Two Systems for Concentrating CO(2) and Bicarbonate during Photosynthesis by Scenedesmus

Scenedesmus cells grown on high CO₂, when adapted to air levels of CO₂ for 4 to 6 hours in the light, formed two concentrating processes for dissolved inorganic carbon: one for utilizing CO₂ from medium of pH 5 to 8 and one for bicarbonate accumulation from medium of pH 7 to 11. Similar results were obtained with assays by photosynthetic O₂ evolution or by accumulation of dissolved inorganic carbon inside the cells. The CO₂ pump with K_0.5 for O₂ evolution of less than 5 micromolar CO₂ was similar to that previously studied with other green algae such as Chlamydomonas and was accompanied by plasmalemma carbonic anhydrase formation. The HCO₃⁻ concentrating process between pH 8 to 10 lowered the K_0.5 (DIC) from 7300 micromolar HCO₃⁻ in high CO₂ grown Scenedesmus to 10 micromolar in air-adapted cells. The HCO₃⁻ pump was inhibited by vanadate (K_i of 150 micromolar), as if it involved an ATPase linked HCO₃⁻ transporter. The CO₂ pump was formed on low CO₂ by high-CO₂ grown cells in growth medium within 4 to 6 hours in the light. The alkaline HCO₃⁻ pump was partially activated on low CO₂ within 2 hours in the light or after 8 hours in the dark. Full activation of the HCO₃⁻ pump at pH 9 had requirements similar to the activation of the CO₂ pump. Air-grown or air-adapted cells at pH 7.2 or 9 accumulated in one minute 1 to 2 millimolar inorganic carbon in the light or 0.44 millimolar in the dark from 150 micromolar in the media, whereas CO₂-grown cells did not accumulate inorganic carbon. A general scheme for concentrating dissolved inorganic carbon by unicellular green algae utilizes a vanadate-sensitive transporter at the chloroplast envelope for the CO₂ pump and in some algae an additional vanadate-sensitive plasmalemma HCO₃⁻ transporter for a HCO₃⁻ pump.     

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.     

High Affinity Transport of CO(2) in the Cyanobacterium Synechococcus UTEX 625

The active transport of CO₂ in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO₃⁻ transport. The substrate concentration required to give one half the maximum rate for whole cell CO₂ transport was determined to be 0.4 ± 0.2 micromolar (mean ± standard deviation; n = 7) with a range between 0.2 and 0.66 micromolar. The maximum rates of CO₂ transport ranged between 400 and 735 micromoles per milligram of chlorophyll per hour with an average rate of 522 for seven experiments. This rate of transport was about three times greater than the dissolved inorganic carbon saturated rate of photosynthetic O₂ evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO₂ transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO₂ transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO₂ transport was caused by HCO₃⁻ or by Na⁺-dependent HCO₃⁻ transport. However, ¹²CO₂ readily interfered with ¹³CO₂ transport. CO₂ transport and Na⁺-dependent HCO₃⁻ transport are separate, independent processes and the high affinity CO₂ transporter is not only responsible for the initial transport of CO₂ into the cell but also for scavenging any CO₂ that may leak from the cell during ongoing photosynthesis.     

Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO(3)-Limited Chemostats

Synechococcus leopoliensis was grown in HCO₃⁻-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO₂). The O₂ to CO₂ ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO₃⁻ rather than CO₂ for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO₃⁻-limited cells and also had a reduced capacity for HCO₃⁻ transport. HCO₃⁻-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO₂ grown cells. However, the low affinity cells of the present study were growing at CO₂ concentrations less than air saturation. This suggests that supranormal levels of CO₂ not required to induce the physiological changes usually ascribed to high CO₂ cells.     

Use of Carbon Oxysulfide, a Structural Analog of CO(2), to Study Active CO(2) Transport in the Cyanobacterium Synechococcus UTEX 625

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO₂. Although hydrolysis of COS (to CO₂ and H₂S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO₂ transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO₂ and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H₂S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H₂S. The active transport of CO₂ was inhibited by COS in an apparently competitive manner. When Na⁺-dependent HCO₃⁻ transport was allowed in the presence of COS, the extracellular [CO₂] rose considerably above the equilibrium level. This CO₂ appearing in the medium was derived from the dehydration of transported HCO₃⁻ and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO₂ was inhibited. These results showed that the Na⁺-dependent HCO₃⁻ transport was not inhibited by COS, whereas active CO₂ transport was inhibited. When COS was removed by gassing with N₂, a normal pattern of CO₂ uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO₃⁻ transport or CO₂ fixation. The steady state rate of CO₂ fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO₂ from the cells that occurred when CO₂ uptake was inhibited by COS. Neither CS₂ nor N₂O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO₂ transport.     

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.     

