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.     

Diurnal variation in light and carbon limitation of photosynthesis by two species of submerged freshwater macrophyte with a differential ability to use bicarbonate

SUMMARY.

• 1 Rates of photosynthetic oxygen evolution by Callitriche cophocarpa and Ranunculus peltatus in stream were measured on live occasions during the light period on 2 days at ambient light and ambient inorganic carbon, ambient light and saturating inorganic carbon, saturating light and ambient inorganic carbon, saturating light and saturating inorganic carbon and air-equilibrium inorganic carbon and ambient light.
• 2 Despite an ambient CO₂ concentration of about 220 μm , which is about ten times air-equilibrium, the concentration of inorganic carbon was more limiting than light on all the occasions that rates were measured. On average, rates of photosynthesis at ambient concentrations of CO₂ were about 130 and 425 μmol O₂ g^?1 DW h^?1 for C. cophocarpa and R. peltatus, respectively. These rates as a percentage of carbon saturated rates were only about 35% for C. cophocarpa and about 60% for R. peltatus. Ambient rates as a percentage of light saturated rates were about 80% for C. cophocarpa and about 95% for R. peltatus. Only in early morning and late evening where the photon irradiance was below 160 μmol m^?2 s^?1 was there evidence for slight light limitation.
• 3 Based on results from pH-drift experiments and from rates of photosynthesis as a function of CO₂ concentration in the presence and absence of HCO₃^?, C. cophocarpa was unable, but R. peltatus able to use HCO₃^? at an ambient HCO₃^? concentration of about 0·84 mm . The greater rates of photosynthesis at ambient CO₂ concentration and the lesser limitation by inorganic carbon shown by R. peltatus compared to C. cophocarpa was the result of HCO₃^?-use as laboratory experiments showed that R. peltatus performed similarly to C. cophocarpa if the HCO₃^? concentration was reduced to 60 μm .

     

Carbonate ions appear to neither inhibit nor stimulate use of bicarbonate ions in photosynthesis by Ulva lactuca

Rates of photosynthesis by the marine macroalga Ulva lactuca were measured in a factorial experiment at five concentrations of HCO₃^? and CO₃^2- between 0·20 and 1·26 mol m^?3, but very low concentrations of CO₂. The results demonstrated that HCO₃^? was available for use, but an analysis of variance showed that CO₃^2- had neither an inhibiting nor a stimulating effect on rates of photosynthesis over this concentration range. Over the experiment, pH varied from 8·46 to 10·06 and this also had no significant effect on rates of photosynthesis. The lack of a stimulatory effect of high concentrations of CO₃^2- on the rate of photosynthesis at low concentrations of HCO₃^? was taken as circumstantial evidence for direct uptake of HCO₃^? rather than proton extrusion and external production of CO₂. In the rockpools in which U. lactuca grows, pH values up to 10·35 have been recorded, and for much of the time, CO₃^2- was the major form of inorganic carbon available. The apparent lack of an ability to use CO₃^2- under these conditions suggests that direct use of CO₃^2- as a source of inorganic carbon for photosynthesis is unlikely to be widespread.     

INDUCIBLE MECHANISMS FOR HCO3– UTILIZATION AND REPRESSION OF PHOTORESPIRATION IN PROTOPLASTS AND THALLI OF THREE SPECIES OF ULVA (CHLOROPHYTA)1

Thalli of Ulva reticulata Forskaal, Ulva rigida C. Ag., and Ulva pulchra Jaasund were incubated at different concentrations of dissolved CO₂. Incubation at a high CO₂ concentration resulted in decreased oxygen evolution rate and lower affinity for inorganic carbon at high pH conditions, i.e. the ability to use HCO₃^– as a carbon source was reduced. This effect was reversible, and plants regained this HCO₃^– uptake capacity when transferred to air concentrations of CO₂. The phytosynthetic oxygen evolution rate of plants grown at high CO₂ concentration was reduced by high O₂ concentrations, whereas thalli and protoplasts from cultures grown at air concentration were not affected. This is interpreted as a deactivation of the carbon-concentrating mechanism during conditions of high CO₂ resulting in high photorespiration when plants are exposed to high O₂ concentrations. Protoplasts were not affected by high O₂ to the same extent and were not able to utilize HCO₃^– from the medium. The algae were able to grow at very low CO₂ concentrations, but growth was suppressed when an inhibitor of external carbonic anhydrase was present. Assay of carbonic anhydrase activities showed that external and internal CA activities were lower in plants grown at a high CO₂ concentration compared to plants grown at a low concentration of CO₂. Possible mechanisms for HCO₃^– utilization in these Ulva species are discussed.     

