CO2-dependent migration and relocation of LCIB,a pyrenoid-peripheral protein in Chlamydomonas reinhardtii

Most microalgae overcome the difficulty of acquiring inorganic carbon (Ci) in aquatic environments by inducing a CO₂-concentrating mechanism (CCM). In the green alga Chlamydomonas reinhardtii, two distinct photosynthetic acclimation states have been described under CO₂-limiting conditions (low-CO₂ [LC] and very low-CO₂ [VLC]). LC-inducible protein B (LCIB), structurally characterized as carbonic anhydrase, localizes in the chloroplast stroma under CO₂-supplied and LC conditions. In VLC conditions, it migrates to aggregate around the pyrenoid, where the CO₂-fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase is enriched. Although the physiological importance of LCIB localization changes in the chloroplast has been shown, factors necessary for the localization changes remain uncertain. Here, we examined the effect of pH, light availability, photosynthetic electron flow, and protein synthesis on the localization changes, along with measuring Ci concentrations. LCIB dispersed or localized in the basal region of the chloroplast stroma at 8.3–15 µM CO₂, whereas LCIB migrated toward the pyrenoid at 6.5 µM CO₂. Furthermore, LCIB relocated toward the pyrenoid at 2.6–3.4 µM CO₂, even in cells in the dark or treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and cycloheximide in light. In contrast, in the mutant lacking CCM1, a master regulator of CCM, LCIB remained dispersed even at 4.3 µM CO₂. Meanwhile, a simultaneous expression of LCIC, an interacting protein of LCIB, induced the localization of several speckled structures at the pyrenoid periphery. These results suggest that the localization changes of LCIB require LCIC and are controlled by CO₂ concentration with ∼7 µM as the boundary.

Algal chloroplast proteins undergo localization changes in response to CO2 concentrations, reflecting their physiological function in survival under fluctuating CO2 environments.     

Isolation and characterization of mutants defective in the localization of LCIB,an essential factor for the carbon-concentrating mechanism in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii acclimates to low-CO₂ (LC) conditions by actively transporting inorganic carbon (Ci) into the cell, resulting in an increase in photosynthetic efficiency. This mechanism is called the carbon-concentrating mechanism (CCM), and soluble protein LCIB is essential for the CCM. LCIB is localized in the vicinity of pyrenoid, a prominent structure in the chloroplast, under LC conditions in the light. In contrast, in the dark or in high-CO₂ conditions, where the CCM is inactive, LCIB diffuses away from the pyrenoid. Although the functional importance of LCIB for the CCM has been shown, the significance and mechanism of the change in suborganellar localization of LCIB remain to be elucidated. In this study, we screened 13,000 DNA-tagged mutants and isolated twelve aberrant LCIB localization (abl) mutants under LC conditions. abl-1 and abl-3 with dispersed and speckled localization of LCIB in the chloroplast showed significant decreases in Ci affinity, Ci accumulation, and CO₂ fixation. Ten abl mutants (abl-1, abl-3, abl-4, abl-5, abl-6, abl-7, abl-8, abl-9, abl-11, and abl-12) showed not only aberrant LCIB localization but also reduced pyrenoid sizes. Moreover, three abl mutants (abl-10, abl-11, and abl-12) showed the increased numbers of pyrenoids per cell. These results suggested that the specific LCIB localization could be related to pyrenoid development.     

CO<Subscript>2</Subscript>-concentrating mechanism in cyanobacterial photosynthesis: organization,physiological role,and evolutionary origin

The cellular and molecular organization of the CO₂-concentrating mechanism (CCM) of cyanobacteria is reviewed. The primary processes of uptake, translocation, and accumulation of inorganic carbon (C_i) near the active site of carbon assimilation by the enzyme ribulose-1,5-bisphosphate carboxylase in the C₃ cycle in cyanobacteria are described as one of the specialized forms of CO₂ concentration which occurs in some photoautotrophic cells. The existence of this form of CO₂ concentration expands our understanding of photosynthetic C_i assimilation. The means of supplying C_i to the C₃ cycle in cyanobacteria is not by simple diffusion into the cell, but it is the result of coordinated functions of high-affinity systems for the uptake of CO₂ and bicarbonate, as well as intracellular CO₂/HCO₃ ^? interconversions by carbonic anhydrases. These biochemical events are under genetic control, and they serve to maintain cellular homeostasis and adaptation to CO₂ limitation. Here we describe the organization of the CCM in cyanobacteria with a special focus on the CCM of relict halo- and alkaliphilic cyanobacteria of soda lakes. We also assess the role of the CCM at the levels of the organism, the biosphere, and evolution.     

