Plant Physiol. (1999) 119: 9-16

UPDATE ON PHOTOSYNTHESIS

How Do Algae Concentrate CO₂ to Increase the Efficiency of Photosynthetic Carbon Fixation?¹

James V. Moroney^* and Aravind Somanchi

Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana 70803

	INTRODUCTION

The ability of photosynthetic organisms to use CO₂ for photosynthesis depends in part on the properties of Rubisco. Rubisco has a surprisingly poor affinity for CO₂, probably because it evolved in an atmosphere that had very high CO₂ levels compared with the present atmosphere. In C₃ plants the K_m(CO₂) of Rubisco ranges between 15 and 25 µM. In cyanobacteria Rubisco has an even lower affinity for CO₂, and the K_m(CO₂) can be greater than 200 µM. In comparison, the concentration of CO₂ in water in equilibrium with air is approximately 10 µM. From these numbers it becomes apparent that Rubisco is operating at no more than 30% of its capacity under standard atmospheric conditions. This is one of the reasons that C₃ plants contain such large amounts of Rubisco. Exacerbating this situation is the fact that O₂ is a competitive substrate with respect to CO₂.

In the atmosphere, where the O₂ level is 21% and the CO₂ level is 0.035%, the competition by O₂ accounts for as much as 30% of the reactions catalyzed by Rubisco. A number of photosynthetic organisms have developed ways to increase the level of CO₂ at the location of Rubisco in the plant. This results in an increase in CO₂ fixation and a decrease in the deleterious oxygenation reaction. An excellent example of a CO₂-concentrating mechanism in higher plants is C₄ photosynthesis, which has arisen independently in a number of plant families. Aquatic photosynthetic organisms such as the microalgae have also adapted to low CO₂ levels by concentrating CO₂ internally. This Update will focus on CO₂-concentrating mechanisms in the microalgae. For more detailed reviews of the CO₂ concentration by algae, the reader is referred to the special issue of the Canadian Journal of Botany (1998, Vol. 76) and the article by Raven (1997).

	TYPES OF CO2-CONCENTRATING MECHANISMS AND THE PROBLEM OF LEAKAGE OF ACCUMULATED CO2

C₄ plants are the best-studied organisms that concentrate CO₂ to enhance the carboxylation reaction of Rubisco. They have high levels of PEP carboxylase in leaf mesophyll cells, whereas Rubisco is located primarily in the bundle-sheath cells. CA within the mesophyll converts CO₂ entering the leaf into HCO₃, which is the substrate for PEP carboxylase. The advantages that PEP carboxylase has over Rubisco are its high affinity for HCO₃ and its insensitivity to O₂. At physiological CO₂ levels and pH, the HCO₃ concentration in the cytoplasm of mesophyll cells is about 50 µM, whereas the K_m(HCO₃) of PEP carboxylase is estimated to be about 8 µM. Therefore, in contrast to Rubisco, PEP carboxylase is saturated for HCO₃ at ambient CO₂ levels. To finish the CO₂-concentrating effect of C₄ metabolism, the C₄ acid generated in the mesophyll cells is then transported to the bundle-sheath cells and decarboxylated, creating an elevated CO₂ level specifically within these cells.

The problem faced by all photosynthetic organisms that concentrate CO₂ is that it can easily diffuse through biological membranes. How can such a slippery substance be accumulated? In C₄ plants CO₂ is concentrated in specific bundle-sheath cells within the leaf. These are the only cells containing significant amounts of Rubisco. Here the thickened cell walls of the bundle sheath prevent the diffusion of the CO₂ generated by decarboxylation reactions. Microalgae face an additional problem in that they are composed of only one or a few cells, all with ready access to the environment; therefore, they must prevent the diffusion of CO₂ out of the cell while allowing the entry of other nutrients.

Microalgae overcome the problem of CO₂ diffusion by accumulating HCO₃. Being a charged species, HCO₃ diffuses through membranes much more slowly than CO₂. However, because CO₂ is the substrate required by Rubisco, the accumulated HCO₃ must be converted to CO₂ before C_i fixation takes place. This appears to be accomplished by packaging Rubisco within the algal cell and generating the CO₂ at that location through the action of a CA. A locally elevated CO₂ environment is thereby created in which CO₂ can out-compete O₂ at the active site of Rubisco. This allows the CO₂ to be used for photosynthesis before it can diffuse out of the cell. Thus, microalgae that concentrate CO₂ package Rubisco in a very specific location, have a means of concentrating HCO₃, and have a means of converting the accumulated HCO₃ to CO₂ rapidly at the location of Rubisco.