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.     

CARBON‐USE STRATEGIES IN MACROALGAE: DIFFERENTIAL RESPONSES TO LOWERED PH AND IMPLICATIONS FOR OCEAN ACIDIFICATION1

Ocean acidification (OA) is a reduction in oceanic pH due to increased absorption of anthropogenically produced CO₂. This change alters the seawater concentrations of inorganic carbon species that are utilized by macroalgae for photosynthesis and calcification: CO₂ and HCO₃^? increase; CO₃^2? decreases. Two common methods of experimentally reducing seawater pH differentially alter other aspects of carbonate chemistry: the addition of CO₂ gas mimics changes predicted due to OA, while the addition of HCl results in a comparatively lower [HCO₃^?]. We measured the short‐term photosynthetic responses of five macroalgal species with various carbon‐use strategies in one of three seawater pH treatments: pH 7.5 lowered by bubbling CO₂ gas, pH 7.5 lowered by HCl, and ambient pH 7.9. There was no difference in photosynthetic rates between the CO₂, HCl, or pH 7.9 treatments for any of the species examined. However, the ability of macroalgae to raise the pH of the surrounding seawater through carbon uptake was greatest in the pH 7.5 treatments. Modeling of pH change due to carbon assimilation indicated that macroalgal species that could utilize HCO₃^? increased their use of CO₂ in the pH 7.5 treatments compared to pH 7.9 treatments. Species only capable of using CO₂ did so exclusively in all treatments. Although CO₂ is not likely to be limiting for photosynthesis for the macroalgal species examined, the diffusive uptake of CO₂ is less energetically expensive than active HCO₃^? uptake, and so HCO₃^?‐using macroalgae may benefit in future seawater with elevated CO₂.     

Continuous Measurements of the Free Dissolved CO(2) Concentration during Photosynthesis of Marine Plants: Evidence for HCO(3) Use in Chondrus crispus

An experimental system consisting of a gas exchange column linked to an assimilation chamber has been developed to record continuously the free dissolved CO₂ concentration in seawater containing marine plants. From experiments performed on the red macroalga Chondrus crispus (Rhodophyta, Gigartinales), this measurement is in agreement with the free CO₂ concentration calculated from the resistance to CO₂ exchanges in a biphasic system (gas and liquid) as earlier reported. The response time of this apparatus is short enough to detect, in conditions of constant pH, a photosynthesis-caused gradient between free CO₂ and HCO₃⁻ pools which half-equilibrates in 25 seconds. Abolished by carbonic anhydrase, the magnitude of this gradient increases with decreasing time of seawater transit from the chamber to the column apparatus. But its maximum magnitude (0.35 micromolar CO₂) is negligible compared to the difference between air and free CO₂ (11.4 micromolar CO₂). This illustrates the extent of the physical limiting-step occurring at the air-water interface when inorganic carbon consumption in seawater is balanced by dissolving gaseous CO₂. The direction of this small free CO₂/HCO₃⁻ gradient indicates that HCO₃⁻ is consumed during photosynthesis.     

