Carbon Concentration Mechanism(s) in Unicellular Green Algae and Cyanobacteria

Unicellular green algae and cyanobacteria have mechanism(s) to actively concentrate dissolved inorganic carbon (DIC) into the cells, only if they are grown with air levels of CO₂. The DIC concentration mechanisms are environmental adaptations to actively transport and accumulate inorganic carbon into the chloroplasts of green algae or into the carboxysomes of cyanobacteria. The current working model of cyanobacterial carbon concentration mechanism consists of at least two basic components: an active C_i transport system and a Rubisco-rich polyhedral carboxysome. In case of unicellular green algae, the working model for DIC concentration mechanism includes several isoforms of carbonic anhydrase (CA), and ATPase driven active bicarbonate transporters at the plasmalemma and at the inner chloroplast envelopes. In the past twenty years, significant progress has been made in isolating and characterizing the isoforms of carbonic anhydrase. However, active transporters are yet to be characterized. This mini-review summarizes the current status of research on DIC-pumps including its significance and possible application to increase the productivity of plants of economic importance.     

Introducing an algal carbon‐concentrating mechanism into higher plants: location and incorporation of key components

Many eukaryotic green algae possess biophysical carbon‐concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO₂ concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H¹⁴CO₃^‐ uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild‐type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.     

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.     

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

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

A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.

A 29.5 kDa intracellular alpha-type carbonic anhydrase, designated Cah3, from the unicellular green alga Chlamydomonas reinhardtii is the first of this type discovered inside a photosynthetic eukaryote cell. We describe the cloning of a cDNA which encodes the protein. Immunoblot studies with specific antibodies raised against Cah3 demonstrate that the polypeptide is associated exclusively with the thylakoid membrane. The putative transit peptide suggests that Cah3 is directed to the thylakoid lumen, which is confirmed further by the presence of mature sized Cah3 after thermolysin treatment of intact thylakoids. Complementation of the high inorganic carbon concentration-requiring mutant, cia-3, with a subcloned cosmid containing the cah3 gene yielded transformants that grew on atmospheric levels of CO2 (0.035%) and contained an active 29.5 kDa alpha-type carbonic anhydrase. Although, cia-3 has reduced internal carbonic anhydrase activity, unexpectedly the level of Cah3 was similar to that of the wild-type, suggesting that the mutant accumulates an inactive Cah3 polypeptide. Genomic sequence analysis of the mutant revealed two amino acid changes in the transit peptide. Results from photosynthesis and chlorophyll a fluorescence parameter measurements show that the cia-3 mutant is photosynthetically impaired. Our results indicate that the carbonic anhydrase, extrinsically located within the chloroplast thylakoid lumen, is essential for growth of C.reinhardtii at ambient levels of CO2, and that at these CO2 concentrations the enzyme is required for optimal photosystem II photochemistry.     

Model of the carbon concentrating mechanism in chloroplasts of eukaryotic algae

A generic chloroplast-based model for the carbon concentrating mechanism (CCM) in eukaryotic algae is presented. The fine structure of chloroplasts is represented by separate compartments: marginal and bulk stroma, pyrenoid, girdle lamella, bulk thylakoids, and central lamella traversing the pyrenoid. The roles of the individual structural elements of the chloroplast with respect to the CCM and the effect of carbonic anhydrase activity in various compartments are analysed. Hypothetical HCO(-)(3)transport into the acidic thylakoid lumen is adjusted by imposing an optimization principle: a given [CO(2)] at the site of RuBisCO is achieved with minimum energy costs for the CCM. Our model is highly efficient in terms of saturation of RuBisCO carboxylase activity and the affinity of the chloroplast for CO(2), if either a girdle lamella or a pyrenoid is present. The highest efficiency is achieved with a pyrenoid. A eukaryotic CCM is not necessarily associated with accumulation of dissolved inorganic carbon (DIC) as in cyanobacteria. Chloroplasts are categorized into four types corresponding to morphological characteristics of all major algal classes with regard to the presence of pyrenoids, girdle lamellae, and the distribution of CA activity.     

