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Photosynthesis Research 77: 139–153, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

139

Regular paper

Historical perspective on microalgal and cyanobacterial acclimation to low- and extremely high-CO2 conditions Shigetoh Miyachi1,∗ , Ikuko Iwasaki2 & Yoshihiro Shiraiwa3 1 Marine

Biotechnology Institute, Kamaishi City, Iwate 026-0001, Japan; 2 Biotechnology Institute, Faculty of Bioresource Science, Akita Prefectural University, Akita, Japan; 3 Institute of Biological Sciences, University of Tsukuba, Tsukuba 305-8572, Japan; ∗ Author for correspondence (e-mail: [email protected]; fax:+81193-266592)

Key words: carbonic anhydrase, Chlorococcum littorale, chlorophyll fluorescence, CO2 acclimation, CO2 concentrating mechanism, cyclic electron flow, extremely high-CO2 tolerance, inorganic carbon transport, photosystems, state transition

Abstract Reports in the 1970s from several laboratories revealed that the affinity of photosynthetic machinery for dissolved inorganic carbon (DIC) was greatly increased when unicellular green microalgae were transferred from high to lowCO2 conditions. This increase was due to the induction of carbonic anhydrase (CA) and the active transport of CO2 and/or HCO3 − which increased the internal DIC concentration. The feature is referred to as the ‘CO2 -concentrating mechanism (CCM)’. It was revealed that CA facilitates the supply of DIC from outside to inside the algal cells. It was also found that the active species of DIC absorbed by the algal cells and chloroplasts were CO2 and/or HCO3 − , depending on the species. In the 1990s, gene technology started to throw light on the molecular aspects of CCM and identified the genes involved. The identification of the active HCO3 − transporter, of the molecules functioning for the energization of cyanobacteria and of CAs with different cellular localizations in eukaryotes are examples of such successes. The first X-ray structural analysis of CA in a photosynthetic organism was carried out with a red alga. The results showed that the red alga possessed a homodimeric β-type of CA composed of two internally repeating structures. An increase in the CO2 concentration to several percent results in the loss of CCM and any further increase is often disadvantageous to cellular growth. It has recently been found that some microalgae and cyanobacteria can grow rapidly even under CO2 concentrations higher than 40%. Studies on the mechanism underlying the resistance to extremely high CO2 concentrations have indicated that only algae that can adopt the state transition in favor of PS I could adapt to and survive under such conditions. It was concluded that extra ATP produced by enhanced PS I cyclic electron flow is used as an energy source of H+ -transport in extremely high-CO2 conditions. This same state transition has also been observed when high-CO2 cells were transferred to low CO2 conditions, indicating that ATP produced by cyclic electron transfer was necessary to accumulate DIC in low-CO2 conditions. Abbreviations: CA – carbonic anhydrase; CCM – CO2 -concentrating mechanism; DIC – dissolved inorganic carbon; Rubisco – ribulose 1, 5-bisphosphate carboxylase/oxygenase Introduction When microalgae and cyanobacteria are transferred from high- (e.g., 5%) to low-CO2 conditions (e.g.,

ordinary air), their photosynthetic activity at a CO2 limiting concentration is increased (Figure 1A). These cells are referred to as low-CO2 cells, low-Ci cells, low-CO2 grown cells or low-CO2 acclimated cells. By

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Figure 1. Relationship between rate of photosynthesis and external DIC concentration in microalgae grown under low, high and extremely high-CO2 conditions. A: low-CO2 cells (grown in air with 0.04% CO2 ); B: low-CO2 cells treated with a CA inhibitor or high-CO2 cells (e.g., Chlamydomonas); C: high-CO2 cells (grown in air containing several percent CO2 ) (e.g., Chlorella); D: extremely high-CO2 cells grown under > 40% CO2 conditions.

contrast, when the cells are grown under high-CO2 conditions (air containing several percent of CO2 ), although the cells lose their ability to efficiently fix CO2 under a limiting concentration of DIC, there is instead an increase in the rate of maximum photosynthesis under saturating DIC conditions in some species of microalgae such as Chlorella (Figure 1C). However, the maximum rate does not change in other species, such as Chlamydomonas (Figure 1B). These cells are referred to as high-CO2 cells, high-Ci cells, high-CO2 grown cells or high-CO2 acclimated cells. It was generally assumed that microalgae were susceptible to a very high concentration of CO2 . However, it has recently been found that some microalgae can grow very rapidly at a CO2 concentration higher than 40%, such cells being referred to as extremely high-CO2 cells (Figure 1D). The mechanism for the acclimation and adaptation of aquatic photoautotrophs to various CO2 environments has been one of the main targets in photosynthesis research during the past two decades or so. A difference in the photosynthetic characteristics between low- and high-CO2 cells was first described by Graham and Whittingham (1968) with Chlorella cells. In respect of Chlamydomonas, Nelson et al. (1969) found that the CA activity was 10–20-fold greater in low-CO2 cells than in high-CO2 (1%) cells. A change in the correlation between CA activity and the rate of photosynthesis induced by the environmental CO2 concentration was first reported by

