• Arabidopsis;
  • Chlamydomonas;
  • cyanobacteria;
  • macro-algae;
  • photosynthesis


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Carbonic anhydrases catalyse the reversible hydration of CO2, increasing the interconversion between CO2 and HCO3 + H+ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C3 dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.


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Carbonic anhydrase (carbonate dehydratase, carbonate hydro-lyase; EC 4·2.1·1) is a zinc metalloenzyme that catalyses the interconversion of CO2 and HCO3 (Khalifah 1971). The enzyme was first discovered in red blood cells but has since been found in most organisms including animals, plants, archaebacteria, and eubacteria (Hewett-Emmett & Tashian 1996). Carbonic anhydrase (CA) is important in many physiological functions that involve carboxylation or decarboxylation reactions, including both photosynthesis and respiration. In addition, it is clear that CA also participates in the transport of inorganic carbon to actively photosynthesizing cells or away from actively respiring cells (Henry 1996).

The known carbonic anhydrases can be grouped into three independent families (Hewett-Emmett & Tashian 1996), called α-CA, β-CA, and γ-CA. Interestingly, these three families have no primary sequence similarities and seem to have evolved independently. The complete distribution of these CAs is uncertain. Plants appear to have all three types of CAs as all three are represented in the Arabidopsis thaliana genome. Cyanobacteria have both α-CA and β-CA and the CcmM protein that bears strong similarity to γ-CAs. Examples of α-CA and β-CA are known in Chlamydomonas reinhardtii (Hewett-Emmett & Tashian 1996; Samuelsson & Karlsson, 2000). However, in animals only the α-type has been found.

Although the primary sequences of these CA families are different, all three types of carbonic anhydrases are Zn+2 metalloenzymes and all appear to share a similar catalytic mechanism (Lindskog 1997). In all cases it appears that a Zn-OH attacks a CO2 molecule residing in a hydrophobic pocket, generating a Zn-bound HCO3 (Eqn 1). The bicarbonate bound to the zinc is then replaced by a water molecule, releasing HCO3. HCO3 in solution can gain a H+ to form H2CO3 or can lose an additional H+ to form CO3−2. The overall relationship between the three forms of dissolved inorganic carbon is shown in Eqn 1.

  • image(1)

The uncatalysed hydration–dehydration reactions are slow, whereas the dissociation reactions are considered instantaneous. Carbonic anhydrase greatly accelerates the hydration of dissolved CO2 in solution thereby increasing the rate at which forms of inorganic carbon interconvert in solution. The equilibrium between the inorganic carbon forms is pH-dependent. At normal intracellular ionic strength, when the pH level is below the first dissociation constant (pK1≈ 6·4) CO2 predominates; at pH between 6·4 and about 10·3 (pK2) HCO3 predominates; whereas above pH of 10·3, CO32− predominates. In this review we will discuss the types of carbonic anhydrases presently known, the intracellular locations of carbonic anhydrases in photosynthetic organisms and the physiological roles of carbonic anhydrases in plants and algae. In each section we will highlight current areas of research and some of the questions being addressed by researchers in this field. We have also collected what is known about the number of putative CA genes from the Arabidopsis genome initiative. We have included in this review putative CA genes encoding open reading frames that contain active site residues. A few of these putative CA genes are known only from genomic sequences, but most of them are clearly expressed and have associated expressed sequence tags (ESTs). In only a few cases has enzymatic activity been measured. For further reading about plant and cyanobacterial CAs readers are referred to earlier excellent reviews by Sültemeyer, Schmidt & Fock (1993), Badger & Price (1994), Raven (1995), Stemler (1997), Sültemeyer (1998) and Kaplan & Reinhold (1999).


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All three known CAs are zinc-containing enzymes that catalyse the same chemical reaction, the reversible hydration of CO2. However, even though these proteins catalyse the same reaction, there are no sequence homologies between the α-, β-and γ-CAs. Apparently, carbonic anhydrase has evolved three different times and may represent an example of convergent evolution of catalytic function (Hewett-Emmett & Tashian 1996). In this section the structures of the three types of CAs are compared. A recent report on a fourth type of CA is also discussed.

