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Eukaryot Cell. Aug 2007; 6(8): 1251–1259.
Published online Jun 8, 2007. doi:  10.1128/EC.00064-07
PMCID: PMC1951128

Proposed Carbon Dioxide Concentrating Mechanism in Chlamydomonas reinhardtii[down-pointing small open triangle]

Aquatic photosynthetic microorganisms account for almost 50% of the world's photosynthesis (19). These organisms face several challenges in acquiring CO2 from the environment. The first challenge is presented by the properties of ribulose bisphosphate carboxylase-oxygenase (Rubisco). Rubisco is an unusually slow enzyme with a low affinity for CO2. At atmospheric levels of CO2, Rubisco can function at only about 25% of its catalytic capacity because the concentration of dissolved CO2 is less than the Km(CO2) of Rubisco and due to the relatively high concentration of O2 which competes with CO2. A second challenge these organisms face is that the diffusion of CO2 in an aqueous solution is 10,000 times slower than the diffusion of CO2 in air. Thus, the ability to scavenge CO2 as quickly as it becomes available is highly advantageous to aquatic photosynthetic organisms. Third, algae often experience significant fluctuations in inorganic carbon (Ci = CO2 + HCO3) levels and pH, which change the availability of CO2 and HCO3 for photosynthesis. At an acidic pH, the vast majority of Ci is in the form of CO2, while at an alkaline pH, Ci is mostly in the form of HCO3, with CO2 making up only a small fraction of the available Ci (8, 25).

Algae have adapted to these challenges through the development of a CO2 concentrating mechanism (CCM). The CCM is a biological adaptation to low carbon dioxide concentrations in the environment. It is a mechanism which augments photosynthetic productivity in algal cells by increasing levels of inorganic carbon many times over the environmental concentration of carbon dioxide. In this minireview, we aim to provide an update on the CCM and present a model on how the green alga Chlamydomonas reinhardtii concentrates CO2.

TYPES OF CCMs

CCMs can be based on biochemical mechanisms such as C4 photosynthesis and crassulaceous acid metabolism (CAM), on active transport of Ci across membranes, or on processes involving localized enhancement of the CO2 concentration by acidification of a particular cellular compartment (28). The role of the CCM is to increase the concentration of CO2 for Rubisco, the enzyme responsible for the initial fixation of CO2. While three different mechanisms are discussed below, it is likely that aquatic photosynthetic organisms display a variety of ways to concentrate CO2. Algae comprise a very diverse group of organisms and have been adapting to the slow diffusion of inorganic carbon in the water for a long time.

C4 mechanism.

C4 photosynthesis and CAM in terrestrial higher plants were the first photosynthetic CCMs to be described in detail. They involve a spatial (C4) or temporal (CAM) separation of the fixation of CO2 by phosphoenolpyruvate (PEP) carboxylase to produce a four-carbon dicarboxylic acid which is transported and decarboxylated, increasing the CO2 available to Rubisco (44, 74). In higher plants, the CCM is dependent on a specialized operation and the interaction of leaf mesophyll and bundle sheath photosynthetic cells. The primary CO2 capture mechanism is through PEP carboxylase located in the cytosol of the mesophyll cells. PEP carboxylase uses HCO3 as its primary substrate for fixation of CO2 into oxaloacetate, so CO2 entering from the external environment must be hydrated rapidly by a carbonic anhydrase (CA) and converted to HCO3. Thus, in C4 plants, the predominant CA activity is found in the mesophyll cell cytosol in order to make this HCO3, in contrast to C3 plants, where the highest levels of CA activity are associated with the stroma of the mesophyll cell chloroplasts (6, 13, 40, 59). C4 carboxylic acids such as malate or aspartate formed in the mesophyll cell cytosol serve as the intermediate CO2 pool.

