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ENVIRONMENTAL STRESS AND ADAPTATION

An Anaplerotic Role for Mitochondrial Carbonic Anhydrase in Chlamydomonas reinhardtii¹

Dipartimento di Scienze del Mare, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy (M.G., A.N.); Umeå Plant Science Center, Umeå University, S901 87 Umeå, Sweden (M.F., M.E.); and Division of Environmental and Applied Biology, School of Life Sciences, University of Dundee, Dundee DD1 4HN, United Kingdom (J.A.R.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
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.

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 -CAs (pCA1 and pCA2) encoded by two similar genes (Spalding, 1998). Intracellularly, another -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 -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).

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Effect of C and N Growth Regimes on C and N Assimilation

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⁺.

-Carboxylases

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 Intracellular DIC System

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 -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 -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.

Carbonic Anydrases

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.

View larger version (34K): [in this window] [in a new window]   Figure 1. Immunoblot of Rubisco and lumenal CA (lumCA) from C. reinhardtii cells cultured in the presence of 0.1, 1, and 10 mM NH4+. The figure shows the changes of expression of the two proteins over a period of 12 h since the time cells were transferred from high CO2 (5% [v/v] in air) to low CO2 (0.036% [v/v] in air) growth condition.

View larger version (43K): [in this window] [in a new window]   Figure 2. Western blot (W) and northern blot (N) of mtCA from C. reinhardtii cells cultured in the presence of 0.1, 1, and 10 mM NH4+. The figure shows the changes in the expression of the protein over a period of 12 h since the time cells were transferred from high CO2 (5% [v/v] in air) to low CO2 (0.036% [v/v] in air) growth condition. The changes in 25S RNA abundance for the same cells are also shown.

View larger version (61K): [in this window] [in a new window]   Figure 3. Western blot of mtCA from C. reinhardtii cells cultured in the presence of 1, 10, and 100 mM NH4+ and bubbled with air containing either 0.1% or 0.2% (v/v) CO2.

Photosynthesis

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.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Interactions between Assimilation of Inorganic C and Assimilation of Inorganic N

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 Role of C and N Supply in the Regulation of mtCA Expression and Implications for the Role of mtCA in the Provision of DIC to the Cytosol

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 -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, -carboxylation takes place in the mitochondria, bicarbonate transport across the mitochondrial membranes is not required to supply anaplerosis with DIC.

Anaplerotic HCO₃^- Supply in Cells Grown at High CO₂ Concentrations

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.

The Role of mtCA in the Assimilation of NH₄⁺ in Cells Grown at Low CO₂ Concentrations

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.

Case 1: HCO₃^- Is the DIC Species Delivered to the Cytosol from the Medium by the CCM
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).

Case 2: CO₂ Is the DIC Species Delivered to the Cytosol from the Medium by the CCM
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.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Cultures

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 = /6(ab²⁾, where a and b are, respectively, the major (longitudinal) and minor (transverse) radii of the cell (Hillebrand et al., 1999).

Cell Constituents

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.

Gas-Exchange Measurements

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 x 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.

Extraction and Assay of Enzymes

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.

SDS-PAGE and Western Blots

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).

Northern Blots

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.

Calculations of the Steady-State Concentrations of CO₂ in the Cytosol and the Mitochondrial Matrix

Estimates of CO₂ in the compartment where -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.

Received March 20, 2003; returned for revision April 24, 2003; accepted May 6, 2003.

	FOOTNOTES

 
¹ Research on mechanisms of DIC acquisition by algae in J.A.R.'s laboratory was funded by the Natural Environment Research Council (UK).

² These authors contributed equally to this paper.