Adaptation of Chlamydomonas reinhardtii High-CO(2)-Requiring Mutants to Limiting CO(2)

Photosynthetic characteristics of four high-CO₂-requiring mutants of Chlamydomonas reinhardtii were compared to those of wild type before and after a 24-hour exposure to limiting CO₂ concentrations. The four mutants represent two loci involved in the CO₂-concentrating system of this unicellular alga. All mutants had a lower photosynthetic affinity for inorganic carbon than did the wild type when grown at an elevated CO₂ concentration, indicating that the genetic lesion in each is expressed even at elevated CO₂ concentrations. Wild type and all four mutants exhibited adaptive responses to limiting CO₂ characteristic of the induction of the CO₂-concentrating system, resulting in an increased affinity for inorganic carbon only in wild type. Although other components of the CO₂-concentrating system were induced in these mutants, the defective component in each was sufficient to prevent any increase in the affinity for inorganic carbon. It was concluded that the genes corresponding to the ca-1 and pmp-1 loci exhibit at least partially constitutive expression and that all components of the CO₂-concentrating system may be required to significantly affect the photosynthetic affinity for inorganic carbon.     

The CO2-concentrating mechanism in a starchless mutant of the green unicellular alga Chlorella pyrenoidosa

The CO₂-concentrating mechanism (CCM) was induced in the green unicellular alga Chlorella when cells were transferred from high (5% CO₂) to low (0.03%) CO₂ concentrations. The induction of the CCM correlated with the formation of a starch sheath specifically around the pyrenoid in the chloroplast. With the aim of clarifying whether the starch sheath was involved in the operation of the CCM, we isolated and physiologically characterized a starchless mutant of Chlorella pyrenoidosa, designated as IAA-36. The mutant strain grew as vigorously as the wild type under high and low CO₂ concentrations, continuous light and a 12 h light/12 h dark photoperiod. The CO₂ requirement for half-maximal rates of photosynthesis [K_0.5(CO₂)] decreased from 40 μM to 2–3 μM of CO₂ when both wild type and mutant were switched from high to low CO₂. The high affinity for inorganic carbon indicates that the IAA-36 mutant is able to induce a fully active CCM. Since the mutant does not have the pyrenoid starch sheath, we conclude that the sheath is not involved in the operation of the CCM in Chlorella cells.     

Changes in Photorespiratory Enzyme Activity in Response to Limiting CO(2) in Chlamydomonas reinhardtii

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO₂-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO₂. Adaptation to air levels of CO₂ by Chlamydomonas involves induction of a CO₂-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO₂-enriched cells. Unlike WT, the high-CO₂-requiring mutant, cia-5, does not respond to limiting CO₂ conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO₂ which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.     

Modeling and mutagenesis of amino acid residues critical for CO2 hydration by specialized NDH-1 complexes in cyanobacteria

The uptake of inorganic carbon in cyanobacteria is facilitated by an energetically intensive CO₂-concentrating mechanism (CCM). This includes specialized Type-1 NDH complexes that function to couple photosynthetic redox energy to CO₂ hydration forming the bicarbonate that accumulates to high cytoplasmic concentrations during the operation of the CCM, required for effective carbon fixation. Here we used a Synechococcus PCC7942 expression system to investigate the role of conserved histidine and cysteine residues in the CupB (also designated, ChpX) protein, which has been hypothesized to participate in a vectoral CO₂ hydration reaction near the interface between CupB protein and the proton-pumping subunits of the NDH-1 complex. A homology model has been constructed and most of the targeted conserved residues are in the vicinity of a Zn ion modeled to form the catalytic site of deprotonation and CO₂ hydration. Growth and CO₂ uptake assays show that the most severe defects in activity among the targeted residues are due to a substitution of the predicted Zn ligand, CupB-His86. Mutations at other sites produced intermediate effects. Proteomic analysis revealed that some amino acid substitution mutations of CupB caused the induction of bicarbonate uptake proteins to a greater extent than complete deletion of CupB, despite growth under CO₂-enriched conditions. The results are discussed in terms of hypotheses on the catalytic function of this unusual enzyme.     

