Previous studies of the mitochondrial carbonic anhydrase (mtCA) of Chlamydomonas reinhardtii showed that expression of the two genes encoding this enzyme activity required photosynthetically active radiation and a low CO₂ concentration. These studies suggested that the mtCA was involved in the inorganic carbon-concentrating mechanism. We have now shown that the expression of the mtCA at low CO₂ concentrations decreases when the external NH₄⁺ concentration decreases, to the point of being undetectable when NH₄⁺ supply restricts the rate of photoautotrophic growth. The expression of mtCA can also be induced at supra-atmospheric partial pressure of CO₂ by increasing the NH₄⁺ concentration in the growth medium. Conditions that favor mtCA expression usually also stimulate anaplerosis. We therefore propose that the mtCA is involved in supplying HCO₃^- for anaplerotic assimilation catalyzed by phosphoenolpyruvate carboxylase, which provides C skeletons for N assimilation under some circumstances.

In algae and plants, the tricarboxylic acid (TCA) cycle plays a fundamental biosynthetic role (Beardall and Raven, 1990). The removal of intermediates from this cycle to feed other biosynthetic pathways (of which amino acid synthesis is often quantitatively the most important) requires that the cycle is replenished of its intermediates via anaplerotic reactions (Beardall and Raven, 1990; Norici and Giordano, 2002). The anaplerotic reactions make use of inorganic carbon to build the C4 compounds in demand in the TCA cycle, via β-carboxylation (Beardall and Raven, 1990; Norici and Giordano, 2002). The provision of inorganic carbon for these reactions can be crucial to sustain amino acid and protein synthesis (among others; Norici and Giordano, 2002). Anaplerotic β-carboxylation therefore represents a pivotal intersection among the metabolisms of C and N. Consequently, mechanisms must exist to ensure that there is sufficient inorganic carbon to maintain anaplerosis at an appropriate rate, especially in conditions in which the dissolved inorganic carbon (DIC) in the cytosol may be limited, competition with other DIC-requiring pathways (mostly photosynthesis) is significant, and N assimilation is fast. Respiration and photorespiration are conveniently located (spatially and functionally) sources of CO₂ to supply reactions that replenish the TCA cycle. A mechanism that recovers respiratory CO₂ would therefore be a very effective way to ensure an appropriate flux of C to the TCA cycle via anaplerosis. However, respiration and photorespiration produce DIC in the form of CO₂, whereas many β-carboxylases such as phosphoenolpyruvate carboxylase (PEPc) and pyruvate carboxylase (PC) require HCO₃^- (Chollet et al., 1996; Norici and Giordano, 2002). Thus, if the uncatalyzed rate of CO₂ conversion to HCO₃^- is not sufficiently high, enzymatic hydration of CO₂ to HCO₃^- may be necessary (Raven and Newman, 1994; Huang and Chapman, 2002). Reactions of this sort are catalyzed by carbonic anhydrases (CAs), whose activity is widespread in photosynthetic organisms (Badger and Price, 1994; Sültemeyer, 1998; Moroney et al., 2001; Badger et al., 2002).

Although a large number of papers has been published on photosynthetic roles of CAs, very few studies (Raven and Newman, 1994; Raven, 1995; Huang and Chapman, 2002) have considered the possibility that CAs are involved in the supply of C in non-photosynthetic biosynthetic pathways.

In this paper, we investigate a possible role of CAs in NH₄⁺ assimilation, using the model organism Chlamydomonas reinhardtii, a unicellular green alga. C. reinhardtii, one of the best-investigated eukaryotic algae with respect to genetics, cell biology, biochemistry, and physiology (Harris, 2001), has CAs in four extra- and intracellular locations. The extracellular CA activity is catalyzed by two α-CAs (pCA1 and pCA2) encoded by two similar genes (Spalding, 1998). Intracellularly, another α-CA (CAH3) is expressed in the thylakoid lumen (Karlsson et al., 1998; van Hunnik et al., 2001; Villarejo et al., 2002). A third CA is associated with the chloroplast envelope and is probably involved in DIC transport into the plastid stroma as part of the CO₂-concentrating mechanism (CCM; Villarejo et al., 2001). A fourth CA is present in the mitochondrial matrix (mtCA); it is a β-CA, it is encoded by two almost identical genes (Eriksson et al., 1996), and it is expressed in the light at low external DIC concentrations (Villand et al., 1997; Eriksson et al., 1998). The role of the mtCA has been suggested as buffering matrix H⁺ upon the initiation of photorespiration when the cells are transferred from high to low CO₂ conditions (Eriksson et al., 1996). Raven (2001) has suggested a role, with (hypothesized) HCO₃^- channels in the inner mitochondrial membrane, in limiting loss to medium of photorespired (and respired) CO₂ in the light and in promoting recycling of this CO₂ to photosynthetic CO₂.