^* Corresponding author; e-mail giordano{at}unian.it; fax 39–071–220–4650.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Amoroso G, Sültemeyer D, Thyssen C, Fock HP (1998) Uptake of HCO₃^- and CO₂ in cells and chloroplasts from the microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta. Plant Physiol 116: 193-201[Abstract/Free Full Text]

Badger MR, Hansen D, Price GD (2002) Evolution and diversity of CO₂ concentrating mechanisms in cyanobacteria. Funct Plant Biol 29: 161-173[CrossRef]

Badger MR, Price GD (1994) The role of carbonic anhydrase in photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 45: 369-392[CrossRef][Web of Science]

Beardall J, Giordano M (2002) Ecological implications of algal CO₂ concentrating mechanisms and their regulation. Funct Plant Biol 29: 333-347

Beardall J, Raven JA (1990). Pathways and mechanisms of respiration in microalgae. Mar Microb Food Webs 4: 7-30

Beardall J, Roberts S, Millhouse J (1991) Effects of nitrogen limitation on uptake of inorganic carbon and specific activity of ribulose-1,5-bisphosphate carboxylase/oxygenase in green microalgae. Can J Bot 69: 1146-1150

Beardall J, Roberts S, Millhouse J (1998) Environmental regulation of CO₂-concentrating mechanisms in microalgae. Can J Bot 76: 1010-1017[CrossRef]

Blank R, Arnold CG (1980) Variability of mitochondrial population in Chlamydomonas reinhardtii. Protoplasma 104: 187-191

Blank R, Hauptmann E, Arnold CG (1980) Variety of mitochondrial shapes, sizes and volumes in Chlamydomonas reinhardtii. Planta 50: 236-241

Britto DT, Siddiqui MY, Glass ADM, Kronzucker HJ (2001) Futile membrane ion cycle cycling: a new cellular hypothesis to explain ammonium toxicity in plants. Proc Natl Acad Sci USA 98: 4255-4258[Abstract/Free Full Text]

Britto DT, Siddiqi MY, Glass ADM, Kronzucker HJ (2002) Subcellular NH₄⁺ flux analysis in leaf segments of wheat (Triticum aestivum). New Phytol 155: 373-380

Chollet R, Vidal J, O'Leary MH (1996) Phosphoenolpyruvate carboxylase: a ubiquitous, highly regulated enzyme in plants. Annu Rev Plant Physiol Plant Mol Biol 47: 273-298[CrossRef][Web of Science][Medline]

Ehana T, Osafune T, Hase E (1995) Behavior of mitochondria in synchronized cells of Chlamydomonas reinhardtii (Chlorophyta). J Cell Sci 108: 499-507[Abstract]

Eriksson M, Karlsson J, Ramazanov Z, Gardeström P, Samuelsson G (1996) Discovery of an algal mitochondrial carbonic anhydrase: molecular cloning and characterization of a low-CO₂-induced polypeptide in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 93: 12031-12034[Abstract/Free Full Text]

Eriksson M, Villand P, Gardeström P, Samuelsson G (1998) Induction and regulation of expression of a low-CO₂-induced mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Plant Physiol 116: 637-641[Abstract/Free Full Text]

Fernandez-Sala E, Sagan M, Cheng C, Yusha SH, Weinberg WC (1999) p53 and tumor necrosis factor alpha regulate the expression of a mitochondrial chloride channel protein. J Biol Chem 274: 36488-36498[Abstract/Free Full Text]

Gainutdinov MK, Konov VV, Luchenko MV, Turakulov IK (1985) Electrogenic transport of bicarbonate ions through the internal mitochondrial membrane induced by cytosplasmic glycopeptide. Biul Ecksp Biol Med 100: 561-563

Geraghty AM, Spalding MH (1996) Molecular and structural changes in Chlamydomonas under limiting CO₂: a possible mitochondrial role in adaptation. Plant Physiol 11: 1339-1347

Giordano M, Hell R (2001) Mineral nutrition in photolithotrophs: cellular mechanisms controlling growth in terrestrial and aquatic habitats. Recent Res Dev Plant Physiol 2: 95-123