The presence of C4- or CAM-like metabolism has been observed in submerged aquatic plants and algae. Examples include Isoetes howellii and Sagittaria subulata (39), the green ulvophycean benthic macroalga Udotea flabelum (79, 80), and the planktonic diatom Thalassiosira weissflogii (77, 78) grown under inorganic CO2-limited conditions. Evidence of a CAM-like mechanism has also been proposed for brown macroalgae, where high levels of PEP carboxykinase and diel fluctuations in titratable acidity and malate have been observed (33, 72).

Active transport of inorganic carbon.

Examples of active transport of HCO3 come primarily from studies using cyanobacteria. Cyanobacteria have a sophisticated CCM which involves a variety of active CO2 and HCO3 uptake systems and an internal microcompartment, the carboxysome (7, 63). At least five distinct Ci transport systems are known for cyanobacteria (Fig. (Fig.1).1). An interesting feature of the cyanobacterial CCM is the induction of multiple transporters under Ci limitation. Cyanobacteria appear to utilize pairs of Ci transporters with complementary kinetics for the same Ci species. For example, two complementary HCO3 transporters are present in Synechococcus PCC7002. The BicA transporter has a relatively low transport affinity of around 38 μM but is able to support a high flux rate. It pairs with the SbtA transporter, which has a high transport affinity of 2 μM but possesses a lower flux rate (65). This strategy of employing a high-flux/low-affinity transporter with a low-flux/high-affinity transporter appears to be a common theme in freshwater and estuarine cyanobacteria (7).

FIG. 1.
Model for inorganic carbon acquisition by cyanobacteria. This model is based on the article by Woodger et al. (102). Most cyanobacteria do not contain all of the transporters depicted. In addition, any given cyanobacteria would have only one type of carboxysome ...

In cyanobacteria, the carboxysome is the specialized compartment in which accumulated HCO3 is converted to CO2 through the action of specific carboxysomal CAs (21, 106). Two types of carboxysomes are recognized in cyanobacteria. In α-type carboxysomes, bicarbonate is converted to CO2 via the action of the CA CcaA (21, 85, 106). In β-type carboxysomes, a component of the carboxysome shell, CsoS3, is responsible for the dehydration of bicarbonate (84, 102). CO2 is elevated due to diffusion restrictions on efflux by the carboxysome protein shell structure (35, 36, 63). Thus, the overall mechanism elevates HCO3 in the cytosol of the cell and converts this accumulated Ci back to CO2 in the carboxysome, the location of Rubisco (102).

CO2 concentration following acidification in a compartment adjacent to Rubisco.

A third type of CCM found in eukaryotic algae relies on the pH gradient set up across the chloroplast thylakoid membrane in the light. In the light, a large change in pH is established across the thylakoid membrane; the chloroplast stroma has a pH of close to 8.0, and the thylakoid lumen has a pH of between 4 and 5. This pH differential is significant because the pKa of the bicarbonate-to-CO2 interconversion is about 6.3 (HCO3 + H+↔H2CO3↔CO2 + H2O). Under these conditions, HCO3 is the predominant species of Ci in the chloroplast stroma while CO2 is the most abundant form of Ci in the thylakoid lumen. Any bicarbonate transported into the thylakoid lumen would be converted to CO2, thus elevating the CO2 concentration above ambient levels. This mechanism, first suggested by Semenenko, Pronina, and colleagues, requires a CA in the acidic thylakoid lumen to rapidly convert the entering HCO3 to CO2 (66, 68). In addition, since HCO3 cannot rapidly cross biological membranes (29), there must be a transport protein or complex that allows HCO3 to enter the thylakoid lumen. This model predicts that CO2 accumulation would not occur in the dark, as light-driven photosynthetic electron transport is required to set up these pH gradients. As discussed below, evidence for this type of CCM comes primarily from work using the model eukaryotic green alga Chlamydomonas reinhardtii.

CHLAMYDOMONAS REINHARDTII CCM

The C. reinhardtii CCM.

