The regulation of photosynthetic rate and activation of extracellular carbonic anhydrase under CO2-limiting conditions in the marine diatom Skeletonema costatum

In the marine diatom Skeletonema costatum , carbonic anhydrase activity exterior to the plasma membrane (CA_ext) was detected only when the available CO₂ concentration was less than 5·0 mmol m^–3, this activity being unaffected by the total dissolved inorganic carbon concentration. The inhibition of CA_ext by dextran bound sulphonamide (DBS) demonstrated the key role of this enzyme in maintaining photosynthetic rate under CO₂-limited conditions. Treatment with trypsin followed by affinity chromatography on p-aminomethylbenzene-sulphamide agarose and subsequent SDS-PAGE analysis revealed a polypeptide from carbon-replete cells of identical molecular mass to the CA_ext released by trypsin from CO₂-limited cells. Redox activity in the plasma membrane of intact cells was measured by following the light-dependent reduction of ferricyanide or NADP, the greatest activity being shown by CO₂-limited cells. Overall the results suggest that high rates of redox activity under conditions of CO₂-limitation were required for the activation of CA_ext.     

Carbonic anhydrases in higher plants and aquatic microorganisms

At physiological pH-values CO₂ and HCO⁻₃are the dominant inorganic carbon species and the interconversion between both is catalyzed by carbonic anhydrase (EC 4.2.1.1). This enzyme is widely distributed among photosynthetic organisms. In the first part of the review, the similarities and the differences of carbonic anhydrases from plants and animals are briefly described. In the second part recent advances in molecular biology to understand the structure of carbonic anhydrase from higher terrestrial plants as well as its involvement in photosynthetic CO₂ fixation are summarized. Lastly, the review deals with the presence of carbonic anhydrase in aquatic organisms including cyanobacteria, microalgae, macroalgae and angiosperms. Evidence for the presence of extracellular and intracellular isozymes in these organisms are discussed. The properties and function(s) of carbonic anhydrase during the operation of the inorganic carbon concentrating mechanism are also described.     

Inorganic carbon limitation of photosynthesis in lake phytoplankton

1. Inorganic carbon availability influences species composition of phytoplankton in acidic and highly alkaline lakes, whereas the overall influence on community photosynthesis and growth is subject to debate.
2. The influence of total dissolved inorganic carbon (DIC) and free CO₂ on community photosynthesis was studied in six Danish lakes during the summer of 1995. The lakes were selected to ensure a wide range of chlorophyll a concentrations (1–120 μg l^–1), pH (5.6–9.6) and DIC concentration (0.02–2.5 m m ). Photosynthesis experiments were performed using the ¹⁴C technique in CO₂-manipulated water samples, either by changing the pH or by adding/removing CO₂.
3. Lake waters were naturally CO₂ supersaturated during most of the experimental period and inorganic carbon limitation of photosynthetic rates did not occur under ambient conditions. However, photosynthesis by phytoplankton in lakes with low and intermediate DIC concentrations was seriously restricted when CO₂ concentrations declined. Similarly, photosynthesis was limited by low CO₂ concentrations during phytoplankton blooms in the hardwater alkaline lakes.     

Photosynthesis of Ulva fasciata. IV. pH, carbonic anhydrase and inorganic carbon conversions in the unstirred layer

Abstract. The common marine macroalga Ulva was found to have a surface pH of about 10 during photosynthesis. Under such a condition, the equilibrium CO₂ concentration within the unstirred layer would be below reported CO₂ compensation points, and dehydration of HCO₃ could not occur. Even at a compensation point approaching zero, uncatalysed rates of HCO₃ to CO₂ conversion within the unstirred layer volume could not support photosynthetic rates as measured under stirred conditions in the presence of a carbonic anhydrase inhibitor. Based on this, it is concluded that Ulva takes up HCO₃. It is likely that HCO₃ uptake leads to high internal CO₂ levels which, in turn, suppress photorespiration and thus cause this plant's efficient gas exchange features. Carbonic anhydrase activity was measurable only in plant extracts. However, inhibitor studies suggest the presence of a surface enzyme. The possible functions of extracellular carbonic anhydrase in Ulva are assessed in terms of CO₂ hydration during emergence and a possible HCO₃, transport system.     

