The CO2 permeability of the plasma membrane of Chlamydomonas reinhardtii: mass-spectrometric 18O-exchange measurements from 13C18O2 in suspensions of carbonic anhydrase-loaded plasma-membrane vesicles

The unicellular green alga Chlamydomonas reinhardtii possesses a CO₂-concentrating mechanism. In order to measure the CO₂ permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO₂ · L?1} (high-C_i cells) or in ambient air (350 μL CO₂ · L?1}; low-C_i cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-C_i cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [¹⁴C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-C_i cells of C. reinhardtii were used to measure the exchange of ¹⁸O between doubly labelled CO₂ (¹³C¹⁸O₂) and H₂O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the ¹⁸O depletion from ¹³C¹⁸O₂ in the external medium provides a powerful tool to study CO₂ diffusion across the PM to the active site of CA which catalyses ¹⁸O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the ¹⁸O loss to H₂O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the ¹⁸O-depletion kinetics performed at pH 7.3 and 7.8, CO₂ permeability coefficients of 0.76 and 1.49·10?3} cm·s?1}, respectively, were calculated for high C_i cells. The corresponding values for low-C_i cells were 1.21 and 1.8·10?3} cm·s?1}. The implications of the similar and rather high CO₂ permeability coefficients (low CO₂ resistance) in high and low-C_i cells for the CO_i-concentrating mechanism of C. reinhardtii are discussed.     

Photosynthesis and inorganic carbon acquisition in the cyanobacterium Chlorogloeopsis sp. ATCC 27193

The ability of the morphologically complex cyanobacterium Chlorogloeopsis sp. ATCC 27193 to actively transport and accumulate inorganic carbon (C₁= CO₂+ HCO₃^?+ CO₃^2?) for photosynthetic CO₂ fixation was investigated. Mass-spectrometric assays revealed that Chlorogloeopsis cells grown under C₁ limitation rapidly took up CO₂ from the medium in a light-dependent reaction which was independent of CO₂ fixation. Ethoxyzolamide, a carbonic anhydrase (CA) inhibitor, inhibited CO₂ transport. Since electrometric and mass-spectrometric assays did not detect the presence of a periplasmic CA, it is suggested that CO₂ transport was mediated by a CA-like activity which converted CO₂ to HCO₃^? during passage across the membrane. Radiochemical assays, using H¹⁴CO₃ as substrate, showed that C₃-limited cells also had a high affinity (K_0.5 HCO₃^?= 37 μM), Na⁺-independent HCO₃^? uptake mechanism. HCO₃^?uptake was light dependent and occurred against its electrochemical potential indicating a carrier-mediated, active transport process. The rate of Na⁺-independent HCO₃^? transport was sufficient to account for the steady state rate of CO₂ fixation. Although not absolutely required. Na⁺ did specifically enhance the rate of HCO₃^? transport by up to 2-fold, but had no effect on the apparent affinity of the transport system for HCO₃^? Combined CO₂ and HCO₃^? transport resulted in C₁ accumulation as high as 25 mM and in excess of 300 times the external concentration. The C₁ pool was the source of CO₂ for photo-synthetic fixation and was generated, presumably, by the dehydration of HCO₃^? catalyzed by an intracellular CA. The collective evidence indicates that Chlorogloeopsis has a physiologically functional CO₂-concentrating mechanism which is essential for photosynthesis.     

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO₂ (high inorganic carbon, C_i) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-C_i grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO₂ supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-C_igrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-C_i cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-C_i algae. After that time their activities were similar to the corresponding CA values in low-C_i-grown cells. In contrast, plastids from high-C_i cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-C_i and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-C_i air-adapted and low-C_i cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.This work is dedicated to Professor A. Wild on the occasion of his 65th birthday     

