Transport and Fixation of Inorganic Carbon during Photosynthesis of Anabaena Grown under Ordinary Air. I. Active Species of Inorganic Carbon Utilized for Photosynthesis

Inorganic carbon transport during photosynthesis of cyanobacteriumAnabaena variabilis grown under ordinary air was investigatedby supplying ¹⁴CO₂ or H¹⁴CO₃^– solution to three differentstrains. Both CO₂ and HCO₃^– were accumulated within thealgal cells. In the cell suspension from which dissolved inorganiccarbon had been depleted by pre-illumination, CO₂ was transportedand accumulated faster than HCO₃^–. When the concentrationof HCO₃^– injected into the cell suspension of A. variabilisM3 was 25 times as high as that of CO2 (the expected ratio atequilibrium at pH 7.8), the initial rates of fixation of bothinorganic carbon species were practically the same. On the otherhand, when ¹⁴CO₂ or H¹⁴CO₃^– was added under steady statephotosynthetic conditions, both carbon species were transportedat similar rates. The ratio of fixed to transported carbon measuredafter the initial 5 s was only 23–27% regardless of thecarbon species supplied. This percentage is much lower thanthat reported for Chlorella cells. ¹ To whom reprint requests should be addressed (Received June 30, 1986; Accepted December 16, 1986)     

Form of Inorganic Carbon Utilized for Photosynthesis in Chlamydomonas reinhardtii1

The effect of carbonic anhydrase (CA) on time courses of photosynthetic¹⁴C incorporation in the presence of ¹⁴CO₂ or NaH¹⁴CO₃ was studiedwith cells of Chlamydomonas reinhardtii which had been grownunder ordinary air (low-CO₂ cells) or air enriched with 4% CO₂(high-CO₂ cells). Experimental data obtained at 20°C andpH 8.0 suggested that the major form of inorganic carbon utilizedby high-CO₂ cells was CO₂, while that utilized by low-CO₂ cellswas HCO₃^–. The cell suspension showed CA activity which was comparableto that observed in the sonicate of cells. Both activities werehigher in low-CO₂ cells than in high-CO₂ cells. The mechanism by which HCO₃^– is utilized by low-CO₂ cellsof C. reinhardtii is discussed. ³Present address: Department of Biology, Faculty of Science,University of Niigata, Niigata 950-21, Japan. (Received August 4, 1982; Accepted January 19, 1983)     

Carbonic Anhydrase and the Regulation of Photosynthesis

THE role of CO₂ in the regulation of photosynthetic and respiratory metabolism in plants is little understood in the unicellular alga Chlorella pyrenoidosa; for example, after autotrophic growth in high CO₂ (5·5% by volume), transfer to a CO₂ concentration about ten times less than the concentration in air results initially in low rates of photosynthesis characterized by the virtual absence of the Calvin cycle¹ of CO₂ fixation². An induction period of about 2 h is necessary before normal photosynthetic rates are established. Cells grown in air (0.03% CO₂) do not show this effect and photosynthesize at comparatively high rates even in very low concentrations of CO₂.     

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.     

Extracellular Carbonic Anhydrase Facilitates Carbon Dioxide Availability for Photosynthesis in the Marine Dinoflagellate Prorocentrum micans

This study investigated inorganic carbon accumulation in relation to photosynthesis in the marine dinoflagellate Prorocentrum micans. Measurement of the internal inorganic carbon pool showed a 10-fold accumulation in relation to external dissolved inorganic carbon (DIC). Dextran-bound sulfonamide (DBS), which inhibited extracellular carbonic anhydrase, caused more than 95% inhibition of DIC accumulation and photosynthesis. We used real-time imaging of living cells with confocal laser scanning microscopy and a fluorescent pH indicator dye to measure transient pH changes in relation to inorganic carbon availability. When steady-state photosynthesizing cells were DIC limited, the chloroplast pH decreased from 8.3 to 6.9 and cytosolic pH decreased from 7.7 to 7.1. Re-addition of HCO₃⁻ led to a rapid re-establishment of the steady-state pH values abolished by DBS. The addition of DBS to photosynthesizing cells under steady-state conditions resulted in a transient increase in intracellular pH, with photosynthesis maintained for 6 s, the amount of time needed for depletion of the intracellular inorganic carbon pool. These results demonstrate the key role of extracellular carbonic anhydrase in facilitating the availability of CO₂ at the exofacial surface of the plasma membrane necessary to maintain the photosynthetic rate. The need for a CO₂-concentrating mechanism at ambient CO₂ concentrations may reflect the difference in the specificity factor of ribulose-1,5 bisphosphate carboxylase/oxygenase in dinoflagellates compared with other algal phyla.     