Giordano M, Pezzoni V, Hell R (2000) Strategies for the allocation of resources under sulphur limitation in the green alga Dunaliella salina. Plant Physiol 124: 857-864[Abstract/Free Full Text]

Harris EH (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol 52: 363-406[CrossRef][Web of Science][Medline]

Hillebrand H, Durselson CD, Kirschtel D, Pollinger U, Zohary T (1999) Biovolume calculation for pelagic and benthic microalgae. J Phycol 35: 402-424

Holdsworth ES, Bruck J (1977) Enzymes concerned with -carboxylation in marine phytoplankton: purification and properties of PEPck. Arch Biochem Biophys 182: 87-94[Medline]

Huang CV, Chapman KD (2002) Biochemical and molecular inhibition of plastidial carbonic anhydrase reduces the incorporation of acetate into lipids in cotton embryos and tobacco cell suspensions and leaves. Plant Physiol 128: 1417-1427[Abstract/Free Full Text]

Huet C, Menendez J, Gancedo C, Francois JM (2000). Regulation of pyc1 encoding pyruvate carboxylase isozyme I by nitrogen sources in Saccharomyces cerevisiae. Eur J Biochem 267: 6817-6823[Medline]

Hutner SH, Provasoli L, Schatz A, Haskins CP (1950). Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94: 152-170

Ishikawa T (1996) A bicarbonate- and weak acid-permeable chloride conductance controlled by cytosolic Ca²⁺ and ATP in rat submandibular acinar cells. J Membr Biol 153: 147-159[CrossRef][Web of Science][Medline]

Jeffrey SW, Humphrey GE (1975) New spectrophotometric equations for determining chlorophylls a, b, c₁ and c₂ in higher plants and natural phytoplankton. Biochem Physiol Plants 167: 191-194

Jelenska J, Crawford MJ, Harbs OS, Zuther E, Haselkorn R, Roos DS, Gornicki P (2001) Subcellular localization of acetyl-CoA carboxylase in the apicomplexan parasite Toxoplasma gondii. Proc Natl Acad Sci USA 98: 2723-2728[Abstract/Free Full Text]

Jones RF, Kates JR, Keller SJ (1968) Protein turnover and macromolecular synthesis during growth and gametic differentiation in Chlamydomonas reinhardtii. Biochim Biophys Acta 157: 589-598[Medline]

Karlsson J, Clarke AK, Chen Z-Y, Hugghins SY, Park Y-L, Husic HD, Moroney JV, Samuelsson G (1998) A novel -type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO₂. EMBO J 17: 1208-1216[CrossRef][Web of Science][Medline]

Kates JR, Jones RF (1967) Periodic increases in enzyme activity in synchronized cultures of Chlamydomonas reinhardtii. Biochim Biophys Acta 145: 153-158[Medline]

Kikinska A, Debska G, Kunz W, Szewczyk A (2000) Mitochondrial potassium and chloride channels. Acta Biochim Pol 47: 541-551[Medline]

Kleiner D (1985a) Energy expenditure for cyclic retention of NH₃/NH₄⁺ during N₂ fixation by Klebsiella pneumoniae. FEBS Lett 187: 237-239[CrossRef][Medline]

Kleiner D (1985b) Bacterial ammonium transporters. FEMS Microbiol Rev 32: 87-100[CrossRef]

Kronzucker HJ, Britto DT, Davenport RJ, Tester M (2001) Ammonium toxicity and the real cost of transport. Trends Plant Sci 6: 335-337[CrossRef][Web of Science][Medline]

Kunzelman K, Gerlach L, Frobe U, Gregan R (1991) Bicarbonate permeability of epithelial chloride channels. Pflugers Arch Eur J Physiol 417: 616-621[CrossRef][Web of Science][Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680-685

Liao VHC, Freedman JH (2001) Characterization of a cadmium-inducible isoform of pyruvate carboxylase from Caenorhabditis elegans. DNA Sequence 12: 137-145[Medline]