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Mass-spectrometric measurements of ¹⁶O₂ and ¹⁸O₂ were made to compare the rates of light-dependent O₂ evolution and uptake by Chlamydomonas reinhardtii Dang. grown in air (0.035% CO₂; low-C_i cells) or CO₂-enriched air (5% CO₂; high-C_i cells) at pH 5.5 and 8.0. While at pH 5.5, no differences were observed in the isotopic O₂-gas exchange of high- and low-C_i cells, at pH 8.0 the rates of true O₂ evolution and uptake were considerably higher in low-C_i than in high-C_i cells. The enhanced rates of O₂ uptake and evolution by low-C_i cells were completely inducible within 6 h after transferring high-C_i cells to ambient air. At pH 8.0, O₂ uptake in the light was inhibited by 2 M 3-(3,4-dichlorophenyl)-1,1 dimethylurea in both types of alga, but this effect was more pronounced in low-C_i than in high-C_i cells.When the cells were grown at pH 5.5 the activities of the superoxide-radical-degrading enzymes, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were similar regardless of the CO₂ concentration provided during growth. At pH 8.0, however, the activities of these enzymes were 4 to 20 times higher in low-C_i than in high-C_i cells. When high-C_i cells were allowed to acclimate to ambient air for 6 h at pH 8.0, the activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate dehydrogenase increased to more than 50% of the level observed with low-C_i cells. These results are consistent with an enhanced operation of O₂ photoreduction which could provide energy to the inorganic-carbon-concentrating mechanism via pseudo-cyclic photophosphorylation.     

Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity

In high inorganic carbon grown (1% CO₂ [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO₂ uptake and to reduce the final internal inorganic carbon (C_i) pool size reached. The relationship between CO₂ fixation rate and internal C_i concentration in high C_i grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High C_i grown cells readily took up CO₂ but had little or no capacity for HCO₃⁻ uptake. These cells appear to possess a CO₂ utilizing C_i pump that has a CA-like function associated with the transport step such that HCO₃⁻ is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low C_i grown cells possess both CO₂ uptake and HCO₃⁻ uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO₂ and HCO₃⁻ uptake (such as the C_i pump) may have been affected. The inhibitor had no apparent effect on internal CO₂/HCO₃⁻ equilibria (internal CA function) in low C_i grown cells.     

Photorespiratory ammonium assimilation in chloroplasts of Chlamydomonas reinhardtii

The effect of CO₂ concentration on the rate of photorespiratory ammonium excretion and on glutamine synthetase (GS) and carbonic anhydrase (CA) isoenzymes activities has been studied in Chlamydomonas reinhardtii cw-15 mutant (lacking cell wall) and in the high CO₂-requiring double mutant cia-3/cw-15 (lacking cell wall and chloroplastic carbonic anhydrase). In cw-15 cells, both the extracellular (CA_ext) and chloroplastic (CA_chl) CA activities increased after transferring cells from media bubbled with 5% CO₂ in air (v/v, high-C_i cells) to 0.03% CO₂ (low-C_i cells), whereas in cia-3/cw-15 cells only the CA_ext was induced after adaptation to low-C_i conditions and the CA_chl activity was negligible. During adaptation to low-C_i conditions in the presence of 1 mM of l-methionine-D,L-sulfoximine (MSX), a specific inhibitor of GS activity, both mutant strains excreted photorespiratory ammonium into nitrogen free medium. In addition, the ammonium excretion rate by cw-15 in the presence of MSX was lower in cells grown and kept at 5% CO₂ than in high-C_i cells adapted to 0.03% CO₂. The double mutant cia-3/cw-15 excreted photorespiratory ammonium at a higher rate than did cw-15. Total GS activity (GS-1 plus GS-2) increased during adaptation to 0.03% CO₂ in both strains of C. reinhardtii. However, only the activity GS-2, which is located in the chloroplast, increased during the adaptation to low CO₂, whereas the cytosolic GS-1 levels remained similar in high and low-C_i cells. We conclude that: (1) cia-3/cw-15 cells lack chloroplastic CA activity; (2) in C. reinhardtii photorespiratory ammonium is refixed in the chloroplasts through the GS-2/GOGAT cycle; and (3) chloroplastic GS-2 concentration changes in response to the variation of environmental CO₂ concentration.     