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.     

碳酸酐酶在中肋骨条藻光合作用中的作用

探讨了在正常空气条件下生长的中肋骨条藻(Skeletonema costatum)的碳酸酐酶(CA)在其光合固碳中的作用.在中肋骨条藻的胞内和胞外均有CA活性,但胞外CA活性很低.CA抑制剂AZ(乙酰唑磺胺)对中肋骨条藻的光合放氧速率没有明显影响,而CA抑制剂EZ(乙氧苯唑胺)对其光合放氧速率有强烈的抑制作用.EZ的抑制作用使细胞最大光合速率、饱和光强和无机碳亲和力下降,无机碳的补偿点和光呼吸提高,使强光下光抑制作用增强.这些结果表明:中肋骨条藻的胞外CA在其光合作用中所起的作用较小,而其胞内CA通过催化胞内碳库中的HCO-3快速转化成CO2,提高胞内CO2的有效供给,从而提高细胞光合固碳能力和对逆境(高O2、强光和低CO2)的适应能力.     

Correlation between Carbonic Anhydrase Activity and Inorganic Carbon Internal Pool in Strain Synechocystis PCC 6174

Existence of an internal carbonic anhydrase was demonstrated in the cyanobacterium Synechocystis PCC 6714. The enzyme, present at a low specific activity, was inducible by limitation in inorganic carbon and inhibited both in vivo and in vitro by acetazolamide. The internal inorganic carbon pool as determined by mass spectrometry, was similarly modulated by the actual inorganic carbon growth regime; its building up was also sensitive to acetazolamide. A possible role of carbonic anhydrase in inorganic carbon metabolism regulation through the control of the dimension and nature of the inorganic carbon pool is 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.     

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.     

Utilization Modes of Inorganic Carbon for Photosynthesis in Various Species of Chlorella

Rates of CO₂ and HCC₃^– fixation in cells of various Chlorellaspecies in suspension were compared from the amounts of ¹⁴Cfixed during the 5 s after the injection of a solution containingonly ¹⁴CO₂ or H¹⁴CO₃^–. Results indicated that irrespectiveof the CO₂ concentration during growth, Chlorella vulgaris 11h and C. miniata mainly utilized CO₂, whereas C. vulgaris C-3,C. sp. K. and C. ellipsoidea took up HCO₃^– in additionto CO₂. Cells of C. pyrenoidosa that had been grown with 1.5%CO₂ (high-CO₂ cells) mainly utilized CO₂, whereas those grownwith air (low-CO₂ cells) utilized HCO₃^– in addition toCO₂. Cells that utilized HCO₃^– had carbonic anhydrase(CA) on their surfaces. The effects of Diamox and CA on the rates of CO₂ and HCO₃^–fixation are in accord with the inference that HCO₃^– wasutilized after conversion to CO₂ via the CA located on the cellsurface. CA was found in both the soluble and insoluble fractions;the CA on the cell surface was insoluble. Independent of the modes of utilization, the apparent K_m (NaHCO₃)for photosynthesis was much lower in low-CO₂ cells than in high-CO₂ones. The fact that the CA in the soluble fraction in C. vulgarisC-3 was closely correlated with the K_m(NaHCO₃) indicates thatsoluble CA lowers the K_m. ¹ Dedicated to the late Professor Joji Ashida, one of the foundersand first president of the Japanese Society of Plant Physiologists. ⁴ On leave from Research and Production Laboratory of Algology,Bulgarian Academy of Sciences, Sofia. (Received September 14, 1982; Accepted March 1, 1983)     

Mechanism of Inorganic Carbon Uptake in Chlorella saccharophila: The Lack of Involvement of Carbonic Anhydrase