Discrimination between12C and13C by marine plants

Summary The natural abundance¹³C/¹²C ratios (as δ¹³C) of organic matter of marine macroalgae from Fife and Angus (East Scotland) were measured for comparison with the species' ability to use CO₂ and HCO ₃ ^- for photosynthesis, as deduced from previously published pH-drift measurements. There was a clear difference in δ¹³C values for species able or unable to use HCO ₃ ^- . Six species of Chlorophyta, 12 species of Phaeophyta and 8 species of Rhodophyta that the pH-drift data suggested could use HCO ₃ ^- had δ¹³C values in the range -8.81‰ to -22.55‰. A further 6 species of Rhodophyta which the pH-drift data suggested could only use CO₂ had δ¹³C values in the range -29.90‰ to-34.51‰. One of these six species (Lomentaria articulata) is intertidal; the other five are subtidal and so have no access to atmospheric CO₂ to complicate the analysis. For these species, calculations based on the measured δ¹³C of the algae, the δ¹³C of CO₂ in seawater, and the known¹³C/¹²C discrimination of CO₂ diffusion and RUBISCO carboxylation suggest that only 15–21% of the limitation to photosynthesisin situ results from CO₂ diffusion from the bulk medium to the plastids; the remaining 79–85% is associated with carboxylation reactions (and, via feedback effects, down-stream processes). This analysis has been extended for one of these five species,Delesseria sanguinea, by incorporating data onin situ specific growth rates, respiratory rates measured in the laboratory, and applying Fick's law of diffusion to calculate a boundary layer thickness of 17–24 μm. This value is reasonable for aDelesseria sanguinea frondin situ. For HCO ₃ ^- -using marine macroalgae the range of δ¹³C values measured can be accommodated by a CO₂ efflux from algal cells which range from 0.306 of the gross HCO ₃ ^- influx forEnteromorpha intestinalis (δ¹³C=-8.81‰) in a rockpool to 0.787 forChondrus crispus (δ¹³C=-22.55‰). The relatively high computed CO₂ efflux for those HCO ₃ ^- -users with the more negative δ¹³C values implies a relatively high photon cost of C assimilation; the observed photon costs can be accommodated by assuming coupled, energy-independent inorganic carbon influx and efflux. The observed δ¹³C values are also interpreted in terms of water movement regimes and obtaining CO₂ from the atmosphere. Published δ¹³C values for freshwater macrophytes were compared with the ability of the species to use CO₂ and HCO ₃ ^- and again there was an apparent separation in δ¹³C values for these two groups. δ¹³C values obtained for marine macroalgae for which no pH-drift data are available permit predictions, as yet untested, as to whether they use predominantly CO₂ or HCO ₃ ^-     

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.

     

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.     

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.     

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.     

Affinity, capacity and oxygen sensitivity of two different mechanisms for bicarbonate utilization in Ulva lactuca L. (Chlorophyta)

Ulva lactuca, collected on the west coast of Sweden at the end of May, was able to utilize the HCO₃ ^? pool of seawater only through extracellular dehydration via carbonic anhydrase, followed by uptake of the CO₂ formed. A decrease in the CO₂ supply via this mechanism resulted in the gradual development of an additional method of HCO₃ ^? utilization, namely a direct uptake of HCO₃ ^? . Photosynthesis could then be supported by either a ‘HCO₃ ^? dehydration mechanism’ or a ‘HCO₃ ^? uptake mechanism’. Through selective inhibition of either of these mechanisms, the physiological properties of the other could be assessed. These properties suggest that the HCO₃ ^? uptake mechanism of U. lactuca is important under conditions when low concentrations of inorganic C, high pH and high external O₂ concentrations would limit photosynthesis supported by the HCO₃ ^? dehydration mechanism. Such conditions may occur during intense irradiation of the alga in rockpools or in shallow bays with low rates of water exchange. The results are discussed in relation to a possible coupling between mechanisms for inorganic C acquisition and cell structure (or even morphology) of green macroalgae. They also illustrate some necessary precautions when using Michaelis–Menten kinetics for estimations of V_max and K_1/2 values.     

INORGANIC CARBON ACQUISITION BY CHRYSOPHYTES1

Twelve species, representing 12 families of the chrysophytes sensu lato, were tested for their ability to take up inorganic carbon. Using the pH‐drift technique, CO₂ compensation points generally varied between 1 and 20 μmol · L^?1 with a mean concentration of 5 μmol · L^?1. Neither pH nor alkalinity affected the CO₂ compensation point. The concentration of oxygen had a relatively minor effect on CO₂‐uptake kinetics, and the mean CO₂ compensation point calculated from the kinetic curves was 3.6 μmol · L^?1 at 10–15 kPa starting oxygen partial pressure and 3.8 μmol · L^?1 at atmospheric starting oxygen partial pressure (21 kPa). Similarly, uptake kinetics were not affected by alkalinity, and hence concentration of bicarbonate. Membrane inlet mass spectrometry (MIMS) in the presence and absence of acetazolamide suggested that external carbonic anhydrase in Dinobryon sertularia Ehrenb. and Synura petersenii Korschikov was either very low or absent. Rates of net HCO₃^? uptake were very low (～5% of oxygen evolution) using MIMS and decreased rather than increased with increasing HCO₃^? concentration, suggesting that it was not a real uptake. The CO₂ compensation points determined by MIMS for CO₂ uptake and oxygen evolution were similar to those determined in pH‐drift and were >1 μmol · L^?1. Overall, the results suggest that chrysophytes as a group lack a carbon‐concentrating mechanism (CCM), or an ability to make use of bicarbonate as an alternative source of inorganic carbon. The possible evolutionary and ecological consequences of this are briefly discussed.     