α-Carbonic anhydrases

The α-type is the most studied CA and is very widely distributed. α-CAs have been found in animals, plants, eubacteria and viruses. Although α-CAs have been known to occur in animals for many years, they have only recently been identified in plants. Humans have at least 10 isoforms of α-CA as well as a number of carbonic anhydrase-related proteins (CA-RPs) that appear not to have CA activity (Sly & Hu 1995). All of the enzymatically active CAs have three histidines coordinating the Zn atom. In Human CAII, these histidines correspond to H94, H96 and H119. X-ray crystallographic studies show the zinc atom liganded to the side chains of three histidines and a hydroxyl ion (Christianson & Cox 1999). These histidines, as well as a number of other residues found in the active site, are conserved in all active α-CAs. The α-CA structure is dominated by antiparallel β-sheets forming a spherical molecule with two halves. The active site is a funnel-shaped crater with the zinc atom located near the bottom. Most α-CAs are active as monomers of about 30 kDa although the C. reinhardtii periplasmic CA, CAH1, is a heterotetramer with two 37 kDa subunits and two 4 kDa subunits held together by disulphide bonds (Kamo et al. 1990). One of the CA genes of Dunaliella salina encodes a protein of about 63 kDa that appears to have two active sites, possibly the result of gene duplication/fusion events (Fisher et al. 1996).

Vertebrate α-CAs can be divided into two distinct groups (Jiang & Gupta 1999). One group includes the soluble isoforms such as CA I, CA II, CA III and CA VII. Another type of α-CA includes the membrane-associated and secreted CAs. Vertebrate isoforms that fall into this class include CA IV, CA VI, CA XII, CA XIV and CA XVI. The membrane-associated CAs are characterized by hydrophobic C-terminal extensions. As an example, CA XVI appears to be associated with the plasma membrane and is localized to the proximal convoluted tubule of the kidney, suggesting a role in the re-absorption of HCO3 by that organ.

The α-CAs have also been found in eubacteria, algae and higher plants. In photosynthetic organisms only a few α-CA have been identified at this time. One has been found in a cyanobacterium (Soltes-Rak, Mulligan & Coleman 1997). The green algae C. reinhardtii (Karlsson et al. 1998) and Dunaliella salina (Fisher et al. 1996; Yang, Zhang & Xu 1999) have three α-CAs. In C. reinhardtii, two of the α-CAs are localized to the periplasmic space and one to the thylakoid membrane. Furthermore, the sequence of a novel α-CA cDNA from A. thaliana was recently reported (accession no. U73462, Susanne Larsson, personal communication). All of the α-CAs observed to date in plants appear to be soluble proteins although they are localized to distinct cellular compartments.

β-Carbonic anhydrases

The distribution of β-CAs does not appear to be as broad as the α-CAs at this time. β-CAs have been found in plants, algae, eubacteria (Hewett-Emmett & Tashian 1996), archaebacteria (Smith & Ferry 1999) the fungi Saccharomyces cerevisiae (Gotz, Gnann & Zimmermann 1999 and Schizosaccharomyces pombe but not in Caenorhabditis elegans, Drosophila melanogaster or vertebrates. In C. reinhardtii, two β-CAs have been found and both appear to be localized to mitochondria (Eriksson et al. 1996). In C3 plants the abundant β-CA localized to the chloroplast stroma is usually the CA isoform with the highest total activity in the plant. β-CAs have been found in both the cytosol and chloroplast in higher plants and in the symbiotic alga Coccomyxa (Hiltonen et al. 1998).

The zinc ligands in β-CAs are quite different to those of α-CAs. X-ray absorption spectroscopy (EXAFS) studies on spinach β-CA indicated that a histidine and two cysteines were likely to be the zinc ligands (Rowlett et al. 1994; Bracey et al. 1994). Very recently, the crystal structures of a β-CAs from Porphyridium purpureum (Mitsuhashi et al. 2000) and Pisum sativum (Kimber & Pai, 2000) were resolved. The pea CA is an octamer in which dimers form tetramers that form octamers. Active sites are at the subunit interfaces and as suggested by EXAFS, mutagenesis and elemental analysis (Rowlett et al. 1994; Bracey et al. 1994), the zinc is bound by two cysteines and one histidine. In the algal CA an aspartate rather than water occupies the fourth co-ordination position (Mitsuhashi et al. 2000). The algal CA was crystallized in the absence of substrate (or inhibitor) whereas the pea enzyme was crystallized in the presence of acetate. Possibly the difference between the zinc ligands of the two enzymes is due to the conditions under which the crystals were formed. The idea that the CO2 residing in a hydrophobic pocket is required for activity is underscored by the recent comparison between the α-CA and β-CA crystal structures (Kimber & Pai, 2000). It turns out that amino acids in the active site of the β-CA are a mirror image of the amino acids in the active site of the α-CA hydrophobic pocket (Kimber & Pai, 2000).