In green algae, the provision of carbon skeletons used in N assimilation involves the use of PEPc, with 0.30 to 0.35 mol HCO₃^- fixed per mol NH₄⁺ assimilated (Raven and Farquhar, 1990; Vanlerberghe et al., 1990). An involvement of CA in the provision of this HCO₃^- requires that the inorganic carbon is supplied as CO₂, and that the CO₂ to HCO₃^- conversion occurs in a small volume. By small here is meant a volume in which the uncatalyzed rate of CO₂ conversion to HCO₃^- is insufficient to supply HCO₃^- at the rate required by NH₄⁺ assimilation. PEPc is a cytosolic enzyme, so the mechanism proposed by Raven (2001) for the conversion of photorespiratory and respiratory CO₂ to HCO₃^- in the mitochondrial matrix using mtCA with efflux of the resulting HCO₃^- to the cytosol could be involved in NH₄⁺ assimilation.

The work reported here involves determination of the effects of growth at a range of NH₄⁺ concentrations on the expression of mtCA and proposes an explanation for the observed pattern of mtCA expression. The experiments were planned in the context of known interactions between C and N metabolism in algae and, especially, interactions between N supply and expression of CCMs (Beardall et al., 1991, 1998; Giordano and Hell, 2001; Beardall and Giordano, 2002).

Growth of C. reinhardtii CC-125 in air was no higher at 10 mm NH₄⁺ than at 1 mm NH₄⁺, but was considerably lower at 0.1 mm NH₄⁺ (Table I). By contrast, cell volume, N per cell, and protein per cell all increased with NH₄⁺ concentration over the complete range of NH₄⁺ concentrations used for growth (Table I), as did light-saturated photosynthetic rate and dark respiration rate (Table II). However, the C to N ratio was the same for 1 mm as for 10 mm NH₄⁺-grown cells (Table I). This shows that there are parallel increases in the rates of net C and N acquisition as NH₄⁺ increases from 1 to 10 mm producing larger cells with the same generation time. At 0.1 mm NH₄⁺, the rate of net N acquisition is decreased more than that of C relative to the rate at 1 mm NH₄⁺. For cells grown in 5% (v/v) CO₂, the growth rate in 0.1 mm NH₄⁺ is the same as that of cells grown in air, but the growth rates in 5% (v/v) CO₂ are higher than those in air for the two higher NH₄⁺ concentrations (Table I). For cells grown in air, the Gln synthetase activity was circa 2 order of magnitudes higher when the medium contained 1 or 10 mm NH₄⁺ than when the concentration was 0.1 mm NH₄⁺ (Table I). This is consistent with the higher rates of primary NH₄⁺ assimilation at the two higher NH₄⁺ concentrations, although it is likely that the recycling of NH₄⁺ from the photorespiratory carbon oxidation cycle is greater at the lowest NH₄⁺ concentration, because the data in Table II suggest less effective CCM functioning, permitting more expression of Rubisco oxygenase activity, at low NH₄⁺.

The higher rate of NH₄⁺ assimilation on a cell basis, with increasing external NH₄⁺, requires a higher rate of anaplerotic DIC fixation for the synthesis of C skeletons. Most of this DIC is generally held to be fixed by PEPc, although Table I shows data for PC activities that are higher than those of PEPc. In agreement with anaplerotic roles, Table I shows that PEPc activity per cell, for cultures grown at air levels of CO₂, increases with NH₄⁺ concentration for growth and hence with the requirement for anaplerotic fixation of DIC (Table III), over the entire range tested.