The induction of inorganic carbon transport and external carbonic anhydrase in Chlamydomonas reinhardtii is regulated by external CO2 concentration

Induction of the carbon concentrating mechanism (CCM) has been investigated during the acclimation of 5% CO₂‐grown Chlamydomonas reinhardtii 2137 mt + cells to well‐defined dissolved inorganic carbon (C_i) limited conditions. The CCM components investigated were active HCO₃^? transport, active CO₂ transport and extracellular carbonic anhydrase (CA_ext) activity. The CA_ext activity increased 10‐fold within 6 h of acclimation to 0·035% CO₂ and there was a further slight increase over the next 18 h. The CA_ext activity also increased substantially after an 8 h lag period during acclimation to air in darkness. Active CO₂ and HCO₃^? uptake by C. reinhardtii cells were induced within 2 h of acclimation to air, but active CO₂ transport was induced prior to active HCO₃^? transport. Similar results were obtained during acclimation to air in darkness. The critical C_i concentrations effecting the induction of active C_i transport and CA_ext activity were determined by allowing cells to acclimate to various inflow CO₂ concentrations in the range 0·035–0·84% at constant pH. The total C_i concentration eliciting the induction and repression of active C_i transport was higher during acclimation at pH 7·5 than at pH 5·5, but the external CO₂ concentration was the same at both pHs of acclimation. The concentration of external CO₂ required for the full induction and repression of C_i transport and CA_ext activity were 10 and 100 μM , respectively. The induction of CA_ext and active C_i transport are not correlated temporally, but are regulated by the same critical CO₂ concentration in the medium.     

Intracellular carbonic anhydrase activities in Dunaliella tertiolecta (Butcher) and Chlamydomonas reinhardtii (Dangeard) in relation to inorganic carbon concentration during growth: further evidence for the existence of two distinct carbonic anhydrases associated with the chloroplasts

Using mass-spectrometric measurements of ¹⁸O exchange from ¹³C¹⁸O₂ intracellular carbonic anhydrase (CA) activity was investigated in the unicellular green algae Dunaliella tertiolecta and Chlamydomonas reinhardtii which were either grown on air enriched with 5% CO₂ (high-C_i cells) or on air (low-C_i cells). In D. tertiolecta high- and low-C_i cells had detectable levels of internal CA activity when measured under in-vivo conditions and this activity could be split up into three distinct forms. One CA was not associated with the chloroplasts, while two isozymes were found to be located within the plastids. The activities of all intracellular CAs were always about twofold higher in low than in high-C_i cells of D. tertiolecta and the chloroplastic enzymes were completely induced within 4 h of adaptation to air. One of the chloroplastic CAs was found to be soluble the other was insoluble. In addition to the physical differences, MgSO₄ in vitro caused a more than twofold stimulation of the soluble activity while the insoluble form of CA remained rather unaffected. In C. reinhardtii, MgSO₄ increased the soluble CA activity by 346% and the concentration of MgSO₄ required for half-maximum stimulation was between 10 and 15 mM. Again, the insoluble CA activity was not affected by MgSO₄. Furthermore, the soluble isoenzyme was considerably more sensitive to ethoxyzolamide, a potent inhibitor of CA, than the insoluble enzyme. The concentration of inhibitor causing 50% inhibition of soluble CA activity was 110 and 85 μM ethoxyzolamide for D. tertiolecta and C. reinhardtii, respectively. From these data we conclude that the two chloroplast-associated CAs are distinct enzymes.     

Effect of external CO2 concentrations on protein synthesis in the green algae Scenedesmus obliquus (Turp.) Kütz and Chlorella vulgaris (Kosikov)

Unicellular algae grown under low-CO₂ conditions (0.03% CO₂) have developed a means of concentrating CO₂ at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Cells with the CO₂-concentrating mechanism (CCM) acquire the ability to accumulate inorganic carbon to a level higher than that obtained by simple diffusion. To identify proteins which are involved in the organization of the CCM, cells of Scenedesumus obliquus and Chlorella vulgaris grown in high CO₂ (5% CO₂ in air) were transferred to low-CO₂ (0.03%) conditions in the presence of ³⁵SO _inf4 ^sup2? and, thereafter, polypeptides labeled with ³⁵S were detected. Under low-CO₂ conditions the inducton of 36-, 39-, 94- and 110- to 116kDa polypeptides were particularly observed in S. obliquus and 16-, 19-, 27-, 36-, 38- and 45-kDa polypeptides were induced in C. vulgaris. Western blots with antibodies raised against 37-kDa subunits of the periplasmic carbonic anhydrase (CA) of Chlamydomonas reinhardtii showed immunoreactive bands with the 39-kDa polypeptide in the whole-cell homogenates from S. obliquus and with 36 and 38-kDa polypeptides in both high- and low-CO₂grown cells of C. vulgaris. Anti-pea-chloroplast CA antibodies cross-reacted with a single polypeptide of 30 kDa in the whole-cell homogenates but not with thylakoid membranes. The CA activity was associated with soluble and membrane-bound fractions, except thylakoid membranes.     