A proposed model for concentrating CO2 in C. reinhardtii is shown in Fig. Fig.2.2. In this model, the CCM can be divided into two phases. The first phase involves acquiring inorganic carbon from the environment and delivering CO2 and HCO3 to the chloroplast. The components of this part of the CCM would include CAs in the periplasmic space (CAH1 and possibly CAH8) and a CA in the cytoplasm (CAH9) as well as HCO3 transporters and CO2 channels on both the plasma membrane and the chloroplast envelope. The second part of the proposed model entails the generation of elevated levels of HCO3 in the chloroplast stroma, utilizing the pH gradient across the thylakoid membrane. This part of the CCM includes the CA located in the chloroplast stroma (CAH6) and the CA located within the thylakoid lumen (CAH3) as well as a proposed but still hypothetical HCO3 transporter on the thylakoid membrane.

FIG. 2.
Model of the CCM of Chlamydomonas reinhardtii. The figure depicts an algal cell with a single chloroplast containing a single pyrenoid. As indicated by the size of the lettering, the concentrations of bicarbonate and carbon dioxide within the chloroplast ...

It should be emphasized that C. reinhardtii has a strictly C3 biochemistry, since unlike the C4 pathway, wherein transported carbon is stored as organic C4, C. reinhardtii accumulates inorganic carbon, specifically HCO3, in the chloroplast stroma. In addition, while experiments indicate that the marine diatom Thalassiosira weisflogii has a C4-like pathway, the same researchers concluded that a C4-like pathway is unlikely to operate in green algae (78). Although C. reinhardtii has two PEP carboxylase genes, CRPPC1 and CRPPC2 (45), the PEP carboxylase activity in C. reinhardtii is never higher than 20% of the Rubisco activity or the maximal rate of CO2 fixation (9). Mamedov et al. concluded that CRPPC1 and CRPPC2 have an anaplerotic, nonphotosynthetic role in C. reinhardtii (45).

Physiological evidence for Ci uptake in C. reinhardtii.

The physiological evidence that C. reinhardtii can accumulate Ci and enhance CO2 fixation is twofold. First, C. reinhardtii has the ability to efficiently fix CO2 even when the external CO2 concentration is well below the Km(CO2) for Rubisco (4, 55, 91). For example, whole-cell photosynthesis rates are saturated at about 2 to 3 μM CO2, while the Km(CO2) of C. reinhardtii Rubisco is about 20 μM (34). In addition, Ci uptake has been measured directly in a number of laboratories (3, 4, 6, 55, 92, 93), and the Ci concentration inside the cell is higher than can be accounted for by diffusion alone.

Further evidence for the existence of a CCM in C. reinhardtii comes from mutant studies. In these studies, mutagenized cells were screened for growth on high (5% CO2 in air) and low (air levels of CO2 or lower) CO2. Mutant strains that grew well on elevated CO2 but poorly on low CO2 were then selected for further studies. This approach has yielded mutants in Ci uptake (91, 100), CA activity (24, 58, 90), the photorespiratory chain (94, 104), and novel proteins. Another interesting class of mutants are those that fail to respond fully to changes in the CO2 environment (22, 57, 105). In particular, the CIA5 (CCM1) gene appears to encode a protein required for the induction of the CCM (22, 57). A listing of these mutant strains is shown in Table Table11.

TABLE 1.
C. reinhardtii CCM mutants and other mutants that grow poorly on low CO2

In this model for Ci uptake (Fig. (Fig.2),2), it is predicted that the pH gradient across the thylakoid membrane is an essential part of the CCM. Since light-driven electron transport is required to set up the pH gradient, Ci uptake should occur only in the light. To date, inorganic carbon concentration in C. reinhardtii has been observed only in cells or chloroplasts exposed to light. The strongest evidence in support of the light requirement comes from the pioneering work of Spalding and Ogren (88), who showed that electron transport inhibitors as well as mutants in the electron transport chain also inhibited the CCM in C. reinhardtii. While this work does not prove that a pH gradient across the thylakoid membrane is required for the CCM to operate, their data are fully consistent with this model.

