CO2 is the main inorganic C species entering photosynthetically active leaf protoplasts of the freshwater macrophyte Ranunculus penicillatus ssp. pseudofluitans

Submerged aquatic macrophytes growing in water where free CO₂ is unavailable (above pH 8·2) must use mechanisms to supply external dissolved inorganic carbon in a form available to chloroplasts (CO₂). Active transport of HCO₃^– across the plasmalemma has not been proven to be widespread in aquatic macrophytes and catalytic conversion of HCO₃^– to CO₂ is the usual supply mechanism in submerged macrophytes. The interaction of leaf form and function in this respect was investigated in the linear, submerged leaves of Ranunculus penicillatus (Dumort.) Bab ssp. pseudofluitans (Syme) S.Webster. Viable protoplasts were isolated using a mixture of cell wall degrading enzymes optimized for this species. Protoplast viabilities greater than 80% after 5 h of isolation were achieved. Photosynthetic rates of isolated protoplasts were comparable with that of intact plant tissue. Results of carbon isotopic disequilibrium experiments showed that CO₂ was the preferred species of dissolved inorganic carbon for photosynthesis by protoplasts and that HCO₃^– which predominates in the plant’s natural environment mainly contributes by supplying CO₂ outside the cells.     

A NEW METHOD FOR ESTIMATING EXTERNAL,CARBONIC ANHYDRASE ACTMTY IN MACROALGAE1,2

The effects of external carbonic anhydrase (CA, E.C. 4.2.1.1) on HCO^?₃and CO₂use under disequilibnum conditions were examined in 14 species of macroalgae. CO₂ was added to the algae in synthetic seawater free from inorganic carbon and buffered to pH 8.7. This resulted in a transient O₂ evolution, which was similar for most of the species tested: an initial rapid increase in the rate was followed by a gradual decrease, approaching a steady state within 10 min. The initial high rate of O₂evolution was attributed to diffusive entry of CO₂into the cells and the steady state to use of HCO^?₃. An enhancement of CO₂diffusive entry was obtained with acetazolamide, an inhibitor of external CA activity. Using this enhancement, a variable was developed to quantify the degree to which CO₂entry into the cell was prevented by the external CA. This variable, CA (%), was used as a measure of the external CA activity of the alga. Comparative measurements of the external CA activity using a potentiomtric method revealed that the new method was able to detect low levels of external CA activity, where the potentiometric method failed. These two different methods could be used together to increase the reliability of the measurements. The new method was useful for red and brown macroalgae and for those green macroalgae that lacked direct HCO₃uptake. Thus prominent external CA activity was found for Valonia utricularis (green), Halopteris scoparia (brown), and Osmunda pinnatifida (red), where the potentiometric method failed, or nearly failed, to indicate any external CA activity. Direct uptake of HCO^?₃interfered with the new method and the degree of this interference was dependent on the magnitude of the uptake.     

PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA,HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (C_i) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO₂ production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO₃^? as a source of C_i for its photosynthesis via the mediation of the external CA. CO₂ concentrations in the culture media affected its photosynthetic properties. A high level of CO₂ (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO₂ level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO₂ was a reduction in the photosynthetic CO₂ affinity. Decreased activity of the external CA in the high CO₂ grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO₂‐limited photosynthesis with the half‐saturating concentration of C_i exceeding that of seawater, did not operate active HCO₃^? uptake but used it via the extracellular CA for its photosynthetic carbon fixation.     

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.     

Carbonic anhydrase inhibitors that directly inhibit anion transport by the human Cl−/HCO3− exchanger,AE1

Carbonic anhydrases (CA, EC 4.2.1.1.) catalyze reversible hydration of CO₂ to HCO₃^?+H⁺. Bicarbonate transport proteins, which catalyze the transmembrane movement of membrane-impermeant bicarbonate, function in cooperation with CA. Since CA and bicarbonate transporters share the substrate, bicarbonate, we examined whether novel competitive inhibitors of CA also have direct inhibitory effects on bicarbonate transporters. We expressed the human erythrocyte membrane Cl^?/HCO₃^? exchanger, AE1, in transfected HEK293 cells as a model bicarbonate transporter. AE1 activity was assessed in both Cl^?/NO₃^? exchange assays, which were independent of CA activity, and in Cl^?/HCO₃^? exchange assays. Transport was measured by following changes of intracellular [Cl^?] and pH, using the intracellular fluorescent reporter dyes 6-methoxy-N-(3-sulfopropyl)quinolinium and 2′,7′-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, respectively. We examined the effect of 16 different carbonic anhydrase inhibitors on AE1 transport activity. Among these 12 were newly-reported compounds; two were clinically used non-steroidal anti-inflammatory drugs (celecoxib and valdecoxib) and two were anti-convulsant drugs (topiramate and zonisamide). Celecoxib and four of the novel compounds significantly inhibited AE1 Cl^?/NO₃^? exchange activity with EC₅₀ values in the range 0.22–2.8 μM. It was evident that bulkier compounds had greater AE1 inhibitory potency. Maximum inhibition using 40 μM of each compound was only 22–53% of AE1 transport activity, possibly because assays were performed in the presence of competing substrate. In Cl^?/HCO₃^? exchange assays, which depend on functional CA to produce transport substrate, 40 μM celecoxib inhibited AE1 by 62±4%. We conclude that some carbonic anhydrase inhibitors, including clinically-used celecoxib, will inhibit bicarbonate transport at clinically-significant concentrations.     