BICARBONATE UTILIZATION BY MARINE PHYTOPLANKTON SPECIES

The contribution of bicarbonate to total dissolved inorganic carbon (DIC) utilization was investigated using 18 marine phytoplankton species, including members of Bacillariophyceae, Dinophyceae, Prymnesiophyceae, and Raphidophyceae, under carbon-replete or -limited conditions. Extracellular carbonic anhydrase (CA) was assayed as an indicator of extracellular CA-catalyzed HCO⁻₃ utilization. For some species, extracellular CA was constitutive, in others activity was detected under conditions of carbon limitation, and in others, even under carbon-limited conditions, activity was not detected. In species without extracellular CA, direct HCO⁻₃ uptake was investigated using a pH drift technique in a closed system, DIC measurements, and the use of the anion exchange inhibitor 4'4'-diisothiocyanatostilbene-2,2-disulfonic acid (DLDS). Three of these species (Chaetoceros compressus, Thalassiosira pseudonana, and Glenodinium foliaceum) gave a pH drift not inhibited by DIDS, but cultures of Chrysochromulina kappa, Gephrocapsa oceanica, and Coccolithus pelagicus, in which DLDS inhibited DIC uptake, did not give a pH drift. This result shows that direct HCO₃⁻ transport may occur by an anion exchange-type mechanism in some species but not others. Of the eighteen species investigated, only Heterosigma akashiwo did not have the potential for direct uptake or extracellular CA-catalyzed HCO⁻₃ utilization.     

The active uptake of carbon dioxide by the unicellular green algae Chlorella saccharophila and C. ellipsoidea

Abstract. Mass spectrometry has been used to measure the rates of CO₂ uptake of acid- and alkali-grown cells of the green algae Chlorella ellipsoidea (UTEX 20) and C. saccharophila (UTEX 27). The time course of CO₂ formation on addition of 100mmol m⁻³ K₂CO₃ to cells in the dark was used as an assay for external carbonic anhydrase (CA). No external CA was detected in acid-grown cells of either species or in alkali-grown cells of C. ellipsoidea but was present in alkali-grown C. saccharophila . In the absence of external CA, or when it was inhibited by 5mmol m⁻³ acetazolamide, cells of both species, on illumination, rapidly depleted the free CO₂ in the medium at pH 7.5 to near zero concentrations before maximum photosynthetic O₂ evolution rates were established. Addition of bovine CA rapidly restored the equilibrium CO₂ concentration in the medium, indicating that the cells were selectively taking up CO₂. Transfer of cells to the dark caused a rapid increase in the CO₂ concentration in the medium largely due to the efflux of inorganic carbon from the cells as CO₂. This rapid light-dependent CO₂ uptake takes place against pH and concentration gradients and, thus, has the characteristics of active transport.     

CARBONIC ANHYDRASE ACTIVITY IN THE BLOOM-FORMING DINOFLAGELLATE PERIDINIUM GATUNENSE

The response of carbonic anhydrase (CA) activity in Peridinium gatunense Nygaard, the natural bloom-forming dinoflagellate in Lake Kinneret, to diel and seasonal variations in environmental conditions was characterized under controlled laboratory experiments. Simulated diel cycles demonstrated large changes in the ambient concentration of dissolved CO₂ and parallel changes in CA activity. The CA activity depended on the total concentrations of inorganic carbon (C₁) and in particular on the dissolved CO₂. Lowering the C₁ concentrations resulted in a large increase in CA activity within several hours. Light and photosynthesis were both required for the induction of CA activity. Under CO₂ -limited conditions, the dependence of the photosynthetic rate on CA (estimated from the ratio of photosynthetic rates in the presence or absence of CA inhibitors) was greater in P. gatunense than in other eukaryotic microalgae. This points to the ecological significance of CA in photosynthetic carbon uptake mechanisms of a large, dominant alga in a natural ecosystem .     