Evidence for the functioning of photosynthetic CO2-concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts

The photosynthetic properties of a range of lichens containing both green algal (11 species) and cyanobacterial (6 species) photobionts were examined with the aim of determining if there was clear evidence for the operation of a CO₂-concentrating mechanism (CCM) within the photobionts. Using a CO₂-gas-exchange system, which allowed resolution of fast transients, evidence was obtained for the existence of an inorganic carbon pool which accumulated in the light and was released in the dark. The pool was large (500–1000 nmol · mg Chl) in cyanobacterial lichens and about tenfold smaller in green algal lichens. In Hypogymnia physodes (L.) Nyl., which contains the green alga Trebouxia jamesii, a small inorganic carbon pool was rapidly formed in the light. Carbon dioxide was released from this pool into the gas phase upon darkening within about 20 s when photosynthesis was inhibited by the carbon-reduction-cycle inhibitor glycolaldehyde. In the absence of this inhibitor, release appeared to be obscured by carboxylation of ribulose bisphosphate. The kinetics of CO₂ uptake and release were monophasic. The operation of an active CCM could be distinguished from passive accumulation and release accompanying the reversible light-dependent alkalization of the stroma by the presence of saturation characteristics with respect to external CO₂. In Peltigera canina (L.) Willd., which contains the cyanobacterium Nostoc sp., a larger CO₂ pool was taken up over a longer period in the light and the release of this pool in the dark was slow, lasting 3–5 min. This pool also accumulated in the presence of glycolaldehyde, and under these conditions the CO₂ release was biphasic. In both species, photosynthesis at low CO₂ was inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide (EZ). Inhibition could be reversed fully or to a considerable extent by high CO₂. In Peltigera, EZ decreased both the accumulation of the CO₂ pool by the CCM and the rate of photosynthesis. Free-living cultures of Nostoc sp. showed a similar effect of EZ on photosynthesis, although it was more dramatic than that seen with the lichen thalli. In contrast, in Hypogymnia, EZ actually increased the size of the CO₂ pool, although it inhibited photosynthesis. This effect was also seen when glycolaldehyde was present together with EZ. Surprisingly, EZ did not alter the kinetics of either CO₂ uptake or release. Taken together, the evidence indicates the operation in cyanobacterial lichens of a CCM which is capable of considerable elevation of internal CO₂ and is similar to that reported for free-living cyanobacteria. The CCM of green algal lichens accumulates much less CO₂ and is probably less effective than that which operates in cyanobacterial lichens.     

Membrane-Associated Polypeptides Induced in Chlamydomonas by Limiting CO(2) Concentrations

Chlamydomonas reinhardtii and other unicellular green algae have a high apparent affinity for CO₂, little O₂ inhibition of photosynthesis, and reduced photorespiration. These characteristics result from operation of a CO₂-concentrating system. The CO₂-concentrating system involves active inorganic carbon transport and is under environmental control. Cells grown at limiting CO₂ concentrations have inorganic carbon transport activity, but cells grown at 5% CO₂ do not. Four membrane-associated polypeptides (M_r 19, 21, 35, and 36 kilodaltons) have been identified which either appear or increase in abundance during adaptation to limiting CO₂ concentrations. The appearance of two of the polypeptides occurs over roughly the same time course as the appearance of the CO₂-concentrating system activity in response to CO₂ limitation.     

Photosynthetic Light Utilization Efficiency, Photosystem II Heterogeneity, and Fluorescence Quenching in Chlamydomonas reinhardtii during the Induction of the CO(2)-Concentrating Mechanism

The photosynthetic light-response curve, the relative amounts of the different photosystem II (PSII) units, and fluorescence quenching were altered in an adaptive manner when CO₂-enriched wild-type Chlamydomonas reinhardtii cells were transferred to low levels of CO₂. This treatment is known to result in the induction of an energy-dependent CO₂-concentrating mechanism (CCM) that increases the internal inorganic carbon concentration and thus the photosynthetic CO₂ utilization efficiency. After 3 to 6 h of low inorganic carbon treatment, several changes in the photosynthetic energy-transducing reactions appeared and proceeded for about 12 h. After this time, the fluorescence parameter variable/maximal fluorescence yield and the amounts of both PSIIα and PSIIβ (secondary quinone electron acceptor of PSII-reducing) centers had decreased, whereas the amount of PSIIβ (secondary quinone electron acceptor of PSII-nonreducing) centers had increased. The yield of noncyclic electron transport also decreased during the induction of the CCM, whereas both photochemical and nonphotochemical quenching of PSII fluorescence increased. Concurrent with these changes, the photosynthetic light-utilization efficiency also decreased significantly, largely attributed to a decline in the curvature parameter θ, the convexity of the photosynthetic light-response curve. Thus, it is concluded that the increased CO₂ utilization efficiency in algal cells possessing the CCM is maintained at the cost of a reduced light utilization efficiency, most probably due to the reduced energy flow through PSII.     

