Induction of CO2 and Bicarbonate Transport in the Green Alga Chlorella ellipsoidea (II. Evidence for Induction in Response to External CO2 Concentration)

The critical species and concentrations of dissolved inorganic carbon (DIC) required for the induction of DIC transport during adaptation to low CO2 were determined for the green alga Chlorella ellipsoidea. The concentration of dissolved CO2 needed for the induction of both CO2 and HCO3- transport was independent of pH during adaptation, whereas the total DIC concentration required increased at alkaline pH. At pH 7.5, the minimum equilibrium DIC concentration at which high CO2 characteristics were maintained, i.e. transport was repressed, was 2100 [mu]M, whereas the maximum equilibrium DIC concentration below which DIC transport was fully induced (DICIND) was 500 [mu]M. Intracellular DIC concentration during adaptation to DICIND decreased temporarily after 2 h to 60% of the maximum level but recovered after 3 h of adaptation. After 3 h of adaptation to DICIND, cells exhibited maximum O2 evolution rate at DICIND. When cells partially adapted to DICIND were returned to high CO2, there was an immediate halt to the induction of transport and a gradual decrease in transport capacity over 23 h. The capacity for the induction of transport was unaffected by the absence of light. These results indicate that changes in the internal DIC pool during adaptation to low CO2 do not trigger the induction of DIC transport and that the induction is not light dependent. Induction of DIC transport in C. ellipsoidea appears to occur in response to the continuous exposure of cells to a critical CO2 concentration in the external medium.     

Active transport of CO(2) and bicarbonate is induced in response to external CO(2) concentration in the green alga Chlorella kessleri

The time-course of induction of CO(2) and HCO(3)- transport has been investigated during the acclimation of high CO(2)-grown Chlorella kessleri cells to dissolved inorganic carbon (DIC)-limited conditions. The rate of photosynthesis of the cells in excess of the uncatalysed supply rate of CO(2) from HCO(3)- was taken as an indicator of HCO(3)- transport, while a stimulation of photosynthesis on the addition of bovine carbonic anhydrase was used as an indicator of CO(2) transport. The maximum rate of photosynthesis (Pmax) was similar for high CO(2)-grown and low CO(2)-grown cells, but the apparent whole cell affinity for DIC and CO(2) of high CO(2)-grown cells was found to be about 30-fold greater than in air-grown cells, which indicates a lower affinity for DIC and CO(2). It was found that HCO(3)- and CO(2) transport were induced in 5.5 h in cells acclimating to air in the light and in the presence and absence of 21% O(2), which indicates that a change in the CO(2)/O(2) ratio in the acclimating medium does not trigger induction of DIC transport. No active DIC transport was detected in high CO(2)-grown cells maintained on high CO(2) for 5.5 h in the presence of 5 mM aminooxyacetate, an aminotransferase inhibitor. These results indicate no involvement of photorespiration in triggering induction. Active DIC transport induction was inhibited in cells treated with 5 microgram ml(-1) cycloheximide, but was unaffected by chloramphenicol treatment, indicating that the induction process requires de novo cytoplasmic protein synthesis. The total DIC concentration eliciting the induction and repression of CO(2) and HCO(3)- transport was higher at pH 7.5 than at pH 6.6. The concentrations of external CO(2) required for the induction and repression of DIC transport were 0 and 120 microM, respectively, and was independent of the pH of the acclimation medium. Prolonged exposure to a critical external CO(2) concentration elicits the induction of DIC transport in C. kessleri.     