The CO2concentrating mechanism in cyanobactiria and microalgae

Over the past 10 years it has become clear that cyanobacteria and microalgae possess mechanisms for actively acquiring inorganic carbon from the external medium and are able to use this to elevate the CO₂ concentration around the active site of the primary photosynthetic carboxylating enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). This results in a vastly enhanced photosynthetic affinity for inorganic carbon (C_i) and improved photosynthetic efficiency. The CO₂ concentrating mechanism is dependent on the existence of membrane bound C_i transport systems, and a microenvironment within the cell where the accumulated C_i can be used to elevate CO₂ at the site of Rubisco. Evidence presented in this review suggests that in cyanobacteria this is achieved by the packaging of Rubisco and carbonic anhydrase (CA) into discrete structures, which are termed carboxysomes. Analogous structures in microalgae, termed pyrenoids, may perform a similar function. The recovery and analysis of high-CO₂-requiring mutants has greatly advanced our understanding of the mechanisms and genes underlying these systems, especially in cyanobacteria, and this review places particular emphasis on the contribution made by molecular genetic approaches.     

The inhibition of the carbon concentrating mechanism of the green alga Chlorella saccharophila by acetazolamide

The effects of the carbonic anhydrase (CA) inhibitors acetazolamide (AZ) and dextran-bound sulfonamide (DBS) on HCO₃⁻-dependent O₂ evolution in Chlorella saccharophila were evaluated. Addition of 4 μ M AZ or 0.4 mg ml⁻¹ DBS to photosynthesizing cells reduced the O₂ evolution rate at low dissolved inorganic carbon (DIC) concentration, decreased the size of the intracellular acid-labile carbon pool, and decreased the apparent affinity of the cells for DIC. Measurement of the whole-cell affinity of cells for CO₂ and HCO₃⁻ in the presence and absence of inhibitors indicated that active HCO₃⁻ transport was inhibited by AZ and DBS. The inhibition of HCO₃⁻ transport was independent of the inhibition of external and internal CA. These results suggest that the active uptake of HCO₃⁻ occurs initially by the interaction of HCO₃⁻ and a CA-like transporter.     

Composition and activity of external carbonic anhydrase of microalgae from karst lakes in China

The species composition, the net photosynthetic O₂ evolution rate and the activity of external carbonic anhydrase (CA) of microalgae from three reservoirs were studied. Carbonic anhydrase activity had a significant positive correlation with the density of Cyanobacteria in Lake Aha. Microalgae's carbonic anhydrase activity in Lakes Baihua and Hongfeng was related to the density of Chlorophyceae. The species abundances of microalgae in Lake Aha, Lake Baihua, and Lake Hongfeng were different. A relationship between carbonic anhydrase activity and net photosynthetic O₂ evolution rate had also been established. Algae with external CA influenced the algal productivity. These results demonstrate the role of external CA in facilitating the availability of CO₂ that limits the photosynthesis of microalgae in karst lakes in China.     

Isolation and characterization of carbonic anhydrases from the marine diatom Phaeodactylum tricornutum

Carbonic anhydrase (CA) isozymes were identified and isolated from three strains of Phaeodactylum tricornutum [University of Texas Culture Collection (UTEX 640), North Eastern Pacific Culture Collection at the University of British Columbia B31 and Culture Collection of Algae and Protozoa 1052/1A]. External (CA_ext) and internal CA activity was detected by potentiometric assay of intact cells and cell homogenates of air and high CO₂-grown cells. CA_ext was detected only in UTEX 640 grown under CO₂-limited conditions and present in trace amounts in cells grown on high CO₂. CA isozymes in cells extracts were separated by cellulose acetate electrophoresis and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All three strains had two CA bands in common, while UTEX 640 had a third, faster-running band which was absent from extracts of high CO₂-grown cells and thus was the external isozyme. The internal CA isoforms of the UTEX 640 strain were shown to have molecular masses of 28 and 25 kDa, and the external 24 kDa. A fourth CA_ext isozyme with a molecular weight of 23.5 kDa was later detected using a polyclonal CA antibody. The CA isozymes were low-CO₂-inducible proteins because Western blot analysis, using a polyclonal antibody, indicated that CA expression was repressed in high CO₂-grown cells. CA localization, using both immunofluorescence and immunogold techniques, with air-grown cells indicated that the CA_ext was located in the periplasmic space and on the cell membrane, whereas in high CO₂-grown cells only internal CA was detected.     