The inorganic carbon substrate for PEPc and PC is HCO₃^- (Norici and Giordano, 2002). Table III shows the concentration of CO₂ required in the steady state to generate HCO₃^- at the rate required for anaplerotic PEPc activity in the cytosol, if uncatalyzed conversion of CO₂ occurs either in the matrix or in the cytosol. The calculations use the assumptions made by Raven (2001), modified as shown in the legend to Table III. Uncatalyzed HCO₃^- production at the required rate in the cytosol (which lacks CA) involves a steady-state CO₂ concentration, which is about 1 order of magnitude higher for 1 and 10 mm NH₄⁺-grown cells than for 0.1 mm NH₄⁺-grown cells. The same is true for uncatalyzed HCO₃^- production in the matrix, where, however, the absolute steady-state CO₂ concentrations needed are much higher.

For low-CO₂ (air)-grown cells, a wide range of values has been suggested for cytosolic steady-state CO₂ concentration (Spalding and Portis, 1985; Yokota et al., 1987; Raven, 1997; Thoms et al., 2001). If no active transport processes occurs between the cytosol and Rubisco, a realistic value for cytosolic CO₂ would be in the order of 50 to 100 μm. Table III shows how this amount of cytosolic CO₂ would be sufficient to support anaplerotic β-carboxylation in the cytosol in the cells cultured at 0.1 mm NH₄⁺, but not in the algae grown at 1 and 10 mm NH₄⁺.

If β-carboxylation occurs in the matrix, the required CO₂ concentration for uncatalyzed CO₂ production is that for the matrix, because HCO₃^- uptake by mitochondria from the cytosol is unlikely (Nicholls and Ferguson, 2002). Our measurements on isolated mitochondria exclude the presence of PEPc activity in these organelles (data not shown), however, it is possible that at least part of the PC activity measured in whole cells is located in mitochondria. This would be in agreement with the known localization of PC in many organisms (e.g., Jelenska et al., 2001; Liao and Freedman, 2001; Liu et al., 2002), although cytosolic PCs are also present in yeasts such as Brewer's yeast (Saccharomyces cerevisiae; Huet et al., 2000). If the steady-state CO₂ concentration in the matrix of air-grown cells at all three NH₄⁺ concentrations is close to the lower 0.1 mm NH₄⁺ value showed in Table III (with the higher respiration rate per cell offset by the larger cell, and possibly mitochondrial volumes at higher NH₄⁺ concentrations for growth; Schötz et al., 1972; Blank and Arnold, 1980; Blank et al., 1980; Ehana et al., 1995), then uncatalyzed HCO₃^- production would not be sufficient at the two higher NH₄⁺ concentrations.

If the DIC for anaplerotic fixation is supplied as CO₂ from the medium or from photorespiration or dark respiration, then it is possible that CA activity is needed to generate HCO₃^-. Catalyzed CO₂ conversion would also be necessary if DIC is supplied as both CO₂ and HCO₃^-, but HCO₃^- supply occurs at a rate lower than the rate of use. PEPc is located in the cytosol, so that any CA involved in generating HCO₃^- from CO₂ must be in the cytosol or in a compartment that is supplied with CO₂ and from which HCO₃^- can move to the cytosol. Neither the periplasmic nor the lumenal CAs could do this (Raven, 1997). The improbability of an anaplerotic role for these two CAs is confirmed by our data: Whereas the N assimilation rate increased over the entire range of N concentrations used for growth, pCA activity had the same value for 1 and 10 mm NH₄⁺-grown cells (Table I); expression of lumenal CA was also very little affected by the N growth regime (Fig. 1). The limited variability of lumenal CA in response to the N growth regime may be linked to the observation of Villarejo et al. (2002), who reported an apparent absolute requirement for active lumenal CA for growth at low N concentrations. The plastid envelope CA has not been sufficiently investigated to determine whether it could be involved. However, the mtCA could use matrix-generated CO₂ to provide anaplerotic carboxylases with HCO₃^-. The mtCA of air-acclimated cells, 24 h after transfer to air from 5% (v/v) CO₂, shows an appropriate response (Fig. 2), with essentially no enzyme message or protein present in cells grown at 0.1 mm NH₄⁺ but with both message and protein present in cells grown with 1 and 10 mm NH₄⁺. Figure 3 shows that expression of mtCA protein occurs even at high CO₂ (0.2% [v/v] in air), if sufficient NH₄⁺ (100 mm) is present.

