Light-induced alkalization of the chloroplast stroma in vivo as estimated from the CO2 capacity of intact sunflower leaves

Rapid CO₂ gas exchange by Helianthus leaves was analysed kinetically using a computer model which distinguished different components of the gas exchange by different time constants. A rapid phase of CO₂ uptake was ascribed to the solubilization of CO₂ in all leaf compartments and to the conversion of the dissolved CO₂ to HCO₃⁻ in the chloroplast stroma which contains carbonic anhydrase. From stromal $\text{HCO}{}_3{}^{\mathrm{-}}\text{CO}{}_2$ ratios the stroma pH of darkened leaves was estimated to be close to 7.5. Occasionally, values as high as 8 or as low as 7 were also obtained. If fast HCO₃⁻ formation also occurs in the cytosol, pH values may be lower by about 0.3 pH units than those calculated under the assumption that carbonic anhydrase is localized in chloroplasts only. Illumination with a light intensity close to saturation of photosynthesis caused an increase in CO₂ solubilization which indicated the alkalization of the chloroplast stroma by about 0.6 pH units. This is an underestimation, if the pH of cytosol decreases in the light liberating CO₂ by the action of carbon anhydrase. An alkalization of the stroma by 0.6 pH units indicates the export of about 450 nmol H⁺/mg chlorophyll from the stroma. This forms the basis of a large transthylakoid pH gradient which drives light-dependent ATP synthesis. A pH gradient between stroma and cytosol is capable of supporting secondary gradients between these compartments in the light, such as a gradient in the $\text{ATP}\text{ADP}$ ratio. On darkening, the stroma alkalization was reversed. The rate of stroma acidification was much higher in the presence of CO₂ than in its absence.     

A role for mitochondrial carbonic anhydrase in limiting CO2 leakage from low CO2-grown cells of Chlamydomonas reinhardtii

A model is presented which quantifies a possible role for the carbonic anhydrase in the mitochondrial matrix of Chlamydomonas reinhardtii which incorporates the observation that the expression of this enzyme is increased under growth conditions in which the expression of the carbon dioxide-concentrating mechanism is increased. It is assumed that the inorganic carbon enters the cytosol from the medium, and leaves the cytosol to the plastids, as HCO₃⁻ and that there is negligible carbonic anhydrase activity in the cytosol. The role of the mitochondrial carbonic anhydrase is suggested to be the conversion to HCO₃^– of the CO₂ produced in the mitochondria in the light from tricarboxylic acid cycle activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity which is not suppressed by the carbon concentrating mechanism. If there is a HCO₃⁻ channel in the inner mitochondrial membrane then almost all of the inorganic carbon leaves the mitochondria as HCO₃⁻, thus limiting the potential for CO₂ leakage through the plasmalemma. This mechanism could increase inorganic C supply to ribulose bisphosphate carboxylase-oxygenase by some 10% at the energetic expense of less than 1% of the total ATP generation by plastids plus mitochondria.     

CO2-concentrating mechanisms: a direct role for thylakoid lumen acidification?

A testable mechanism of CO₂ accumulation in photolithotrophs, originally suggested by Pronina & Semenenko, is quantitatively analysed. The mechanism involves (as does the most widely accepted hypothesis) the delivery of HCO₃^? to the compartment containing Rubisco. It differs in proposing subsequent HCO₃^? entry (by passive uniport) to the thylakoid lumen, followed by carbonic anhydrase activity in the lumen; uncatalysed conversion of HCO₃^? to CO₂, even at the low pH of the lumen, is at least 300 times too slow to account for the rate of inorganic C acquisition. Carbonic anhydrase converts the HCO₃^? to CO₂ at the lower pH maintained in the illuminated thylakoid lumen by the light-driven H⁺ pump, generating CO₂ at 10 times or more the thylakoid HCO₃^? concentration. Efflux of this CO₂ can suppress Rubisco oxygenase activity and stimulate carboxylase activity in the stroma. This mechanism differs from the widely accepted hypotheses in the required location of carbonic anhydrase, i.e. in the thylakoid lumen rather than the stroma or pyrenoid, and in the need for HCO₃^? influx to thylakoids. The capacity for anion (assayed as Cl^?) entry by passive uniport reported for thylakoid membranes is adequate for the proposed mechanism; if the Cl^? channel does not transport HCO₃^?, HCO₃^? entry could be by combination of the Cl^? channel with a Cl^? HCO₃^? antiporter. This mechanism is particularly appropriate for organisms which lack overt accumulation of total inorganic C in cells, but which nevertheless have the gas exchange characteristics of an organism with a CO₂-concentrating mechanism.     

Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

Carbonic anhydrase in Ranunculus penicillatus spp. pseudofluitans: activity, location and implications for carbon assimilation

Levels of carbonic anhydrase activity were determined on a total (60 E_Umg^?1 protein), external (7.36 E_U), internal (50.14 E_U) and protoplast (15.63 E_U) basis for Ranunculus penicillatus (Dumort.) Bab ssp. pseudofluitans (Syme) S. Webster, a freshwater aquatic macrophyte, by conventional electrometric methods. The site of activity of ‘external’ carbonic anhydrase (CA) has been visualized using 5-Dimethylaminonapthalene-1- sulphonamide (DNSA)-CA fluorescent complex formation, and is postulated to be closely associated with the epidermal cell wall. The photosynthetic rate of R. penicillatus ssp. pseudofluitans at pH 9.0 is in excess of the uncatalysed rate of production of CO₂ from HCO^?₃, and this plant is therefore using HCO^?₃ for photosynthesis. The possible contribution of CA activity to inorganic carbon assimilation, and specifically to transport of HCO^?₃, in submerged aquatic plants is discussed.     

Influencing factors and formation mechanism of CaCO3 precipitation induced by microbial carbonic anhydrase

Microbial carbonic anhydrase promotes carbonate deposition, which is important in the formation and evolution of global carbon cycle and geological processes. A kind of bacteria producing extracellular carbonic anhydrase was selected to study the effects of temperature, pH value and Ca²⁺ concentration on bacterial growth, carbonic anhydrase activity and calcification rate in this paper. The results showed that the activity of carbonic anhydrase at 30 °C was the highest, which was beneficial to the calcification reaction, calcification rate of CaCO₃ was the fastest in alkaline environment with the initial pH value of 9.0. When the Ca2+ concentration was 60 mM, compared with other Ca²⁺ concentration, CA bacteria could grow and reproduce best, and the activity of bacteria was the highest, too low Ca²⁺ concentration would affect the generation of CaCO₃, while too high Ca²⁺ concentration would seriously affect the growth of bacteria and reduce the calcification rate. Finally, the mechanism of CaCO₃ precipitation induced by microbial carbonic anhydrase was studied. Carbonic anhydrase can accelerate the hydration of CO₂ into HCO₃⁻, and react with OH⁻ and Ca²⁺ to form CaCO₃ precipitation in alkaline environment and in the presence of calcium source.     

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO₃^?/CO₂ as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α?carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α?carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO₃^?/CO₂. Addition of exogenous HCO₃^? or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

A Model for HCO(3) Accumulation and Photosynthesis in the Cyanobacterium Synechococcus sp: Theoretical Predictions and Experimental Observations

A simple model based on HCO₃⁻ transport has been developed to relate photosynthesis and inorganic carbon fluxes for the marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1). Predicted relationships between inorganic carbon transport, CO₂ fixation, internal carbonic anhydrase activity, and leakage of CO₂ out of the cell, allow comparisons to be made with experimentally obtained data. Measurements of inorganic carbon fluxes and internal inorganic carbon pool sizes in these cells were made by monitoring time-courses of CO₂ changes (using a mass spectrometer) during light/dark transients. At just saturating CO₂ conditions, total inorganic carbon transport did not exceed net CO₂ fixation by more than 30%. This indicates CO₂ leakage similar to that estimated for C₄ plants.

For this leakage rate, the model predicts the cell would need a conductance to CO₂ of around 10⁻⁵ centimeters per second. This is similar to estimates made for the same cells using inorganic carbon pool sizes and CO₂ efflux measurements. The model predicts that carbonic anhydrase is necessary internally to allow a sufficiently fast rate of CO₂ production to prevent a large accumulation of HCO₃⁻. Intact cells show light stimulated carbonic anhydrase activity when assayed using ¹⁸O-labeled CO₂ techniques. This is also supported by low but detectable levels of carbonic anhydrase activity in cell extracts, sufficient to meet the requirements of the model.

     

Carbonic Anhydrase Activity and Localization in Some Plant Species

The aim of this work concerned the study of the differences in the carbonic anhydrase activity and localization between plant species, the photosynthesis of which is carried out according to the C₃ and C₄ pathways respectively. The measurement of enzymatic activity was made with a titrimetric evaluation of the rate of the reaction CO₂+ H₂O ? H⁺+ HCO^?₃. The C₃ plant species showed higher activities than the C₄ species. The localization of carbonic anhydrase was carried out with a histochemical method. The carbonic anhydrase appeared in the chloroplasts both in the mesophyll and the bundle sheath without any difference between C₃ and C₄ plants.     