A New Screening Method for Algal Photosynthetic Mutants (CO2-Insensitive Mutants of the Green Alga Chlorella ellipsoidea)

A new method has been developed for screening algal photosynthetic mutants. This method uses autoradiography to assess gross photosynthetic 14C fixation by green algal colonies on agar plates and allows the identification of clones that differ in photosynthetic characteristics from wild-type cells. Three wild-type cells, high-CO2-grown Chlorella ellipsoidea, air-grown C. ellipsoidea, and air-grown Chlorella saccharophila, had K0.5 values for dissolved inorganic carbon (DIC) of 1083, 250, and 50 [mu]M, respectively, and as plaques on agar plates at Chl densities greater than 25 [mu]g cm-2 exhibited relative amounts of 14C fixation of 15, 55, and 100%, respectively. Cells of C. ellipsoidea were mutagenized with x-rays and screened by this method. Growth of C. ellipsoidea in high CO2 represses DIC transport and thus lowers its affinity for DIC. Five of the mutants detected by this method showed high-affinity photosynthesis similar to air-grown wild-type cells even when grown in high CO2. Seven other mutants when grown in high CO2 showed affinities for DIC intermediate between air-grown and high-CO2-grown wild-type cells. The affinities of high-CO2-grown mutants were reflected precisely in their capacities to accumulate DIC intracellularly. These results indicate that the mutants are fully or partially insensitive to the repressive effect of ambient CO2 concentration on DIC transport.     

Quenching of Chlorophyll a Fluorescence in Response to Na+-Dependent HCO3- Transport-Mediated Accumulation of Inorganic Carbon in the Cyanobacterium Synechococcus UTEX 625

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.     

The influence of elevated rhizosphere dissolved inorganic carbon concentrations on respiratory O2 and CO2 flux in tomato roots

Respiratory CO2 and O2 flux were measured in hydroponically grown Lycopersicon esculentum (L.) Mill. cv. F144 plants at either low (O mol mol^-1) or elevated CO2 concentrations (>2000 mol mol^-1) supplied to the roots. In NO3^- fed plants the consumption of O2 and the engagement of the alternative pathway were increased by elevated dissolved inorganic carbon (DIC = CO2 + HCO3^-) concentrations. This was ascribed to the influence of organic acids on the TCA cycle and electron transport pathways. Inhibition of O2 consumption by elevated DIC in NH4^--fed plants may be due to the reduction requirements of anaplerotic carbon entering the TCA cycle or the removal of carbon from the TCA cycle for amino acid synthesis. In both NO3^- and NH4⁺-fed plants elevated DIC inhibited CO2 release due to high rates of DIC incorporation by phosphoenolpyruvate carboxylase (PEPc) activity. Transient net CO2 consumption due to the inhibition of respiration by salicylhydroxamic acid and KCN, together with high respiratory quotients after the addition of inhibitors of carbonic anhydrase (CA) activity, were also ascribed to high rates of DIC incorporation at elevated DIC concentrations. Ethoxyzolamide, an inhibitor of CA activity, inhibited both DI¹⁴C incorporation into organic products and NO3^- uptake by 81% and 40%, respectively. This, together with a 32% increase in DI¹⁴C accumulation and inhibition of NO3^- uptake upon inhibition of anion transport by diisothiocyanato-stilbene-2,2'-disulphonic acid (DIDS) may indicate the exchange of HCO3^- for NO3^- across the root plasmalemma. It was concluded that dark incorporation of HCO3^- by PEPc increased at elevated rhizosphere DIC concentrations and that the products of DIC incorporation may stimulate respiratory electron transport. Additional reducing energy and carbon skeletons from the tricarboxylic acid (TCA) cycle would therefore be available for respiration and the reduction and incorporation of NO3^- into amino acids.Key words: Tomato, PEPc, respiration, carbon dioxide nitrate.      