Another requirement of this model is a CA in the thylakoid lumen to rapidly convert the bicarbonate entering the lumen to CO2. In C. reinhardtii, CAH3 has been localized to the thylakoid lumen, and mutations in the CAH3 gene result in cells with a nonfunctional CCM (60). An additional requirement of this model is that the CO2 generated in the thylakoid lumen becomes available to Rubisco before being converted back to HCO3 in the basic environment of the chloroplast stroma. The pyrenoid of the chloroplast might serve to separate Rubisco from the CA in the stroma of the chloroplast. The pyrenoid is a proteinaceous structure where most of the Rubisco is located. The pyrenoid undergoes a dramatic morphological change when cells are switched from high- to low-CO2 conditions (76) (Fig. (Fig.3).3). When the CCM is functional, a starch sheath appears around the pyrenoid and 90% of Rubisco is present in the pyrenoid (11, 52, 70).

FIG. 3.
Representative pyrenoids (P) from C. reinhardtii cells acclimated to high CO2 (A) or low CO2 (B). The white areas in the micrographs are starch. Bars, 1 μm.

Notably, most eukaryotic photosynthetic algae have pyrenoids (10), while pyrenoids are almost absent from the chloroplasts of terrestrial higher plants. Exceptions include some strains of Chloromonas which exhibit a CCM but are demonstrated to lack pyrenoids (50). However, pyrenoid-less CCM-containing strains of Chloromonas were demonstrated to have small Ci pools, of 24 to 31 μM, in comparison with the large Ci pools, of 231 to 252 μM, in algae exhibiting typical (dense with a high concentration of Rubisco) pyrenoids. It has been speculated that the formation of a large intracellular Ci pool in algae with a CCM is correlated with the presence of typical pyrenoids exhibiting a high concentration of Rubisco molecules. Algae lacking pyrenoids, such as Chloromonas, are often found in harsh environments, and perhaps the presence of a pyrenoid is not necessary because other factors besides CO2 fixation are limiting growth (51).

Finally, a CA located in the chloroplast stroma would also be required for the operation of this type of CCM. This stromal CA would serve two functions: first, to convert CO2 entering the chloroplast to HCO3 in the basic environment of the chloroplast stroma, and second, to recapture the CO2 coming from the thylakoid lumen before it diffuses from the chloroplast. In C. reinhardtii, most of the required features of this type of CCM have been identified. There are CA isoforms in the thylakoid lumen (37) and the chloroplast stroma (48). What has not been established is how bicarbonate is transported across the thylakoid membrane. However, a number of proteins have been identified as potential HCO3 transporters, and this issue is discussed later in this article.

CAs.

The acclimation of algae, including C. reinhardtii, to limiting CO2 has been correlated with increased levels of CAs (2, 4, 16, 23, 90). In this minireview, we focus only on the α- and β-type CAs found in C. reinhardtii. These two forms of CAs are very different in structure and amino acid sequence. For example, the α-CAs share homology with the mammalian CAs and have three His residues coordinating the Zn ion. α-CAs are usually monomeric. In contrast, β-CAs have one His and two Cys residues coordinating the Zn (12) and are often multimeric. The C. reinhardtii β-CAs are quite similar to higher plant chloroplast CAs and bacterial CAs. As of this time, nine different α- and β-CA genes have been identified in the Chlamydomonas genome (Table (Table2).2). This plethora of CA genes has led to questions about what roles are played by these CAs and which ones are critical to the functioning of the CCM. A number of these proteins are implicated to have possible roles in the CCM.

TABLE 2.
CAs in C. reinhardtii

The role of the periplasmic α-CA CAH1 is to facilitate entry of carbon dioxide into the algal cell. At pHs above 6.3, HCO3 is the predominant inorganic carbon species. This form of Ci, being an anion, cannot readily cross the plasma membrane (29, 53). CAH1, one of the first α-CAs reported for a photosynthetic organism, converts HCO3 to CO2. Two lines of evidence have been presented for this physiological role of CAH1. First, membrane-impermeant CA inhibitors have a strong inhibitory effect on photosynthetic CO2 fixation at high pHs, where HCO3 predominates, but a less pronounced effect at lower pHs, where most of the inorganic carbon is already in the form of carbon dioxide and the activity of periplasmic CA is no longer required (56). This view of the role of CAH1 was challenged by Van and Spalding, who found no evidence of growth inhibition in a mutant missing CAH1 (96). However, the presence of other CA isoforms in the periplasmic space, namely, CAH2 (75, 95) and possibly CAH8, makes the interpretation of these results more complicated. CAH1 biosynthesis is strongly regulated by changes in environmental CO2 concentration as well as light. CAH1 is very strongly induced under limiting CO2 conditions, where the CCM is operational (23).