Active Transport of CO(2) by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry

Mass spectrometry has been used to confirm the presence of an active transport system for CO₂ in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO₃ in the absence of Na⁺ (to prevent HCO₃⁻ transport). Upon illumination the cells rapidly removed almost all the free CO₂ from the medium. Addition of carbonic anhydrase revealed that the CO₂ depletion resulted from a selective uptake of CO₂, rather than a total uptake of all inorganic carbon species. CO₂ transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO₂ fixation but had little effect on CO₂ transport. In iodoacetamide poisoned cells, transport of CO₂ occurred against a concentration gradient of about 18,000 to 1. Transport of CO₂ was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO₂ transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na⁺ (<100 microequivalents per liter), but not of K⁺, stimulated CO₂ transport as much as 2.4-fold. Unlike Na⁺-dependent HCO₃⁻ transport, the transport of CO₂ was not inhibited by high concentrations (30 milliequivalents per liter) of Li⁺. During illumination, the CO₂ concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO₂ transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO₃⁻ to CO₂ would rapidly raise the CO₂ concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO₂ transport was balanced by HCO₃⁻ efflux.     

Selective and Reversible Inhibition of Active CO(2) Transport by Hydrogen Sulfide in a Cyanobacterium

The active transport of CO₂ in the cyanobacterium Synechococcus UTEX 625 was inhibited by H₂S. Treatment of the cells with up to 150 micromolar H₂S + HS⁻ at pH 8.0 had little effect on Na⁺-dependent HCO₃⁻ transport or photosynthetic O₂ evolution, but CO₂ transport was inhibited by more than 90%. CO₂ transport was restored when H₂S was removed by flushing with N₂. At constant total H₂S + HS⁻ concentrations, inhibition of CO₂ transport increased as the ratio of H₂S to HS⁻ increased, suggesting a direct role for H₂S in the inhibitory process. Hydrogen sulfide does not appear to serve as a substrate for transport. In the presence of H₂S and Na⁺ -dependent HCO₃⁻ transport, the extracellular CO₂ concentration rose considerably above its equilibrium level, but was maintained far below its equilibrium level in the absence of H₂S. The inhibition of CO₂ transport, therefore, revealed an ongoing leakage from the cells of CO₂ which was derived from the intracellular dehydration of HCO₃⁻ which itself had been recently transported into the cells. Normally, leaked CO₂ is efficiently transported back into the cell by the CO₂ transport system, thus maintaining the extracellular CO₂ concentration near zero. It is suggested that CO₂ transport not only serves as a primary means of inorganic carbon acquisition for photosynthesis but also serves as a means of recovering CO₂ lost from the cell. A schematic model describing the relationship between the CO₂ and HCO₃⁻ transport systems is presented.     

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.     

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.     

Photosynthesis and Inorganic Carbon Usage by the Marine Cyanobacterium, Synechococcus sp

The marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1) changes its affinity for external inorganic carbon used in photosynthesis, depending on the concentration of CO₂ provided during growth. The high affinity for CO₂ + HCO₃⁻ of air-grown cells (K_½ < 80 nanomoles [pH 8.2]) would seem to be the result of the presence of an inducible mechanism which concentrates inorganic carbon (and thus CO₂) within the cells. Silicone-oil centrifugation experiments indicate that the inorganic carbon concentration inside suitably induced cells may be in excess of 1,000-fold greater than that in the surrounding medium, and that this accumulation is dependent upon light energy. The quantum requirements for O₂ evolution appear to be some 2-fold greater for low CO₂-grown cells, compared with high CO₂-grown cells. This presumably is due to the diversion of greater amounts of light energy into inorganic carbon transport in these cells.

A number of experimental approaches to the question of whether CO₂ or HCO₃⁻ is primarily utilized by the inorganic carbon transport system in these cells show that in fact both species are capable of acting as substrate. CO₂, however, is more readily taken up when provided at an equivalent concentration to HCO₃⁻. This discovery suggests that the mechanistic basis for the inorganic carbon concentrating system may not be a simple HCO₃⁻ pump as has been suggested. It is clear, however, that during steady-state photosynthesis in seawater equilibrated with air, HCO₃⁻ uptake into the cell is the primary source of internal inorganic carbon.

     

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae

Rates of photosynthetic O₂ evolution, for measuring K_0.5(CO₂ + HCO₃⁻) at pH 7, upon addition of 50 micromolar HCO₃⁻ to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K₁(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO₂ uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O₂ evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO₃), and the rate of ¹⁴CO₂ fixation with 100 micromolar [¹⁴C] HCO₃⁻. Salicylhydroxamic acid inhibition of O₂ evolution and ¹⁴CO₂-fixation was reversed by higher levels of NaHCO₃. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO₂ accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.     

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.     

Inorganic Carbon Uptake during Photosynthesis : II. Uptake by Isolated Asparagus Mesophyll Cells during Isotopic Disequilibrium

The species of inorganic carbon (CO₂ or HCO₃⁻) taken up a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO₂ or HCO₃⁻ transport, during steady state photosynthesis, is achieved by monitoring the changes (by ¹⁴C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO₂ or HCO₃⁻ transport) and experimental time-courses of ¹⁴C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO₂, rather than HCO₃⁻, is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO₂ is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO₃⁻ transport, as the incorporation of ¹⁴C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO₂ uptake alone. The contribution of HCO₃⁻ to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO₃⁻ concentration. The evidence for direct HCO₃⁻ transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of ¹⁴C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO₂, which is partially alleviated by a high extracellular concentration of HCO₃⁻.     