Table II shows that the NH₄⁺ concentration supplied for growth has consequences for the affinity for DIC in photosynthesis. The half-saturation value for DIC is twice as high at 0.1 mm NH₄⁺ as at 1 mm NH₄⁺, with no significant further effect at 10 mm NH₄⁺. The results of this study are in general agreement with earlier work (Eriksson et al., 1996; Geraghty and Spalding, 1996; Villand et al., 1997), showing that mtCA was expressed in cells grown under air levels of CO₂, but not under high CO₂ levels. It also agrees with the unpublished work on mtCA-deficient mutants cited by Sültemeyer (1998), showing that the CCM was disabled in mtCA-deficient mutants. However, it is significant that, despite the lower DIC affinity and the much lower initial slope of photosynthetic rate increase per unit increase in DIC concentration, the DIC compensation concentration and DIC saturation points are lower for the 0.1 mm NH₄⁺ cells than those grown at higher NH₄⁺ concentrations.

The lowest NH₄⁺ concentration (0.1 mm) used in the experiments presented in Tables I through III led to a significantly higher C to N ratio in the cells than did the two higher concentrations of NH₄⁺, at least for the low CO₂-grown cells; no data are available for the cells grown at high CO₂. Although this lowest NH₄⁺ concentration used provided evidence of N limitation of growth, the highest NH₄⁺ concentration did not yield NH₄⁺ toxicity because there was no decrease in growth rate relative to 1 mm NH₄⁺ for cells grown in air (Table I). Under the light-saturated conditions of the experiments, any energy cost of recycling of NH₃/NH₄⁺ at high external NH₄⁺ concentrations (Raven, 1980; Kleiner, 1985a, 1985b; Britto et al., 2001, 2002; Kronzucker et al., 2001) does not impact upon growth rate. The supply of CO₂ was limiting for growth in the cultures equilibrated with air at the two higher NH₄⁺ concentrations, but not when the NH₄⁺concentration for growth was 0.1 mm.

The data presented here show that the expression of mtCA is under the control of NH₄⁺ supply as well as of DIC supply, because expression of mtCA at low DIC concentrations for growth is prevented if the NH₄⁺ concentration is also low, i.e. 0.1 mm (Fig. 2), and expression of mtCA can occur even in 0.2% (v/v) CO₂ in air if the NH₄⁺ concentration is very high, i.e. 100 mm (Fig. 3). The fact that the expression of mtCA can be modulated by changing the relative amounts of C and N is compatible with an involvement of this enzyme in anaplerosis. However, anaplerosis is generally believed to be initiated from the cytosol, where the initial β-carboxylation occurs (Norici and Giordano, 2002). The HCO₃^- generated in the matrix by mtCA from respiratory/photorespiratory CO₂ would then have to be transferred to the cytosol. The model presented by Raven (2001) requires that the HCO₃^- produced with the catalysis of mtCA leaves the mitochondria via HCO₃^- channels in the inner mitochondrial membrane. Anion channels in mitochondrial inner membranes are known from a number of organisms. Much of this work has involved chloride as the only anion tested (Fernandez-Sala et al., 1999; Kikinska et al., 2000). However, it is known that a number of anion channels in the plasmalemma of metazoa can transport bicarbonate as well as chloride (Kunzelman et al., 1991; Paulsen et al., 1994; Ishikawa, 1996). Furthermore, there is direct evidence for bicarbonate flux across the inner mitochondrial membrane (Selwyn and Walker, 1977; Simpson, 1983; Gainutdinov et al., 1985). These data confirm that the mitochondrial bicarbonate channel aspect of the hypothesis of Raven (2001) is plausible. If, however, β-carboxylation takes place in the mitochondria, bicarbonate transport across the mitochondrial membranes is not required to supply anaplerosis with DIC.