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

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

Effects of CO2 concentrations on the freshwater microalgae, Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus (Chlorophyta)

In order to investigate the possible impacts of increased atmospheric CO₂ levels on algal growth and photosynthesis, the influence of CO₂ concentration was tested on three planktonic algae (Chlamydomonas reinhardtii, Chlorella pyrenoidosa, and Scenedesmus obliquus). Increased CO₂ concentration enhanced significantly the growth rate of all three species. Specific growth rates reached maximal values at 30, 100, and 60 M CO₂ in C. reinhardtii, C. pyrenoidosa, and S. obliquus, respectively. Such significant enhancement of growth rate with enriched CO₂ was also confirmed at different levels of inorganic N and P, being more profound at limiting levels of N inC. pyrenoidosa and P in S. obliquus. The maximal rates of net photosynthesis, photosynthetic efficiency and light-saturating point increased significantly (p < 0.05) in high-CO₂-grown cells. Elevation of the CO₂ levels in cultures enhanced the photoinhibition of C. reinhardtii, but reduced that of C. pyrenoidosa and S. obliquus when exposed to high photon flux density. The photoinhibited cells recovered to some extent (from 71% to 99%) when placed under dim light or in darkness, with better recovery in high-CO₂-grownC. pyrenoidosa and S. obliquus. Although pH and pCO₂ effects cannot be distinguished from this study, it can be concluded that increased CO₂ concentrations with decreased pH could affect the growth rate and photosynthetic physiology of C. reinhardtii, C. pyrenoidosa, and S. obliquus.     

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O₂ evolution, gross CO₂ uptake and net HCO _inf3 ^su– uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO₂ concentrations. Regardless of the CO₂ concentration during growth, all cells had the capacity to transport both CO₂ and HCO _inf3 ^su– ; however, the activity of HCO _inf3 ^su– transport was more than twofold higher than CO₂ transport even in cyanobacteria grown at high concentration of inorganic carbon (C_i = CO₂ + HCO _inf3 ^su– ). In low-C_i cells, the affinities of CO₂ and HCO _inf3 ^su– transport for their substrates were about 5 (CO₂ uptake) and 10 (HCO _inf3 ^su– uptake) times higher than in high-C_i cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated C_i pool reached 18, 32 and 55 mM in high-C_i, air-grown and low-C_i cells, respectively, when measured at 1 mM external C_i. Photosynthetic O₂ evolution, maximal CO₂ and HCO _inf3 ^su– transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO₂ provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO₂ concentrations. Transport studies with high-C_i cells revealed that CO₂ and HCO _inf3 ^su– uptake were equally inhibited when CO₂ fixation was reduced by the addition of glycolaldehyde. In contrast, in low-C_i cells steady-state CO₂ transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO₂ and HCO _inf3 ^su– uptake as well as O₂ evolution in both cell types. In high-C_i cells, the degree of inhibition was similar for HCO _inf3 ^su– transport and O₂ evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO₂ was much more sensitive to the inhibitor than HCO _inf3 ^su– transport, with an apparent I₅₀ value of around 250 M ethoxyzolamide for CO₂ uptake. The implications of our results are discussed with respect to C_i utilisation in the marine Synechococcus strain.Abbreviations Chl chlorophyll - C_i inorganic carbon (CO₂ + HCO _inf3 ^su– ) - CA carbonic anhydrase - CCM CO₂-concentrating mechanism - EZA ethoxyzolamide - GA glycolaldehyde - K_1/2 concentration required for half-maximal response - Rubisco ribulose-1,5,-bisphosphate carboxylase-oxygenase D.S. is a recipient of a research fellowship from the Deutsche Forschungsgemeinschaft (D.F.G.). In addition, we are grateful to Donald A. Bryant, Department of Molecular and Cell Biology and Center of Biomolecular Structure Function, Pennsylvania State University, USA, for sending us the wild-type strain of Synechococcus PCC7002.     