The acid-tolerant green alga Chlorella saccharophila maintainedphotosynthesis and accumulated intracellular pools of inorganiccarbon over a a range of external pH from 4.0 to 7.5. This accumulationwas unaffected by treatment of cells with 10 mol m^–3 acetazolamide(AZA). Cells grown at alkaline pH had extracellular carbonicanhydrase (CA), but CA activity was repressed when cells weregrown at pH 5.0. Acid-grown cells retained a high affinity forCO₂, both at acid and alkaline pH, and the ability to accumulateinorganic carbon. Rates of photosynthesis of acid-grown cellsand alkaline-grown AZA-treated cells at pH 8.0 were 2.5-foldhigher than the rate of CO₂ supply from the uncatalysed dehydrationof , indicating that the cells can take up as a source of substrate for photosynthesis. Isotopic disequilibrium experiments with acid-grown cells maintainingsteady-state photosynthesis at pH 7.5 demonstrate that ¹⁴C from¹⁴CO₂ was taken up more rapidly than from H¹⁴. This uptake takes place against a concentration gradient. Theseresults demonstrate that C. saccharophila cells have activetransport systems for the uptake of both CO₂ and and both operate without the mediation of CA. Key words: Bicarbonate transport, carbon dioxide, carbonic anhydrase, Chlorella saccharophila, inorganic carbon accumulation     

Quantification of the Contribution of CO2, HCO3-, and External Carbonic Anhydrase to Photosynthesis at Low Dissolved Inorganic Carbon in Chlorella saccharophila

cDNAs encoding the large subunit and a possibly truncated small subunit of the potato tuber (Solanum tuberosum L.) adenosine 5'-diphosphate-glucose pyrophosphorylase have been expressed in Escherichia coli (A.A. Iglesias, G.F. Barry, C. Meyer, L. Bloksberg, P.A. Nakata, T. Greene, M.J. Laughlin, T.W. Okita, G.M. Kishore, J. Preiss, J Biol Chem [1993] 268: 1081-1086). However, some properties of the transgenic enzyme were different from those reported for the enzyme from potato tuber. In this work, extension of the cDNA was performed to elongate the N terminus of the truncated small subunit by 10 amino acids. This extension is based on the almost complete conservation seen at the N-terminal sequence for the potato tuber and the spinach leaf small subunits. Expressing the extended cDNA in E. coli along with the large subunit cDNA yielded a transgenic heterotetrameric enzyme with similar properties to the purified potato tuber enzyme. It was also found that the extended small subunit expressed by itself exhibited high enzyme activity, with lower affinity for activator 3-phosphoglycerate and higher sensitivity toward inorganic phosphate inhibition. It is proposed that a major function of the large subunit of adenosine 5'-diphosphate-glucose pyrophosphorylases from higher plants is to modulate the regulatory properties of the native heterotetrameric enzyme, and the small subunit's major function is catalysis.     

Role of Carbonic Anhydrase in Photosynthesis of Blue-Green Alga (Cyanobacterium) Anabaena variabilis ATCC 29413

The affinity for NaHCO₃ (CO₂) in photosynthesis of Anabaenavariabilis ATCC 29413 was much higher in the cells grown underordinary air (low-CO₂ cells) than in those grown in air enrichedwith 2–4% CO₂ (high-CO₂ cells) (pH 8.0, 25?C). Ethoxyzolamide(50 µM) increased the K_m(NaHCO₃ in low-CO₂ cells aboutnine times (from 14.3 to 125), while the maximum rate of photosynthesisdecreased about 20%. When high-CO₂ cells were transferred tolow-CO₂ conditions, carbonic anhydrase (CA) activity increased,while K_m(NaHCO₃) in photosynthesis decreased from 140 to 30µM within about 5 h. The addition of CA to the suspensionof both high- and low-CO₂ cells enhanced the rates of photosyntheticO₂ evolution under CO₂-limiting conditions. The rate of ¹⁴CO₂fixation was much faster than that of H¹⁴CO₃^– fixation.The former reaction was greatly suppressed, while the latterwas enhanced by the addition of CA. These results indicate thatthe active species of inorganic carbon utilized for photosynthesiswas free CO₂ irrespective of the CO₂ concentration given duringgrowth. It is suggested that CA plays an active role in increasingthe affinity for CO₂ in photosynthesis of low-CO₂ cells of thisblue-green alga. (Received January 24, 1984; Accepted October 22, 1984)     

Effect of Zinc Nutrition on Photosynthesis and Carbonic Anhydrase Activity in Cotton

Photosynthesis, respiration, carbonic anhydrase activity and chlorophyll concentrations were correlated with zinc nutrition in cotton (Gossypium hirsutum L.). The critical zinc level during early plant growth was 13 μg/g dry weight in recently matured leaves (blade three). Photosynthesis and chlorophyll concentration required a minimum Zn of 13 and 14 μg/g dry weight, respectively, in blade three for maximum activity and synthesis. Respiration was not influenced by zinc status. Carbonic anhydrase activity increased curvilinearly as zinc status improved from deficiency to adequacy.     