Liu YQ, Jetton TL, Leahy JL (2002) Beta-cell adaptation to insulin resistance: increased pyruvate carboxylase and malate-pyruvate shuttle activity in islets of nondiabetic Zicker fatty rats. J Biol Chem 277: 39163-39168[Abstract/Free Full Text]

Miyachi S, Tsuuki M, Avramova T (1983) Utilization modes of inorganic carbon for photosynthesis in various species of Chlorella. Plant Cell Physiol 29: 441-445

Moroney JV, Bartlett SG, Samuelsson G (2001) Carbonic anhydrase in plants and algae. Plant Cell Environ 24: 141-153[Medline]

Nicholls DG, Ferguson SJ (2002) Bioenergetics, Ed 3. Academic Press, London

Norici A, Dalsass A, Giordano M (2002) Role of phosphoenolpyruvate carboxylase in anaplerosis in the green microalga Dunaliella salina cultured under different nitrogen regimes. Physiol Plant 116: 186-191[Medline]

Norici A, Giordano M (2002) Anaplerosis in microalgae. Recent Res Dev Plant Physiol 3: 153-164

Oaks A, Stulen J, Jones MJ, Boosel IL (1980) Enzymes of nitrogen assimilation in maize roots. Planta 148: 186-191

Palmqvist K, Ramazanov Z, Samuelsson G (1990). The role of extracellular carbonic anhydrase for accumulation of inorganic carbon in the green alga Chlamydomonas reinhardtii: a comparison between wild-type and cell-wall-less mutant cells. Physiol Plant 80: 267-276

Paulsen JH, Fischer H, Illek B, Machen TE (1994) Bicarbonate conductance and pH regulatory capacity of cystic-fibrosis transmembrane conductance regulation. Proc Natl Acad Sci USA 91: 5340-5344[Abstract/Free Full Text]

Peterson GL (1977) Simplification of the protein assay method of Lowry which is more generally applicable. Arch Biochem Biophys 83: 246-356

Raven JA (1980) Nutrient transport in micro-algae. Adv Microbiol Physiol 21: 47-226[Medline]

Raven JA (1984) Energetics and Transport in Aquatic Plants. AR Liss, New York

Raven JA (1995) Photosynthetic and non-photosynthetic roles of carbonic anhydrase in algae and cyanobacteria. Phycologia 34: 93-101

Raven JA (1997) CO₂ concentrating mechanisms: a direct role for thylakoid lumen acidification? Plant Cell Environ 20: 147-154

Raven JA (2001) A role for mitochondrial carbonic anhydrase in limiting CO₂ leakage from low CO₂-grown cells of Chlamydomonas reinhardtii. Plant Cell Environ 24: 261-265[CrossRef]

Raven JA, Farquhar GD (1990) The influence of N metabolism and organic acid synthesis on the natural abundance of isotopes of carbon in plants. New Phytol 116: 505-529[CrossRef]

Raven JA, Newman JR (1994) Requirement for carbonic anhydrase activity in processes other than photosynthetic inorganic carbon assimilation. Plant Cell Environ 20: 123-130

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Schötz F, Bathelt H, Arnold C-G, Schimmer O (1972) Die Architektur und Organisation der Chlamydomonas-Zelle: Ergebnisse der Elektronmikroskoscopie von Serienschnitten und der darous resulterenden dreidimensional Rekonstruktion. Protoplasma 75: 229-254[CrossRef][Medline]

Selwyn MJ, Walker HA (1977) Permeability of the mitochondrial membrane to bicarbonate ions. Biochem J 166: 137-139[Medline]

Simpson DP (1983) Mitochondrial bicarbonate carrier: a site of regulation of renal substrate metabolism by acid-base changes. Trans Assoc Am Physicians 96: 918-924