	THE LOCATION OF RUBISCO IN MICROALGAE

In higher plants Rubisco appears to act largely as a soluble protein that is distributed throughout the chloroplast stroma. By analogy, one might expect eukaryotic algae to have Rubisco throughout their chloroplast stroma and cyanobacteria to contain Rubisco throughout their cytoplasm, but this is clearly not the case. In most microalgae Rubisco is concentrated in a specific location: in carboxysomes in cyanobacteria and in the pyrenoid in algae (Fig. 1; Table I). Recent studies support the hypothesis that Rubisco localization is required for efficient acquisition of environmental CO₂.

View larger version (159K): [in this window] [in a new window]   Figure 1. Carboxysomes and pyrenoids in different photosynthetic organisms. A, Electron micrograph of the cyanobacteria Anabaena; B, electron micrograph of the green alga C. reinhardtii; C, electron micrograph of the diatom Amphora; D, Immunogold labeling of the pyrenoid of C. reinhardtii with an anti-Rubisco antibody. Bars = 0.5 µm. Cs, Carboxysome; Py, pyrenoid.

Carboxysomes are electron-dense particles that are surrounded by a protein shell. Evidence that they contain large amounts of Rubisco is extensive. In fact, isolated carboxysomes have been found to be composed mostly of Rubisco (Price et al., 1992). Immunolocalization studies using antibodies raised against Rubisco indicate that the carboxysome is the primary location in cyanobacteria (McKay et al., 1993). A mutation that causes a 30-amino acid extension of the Rubisco small subunit leads to a Rubisco that does not pack into the carboxysome, which leaves the carboxysome empty (Schwarz et al., 1995). Mutations in any of the genes affecting the assembly, functioning, or shape of the carboxysome result in cells that cannot grow on air levels of CO₂ (Price et al., 1998).

Rubisco is also packaged in microalgae, where it is the major protein component of the pyrenoid. Pyrenoids have been purified from both Eremosphera (Okada, 1992) and Chlamydomonas reinhardtii (Kuchitsu et al., 1991), and in both cases they consisted primarily of Rubisco. In addition, C. reinhardtii cells with a mutation of the rbcL gene (Rubisco large subunit) that leads to a truncation of the large subunit of Rubisco have no pyrenoids (Rawat et al., 1996). Although it is accepted that Rubisco is the major constituent of the pyrenoid, there are conflicting findings regarding what percentage of the cell's Rubisco is in the pyrenoid. A recent report by Borkhsenious et al. (1998) demonstrated that in C. reinhardtii the amount of Rubisco in the stroma varies with growth conditions.

In all published immunolocalization studies the pyrenoid is densely labeled when an anti-Rubisco antibody is used as the primary probe (Borkhsenious et al., 1998). An example of this immunogold labeling is shown in Figure 1D. In these studies the amount of Rubisco in each subcellular location was estimated by multiplying the density of particles (particles per area) in that location by the average volume of the pyrenoid (2.4 mm³) or the stroma (35.6 mm³) (Lacoste-Royal and Gibbs, 1987). However, this still leaves a fairly broad range of estimates for the amount of Rubisco in the pyrenoid, from 50% to 99%. These differences could be attributed to the growth regime used by the various research groups. Borkhsenious et al. (1998) found that the amount of Rubisco in the stroma varied with growth conditions: about 50% of the Rubisco was localized to the pyrenoid in C. reinhardtii cells grown on elevated CO₂ (5%, v/v). In contrast, they reported that when C. reinhardtii cells were grown under low CO₂ (ambient levels of CO₂ are considered low) more than 90% of the Rubisco was located in the pyrenoid. These results are consistent with those of Morita et al. (1997), who reported that 99% of the Rubisco was located in the pyrenoid in cells grown with ambient levels of CO₂.