Graham and Reed (1971) and Graham et al. (1971) for Chlorella pyrenoidosa. Similar phenomena were also observed in Scenedesmus obliquus (Findenegg 1976), Anacystis nidulans (Döhler 1974) and four cyanobacterial species (Ingle and Colman 1975). With the above-mentioned findings as the background, the subsequent reports published in 1977 from Carnegie Institution of Washington and University of Tokyo aroused interest in the roles played by CCM and carbonic anhydrase in microalgal and cyanobacterial photosynthesis. Berry et al. (1976) reported that the affinity of the photosynthetic mechanism for DIC was much higher in low-CO2 cells than in high-CO2 cells of Chlamydomonas reinhardtii. To explain this finding, they postulated a mechanism that can increase the CO2 concentration at the site of CO2 fixation by Rubisco which includes an active bicarbonate influx pump. Using a filtering centrifugation technique, they showed that the ratio of the internal/external CO2 concentration was higher in low-CO2 cells than in high-CO2 cells of C. reinhardtii and Anabaena variabilis (Badger et al. 1977, 1978). The same changes in Km (CO2 ) and Vmax induced by a change in the CO2 concentration during algal growth were observed in Chlorella vulgaris 11h cells (Hogetsu and Miyachi 1977) (Figure 1). During acclimation to a low-CO2 concentration, both the rate of photosynthesis under limiting CO2 conditions as well as CA activity increased in parallel, while the Rubisco activity did not change (Hogetsu and Miyachi 1979). As a result, the CA activity in high-CO2 cells of C. vulgaris 11h was 20-90 times lower than that in low-CO2 cells. When the high-CO2 cells were transferred to low-CO2 conditions, the CA activity was increased, this increase being accompanied by an increase in the rate of photosynthetic CO2 fixation under the CO2 limiting conditions. The opposite effects on the CA activity and the rate of photosynthesis at a limiting CO2 concentration were observed when low-CO2 cells were transferred to high-CO2 conditions. Diamox (= Acetazolamide), an inhibitor of CA, diminished the affinity of the photosynthetic mechanism for DIC and inhibited photosynthesis only under low-CO2 concentration, a high-CO2 concentration having no effect on the photosynthesis (Figure 1B). Fractionation by density of the low-CO2 cells in a non-aqueous medium indicated that CA was located in the chloroplast. Hogetsu and Miyachi (1979) therefore concluded that CA in the chloroplasts enhanced photosynthesis by stimulating the supply of CO2 under a limiting concentration of CO2 . The mechanism, referred to as ‘the

141 indirect supply of CO2 ’, was proposed to explain the role of CA in photosynthesis under a low-CO2 concentration. While the concept of CCM was accepted soon after the report by Berry et al. (1976), it took several years before the role of CA was accepted. With some cyanobacteria and microalgae, Bürger et al. (1988) and Miyachi et al. (1996) found that the ratio of PS I/PS II fluorescence (77K) was higher in low-CO2 cells than that in high-CO2 cells. A similar change in the ratio was observed when an extremely high-CO2-tolerant alga such as Chlorococcum littorale was transferred from air to extremely high-CO2 conditions, while no such fluorescence change was apparent when Stichococcus sp. cells which were intolerant to extremely high-CO2 conditions were transferred from air to 40% CO2 (Iwasaki et al. 1998; Satoh et al. 2002). Studies along similar lines led to the conclusion that microalgae and cyanobacteria could acclimate to low-CO2 conditions by producing the extra ATP needed to supply necessary DIC via cyclic electron flow. Likewise, only those microalgae which produced extra ATP by the same state transition could grow under extremely high-CO2 conditions.

CCM and CA in eukaryotes CCM operates by a combination of several components, such as energy-dependent DIC transporter(s), subcellular compartmentation and CAs (Badger et al. 1980; Aizawa and Miyachi 1986; Tsuzuki and Miyachi 1989; Sültemeyer et al. 1993; Suzuki et al. 1994; Badger and Price 1994; Spalding 1998). The function of CCM enables cells to acquire a high apparent affinity for CO2 , a low CO2 compensation point, and reduced photorespiration due to a high ratio of CO2 to O2 . Algal species that have CA activity only within the cells, e.g., C. vulgaris 11h and Chlorella miniata, mainly take up CO2 , while those showing extracellular CA activity, e.g., C. vulgaris C-3, C. reinhardtii and Dunaliella tertiolecta, take up HCO3 − in addition to CO2 . It has been assumed that HCO3 − could apparently be used for photosynthesis via dehydration to CO2 due to the functioning of CA (Miyachi and Shiraiwa 1979; Tsuzuki et al. 1980; Miyachi et al. 1983; Imamura et al. 1983; Tsuzuki 1983; Aizawa et al. 1986). The function of CA in CO2 uptake was supported by evidence that CO2 transport through the plasma membrane of erythrocyte was facilitated by CA (Enns 1967). On the other hand, Tsuzuki (1986)

