Planta (1994)192:46-51

Pl~.tl'lt)~ ?) Springer-Verlag1994

Effect of vanadate on photosynthesis and the ATP/ADP ratio in low-CO2-adapted Chlamydomorms reinhardtii cells Jan Karlsson 1, Ziyadin Ramazanov 2, Thomas Hiltonen 1, Per GardestriJm 1, G~iran Samuelsson 1 Department of Plant Physiology,Universityof UmeA,S-90187 Ume~, Sweden 2 Department of Botany, LouisianaState University, Baton Rouge, LA 70803, USA Received: ! February 1993/ Accepted: 12 June 1993

Abstract. We have assessed the effect of vanadate as an inhibitor of plasma-membrane ATPase on photosynthesis and the ATP/ADP ratio in Chlamydomonas reinhardtii CW-92 (a mutant strain lacking a cell wall). This effect was compared in low-CO2-adapted cells grown in media bubbled with air containing 400 or 70 g L . L CO 2. Evidence is presented indicating that cells grown at 70 laL'L J CO2 have a higher rate of photosynthetic 02 evolution than cells grown at 400 ~tL. L-1 CO2, at limiting carbon concentrations. Extracellular and intracellular carbonic-anhydrase activities were, however, similar in cells grown in both of the low-carbon conditions. Vanadate inhibited, to a different extent, the HCO3-dependent 02 evolution in cells grown at 400 and 70 gL. L ~CO2. At 400 gM vanadate, inhibition reached 70-75% in cells grown at 400 g L . L 1 but only 50% in those grown at 70 p L . L i CO2. The ATP/ADP ratios determined with and without vanadate at limiting concentrations of dissolved inorganic carbon indicated that more ATP was hydrolysed in algae grown at 70 pL. L than in those grown at 400 gL.L i CO2. We conclude that the maximal capacity to accumulate dissolved inorganic carbon is inversely related to the CO2 concentration in the medium. Activation and or synthesis of vanadate-sensitive ATPase may be the major explanation for the higher capacity for HCO3-dependent O2 evolution in cells grown under limited CO2 concentrations. Key words: ATPase - Carbon concentrating mechanism - Carbonic anhydrase - Chlamydomonas - Photosynthesis

Introduction Chlamydomonas reinhardtii, as well as many other species of microalgae and cyanobacteria, has an active mechan-

Abbreviations: CA=carbonic anhydrase; DIC=dissolved inorganic carbon Correspondence to: G. Samuelsson; FAX: 46(90)166676

ism for the transport/accumulation of dissolved inorganic carbon (DIC) when growing under limited CO2 concentrations (e.g. in media bubbled with air; Badger et al. 1980; Bedu et al. 1989; Siiltemeyer et al. 1990; Moroney and Mason 1991). Microalgal cells induce this mechanism after transfer from a high (5% CO2 in air) to a low CO2 (0.03% CO2 in air) concentration. An increased affinity for CO2 is often seen within 1-5 h (depending on experimental conditions) after transfer of C. reinhardtii to low-DIC conditions, with K1/2~co2) ranging from 0.2 to 5 laM (Aizawa and Miyachi 1984; Badger 1987; Palmqvist et al. 1990a; Moroney and Mason 1991). The Km for ribulaze-l,5-biphosphate carboxylase-oxygenase (Rubisco) isolated from C. reinhardtii was reported to be 25 ~tM (Berry et al. 1976). This active transport mechanism allows an accumulation of DIC inside the cells, up to concentrations much higher than can be explained by passive diffusion only. The net effect at the metabolic level is a decreased rate of photorespiration and an increased affinity for DIC which, in turn, lead to a more efficient photosynthesis under conditions of low external DIC concentrations (Spalding et al. 1983). Carbonic anhydrase (CA), which catalyses the hydration/dehydration between CO2 and HCO~, has been shown to be involved in the DIC-concentrating mechanism (Findenegg 1976; Aizawa and Miyachi 1984; Moroney et al. 1985; Bedu et al. 1990; Samuelsson et al. 1990; Siiltemeyer et al. 1990; Palmqvist et al. 1990a, b, 1991; Moroney and Mason 1991). There are different isoforms of CA in algae (Husic et al. 1989; Rawat and Moroney 1991) and in higher plants (Burnell 1990). The best-characterised CA is an extracellular glycoprotein bound to the cell wall or plasma membrane by ionic interactions (Coleman and Grossman 1984; Geraghty et al. 1990; Husic and Quigley 1990). The involvement of CA in DIC transport has been studied by using the CA inhibitors acetazolamide and ethoxyzolamide (Moroney et al. 1985) and high-CO2-requiring mutants of C. reinhardtii (Spalding et al. 1983; Moroney et al. 1989). When bound to dextran, acetazolamide cannot penetrate the plasma membrane and selectively inhibits the extracellular CA (Moroney et al. 1985). It has been suggest-