C. reinhardtii concentrates CO₂ only when it is grown under low-CO₂ conditions. Because more than 90% of the Rubisco is localized to the pyrenoid under low-CO₂ conditions, one question is whether pyrenoidal Rubisco is active in CO₂ fixation or whether the pyrenoid is a storage body. In vitro measurements of Rubisco activity imply that the enzyme in the pyrenoid must be active to account for the levels of CO₂ fixation observed in C. reinhardtii. A specific localization of Rubisco to the pyrenoid is also compatible with the view that organisms that have CO₂-concentrating mechanisms specifically package Rubisco. In lichens and bryophytes there is a good correlation between the operation of a CO₂-concentrating mechanism and the presence of a pyrenoid (Smith and Griffiths, 1996). In cyanobacteria it appears that the CO₂ level is elevated within the carboxysome (Price et al., 1998), thus favoring carboxylation activity over the oxygenation activity of Rubisco. The pyrenoid may serve a similar function in C. reinhardtii and other microalgae.

	THE ACCUMULATION OF HCO3

The physiological evidence for the existence of CO₂ concentration in microalgae is 2-fold. First, algae are very efficient at pulling C_i out of the environment. They are much more efficient than would be expected, with cells showing an apparent affinity for CO₂ of about 1 µM versus the K_m(CO₂) of Rubisco of about 20 µM. In some cases the growth conditions of the alga influences the cell's affinity for CO₂. Some species of algae, when grown on elevated CO₂ concentrations (10 times higher than ambient), are not efficient in their acquisition of C_i (Matsuda et al., 1998). However, if these same algae are grown on limiting CO₂ they become very efficient in CO₂ uptake and fixation. This implies that there are inducible transport mechanisms, because the amount of Rubisco does not change during adaptation from high- to low-CO₂ conditions.

Second, the accumulation of C_i within the cell can be measured directly. In the light, cyanobacteria can concentrate HCO₃ within the cell more than 100-fold (Miller et al., 1990). Eukaryotic algae are not as efficient but can accumulate HCO₃ at least 20-fold over ambient CO₂ levels. C_i transporters and CAs may enable the cells to accumulate HCO₃ within the cell. The exact identity of the C_i transporters is still unknown, but recent work has identified some transporters that may play a significant role in the accumulation of C_i (Okamura et al., 1997).

In cyanobacteria difficulty in obtaining CO₂- and HCO₃-transport mutants has been proposed to indicate the presence of multiple transporters for CO₂ and HCO₃. There is physiological evidence for three types of transporters: (a) a Na⁺-independent HCO₃ transporter, (b) a Na⁺-dependent HCO₃ transporter, and (c) a CO₂ transporter.

Na⁺-independent HCO₃ transport under extreme C_i limitation (Espie and Kandasamy, 1992) and a difference in the magnitude of the requirement of Na⁺ for HCO₃ transport versus CO₂ transport (Miller et al., 1990) have been detected in Synechococcus PCC 7942. These data indicate the presence of either a Na⁺/HCO₃ symporter (Espie and Kandasamy, 1994) or the regulation of pH through Na⁺/H⁺ antiport mechanisms.

A mutant of Synechococcus PCC 7942, M42, has been shown to have a reduced affinity for HCO₃. The mutation in M42 has been shown to be in the gene cluster cmpABCD, which codes for a Na⁺-independent, high-affinity HCO₃ transporter induced under low C_i (Okamura et al., 1997). This is the first reported primary transporter for HCO₃, and belongs to the subfamily of ABC transporters also known as traffic ATPases (Higgins, 1992). The presence of an ABC-type transporter indicates that at high pH, when HCO₃ is taken up, ATP may be the energy source for C_i uptake. A high-CO₂-requiring mutant of Synechococcus PCC 7942 has recently been characterized; it has a lesion in the gene dc14 (Ronen-Tarazi et al., 1998), which encodes a putative Na⁺-dependent HCO₃ transporter. This transporter may be responsible for the fast induction response to low CO₂ reported from Synechococcus PCC 7942 and Synechocystis PCC 7002 (Sültemeyer et al., 1997).

