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.     

Structure and function of LCI1: a plasma membrane CO2 channel in the Chlamydomonas CO2 concentrating mechanism

Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO₂ in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO₂ concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C_i) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organism Chlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C_i uptake systems. Fundamental to eukaryotic C_i uptake systems are C_i transporters/channels located in membranes of various cell compartments, which together facilitate the movement of C_i from the environment into the chloroplast, where primary CO₂ assimilation occurs. Two putative plasma membrane C_i transporters, HLA3 and LCI1, are reportedly involved in active C_i uptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO₃^? transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full‐length LCI1 membrane protein to reveal LCI1 structural characteristics, as well as in vivo physiological studies in an LCI1 loss‐of‐function mutant to reveal the C_i species preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO₂ uptake and that LCI1 likely functions as a plasma membrane CO₂ channel, possibly a gated channel.     

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.     

Mitochondrial carbonic anhydrases are needed for optimal photosynthesis at low CO2 levels in Chlamydomonas

Chlamydomonas reinhardtii can grow photosynthetically using CO₂ or in the dark using acetate as the carbon source. In the light in air, the CO₂ concentrating mechanism (CCM) of C. reinhardtii accumulates CO₂, enhancing photosynthesis. A combination of carbonic anhydrases (CAs) and bicarbonate transporters in the CCM of C. reinhardtii increases the CO₂ concentration at Ribulose 1,5-bisphosphate carboxylase oxygenase (Rubisco) in the chloroplast pyrenoid. Previously, CAs important to the CCM have been found in the periplasmic space, surrounding the pyrenoid and inside the thylakoid lumen. Two almost identical mitochondrial CAs, CAH4 and CAH5, are also highly expressed when the CCM is made, but their role in the CCM is not understood. Here, we adopted an RNAi approach to reduce the expression of CAH4 and CAH5 to study their possible physiological functions. RNAi mutants with low expression of CAH4 and CAH5 had impaired rates of photosynthesis under ambient levels of CO₂ (0.04% CO₂ [v/v] in air). These strains were not able to grow at very low CO₂ (<0.02% CO₂ [v/v] in air), and their ability to accumulate inorganic carbon (C_i = CO₂ + HCO3−) was reduced. At low CO₂ concentrations, the CCM is needed to both deliver C_i to Rubisco and to minimize the leak of CO₂ generated by respiration and photorespiration. We hypothesize that CAH4 and CAH5 in the mitochondria convert the CO₂ released from respiration and photorespiration as well as the CO₂ leaked from the chloroplast to HCO3- thus “recapturing” this potentially lost CO₂.

Mitochondrial carbonic anhydrases CAH4 and CAH5 in Chlamydomonas reinhardtii are involved in maintaining optimal photosynthesis.     

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.     

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

The nature of the inorganic carbon (C_i) species actively taken up by cyanobacteria CO₂ or HCO₃⁻ has been investigated. The kinetics of CO₂ uptake, as well as that of HCO₃⁻ uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO₂ was higher than that for HCO₃⁻. Though the calculated V_max was the same in both cases, the maximum rate of uptake actually observed was higher when HCO₃⁻ was supplied. C_i uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO₂ was the species supplied. Observations of photosynthetic rate as a function of intracellular C_i level (following supply of CO₂ or HCO₃⁻ for 5 seconds) led to the inference that HCO₃⁻ is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.

On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C_i transport apparatus binds CO₂ and releases HCO₃⁻ at or near a membrane porter. The latter transports HCO₃⁻ ions to the cell interior.

     

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.     

CO<Subscript>2</Subscript>-concentrating mechanism in cyanobacterial photosynthesis: organization,physiological role,and evolutionary origin

The cellular and molecular organization of the CO₂-concentrating mechanism (CCM) of cyanobacteria is reviewed. The primary processes of uptake, translocation, and accumulation of inorganic carbon (C_i) near the active site of carbon assimilation by the enzyme ribulose-1,5-bisphosphate carboxylase in the C₃ cycle in cyanobacteria are described as one of the specialized forms of CO₂ concentration which occurs in some photoautotrophic cells. The existence of this form of CO₂ concentration expands our understanding of photosynthetic C_i assimilation. The means of supplying C_i to the C₃ cycle in cyanobacteria is not by simple diffusion into the cell, but it is the result of coordinated functions of high-affinity systems for the uptake of CO₂ and bicarbonate, as well as intracellular CO₂/HCO₃ ^? interconversions by carbonic anhydrases. These biochemical events are under genetic control, and they serve to maintain cellular homeostasis and adaptation to CO₂ limitation. Here we describe the organization of the CCM in cyanobacteria with a special focus on the CCM of relict halo- and alkaliphilic cyanobacteria of soda lakes. We also assess the role of the CCM at the levels of the organism, the biosphere, and evolution.     