J. Karlsson et al.: Effect of vanadate on photosynthesis and ATP/ADP ratio in C. reinhardtii

ed that the role of extracellular CA is to speed up the conversion of HCO~ to COz in the extracellular space; CO2 can then either diffuse (Moroney and Mason 1991 ; Ramazanov and Cardenas 1992) or be actively transported into the cells (Siiltemeyer et al. 1990). In green algae, several authors have come to the conclusion that either HCO3 (Beardall 1981; Beardall and Raven 1981; Marcus et al. 1984) or both CO2 and HCO3 (Williams and Turpin 1987; Siiltemeyer et al. 1988, 1990) are actively transported across the algal plasma membrane and the chloroplast envelope. Information regarding intracellular CA is less precise. The enzyme is present in the cytosol (Ramazanov and Cardenas 1992) and in the chloroplast of both algae (Siiltemeyer et al. 1990; Coleman et al. 1991; Ramazanov and Cardenas 1992) and higher plants (Bumell 1990). There is also a low-C02inducible 36-kDa protein in the chloroplast envelope of C. reinhardtii cells growing at low DIC concentrations (Mason et al. 1990; Ramazanov et al. 1993). Activity of CA has also been detected in isolated chloroplasts from C. reinhardtii (Sfiltemeyer et al. 1990) and in Dunaliella salina (Ramazanov and Cardenas 1992), but the molecular structure of CA is still unknown. However, CA activity cannot fully explain the accumulation that takes place under conditions of low external DIC. A mutant strain (pmp-1) of C. reinhardtii that requires high CO2 for growth and cannot induce DIC accumulation, induces CA activity under low-DIC conditions (Spalding et al. 1983). It has further been proposed that a pumping mechanism is required to transport DIC against a concentration mechanism (Spalding et al. 1983; Spalding and Ogren 1983; Goyal and Tolbert 1989; Thielman et al. 1990; Moroney and Mason 1991). Furthermore, the transport of DIC requires ATP (Raven and Lucas 1985). A specific protein for the transport of DIC has not yet been identified and there is some controversy about the localisation of this possible transporter (Marcus et al. 1984; Goyal and Tolbert 1989). The requirement of a plasma-membrane ATPase for the transport and assimilation of CO2 in Scenedesmus obliquus, C. reinhardtii and D. salina has been demonstrated in cells growing under low DIC concentrations (Palmqvist et al. 1988; Goyal and Tolbert 1989; Thielman et al. 1990), although the mechanism by which this ATPase is involved in DIC transport is still unknown. In this paper we have extended previous work (Samuelsson et al. 1990; Palmqvist et al. 1990a) concerning the differences in HCO3-dependent photosynthetic 02 evolution between cells adapted to low CO2 (400 gL 9L-1 CO2 in air) and very low COz (70 ~tL 9 L-1 C O / i n air) concentrations. We found that there was a difference in the efficiency of DIC uptake at very low external CO/ concentrations between cells grown at 400 and 70 ~tL 9 L- ~ COz. Measurement of CA activities suggested that these were not responsible for the different rates of DIC uptake. We therefore concluded that the different rates of photosynthesis were mainly dependent on differences in pumping capacity. In this paper we have investigated this hypothesis further.