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     

The utilization of bicarbonate ions by the marine microalga Nannochloropsis oculata (Droop) Hibberd

HCO₃^? utilization by the marine microalga Nannochloropsis oculata was investigated using a pH drift technique in a closed system. Light-dependent alkalization of the medium resulted in a final pH of 10.5, confirming substantial HCO₃^? use by this alga. Alkalinity remained constant throughout the pH drift. Measurement of dissolved inorganic carbon (DIC) or the uptake of H¹⁴CO₃^? showed that nearly 50% of the total DIC remained external to the plasma membrane on completion of a pH drift. The rate of light-driven alkalization was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and thus was dependent on photosynthesis. Light-driven alkalization was not inhibited by a membrane-impermeable inhibitor of carbonic anhydrase (CA), dcxtran-bound sulphonamide (DBS), indicating that external CA was not involved in HCO₃^? utilization. The anion-cxchangc inhibitor 4′,4′-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) completely inhibited light-driven alkalization of the medium and H¹⁴CO₃^? uptake, providing unequivocal support for a direct uptake of H¹⁴CO₃^?. Chloride ions were essential for DIC-dependent photosynthetic oxygen evolution, suggesting that bicarbonate transport occurs by HCO₃^?/CI^? exchange.     

Governing effect of regulatory proteins for Cl−/HCO3− exchanger 2 activity

Anion exchanger 2 (AE2) has a critical role in epithelial cells and is involved in the ionic homeostasis such as Cl^? uptake and HCO₃^? secretion. However, little is known about the regulatory mechanism of AE2. The main goal of the present study was to investigate potential regulators, such as spinophilin (SPL), inositol-1,4,5-trisphosphate [IP₃] receptors binding protein released with IP₃ (IRBIT), STE20/SPS1-related proline/alanine-rich kinase (SPAK) kinase, and carbonic anhydrase XII (CA XII). We found that SPL binds to AE2 and markedly increased the Cl^?/HCO₃^? exchange activity of AE2. Especially SPL 1–480 domain is required for enhancing AE2 activity. For other regulatory components that affect the fidelity of fluid and HCO₃^? secretion, IRBIT and SPAK had no effect on the activity of AE2 and no protein-protein interaction with AE2. It has been proposed that CA activity is closely associated with AE activity. In this study, we provide evidence that the basolateral membrane-associated CA isoform CA XII significantly increased the activity of AE2 and co-localized with AE2 to the plasma membrane. Collectively, SPL and CA XII enhanced the Cl^?/HCO₃^? exchange activity of AE2. The modulating action of these regulatory proteins could serve as potential therapeutic targets for secretory diseases mediated by AE2.     

The effects of pH and dissolved inorganic carbon on external carbonic anhydrase activity in Chlorella saccharophila

External carbonic anhydrase (CA) activity in Chlorella saccharophila is suppressed by growth at high dissolved inorganic carbon and at acid pH. External CA activity was shown to be suppressed by growth at pHs below 7.0, with total repression at pH5.0. Growth in the presence of the buffer 3-[N-Morpholino]propane-sulphonic acid (MOPS) between pH 7 and 8 suppressed CA activity. Cells grown at pH8.0 aerated at 6 dm³ h^?1 exhibited external CA activity of 5 units mg^?1 Chl once the dissolved inorganic carbon (DIC) was reduced to 300 mmol m^?3, and this increased to 30 units mg^?1 Chl over a period of 3d while the DIC dropped to 30mmol m^?3. Cells aerated at 180 dm³ h^?1 showed a similar trend in CA activity, although the onset was delayed by 1 d and the DIC did not drop below 300 mmol m^?3. Cells grown at pH 7.8 near an air equilibrium DIC of 300 mmol m^?3had no detectable external CA activity. It is probable that it is the CO² supply to the cell, and not total DIC or HCO^?₃ which controls external CA activity. Cells grown at pH 5.0 had no detectable activity, although they reduced the CO² concentration to 0.6 mmol m^?3. The loss of CA upon transfer of air-grown cells to 10 mmol mol^?1 CO² took place over 48 h and was light dependent, while the loss upon transfer from alkaline pH to acid pH look place over 12 h and was independent of light. The effects of pH are independent of the response to CO₂.     