γ-Carbonic anhydrases

A third type of CA, the γ-CA, was discovered in the archaebacterium Methanosarcina thermophila (Alber & Ferry 1994). Genes encoding putative proteins with sequences similar to γ-CA have been found in eubacteria and plants (Newman et al. 1994). The γ-CA from M. thermophila was crystallized and its structure solved (Kisker et al. 1996). The structure of γ-CA is strikingly different from either the α-CA or β-CA. The γ-CA functions as a trimer of identical subunits. The structure of each monomer is dominated by a left-handed β-helix (Kisker et al. 1996). The trimer contains three Zn atoms, one each at the three subunit interfaces. As in the α-CAs, three histidines and a water molecule coordinate the Zn, but the histidines are provided by two separate subunits. For the M. thermophila protein, His81 and His122 from one subunit act as ligands and His117 from a different subunit is the third ligand. In spite of the fact that the active site is at the subunit interface, the architecture of the active site of γ-CA is similar to that of α-CA (Kisker et al. 1996).

A γ-CA homologue, CcmM, was discovered earlier in Synechococcus PCC7942 (Price et al. 1993). While there has been no clear demonstration that CcmM has any CA activity it is clearly required for optimal growth on low CO2. If this protein is deleted by mutation, the cyanobacteria cannot grow on air levels of CO2, indicating CcmM functions as part of the CO2 concentrating mechanism (Price et al. 1993). The sequence of CcmM from cyanobacteria is striking. It is over 300 amino acids longer than the M. thermophila protein. However, the N-terminal portion of CcmM has a high homology to the archaebacterial γ-CA. The C-terminal portion of CcmM has three to four 87 amino acid repeats that are very similar to the small subunit of Rubisco protein from the cyanobacteria (Price et al. 1998). The exact role of CcmM in CO2 concentration is not clear.

Unresolved questions: are there more carbonic anhydrase gene families?

Clearly there are at least three major classes of carbonic anhydrases. Recently a cDNA encoding a carbonic anhydrase from the diatom Thalassiosira weissflogii was described (Roberts, Lane & Morel 1997). In this work a CA from T. weissflogii was isolated and a partial amino acid sequence obtained from the purified protein. Using this sequence information, a fragment of DNA was amplified by polymerase chain reaction (PCR) and this was used to screen a T. weissflogii cDNA library. The putative CA cDNA obtained in this fashion was sequenced and the sequence does not match a known CA from any of the three gene families. This raises the possibility that there might be yet an additional CA gene family. Unfortunately the authors could not express the protein in Escherichia coli and obtain CA activity. In addition, there have been no further reports of CA cDNAs that match the cDNA obtained from T. weissflogii. Future work is needed to support the notion that there is a δ-CA gene family in addition to the other three gene families.

An additional question raised by work from Morel's group is whether Cd can substitute for Zn in CA or whether there is a separate Cd-dependent CA (Lane & Morel, 2000). Recent work with T. weissflogii indicates that this diatom might be able to substitute Cd for Zn under Zn-limiting environmental conditions (Lane & Morel, 2000). They demonstrated that the Cd-CA was different than the other CA they had previously found in T. weissflogii (Roberts et al. 1997). This raises the possibility that there are Cd-requiring CAs in diatoms, and possibly, in algae and higher plants.


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α-Carbonic anhydrases

The first α-CA genes cloned from a photosynthetic organism were Cah1 and Cah2 that encode two periplasmic CAs in C. reinhardtii (Fukuzawa et al. 1990; Fujiwara et al. 1990). These two genes encode very similar proteins although they are differentially regulated. Cah1 is expressed under low CO2 but not under elevated CO2 conditions. In contrast Cah2 is poorly expressed under low CO2 conditions and only very slightly up-regulated under increased CO2 partial pressure. In addition, the expression of Cah2 under high CO2 conditions appears low when compared with the expression of Cah1 under low CO2 conditions (Fujiwara et al. 1990). Possibly Cah2 resulted from a gene duplication event and has a poorly functioning promoter.