47

Materials and methods Culture conditions. The cell-wall-less mutant strain (CW-92) of Chlamydomonas reinhardtii, kindly donated by Dr. D.D. Kaska (Dept. of Biol. Sci., University of California, Santa Barbara, Cal. USA), was grown in continuous light with an incident photosynthetic photon flux density (PPFD) of 1301amol "m - 2 ' s -1, obtained from fluorescent tubes (TL 40W/55; Philips, Eindhoven, The Netherlands), in a medium buffered with potassium phosphate (0.1 M, pH 7.5 at 26 ~ C). The medium used was that of Solter and Gibor (1977) for macronutrients and it was supplemented with micronutrients as in Surzycki (1971). Fresh cultures were inoculated from algae axenically grown on agar slants. Media were gently stirred, bubbled with air containing 5 - 104 laL" L -1 CO2~gl in air (5%), (AGA Specialgas AB, Skellefte~t, Sweden) in 1-L glass vials and maintained in the logarithmic growth phase by daily dilution with fresh medium. Light was measured with a quantum sensor (Li-Cor, Lincoln, Neb., USA).

Adaptation of algae to low DIC concentrations. High-DIC-grown cells were harvested by centrifugation at 1000-9' for 2 min and resuspended to 2-4 lag C h l - m L ~ in a low-DIC-equilibrated medium, either bubbled with ambient air (350-400 laL 9 L 1 CO2~g) (400laL- L 1)) or with air containing < 1 0 0 l a L - L -1 CO2(g ) (70laL" L - l ) . The gas flow was kept at a high rate (between 1500-2000 laL. min -~) to minimise Oz accumulation in the vials and to favour an equilibrium concentration of DIC. This flow rate was high enough to reach equilibrium of DIC in the medium, as confirmed by mass-spectrometric measurements (K. Palmqvist, Department of Plant Physiology, University of Ume~., personal communication). Air containing < 100 laL. L 1 CO2~g~was obtained by mixing two streams of air, one with ambient air and one with COz-free air, in a ratio 1:4. Chlorophyll determination. Chlorophyll was extracted in hot methanol and the concentration was calculated using the absorption coefficient of MacKinney (1941). Determination of CA activity. Algae were harvested by centrifugation at 300"9 for 10 min and pelleted cells were resuspended in ice-cold CA buffer [30 mM Epps (hydroxyethyl piperazine propanesulfonic acid) buffer, pH 8.2, containing 1 mM MgCI 2 and 1 mM EDTA], to a chlorophyll concentration of 20 lag Chl. L -1. To remove extracellular CA, pelleted cells were washed once as described by Williams and Turpin (1987) and centrifuged as above. Pelleted intact cells without measurable CA activity were resuspended in 10 mL of CA buffer and disrupted with a Parr bomb (3.5 MPa at a flow rate of 2 m L . min-1), and the homogenate was used to measure the activity of intracellular CA. Carbonic anhydrase (EC 4.2.1) activity was determined in CA buffer by determining the time taken for the pH to drop from 8.2 to 7.4 at 2~ C in a 2-mL sample. The reaction was started by rapid injection of 2 mL of ice-cold CO2-saturated distilled H20 (Ramazanov and Cardenas 1992). One unit of enzymatic activity (Wilbur Anderson, WA, unit) is defined as 10 ((To/T~I), where To and T are the times (s) for the pH change to occur in the nonenzymatic and enzymatic reactions, respectively.

Measurement of photosynthetic 0 2 evolution. Photosynthesis was measured on l-mL algal samples with an oxygen electrode (Hansatech King's Lynn, Norfolk, UK). Algae were transferred to Eppendorf centrifugation tubes (1.5 mL) and spun down during an approx 20-s centrifugation. Pelleted algae were resuspended in 1 mL of a 20 mM Hepes-KOH buffer, pH 8.0 (3-5 lag Chl 9m L - 1) and transferred to the electrode, where they were allowed to consume the inorganic carbon of the buffer and the intracellular pool of DIC until no net photosynthesis was observed. Bicarbonate and vanadate were added when net 02 evolution had levelled off. The PPFD was 400 lamol 9m - 2. s- 1. Orthovanadate was prepared as reported by Goyal and Tolbert (1989). Further details are given in the figures.