Active CO(2) Transport by the Green Alga Chlamydomonas reinhardtii

Mass spectrometric measurements of dissolved free ¹³CO₂ were used to monitor CO₂ uptake by air grown (low CO₂) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the ¹³CO₂ concentration in the medium to close to zero. Similar results were obtained with low CO₂ cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO₂ uptake revealed that the removal of CO₂ from the medium in the light was due to selective and active CO₂ transport rather than uptake of total dissolved inorganic carbon. In the light, low CO₂ cells and protoplasts incubated with carbonic anhydrase took up CO₂ at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO₂ uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO₂ cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO₂ compensation point. During the linear uptake of CO₂, low CO₂ cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O₂ evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O₂ evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO₂ uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO₂ had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O₂ evolution. These results show low CO₂ cells of Chlamydomonas are able to transport both CO₂ and HCO₃⁻ but CO₂ is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO₂ from the dehydration of HCO₃⁻.     

The roles of carbonic anhydrases in photosynthetic CO2 concentrating mechanisms

Cyanobacteria, algae, aquatic angiosperms and higher plants have all developed their own unique versions of photosynthetic CO₂ concentrating mechanisms (CCMs) to aid Rubisco in efficient CO₂ capture. An important aspect of all CCMs is the critical roles that the specialised location and function that various carbonic anhydrase enzymes play in the overall process, participating the interconversion of CO₂ and HCO₃ ⁻ species both inside and outside the cell. This review examines what we currently understand about the nature of the carbonic anhydrase enzymes, their localisation and roles in the various CCMs that have been studied in detail. This revised version was published online in August 2006 with corrections to the Cover Date.     

Oxygen-18 Exchange as a Measure of Accessibility of CO(2) and HCO(3) to Carbonic Anhydrase in Chlorella vulgaris (UTEX 263)

We have measured the exchange of ¹⁸O between CO₂ and H₂O in stirred suspensions of Chlorella vulgaris (UTEX 263) using a membrane inlet to a mass spectrometer. The depletion of ¹⁸O from CO₂ in the fluid outside the cells provides a method to study CO₂ and HCO₃⁻ kinetics in suspensions of algae that contain carbonic anhydrase since ¹⁸O loss to H₂O is catalyzed inside the cells but not in the external fluid. Low-CO₂ cells of Chlorella vulgaris (grown with air) were added to a solution containing ¹⁸O enriched CO₂ and HCO₃⁻ with 2 to 15 millimolar total inorganic carbon. The observed depletion of ¹⁸O from CO₂ was biphasic and the resulting ¹⁸C content of CO₂ was much less than the ¹⁸O content of HCO₃⁻ in the external solution. Analysis of the slopes showed that the Fick's law rate constant for entry of HCO₃⁻ into the cell was experimentally indistinguishable from zero (bicarbonate impermeable) with an upper limit of 3 × 10⁻⁴ s⁻¹ due to our experimental errors. The Fick's law rate constant for entry of CO₂ to the sites of intracellular carbonic anhydrase was large, 0.013 per second, but not as great as calculated for no membrane barrier to CO₂ flux (6 per second). The experimental value may be explained by a nonhomogeneous distribution of carbonic anhydrase in the cell (such as membrane-bound enzyme) or by a membrane barrier to CO₂ entry into the cell or both. The CO₂ hydration activity inside the cells was 160 times the uncatalyzed CO₂ hydration rate.     

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.     

Carbonic Anhydrase and the Uptake of Inorganic Carbon by Synechococcus sp. (UTEX-2380)

We report the changes in the concentrations and ¹⁸O contents of extracellular CO₂ and HCO₃⁻ in suspensions of Synechococcus sp. (UTEX 2380) using membrane inlet mass spectrometry. This marine cyanobacterium is known to have an active uptake mechanism for inorganic carbon. Measuring ¹⁸O exchange between CO₂ and water, we have found the intracellular carbonic anhydrase activity to be equivalent to 20 times the uncatalyzed CO₂ hydration rate in different samples of cells that were grown on bubbled air (low-CO₂ conditions). This activity was only weakly inhibited by ethoxzolamide with an I₅₀ near 7 to 10 micromolar in lysed cell suspensions. We have shown that even with CO₂-starved cells there is considerable generation of CO₂ from intracellular stores, a factor that can cause errors in measurement of net CO₂ uptake unless accounted for. It was demonstrated that use of ¹³C-labeled inorganic carbon outside the cell can correct for such errors in mass spectrometric measurement. Oxygen-18 depletion experiments show that in the light, CO₂ readily passes across the cell membrane to the sites of intracellular carbonic anhydrase. Although HCO₃⁻ was readily taken up by the cells, these experiments shown that there is no significant efflux of HCO₃⁻ from Synechococcus.     