Growth of C. reinhardtii at high (i.e. 5% [v/v]) CO₂ levels corresponds to equilibrium CO₂ concentrations in a solution of 1 to 2 mm (varying with temperature). With the permeability coefficients of the C. reinhardtii plasmalemma found by Sültemeyer and Rinast (1996), the steady-state concentrations of CO₂ in all cell compartments during photosynthesis with diffusive CO₂ entry for the cells grown at high CO₂ would be within 10% of the external concentration. Under these conditions, the expression of mtCA was not observed, and the supply of HCO₃^- to PEPc in providing C skeletons for NH₄⁺ assimilation cannot depend on mtCA activity. Using the values in Table III, it is clear that cultures in high CO₂ conditions yield intracellular CO₂ concentrations in either light or dark that give uncatalyzed rates of HCO₃^- production in either the cytosol (at all NH₄⁺ concentrations) or the matrix (for 0.1 mm NH₄⁺) in excess of those needed for anaplerotic PEPc activity. Although the uncatalyzed rate of HCO₃^- production in the matrix is insufficient to meet the requirements of PEPc at the two high NH₄⁺ concentrations, this shortfall could readily be met by uncatalyzed cytosolic HCO₃^- production. However, if the anaplerotic carboxylase is mitochondrial, then HCO₃^- supply at the two higher NH₄⁺ concentrations could not be maintained either by uncatalyzed production in the matrix (Table III) or by supply, via anion channels, of HCO₃^- produced by uncatalyzed conversion of CO₂ in the cytosol.

Two main scenarios can be envisaged for low CO₂-grown cells: (a) the CCM delivers HCO₃^- to the cytosol; (b) the CCM delivers CO₂ to the cytosol.

Under these conditions, delivery to the cytosol of the HCO₃^- generated by uncatalyzed (0.1 mm NH₄⁺) or mtCA-catalyzed (1 and 10 mm NH₄⁺) hydroxylation of CO₂ would be a relatively minor component of HCO₃^- supply to the cytosol. At light saturation, HCO₃^- flux to the cytosol from DIC in the medium is 1 order of magnitude greater than the flux that could occur from the mitochondrial matrix in C. reinhardtii cells (Raven, 2001). This suggests that any HCO₃^- produced in the mitochondrial matrix and transported to the cytosol would not be significant, provided that HCO₃^- uptake systems at the chloroplast envelope do not scavenge cytosolic HCO₃^- to the point of reducing it below the requirement of PEPc. Such a situation, although unlikely, cannot be excluded for the cells cultured at 1 and 10 mm NH₄⁺, whose growth was DIC-limited and photosynthesis (saturation [CO₂] = 150 and 214 μm, respectively), at the CO₂ levels found in air, was strongly DIC-limited at saturating light (Table II; air CO₂ = 360 μmol mol^-1 ≈ 12.5 μm, with a Henry's constant, K_H = 0.03458, 25°C, in freshwater). It is possible that cells grown in air use mtCA for NH₄⁺ assimilation with PEPc as the anaplerotic carboxylase in the dark phase of the natural light cycle, when the CCM activity is greatly diminished or absent. Although the mtCA is only produced in the light, the mtCA produced in the light phase of the natural light-dark cycle would remain significantly functional during the dark phase. A crucial requirement of this suggestion is that NH₄⁺ (more generally, inorganic N) assimilation shall occur in the dark phase of the diel cycle in C. reinhardtii. It appears that the assimilation of NH₄⁺ in the dark phase is most likely to occur when NH₄⁺ availability is low (Kates and Jones, 1967; Jones et al., 1968; Weger et al., 1990), i.e. the conditions under which mtCA expression in air-grown C. reinhardtii cells is minimal. Further work is needed, using C. reinhardtii growing at air levels of CO₂ in a range of NH₄⁺ concentrations with light-dark cycles, to test this suggestion as to the role of mtCA in NH₄⁺ assimilation.

For a mitochondrial carboxylase in cells growing in air, a role for mtCA in supplying HCO₃^- in the matrix can readily be seen for cells growing in 1 or 10 mm NH₄⁺. The mtCA activity is needed because the uncatalyzed rate of conversion of CO₂ to HCO₃^- is much too slow to supply the carboxylase with HCO₃^- (Table I), and the transfer of HCO₃^- from the cytosol to the matrix via anion channels would not yield an adequate steady-state HCO₃^- concentration in the matrix (see “Results”). For cells growing in 0.1 mm NH₄⁺, with negligible mtCA expression, carboxylase function requires uncatlayzed HCO₃^- production in the matrix at 10 times the rate shown in Table III, i.e. a 10 times higher steady-state concentration of CO₂ in the matrix resulting from a 10 times higher rate of mitochondrial decarboxylation (see “Materials and Methods”). A contribution to such an increase could result from faster photorespiration with a less effective CCM in cells growing in 1 mm NH₄⁺ (Table II).