48

J. Karlsson et al.: Effect of vanadate on photosynthesis and A T P / A D P ratio in C. reinhardtii

Chloroplast isolation. One litre of exponentially growing cells was harvested by centrifugation at 4000- 9 for 5 rain at room temperature. Cells were washed with 20 mM Hepes-KOH (pH 7.5), centrifuged as above and the pellet resuspended in 30 mL of ice-cold breaking buffer [50 mM Hepes-KOH, pH 7.2; 300 mM sorbitol; 2mM EDTA; 1 mM MgC12; 1% (w/v) bovine serum albumin (BSA); Togasaki et al. 1987]. Cells were then transferred to a precooled Yeda Press bomb (Yeda Research and Development Co., Rehovot, Israel), equilibrated at 1.0-1.5 MPa (nitrogen gas) for 4 rain. The lysate was let out at an even flow of about 2 mL 9s- 1. After centrifugation at t000"g for 30 s, the pellet was resuspended in the disruption buffer and was layered onto a discontinuous Percoll gradient consisting of 45 and 70% Percoll solution [Percoll; 3% polyethyleneglycol (PEG) 6000; 5% Ficoll; 1% BSA] in a buffer (45 mM Hepes-KOH, pH 6.8; 300 mM sorbitol; 0.9 mM Na,,P207; 1.8 mM EDTA; 0.9 mM MgC12; 0.9 mM MnCI2; Goldschmidt-Clermont et al. 1989). Ascorbate and dithiothreitol (DTT) were added to the gradient solution to a final concentration of 5 mM each. The gradient was centrifuged using 50-mL Falcon tubes in a HERMLE (Gosheim, FRG) swing-out rotor at 3700 90, 0~ C for 20 min. After centrifugation, the chloroplast fraction was collected from the 45-70% interface and diluted fourfold with disruption buffer. Chloroplasts were sedimented by allowing the rotor to speed up to 500 99 and then stopping the run. The pellet was washed once in breaking buffer, resuspended in 1-5 mL of storage buffer (50 mM Hepes-KOH, pH 8.0; 300 mM sorbitol) and stored on ice. Measurements of ATP/ADP. Aliquots (0.5 mL) of cells from the different growth conditions (400 and 70 IlL 9 L-1 CO2), and with a chlorophyll concentration between 4-5 lag-mL 1, were incubated in light in a water bath at 25 ~ C. After 5 rain of illumination at 400 lamol" m - 2 - s - 1, vanadate and-or bicarbonate was added and the illumination was continued for another 5 min after which 0.5 mL of 6 M perchloric acid was added to kill the cells. The perchloric acid was always added during illumination since very quick fluctuations in A T P / A D P ratios can occur when the light is turned off ( G a r d e s t r r m and Wigge 1988). The samples were frozen and stored in the freezer up to one week before measurements of adenylates. After thawing, the samples were neutralised (3 M KOH, 0.2 M Hepes) immediately before measurement of adenylates. This was done by the firefly luciferase method essentially as described earlier (Gardestr6m and Wigge 1988).

Results

Photosynthesis of C. reinhardtii cells 9rown at 400 and 70 HL 9 L - ~ C02. Figure 1 shows the D I C - d e p e n d e n t 0 2 evolution in cells g r o w n at 400 and 70 g L 9 L - 1 CO2. Cells g r o w n at 70 g L 9 L - 1 CO2 d e m o n s t r a t e d a higher affinity for D I C than those g r o w n at 400 p L 9 L - ~ CO2. The p h o t o s y n t h e t i c response was c o m p a r e d at different concentrations o f D I C f r o m 10 g M to 4 m M at p H 8.0. At low D I C concentrations the cells g r o w n at 70 gL 9 L - 1 CO2 exhibited a higher rate o f p h o t o s y n t h e t ic 0 2 evolution than those g r o w n at 400 pL 9 L - ~ CO2, but at higher concentrations o f D I C this difference was reduced. This is in a c c o r d a n c e with the data o f Samuelsson et al. (1990) and Palmqvist et al. (1990a), a l t h o u g h the difference in affinities for CO2 between the two types o f cell was larger in previous work.