Mechanisms of inorganic carbon acquisition in Gracilaria gaditana nom. prov. (Rhodophyta)

The mechanisms for acquisition of dissolved inorganic carbon (DIC) in the red macroalga Gracilaria gaditana nom. prov. have been investigated. The capacity for HCO₃ ⁻ use by an extracellular carbonic anhydrase (CA; EC 4.2.1.1), and by an anion exchanger with similar properties to that of red blood cells (AE1), has been quantified. It was illustrated by comparing O₂ evolution rates with those theoretically supported by CO₂, as well as by photosynthesis-pH curves. Both external and internal CA, and a direct uptake were involved in HCO₃ ⁻ use, since photosynthesis and pH evolution were affected by acetazolamide, 6-ethoxyzolamide (inhibitors of external and total CA, respectively) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate, (DIDS; an inhibitor of HCO₃ ⁻ exchanger protein). The activity of the external CA was detected by a potentiometric method and by an alternative method based on the study of O₂ evolution after addition of CO₂ and acetazolamide. The latter method showed a residual photosynthetic rate due to direct HCO₃ ⁻ use. Inhibitors caused a reduction in the pH compensation points in pH-drift experiments. The CO₂ compensation points for photosynthesis increased when the inhibitors were applied, indicating a suppresion of the pathways involved in the carbon-concentrating mechanism. The net photosynthesis rates as a function of DIC concentration displayed a biphasic pattern that could be supported by the occurrence of the two mechanisms of HCO₃ ⁻ use. The potential contribution to HCO₃ ⁻ acquisition by the DIDS-sensitive mechanism was higher after culturing at a high pH. Our results suggest that the HCO₃ ⁻ use by Gracilaria gaditana is carried out by the two DIC uptake mechanisms. These operate simultaneously with different affinities for DIC, the indirect HCO₃ ⁻ use by an external CA activity being the main pathway. The presence of a carbon-concentrating mechanism confers eco-physiological advantages in a fluctuating ecosystem subjected daily to high pHs and low DIC concentrations. Received: 3 July 1998 / Accepted: 30 November 1998     

The effect of pH on the inorganic carbon source for photosynthesis in the seagrass Zostera muelleri irmisch ex aschers

The ability of the seagrass Zostera muelleri Irmisch ex Aschers. to use HCO₃⁻ as well as CO₂ for photosynthesis was investigated by measuring photosynthetic O₂ evolution over a range of pH values. It was found that the apparent K_m CO₂ fell from 0.128 mM at pH 7.9 to 0.016 mM at pH 9.1 indicating that HCO₃⁻ as well as CO₂ may act as a substrate for photosynthesis.The true K_m CO₂ could not be determined due to inhibition of photosynthesis at pHs less than 7.8 K_m CO₂ must be at least 0.128 mM, the apparent K_m at pH 7.9, and is probably of the order of 0.200 mM CO₂, the same as that reported for other marine plants. K_m HCO₃⁻¹ is about 20 mM when CO₂-dependent photosynthesis is minimal. Such a high K_m HCO₃⁻ resembles values reported for freshwater, rather than marine plants.Photosynthetic O₂ evolution is not saturated with respect to total inorganic carbon in natural seawater (pH 8.2). It is suggested that the distinctive shoulder from pH 8.1 to 8.5 in the pH profile of photosynthetic O₂ evolution at a constant concentration of inorganic carbon is caused by an effect of pH on HCO₃⁻ uptake. The effect of pH on HCO₃⁻ uptake was determined by constructing a pH profile of photosynthesis at constant HCO₃⁻ concentration, and subtracting the estimated contribution of CO₂ to photosynthesis from this rate. The resultant curve has a maximum at pH 8.4 and declines sharply at pHs less than 8.     

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.     

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.     

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.     

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.