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

An active CO₂-concentrating mechanism is induced when Chlamydomonas reinhardtii acclimates to limiting inorganic carbon (Ci), either low-CO₂ (L-CO₂; air level; approximately 0.04% CO₂) or very low-CO₂ (VL-CO₂; approximately 0.01% CO₂) conditions. A mutant, ad1, which is defective in the limiting-CO₂-inducible, plastid-localized LCIB, can grow in high-CO₂ or VL-CO₂ conditions but dies in L-CO₂, indicating a deficiency in a L-CO₂-specific Ci uptake and accumulation system. In this study, we identified two ad1 suppressors that can grow in L-CO₂ but die in VL-CO₂. Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of L-CO₂-acclimated suppressors under acclimation CO₂ concentrations were more than 2-fold higher than ad1, apparently resulting from a more than 20-fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. However, photosynthetic rates of VL-CO₂-acclimated cells under acclimation CO₂ concentrations were too low to support growth in spite of a significantly elevated intracellular Ci concentration. We conclude that LCIB functions downstream of CAH3 in the CO₂-concentrating mechanism and probably plays a role in trapping CO₂ released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO₂ to support growth in L-CO₂-acclimated cells, but not in VL-CO₂-acclimated cells, even in the absence of LCIB.CO₂ serves both as the substrate for photosynthesis and as an important signal to regulate plant growth and development, so variable CO₂ concentrations can impact photosynthesis, growth, and productivity of plants. Terrestrial C₄ plants have developed a CO₂-concentrating mechanism (CCM) involving anatomical and biochemical adaptations to accumulate a higher concentration of CO₂ as substrate Rubisco and to suppress oxygenation of ribulose-1,5-bisP, a wasteful side reaction. In contrast, a different type of CCM is induced in the unicellular green microalga Chlamydomonas reinhardtii when the supply of dissolved inorganic carbon (Ci; CO₂ and HCO₃⁻) for photosynthesis is limited (Beardall and Giordano, 2002; Giordano et al., 2005; Moroney and Ynalvez, 2007; Spalding, 2008). In response to limiting CO₂, the CCM uses active Ci transport, both at the plasma membrane and the chloroplast envelope, to accumulate a high concentration of HCO₃⁻ within the chloroplast (Palmqvist et al., 1988; Sültemeyer et al., 1988). The thylakoid lumen carbonic anhydrase (CAH3) plays an essential role in the rapid dehydration of the accumulated HCO₃⁻ to release CO₂ into the pyrenoid, a Rubisco-containing internal compartment of the chloroplast, for assimilation by Rubisco (Price et al., 2002; Spalding et al., 2002).While a number of genes and proteins essential to the operation of the CCM in C. reinhardtii have been identified, our understanding of Ci uptake and its regulation, as well as other aspects of CCM function is limited. A better understanding of the similar CCM in prokaryotic organisms, specifically the cyanobacteria Synechocystis and Synechococcus, has been gained. At least five different types of Ci transporters have been identified in cyanobacteria, including three HCO₃⁻ transporters and two active CO₂ uptake systems (Price et al., 2002, 2004).Recently, at least three distinct CO₂-regulated acclimation states were identified in C. reinhardtii based on growth, photosynthesis and gene expression characteristics, a high-CO₂ (H-CO₂) state (5%–0.5% CO₂), low-CO₂ (L-CO₂) state (air level; 0.4%–0.03% CO₂), and very low-CO₂ (VL-CO₂) state (0.01%–0.005% CO₂; Vance and Spalding, 2005). Two allelic HCR (H-CO₂-requiring) mutants, pmp1 and ad1, grow as well (pmp1) or nearly as well (ad1) as wild-type cells in both H-CO₂ and VL-CO₂ conditions while only dying in L-CO₂, indicating a deficient Ci transport and/or accumulation system only in the L-CO₂ acclimation state (Spalding et al., 1983b, 2002). The defective gene responsible for the pmp1/ad1 phenotype was identified as LCIB, a limiting CO₂-inducible gene, the product of which is predicted to be located in the chloroplast stroma and proposed to be involved with chloroplast Ci uptake in L-CO₂ conditions (Wang and Spalding, 2006). The LCIB gene product is a member of a small gene family so far only found in a few microalgae species (Spalding, 2008).To investigate the roles of LCIB in eukaryotic photosynthetic organisms and identify other functional components involved in chloroplast Ci accumulation in C. reinhardtii, we used an insertional mutagenesis approach to select suppressors of the air-dier phenotype of the LCIB mutant ad1. In this study, we describe two ad1 suppressors, ad-su6 and ad-su7, that grow normally in L-CO₂ but, unlike ad1, die in VL-CO₂. This report also presents data suggesting that the air-dier phenotype of ad1 is suppressed by increased intracellular Ci concentrations in the two suppressors, and suggesting a possible role for LCIB as a CO₂ trap rather than having any direct role in chloroplast envelope Ci transport.     