Such a mechanism of inorganic C delivery to the cytosol is consistent with the models of the eukaryotic algal CCM presented by Thoms et al. (2001) and the conclusion of Palmqvist et al. (1990). In this case, a carbonic-anhydrase-like activity must be present in the plasmalemma to convert HCO₃^- to CO₂ when HCO₃^- is the form of inorganic C taken up from the medium. Because the plastid of C. reinhardtii can accumulate DIC (Amoroso et al., 1998), it is not possible to use Rubisco kinetics to estimate the steady-state CO₂ concentration in the cytosol during photosynthesis by air-acclimated cells. Certainly a value of (50 to) 100 μm would be an upper limit, because this concentration could, without further DIC accumulation into the plastid, account for the carboxylase to oxygenase activity ratio of Rubisco in air-grown C. reinhardtii expressing a CCM (see Raven, 2001). Such a steady-state CO₂ concentration would account for the PEPc activity required in N assimilation in air-grown C. reinhardtii with 0.1 mm NH₄⁺ using only uncatalyzed conversion of CO₂ to HCO₃^- in the cytosol (Table III). However, such uncatalyzed conversion would not be adequate for anaplerotic PEPc activity at the two higher NH₄⁺ concentrations (Table III).

This alternative model for the CCM of C. reinhardtii could thus provide a rationale for the involvement of mtCA in supplying HCO₃^- to PEPc for NH₄⁺ assimilation in air-grown cells at 1 and 10 mm NH₄⁺ but not with 0.1 mm NH₄⁺ (provided the steady-state CO₂ concentration in the light is 50–100 μm), nor at any NH₄⁺ concentration for growth at 5% (v/v) CO₂ with diffusive CO₂ entry.

Chlamydomonas reinhardtii CC-125 cells were cultured at 25°C in continuous light (100 μmol m^-2 s^-1, 400–700 nm) in a minimal medium based on the recipe of Sueoka (1960), with the trace element composition modified according to Hutner et al. (1950). Nitrogen was added as NH₄Cl at 0.1, 1, or 10 mm, unless otherwise stated. To achieve high CO₂ concentrations, the cultures were bubbled with air enriched with CO₂ to 5% by volume (50 mmol CO₂ mol^-1 total gas). Low CO₂ concentrations were attained by bubbling the cultures with air (0.36 mmol CO₂ mol^-1 total gas). Cells were acclimated to each N and CO₂ concentration for at least 3 weeks before the experiments, except in the case of the experiments on induction of the CCM by changing from high to low CO₂ in the gas stream. All experiments were carried out on cells in the mid-exponential growth phase.

Growth was followed on three different cultures by daily counts using a Burker hemocytometer. The cell volume and surface area were determined from the longitudinal and transverse axes of 50 cells from each culture with an ocular micrometer, using a graduated slide as a reference. C. reinhardtii cell volume (V) was calculated using the equation for a prolate ellipsoid, V = π/6(ab²⁾, where a and b are, respectively, the major (longitudinal) and minor (transverse) radii of the cell (Hillebrand et al., 1999).

Chlorophyll was extracted in a mixture of 9 volumes of acetone and 1 volume of water and was determined according to Jeffrey and Humphrey (1975). Total proteins were measured as described by Peterson (1977); amino acids were determined from crude extracts as described by Stocchi et al. (1989). The starch content of cells was estimated enzymatically using a commercial kit (kit 207748, Roche Diagnostics, Armstadt, Germany). Total C and N were measured on lyophilized cells using a CHN elemental analyzer (FlashEA 1112, ThermoFinnegan, Waltham, MA). The data are expressed as the mean ± sd calculated from triplicates.