demonstrated that low-CO2 cells of C. vulgaris 11h could also absorb HCO3 − , although the rate was much lower than that of CO2 . In addition, a direct HCO3 − uptake has also been demonstrated by low-CO2 cells of C. reinhardtii (Williams and Turpin 1987; Sültemeyer et al. 1991), Chlorella saccharophila (Gehl et al. 1990), Skeltonema costatum (Korb et al. 1997), Phaeodactylum tricornutum (Nimer et al. 1997) and Thalassiosira weissflogii (Nimer et al. 1997; Lane and Morel 2000). The localization of the active transport system for inorganic carbon in the plasma membrane and in both the plasma membrane and the chloroplast envelope has been suggested for Chlorella (Rotatore and Colman 1991), and for Chlamydomonas, Scenedesmus and Dunaliella, respectively (Goyal and Tolbert 1989; Sültemeyer et al. 1989, 1991; Ramazanov and Cardenas 1992; Badger and Price 1994; Palmqvist et al. 1994; Amoroso et al. 1998). The putative bicarbonate transporter had both chemical and immunological similarity to mammalian erythrocyte HCO3 − /Cl− exchangers (the anion-exchange type of transporter) sensitive to 4,4 -diisothiocyano-2,2-stilbenedisulphonate (DIDS) in macrophytes (Drechsler et al. 1993). The involvement of transmembrane ATPase proteins was reported in DIC uptake by chlorophytes (Rotatore et al. 1992; Karlsson et al. 1994; Ramazanov et al. 1995). Inhibition of DIC acquisition by DIDS was speciesdependent in chromophyte algae (Merett et al. 1996; Nimer et al. 1996). Studies on changing photochemical properties during the process of acclimation in Chlamydomonas from high to low-CO2 conditions suggested that the active transport of DIC required ATP (Spalding et al. 1984; Palmqvist et al. 1990). The distribution, cellular location and activity of CA vary according to the species of organisms (Reed and Graham 1981; Aizawa and Miyachi 1986; Fukuzawa et al. 2000; Hewett-Emmett 2000). The extracellular activity of CA has been found on the surface of low-CO2 cells of Scenedesmus (Findenegg 1976). In the wall-less mutant of C. reinhardtii, cw-15, extracellular CA was released into the medium, suggesting that CA was located in the periplasmic space (Kimpel et. al. 1983). The presence of periplasmic CA was also shown by the inhibitory effect of the membraneimpermeable CA inhibitor, dextran-bound acetazolamide, in Chlamydomonas (Moroney et al. 1985), of subtilisin in Dunaliella (Aizawa and Miyachi 1984) and of Pronase P in Chlorella (Nara et al. 1990). Low-CO2 -inducible periplasmic CA in Chlamydomo-

142 nas was found to be a glycoprotein consisting of two large (35 kD) and two small (4 kD) subunits linked by an S-S bond (Kamo et al. 1990). During the synthesis of one of the large subunits of CA, a 42-kD polypeptide was transiently synthesized and then converted to a 35-kD polypeptide (Toguri et al. 1986). In respect of other eukaryotic algae, only a few α-CAs have been cloned in Chlorella and Dunaliella (Satoh et al. 1998; Fisher et al. 1996). There have been some conflicting reports on the functions of periplasmic CA in Chlamydomonas. Moroney et al. (1985) reported that the enzyme affected the enhancement of photosynthesis under limiting CO2 concentrations in an alkaline pH range, but Williams and Turpin (1987) could not confirm their results. More recently, Van and Spalding (1999) have shown that there was no difference in the rate of photosynthetic O2 evolution between wild-type cells and periplasmic-CA-deficient mutant cells, even in an alkaline pH range, indicating that periplasmic CA was not essential for either CCM or the growth of C. reinhardtii at limiting CO2 concentrations (see also the review by Spalding 1998). For periplasmic CA to be essential, the conversion between CO2 and HCO3 − should be a critical step in photosynthesis under lowCO2 conditions. On the other hand, it is unanimously agreed that internal CA is essential for photosynthesis, although its activity is much lower than the external activity in Chlamydomonas. In this context, it has been shown that CA was mainly located in the chloroplast of such eukaryotic algae, as Chlorella vulgaris 11h (Hogetsu and Miyachi 1979), C. ellipsoidea (Rotatore and Colman 1990), Dunaliella salina (Ramazanov and Cardenas 1992), and the coccolithophorid, Pleurocrysis carterae (Quiroga and Gonzalez 1993). The application of immunogold labeling with a polyclonal antibody raised against recombinant CA derived from cloned cDNA of CA in Porphyridium purpureum, proved that CA is associated with the cytoplasmic periphery of the plasma membrane, although not in any membrane-spanning capacity (Mitsuhashi et al. 2001). P. tricornutum had only internal β-type CA with a molecular mass of 28 kD (Satoh et al. 2001b), and no external CA activity. An isozyme of CA with a molecular mass of 29.5 kD (ctCA1 encoded by the Cah3 gene) has recently been found in Chlamydomonas. This CA is of α-type and functions to efficiently provide CO2 to Rubisco from actively accumulated HCO3 − in lowCO2 cells (Karlsson et al. 1995). This CA was soluble