Spalding MH (1998) CO₂ acquisition: acclimation to changing carbon availability. In J-D Rochaix, M Goldschmidt-Clermont, S Merchant, eds, The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 529-547

Spalding MH, Portis AR Jr (1985) A model for carbon dioxide assimilation in Chlamydomonas reinhardtii. Planta 164: 308-320

Stocchi V, Piccoli G, Magnani M, Palma F, Biagiarelli B, Cucchiarini L (1989) Reversed-phase high-performance liquid-chromatography separation of dimethylaminoazobenzene sulfonylaminoazobenzene and dimethylaminoazobenzene thiohydantoin amino-acid derivatives for amino-acid analysis and microsequencing studies at the picomole level. Anal Biochem 178: 107-117[CrossRef][Web of Science][Medline]

Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 46: 83-91[Free Full Text]

Sültemeyer D (1998) Carbonic anhydrase in eukaryotic algae: characterization, regulation and possible function during photosynthesis. Can J Bot 76: 962-972[CrossRef]

Sültemeyer D, Rinast K-A (1996) The CO₂ permeability of the plasmalemma of Chlamydomonas reinhardtii: mass spectrometric ¹⁸O exchange measurements from ¹³C¹⁸O₂ in suspensions of carbonic anhydrase-loaded plasma membrane vesicles. Planta 200: 258-268

Thoms S, Pahlow M, Wolf-Gladrow DA (2001) Model of the carbon concentrating mechanism in chloroplast of eukaryotic algae. J Theor Biol 208: 201-260[CrossRef][Web of Science][Medline]

van Hunnik E, Livne A, Pogenberg V, Spijkerman E, van den Ende H, Garcia EM, Sültemeyer D, de Leeuw JW (2001) Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta). Planta 212: 454-459[Medline]

Vanlerberghe GC, Schuller KA, Smith RG, Feil R, Plaxton WC, Turpin DH (1990) Relationship between NH₄⁺ assimilation rate and in vivo phosphoenolpyruvate carboxylase activity. Regulation of anaplerotic carbon flow in the green alga Selenastrum minutum. Plant Physiol 94: 284-290[Abstract/Free Full Text]

Villand P, Eriksson M, Samuelsson G (1997) Carbon dioxide and light regulation of promoters controlling the expression of mitochondrial carbonic anhydrase in Chlamydomonas reinhardtii. Biochem J 327: 51-57[Medline]

Villarejo A, Rolland N, Martinez F, Sültemeyer DF (2001) A new chloroplast envelope carbonic anhydrase activity is induced during acclimation to low carbon dioxide concentrations in Chlamydomonas reinhardtii. Planta 213: 286-295[CrossRef][Medline]

Villarejo A, Shutora T, Moshkin O, Forssén M, Klimov VV, Samuelsson G (2002) A photosystem II-associated carbonic anhydrase regulates the efficiency of photosynthetic oxygen evolution. EMBO J 21: 1930-1938[CrossRef][Web of Science][Medline]

Weger HG, Chadderton AR, Lin M, Guy RD, Turpin DH (1990) Cytochrome and alternative pathway respiration during transient ammonium assimilation by N-limited Chlamydomonas reinhardtii. Plant Physiol 94: 1131-1136[Abstract/Free Full Text]

Wilbur KM, Anderson NG (1948) Electrometric and colorimetric determination of carbonic anhydrase. J Biol Chem 176: 147-154[Free Full Text]

Wurtele ES, Nikolau BJ (1992) Differential accumulation of biotin enzymes during carrot somatic embryogenesis. Plant Physiol 99: 1699-1703[Abstract/Free Full Text]

Yokota A, Iwaka T, Miura K, Wadano A, Kitaoka S (1987) Model for the relationship between CO₂-concentrating mechanism, CO₂ fixation, and glycolate synthesis during photosynthesis in Chlamydomonas reinhardtii. Plant Cell Physiol 28: 1363-1376[Abstract/Free Full Text]

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