The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO₂ and HCO₃ ^?, the utilizable forms of inorganic carbon (C_i). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of C_i for growth and development. This CCM is induced when the CO₂ concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of C_i into the cell as well as its directed transport to the site where Rubisco fixes CO₂ into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed.     

The effect of pCO2 on carbon acquisition and intracellular assimilation in four marine diatoms

The effect of pCO₂ on carbon acquisition and intracellular assimilation was investigated in the three bloom-forming diatom species, Eucampia zodiacus (Ehrenberg), Skeletonema costatum (Greville) Cleve, Thalassionema nitzschioides (Grunow) Mereschkowsky and the non-bloom-forming Thalassiosira pseudonana (Hust.) Hasle and Heimdal. In vivo activities of carbonic anhydrase (CA), photosynthetic O₂ evolution, CO₂ and HCO₃⁻ uptake rates were measured by membrane-inlet mass spectrometry (MIMS) in cells acclimated to pCO₂ levels of 370 and 800 μatm. To investigate whether the cells operate a C₄-like pathway, activities of ribulose-1,5-bisphosphate carboxylase (RubisCO) and phosphoenolpyruvate carboxylase (PEPC) were measured at the mentioned pCO₂ levels and a lower pCO₂ level of 50 μatm. In the bloom-forming species, extracellular CA activities strongly increased with decreasing CO₂ supply while constantly low activities were obtained for T. pseudonana. Half-saturation concentrations (K_1/2) for photosynthetic O₂ evolution decreased with decreasing CO₂ supply in the two bloom-forming species S. costatum and T. nitzschioides, but not in T. pseudonana and E. zodiacus. With the exception of S. costatum, maximum rates (V_max) of photosynthesis remained constant in all investigated diatom species. Independent of the pCO₂ level, PEPC activities were significantly lower than those for RubisCO, averaging generally less than 3%. All examined diatom species operate highly efficient CCMs under ambient and high pCO₂, but differ strongly in the degree of regulation of individual components of the CCM such as C_i uptake kinetics and extracellular CA activities. The present data do not suggest C₄ metabolism in the investigated species.     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

Regulation of the mechanism for HCO 3 − use by the inorganic carbon level in Porphyra leucosticta Thur. in Le Jolis (Rhodophyta)

The capacity for HCO₃ ⁻ use by Porphyra leucosticta Thur. in Le Jolis grown at different concentrations of inorganic carbon (C_i) was investigated. The use of HCO₃ ⁻ at alkaline pH by P. leucosticta was␣demonstrated by comparing the O₂ evolution rates measured with the O₂ evolution rates theoretically supported by the CO₂ spontaneously formed from HCO₃ ⁻ . Both external and internal carbonic anhydrase (CA; EC 4.2.1.1) were implied in HCO₃ ⁻ use during photosynthesis because O₂ evolution rates and the increasing pH during photosynthesis were inhibited in the presence of azetazolamide and ethoxyzolamide (inhibitors for external and total CA respectively). Both external and internal CA were regulated by the C_i level at which the algae were grown. A high C_i level produced a reduction in total CA activity and a low C_i level produced an increase in total CA activity. In contrast, external CA was increased at low C_i although it was not affected at high C_i . Parallel to the reduction in total CA activity at high C_i is a reduction in the affinity for C_i, as estimated from photosynthesis versus C_i curves, was found. However, there was no evident relationship between external CA activity and the capacity for HCO₃ ⁻ use because the presence of external CA became redundant when P. leucosticta was cultivated at high C_i. Our results suggest that the system for HCO₃ ⁻ use in P. leucosticta is composed of different elements that can be activated or inactivated separately. Two complementary hypotheses are postulated: (i) internal CA is an absolute requirement for a functioning C_i-accumulation mechanism; (ii) there is a CO₂ transporter that works in association with external CA. Received: 20 April 1996 / Accepted: 5 August 1996     