(Funke et al. 1997) and located in the thylakoid lumen of Chlamydomonas cells (Karlsson et al. 1998). In addition, insoluble chloroplast CA has also been found in the ctCA1-deficient mutant of Chlamydomonas (Sültemeyer et al. 1995). Co-localization of pyrenoid CA with Rubisco was shown in Chlamydomonas and it was, therefore, suggested that it functions in efficiently supplying CO2 to Rubisco (Kuchitsu et al. 1991; Funke et al. 1997). This function might be similar to that of carboxysomal CA in cyanobacteria reported by Badger and Price (1992, 1994). Based on comparative morphological, physiological and phylogenetic studies on Chlamydomonas and Chloromonas strains, Morita et al. (1998, 1999) have shown that the presence of typical pyrenoids with a high concentration of Rubisco molecules was related to the formation of a large internal DIC pool. However, some opposing results, indicating that the pyrenoid and pyrenoid starch sheath did not play any essential role in CCM, have been presented for Chlorella and Chlamydomonas (Plumed et al. 1996; Villarejo et al. 1996). Further investigations are necessary on the role of pyrenoid CA. The 36 kD polypeptide, LIP-36, that consists of Ccp1 and Ccp2 has been induced under low-CO2 conditions in Chlamydomonas (Geraghty et al. 1990; Ramazanov et al. 1993; Chen et al. 1997). The polypeptide was located in the chloroplast envelope and the gene sequence is quite similar to that of the mitochondrial carrier protein superfamily, suggesting that the protein may function as a carrier through the chloroplast envelope (Chen et al. 1997). In a wall-less mutant of C. reinhardtii, the isoenzyme of CA with molecular mass of 110 kD has been identified as a cytoplasmic CA (Husic et al. 1989). This molecular mass is similar to that of intracellular and extrachloroplastic β-CA, the mature protein of which is a homotetramer with an estimated molecular mass of 100 kD in the unicellular green alga, Coccomyxa (Hiltonen et al. 1998). The mitochondrial CAs, mtCA1 and mtCA2 (βCAs) encoded in Mca1 and Mca2 genes, which are identical to LIP-21 reported by Geraghty and Spalding (1996) have been found in Chlamydomonas (Eriksson et al. 1996). It has been suggested that mitochondrial CAs function in pH regulation via the hydration of photorespiratory CO2 to prevent alkalization of the mitochondrial matrix during the rapid production of ammonia and to facilitate the diffusion of CO2 from the matrix (Eriksson et al. 1996). The conversion to HCO3 − of the CO2 produced in the mitochondria under illumination, from tricarboxylic acid cycle

143 activity and from decarboxylation of glycine in any photorespiratory carbon oxidation cycle activity by the mitochondrial CA was also suggested (Raven 2001). Spalding (1998) has assumed that it functions by converting into bicarbonate any CO2 produced in the cytoplasm and leaked from the chloroplasts and mitochondria during photorespiration and respiration. If this CA were to be located in the intermembrane space rather than in the matrix of the mitochondrion, the CA would be more functional. High-CO2 inducible CA has also been found in Chlamydomonas and identified as CA2 (Tachiki et al. 1992; Rawat and Moroney 1991), although its function is still unclear. It was found that two CA genes were clustered in tandem in the genome of Chlamydomonas, the upstream and downstream genes being respectively designated as CAH1 and CAH2 (Fujiwara et al. 1990; Fukuzawa et al. 1990a, b; Ishida et al. 1993). CAH1 and CAH2 consisted of 11 exons and 10 introns and were expressed reciprocally, namely under low- and high-CO2 conditions, respectively. Light was required for CAH1 expression, but not for CAH2 (Tachiki et al. 1994). CA of the red alga P. purpureum contains two zinc atoms per molecule. It was suggested that this CA was derived from duplication of an ancestral CA gene with subsequent fusion of the duplicated CA gene (Mitsuhashi and Miyachi 1996). An X-ray analysis of the structure revealed that the CA monomer of P. purpureum is composed of two internally repeating structures, being folded as a pair of fundamentally equivalent motifs of an α/β domain and three projecting α-helices (Mitsuhashi et al. 2000) (Figure 2). The homodimeric CA appears as a tetramer with pseudo 222 symmetry. The zinc at the active site is coordinated by a Cys-Asp-His-Cys tetrad that is strictly conserved among the β-CAs. CA of the marine diatom Thalassiosira weissflogii has recently been found to be unique, since it has no significant homology with other CAs, although the active site structure is similar to that of mammalian α-CAs (Cox et al. 2000). This example may provide evidence for convergent evolution at the molecular level.