Acclimation of wild-type cells and CO2-insensitive mutants of the green alga Chlorella ellipsoidea to elevated [CO2

CO(2)-insensitive mutants of the green alga Chlorella ellipsoidea were previously shown to be unable to repress an inorganic carbon-concentrating mechanism (CCM) when grown under 5% CO(2). When air-grown, wild-type (WT) cells were transferred to 5% CO(2), an abrupt drop of P(max) to 43% the original level of air-grown cells was observed within the initial 12 h. Photosynthetic affinities of WT cells to dissolved inorganic carbon (DIC) were maintained at high levels for the initial 4 d of acclimation, and then decreased gradually to lower levels over the next 6 d. In contrast to WT cells, the CO(2)-insensitive mutant, ENU16, exhibited a constant P(max) at maximum levels and a low K(1/2)[DIC] throughout the acclimation period. The rapid P(max) drop within 12 h of acclimation in WT cells was significantly reduced by treatment with 0.5 mm of 6-ethoxybenzothiazole-2-sulphonamide (EZA), a specific membrane-permeable inhibitor of carbonic anhydrase (CA), suggesting the participation of internal CAs in the temporary drop in P(max) in WT cells. WT and ENU16 cells were grown in controlled equilibrium [CO(2)], and the photosynthetic rate of each acclimated cell type was measured under equilibrated growth [DIC] conditions. In WT cells acclimated to 0.14-0.4% [CO(2)], K(1/2)[DIC] values increased as [CO(2)] increased, and the photosynthetic rates at growth DIC conditions were shown to decrease to about 70% the P(max) level in this intermediate [CO(2)] range. Such decreases in the net photosynthetic rates were not observed in ENU16. These results suggest that algal primary production could be depressed significantly under moderately enriched CO(2) conditions as a result of acquiring intermediate affinities for DIC because of their sensitive responses to changes in the ambient [CO(2)].     

A Mutant Isolated from the Cyanobacterium Synechococcus PCC7942 Is Unable to Adapt to Low Inorganic Carbon Conditions

Using a novel screening procedure, we have selected a new class of mutant from the cyanobacterium Synechococcus PCC7942 that fails to adapt to growth at an extremely low inorganic carbon (Ci) concentration. The mutant (Tm17) reported in this study grows normally at or above air levels of CO2 (340 [mu]L L-1) but does not survive at 20 [mu]L L-1 CO2 in air. Air-grown Tm17 cells showed properties similar to wild-type cells in various aspects of the CO2-concentrating mechanism examined. Following transfer from air levels to 20 [mu]L L-1 CO2, however, the mutant cells failed to increase their photosynthetic affinity for Ci. This results in an approximately 10-fold difference in photosynthetic affinity between the wild-type and Tm17 cells under Ci-limiting conditions [the K0.5(Ci) values were 11 and 136 [mu]M, respectively]. Further examination of factors possibly contributing to this low photosynthetic affinity showed that Tm17 cells have no inducible high-affinity HCO3- transport and do not appear to show induction of increased carboxysomal carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase activities. It appears that a common factor, possibly relating to CO2 detection and/or induction signal, or the HCO3-transport mechanism may have been impaired in the mutant. Complementation results indicate that the mutation responsible for the phenotype has occurred in an 8- to 10-kb EcoRI genomic DNA fragment.     

Active transport and accumulation of bicarbonate by a unicellular cyanobacterium

The rates of inorganic carbon accumulation and carbon fixation in light by the unicellular cyanobacterim Coccohloris peniocystis have been determined. Cells incubated in the light in medium containing H14CO3- were rapidly separated from the medium by centrifugation through silicone oil into a strongly basic terminating solution. Samples of these inactivated cells were assayed to determine total 14C accumulation, and acid-treated samples were assayed to determine 14C fixation. The rate of transport of inorganic into illuminated cells was faster than the rate of CO2 production in the medium from HCO3- dehydration. This evidence for HCO3- transport in these cells is in agreement with our previous results based upon measurements of photosynthetic O2 evolution. A substantial pool of inorganic carbon was bulit up within the cells presumably as HCO3- before the onset of the maximum rate of photosynthesis. Large accumulation ratios were observed, greater than 1,000 times the external HCO3- concentration. Accumulation did not occur in the dark and was greatly suppressed by the photosynthesis inhibitors 3-(3,4-dichlorophenyl)-1,1-dimethyl urea and 3-chloro-carbonylcyanide phenylhydrazone. These results indicate that the accumulation of inorganic carbon in these cells involves a light-dependent active transport process.     