CAH1 has been shown to be controlled by two regulatory regions, namely, a silencer region, which represses transcription under high-CO2 conditions or in the dark, and an enhancer region, which activates it under low-CO2 conditions in the light (42). These sites may be important cis-acting elements that constitutively bind one or more proteins that assist in the regulated transcription of CAH1 (43). LCR1 has also been identified as a regulatory gene of CAH1. LCR1 is a Myb transcription factor that functions in amplification and maintenance of CAH1 mRNA levels in response to limiting CO2 (105).

CAH2 is also a periplasmic α-CA but is not thought to have an important role in the CCM. CAH2 is an active CA (75, 95) but is poorly expressed. In fact, CAH2 expression is down-regulated under limiting CO2 conditions, the growth conditions under which the CCM is operational (75). CAH2 is only 1.4 kb away from the CAH1 gene (20) and may be the result of a recent gene duplication.

CAH3, the third CA gene described for C. reinhardtii, codes for an α-CA that has a leader sequence consistent with targeting CAH3 to the thylakoid lumen (24, 38). Immunoblot studies using antibodies raised against CAH3 demonstrated that CAH3 is associated with the thylakoid membrane (37). More specifically, immunolocalization studies indicated that CAH3 is localized on the luminal side of the thylakoids and inside the pyrenoid tubules (47). The evidence that CAH3 plays an essential role in the CCM is persuasive. C. reinhardtii strains defective in CAH3 cannot grow in air levels of CO2 even though they grow normally on elevated levels of CO2 (37, 58, 67, 90). Putting the wild-type CAH3 gene back into these strains restores normal photosynthesis (24, 37). Strains defective in CAH3 also accumulate large pools of Ci but are unable to use Ci efficiently for photosynthesis (58, 90). Therefore, CAH3 appears to convert accumulated HCO3 to CO2, the form of Ci that Rubisco can use. Its location suggests that CAH3 catalyzes the formation of CO2 from HCO3 in the acidic lumen of thylakoids and that this CO2 diffuses through the thylakoid membrane to the pyrenoid, where the CO2 will be fixed by Rubisco (5, 53, 54, 60, 71). CAH3 is expressed under both high- and low-CO2 growth conditions, although there is a twofold increase in message abundance under low-CO2 conditions.

CAH3 has also been proposed to be associated with photosystem II (PSII) and to help to stabilize the PSII manganese cluster and the catalytic function of PSII reaction centers (60, 99). This hypothesis is reinforced by the evidence that at low Ci concentrations, the cah3 mutant, cia3, is impaired in maintaining high rates of electron transport and/or coupling the residual electron transport to ATP formation (31). However, subsequent studies with the Chlamydomonas cah3 mutant have shown that as CO2 becomes limiting, the chloroplast ribulose 1,5-bisphosphate pool is increased compared with that in the wild type, which indicates a CO2 supply limitation rather than a PSII energy supply defect (30).