TRANSPORT OF INORGANIC CARBON AND THE ‘CO2 CONCENTRATING MECHANISM’ IN CHLORELLA EMERSONII (CHLOROPHYCEAE)1

Chlorella emersonii Shihira et Krauss var. emersonii exhibits ‘C₄-like’ gas exchange characteristics when grown at air levels of CO₂, but is ‘C₃-like’ when grown with extra CO₂. The total inorganic carbon concentration, and the free CO₂ concentration, averaged over the cell interior are higher in air-adapted cells than can be accounted for by passive CO₂ equilibration from the medium and the mean intracellular pH value. The ‘extra’ inorganic C in the air-grown cells probably cannot all be accounted for in terms of binding to proteins and requires an active transport process to account for it. The electrical potential of the cell interior becomes more negative when the ‘CO₂ concentrating mechanism’ is operative; this is most readily explained if the active step in inorganic C accumulation is primary active uniport of HCO₃^?. Since the ‘CO₂ concentrating mechanism’ can operate when CO₂ is the species crossing the outer permeation barrier, it is suggested that the site of active HCO₃^? transport in Chlorella (and other eukaryotes) is the chloroplast envelope, and the plasmalemma in cyanobacteria. This scheme explains the obligatory role of the de-repressed carbonic anhydrase in C₄-like photosynthesis in algae, but some other data support an explanation of C₄-like photosynthesis in terms of special properties of carbonic anhydrase as a carbon donor to RuBP carboxylase-oxygenase.     

A new chloroplast envelope carbonic anhydrase activity is induced during acclimation to low inorganic carbon concentrations in Chlamydomonas reinhardtii

Using mass-spectrometric measurements of 18O exchange from 13C18O2 we determined the activity of carbonic anhydrase (CA; EC 4.2.1.1) in chloroplast envelope membranes isolated from Chlamydomonas reinhardtii cw-15. Our results show an enrichment of CA activity in these fractions relative to the activity in the crude chloroplast. The envelope CA activity increased about 8-fold during the acclimation to low-CO2 conditions and was completely induced within the first 4 h after the transfer to air levels of CO2. The CA-activity was not dissociated from envelope membranes after salt treatment. In addition, no cross-reactivity with other CA isoenzymes of Chlamydomonas was observed in our chloroplast envelope membranes. All these observations indicated that the protein responsible for this activity was a new CA isoenzyme, which was an integral component of the chloroplast envelopes from Chlamydomonas. The catalytic properties of the envelope CA activity were completely different from those of the thylakoid isoenzyme, showing a high requirement for Mg2+ and a high sensitivity to ethoxyzolamide. Analysis of the integral envelope proteins showed that there were no detectable differences between high- and low-inorganic carbon (Ci) cells, suggesting that the new CA activity was constitutively expressed in both high- and low-Ci cells. Two different high-Ci-requiring mutants of C. reinhardtii, cia-3 and pmp-1, had a reduced envelope CA activity. We propose that this activity could play a role in the uptake of inorganic carbon at the chloroplast envelope membranes.     

绿藻CO2浓缩机制的研究进展

单细胞绿藻是淡水水体中浮游植物的重要组成部分,也是淡水生态系统中主要的初级生产者,其在适应外界CO2浓度变化的过程中,细胞内形成了一种主动转移无机碳的机制－CO2浓缩机制（CO2 concentrating mechanism,CCM）。该机制能使细胞在核酮糖－2－磷酸羧化氧化酶（rubiscol)固碳位点提高CO2浓度,以增加光合作用和减少光吸收。本文综述了这种机制中的无机碳转移模型和不同环境因子（光,温度,CO2浓度和营养水平）对它的调控作用,以期促进深入开展浮游植物对大气CO2浓度升高响应的研究。     

Carbonic anhydrases in plants and algae

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

干出和沉水不同条件下羊栖菜光合作用碳源获得机制比较(英文)

经济海洋褐藻羊栖菜(Hizikia fusiforme(Harv.)Okamura)低潮时常常周期性地暴露于空气中。为了认识这种海藻在潮汐循环背景下的光合特征,对其在高潮沉水和低潮干出不同条件下的光合作用碳素获得机制进行了比较。沉水时,羊栖菜主要利用海水中HCO_3~-作为外源无机碳源驱动光合作用;而在干出条件下,其光合作用的主要碳源为空气中的CO_2。在这两种不同环境条件下,光合作用与pH值的关系不同:沉水状态时,羊栖菜在高pH值(10.0)下光合活性很弱;而在干出条件下,羊栖菜在高pH值时仍有较高的光合活性。然而,光合作用无论是在沉水还是在干出条件下,对外源碳源的获得都表现出对胞外碳酸酐酶(CA)强烈的依赖性,并且其光合速率都受周围环境中无机碳源水平的限制。此外,在沉水和干出两种环境条件下,羊栖菜光合作用都表现出对氧气的敏感性。这表明,在羊栖菜中,依赖胞外CA的碳源获得机制不能使细胞内CO_2浓度提高到阻碍其光呼吸的程度。增加空气中或海水中无机碳的浓度,能促进羊栖菜的光合作用,进而增加这种海藻的水产养殖产量。     