Ethoxyzolamide Differentially Inhibits CO2 Uptake and Na+-Independent and Na+-Dependent HCO3- Uptake in the Cyanobacterium Synechococcus sp. UTEX 625

The effects of ethoxyzolamide (EZ), a carbonic anhydrase inhibitor, on the active CO2 and Na+-independent and Na+-dependent HCO3- transport systems of the unicellular cyanobacterium Synechococcus sp. UTEX 625 were examined. Measurements of transport and accumulation using radiochemical, fluorometric, and mass spectrometric assays indicated that active CO2 transport and active Na+-independent HCO3- transport were inhibited by EZ. However, Na+-independent HCO3- transport was about 1 order of magnitude more sensitive to EZ inhibition than was CO2 transport (50% inhibition = 12 [mu]M versus 80 [mu]M). The data suggest that both the active CO2 (G.D. Price, M.R. Badger [1989] Plant Physiol 89: 37-43) and the Na+ -independent HCO3 - transport systems possessed carbonic anhydrase-like activity as part of their mechanism of action. In contrast, Na+-dependent HCO3- transport was only partially (50% inhibition = 230 [mu]M) and noncompetitively inhibited by EZ. The collective evidence suggested that EZ inhibition of Na+ -dependent HCO3- transport was an indirect consequence of the action of EZ on the CO2 transport system, rather than a direct effect on HCO3- transport. A model is presented in which the core of the inorganic carbon translocating system is formed by Na+-dependent HCO3- transport and the CO2 transport system. It is argued that the Na+-independent HCO3 - utilizing system was not directly involved in translocation, but converted HCO3- to CO2 for use in CO2 transport.     

Limiting CO2 levels induce a blue light-dependent HCO3- uptake system in Monoraphidium braunii

The in situ photoactivation of an HCO3- uptake system in the green alga Monoraphidium braunii requires the irradiation of the cell suspensions with short wavelength radiation (blue, UVA and/or UVC). Plasma membrane ATPase inhibitors block the uptake of this monovalent anion at pH 9. M. braunii cells grown in high CO2 lack an HCO3- uptake system in their plasma membrane, but those grown in low CO2 can take up this anion at high rates. Cells grown in high CO2, transferred to CO2-limiting conditions in the light, start taking up HCO3- in 30 min, although they take 90 min to reach maximum rates of HCO3- transport. Therefore, this induction process seems to be triggered by low external CO2 concentration. In fact, increasing or decreasing the external HCO3- concentration does not induce the uptake system and only a decrease in CO2 concentration in the medium triggers the induction process. The appearance of the HCO3- transport activity is sensitive to cycloheximide, indicating that cytoplasmic protein biosynthesis is necessary for the induction of the uptake system. Photosynthetically active radiation, but not particularly blue light, is essential for induction of the uptake system to occur and the inhibition of photosynthesis by DCMU blocks it. From these results it can be inferred that when M. braunii cells detect a drop in CO2 concentration, they induce a blue light-dependent HCO3- uptake system.     

Monensin Inhibition of Na+-Dependent HCO3- Transport Distinguishes It from Na+-Independent HCO3- Transport and Provides Evidence for Na+/HCO3- Symport in the Cyanobacterium Synechococcus UTEX 625

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.     

Characterization of a mutant lacking carboxysomal carbonic anhydrase from the cyanobacterium Synechocystis PCC6803

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.     