Much less is known about the transport of C_i in microalgae. Extracellular C_i has to pass through at least two membrane systems to reach the site of carboxylation, which makes transport more complex than in cyanobacteria. At least two types of C_i uptake can be observed in microalgae. There is evidence for both direct transport of HCO₃ and CA-facilitated diffusion of CO₂ across the membrane. The two membranes that we will consider as possible sites of C_i transport are the plasma membrane and the chloroplast envelope.

At the plasma membrane there is evidence for both HCO₃ uptake and CA-facilitated diffusion. In Scenedesmus obliquus there is very good evidence that HCO₃ is taken up directly by the cell (Thielmann et al., 1990). These cells can photosynthesize even when the pH is greater than 10 and HCO₃ and CO₃² are the major C_i species. Chlorella saccharophila also appears to take up HCO₃, although CO₂ is its preferred C_i source (Williams et al., 1995).

The other major process by which microalgae take up C_i is through the uptake of CO₂. Many microalgae produce large amounts of CA when grown on limiting CO₂ (Raven, 1997). CA is a zinc metalloprotein, often located in the periplasmic space of the cell, that catalyzes the interconversion of CO₂ and HCO₃ according to the following formula: Genes encoding periplasmic CAs have been identified in both Dunaliella salina and C. reinhardtii (Fujiwara et al., 1990). CA1, the periplasmic CA, has been identified as one of the prominent low-CO₂-inducible proteins in C. reinhardtii. The ability of microalgal cells to use external HCO₃ for photosynthesis has been correlated with the presence of periplasmic CA. The presence of external CA inhibitors decreased the use of external C_i for photosynthesis (Moroney et al., 1985). The periplasmic CA probably increases the efficiency with which the cells can take in external C_i. This includes both the supply of CO₂ for diffusion across the plasma membrane and the supply of HCO₃ for the plasma membrane's HCO₃-transport system.

The chloroplast envelope is another possible location of HCO₃ accumulation (Beardall, 1981). Intact chloroplasts isolated from C. reinhardtii and Dunaliella tertiolecta retain the ability to accumulate HCO₃ when grown on low CO₂, and have the ability to concentrate CO₂. At low CO₂, C. reinhardtii induces the synthesis of LIP-36, a transport protein that is localized to the chloroplast envelope (Chen et al., 1997). LIP-36 belongs to a family of transport proteins that often act as exchangers (e.g. ATP for ADP transporters). It is possible that LIP-36 plays a role in HCO₃ accumulation by the chloroplast, because chloroplasts with LIP-36 accumulate HCO₃ and those without LIP-36, isolated from high-CO₂-grown cells, do not. The fact that LIP-36 is encoded by two separate genes (Chen et al., 1997) has made it difficult to obtain mutants devoid of this protein.

	THE GENERATION OF CO2 AT THE LOCATION OF RUBISCO

The generation of CO₂ at the location of Rubisco is accomplished through the action of a CA located at or near Rubisco. In cyanobacteria a CA is localized to the carboxysome (Price et al., 1992). Carboxysomes purified from Synechocystis species have significant CA activity. In Synechocystis 6803, for which the complete genome has been sequenced, only one CA gene has been identified. The role of this CA is the dehydration of accumulated HCO₃ to form a localized, elevated concentration of CO₂ in the carboxysome. Loss of the carboxysomal CA through mutation leads to a cell that cannot grow well on limiting levels of CO₂ (Fukuzawa et al., 1992). In addition, cells missing the carboxysomal CA actually accumulate HCO₃ to higher levels than wild-type cells, presumably because the cell can no longer convert the HCO₃ to CO₂ for photosynthesis. In these CA-deficient cells, the CO₂-concentrating mechanism is still operational, but the final conversion of HCO₃ to CO₂ is too slow.

It is noteworthy that CA activity is not found in the cytoplasm of cyanobacteria. Price and Badger (1989) demonstrated that transforming Synechococcus species with a human CA actually "short-circuits" HCO₃ accumulation, and this transformant requires high CO₂ for growth. The human CA was localized to the cytoplasm and converted the accumulated HCO₃ to CO₂. The CO₂ thus formed then leaked from the cell and could not be used efficiently for photosynthesis. From these studies it appears that the location of the internal CA is as important as the packaging of Rubisco.