A pyrenoid-based carbon-concentrating mechanism is present in terrestrial bryophytes of the class Anthocerotae

It has been widely accepted that carbon assimilation in bryophytes is exclusively based on the conventional C₃ photosynthetic pathway. The occurrence of biochemical CO₂-concentrating mechanisms (C₄ or Crassulacean acid metabolism), which have developed in plants in the last 20–100 million years, has been discounted for bryophytes from studies of the carbon isotope composition (¹³C) of organic material. In contrast cyanobacteria and many algae show active accumulation of dissolved inorganic carbon via biophysical CO₂-concentrating mechanisms which are also found in the photobiont partners in certain lichens. The presence of a pyrenoid, a granular particle within the chloroplast, has been linked with CO₂-concentrating mechanism activity in green algae and lichens and we now show that such a mechanism is categorically associated with the occurrence of a pyrenoid in bryophytes belonging to the class of Anthocerotae. These observations have significant evolutionary implications for the development of terrestrial photosynthesis during the colonisation of the land, raising the intriguing question of why the pyrenoid-based CO₂-concentrating mechanism did not persist in the terrestrial environment.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIG dissolved inorganic carbon (CO₂+HCO ₃ ^- +CO ₂ ^- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - Rubisco ribulose-l,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This work was supported by the Natural Environment Research Council (GR3/8813) and the Leverhulme Trust. We thank Prof. A. Roy Perry (National Museum of Wales, Cardiff), Dr. B. Coppins and Mr. D. Long (Royal Botanic Garden Edinburgh) for access to herbarium specimens and Mr. M. Fletcher for providing living bryophytes.     

High-resolution suborganellar localization of Ca<Superscript>2+</Superscript>-binding protein CAS,a novel regulator of CO<Subscript>2</Subscript>-concentrating mechanism

Many aquatic algae induce a CO₂-concentrating mechanism (CCM) associated with active inorganic carbon transport to maintain high photosynthetic affinity using dissolved inorganic carbon even in low-CO₂ (LC) conditions. In the green alga Chlamydomonas reinhardtii, a Ca²⁺-binding protein CAS was identified as a novel factor regulating the expression of CCM-related proteins including bicarbonate transporters. Although previous studies revealed that CAS associates with the thylakoid membrane and changes its localization in response to CO₂ and light availability, its detailed localization in the chloroplast has not been examined in vivo. In this study, high-resolution fluorescence images of CAS fused with a Chlamydomonas-adapted fluorescence protein, Clover, were obtained by using a sensitive hybrid detector and an image deconvolution method. In high-CO₂ (5% v/v) conditions, the fluorescence signals of Clover displayed a mesh-like structure in the chloroplast and part of the signals discontinuously overlapped with chlorophyll autofluorescence. The fluorescence signals gathered inside the pyrenoid as a distinct wheel-like structure at 2 h after transfer to LC-light condition, and then localized to the center of the pyrenoid at 12 h. These results suggest that CAS could move in the chloroplast along the thylakoid membrane in response to lowering CO₂ and gather inside the pyrenoid during the operation of the CCM.     