Inorganic carbon acquisition in red tide dinoflagellates

Carbon acquisition was investigated in three marine bloom-forming dinollagellates-Prorocentrum minimum, Heterocapsa triquetra and Ceratium lineatum. In vivo activities of extracellular and intracellular carbonic anhydrase (CA), photosynthetic O2 evolution, CO2 and HCO3- uptake rates were measured by membrane inlet mass spectrometry (MIMS) in cells acclimated to low pH (8.0) and high pH (8.5 or 9.1). A second approach used short-term 14C-disequilibrium incubations to estimate the carbon source utilized by the cells. All three species showed negligible extracellular CA (eCA) activity in cells acclimated to low pH and only slightly higher activity when acclimated to high pH. Intracellular CA (iCA) activity was present in all three species, but it increased only in P. minimum with increasing pH. Half-saturation concentrations (K1/2) for photosynthetic O2 evolution were low compared to ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) kinetics. Moreover, apparent affinities for inorganic carbon (Ci) increased with increasing pH in the acclimation, indicating the operation of an efficient CO2 concentration mechanism (CCM) in these dinoflagellates. Rates of CO2 uptake were comparably low and could not support the observed rates of photosynthesis. Consequently, rates of HCO3- uptake were high in the investigated species, contributing more than 80% of the photosynthetic carbon fixation. The affinity for HCO3- and maximum uptake rates increased under higher pH. The strong preference for HCO3- was also confirmed by the 14C-disequilibrium technique. Modes of carbon acquisition were consistent with the 13C-fractionation pattern observed and indicated a strong species-specific difference in leakage. These results suggest that photosynthesis in marine dinoflagellates is not limited by Ci even at high pH, which may occur during red tides in coastal waters.     

Diversity of inorganic carbon acquisition mechanisms by intact microbial mats of Microcoleus chthonoplastes (Cyanobacteriae, Oscillatoriaceae)

The dissolved inorganic carbon (DIC) acquisition mechanisms were researched in intact microbial mats dominated by the cyanobacteria Microcoleus chthonoplastes Thuret, by determining the effect on photosynthesis of different inhibitors. The microbial mats exhibited high affinity for DIC at alkaline pH, with K(m(DIC)) values similar to the ones described for pure cultures of cyanobacteria and algae in which carbon concentrating mechanisms have been researched. Besides, the photosynthesis was non-sensitive to pH changes within the range of 5.6-9.6, indicating that HCO(3)(-) was the main DIC source used for photosynthesis. The M. chthonoplastes mats featured external and internal carbonic anhydrase (CA) activity as measured in intact cells and cell extracts, respectively. Acetazolamide (AZ, which slowly enters the cell and then inhibits mainly the external CA) and ethoxyzolamide (EZ, which inhibits both external and internal CA) reduced significantly the oxygen evolution rates, demonstrating that the CA was implied in the DIC acquisition. Vanadate inhibited photosynthesis by 60% although its application, when CA being inhibited (i.e. after applying AZ + EZ), did not produce any additional effect. It could indicate that ATPase-dependent HCO(3)(-) use occurred and also that this putative mechanism was coupled with CA-like activity at the plasma membrane. The involvement of Na(+)-dependent HCO(3)(-) transporters in DIC acquisition was also inferred as monensin and 4-4'-diisothiocyanatostibilene-2,2'-disulfonate (DIDS) reduced photosynthesis by 70%. DIDS produced a strong inhibitory effect even after application of AZ + EZ + vanadate, indicating that this mechanism was not related to CA activity. The microbial mats become subject to very unfavourable conditions for Rubisco carboxylation at their natural habitats (e.g. external pH of 10.5 and O(2) concentration doubled with respect to saturation concentration); therefore, this putative diversity of DIC acquisition mechanisms could ensure their growth under these extreme conditions.     