A third C. reinhardtiiα-CA was discovered in 1995 (Karlsson et al. 1995). Later Karlsson et al. (1998) published the sequence of the cDNA encoding this intracellular α-CA, designated Cah3. The presence of a bipartite presequence and immunolocalization study indicate that this CA is located in the lumen of the thylakoid membrane. No more than one α-CA has been found in any given cyanobacterium. An α-CA has been found in both in Anabaena and Synechococcus (Soltes-Rak et al. 1997) and in each organism the protein is localized to the periplasmic space. In contrast, Synechocystis 6803 does not have a gene with high similarity to α-CAs.

It is clear that a number of α-CAs remain to be identified in higher plants. As of September 2000, the Arabidopsis database contained sequences representing six different genes that align with α-CAs (Table 1). In contrast, no sequences in the Synechocystis 6803 genome align strongly with α-CAs. To our knowledge only one α-CA cDNA from a higher plant, ACAH1 from A. thaliana, has been completely sequenced (accession number U73462, Susanne Larsson personal communication). However some ESTs in the Arabidopsis database do align with some of the other α-CAs identified by the genome project, implying that these are functional genes.

Table 1.  Summary of putative CA genes in the higher plant Arabidopsis thaliana compiled from the information in gene bank as of September 2000. The open reading frame encoded in each gene can be translated to yield a protein containing active site residues appropriate for its CA family. The database contains ESTs corresponding to most of these genes, and so they appear to be expressed. Full-length cDNA clones have only been described for three of the putative genes
Gene familyGene numbercDNA accessionEST accessionGenomic accessionChromosome locationReferences
  1. AI992681

α1U73462AV442219AL353912III Larsson et al. 1997
α2 T45411AC006202II
α3,4  AL161554IV
α5,6  AC026875I
β1X65541AV440467AC009325IIIRaines et al. 1992
β2L18901AV442315AL391149VFett and Coleman 1994
β3 AA597643AC003671I
β4 Z235745AC005990I
β5 AA598084AL031394IV
γ1 T04294AC024609I
γ2 AV564979AC074226?
γ3 AA72011AB013389V

β-Carbonic anhydrases

β-CAs were first recognized to be carbonic anhydrases in photosynthetic organisms (Burnell, Gibbs & Mason 1990; Fawcett et al. 1990). Once recognized as a CA, β-CAs have been found in eubacteria (Hewett-Emmett & Tashian 1996), archaebacteria (Smith & Ferry 1999), cyanobacteria, micro-algae, yeast (Gotz et al. 1999) and higher plants. In cyanobacteria one type of β-CA has been described and it appears to be localized to the carboxysome (Fukuzawa et al. 1992; Yu et al. 1992). Loss of the carboxysomal β-CA leads to a high CO2-requiring phenotype (Price & Badger 1989b). The Synechocystis 6803 genome contains no other β-CA, indicating that the carboxysomal β-CA is the only β-CA in at least this cyanobacterium. Two β-CAs have been identified in the micro-alga, C. reinhardtii (Eriksson et al. 1996). Both β-CAs in C. reinhardtii are localized to the mitochondria. In addition, expression of the β-CAs in C. reinhardtii is strongly influenced by the CO2 concentration (Eriksson et al. 1998). Under elevated CO2 conditions very little β-CA mRNA is made but under low CO2 the mRNA is quite abundant. β-CAs have been found in both the cytosol and chloroplast in higher plants (Fett & Coleman 1994) and in the cytosol of the symbiotic alga Coccomyxa (Hiltonen et al. 1998).

In red algae, the β-CA of P. purpureum is the only CA described to date. The P. purpureum protein has two active sites per polypeptide instead of the one found in other β-CAs from algae and higher plants suggesting a gene duplication event occurred (Mitsuhashi & Miyachi 1996; Mitsuhashi et al. 2000). In C3 plants, the chloroplast stroma has the highest levels of CA activity and that activity is due to a β-CA. In A. thaliana cDNAs encoding cytosolic and chloroplastic forms of β-CA have been described (Fett & Coleman 1994). At this time sequences encoding at least five β-CA genes from A. thaliana are in the database (Table 1), and it is possible that other β-CAs might be found in the near future. The C4 plants Zea Mays (Burnell, Ludwig, & Sugiyama 1999) and Urochloa panicoides (Ludwig & Burnell 1995), also have β-CAs. Interestingly, conceptual translation of the two CA cDNAs for maize β-CA in the database yields significantly larger proteins of 74 and 60 kDa (Burnell et al. 1999). This large polypeptide appears to be a fusion of two monomers since it contains two sets of active site residues. The quaternary structure of these maize CAs has not been investigated. The unusual structure of the maize polypeptide appears to be unique among higher plant (and possibly its close relatives), since the cDNAs of Urochloa paniculata and Flaveria bidentis, both of which are C4 plants, appear ‘normal’ in size for β-CAs having deduced molecular weights of between 24 and 30 kDa. However the fused duplicated gene seen in maize is reminiscent of the P. purpureum CA (Mitsuhashi et al. 2000).