CCM and CA in prokaryotes Many reviews have been published on the action of CCM in cyanobacteria, e.g., Badger (1987), Pierce and Omata (1988), Kaplan et al. (1990), Coleman (1991), Badger and Price (1992) and Ogawa (1993).

In respect of DIC transport, although both CO2 and HCO3 − were taken up by low-CO2 cells of Synechococcus sp., the rate of CO2 uptake was faster than that of the HCO3 − uptake (Badger and Andrews 1982; Volokita et al. 1984; Abe et al. 1987a). The results of mass spectrometric analysis have suggested the operation of CA activity during DIC transport (Badger et al. 1985). Irrespective of the DIC species taken up, HCO3 − was the species that penetrated the cell (Volokita et al. 1984; Abe et al. 1987b). Some cyanobacteria, i.e., freshwater species Synechococcus PCC7942 and marine species Synechococcus PCC7002, are capable of absorbing both CO2 and HCO3 − with constitutive capacity, but the ability was stimulated about 10-fold after low-CO2 acclimation (Yu et al. 1994; Sültemeyer et al. 1995). CCM in cyanobacteria involves the active transport of HCO3 − that is induced under limiting CO2 conditions and maintains a high HCO3 − concentration in the cytoplasm (see the review by Kaplan and Reinhold 1999). In Synechococcus PCC7942, IctB was identified as a candidate for HCO3 − transporter (Bonfil et al. 1998). However, as the IctB-deficient mutant still showed saturable kinetics for bicarbonate transport activity, the operation of multiple HCO3 − transporters was suggested. Omata et al. (1999) have identified the active HCO3 − transporter in Synechococcus sp. PCC7942 as an ATP-binding cassette similar to the nitrate/nitrite transporter. In addition, a gene (sbtA), which is essential to Na+ -dependent HCO3 − transport, was identified in Synechocystis sp. PCC6803 (Shibata et al. 2002a, b). Synechocystis PCC6803 possesses two systems for CO2 uptake, namely one induced by low CO2 conditions and the other being constitutive (Shibata et al. 2001). The inducible CO2 transport showed higher activity than the other, while both systems are driven by NAD(P)H dehydrogenase complexes. At the time of writing, four systems have been identified for DIC acquisition in Synechocystis sp. PCC6803 (Shibata et al. 2002b). From their 18 O exchange experiments with chp mutants of Synechococcus sp. PCC7942, Maeda et al. (2002) reported that novel ChpX and ChpY proteins with CO2 hydration activity and/or binding an active Zn center, but no homology with CA, are coupled with electron flow within the NdhD3/D4-containing NDH-1 complexes on the thylakoid membrane. In the early stage of CA studies, it had been assumed that CA played a significant role in unicellular green algae, but the function in cyanobacteria was questionable, since no or only very low activity

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Figure 2. Schematic diagrams of the structure of P. purpureum CA. A: ribbon diagram along the pseudo two-fold axis of a monomer. The N- and C-terminal halves and the other parts are respectively colored blue, green and gray. α-Helices and β-strands are respectively shown as ribbons and arrows. The positions of zinc are shown as red spheres, and the N and C termini are marked. B: trace of the dimer. One monomer is shown in blue, the other in red. The positions of zinc are shown as green spheres. Left, view looking down along the pseudo two-fold axis of a dimer. Right, view looking down along the 2-fold axis of a dimer. Note that a long-turn segment (residues 310-339) connecting the N- and C-terminal halves protrudes out from one monomer to the surface of the counter monomer and has no symmetrical counterpart. Filled circles, unfilled black circles, and unfilled red circles respectively represent the two-fold axis between the dimer, pseudo two-fold axis between the dimmer, and pseudo axis within a monomer (Mitsuhashi et al. 2000). Adapted from Mitsuhashi et al. (2000)

had been detected by the electrometric method (Kaplan et al. 1980; Badger et al. 1985; Lanaras et al. 1985). However, since the formation of an internal DIC pool in Anabaena variabilis M3 was inhibited by the CA inhibitor, ethoxyzolamide, although CA activity was undetectable, the involvement of a CAlike moiety, and not CA itself, was postulated in CCM

of cyanobacteria (Volokita et al. 1984). Under similar circumstances, Yagawa et al. (1984) detected CA in A. variabilis ATCC29413, and revealed that its function is to increase the affinity for DIC in photosynthesis (Shiraiwa and Miyachi 1985a). Lanaras et al. (1985) also found CA activity in the particulate fraction of the cyanobacterium, Chlorogloeopsis fritschii. These res-