Characterization of the na-requirement in cyanobacterial photosynthesis

The Na⁺ requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li⁺ concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O₂ evolution) as a function of Na⁺ concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na⁺ (K_½[Na⁺]) were calculated. The value of both the low and the high K_½(Na⁺) was dependent upon extracellular DIC concentration. The low K_½(Na⁺) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K_½(Na⁺) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na⁺ and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na⁺ and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO₂ and HCO₃⁻ transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO₂ and HCO₃⁻ transport systems required Na⁺, directly or indirectly, for full activity. However, micromolar levels of Na⁺ were required for CO₂ transport while millimolar levels were required for HCO₃⁻ transport. These levels corresponded to those found for the low and high K_½(Na⁺) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO₂ transport increased with increased DIC concentration relative to HCO₃⁻ transport. This change was reflected in a decrease in the Na⁺ concentration required for maximum photosynthesis, in accord with the lower Na⁺-requirement for CO₂ transport. Lithium competitively inhibited Na⁺-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na⁺-dependent HCO₃⁻ transport. Lithium had little effect on CO₂ transport and only a small effect on the size of the pool it generated. Thus, CO₂ transport did not require a functional HCO₃⁻ transport system for full activity. Based on these observations and the differential requirement for Na⁺ in the CO₂ and HCO₃⁻ transport system, it was proposed that CO₂ and HCO₃⁻ were transported across the membrane by different transport systems.     

Light-Induced Proton Release by the Cyanobacterium Anabaena variabilis: Dependence on CO(2) and Na

Light-induced acidification by the cyanobacterium Anabaena variabilis is biphasic (a fast phase I and slow phase II) and shown to be sodium-dependent with an optimum concentration of 40 to 60 millimolar Na⁺. Cells grown under low CO₂ concentrations at pH 9 (i.e. mainly HCO₃⁻ present in the medium) exhibited the slow phase II of proton efflux only, while cells grown under low CO₂ concentrations at pH 6.3 (i.e. CO₂ and HCO₃⁻ present) exhibited both phases. Light-induced proton release of phase I was dependent on inorganic carbon available in the bathing medium with an apparent K_m for CO₂ of 20 to 70 micromolar. As was concluded from the CO₂ dependence of acidification measured at different pH of the bathing medium, bicarbonate inhibited phase-I acidification noncompetetively. Acidification was inhibited by acetazolamide, an inhibitor of carbonic anhydrase. Apparently, acidification of phase I is due to a light-dependent uptake of CO₂ being converted to HCO₃⁻ by a carbonic anhydrase-like function of the HCO₃⁻-transport system (M Volokita, D Zenvirth, A Kaplan, L Reinhold 1984 Plant Physiol 76: 599-602) before or during entering the cell, thus releasing one proton per CO₂ converted to HCO₃⁻.     

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.     

Inorganic Carbon Uptake by Chlamydomonas reinhardtii

The rates of CO₂-dependent O₂ evolution by Chlamydomonas reinhardtii, grown with either air levels of CO₂ or air with 5% CO₂, were measured at varying external pH. Over a pH range of 4.5 to 8.5, the external concentration of CO₂ required for half-maximal rates of photosynthesis was constant, averaging 25 micromolar for cells grown with 5% CO₂. This is consistent with the hypothesis that these cells take up CO₂ but not HCO₃⁻ from the medium and that their CO₂ requirement for photosynthesis reflects the K_m(CO₂) of ribulose bisphosphate carboxylase. Over a pH range of 4.5 to 9.5, cells grown with air required an external CO₂ concentration of only 0.4 to 3 micromolar for half-maximal rates of photosynthesis, consistent with a mechanism to accumulate external inorganic carbon in these cells. Air-grown cells can utilize external inorganic carbon efficiently even at pH 4.5 where the HCO₃⁻ concentration is very low (40 nanomolar). However, at high external pH, where HCO₃⁻ predominates, these cells cannot accumulate inorganic carbon as efficiently and require higher concentrations of NaHCO₃ to maintain their photosynthetic activity. These results imply that, at the plasma membrane, CO₂ is the permeant inorganic carbon species in air-grown cells as well as in cells grown on 5% CO₂. If active HCO₃⁻ accumulation is a step in CO₂ concentration by air-grown Chlamydomonas, it probably takes place in internal compartments of the cell and not at the plasmalemma.     

Evidence for HCO(3) Transport by the Blue-Green Alga (Cyanobacterium) Coccochloris peniocystis

The possibility of HCO₃⁻ transport in the blue-green alga (cyanobacterium) Coccochloris peniocystis has been investigated. Coccochloris photosynthesized most rapidly in the pH range 8 to 10, where most of the inorganic C exists as HCO₃⁻. If photosynthesis used only CO₂ from the external solution the rate of photosynthesis would be limited by the rate of HCO₃⁻ dehydration to CO₂. Observed rates of photosynthesis at alkaline pH were as much as 48-fold higher than could be supported by spontaneous dehydration of HCO₃⁻ in the external solution. Assays for extracellular carbonic anhydrase were negative. The evidence strongly suggests that HCO₃⁻ was a direct C source for photosynthesis.