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is dependent not only on the c o n c e n t r a t i o n o f CO2 in the medium, but also on the CO2/O2 ratio ( R a m a z a n o v and Semenenko 1988) as well as the light intensity (Aizawa

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grown in low-DIC medium equilibrated by bubbling with air containing 400 laL 9L- x (E3) or 70 laL 9L 1 (m) CO2. The irradiance during the measurement was 400 lamol 9m-2. s-1, the chlorophyll content was 3-5 lag 9mL-1 and the pH was 8.0. The bars indicate SD Table 1. Activities of CA (WA units 9mg Cht-:) in fractions containing a homogenate of washed C. reinhardtii ceils, intact chloroplasts or solubilized chloroplasts (0.05% Triton X-100) from algae grown at high (5 9 104 laL 9L- 1) and low (400 and 70 laL- L- 1 CO2) concentrations of DIC

Homogenate Intact chloroplasts Solubilized chloroplasts

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and Miyachi 1984; R a m a z a n o v and Semenenko 1988). The activity o f C A is higher under l o w - C O 2 conditions and u n d e r conditions o f a low CO2/O2 ratio than u n d e r h i g h - D I C conditions or a higher CO2/O2 ratio. The increase in C A activity is correlated with an increased affinity for CO2 in l o w - D I C - g r o w n cells (Aizawa and Miyachi 1984; Palmqvist et al. 1990a; Sfiltemeyer et al. 1990). The total C A activity in the h o m o g e n a t e and in the intact chloroplast fractions isolated f r o m C. reinhardtii C W - 9 2 cells g r o w n at 400 and 70 p.L 9 L - 1 CO2 was similar (Table 1), In b o t h types o f l o w - D I C - g r o w n cell the intracellular C A was higher then in the h i g h - D I C g r o w n cells. There was no measurable C A activity in intact washed cells (data not shown). Therefore, the higher C A activity in the h o m o g e n a t e o f b o t h types o f l o w - D I C - g r o w n cell is due to increased intracellular CA. C a r b o n i c - a n h y d r a s e activity increased in the chloroplasts during a d a p t a t i o n to l o w - D I C conditions, but there was no significant difference in the C A activities o f chloroplasts f r o m cells g r o w n at 400 or 70 pL 9 L - 1 CO2.

Effect of vanadate on photosynthesis.

Carbonic-anhydrase activity in cells 9rown at 400 and 70 IlL" L - 1 C02. It is well k n o w n that the activity o f C A

160

Inhibition o f A T P a s e activity by o r t h o v a n a d a t e reduced the D I C dependent O2 evolution c o m p a r e d to control cells (Fig. 2). The effect o f v a n a d a t e was inversely to the c o n c e n t r a t i o n o f D I C in the assay medium. Cells g r o w n at 400 pL 9 L - 1 CO2 were m o r e sensitive to v a n a d a t e than those g r o w n

J. Karlsson et al.: Effect of vanadate on photosynthesis and ATP/ADP ratio in C. reinhardtii 80

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1000 ' Vonodate (p.M) Fig. 2. Inhibition of O2 evolution by different concentrations of orthovanadate. Cells of C. reinhardtfi were grown at 400 (D) or 70 ( m ) g L . L -~ CO2. Prior to the measurements, 40 laM HCOs was added to the electrode chamber. Other conditions as in Fig 1. The bars indicate SD 0

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HCO~(~M) Fig. 3. Inhibition of 02 evolution as a function of the HCO~concentration at limiting (250 gM) orthovanadate concentration. Cells of C. reinhardtii were grown at 400 (n) or 70 (o) pL 9L -1 CO2. Other conditions as in Fig. 1. Representative results from three repeated experiments are shown

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Fig. 4. Ratios of A T P / A D P in C. reinhardtii cells grown at 70 or 400 p L - L -x CO2. Control cells were incubated in light with 100 pM HCO~- ([:3) added. Treated cells were incubated either with vanadate alone (1 raM) (~]) or with vanadate (1 mM) plus HCO~(100 pM) (11). The bars indicate SD

at 70 g L . L -1 C02 (Fig. 3). The inhibitory effect increased with increasing vanadate concentration.