145 ults suggest that CA is one of the indispensable factors for DIC transport in prokaryotes. In A. variabilis both CO2 and HCO3 − were transported into low-CO2 cells that had CA activity in their intact spheroplast, but only CO2 was transported into high-CO2 cells (Abe et al. 1987b). The transport of CO2 , and not bicarbonate, in A. variabilis was significantly enhanced by Na+ and inhibited by the CA inhibitor, ethoxyzolamide. Abe et al. (1987a, b) concluded that DIC accumulated in cyanobacterial cells was the bicarbonate ion, irrespective of the external DIC species supplied. Prior to its transportation across the plasma membrane, CO2 was converted into bicarbonate ion by CA, and the transport was mediated by Na+ as well as by proton motive force. CA activity was later demonstrated in the isolated carboxysomal fraction of Synechococcus PCC7942 (Price et al. 1992). In respect of cyanobacteria, CA in Synechococcus PCC7492 has been reported to be of β-type which was encoded in icfA (Fukuzawa et al. 1992), showing a similarity to higher-plant chloroplast CA (Hewett-Emmett and Tashian 1996) and carboxysomal CA (Fukuzawa et al. 1992). Unlike α-CA, which is insensitive to or activated by dithiothreitol (DTT) but not affected by Mg2+ , carboxysomal CA is inactivated by DTT and stimulated by Mg2+ (Price et al. 1992). Carboxysomal CA in cyanobacteria functions in the supply of CO2 to Rubisco (Kaplan and Reinhold 1999). A CcmM protein of Synechococcus, the N-terminal region of which is similar to γ -type CA of Methanosarcia thermophila (Hewett-Emmett and Tashian 1996), has been shown to be essential for carboxysome formation and the maintenance of CCM (Ludwig et al. 2000). The expression of human CA in the cytoplasm of cyanobacteria was associated with the leakage of CO2 to the medium (Price and Badger 1989). These results indicate that carboxysomal CA might be most important for CCM in cyanobacteria. In contrast to autotrophically grown cyanobacteria, the CA activity in the prokaryotic symbiotic alga, Prochloron sp., isolated from ascidian hosts, was located mostly outside the cells (Dionisio-Sese et al. 1993). The CA might function to enhance CO2 absorption from the host cells. Smith et al. (1999) have stated that CA is widely distributed in the Archaea and Bacteria domains and proposed that CA plays a more fundamental role in prokaryotes than had previously been recognized.

Induction of CA and CCM The changes in CA activity and apparent K1/2 (CO2 ) value for photosynthesis in C. vulgaris 11h are linearly dependent on the concentration of aerated CO2 up to 1%, the half-saturated concentration of CO2 being about 0.5% (Shiraiwa and Miyachi 1985b). The regulating factors suggested for the induction of CA and CCM were photorespiratory carbon metabolism in C. reinhardtii (Spalding and Ogren 1982), photorespiratory N metabolism in C. regularis (Umino and Shiraiwa 1991), glycolate and other intermediates of the glycolate pathway (Ramazanov et al. 1984; Ramazanov and Semenenko 1986) and light intensity in Chlorella sp. K (Semenenko et al. 1979; Pronina et al. 1981), phosphoglycolate in A. variabilis M-3 (Marcus et al. 1983), and O2 concentration and/or O2 /CO2 ratio in C. vulgaris 11h (Shiraiwa et al. 1988; Shiraiwa and Umino 1991). CA induction in C. regularis was suppressed by metabolizable organic carbon sources such as glucose, acetate and glycine (Umino and Shiraiwa 1991), C. vulgaris 11h (Shiraiwa and Umino 1991) and C. vulgaris UAM101 isolated from the effluent of a sugar refinery (Martinez and Orus 1991). Suppression by acetate was reported for C. reinhardtii (Spalding and Ogren 1982). Chlorella species did not require high photoenergy for the induction of CA and CCM (Shiraiwa et al. 1981; Shiraiwa and Miyachi 1983; Umino et al. 1991). In Chlamydomonas, the induction of CA was regulated by both a photosynthesis-dependent step and the subsequent photosynthesis-independent step that required blue light as a photosignal at the post-transcriptional level (Dionisio et al. 1989a). The results of an experiment with a quencher of the flavin triplet-excited state (Dionisio et al. 1989b) suggested that flavin might be involved in CA induction (Dionisio-Sese et al. 1990). Rythmic changes in the activity of the CO2 -concentrating mechanism, the K1/2 (CO2 ) value for photosynthesis, CA activity and the maximum rate of photosynthesis were monitored during the cell cycle of Chlamydomonas (Marcus et al. 1986). At a constant concentration of dissolved CO2 , intracellular CA activity showed obvious fluctuation in the light period, while extracellular CA activity showed only minor fluctuations (Nara et al. 1989). In a synchronous culture of C. reinhardtii C-9, Toguri et al. (1989) have shown that the translation of CA-mRNA and the subsequent processing of the 42-kD precursor to yield the 35-kD polypeptide of mature CA were performed in the light. In C. regularis and C. ellipsoidea