Bicarbonate-Reversible and Irreversible Inhibition of Photosystem II by Monovalent Anions

We tested a number of inhibitory monovalent anions for their primary site of action on photosystem II(PSII) in chloroplasts. We find that the inhibitory effects of F⁻, HCO₂⁻, NO₂⁻, NO₃⁻, and CH₃CO₂⁻ are all reversed by addition of a high concentration of HCO₃⁻. This class of anions competitively inhibits H¹⁴CO₃⁻ binding to PSII. All of those anions tested reduced H¹⁴CO₃⁻ binding more in the light than in the dark. We conclude that the primary inhibitory site of action of a number of monovalent anions is at the HCO₃⁻ binding site(s) on the PSII complex. The carbonic anhydrase inhibitor gold cyanide, and also azide, inhibit PSII but at a site other than the HCO₃⁻ binding site. We suggest that the unique ability of HCO₃⁻ to reverse the effects of inhibitory anions reflects its singular ability to act as a proton donor/acceptor at the anion binding site. A similar role has been proposed for non-substrate-bound HCO₃⁻ on carbonic anhydrase by Yeagle et al. (1975 Proc Natl Acad Sci USA 72: 454-458).     

Carbonic acid as the host signal for the development of parasitic stages of nematodes

Petronijevic T., Rogers W. P. and Sommerville R. I. 1985. Carbonic acid as the host signal for the development of parasitic stages of nematodes. International Journal for Parasitology15: 661–667. This paper gives results on which may be based an identification of the component of the system CO₂ + H₂O ai H₂CO₃ ai H⁺ HCO₃^? which acts as the stimulus from the animal host for some nematodes. Using infective juveniles of Nematospiroides dubius and Haemonchus contortus, the effects on exsheathment of (1) low pCO₂ values, (2) the presence of carbonic anhydrase in the stimulating medium, and (3) the inhibition of carbonic anhydrase within the juveniles have been examined. The results lead to the suggestion that it is the “readily available” undissociated H₂CO₃, or H₂CO₃ + HCO₃^? which is the critical factor in the stimulus for development. The wide range of [H⁺]s over which “readily available” H₂CO₃ is present in physiological environments suggests that this host signal may be important for infection with many species.     

PHOTOSYNTHETIC PROPERTIES OF PROTOPLASTS,AS COMPARED WITH THALLI,OF ULVA FASCIATA (CHLOROPHYTA)1

Protoplasts were prepared from Ulva fasciata Delile, and their photosynthetic performance was measured and compared with that of thalli discs. These protoplasts maintained maximal rates of photosynthesis as high as those of thalli (up to 300 μmol O₂·mg chlorophyll^?1·h^?1) for several hours after preparation and were therefore considered suitable for kinetic studies of inorganic carbon utilization. The photosynthetic K_1/2(inorganic carbon) at pH 6.1 was 3.8 μM and increased to 67, 158, and 1410 μM at the pH values 7.0, 7.9, and 8.9, respectively. Compared with these protoplasts, thalli had a much lower affinity for CO₂ but approximately the same affinity for HCO₃^?. Comparisons between rates of photosynthesis and the spontaneous dehydration of HCO₃^? (at 50 μM inorganic carbon) revealed that photosynthesis of both protoplasts (which lacked apparent activity of extracellular/surface-bound carbonic anhydrase) and thalli (which were only 25% inhibited by the external carbonic anhydrase inhibitor acetazolamide) could not be supported by CO₂ formation in the medium at the higher pH values, indicating HCO₃^? uptake. Since both protoplasts and thalli were sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulfonate, we suggest that HCO₃^? transport was facilitated by the membrane-located anion exchange protein recently reported to function in certain Ulva thalli. These findings suggest that the presence of a cell wall may constitute a diffusion barrier for CO₂, but not for HCO₃^?, utilization under natural seawater conditions.     

Carbonic anhydrase activity in leaves and its role in the first step of c(4) photosynthesis

In C₄ plants carbonic anhydrase catalyzes the critical first step of C₄ photosynthesis, the hydration of CO₂ to bicarbonate. The maximum activity of this enzyme in C₄ leaf extracts, measured by H⁺ production with saturating CO₂ and extrapolated to 25°C, was found to be 3,000 to 10,000 times the maximum photosynthesis rate for these leaves. Similar activities were found in C₃ leaf extracts. However, the calculated effective activity of this enzyme at in vivo CO₂ concentrations was apparently just sufficient to prevent the rate of conversion of CO₂ to HCO₃⁻ from limiting C₄ photosynthesis. This conclusion was supported by the mass spectrometric determination of leaf carbonic anhydrase activities.