Effect of vanadate and HCO~ on the A TP/ADP ratio. The ATP/ADP ratio of the total extract of C. reinhardtii was determined in the light (Fig. 4). The algae were first illuminated for 5 min without additions. Since no H C O ;

49

was added, the carbon in the medium was depleted during the illumination, as determined by photosynthetic 02 production (result not shown). After the initial 5 min illumination, vanadate and-or HCO3 was added and illumination was continued for 5 min before the cells were killed with perchloric acid. In the presence of HCO3, ATP/ADP ratios of 2.5 and 2.6 were obtained in cells grown at 70 and 400 gL 9L- 1 CO2, respectively. In cells grown at 70 gL 9L- 1 C02, vanadate together with HCO3 increased the ATP/ADP ratio to about 4.1 but this ratio was only 3.2 in cells grown in 400 gL 9L-1 C02. When only vanadate was added the ratios obtained were very similar to those found with vanadate plus HC03. Discussion Green algae have an active DIC-accumulating mechanism when grown under conditions of limiting carbon supply. However, no study has been undertaken to carefully correlate the extent of accumulation with the DIC concentration in the medium during growth. There is no reason to assume that there is a threshold DIC concentration from which induction of a full DICconcentrating mechanism suddenly starts. It has been suggested that there is a continuous activation of different components of the active DIC-accumulating mechanism (Badger 1987). In previous reports (Palmqvist et al. 1990a; Samuelsson et al. 1990), it has been shown that in cultures of C. reinhardtii grown under very low CO2 levels, the cellular affinity for DIC increased, as compared to cells grown in media in equilibrium with air. In this work we employed a conventional oxygen-electrode technique and maintained a constant pH of 8.0 using a Hepes-KOH buffer. As seen in Fig. 1, cells grown in media equilibrated with air containing 400 or 70 gL" L -1 CO2 have different affinities for DIC. Photosynthesis was measured at DIC concentrations up to 4 mM HCOs which is enough to fully equalise the differences in DIC affinity. One possible explanation of the differences in affinity for DIC between cells grown at 400 and 70 gL 9 L-1 CO2 is that it could be caused by differences in CA activity. Actually it has been shown that CA activity in Chlorella sp. depended on the Oz/CO2 ratio and that CA activity increased when this ratio was above 1 (Ramazanov and Semenenko 1984). In Table 1, the CA activity is shown for homogenates of cells grown at 5 9 104 pL 9L- 1, 400 and 70 p,L 9L- 1 CO2. Carbonic-anhydrase activity increased to a similar extent in cells grown at 400 and 7 0 p L . L -1 CO2 as compared with those grown at 5 9 104 pL 9L-~ CO2. The increase of CA activity in intact cells was relatively small, but it should be remembered that the cells were washed once in buffer before the experiment. It has been shown that the cell-wall-less mutant normally excretes extracellular CA into the growth medium (Coleman and Grossman 1984). Therefore, the relatively higher CA activity in cells grown at 400 and 70 gL 9L- 1 COz is due to increased intracellular CA activity. Interestingly, chloroplastic CA activity increased after transferring the