146 CA induction preferred alkaline rather than acidic pH conditions, irrespective of its localization in the cells (Shiraiwa et al. 1991; Patel and Merrett 1986). A key factor pertaining to sensing the concentration of CO2 and triggering the gene expression of CO2 -responsive genes associated with CA and CCM has recently been found in Chlamydomonas (Fukuzawa et al. 2001; Xiang et al. 2001). The genes designated Ccm1 (Fukuzawa et al. 2001) and Cia5 (Xiang et al. 2001) were the same and were shown to contain the zinc-finger motif in the N-terminal region and a glutamine repeat of the transcription factor. As Ccm1 is constitutively expressed under both high- and low-CO2 conditions, sensing the CO2 concentration or CO2 -signal transduction might require modification of the CCM1 protein and activation of the genes for the CO2 -concentrating mechanism (Miura et al. 2002). The photosynthetic electron transport system is also affected during the acclimation of cells to lowand high-CO2 conditions. The PS I/PS II fluorescence (77K) ratio and the quantum requirement for photosynthetic O2 evolution have been found to be higher in low-CO2 cells than in high-CO2 cells of some unicellular green algae and cyanobacteria (Bürger et al. 1988; Miyachi et al. 1996). In the cyanobacterium Anacystis nidulans, the PS I/PS II reaction center ratio was higher in low-CO2 cells than in high-CO2 cells (Manodori and Melis 1984). These phenomena are thought to reflect the requirement of excess energy via PS I for cyclic electron flow to drive the DIC pump, as suggested by Ogawa and Ogren (1985) and Ogawa et al. (1985). Satoh et al. (2002) have reported that a similar pattern for the increase in the PS I/PS II fluorescence ratio was also observed in the extremely high-CO2-tolerant unicellular green alga, Chlorococcum littorale, even when high-CO2 (3%) cells were transferred to the stress conditions of low-CO2 and extremely high-CO2 under a low light intensity.

Tolerance and acclimation to extremely high-CO2 conditions Although microalgae and cyanobacteria proliferate at a high-CO2 concentration, it has generally been assumed that an extremely high concentration of CO2 would inhibit photosynthesis and thus growth. The mechanism underlying the effect is not well understood, except for the proposal of a narcotic effect (e.g., Nielsen 1955).

A new species of the unicellular marine green alga, Chlorococcum littorale, which had been isolated from a saline pond in Kamaishi City (Kodama et al. 1993; Chihara et al. 1994), has recently been found to grow rapidly at a CO2 concentration as high as 40%, and even at 60% CO2 if the concentration was elevated stepwise in 10% increments (Kodama et al. 1993). Several other species of algae have also been found that were endowed with the capability to grow in extremely high-CO2 conditions; for example, Chlorella sp. UK001 (Murakami et al. 1998). The cyanobacterium, Synechocystis aquatilis SI-2, has been reported to grow in 40% CO2 (Zhang et al. 1999), while the red microalgae, Cyanidium caldarium (Seckbach et al. 1970), and Cyanidioschyzon merollae and Galdieria partite (Kurano et al. 1995) could grow in 100% CO2 , although the growth rates were much lower than those of green or blue-green algae. These findings opened a new research topic on the tolerance of microalgae and cyanobacteria to extremely high-CO2 concentrations. When low-CO2 cells of C. littorale were transferred to extremely high-CO2 conditions, the cells started to grow after a lag period. During the initial period of the lag phase, the activities of photosynthetic CO2 fixation, O2 evolution and the quantum yield of PS II measured by a quenching analysis of chlorophyll fluorescence using intact cells were initially suppressed and then increased. In contrast, the PS I activity, mainly of cyclic electron transfer around PS I, was greatly enhanced (Pesheva et al. 1994; Iwasaki et al. 1996, 1998). The CA activities decreased to zero after transfer from a low- to high-CO2 concentration (Pesheva et al. 1994). During this temporal inactivation of PS II, it was concluded that down-regulation of the linear electron transfer between the two photosystems occurs (Iwasaki et al. 1998). This kind of state-II transition, which is usually caused by a highly reduced state of the plastoquinone pool, is due to an accumulation of such reductants as NADPH (Schreiber 1986; Schreiber et al. 1986; Genty et al. 1989; Krause and Weis 1991). Several key enzymes involved in the assimilation of CO2 , such as Rubisco, are readily inactivated by a decrease of pH in the stroma (Bailey and Ollis 1986; Krause and Weis 1991). This decreased pH value seems to be one of the main factors governing the inactivation of the Calvin-Benson cycle. Stichococcus bacillaris, which is intolerant to such extremely high-CO2 conditions, did not show any significant state transition after being transferred from low- to extremely high-CO2 conditions (Iwasaki et al. 1998).