Genetic and physiological analysis of the CO2-concentrating system of Chlamydomonas reinhardii

When grown photoautotrophically at air levels of CO₂, Chlamydomonas reinhardii possesses a system involving active transport of inorganic carbon which increases the intracellular CO₂ concentration considerably above ambient, thereby stimulating photosynthetic CO₂ assimilation. In previous investigations, two mutant strains of this unicellular green alga deficient in some component of this CO₂-concentrating system were recovered as strains requiring high levels of CO₂ to support photoautotrophic growth. One of the mutants, ca-1-12-1C, is a leaky (nonstringent) CO₂-requiring strain deficient in carbonic anhydrase (EC 4.2.1.1) activity, while the other, pmp-1-16-5K, is a stringent CO₂-requiring strain deficient in inorganic carbon transport. In the present study a double mutant (ca pmp) was constructed to investigate the physiological and biochemical interaction of the two mutations. The two mutations are unlinked and inherited in a Mendelian fashion. The double mutant was found to have a leaky CO₂-requiring phenotype, indicating that the mutation ca-1 overcomes the stringent CO₂-requirement conferred by the mutation pmp-1. Several physiological characteristics of the double mutant were very similar to the carbonic-anhydrase-deficient mutant, including high CO₂ compensation concentration, photosynthetic CO₂ response curve, and deficiency of carbonic-anhydrase activity. However, the labeling pattern of metabolites during photosynthesis in ¹⁴CO₂ was more like that of the bicarbonatetransport-deficient mutant, and accumulation of internal inorganic carbon was intermediate between that of the two original mutants. These data indicate a previously unsuspected complexity in the Chlamydomonas CO₂-concentrating system.     

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.     

CO2 acquisition in Chlamydomonas acidophila is influenced mainly by CO2, not phosphorus,availability

The extremophilic green microalga Chlamydomonas acidophila grows in very acidic waters (pH 2.3–3.4), where CO₂ is the sole inorganic carbon source. Previous work has revealed that the species can accumulate inorganic carbon (Ci) and exhibits high affinity CO₂ utilization under low-CO₂ (air-equilibrium) conditions, similar to organisms with an active CO₂ concentrating mechanism (CCM), whereas both processes are down-regulated under high CO₂ (4.5 % CO₂) conditions. Responses of this species to phosphorus (Pi)-limited conditions suggested a contrasting regulation of the CCM characteristics. Therefore, we measured external carbonic anhydrase (CA_ext) activities and protein expression (CAH1), the internal pH, Ci accumulation, and CO₂-utilization in cells adapted to high or low CO₂ under Pi-replete and Pi-limited conditions. Results reveal that C. acidophila expressed CA_ext activity and expressed a protein cross-reacting with CAH1 (the CA_ext from Chlamydomonas reinhardtii). Although the function of this CA remains unclear, CA_ext activity and high affinity CO₂ utilization were the highest under low CO₂ conditions. C. acidophila accumulated Ci and expressed the CAH1 protein under all conditions tested, and C. reinhardtii also contained substantial amounts of CAH1 protein under Pi-limitation. In conclusion, Ci utilization is optimized in C. acidophila under ecologically relevant conditions, which may enable optimal survival in its extreme Ci- and Pi-limited habitat. The exact physiological and biochemical acclimation remains to be further studied.     

Physiology of inorganic C acquisition and implications for resource use efficiency by marine phytoplankton: relation to increased CO2 and temperature

Abstract. Photosynthesis by many marine phytoplankton algae is saturated by the inorganic C concentration in air-equilibrated sea water. These organisms appear to use an active inorganic C transport process (CO₂-concentrating mechanism) which increases the CO₂ concentration around rubisco and saturates this enzyme with CO₂ and suppresses its oxygenase activity. A minority of marine phytoplankton algae have photosynthetic characteristics more suggestive of diffusive CO₂ entry; the inorganic C concentration present in sea water does not saturate photosynthesis by these organisms. Theoretical considerations, tested when possible against observation, suggest that the organisms with a CO₂-concentrating mechanism could have a lower cost of photons, nitrogen, iron, manganese and molybdenum to achieve a given rate of carbon accumulation by the cells than is the case for the organisms with diffusive CO₂ entry. Zinc and selenium costs may show the reverse effect. The increased sea-surface inorganic C, and CO₂ concentrations which will result from anthropogenic increases in atmospheric CO₂ content are predicted to increase the rate of photosynthesis, and of growth when other resources are abundant, and to reduce, or reverse, the higher resource (photons, nitrogen, iron, manganese and molybdenum) cost of a given rate of CO₂ assimilation in organisms with CO₂ diffusion relative to those which have CO₂ concentrating mechanisms and do not repress them at higher inorganic C concentrations. These effects may well alter species composition, and overall resource cost of growth, of phytoplankton; any influence that these effects may have on CO₂ removal from the atmosphere are severely constrained by other trophic levels and, especially, oceanic circulation patterns. Changed sea-surface temperatures are unlikely to qualitatively alter these conclusions.