Inorganic carbon acquisition in Riccia fluitans L

The response of photosynthetic rate to pH indicates that CO2 is the inorganic carbon (Ci) species preferentially used by the liverwort Riccia fluitans for photosynthesis. The absence of external carbonic anhydrase (CA) activity and insensitivity to the anion-exchanger inhibitor 4,4-diisothiocyanostilbene-2,2-disulphonate (DIDS) suggest that bicarbonate is not taken up. Cultivation with bicarbonate produces a decrease in the semi-saturation constant for Ci and an increase in soluble CA activity, but maximum photosynthetic rate decreases and no significant change in the Ci compensation point occurs. Plants cultivated at 1% CO2 show no significant differences in photosynthetic characteristics and CA activity from control plants. Electrophysiological measurements also suggest that CO2 is the form that crosses the plasmalemma. Application of 1% CO2 results in a transient hyperpolarization of the membrane potential (Em) and also a transient acidification of the cytoplasmic pH (pHc). Addition of 1 mM bicarbonate at pH 7.3 produces a similar but less marked response; at an external pH of 8.3 no acidification is observed. These results suggest that bicarbonate is not transported, because its effect mimics the response caused by CO2 which enters the cell inducing a fall in cell pH, and a hyperpolarization of Em probably due to stimulation of the proton pump.Key words: Riccia fluitans, inorganic carbon, membrane potential, cytoplasmic pH, photosynthesis.      

Regulation of carbonic-anhydrase activity,inorganic-carbon uptake and photosynthetic biomass yield inChlamydomonas reinhardtii

The regulation of carbonic anhydrase by environmental conditions was determined forChlamydomonas reinhardtii. The depression of carbonic anhydrase in air-grown cells was pH-dependent. Growth of cells on air at acid pH, corresponding to 10 m CO₂ in solution, resulted in complete repression of carbonic-anhydrase activity. At pH 6.9, increasing the CO₂ concentration to 0.15% (v/v) in the gas phase, corresponding to 11 M in solution, was sufficient to completely repress carbonic-anhydrase activity. Photosynthesis and intracellular inorganic carbon were measured in air-grown and high-CO₂-grown cells using a silicone-oil centrifugation technique. With carbonic anhydrase repressed cells limited inorganic-carbon accumulation resulted from non-specific binding of CO₂. With air-grown cells, inorganic-carbon uptake at acid pH, i.e. 5.5, was linear up to 0.5 mM external inorganic-carbon concentration whereas at alkaline pH, i.e. 7.5, the accumulation ratio decreased with increase in external inorganic-carbon concentration. It is suggested that in air-grown cells at acid pH, CO₂ is the inorganic carbon species that crosses the plasmalemma. The conversion of CO₂ to HCO ₃ ^- by carbonic anhydrase in the cytosol results in inorganic-carbon accumulation and maintains the diffusion gradient for carbon dioxide across the cell boundary. However, this mechanism will not account for energy-dependent accumulation of inorganic carbon when there is little difference in pH between the exterior and cytosol.     

不同理化因子对雨生红球藻CG-11碳酸酐酶活性的影响

以雨生红球藻CG-11为实验藻株,探讨在不同CO2、HCO3-、Zn2＋浓度以及pH和氮磷比例条件下,藻细胞的碳酸酐酶活性对这些理化因子的响应。结果表明,通入空气实验组的碳酸酐酶活性最高,为（75.20±1.53）U·mg-1（Chla）,通入5%CO2条件下的碳酸酐酶活性为（9.96±1.43）U·mg-1（Chla）;高浓度HCO3-对碳酸酐酶活性亦具有明显抑制作用,培养液中可溶性无机碳的浓度与碳酸酐酶活性呈负相关;在实验设置的pH范围内,pH9.0时碳酸酐酶活性最高,为（62.32±3.25）U·mg-1（Chla）;适当的氮磷比与Zn2＋浓度显著提高了雨生红球藻CG-11的生长速率,碳酸酐酶的活性亦有明显提高。     

The Location of the Transporting System for Inorganic Carbon and the Nature of the Form Translocated in Chlamydomonas reinhardtii