Photosynthetic O₂ evolution was measured using an O₂ electrode system (Chloroview 2, Hansatech, Kings Lynn, Norfolk, UK). Cells were harvested by centrifugation at 1,200g for 10 min and washed in DIC-free growth medium. All experiments were carried out at a cell density of 2 × 10⁶ cells mL^-1 at a temperature of 25°C and with a photon flux density (400–700 nm) of 100 μmol photons m^-2 s^-1. The measurements were started with a dissolved O₂ concentration equivalent to 20% of the air equilibrium value and were performed according to Giordano et al. (2000). Respiratory rates were determined by measuring O₂ consumption in the dark in the same system (Giordano et al., 2000). The results are shown as the mean ± sd of measurements obtained with triplicates.

Cells in the mid-exponential phase of growth were harvested by centrifugation at 1,200g for 10 min and were resuspended in extraction medium as described by Norici et al. (2002). The samples were sonicated for three cycles of 20 s each at 12 J (Soniprep 150, MSE Sanyo, Loughborough, UK) and then centrifuged at 12,000g for 5 min. The supernatant was collected and used for enzyme activity assays. All of the extraction steps were performed on ice. PEPc (EC.4.1.1.31) activity was assayed spectrophotometrically following the method by Holdsworth and Bruck (1977) and PC (EC 6.4.1.1) activity was assayed according to Wurtele and Nikolau (1992). The activity of pCA (EC 4.2.1.1) was measured on intact cells using the potentiometric method of Wilbur and Anderson (1948) as modified by Miyachi et al. (1983). Gln synthetase (EC 6.3.1.2) activity was assayed spectrophometrically (Oaks et al., 1980). All results are shown as the mean ± sd of measurements from triplicates.

Immunodetection of proteins was effected on extracts of C. reinhardtii acclimated to 5% (v/v) CO₂ and 0.1, 1, or 10 mm NH₄⁺, obtained at intervals of 0, 1, 2, 4, 8, 12, and 24 h after transfer to low CO₂ conditions for growth. No nitrogen was added to the cultures during the experiment. Similar experiments were also carried out on C. reinhardtii cells acclimated to 5% (v/v) CO₂ in the presence of 1, 10, and 100 mm of NH₄Cl. These cells were sampled 24 h after they were transferred to 0.1% (v/v) or 0.2% CO₂.

Cell-free extracts obtained as described above were loaded on 12% (v/v) SDS-PAGE gels (Laemmli, 1970). Western blots were produced according to Norici et al. (2002). Immunodetection was performed using anti-mitochondrial CA and anti-luminal CA IgGs raised against the respective proteins of C. reinhardtii. Anti-Rubisco IgGs were raised against Rubisco from spinach (Spinacia oleracea). The hybridization of the antibodies was detected using the Immunostar kit (Bio-Rad Laboratories, Hercules, CA).

C. reinhardtii cultures were grown and sampled as described above for low CO₂ induction experiments. Total RNA was isolated immediately after cell collection using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Glyoxal-denatured northern-blot analyses were performed as described by Sambrook et al. (1989). Hybridization and washing were done at 65°C. The probes were ³²P-labeled DNA sequences amplified from the following genes: Mca1/Mca2 and Cah3 from C. reinhardtii and rbcS1 and 25S from Arabidopsis.

Estimates of CO₂ in the compartment where β-carboxylation may take place are required to account for the anaplerotic use of HCO₃^- in the absence of CA activity in these compartments. Calculations were therefore made following the methods and assumptions of Raven (2001), so that the rate of CO₂ production in mitochondria in the light was computed from the rate of decarboxylation required to support the observed rate of growth rather than from the measured rates of respiratory O₂ uptake in the dark. In brief, the rate of CO₂ production from photorespiration as a fraction of the organic carbon accumulation rate during growth of C. reinhardtii acclimated to low CO₂, at light saturation, in continuous illumination, was computed from the CCM models of Spalding and Portis (1985) and of Yokota et al. (1987). The methods of Raven (1984) were used to compute the rate of CO₂ production for the synthesis of those carbon skeletons required for biosynthesis, which can only be produced by the metabolism of pyruvate in the mitochondria as a fraction of the organic carbon accumulation during growth of C. reinhardtii in continuous light. These are minimal estimates, assuming a minimal involvement of mitochondrial respiratory reactions in maintenance and repair processes in continuously illuminated cells.