In eukaryotic algae CA is often found inside the cell and in the periplasmic space. It is now clear that C. reinhardtii has at least five genes that encode CAs. Two of these genes, Cah1 and Cah2, encode CAs that are directed to the periplasmic space (Fujiwara et al., 1990). Two more genes encode mitochondrial CAs (Eriksson et al., 1996). Recently, a fifth gene, Cah3, was found to encode a chloroplast CA (Karlsson et al., 1998). This CA has a leader sequence that directs the protein into the lumen of the thylakoid membrane. Pharmacological and genetic evidence indicates that Cah3 is essential in generating an elevated CO₂ concentration for Rubisco. It appears to play a role similar to that of the carboxysomal CA of cyanobacteria. This thylakoid CA is sensitive to sulfonamides, pharmaceuticals often used to inhibit mammalian CAs. Treatment of C. reinhardtii with sulfonamides that can enter the cell results in repression of CO₂ fixation (Moroney et al., 1985). Sulfonamides also severely inhibit photosynthesis in many other algae at low CO₂ concentrations, indicating that this thylakoid CA may be found in many algae. Furthermore, mutant strains of Cah3 are unable to grow at low CO₂, although the ability of these strains to accumulate HCO₃ is not impaired. The thylakoid CA is thought to increase the concentration of CO₂ in the chloroplast by dehydration of the high concentration of HCO₃ the cell accumulates there.

Chloroplast CAs from higher plants are quite different from the Cah3 protein of C. reinhardtii. Cah3 does not share any sequence similarity with higher-plant chloroplast CAs. The higher-plant enzymes are of the -type and are found in the chloroplast stroma (Badger and Price, 1994). In contrast, Cah3 is of the -type and is found in the thylakoid lumen (Karlsson et al., 1998). At this point no stromal CA has been found in an algal species that actively concentrates CO₂. It appears that a stromal CA might short-circuit the active accumulation of HCO₃. If CA were present in the chloroplast stroma it might convert accumulated HCO₃ back to CO₂, allowing it to leak out of the cell before being fixed by Rubisco.

	A MODEL FOR CO2 CONCENTRATION

Even though the types of cells that possess CO₂-concentrating abilities are very different, they have certain properties in common that allow them to use CO₂ efficiently. The first property is the ability to accumulate HCO₃ in some fashion. For most cyanobacteria and many eukaryotic algae, HCO₃ can be transported into the cell directly. For other eukaryotic algae, particularly those that live in acidic environments, where the concentration of HCO₃ is low, CO₂ is the C_i species that enters the cell and HCO₃ is accumulated in the chloroplast. A second property is that Rubisco is usually packaged in a very specific way within the photosynthetic cell. Although it is possible that not every microalgal cell that concentrates CO₂ has a carboxysome or a pyrenoid, most cyanobacteria have carboxysomes and most microalgae have pyrenoids. The third property that appears to be common among these types of cells is the presence of a CA near the location of Rubisco. The CA supplies the Rubisco with CO₂ from the pool of HCO₃. Loss of this CA through mutation or inhibition greatly impairs the cell's ability to use external C_i for photosynthesis (Price et al., 1992; Karlsson et al., 1998).

A general model for CO₂ concentration in cyanobacteria is shown in Figure 2. Evidence for this model comes from physiological experiments and mutant analysis (Table II). In Figure 2 three different types of transporters are shown at the plasma membrane. It is very likely that there are a number of transporters important in HCO₃ accumulation, because no single mutation has totally inhibited it. Recent work with the Cmp gene cluster of cyanobacteria has shown that high-affinity HCO₃-transporter activity is lost if genes within this operon are deleted (Okamura et al., 1997). The Cmp operon appears to encode an ABC transporter with significant similarity to proteins known to transport small anions such as NO₃ (Ogawa et al., 1998; Ohkawa et al., 1998). The fact that Cmp deletion mutants still retain the ability to grow on low HCO₃ concentrations implies that other transporters remain to be identified. This is consistent with the multiple transport activities detected in the physiological experiments.

View larger version (27K): [in this window] [in a new window]   Figure 2. A model for CO2 concentration in cyanobacteria. The font sizes of CO2 and HCO3 indicate the relative concentrations of these Ci species. PGA, 3-Phosphoglyceric acid.