50

J. Karlsson et al.: Effect of vanadate on photosynthesis and ATP/ADP ratio in C. reinhardtii

cells to l o w - C O 2 conditions compared with CA in chloroplasts isolated from the cells grown in high-CO2 conditions, which agrees with the data obtained by Sfiltemeyer et al. (1990) for C. reinhardtii, Coleman et al. (1991) for Chlorella and Ramazanov and Cardenas (1992) for Dunaliella saling. It should be noted that there is also an increased intracellular non-chloroplastic CA activity. However, the intracellular CA activities were very similar in the 400- and the 70-1~L 9 L - 1 grown cells. Vanadate binds to ATPases and is known to inhibit the P-type ATPase activity of higher-plant and fungal plasma membranes (Serrano 1989). Since the permeation of vanadate through the membrane is slow, there is little inhibition of intracellular ATPases (Goyal and Tolbert 1989; Thielmann et al. 1990). Vanadate inhibition was dependent on the inhibitor concentration (Fig. 2) and, within the range of concentrations studied, the strongest effect was always seen in algae grown at 400 ktL 9 L CO2. For both growth conditions, the effect of the inhibitor was reduced when the HCO3 concentration was increased. To determine A T P / A D P ratios, the vanadate concentration used (1 raM) was high enough to cause 80% inhibition of the HCO3-stimulated O2 evolution in cells grown at both 400 and 70 laL 9 L -~ CO2 (see also Fig. 2). The increased A T P / A D P ratio after vanadate treatment indicates that the balance between ATP production and consumption is being affected. Generally, an inhibition of photosynthesis would give an increased A T P / A D P ratio. This was observed in cells grown at both 400 and 70 pL 9L-~ COz (Fig. 4). However, a considerably bigger increase in the A T P / A D P ratio was observed in algae adapted to 7 0 p L . L -z CO2 as compared to 400 ~ L . L -~ CO2. This might indicate that a bigger proportion of the cellular ATP consumption is occurring at the plasma membrane ATPase in cells grown at 70 p L . L-~ CO 2 than in those grown at 400 ~ L . L CO2. We could only determine the A T P / A D P ratio in the total cell extracts because, in contrast to protoplasts from higher plants (Gardestr6m and Wigge 1988), there is not a method available to rapidly separate subcellular fractions of algae. Thus, we can not determine whether the increase in the total A T P / A D P ratio reflects an increase in the chloroplast or in the cytosol. The big effect on the cellular A T P / A D P ratio might indicate that the DIC accumulation at the plasma membrane is an important energy-requiring step in the DIC-transporting mechanism. Based on the results in this work we therefore suggest that the different responses to DIC in C. reinhardtii CW-92 cells are caused by a difference in a pumping mechanism located in the plasma membrane. Probably, this mechanism is induced by low DIC concentration in the growth medium, because photosynthesis in high-DIC cells is not inhibited by vanadate (Thielman et al. 1990). However, differences in the chloroplast envelopes of cells grown at 400 or 70 I~L 9 L-~ CO2 cannot be excluded, since inhibition of intracellular ATPases is possible. Goyal and Tolbert (1989) demonstrated that intact chloroplasts isolated from low-DIC cells of Dunaliella exhibited the ability to transport HCO3 actively, but that this transport was suppressed by vanadate.

The mechanism by which the ATPase-dependent carbon transport across plasma membrane works is still unknown. In the light of our results, two possible explanations can be proposed: (i) either the pumping of protons following hydrolysis of ATP acidifies the surroundings of the membranes, thus increasing the local concentration of CO2 around the cell, or (ii) a proton gradient, via a secondary mechanism, coupled to DIC transport into the cells (Badger 1987). Thus, adaptation of algae to low-DIC conditions represent a process with a complex organisation. The mechanism includes induction of synthesis of extracellular and intracellular CAs and, probably, vanadate-sensitive ATPases. Inhibition of the activity of one step leads to suppression of the whole mechanism governing adaptation of the photosynthesizing cell to conditions of CO2 limitation. We are grateful to Prof. J Cardenas (Dept de Bioquimica y Biologia Moleculary Fisiologia, University of Cordoba, Spain) and Prof. J.V. Moroney (Dept of Botany, Louisiana State University, USA) for critically reading the manuscript. We are also grateful to Gunilla Malmberg for expert assistance in the measurements of ATP/ADP ratios and Dr. C. Santos (The University of Cordoba, Spain) for skiful secretarial assistance. This work was supported by the Swedish Natural Research Council, Seth M. Kempes Memorial foundation and the Swedish Institute.

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