Bicarbonate uptake via an anion exchange protein is the main mechanism of inorganic carbon acquisition by the giant kelp Macrocystis pyrifera (Laminariales,Phaeophyceae) under variable pH

Macrocystis pyrifera is a widely distributed, highly productive, seaweed. It is known to use bicarbonate (HCO₃^?) from seawater in photosynthesis and the main mechanism of utilization is attributed to the external catalyzed dehydration of HCO₃^? by the surface‐bound enzyme carbonic anhydrase (CA_ext). Here, we examined other putative HCO₃^? uptake mechanisms in M. pyrifera under pH_T 9.00 (HCO₃^?: CO₂ = 940:1) and pH_T 7.65 (HCO₃^?: CO₂ = 51:1). Rates of photosynthesis, and internal CA (CA_int) and CA_ext activity were measured following the application of AZ which inhibits CA_ext, and DIDS which inhibits a different HCO₃^? uptake system, via an anion exchange (AE) protein. We found that the main mechanism of HCO₃^? uptake by M. pyrifera is via an AE protein, regardless of the HCO₃^?: CO₂ ratio, with CA_ext making little contribution. Inhibiting the AE protein led to a 55%–65% decrease in photosynthetic rates. Inhibiting both the AE protein and CA_ext at pH_T 9.00 led to 80%–100% inhibition of photosynthesis, whereas at pH_T 7.65, passive CO₂ diffusion supported 33% of photosynthesis. CA_int was active at pH_T 7.65 and 9.00, and activity was always higher than CA_ext, because of its role in dehydrating HCO₃^? to supply CO₂ to RuBisCO. Interestingly, the main mechanism of HCO₃^? uptake in M. pyrifera was different than that in other Laminariales studied (CA_ext‐catalyzed reaction) and we suggest that species‐specific knowledge of carbon uptake mechanisms is required in order to elucidate how seaweeds might respond to future changes in HCO₃^?:CO₂ due to ocean acidification.     

UPTAKE,EFFLUX, AND PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON BY THE MARINE EUSTIGMATOPHYTE NANNOCHLOROPSIS SP.1

Uptake, efflux and utilization of inorganic carbon were investigated in the marine eustigmatophyte Nannochloropsis sp. grown under an air level of CO₂. Maximal photosynthetic rate was hardly affected by raising the pH porn 5.0 to 9.0. The apparent photosynthetic affinity for dissolved inorganic carbon (DIC) was 35 μM DIC between pH 6.5 to 9.0, but increased approximately threefold at pH 5.0 suggesting that HCO₃- was the main DIC species used from the medium. No external carbonic anhydrase (CA) activity could be detected by the pH drift method. However, application of ethoxyzolamide (an inhibitor of CA) resulted an a significant inhibition of photosynthetic O₂ evolution and carbon utilization, suggesting involvement of internal CA or CA-like activity in DIC utilization. Under high light conditions, the rate of HCO₃^? uptake and its internal conversion to CO₂ apparently exceeded the rate of carbon fixation, resulting in a large leak of CO₂ from the cells to the external medium. When the cells were exposed to low DIC concentrations, the ratio of internal to external DIC concentration was about eight. On the other hand, in the presence of 2 mM DIC, conditions prevailing in the marine environment, the internal concentration of DIC was only 50% higher than the external one.     

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.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

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.     

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.     

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.     

Studies of Elodea nuttallii grown under photorespiratory conditions. I. Photosynthetic characteristics