CO2 uptake and transport in leaf mesophyll cells

Abstract The acquisition of inorganic carbon for photosynthetic assimilation by leaf mesophyll cells and chloroplasts is discussed with particular reference to membrane permeation of CO₂ and HCO^?₃. Experimental evidence indicates that at the apoplast pH normally experienced by leaf mesophyll cells (pH 6–7) CO₂ is the principal species of inorganic carbon taken up. Uptake of HCO^?₃ may also occur under certain circumstances (i.e. pH 8.5), but its contribution to the net flux of inorganic carbon is small and HCO^?₃ uptake does not function as a CO₂-concentrating mechanism. Similarly, CO₂ rather than HCO^?₃ appears to be the species of inorganic carbon which permeates the chloroplast envelope. In contrast to many C₃ aquatic plants and C₄ plants, C₃ terrestrial plants lack specialized mechanisms for the acquisition and transport of inorganic carbon from the intercellular environment to the site of photosynthetic carboxylation, but rely upon the diffusive uptake of CO₂.     

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

The role of extracellular carbonic anhydrase (CA_ex) for dissolved inorganic carbon (DIC) accumulation in the green alga Chlamydomonas reinhardtii was investigated. It was found that when algal cells were bubbled with ambient air, cell-wall-less mutant cells exhibited the same high photosynthetic affinity for CO₂ as wild-type cells despite a 10 times lower activity of CA^ex. It was also found that the affinity for CO₂ was further increased when the total DIC concentration of the algal medium was reduced from that in equilibrium with ambient air to even lower levels. This increased affinity was not correlated with any further increase in the CA_ex activity. Dextran-bound sulfonamide (DBS. 100 μM bound ligand) completely inhibited the activity of CA_ex in intact, low-DIC grown, wild-type cells, while photosynthesis at <2 μM CO₂(aq) proceeded at a far greater rate than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO^?₃. DBS-inhibition of CA_ex, during the induction of the DIC-accumulating mechanism in previously high-DIC grown cells, only caused a 50% inhibition of photosynthesis at 10 μM CO₂(aq) after 1 h of low-DIC acclimation. It was also shown that 50 μM acetazolamide (AZ) inhibited photosynthesis at low DIC concentrations to a relatively higher degree than DBS, suggesting that AZ inhibited intracellular CA as well. Taken together, these results suggest that low-DIC grown cells of C. reinhardtii have the ability to transport HCO^?₃ across the plasma membrane in addition to the CA_ex-mediated, facilitated diffusion and/or transport of CO₂. It is also suggested that the relative importance of these two fluxes (CO₂ or HCO^?₃) is dependent on the growth and experimental conditions. Facilitated CO₂ uptake seems to be most prevalent, supported by HCO^?₃-transport under more or less extreme situations, such as a reduction of CO₂ to extremely low concentrations, leakage of CA_ex to the medium as in cultures of cell-wall-less mutant cells or when the activity of CA_ex has been artificially inhibited.     

The active uptake of carbon dioxide by the marine diatoms Phaeodactylum ticornutum and Cyclotella sp.

Mass spectromelry has been used to investigate the uptake of CO₂ by two marine diatoms, Phaeodactylum tricornutum and Cyclotella sp. The time course of CO₂ formation in the dark after addition of 100 mmol m^?3 dissolved inorganic carbon (DIC) to cell suspensions showed that external carbonic anhydrase (CA) was not present in cells of P. tricornutum but was present in Cyclotella sp. In the absence of external CA, or when it was inhibited by 5 mmol m^?3 acetazolamide, cells of both species preincubated with 100 mmol m^?3 DIG rapidly depleted almost all of the free CO₂ (3·2mmol m^?31 at pH7·5) from the suspending medium within seconds of illumination and prior to the onset of steady-state photosynthesis. Addition of bovine CA quickly restored the HCO₃^?–CO₂ equilibrium in the medium, indicating that the initial depletion of CO₂ resulted from the selective uptake of CO₂ rather than uptake of all DIG species. Transfer of cells to the dark caused a rapid increase in the CO₂ concentration in the medium, largely as a result of the efflux of unfixed inorganic carbon from the cells. The measured CO₂ uptake rates for both species accounted for 50% of the total DIG uptake at HCO₃^?–CO₂ equilibrium, indicating that HCOHCO₃^? was also being taken up. These results indicate that both Phaeodactylum tricornutum and Cyclotella sp. have the capacity to transport CO₂ actively against concentration and pH gradients.     