Involvement of photorespiration and glycolate pathway in carbonic anhydrase induction and inorganic carbon concentration in Chlamydomonas reinhardtii

Interaction between induction of carbonic anhydrase (CA) activity, induction of inorganic carbon (C_i) concentrating mechanisms and the photorespiratory glycolate pathway has been studied in wild type 6145c and photorespiratory mutant 18–7F (low in phosphoglycolate phosphatase activity) cells of C. reinhardtii . Cell transfer from high CO₂ (5%, v/v) to low CO₂ (0.03%) provoked an increase of extracellular and total (extracellular plus intracellular) CA in both wild type and mutant cells. During adaptation to low CO₂ conditions, both strains excreted ammonium to the medium at a similar rate in the presence of l -methionine- d-l -sulfoximine (MSX), an inhibitor of glutamine synthetase (GS). MSX also provoked ammonium excretion by air adapted wild type and mutant cells, even though both strains had high levels of CA activity and of C_i concentrating activities.
GS increased in both strains after transfer from high to low CO₂ conditions. However, this increase was abolished by aminooxyacetate, an inhibitor of the glyoxylate-serine aminotransferase, and by glycolaldehyde, an inhibitor of triose phosphate to ribulose 1,5-bisphosphate conversion. CA synthesis did not occur in the presence of either aminooxyacetate or glycolaldehyde. Algae grown in high CO₂ in the presence of aminooxyacetate did not induce C_i concentrating mechanisms. Integration of these three processes, i.e., CA synthesis, C_i-concentration, and photorespiratory glycolate pathway is proposed in the framework of carbon metabolism of the alga.     

Limited acclimation of photosynthesis to blue light in the seaweed Gracilaria tenuistipitata

Changes in photosynthetic capacity of the seaweed Gracilaria tenuistipitata Zhang et Xia acclimated to monochromatic blue light were studied. For this purpose, affinity for external inorganic carbon, light use efficiency, carbonic anhydrase (CA; EC 4.2.1.1) activity and content of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) were determined in thalli acclimated to 45 µmol m⁻² s⁻¹ of blue light. Thalli cultured in white light of the same photon fluence rate were used as a control. Lower maximal photosynthetic rates (i.e. at light and carbon saturation) were obtained in the thalli cultured in blue light. Apparently, this lower photosynthetic capacity was not due to differences in affinity and/or capacity for use of external dissolved inorganic carbon (DIC) since (1) CA activity did not change significantly and (2) similar values of photosynthetic conductance for DIC at alkaline pH were obtained (0.95 × 10⁻⁶ m s⁻¹). In addition, the pool size of Rubisco was not modified by the blue light treatment since there were no significant differences in Rubisco content between white (12.14% of soluble proteins) and blue light (12.13% of soluble proteins) treatments. In contrast, F _v/ F _m was increased by 11% and photosynthetic efficiency for oxygen production was reduced by 50% in blue light. This absence of correlation between quantum yields for maximum stable charge separation of photosystem II and oxygen evolution suggests that blue light promote changes in rates of photosynthetic electron flow.     