C. reinhardtii contains identical mitochondrial β-CAs (mtCAs), CAH4 and CAH5, that exhibit a pattern of expression which correlates with the expression of the CCM. The genes encoding CAH4 and CAH5 are adjacent to each other in the C. reinhardtii genome (18). They are highly induced at both the transcriptional and translational levels under low-CO2 conditions (17, 18, 26, 49) and may have an important role in the acclimation of C. reinhardtii to low-CO2 conditions. However, the exact role of these CAs is still not clear. One suggested function of mtCAs is to buffer the mitochondrial matrix, since prior to the complete induction of the CCM, photorespiratory glycine decarboxylation produces equivalent amounts of NH3 and CO2. The mtCA might serve to catalyze the hydration of CO2, producing H+, which would prevent alkalinization in the mitochondrial matrix as a result of the generation of NH3 by glycine decarboxylation (18). Alternatively, the mtCAs have been proposed to play a role in converting the CO2 generated by respiration and photorespiration to HCO3. This would effectively “recapture” the CO2 generated by the photorespiratory pathway (73). More recently, it has been shown that even under low-CO2 conditions, but with increasing NH4+ concentrations in the growth medium, the expression of mtCAs decreases at both the transcriptional and translational levels. Thus, it has been proposed that mtCAs are involved in supplying HCO3 to PEP carboxylase for NH4+ assimilation under certain conditions (27). As of this writing, there are no mutants of C. reinhardtii missing these mtCAs.

CAH6 is a constitutively expressed β-CA in the chloroplast stroma (47, 48). This CA might be involved in recapturing CO2 as it effluxes from the thylakoid lumen and in helping to maintain a high concentration of inorganic carbon in the stroma. Likewise, it might be another CA responsible for supplying CO2 for Rubisco. It might shuttle HCO3 to CO2 in the stroma as CO2 is depleted by the action of Rubisco. This is the same role proposed for chloroplast CAs of higher plants. The generation of mutants of CAH6 could help to confirm the physiological role of CAH6 in photosynthesis and the CCM.

Two additional β-CAs, designated CAH7 and CAH8, are closely related CAs with 63% similarity. They are constitutively expressed at moderate levels in C. reinhardtii. An interesting feature of these two CAs is the presence of long, relatively hydrophobic C-terminal extensions that are unusual among β-CAs described to date. CAH7 has been localized to the chloroplast, while CAH8 has been localized to the periplasmic space close to the cell membrane (R. A. Ynalvez et al., unpublished data). The location of CAH8 at or near the plasma membrane suggests that it might help to facilitate Ci entry into the cell. The most recently discovered CA is CAH9. This β-CA has no leader sequence and has tentatively been assigned to be localized to the cytoplasm. An interesting point is that the sequence of this β-CA aligns more closely with bacterial CAs than with the other C. reinhardtii β-CAs (D. Deb and J. V. Moroney, unpublished results). The role, if any, of CAH9 in CO2 acquisition remains to be determined.

Putative transporters.

While a number of CAs have been shown to be part of the CCM in C. reinhardtii, no transporter has been linked definitively to the CCM. However, some promising candidate genes and proteins have been identified, and it is likely that one or more of the following proteins may participate in Ci uptake in C. reinhardtii. The candidate proteins are CCP1, CCP2, LCI1, NAR1.2 (LCIA), LCIB, HLA3, RH1, and YCF10. All of these proteins are encoded in the nucleus, with the exception of YCF10, which is encoded by the chloroplast genome. Most of these proteins, or the corresponding genes, were first identified because the protein or mRNA dramatically increases in abundance when C. reinhardtii is grown under limiting CO2 growth conditions. For example, CCP1, CCP2, LCI1, NAR1.2, LCIB, and HLA3 are all strongly induced when C. reinhardtii is making a functional CCM. In addition, mutations in the putative transcription factor CIA5/CCM1 (22, 103) reduce the expression of many of these proteins (15, 46, 49, 57).