The amount of energy required for HCO₃ uptake is not clear at present. Because ABC transporters require ATP, it is reasonable to assume that some ATP is used in HCO₃ uptake (Fig. 2). Ogawa et al. (1998) have provided support for this contention by identifying a number of mutations that encode subunits of a NAD(P)H dehydrogenase. Deletions of these ndh genes lead to cells that require high CO₂ for photoautotrophic growth. The explanation for these mutants is that cyclic electron transport is disrupted in these cells such that too little ATP is made to support HCO₃ transport. Mi et al. (1992) have also provided evidence that cyclic electron transport around PSI is required for HCO₃ uptake.

Because Rubisco uses CO₂ and not HCO₃, the HCO₃ accumulated by the cyanobacteria must be converted to CO₂ for fixation. As indicated in Table II, any disruption of the proper localization of Rubisco to the carboxysome in cyanobacteria leads to a cell that requires high CO₂ for photoautotrophic growth. One example of this is the loss of carboxysomes through loss of the carboxysomal shell proteins (Orús et al., 1995), in which case Rubisco is distributed in the cytoplasm. A similar situation occurs in the mutant EK6, which contains a 30-amino acid extension of the small subunit of Rubisco and has empty carboxysomes (Schwarz et al., 1995). Even though the kinetics of this Rubisco appear normal, this strain requires high concentrations of CO₂ for normal growth. Again, the explanation appears to be that without an elevated CO₂ supply, the Rubisco is not packaged correctly into the carboxysome and ends up in the cytoplasm. Finally, the substitution of a bacterial Rubisco in place of the normal enzyme (Pierce et al., 1989) results in Rubisco free in the cytoplasm and in cells that require high CO₂ for growth.

The location of CA in cyanobacteria is also critical to the operation of the CO₂-concentrating mechanism. If the carboxysomal CA is inhibited or lost through mutation, the cell loses its ability to grow on low CO₂ concentrations. Therefore, the CA indicated in the carboxysome in Figure 2 is essential for the CO₂-concentrating mechanism, and its packaging is as important as the packaging of Rubisco.

A model of CO₂ concentration in eukaryotic algae is shown in Figure 3. This system is less understood because eukaryotic algae have more cellular compartments, are a very diverse group of organisms, and there are a limited number of systems in which molecular and genetic tools are available. However, the overall scheme of CO₂ concentration retains many similarities to the cyanobacterial model of active HCO₃ accumulation, Rubisco packaging, and HCO₃ dehydration in the chloroplast. In Figure 3 we have indicated both active uptake of HCO₃ and diffusion of CO₂ across the plasma membrane, an uptake facilitated by the periplasmic CA. Microalgae also package their Rubisco in the pyrenoid, and deletion of the rbcL gene results in a strain without a pyrenoid (Table III).

View larger version (26K): [in this window] [in a new window]   Figure 3. A model for CO2 concentration in eukaryotic microalgae. The font sizes of CO2 and HCO3 indicate the relative concentrations of these Ci species. cCA, Chloroplastic CA; pCA, periplasmic CA; PGA, 3-phosphoglyceric acid.

The generation of CO₂ for Rubisco is also catalyzed by a specific CA, Cah3. Mutations in the gene encoding this chloroplastic CA require high CO₂ for photoautotrophic growth, and these mutants can be complemented by transforming the strain with the wild-type gene (Funke et al., 1997; Karlsson et al., 1998). In Figure 3 this CA is shown in the pyrenoid near Rubisco, but its exact location in relation to the pyrenoid has not been clearly established.

One important difference between algae, which concentrate CO₂, and C₃ plants, which do not, is the amount of CA activity in the stroma of the chloroplasts. In C₃ plants, there is a highly active, -type CA in the chloroplast stroma (Badger and Price, 1994). In C. reinhardtii and other green algae there is very little, if any, stromal CA activity. In fact, the only chloroplast CA known is located in the thylakoid lumen (Karlsson et al., 1998). If the chloroplast is analogous to the cyanobacterial cytoplasm, a stromal CA might short-circuit the CO₂-concentrating mechanism. In cyanobacteria the insertion of human CA in the cytoplasm defeated the activity of the HCO₃-accumulation system (Price and Badger, 1989). One prediction of the model shown in Figure 3 is that the presence of a CA in the chloroplast stroma might result in a cell that requires high CO₂ for growth.