The permeability of the plasmalemma of Chlamydomonas reinhardtiicells was increased by treatment with poly-L-lysine or dimethylsulphoxideas indicated by 3-phosphoglyceric acid dependent O₂ evolution.These treatments decreased the ability of the cells to accumulateinorganic carbon internally and hence their photosynthetic affinityfor inorganic carbon in the medium. With saturating light andinorganic carbon, the photosynthetic rate was less affectedby the poly-L-lysine and dimethylsulphoxide treatments. Thusthe poly-L-lysine and dimethylsulphoxide did not alter the activityof the chloroplasts but rather made the intracellular inorganiccarbon pool more freely exchangeable with the medium. It isconcluded that the transporting system for inorganic carbonis located at the plasmalemma. Treatment with Diamox, an inhibitor of carbonic anhydrase, didnot affect photosynthetic rate and accumulation of inorganiccarbon when CO₂ was supplied but strongly inhibited both parameterswhen HCO^–₃ was supplied. In a mutant of Chlamydomonasreinhardtii lacking a cell wall, carbonic anhydrase leaks tothe medium and uptake of inorganic carbon is much faster whenCO₂ is supplied than when HCO^–₃ is supplied. These resultssuggest that CO₂ rather than HCO^–₃ is the inorganic carbonspecies that is actively translocated across the plasmalemma. Key words: Chlamydomonas, Inorganic carbon uptake     

Sensing inorganic carbon: CO2 and HCO3-

Enzymes and transporters that catalyse reactions involving inorganic carbon are well characterized with respect to the species of inorganic carbon (CO2 or HCO3-) with which they interact. There is less information on the species recognized by proteins that sense inorganic carbon. In this issue of the Biochemical Journal, Hammer and colleagues show conclusively that cyanobacterial adenylyl cyclases are activated by CO2 and not HCO3-, as was believed previously. While in some circumstances a similar in vivo regulatory outcome is achieved from sensing HCO3- as from sensing CO2, there are cases in which the outcomes are significantly different. The most striking example is where a compartment lacks carbonic anhydrase yet supports large metabolic fluxes of inorganic carbon species so that CO2 and HCO3- are not at equilibrium. Other examples involve changes in pH, or temperature, of a compartment containing an equilibrium mixture of CO2 and HCO3-.     

Microalgal carbon-dioxide-concentrating mechanisms: Chlamydomonas inorganic carbon transporters

Aquatic photosynthetic micro-organisms have adapted to the variable and often-limiting availability of CO(2), and inorganic carbon (Ci) in general, by development of inducible CO(2)-concentrating mechanisms (CCMs) that allow them to optimize carbon acquisition. Both microalgal and cyanobacterial CCMs function to facilitate CO(2) assimilation when Ci is limiting via active Ci uptake systems to increase internal Ci accumulation and carbonic anhydrase activity to provide elevated internal CO(2) concentrations through the dehydration of accumulated bicarbonate. These CCMs have been studied over several decades, and details of the cyanobacterial CCM function have emerged over recent years. However, significant advances in understanding of the microalgal CCM have been more recent. With the aid of mutational approaches and the availability of multiple microalgal genome sequences, an integrated picture of the functional components of the microalgal CCMs is emerging, together with the molecular details regarding the function and regulation of the CCM. This review will focus on the recent advances in identifying and characterizing the Ci transport components of the microalgal CCM, especially in the model organism Chlamydomonas reinhardtii Dangeard.     

Carbonic anhydrase of spinach: studies on its location, inhibition, and physiological function

Carbonic anhydrase activity was determined in spinach (Spinacia oleracea) leaf organelles isolated on sucrose density gradients and was found to be predominantly in the intact chloroplast fraction. The small amount of activity associated with the mitochondrial fractions was probably due to intact chloroplast contamination. No activity could be associated with the broken chloroplast or microbody fractions. Based upon inhibitor studies, carbonic anhydrase was found to be around 2 mm in the chloroplast. Ethoxzolamide, an inhibitor of carbonic anhydrase, reduced CO(2) fixation in intact chloroplasts. The concentration required to inhibit CO(2) fixation 20 to 40% was in excess of that required to inhibit the purified enzyme. The inhibition was partially reversed by CO(2). Ethoxzolamide had no effect on photosynthetic NADP reduction or photophosphorylation measured by methyl viologen reduction. The physiological role of carbonic anhydrase was shown not to be associated with CO(2) diffusion or CO(2) concentration. It is proposed that other functions of carbonic anhydrase could be the protection against denaturation by transient localized changes in pH or the hydration of compounds other than CO(2).