Abstract. The photosynthetic characteristics of Elodea nuttallii grown in wastewater in continuous flow reactors in a greenhouse were investigated. The diurnal changes in dissolved inorganic carbon (DIC), dissolved oxygen (DO) and pH were monitored. Photosynthesis removed both CO₂(aq) and HCO₃^? from the reactors. A stoichiometry of 1.19:1 was observed between HCO₃^? removal during photosynthesis and OH^? production during photosynthesis, consistent with theories regarding direct bicarbonate utilization. In laboratory experiments, the light compensation points (г_PPFD) were similar (31–35μmol m^?2 s^?1) to reported values for other macrophytes; however, the light saturation level was high (1100μmol m^?2 s^?1) and similar to values reported for aerial portions Of heterophyllous macrophytes. The kinetics of photosynthetic oxygen evolution (K_m (CO₂) = 96mmol m^?3; V_max= 133mmol g^?1 Chl h^?1) and the CO₂ compensation point (г= 44cm³ m^?3) suggested an adaptive, low photorespiratory state in response to low carbon concentrations. Photosynthetic V_max values were slightly, but significantly higher (P 0.001) at pH 8.0 compared to pH 4.5. While CO₂ utilization at pH 8 could account for most of the observed phototsynthetic rates, an HCO₃^? component was present, suggesting two separate transport systems for HCO₃^? and CO₂(aq) in E. nuttallii. The activity of RUBISCO (160.3 mmol g^?1 Chl h^?1 was one of the highest reported values for aquatic macrophytes. Compared to RUBISCO, we observed lower activities of the β-carboxylating enzymes phopho enolpyruvate carboyxlase (PEPcase), 24.1 mmol g^?1 Chl h^?1; phosphor enol pyruvate carboxykinase (PEPCKase), 14 mmol g^?1 Chl h^?1. This suggests that the potential light-independent fixation of carbon in E. nuttallii was much less than RUBISCO-dependent fixation. The RUBISCO/PEPcase ratio was 6.6, indicating that E. nuttallii was similar to Myriophyllum sp. in possessing a physiological adaptation to low CO₂ levels which is hypothesized to include carbonic anhydrase (CA) and an active transport system for HCO₃^?. CA levels were surprisingly low in E. nuttallii (14.2 EUmg Chl^?).     

The effects of carbonic anhydrase inhibitors formate,bicarbonate, acetazolamide,and imidazole on photosystem II in maize chloroplasts

Inhibitors of carbonic anhydrase were tested for their effects on Photosystem II (PS II) activity in chloroplasts. We find that formate inhibition of PS II turnover rates increases as the pH of the reaction medium is lowered. Bicarbonate ions can inhibit PS II turnover rates. The relative potency of the anionic inhibitors N₃^?, I^?, OA_c^?, and Cl^? is the same for both carbonic anhydrase and PS II. The inhibitory effect of acetazolamide on PS II increases as light intensity decreases, indicating a lowering of quantum yields in the presence of the inhibitor. Imidazole inhibition of PS II increases with pH in a manner suggesting that the unprotonated form of the compound is inhibitory. Formate, bicarbonate, acetazolamide, and imidazole all inhibit DCMU-insensitive, silicomolybdate-supported oxygen evolution, indicating that the site(s) of action of the inhibitors is at, or before, the primary stable PS II electron acceptor Q. This inhibitory effect of low levels of HCO₃^? along with the known enhancement by HCO₃^? of quinone-mediated electron flow suggests an antagonistic control effect on PS II photochemistry. We conclude that the responses of PS II to anions (formate, bicarbonate), acetazolamide, and imidazole are analogous to the responses shown by carbonic anhydrase. These findings suggest that the enzyme carbonic anhydrase may provide a model system to gain insight into the “bicarbonate-effect” associated with PS II in chloroplasts.     

The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH

The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO₃⁻ thereby showing that C. reinhardii has the ability to utilize exogenous HCO₃⁻. Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K_½^DIC from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO₃ addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO₂ transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO₂ and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga.     

Requirement for carbonic anhydrase activity in processes other than photosynthetic Inorganic carbon assimilation

A number of non-green plant tissues have high rates of HCO₃⁻-consuming reactions in the cytosol, i.e. C₄ dicarboxylic acid production preceding organic acid anion transport into dicarboxylate consuming compartments in N₂-fixing root nodules, in lipogenic tissues, and in thermogenic aroid spadices and, in the case of lipogenic tissues, in acetyl CoA incorporation into lipid in plastid stroma. Since inorganic C supply to the cytosol or stroma by decarboxylation reactions, and by transmembrane fluxes, involves only CO₂, the HCO₃⁻ consumed in the rapid metabolic processes must originate from hydration (hydroxylation) of CO₂. Computations based on the first-order rate constant for uncatalysed conversion of CO₂ to HCO₃⁻ and the most likely in vivo CO₂ concentration show that the uncatalysed reaction is possibly adequate to supply the observed HCO₃⁻ requirement in the HCO₃⁻-consuming compartments. However, carbonic anhydrase activity is well established in legume root nodules, and also appears to occur in aroid spadices. In addition to coping with any heterogeneities in HCO₃⁻, consumption in the cytosol, the root nodule activity may be involved in optimizing haemoglobin function. Further work is needed on carbonic anhydrase expression is tissues with rapid HCO₃⁻ consumption, especially in view of reports of negligible carbonic anhydrase activity in some non-green plant tissues. Other possible roles of carbonic anhydrase in non-green plant tissues are briefly discussed.