The case for chloroplast thylakoid carbonic anhydrase

Washed thylakoid membranes and photosystem II-enriched membrane fragments from cyanobacteria, green algae, and chloroplasts from both C₃ and C₄ plants possess the ability to reversibly hydrate CO₂. That is, the membranes have an intrinsic carbonic anhydrase activity. The present review outlines the discovery of thylakoid carbonic anhydrase and presents the evidence that it is a unique isozyme, distinct from other cellular carbonic anhydrases. It appears that at least some thylakoid carbonic anhydrase is closely associated with photosystem II and may be required for electron transport. This would explain why all inhibitors of carbonic anhydrase also inhibit photosystem II. Several speculative functions of thylakoid carbonic anhydrase are discussed. These include a possible role in carbon metabolism, in the protonation of plastoquinone, and/or in oxygen evolution.     

ISOLATION OF ccmKLMN GENES FROM THE MARINE CYANOBACTERIUM, SYNECHOCOCCUS SP. PCC7002 (CYANOPHYCEAE), AND EVIDENCE THAT CcmM IS ESSENTIAL FOR CARBOXYSOME ASSEMBLY

A high CO₂ requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene-tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (C_i) and accumulated high internal levels of C_i that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO₂ concentrating mechanism (CCM), the so-called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN , are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N-terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C-terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM -defined mutant showed that these cells were essentially identical to the original mutant; they required high CO₂ concentrations for growth, they had a low photosynthetic affinity for C_i, and they internalized C_i to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.     

Calcification in aquatic plants

Abstract. The CaCO₃ deposits of aquatic plants may be intra-, inter- and extracellular. Calcification is mainly the result of photosynthetic CO₂ or HCO⁻₃ assimilation. This raises the local pH and CO²⁻₃ concentration resulting from shifts in the dissolved inorganic carbon equilibrium, due to either net CO₂ depletion as in Halimeda or localized OH⁻ efflux (or H⁺ influx) as in Chara. The plant cell wall may be important in CaCO₃ nucleation by acting as an epitaxial substratum or template, or by creating a microenvironment enriched in Ca²⁺ compared to Mg²⁺. Hypotheses on the reason for the lack of calcification in many aquatic plants are presented.     

Regulation of the CO2-concentrating mechanism in Chlorella vulgaris UAM 101 by glucose

In the green alga Chlorella vulgaris UAM 101, a CO₂-concentrating mechanism is induced when the cells are growing under low CO₂ conditions. We have investigated the effect of glucose on the induction of this mechanism. Cells adapted to low CO₂ in the presence of glucose showed a reduced ability to transport and fix external inorganic carbon. This reduction was correlated with a decrease in internal carbonic anhydrase activity. 3- O -methyl-glucose, a nonmetabolizable analog of glucose, caused a more dramatic repression of these phenomena. Immunoblot analyses of total cell protein of Chlorella vulgaris UAM 101 against large subunit of ribulose-1.5-bisphosphate carboxylase/oxygenase and ribulose 1.5-bisphosphate-carboxylase/oxygenase activase polyclonal antibodies showed that the expression of these two polypeptides was affected by neither CO₂ level, nor glucose or 3- O -methyl-glucose. Ultrastructure studies showed that the low CO₂-induced development of the pyrenoid was also affected by glucose. Immunocytochemical data demonstrated that ribulose-1.5-bisphosphate carboxylase/oxygenase was exclusively located in the pyrenoid matrix. This localization and the density of labeling of the pyrenoid region were affected by neither CO₂ level nor the presence of glucose.     

The acquisition and accumulation of inorganic carbon by the unicellular green alga Chlorella ellipsoidea