γ-Carbonic anhydrases

Several Arabidopsis ESTs in the databases have homology with the γ-CA from Methanosarcina thermophila. When the databases are searched using sequences around the putative active site of one of these ESTs, three different genomic sequences are obtained. Two of them, one from chromosome I, and one from chromosome V, have very similar sequences around the active site. The other, a shotgun clone of unknown chromosomal location, is much less similar but retains the histidines at the active site. Thus, the Arabidopsis genome contains at least three genes encoding γ carbonic anhydrase homologues (Table1). The Chlamydomonas EST database also has an EST that aligns well with the M. thermophilaγ-CA. Cyanobacteria have the CcmM gene that encodes a protein with an N-terminus that has homology with M. thermophilaγ-CA. It is not yet known whether any of the gene products actually have carbonic anhydrase activity or what their physiological functions might be.

Unresolved questions

Clearly the key unresolved question for plants and algae is ‘how many carbonic anhydrase do they have and what is the function of all of these isoforms’. To date the A. thaliana genome sequencing initiative has revealed at least 14 putative CA genes (Table 1). The location and organ specificity of only a few of these CAs is known at this time but there are enough different genes to indicate that they may be located in many different organelles or compartments within the plant cell. Perhaps some of the genes will be expressed only under certain growth conditions or environmental stresses. It seems safe to say that higher plants will have a large number of CA genes and that more than one CA gene family will be present.

The situation is clearer in cyanobacteria, probably due to their simple cellular architecture. However, even these bacteria have more than one CA and all three CA types are represented in some cyanobacteria. Micro-algae also have a number of CA genes. For example, in C. reinhardtii, a relatively simple unicellular alga, there are already five carbonic anhydrase genes documented; three α-CAs and two β-CAs. Perhaps most organelles will have specific CAs associated with them. It appears likely that the chloroplast stroma and the thylakoid lumen both have distinct CAs. Although there is very little data from macrophytes, the safe assumption is that they, too, have multiple isoforms of CA. It appears to be dangerous to run CA assays on plant homogenates and ascribe the activity to a single isoform because an abundant CA might obscure the activity of less abundant isoforms. Another unresolved question is if the three types of CA represent convergent evolution is it surprising to find all three in a single organism? In fact, in the case of C. reinhardtii and cyanobacteria the different types of CA are present in the same cell type. In cyanobacteria both the β-CA and γ-CA analog, CcmM, appear to be associated with the carboxysome. The fact that two different types of CA proteins are associated with the same organelle implies that there are additional structural and physiological constraints guiding the evolution of these different carbonic anhydrases.


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Clearly the expression of different isoforms of carbonic anhydrase studied to date is regulated and each individual CA isozyme has a specific location in the cell or periplasmic space. Not only are members of all three CA gene families found in some organisms, but individual cells may contain CAs from all three families or multiple isoforms of a single CA family. For example, vertebrates have over 10 forms of CA and the red blood cells of many vertebrates have two α-CAs (CAI and CAII) in the cytoplasm. In plants, the identification of CA isozymes has been slower but during the last 5 years many new CA cDNAs have been cloned and sequenced and some of the corresponding proteins isolated. Many plant cells, such as leaf cells of A. thaliana, contain multiple forms of CA. Even unicellular algae such as C. reinhardtii and D. salina have multiple isoforms of CA. Clearly the cellular locations of CA in plants are still an open question since not all CA genes and proteins have been discovered. However, a picture is emerging and it seems likely that CA will be found in most compartments of plant cells, as well as in different organs and tissues. The localization of CA to different compartments in different species may correlate with the difference in physiology between plants. It should also be pointed out here that not all CAs are enzymes with high activities. There is at least a 1000-fold difference in the specific activity between some of the animal CAs (Khalifah 1971) and there are reasons to believe that this is the same for plant CAs.