LCI1, a Chlamydomonas reinhardtii plasma membrane protein,functions in active CO2 uptake under low CO2

In response to high CO₂ environmental variability, green algae, such as Chlamydomonas reinhardtii, have evolved multiple physiological states dictated by external CO₂ concentration. Genetic and physiological studies demonstrated that at least three CO₂ physiological states, a high CO₂ (0.5–5% CO₂), a low CO₂ (0.03–0.4% CO₂) and a very low CO₂ (< 0.02% CO₂) state, exist in Chlamydomonas. To acclimate in the low and very low CO₂ states, Chlamydomonas induces a sophisticated strategy known as a CO₂‐concentrating mechanism (CCM) that enables proliferation and survival in these unfavorable CO₂ environments. Active uptake of C_i from the environment is a fundamental aspect in the Chlamydomonas CCM, and consists of CO₂ and HCO₃^– uptake systems that play distinct roles in low and very low CO₂ acclimation states. LCI1, a putative plasma membrane C_i transporter, has been linked through conditional overexpression to active C_i uptake. However, both the role of LCI1 in various CO₂ acclimation states and the species of C_i, HCO₃^– or CO₂, that LCI1 transports remain obscure. Here we report the impact of an LCI1 loss‐of‐function mutant on growth and photosynthesis in different genetic backgrounds at multiple pH values. These studies show that LCI1 appears to be associated with active CO₂ uptake in low CO₂, especially above air‐level CO₂, and that any LCI1 role in very low CO₂ is minimal.     

Influx and efflux of inorganic carbon in Synechococcus UTEX625

The CO₂ and HCO₃^? fluxes in air-grown cells of Synechococcus UTEX 625 al pH 8-0 were measured during dark to light and light to dark transitions using a mass spectrometer and sampling of the reaction medium. The kinetic parameters for initial uptake of CO₂ and HCO₃^? were determined during the initial period of illumination. The development of the internal C_i pool was followed up to steady-state photosynthesis, which occurred when the size of the internal inorganic carbon pool remained apparently constant for a limited period. The experimental procedure confirmed that only CO₂ transport occurred with 100mmolm^?3 Na⁺ and that both CO₂ and HCO^?3 transport occurred with 25molm^?3 Na⁺. The K_1/2 values of initial CO₂ and HCO₃ uptake were 0.7 and 17.2 mmolm^?3respectively and agreed closely with the K_1/2 values of net CO₂ and HCO₃^? transport during steady-state photosynthesis, which were 0.66 and 17.1 mmolm^?3 respectively. Maximum rates of CO₂and HCO₃^? transport were 423 and 219mmolh^?1 g^?1 Chl. Maximum CO₂ efflux observed upon darkening was 118mmolh^?1 g^?1 Chl. A permeability coefficient of the cell for CO₂ of 3 × 10^?8 m s^?1 was determined from the dark CO₂ efflux assuming an internal pH of 7.2 in the dark. Following the initial CO₂ uptake in the light, the extracellular [CO₂] steadily declined when only CO₂ transport was allowed, but an increase in the extracellular [CO₂] when HCO₃^? transport was allowed to proceed suggested that an enhanced CO₂ efflux occurred as a result of the larger size of the intracellular C_i pool.     

Utilization of inorganic carbon in the edible cyanobacterium Ge-Xian-Mi (Nostoc) and its role in alleviating photo-inhibition