海洋浮游藻类无机碳利用机理的研究

为了认识海洋浮游藻类在碳充足和碳受限条件下对水体中溶解无机碳(DIC)的利用方式与可能机理,对13种海洋浮游藻类在不同pH和CO2浓度及不同DIC条件下细胞外碳酸酐酶(CA)的活性进行了分析测定.结果显示:13种藻中,只有Amphidinium carterae和Prorocentrum minimum在碳充足条件下具细胞外CA活性.Melosira sp.、Phaeodactylum tricornutum、Skeletonema costatum、Thalassiosira rotula、Emiliania huxleyi和Pleurochrysis carterae则在碳受限条件下才具细胞外CA活性.Chaetoceros compressus、Glenodinium foliaceum、Coccolithus pelagicus、 Gephrocapsa oceanica和Heterosigma akashiwo即使在碳受限条件下也未检测到细胞外CA活性.应用封闭系统中pH漂移技术和阴离子交换抑制剂4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS)等的研究表明,Coc. pelagicus和G. oceanica可通过阴离子交换机制进行HCO-3的直接利用.H. akashiwo没有潜在的HCO-3直接利用或细胞外CA催化的HCO-3利用.     

Carbon concentration mechanisms in photosynthetic microorganisms

Unicellular green algae and cyanobacteria have mechanism to actively concentrate dissolved inorganic carbon into the cells, only if they are grown with air levels of CO2. The carbon concentration mechanisms are commonly known as "CCM" or "DIC-pumps". The DIC-pumps are environmental adaptation that function to actively transport and accumulate inorganic carbon (HCO3- and CO2; Ci) within the cell and then uses this Ci pool to actively increase the concentration of CO2 at the site of ribulose bisphosphate carboxylase-oxygenase (Rubisco), the primary CO2-fixing enzyme. The current working model for dissolved inorganic carbon concentration mechanism in unicellular green algae includes several isoforms of carbonic anhydrase (CA), and ATPase driven active transporters at the plasmalemma and at the inner chloroplast envelopes. In the past fifteen years, significant progress has been made in isolating and characterizing the various isoforms of carbonic anhydrase at the biochemical and molecular level. However, we have an inadequate understanding of active transporters that are located on the plasmalemma and at the chloroplast envelopes. In this mini-review we focus on certain aspects of the induction, function and significance of the dissolved inorganic carbon concentration mechanisms in aquatic photosynthetic microorganisms.     

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 K+-dependent HCO3- utilization in the marine diatom Phaeodactylum tricornutum

Photosynthetic utilization of inorganic carbon in the marine diatom Phaeodactylum tricornutum was investigated by the pH drift experiment, measurement of K(1/2) values of dissolved inorganic carbon (DIC) with pH change, and comparison of the rate of photosynthesis with the rate of the theoretical CO(2) formation from uncatalyzed HCO(3)(-) conversion in the medium. The higher pH compensation point (10.3) and insensitivity of the photosynthetic rate to acetazolamide indicate that the alga has good capacity for direct HCO(3)(-) utilization. The photosynthetic rate reached 150 times the theoretical CO(2) supply rate at 100 micromol L(-1) DIC (pH 9.0) in the presence of 10 mmol L(-1) K(+) and 46 times that in the absence of K(+), indicating that for pH 9.4-grown P. tricornutum, HCO(3)(-) in the medium is taken up through K(+)-dependent and -independent HCO(3)(-) transporters. The K(1/2) (CO(2)) values at pH 8.2 were about 4 times higher than those at pH 9.0, whereas the K(1/2) (HCO(3)(-)) values at pH 8.2 were slightly lower than those at pH 9.0 whether without or with K(+), providing further evidence for the presence of the two HCO(3)(-) transport patterns in this alga. Photosynthetic rate and affinity for HCO(3)(-) in the presence of K(+), respectively, were about 2- and 7-fold higher than those in the absence of K(+), indicating that K(+)-dependent HCO(3)(-) transport is a predominant pattern of HCO(3)(-) cellular uptake in low DIC concentration. However, as P. tricornutum was cultured at pH 7.2 or 8.0, photosynthetic affinities to HCO(3)(-) were not affected by K(+), implying that K(+)-dependent HCO(3)(-) transport is induced when P. tricornutum is cultured at high alkaline pH.