Established locations of CA in photosynthetic organisms


Although cyanobacteria are not eukaryotes they are brought up here because they serve as a model system for plants and they perform oxygenic photosynthesis in much the same way as green plants do. Studies of the CO2 concentrating mechanism (CCM) in the cyanobacterium Synechococcus PCC7942 led to the simultaneous identification of the first cyanobacterial CA in 1992 by two groups (Fukuzawa et al. 1992; Yu et al. 1992). This CA was then found to be located in the carboxysome, a proteinaceous structure in the cytoplasm of the cyanobacterial cell containing the majority of Rubisco. This carboxysomal CA was identified, based on its amino acid sequence, as a protein belonging to the β-CA family. Later a homologue was described from another cyanobacterium, the closely related Synechocystis (So & Espie 1998). A member of the α-CA family was discovered in each of two different cyanobacteria by Soltes-Rak et al. (1997). Both Anabaena sp. strain PCC7120 and Synechococcus sp. strain PCC7942 were found to contain CA of the α-type and immunogold localization studies revealed that these CAs were located in the periplasmic space. Cyanobacteria also contain the γ-CA-like protein CcmM. Immunolocalization studies and physiological studies are consistent with a carboxysomal location for CcmM (Price et al. 1998). In conclusion, cyanobacteria have all three types of CA with the α-CA being periplasmic, the β-CA found in the carboxysome, and the γ-CA also associated with the carboxysome.

Chloroplast stroma

The most abundant form of CA in higher plants is the CA found in the chloroplast stroma of leaf mesophyll cells in C3 plants. This nuclear-encoded CA accounts for up to 2% of total leaf protein (Okabe et al. 1984). Although the chloroplast β-CA migrates as a hexamer in sieving gels, the X-ray structure of the pea chloroplast β-CA reveals an octamer (Kimber & Pai, 2000). The activity of this enzyme is almost as high as that in the human CAII isoform. However, unlike animal CAs, the plant enzyme is sensitive to oxidation, and reducing conditions in vitro are required to maintain the enzyme in its most active form (Johansson & Forsman 1993; Björkbacka et al. 1997). Monocot CAs have not been characterized using physical methods. A CA from Tradescantia leaves, presumably the chloroplast β-CA, appears to be a monomer or dimer when subjected to gel filtration (Atkins, Patterson & Graham 1972). The chloroplast β-CA from barley appears to be a dimer or trimer using the same method (M.H. Bracey and S.G. Bartlett, unpublished observation). Interestingly, all of the monocot CAs for which sequence information is available lack 12 carboxy-terminal residues which are conserved in all dicot CAs and play a role in the oligomerization of the pea protein (Kimber & Pai, 2000). Thus, it would appear that the smaller number of subunits in the monocot β-CA is due, at least in part, to lack of these carboxy-terminal residues, although it would be easy to imagine that loss of these carboxy-terminal residues would result in a dimeric or tetrameric monocot enzyme.

Additional evidence that the abundant β-type CA is located in the chloroplast comes from analyses of leader sequences. They all have features of cleavable leader sequences that target proteins to the chloroplast stroma (Fawcett et al. 1990; Forsman & Pilon 1995). Biochemical studies demonstrating the uptake of the precursor CA into isolated chloroplasts is further evidence for a chloroplastic localization (Forsman & Pilon 1995). It is likely that a similar β-CA will be found in chloroplasts of all higher C3-type plants.

Until recently it was assumed that the β-CA was the only CA in the chloroplast stroma of higher plants. However using an EST clone as a probe to screen an Arabidopsis thaliana library, a cDNA was obtained having a deduced amino acid sequence with significant sequence homology with low activity α-CAs from other organisms. This clone (Z18493) denoted ACAH1, represents the first α-CA from a higher plant (Susanne Larsson personal communication). Although the leader sequence of ACAH1 is atypical of chloroplast leader sequences, immunogold localization experiments point to a location in the chloroplast stroma for this 35 kDa CA. The C-terminus of ACAH1, which is rich in lysine might facilitate an association with membranes or with other proteins in the stroma.