Mechanism of Photosynthetic Carbon Dioxide Uptake by the Red Macroalga, Chondrus crispus

The aim of this study was to determine how Chondrus crispus, a marine red macroalga, acquires the inorganic carbon (C_i) it utilizes for photosynthetic carbon fixation. Analyses of C_i uptake were done using silicone oil centrifugation (using multicellular fragments of thallus), infrared gas analysis, and gas chromatography. Inhibitors of carbonic anhydrase (CA), the band 3 anion exchange protein and Na⁺/K⁺ exchange were used in the study. It was found that: (a) C. crispus does not accumulate C_i internally above the concentration attainable by diffusion; (b) the initial C_i fixtion rate of C. crispus fragments saturates at approximately 3 to 4 millimolar C_i; (c) CA is involved in carbon uptake; its involvement is greatest at high HCO₃⁻ and low CO₂ concentration, suggesting its participation in the dehydration of HCO₃⁻ to CO₂; (d) C. crispus has an intermediate C_i compensation point; and (e) no evidence of any active or facilitated mechanism for the transport of HCO₃⁻ was detected. These data support the view that photosynthetic C_i uptake does not involve active transport. Rather, CO₂, derived from HCO₃⁻ catalyzed by external CA, passively diffuses across the plasma membrane of C. crispus. Intracellular CA also enhances the fixation of carbon in C. crispus.     

The Stoichiometry between CO(2) and H Fluxes Involved in the Transport of Inorganic Carbon in Cyanobacteria

The pH of the medium during CO₂ uptake into the intracellular inorganic carbon (C_i) pool of a high CO₂-requiring mutant (E₁) and wild type of Anacystis nidulans R2 was measured. Experiments were performed under conditions where photosynthetic CO₂ fixation is inhibited. There was an acidification of the medium during CO₂ uptake in the light and an alkalization during CO₂ efflux after darkening. A one to one stoichiometry existed between the amounts of H⁺ appearing in the medium and CO₂ taken up into the intracellular C_i pool, regardless of the carbon species transported. The results indicate that (a) CO₂ is taken up simultaneously with an efflux of equimolar H⁺, probably produced as a result of CO₂ hydration during transport and (b) HCO₃⁻ produced by hydration of CO₂ in the medium was transported into the cells without accompanying net flux of H⁺ or OH⁻. The influx and efflux of C_i during C_i transport produced nonequilibrium between CO₂ and HCO₃⁻ in the medium, with the concentration of HCO₃⁻ being higher than that expected under equilibrium conditions. The nonequilibrium was present even under the conditions where the influx of C_i is compensated by its efflux. The direction of this nonequilibrium suggested that efflux of HCO₃⁻ occurs during uptake of C_i.     

Photosynthetic inorganic carbon uptake and accumulation in two marine diatoms

Some physiological characteristics of photosynthetic inorganic carbon uptake have been examined in the marine diatoms Phaeodactylum tricornutum and Cyclotella sp. Both species demonstrated a high affinity for inorganic carbon in photosynthesis at pH7.5, having K_1/2(CO₂) in the range 1.0 to 4.0mmol m^?3 and O_2? and temperature-insensitive CO₂ compensation concentrations in the range 10.8 to 17.6 cm³ m^?3. Intracellular accumulation of inorganic carbon was found to occur in the light; at an external pH of 7.5 the concentration in P. tricornutum was twice, and that in Cyclotella 3.5 times, the concentration in the suspending medium. Carbonic anhydrase (CA) was detected in intact Cyclotella cells but not in P. tricornutum, although internal CA was detected in both species. The rates of photosynthesis at pH 8.0 of P. tricornutum cells and Cyclotella cells treated with 0.1 mol m^?3 acetazolamide, a CA inhibitor, were 1.5- to 5-fold the rate of CO₂ supply, indicating that both species have the capacity to take up HCO₃^? as a source of substrate for photosynthesis. No Na⁺ dependence for HCO₃^? could be detected in either species. These results indicate that these two marine diatoms have the capacity to accumulate inorganic carbon in the light as a consequence, in part, of the active uptake of bicarbonate.     