The present work investigated the inorganic carbon (C_i) uptake, fluorescence quenching and photo‐inhibition of the edible cyanobacterium Ge‐Xian‐Mi (Nostoc) to obtain an insight into the role of CO₂ concentrating mechanism (CCM) operation in alleviating photo‐inhibition. Ge‐Xian‐Mi used HCO₃^– in addition to CO₂ for its photosynthesis and oxygen evolution was greater than the theoretical rates of CO₂ production derived from uncatalysed dehydration of HCO₃^–. Multiple transporters for CO₂ and HCO₃^– operated in air‐grown Ge‐Xian‐Mi. Na⁺‐dependent HCO₃^– transport was the primary mode of active C_i uptake and contributed 53–62% of net photosynthetic activity at 250 µmol L^?1 KHCO₃ and pH 8.0. However, the CO₂‐uptake systems and Na⁺‐independent HCO₃^– transport played minor roles in Ge‐Xian‐Mi and supported, respectively, 39 and 8% of net photosynthetic activity. The steady‐state fluorescence decreased and the photochemical quenching increased in response to the transport‐mediated accumulation of intracellular C_i. Inorganic carbon transport was a major factor in facilitating quenching during the initial stage and the initial rate of fluorescence quenching in the presence of iodoacetamide, an inhibitor of CO₂ fixation, was 88% of control. Both the initial rate and extent of fluorescence quenching increased with increasing external dissolved inorganic carbon (DIC) and saturated at higher than 200 µmol L^?1 HCO₃^–. The operation of the CCM in Ge‐Xian‐Mi served as a means of diminishing photodynamic damage by dissipating excess light energy and higher external DIC in the range of 100–10000 µmol L^?1 KHCO₃ was associated with more severe photo‐inhibition under strong irradiance.     

Inorganic carbon transport across cell compartments of the halotolerant alga Dunaliella salina

The inorganic carbon (C_i) accumulation and the intracellular location of carbonic anhydrase (CA, EC 4.2.1.1) in the halotolerant unicellular alga Dunaliella salina have been investigated. The rate of HCO₃ -dependent O₂ evolution was determined by growth conditions. Algae grown under high CO₂ conditions (5% CO₂ in air, v/v; high C_i cells) had a very low affinity for HCO₃^? at pH 7.0 and 8.2, whereas algae grown under low CO₂ conditions (0.03% CO₂ in air; low C_i cells) showed a high affinity for HCO₃^? at both pH values and were sensitive to Dextran-bound sulfonamide (DBS), an inhibitor of extracellular CA. The photosynthetic rate or HCO₄^? dependent O₂ evolution was always higher at pH 7.0 than at pH 8.2. Ethoxyzolamide (EZ), an inhibitor of total (extacellular plus intracellular) CA activity, strongly inhibited photosynthesis at both pH values. During adaptation from high to low CO₂ conditions CA activity increased in chloroplasts in a process dependent on the novo protein synthesis. Carbonic anhydrase activity was found in the supernatant and pellet fractions of chloroplast homogenates. The rate of photosynthesis of chloroplasts from low C_i cells was higher at pH 7.0 than at pH 8.2. The alkalinization of the growth medium, which took place only in the presence of C_i, was partially inhibited by DBS and completely by EZ. We suggest that in D. salina CO₂ is the general form of C_i transported across the plasma membrane and the chloroplast envelope and that bicarbonate enters the cell mainly, although not entirely, by an ‘indirect’ mechanism after dehydration to CO₂.     

Active transport of CO2 by three species of marine microalgae

The occurrence of an active CO₂ transport system and of carbonic anhydrase (CA) has been investigated by mass spectrometry in the marine, unicellular rhodophyte Porphyridium cruentum (S.F. Gray) Naegeli and two marine chlorophytes Nannochloris atomus Butcher and Nannochloris maculata Butcher. Illumination of darkened cells incubated with 100 μM H¹³CO₃^? caused a rapid initial drop, followed by a slower decline in the extracellular CO₂ concentration. Addition of bovine CA to the medium raised the CO₂ concentration by restoring the HCO₃^?–CO₂ equilibrium, indicating that cells were taking up CO₂ and were maintaining the CO₂ concentration in the medium below its equilibrium value during photosynthesis. Darkening the cell suspensions caused a rapid increase in the extracellular CO₂ concentration in all three species, indicating that the cells had accumulated an internal pool of unfixed inorganic carbon. CA activity was detected by monitoring the rate of exchange of ¹⁸O from ¹³C¹⁸O₂ into water. Exchange of ¹⁸O was rapid in darkened cell suspensions, but was not inhibited by 500 μM acetazolamide, a membrane‐impermeable inhibitor of CA, indicating that external CA activity was not present in any of these species. In all three species, the rate of exchange was completely inhibited by 500 μM ethoxyzolamide, a membrane‐permeable CA‐inhibitor, showing that an intracellular CA was present. These results demonstrate that the three species are capable of CO₂ uptake by active transport for use as a carbon source for photosynthesis.