Very few mutants that affect the expression of the genes encoding putative Ci transport proteins have been found. However, the pmp1 mutant does have a mutation in LCIB, and this mutant is defective in Ci transport (91, 97). Recently, the allelic mutant ad1, air dier 1, was also described, and this strain also cannot grow in low CO2 (350 ppm) but can grow either in high CO2 (5% CO2) or in very low CO2 (200 ppm). The fact that the pmp1/ad1 mutant fails to grow on air levels of CO2 but manages to survive on very low levels of CO2 has been interpreted as indicative of the existence of multiple Ci transport systems in C. reinhardtii corresponding to multiple CO2 level-dependent acclimation states (89, 98, 100). This would be similar to the multiple Ci uptake systems seen in cyanobacteria. pmp1/ad1 was found to be identical to the previously identified CO2-responsive gene LCIB (49). LCIB does not have any significant homology to proteins from other organisms, but its predicted amino acid sequence has similarity with the predicted amino acid sequence of three genes, LCIC, LCID, and LCIE, in the C. reinhardtii genome. LCIC and LCID are also upregulated under low-CO2 conditions. While these observations point to a role for LCIB in the adaptation to low CO2, it is unlikely that LCIB is a transport protein by itself, as it lacks any hydrophobic transmembrane domains. Therefore, LCIB more likely has a regulatory role or might be part of a complex that transports Ci (97).

Another promising candidate protein to be a Ci transporter is LCI1. The LCI1 gene was first identified as being very highly expressed in cells growing under low-CO2 conditions (14). LCI1 contains four predicted transmembrane helices and also shows very little homology to any other protein in the NCBI database. Recent work with strains showing reduced expression of LCI1 due to the presence of an LCI1-RNA interference (LCI1-RNAi) insert showed reduced growth on low CO2 (Mason and Moroney, unpublished observations), but the physiological role of LCI1 remains to be determined.

Two other genes encoding putative Ci transport proteins are CCP1 and CCP2. These genes encode the low-CO2-inducible proteins LIP-36 G1 and LIP-36 G2 (26). These two proteins are 96% identical, have six transmembrane domains, are localized in the chloroplast envelope (54, 69), and have a high degree of similarity to the mitochondrial carrier family of proteins (15). When the abundance of CCP1 and CCP2 messages was reduced using RNAi, the resultant strains grew poorly with low CO2 levels but normally with elevated levels of CO2 (62). However, Ci uptake was normal in these strains (61). This might indicate that CCP1 and CCP2 are transporters of metabolic intermediates of photorespiration or transporters of other metabolic intermediates (61) or that these proteins are part of a redundant system of Ci transport, as seen in cyanobacteria.

Another putative Ci transporter, LCIA, was also first discovered using expression analysis (49). LCIA is also called NAR1.2. LCIA/NAR1.2 was first annotated as a nitrite transporter and has strong similarity to the bacterial nitrite/formate family of transporters. NAR1.2 belongs to a gene family consisting of six NAR genes in C. reinhardtii, and surprisingly, these genes have no obvious homolog in Arabidopsis. The expression of NAR1.2 is induced under low-CO2 conditions and is partially under the control of CIA5, a transcription factor that is required for the expression of other CCM genes (49). NAR1.2 is predicted to be localized to the chloroplast thylakoid or chloroplast envelope and has six transmembrane domains. The functional expression of NAR1.2 in Xenopus oocytes has shown that the presence of NAR1.2 increases the bicarbonate entry into oocytes twofold compared to that of the control (46). These features suggest that NAR1.2 is an attractive candidate to be a bicarbonate transporter.

Three other proteins suggested to be part of the Ci uptake system include HLA3 (32), RH1 (86), and YCF10 (81). HLA3 was first identified as a gene showing expression when C. reinhardtii cells were exposed to high light. Subsequent work showed that HLA3 expression is also controlled by the CO2 concentration. HLA3 has strong sequence similarity to an ABC transporter and was first predicted to be localized to the chloroplast membrane (32). However, more recent versions of the prediction servers give much less clear predictions as to the location of HLA3. HLA3 might be a potential transporter in the acclimation of cells to low CO2 or might be involved in redox control and only indirectly involved in the control of CCM expression (32). Another chloroplast envelope protein that has been implicated in Ci uptake is the product of the ycf10 gene. It can form two or three transmembrane domains and has been localized in the inner chloroplast envelope membrane (82). Disruption of the open reading frame affected the uptake of inorganic carbon (81). These observations raise the possibility that this protein is a Ci transporter. However, subsequent experiments provided evidence that YCF10 may not be involved directly in Ci uptake but rather may regulate the Ci transport system. It could be associated with a system in the chloroplast envelope involved in HCO3 and/or CO2 uptake (81).