Thylakoid membrane

There has been a debate about a possible thylakoid CA over a period of many years (see Stemler 1997). In 1995 Karlsson et al. described the purification of an intracellular CA from the unicellular green alga C. reinhardtii and partial sequencing of the polypeptide showed that it belongs to the α-CA family. The gene encoding this CA was cloned and sequenced and found to contain an N-terminal extension characteristic of a bipartite leader sequence. This information, together with biochemical evidence, indicated that the α-CA was located inside the thylakoid lumen (Karlsson et al. 1998). In addition, uptake of this α-CA into the thylakoid lumen was powered by the ΔpH across the thylakoid membrane as outlined by Robinson & Mant (1997). This α-CA, CAH3, is constitutively expressed but increases in abundance when C. reinhardtii cells are shifted from high CO2 to air growth conditions. CAH3 is a 30 kDa hydrophobic protein with a calculated pI of ~9·4. The fact that CAH3 is so hydrophobic and co-purifies with the thylakoid membrane fraction has led to the hypothesis that it is attached to the interior of the thylakoid membrane by hydrophobic interactions. CAH3 is not a membrane spanning protein as it can be released into the soluble fraction by washing the thylakoids with 200 mM KCl. Results by Park et al. (1999) indicate that CAH3 is associated with photosystem II (PSII) particles but this needs to be further confirmed. To date there is no firm data to support a thylakoid localization for CA in any other higher plant or alga (see also below under unresolved questions).


In higher animals, liver cells express a mitochondrial CA (CAVI) but no corresponding CA has been isolated from higher plants. However, Geraghty & Spalding (1996) showed that a 21 kDa polypeptide expressed in C. reinhardtii cells grown on low CO2 was exclusively located in the mitochondria. Eriksson et al. (1996) later identified this polypeptide as a β-CA. Two virtually identical genes encode this mitochondrial CA and the gene product is approximately 21 kDa in molecular size. The mitochondrial β-CA is a soluble protein but is peripherally associated with membranes in vitro (Geraghty & Spalding 1996). No other mitochondrial CAs have been reported in algae or higher plants.

Cytoplasmic CA.

Only a few publications report that a CA is located in the cytosol in a C3 plant. Fett & Coleman (1994) sequenced a clone, CAH2, from A. thaliana and postulated it to be located in the cytoplasm since the deduced amino acid sequence lacked a leader sequence. Later Rumeau et al. (1996) reported that potato leaves have CA activity in the cytosol (13% of total CA activity). This cytosolic CA is an octamer of 255 kDa and its presence in the cytosol was confirmed by immunocytolocalization experiments. The green unicellular alga Coccomyxa sp. was recently found to contain a cytosolic CA as indicated by activity measurements of various cell fractions (Hiltonen et al. 1998). This observation was confirmed by immunogold localization experiments with purified antibodies raised against the overexpressed protein (Hiltonen et al. personal communication). To date all cytosolic CAs reported in plants belong to the β-CA family. Presently these are the only documented CAs in the cytosol of photosynthetic cells.

Periplasmic CA

The model organism C. reinhardtii has two periplasmic α-CAs (Fukuzawa et al. 1990; Fujiwara et al. 1990; Rawat & Moroney 1991). The periplasmic CAs are encoded by two structurally very similar but differentially regulated genes, CAH1 and CAH2. CAH1 is the major periplasmic CA and its transcription is rapidly induced under low CO2 conditions whereas CAH2 is mainly expressed under high CO2 conditions (Fukuzawa et al. 1990; Fujiwara et al. 1990). Unlike the monomeric animal α-CAs, these are heterotetramers composed of two large subunits (35–37 kDa) and two small subunits (4 kDa) linked by disulphide bonds (Kamo et al. 1990). However the large and small subunits are encoded as a proprotein and cleaved in two places resulting in the two different types of subunits (Fujiwara et al. 1990). In addition to C. reinhardtii, extracellular CAs have been documented in D. salina (Fisher et al. 1996) and Chlorella sorokiniana (Satoh et al. 1998). Periplasmic CA activity has also been reported for a large number of other algae, both micro-algae and macro-algae, indicating that this localization may be very common in aquatic photosynthetic organisms.

Root nodules

A number of non-green plant tissues also appear to have CA activity based on the observed high rates of HCO3 consumption (Raven & Newman 1994). In 1974, Atkins detected CA activity in root nodules (Atkins 1974). Recently CA mRNAs were localized in the nodules of two legumes. For example, Coba de la Pena et al. (1997) showed that an α-carbonic anhydrase transcript is present in both spontaneously formed and in Rhizobium meliloti-induced root nodules of alfalfa. This transcript, Msca1, is expressed early in the nodule primordium. Later in nodule development Msca1 is found in the peripheral envelopes of cells in both developing and mature nodules. A similar α-CA cDNA was recently found in soybean nodules. This CA was found throughout young nodules but mainly in the cortical region of old nodules (Kavroulakis et al. 2000), suggesting that the role of CA early in nodule development is recycling of CO2, whereas later in development it is to facilitate diffusion of CO2 from the nodule.