Abstract. The uptake and accumulation of inorganic carbon has been investigated in Chlorella ellipsoidea cells grown at acid or alkaline pH. Carbonic anhydrase (CA) was detected in ceil extracts but not in intact cells and CA activity in acid-grown cells was considerably less than that in alkali-grown cells. Both cell types demonstrates low K_1/2 (CO₂) values in the range pH 7.0–8.0 and these were unaffected by O₂ concentration. The CO₂ compensation concentrations of acid- and alkali-grown cells suspended in aqueous media were not significantly different in the range of pH 6.0–8.0, but at pH 5.0, the CO₂ compensation concentrations of acid-grown cells (57.4cm³ m⁻³) were lower than those of alkali-grown cells (79.2cm³ m⁻³). The rate of photo-synthetic O₂ evolution in the range pH 7.5–8.0 exceeded the calculated rate of CO₂ supply two- to three-fold, in both acid- and alkali-grown cells, indicating that HCO₃⁻ was taken up by the cells. Accumulation of inorganic carbon was measured at pH 7.5 by silicone-oil centri-fugation, and the concentration of unfixed inorganic carbon was found to be 5.1 mol m⁻³ in acid-grown and 6.4mol m⁻³ in alkali-grown cells. These concentrations were 4.6- and 5.9-fold greater than in the external medium. These results indicate that photorespiration is suppressed in both acid- and alkali-grown cells by an intracellular accumulation of inorganic carbon due, in part, to an active uptake of bicarbonate.     

The effect of different growth conditions on dark and light carbon assimilation in Littorella uniflora

The effect of long-term exposure to different inorganic carbon, nutrient and light regimes on CAM activity and photosynthetic performance in the submerged aquatic plant, Littorella uniflora (L.) Aschers was investigated. The potential CAM activity of Littorella was highly plastic and was reduced upon exposure to low light intensities (43 μmol m⁻² s⁻¹), high CO₂ concentrations (5.5 mM, pH 6.0) or low levels of inorganic nutrients, which caused a 25–80% decline in the potential maximum CAM activity relative to the activity in the control experiments (light: 450 μmol m⁻² s⁻¹; free CO₂: 1.5 mM). The CAM activity was regulated more by light than by CO₂, while nutrient levels only affected the activity to a minor extent. The minor effect of low nutrient regimes may be due to a general adaptation of isoetid species to low nutrient levels.
The photosynthetic capacity and CO₂ affinity was unaffected or increased by exposure to low CO₂, irrespective of nutrient levels. High CO₂, low nutrient and low light, however, reduced the capacity by 22–40% and the CO₂ affinity by 35-45%, relative to control.
The parallel effect of growth conditions on CAM activity and photosynthetic performance of Littorella suggest that light and dark carbon assimilation are interrelated and constitute an integrated part of the carbon assimilation physiology of the plant. The results are consistent with the hypothesis that CAM is a carbon-conserving mechanism in certain aquatic plants. The investment in the CAM enzyme system is beneficial to the plants during growth at high light and low CO₂ conditions.     

A relationship between carbon dioxide, photosynthetic efficiency and shade tolerance

Net photosynthesis and transpiration of seedlings from shade tolerant, moderately tolerant and intolerant tree species were measured in ambient carbon dioxide (CO₂) concentrations ranging from 312 to 734 ppm. The species used, Fagus grandifolia Ehrh. (tolerant), Quercus alba L., Q. rubra L., Liriodendron tulipifera L. (moderately tolerant), Liquidambar styraciflua L. and Pinus taeda L. (intolerant), are found co-occurring in the mixed pine-hardwood forests of the Piedmont region of the southeastern United States. When seedlings were grown in shaded conditions, photosynthetic CO₂ efficiency was significantly different in all species with the highest efficiency in the most shade tolerant species, Fagus grandifolia , and progressively lower efficiencies in moderately tolerant and intolerant species. Photosynthetic CO₂ efficiency was defined as the rate of increase in net photosynthesis with increase in ambient CO₂ concentration. When plants which had grown in a high light environment were tested, the moderately tolerant and intolerant deciduous species had the highest photosynthetic CO₂ efficiencies but this capacity was reduced when these species grew in low light. The lowest CO₂ efficiency and apparent quantum yield occurred in Pinus taeda in all cases. Water use efficiency was higher for all species in enriched CO₂ environments but transpiration rate and leaf conductance were not affected by CO₂ concentration. High photosynthetic CO₂ efficiency may be advantageous for maintaining a positive carbon balance in the low light environment under a forest canopy.