultures were continuously illuminated at a photon flux density of 75 µmol quanta m² s¹ provided by a combination of cool-white and Gro-lux fluorescent lamps. Cells were maintained in exponential growth phase by daily dilution.

Experimental Conditions

Cells were harvested by centrifugation (2,800g, 10 min), washed twice with and resuspended in a Ci-free medium containing 450 mM NaCl, 40 mM MgCl₂, 10 mM KCl, 10 mM Na₂SO₄, and 5 mM CaCl₂, and buffered at pH 8.0 with 25 mM TRIZMA-Base. The medium was depleted of Ci by gassing with N₂. The cell suspension (6 mL) was placed in a glass reaction vessel at 25°C and 1 mmol quanta m² s¹ light (photosynthetically active radiation) and was allowed to fix residual Ci in the medium until the CO₂ compensation point was reached. The final cell density was 2 × 10⁸ cells mL¹ and corresponded to 30 µg chlorophyll a mL¹.

Mass Spectrometry

Washed cell suspensions (6 mL) were transferred to a glass reaction vessel containing a magnetic stirrer, and the chamber was closed with a plexiglass stopper leaving no head space. Gases and treatment solutions were introduced into the chamber through a capillary bore in the stopper. The reaction chamber was connected to the ion source of a magnetic sector mass spectrometer (model MM 14-80 SC, VG Gas Analysis, Middlewich, UK) by an inlet covered with a dimethyl silicone membrane that allowed dissolved gases to pass freely but not ions like HCO₃ (Espie et al., 1991). The illuminated cells were allowed to consume residual Ci before experiments commenced and then were darkened and treated with inhibitors, if necessary. When used, ¹³Ci was supplied to the cells from a stock solution of K₂¹³CO₃ (300 mM). Concentrations of ¹⁶O₂, ¹²CO₂, and ¹³CO₂ (m/z = 32, 44, and 45, respectively) in cell suspensions were measured simultaneously. The mass spectrometer was calibrated for O₂ and CO₂ as described previously (Espie et al., 1991). Rates of O₂ evolution or consumption were derived from the slopes of the m/z = 32 signal. In some experiments, net photosynthetic O₂ evolution was measured using a temperature-controlled Clarke-type electrode (Hansatech, King's Lynn, UK; Huertas et al., 2000).