RH1 has been implicated in CO2 transport because it is very similar to bacterial proteins shown to be ammonia and/or CO2 channels (86). However, the expression of this protein is not consistent with it being part of the CCM, as RH1 is expressed at high levels of CO2 when cells are grown on elevated CO2 and not when cells are grown on low CO2. In addition, when RH1 expression is reduced by mutation, C. reinhardtii can still grow on low levels of CO2 but shows reduced growth on elevated levels of CO2 (87). Likewise, RH1 is not regulated by CIA5 (101). The possible physiological role of this protein is to facilitate CO2 entry into the cell when the CO2 level is high. The role of RH1 in CO2 transport remains a very interesting question in this field.

CHALLENGES AND FUTURE DIRECTIONS

The biggest challenge facing researchers studying the CCMs in eukaryotic algae is identifying the transport components involved in inorganic carbon accumulation. This is especially true for the proposed thylakoid HCO3 transporter. In the case of the thylakoid, experiments need to be done to demonstrate whether HCO3 can cross the membrane at all, as the only report on HCO3 transport was negative (31). In an effort to identify additional components of the CCM in C. reinhardtii, a number of insertional mutants have been generated (Table (Table1).1). While a number of candidate transport proteins have been identified in C. reinhardtii, none of these proteins has been proven conclusively to be an essential part of the CCM. One issue that may be hampering these efforts is that there may be a number of transporter proteins and eliminating only one through mutation may not lead to an obvious growth phenotype. This is the case for cyanobacteria. One frustrating point has been that none of the transport proteins identified in cyanobacteria aligns well with an annotated gene product in C. reinhardtii. This lack of homology underscores both the evolutionary distance between green algae and cyanobacteria and the possibility that the CCM may have evolved independently in these different lineages.

In contrast, the number and location of the CA isoforms are becoming clearer. While mutants exist for only two of the CA genes (CAH1 and CAH3), RNAi studies should help to clarify the physiological roles of the other isoforms. It will be interesting to see if the mtCAs are important to the CCM. The two mitochondrial proteins, CAH4 and CAH5, dramatically increase in abundance when C. reinhardtii is in a low-CO2 environment. This induction implies that CAH4 and CAH5 are important to the cells' acclimation to limiting CO2 conditions. However, whether these mitochondrial proteins are important in CO2 recapture, the photorespiratory pathway, or some other anaplerotic function remains to be established.

The role of the pyrenoid remains another important topic of research. In C. reinhardtii, there is a dramatic rearrangement of starch granules when the cells are shifted from high- to low-CO2 growth conditions (Fig. (Fig.3).3). When the cells experience high CO2, the starch granules are evenly distributed throughout the chloroplast stroma. When they are switched to low CO2, the starch strongly associates with the pyrenoid, forming a “shell” or “sheath” around the pyrenoid (11). Since almost all of the Rubisco is contained within the pyrenoid, that means that all of the Rubisco is encased in this carbohydrate shell (41, 52). This observation has evoked the speculation that the starch sheath might be an important acclimation to low-CO2 growth conditions. However, when mutants unable to make starch were tested for growth with low CO2, they were still able to grow at a rate indistinguishable from that of wild-type cells (Mason and Moroney, unpublished observations). For cyanobacteria, mutations that disrupt the carboxysome or cause Rubisco not to package in the carboxysome (64, 83) cause the bacteria to grow slowly on low levels of CO2. To date, no mutations that disrupt the pyrenoid structure in C. reinhardtii are known, except for mutations in rbcL itself, which eliminate Rubisco and also eliminate the pyrenoid altogether (76).

Acknowledgments

We thank David Longstreth, Catherine Mason, and Patricia Moroney for helpful comments and suggestions on the manuscript. We also thank Cindy Henk for help with electron microscopy.

This work was supported by NSF grant IOB-0516810 to J.V.M.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 June 2007.

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