	AREAS OF CURRENT INTEREST

From the discussion above it is clear that there are still many unanswered questions about the mechanism by which microalgae accumulate C_i. The first challenge is to identify the other transport components of cyanobacteria and microalgae. In cyanobacteria a combination of better screening strategies for insertional mutants and the availability of the complete genome database for Synechocystis PCC 6803 should facilitate identification of the other C_i transporters and their mechanisms of operation. In microalgae the role of LIP-36 is being investigated. The recent development of several positive selectable markers for insertional mutagenesis in C. reinhardtii provides a powerful tool that will aid these studies. Insertional mutagenesis may be used not only for the identification of the C_i transporter, but also for the identification of other components involved in HCO₃ accumulation, as well as the characterization of the roles played by these proteins.

A second important area of future interest is the role of the carboxysome and pyrenoid in CO₂ concentration. Carboxysomes are relatively well characterized in terms of their constituents, the genes that encode the proteins involved, and the effect of inactivation of these genes. However, the current evidence for the role of the pyrenoid in CO₂ concentration is circumstantial. The identification of mutants with disrupted or aberrant pyrenoids would help to clarify this issue.

A third area is the cost of CO₂ concentration. There is strong evidence for a light requirement in this process (Raven, 1997). In cyanobacteria mutant analysis and antibody studies provide evidence for the energization of C_i accumulation through the NAD(P)H-dependent PSI cyclic electron flow (Mi et al., 1992). In microalgae the light requirement for CO₂ concentration may be attributable in part to the acidification of the lumen, because that is the location of Cah3 and, presumably, the site of the generation of CO₂ for Rubisco. As specific transport proteins are identified, the energy costs can be better estimated. It will be interesting to compare the cost of this process with C₃ and C₄ photosynthesis.

The regulation of the CO₂-concentrating mechanism is also an interesting challenge for future research. It is clear that the synthesis of many of the components of the CO₂-concentrating mechanism increases under low-CO₂ conditions (Beardall et al., 1998). Current evidence indicates that algal cells can "sense" the CO₂ level in the environment (Matsuda et al., 1998). The existence of mutants that fail to respond to low CO₂ (Table III) indicates that this signal transduction pathway can be identified through insertional mutagenesis studies. In addition, there are mutants of Chlorella ellipsoidea that express the CO₂-concentrating mechanism constitutively (Matsuda et al., 1998). A different approach has been taken by investigators who have linked the promoter regions for genes that respond to low-CO₂ conditions to reporter genes in C. reinhardtii (Eriksson et al., 1998). These chimeric genes respond to CO₂, and mutants have been found that fail to induce the reporter gene.

Another important current research topic is how organisms with a CO₂-concentrating mechanism will respond to increasing atmospheric CO₂ levels. For example, how will marine phytoplankton respond? If these organisms already possess an active CO₂-concentrating mechanism, then only a small increase in photosynthesis would be expected. On the other hand, if an algal species does not express the CO₂-concentrating mechanism under present atmospheric conditions, the increase in CO₂ might enhance its growth and photosynthesis. It is known that most algae, including coccoliths, diatoms, and cyanobacteria, have the ability to concentrate CO₂; however, little is known about whether these organism express the CO₂-concentrating mechanism in their native environments.

In conclusion, although much progress has been made in this field of study in the past few years, we are still a long way from complete characterization. The development of new tools and strategies will contribute to further progress in the elucidation of the C_i-accumulation mechanism in the microalgae.

	FOOTNOTES

   Received August 11, 1998; accepted October 12, 1998.

	ABBREVIATIONS

Abbreviations: ABC, ATP-binding cassette. CA, carbonic anhydrase. C_i, inorganic carbon.

	ACKNOWLEDGMENTS

The authors thank Olga Borkhsenious for her help with the electron micrographs and James Adams, Sergio Colombo, Catherine Mason, and Patricia Moroney for critically reading the manuscript.

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