147 A short-term (hour-level) study by Demidov et al. (2000) found that the quantum yield of PS II and the oxygen evolution in C. littorale were quickly suppressed within 5-30 min after the exposure of low-CO2 cells to an extremely high level of CO2 . When the CO2 concentration was reduced, the recovery from suppression took the same time as the suppression itself. Pronina et al. (1993) have shown by 31 P-in vivo NMR spectroscopy that the pH value in the cytoplasm of C. littorale cells remained constant at 7.0 or was raised by 0.1-0.4 pH units when low-CO2 cells were grown under extremely high (40%) CO2 conditions. These results indicate that growth inhibition by an extremely high CO2 concentration is associated with cytoplasmic acidification and probably also with chloroplastic acidification. An increase in the number and size of vacuoles of C. littorale cells has also been observed after exposing the algal cells to extremely high-CO2 conditions (Pronina et al. 1993; Kurano et al. 1998; Sasaki et al. 1999). Sasaki et al. (1999) have pointed out that the development of vacuoles associated with the increasing activity and induction of vacuolar H+ -ATPase (V-ATPase) in C. littorale cells seemed to be well correlated with the recovery of photosynthetic activity during the acclimation of the cells to extremely high-CO2 conditions. Vacuolar proton pumps such as V-ATPase and H+ -pyrophosphatase make secondary transport possible and maintain pH homeostasis in the cytoplasm by generating an electrochemical potential difference across the vacuolar membrane (Sze et al. 1992; Taiz 1992; Maeshima 2001). Two cDNA clones, HCR1 and HCR2, that were induced under 20% CO2 conditions and ferrous iron deficiency have been isolated from C. littorale by differential screening (Sasaki et al. 1998a). The sequence similarity between HCR2 and the yeast ferric reductase proteins, FRE1, FRE2 and Frp1 (Dancis et al. 1992; Georgatsou and Alexandraki 1994; Roman et al. 1993) suggests that the HCR2 protein may catalyze iron reduction in C. littorale cells (Sasaki et al. 1998a, b). According to the induction patterns of HCR2 mRNA and its protein, which were accompanied by that of the cell-surface ferric reductase activity, the gene product of HCR2 would play an important role in C. littorale cells under 20% CO2 conditions (Sasaki et al. 1998a, b). Since iron is essential for photosynthetic and respiratory proteins, iron uptake is very important for stimulating growth during the acclimation of C. littorale cells to an extremely high-CO2 concentration.

Satoh et al. (2001a) found that intracellular acidification occurred upon exposing low-CO2 cells of C. littorale to 40% CO2 for 1 h, and that this intracellular acidification could be avoided by adding ethoxyzolamide (EZA), a membrane-permeable CA inhibitor. This result indicates that the intracellular acidification might have been due to the function of CA. They also showed that when high-CO2-grown (5%) C. littorale cells with neither extracellular nor intracellular CA activity were transferred to 40% CO2 conditions, they could grow without any lag period. It is interesting to note that intracellular CA, which enhanced photosynthetic CO2 fixation under low-CO2 conditions, caused intracellular acidification and hence the inhibition of photosynthetic carbon fixation accompanied by state-transition during the acclimation from low-CO2 to extremely high-CO2 conditions. Regulation of the energy balance during photosynthesis in response to extremely high (40%) and low (0.04%) CO2 stress has been studied in C. littorale and Chlorella sp. UK001 which could grow safely in extremely high-CO2 conditions (Satoh et al. 2002). Under a limited light intensity (20 µmol m−2 s−1 ), the PS I/PS II fluorescence (77K) ratio increased when high-CO2 (3%) cells of C. littorale were transferred to extremely high-CO2 and to low-CO2 conditions. The same change in the fluorescence ratio was observed when low-CO2 cells were transferred to extremely high-CO2 conditions. Another extremely high-CO2tolerant alga, Chlorella sp. UK001, has also shown this increase in the PS I/PS II fluorescence ratio, without any lag period, after transferring low-CO2 cells to extremely high-CO2 (40%) conditions. These observations indicate that the electron flow from relatively increased LHCI toward PS I cyclic electron flow was enhanced under light-limited conditions that resulted in the generation of extra ATP (Satoh et al. 2002). These results are supported by the higher relative quantum yield of PS I in the presence of DCMU in both microalgae acclimated to low- and extremely high-CO2 conditions than those grown in high-CO2 (3%) conditions. This indicates that the regulation of energy balance in photosystems in response to excess-CO2 stress was similar to that in response to low-CO2 stress under light-limited conditions (Satoh et al. 2002). The foregoing observations suggest several important factors that should be taken into account for algal acclimation to extremely high-CO2 conditions. The first factor is the cyclic electron transfer around PS I which leads to the production of extra ATP.

148 The second is the pH regulation controlled by proton pumps driven by such constituents as V-ATPase that can maintain pH homeostasis by using the extra ATP produced by cyclic electron transport. When lowCO2 cells of C. littorale were transferred to extremely high-CO2 conditions under saturating light intensity, the increase in the PS I/PS II fluorescence ratio was only observed during the lag period (Pescheva et al. 1994; Iwasaki et al. 1998; Satoh et al. 2002). During the induction period, H+ production would be very high due to the function of CA present in the lowCO2 cells. The production of extra ATP by cyclic photophosphorylation would be needed only during the induction period, when CA was active. However, extra ATP would not be needed during the subsequent steady-state period if there were enough light energy available, since no CA is present during that period.

Acknowledgements The authors are grateful to Drs H. Fukuzawa of Kyoto University, S. Mitsuhashi of Kyowa Fermentation Co. Ltd. and A. Satoh of the Marine Biotechnology Institute for their valuable suggestions.

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