Proposed Carbon Dioxide Concentrating Mechanism in Chlamydomonas reinhardtii^▿

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 C_i transport systems are known for cyanobacteria (Fig. 1). An interesting feature of the cyanobacterial CCM is the induction of multiple transporters under C_i limitation. Cyanobacteria appear to utilize pairs of C_i transporters with complementary kinetics for the same C_i 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).

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 C_i 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 pK_a of the bicarbonate-to-CO₂ interconversion is about 6.3 (HCO₃⁻ + H⁺↔H₂CO₃↔CO₂ + H₂O). Under these conditions, HCO₃⁻ is the predominant species of C_i in the chloroplast stroma while CO₂ is the most abundant form of C_i 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.

The C. reinhardtii CCM.

A proposed model for concentrating CO₂ in C. reinhardtii is shown in Fig. 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.

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 C_i uptake in C. reinhardtii.

The physiological evidence that C. reinhardtii can accumulate C_i 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 K_m(CO₂) for Rubisco (4, 55, 91). For example, whole-cell photosynthesis rates are saturated at about 2 to 3 μM CO₂, while the K_m(CO₂) of C. reinhardtii Rubisco is about 20 μM (34). In addition, C_i uptake has been measured directly in a number of laboratories (3, 4, 6, 55, 92, 93), and the C_i 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 C_i 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 1.

In this model for C_i uptake (Fig. 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, C_i 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. 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).

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 C_i pools, of 24 to 31 μM, in comparison with the large C_i 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 C_i 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 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.

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 C_i, 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 C_i but are unable to use C_i efficiently for photosynthesis (58, 90). Therefore, CAH3 appears to convert accumulated HCO₃⁻ to CO₂, the form of C_i 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 C_i 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 C_i 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 C_i 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 C_i transport proteins have been found. However, the pmp1 mutant does have a mutation in LCIB, and this mutant is defective in C_i 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 C_i transport systems in C. reinhardtii corresponding to multiple CO₂ level-dependent acclimation states (89, 98, 100). This would be similar to the multiple C_i 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 C_i (97).

Another promising candidate protein to be a C_i 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 C_i 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, C_i 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 C_i transport, as seen in cyanobacteria.

Another putative C_i 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 C_i 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 C_i 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 C_i transporter. However, subsequent experiments provided evidence that YCF10 may not be involved directly in C_i uptake but rather may regulate the C_i 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.