Utilization of Exogenous Inorganic Carbon Species in Photosynthesis by Chlorella pyrenoidosa

The nature of the inorganic carbon utilized during photosynthesis by Chlorella pyrenoidosa was investigated using three experimental techniques (open gas analysis system with “artificial leaf” or “aqueous” chambers and O₂ electrode system) to measure carbon assimilation. Photosynthesis was studied as a function of pH and CO₂ concentration. The CO₂ concentration was inadequate to meet the requirements of photosynthesis only when HCO₃⁻ was added at high pH. Under all other conditions, the low and constant K_m (CO₂), in contrast to the highly variable K_m (HCO₃⁻), suggested that CO₂ was the major species utilized.     

芸薹属中几个物种碳酸酐酶活性的比较

检测芸薹属中几种植物的碳酸酐酶在根、茎、叶中的分布和活性以及叶片净光合速率和锌元素含量的结果表明：碳酸酐酶在甘蓝型油菜的根、茎、叶中均有分布,叶中的碳酸酐酶活性最大,茎中的次之,根中的最小。不同芸薹属植物中碳酸酐酶活性的大小依序为：甘蓝型油菜〉白菜型油菜〉芜菁〉埃塞俄比亚芥。以甘蓝型油菜为例,碳酸酐酶活性的日变化呈双峰曲线,在整个生育期的变化趋势是先上升后下降,抽薹期间的碳酸酐酶活性最高。碳酸酐酶活性与净光合速率以及与锌元素含量之间均呈现显著的正相关。     

Carbonic Anhydrase of a Unicellular Red Alga Porphyridium cruentum R-l. II. Distribution and Role in Photosynthesis

Antibody was raised against Porphyridium carbonic anhydrase(CA) which was electrophoretically recovered from the gel afterSDS-polyacrylamide slab gel electrophoresis (SDS-PAGE) of thepartially purified enzyme. The antiserum reacted with CA ofPorphyridium, but not with that of Chlamydomonas reinhardtii.Even though the antiserum did not react with CA from P. cruentumR-l in Ouchterlony's double immunodiffusion, it blocked theenzyme activity in the presence of 1% Nonidet P-40 and 1% TritonX-100. After Western blotting and enzyme-linked immunostaining(ELIS), only one band which reacted with the antiserum was detectedin the extract of low-CO₂ cells (grown under ordinary air) ofP cruentum, while no significant band was detected in that ofhigh-CO₂ cells (grown under air enriched with 1–5% CO₂).Immunogold electron microscopy of low-CO₂ cells of P. cruentumR-l using this antibody revealed that most of the CA was localizedin the chloroplast, with some in the cytoplasm. No specificbinding of gold particles was observed in the high-CO₂ cells. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan (Received May 18, 1987; Accepted September 7, 1987)     

Effects of Carbonic Anhydrase Inhibitors on Proton Exchange and Photosynthesis in Pea Protoplasts

At concentrations of 100–200 M, ethoxyzolamide, a lipophilic inhibitor of carbonic anhydrase, considerably (by 60%) inhibited light-induced CO₂-dependent oxygen evolution in pea protoplasts at the optimum concentration of inorganic carbon (100 M CO₂) in the medium. At the same concentrations of the inhibitor, electron transport in isolated pea thylakoids was inhibited only by 6–9%. Acetazolamide, a water-soluble inhibitor of carbonic anhydrase, affected neither the rate of CO₂-dependent O₂evolution in protoplasts nor electron transport in thylakoid membranes. A light-dependent proton uptake by protoplasts was demonstrated. At pH 7.2, the induction kinetics and the rate of proton uptake were similar to those for CO₂-dependent O₂evolution. The rate of proton uptake was decreased twofold by 1 mM acetazolamide. This fact agrees with the notion that a membrane-bound carbonic anhydrase is operative in the plasma membrane of higher plant cells. A mechanism of its functioning is suggested. Possible functions of carbonic anhydrases in the cells of C₃-plants are discussed.