Is There a Role for the 42 Kilodalton Polypeptide in Inorganic Carbon Uptake by Cyanobacteria?

Cyanobacterial cells accumulate substantial amounts of a membrane-associated 42 kilodalton polypeptide during adaptation to low CO₂ conditions. The role of this polypeptide in the process of adaptation and in particular in the large increase in the ability to accumulate inorganic carbon (C_i), which accompanies this process, is not yet understood. We have isolated a mutant Synechococcus PCC7942 that does not accumulate the 42 kilodalton polypeptide. The mutant requires a high-CO₂ concentration for growth and exhibits a very low apparent photosynthetic affinity for extracellular C_i. The latter might be attributable to the observed defective ability of the mutant to utilize the intracellular C_i pool for photosynthesis. The 42 kilodalton polypeptide does not appear to participate directly in the active transport of C_i, since the difference between the observed capabilities for CO₂ and HCO₃⁻ uptake of the mutant and the wild type is not sufficient to account for their different growth and photosynthetic performance. Furthermore, high CO₂-grown wild-type cells, where we could not detect the 42 kilodalton polypeptide, transported CO₂ faster than the mutant. An analysis of the curves relating the rate of accumulation of C_i to the concentration of CO₂ or HCO₃⁻ supplied, in the presence or absence of carbonic anhydrase, indicated that under the experimental conditions used here, CO₂ was the preferred C_i species taken up by Synechococcus.     

Uptake of bicarbonate ion in darkness by isolated chloroplast envelope membranes and intact chloroplasts of spinach

Bicarbonate uptake by isolated chloroplast envelope membranes and intact chloroplasts of spinach (Spinacia oleracea L. var. Viroflay) in darkness exhibited a similar dependency upon temperature, pH, time, and concentrations of isolated or attached envelope membranes. This similarity in uptake properties demonstrates the usefulness of the envelope membranes for the study of chloroplast permeability. Maximal rates for dark HCO₃^- uptake by isolated envelope membranes and intact chloroplasts were more than sufficient to account for the maximal rates of photosynthetic CO₂ fixation observed with intact chloroplasts. The active species involved in the uptake process was found to be HCO₃^- and not CO₂. The significance of HCO₃^- uptake and its relationship to carbonic anhydrase and ribulose diphosphate carboxylase is discussed. Conditions for maximal HCO₃^- uptake in darkness by intact chloroplasts were found to be similar to those required for maximal photosynthetic CO₂ fixation, suggesting that HCO₃^- uptake by the envelope membrane may regulate photosynthetic CO₂ fixation.     

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.     

Mutants of Synechocystis PCC6803 Defective in Inorganic Carbon Transport

Eighty mutants of Synechocystis PCC6803 that require high CO₂ for growth were examined with a mass spectrometer for their ability to take up CO₂ in the light. Two of these mutants (type A) did not show any CO₂ uptake while the rest of the mutants (type B) took up CO₂ actively. Type A mutants (RKa and RKb) and one type B mutant (RK11) were partially characterized. At 3% CO₂, growth rates of the mutants and the wild type (WT) were similar. Under air levels of CO₂, growth of RKa and RKb was very slow, and RK11 did not grow at all. The photosynthetic affinities for inorganic carbon (C_i) in these three mutants were about 100 times lower than the affinity in WT. The following characteristics of type A mutants indicated that the mutants have a defect in their CO₂-transport system: (a) the activity of ¹³C¹⁸O₂ uptake in RKa and RKb in the light was less than 5% the activity in WT, and (b) each mutant had only a low level of activity of ¹⁴CO₂ uptake as measured by the method of silicone oil-filtering centrifugation. The HCO₃⁻-transport system was also impaired in these mutants. The activity of H¹⁴CO₃⁻ uptake was negligibly low in RKb and was one-third the activity of WT in RKa. On the other hand, the type B mutant, RK11, transported CO₂ and HCO₃⁻ into the intracellular C_i pool as actively as WT but was unable to utilize it for photosynthesis. Complementation analysis of type A mutants indicated that RKa and RKb have mutations in different regions of the genome. These results suggested that at least two kinds of proteins are involved in the C_i-transport system.     

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.