Unresolved questions

In this review we have tried to describe the localization of CA in plants, algae and cyanobacteria. There is a vast literature on the localization of CA in various photosynthetic organisms and it is not possible to cover all this information in a meaningful way. Many previous studies have relied solely on CA assays for localization information. This was not unreasonable at the time as it was assumed that there was one or at most a few CAs in a given tissue or cell. However, in light of the large number of CA genes now documented, we feel that localization studies based on activity alone are equivocal. We therefore chose to summarize the best data about CA localization in tissues, cells and subcellular compartment, limiting our review to proteins that have been immunolocalized to specific organelles or tissues. To clearly localize a specific CA in the future researchers will need to rely on immunolocalization techniques in conjunction with sequence information. In most cases identifying a polypeptide as a CA belonging to any of the known CA gene families is relatively easy as many active site residues are conserved from bacteria to vertebrates. The sequence can also provide localization information as more and more leader sequences are recognized. We consider strong evidence for proposing a specific localization to be a combination of sequence data various immunological techniques and optimally, CA activity data.

CA localization in two cellular compartments, the periplasmic space and the thylakoid lumen, has been frequently discussed in the literature, but rarely proven. One such compartment is in the periplasm of various macro-algae including green, red and brown algae (Larsson & Axelsson 1999). Most of the evidence for a periplasmic CA is based on physiological experiments using various CA inhibitors. In these studies, the effect of acetazolamide (AZ) on photosynthesis is investigated based on the assumption that AZ penetrates the cell very slowly (Geib, Golldack & Gimmler 1996) so that the only CA inhibited is on the cell surface. If photosynthesis is inhibited by this treatment then a periplasmic CA is thought to play a role in Ci uptake in the macro-alga being investigated. Although we agree that the data support the notion that a CA might be located at the exterior of the cells, we would like to argue that this is not enough to state that a CA really is located there. There remains the possibility that some AZ will leak into the cell, inhibiting a particularly susceptible CA isoform required for CO2 fixation. In our opinion use of impermeant sulphonamides, such as dextran-bound sulphonamide (Moroney, Husic & Tolbert 1985) is a better choice for these types of studies. The current controversy about the physiological role of the periplasmic CA in C. reinhardtii underscores this problem. AZ clearly inhibits photosynthesis at high external pH and low inorganic carbon concentrations in C. reinhardtii (Moroney et al. 1985), yet a C. reinhardtii mutant lacking the periplasmic CAH1 protein appears to grow normally (Van & Spalding 1999). This controversy will be discussed more in the next section.

Another question that still needs to be resolved is whether higher plants contain a CA localized to the thylakoid lumen like the one found in C. reinhardtii. Evidence for a higher plant thylakoid associated CA is mainly based on measurements of CA activity using isolated thylakoids or PSII preparations (Ignatova et al. 1998; for a review see also Stemler 1997). Although CA activity can be detected in isolated PSII-enriched membranes from higher plants, the critical experiments showing that this activity is not due to a contamination by stromal CA still need to be done. Recent experiments demonstrating an association between the lumenal α-CA and PSII in C. reinhardtii (Park et al. 1999) have led to a renewed discussion of existence of a similar α-CA in higher plants.


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Cyanobacteria possess, as do many micro-algae, a carbon concentrating mechanism (CCM). Inorganic carbon (Ci) is accumulated inside the cyanobacterial cells under conditions when the concentration of Ci in the medium is low. Inside the cyanobacterial cell, the carboxylating enzyme Rubisco is located in a proteinous body called the carboxysome. Ci is presumably accumulated in the cytoplasm in the form of HCO3; and to speed up the formation of CO2 from HCO3 in the vicinity of Rubisco, a CA is required. Calculations show that an elevated level of CO2 can occur in the carboxysome when cyanobacterial cells accumulate Ci (Reinhold, Kosloff & Kaplan 1991; Fridlyand, Kaplan & Reinhold 1996). Price, Coleman & Badger (1992), Fukuzawa et al. (1992) and So & Espie (1998) were able to identify a β-CA in the carboxysome of the cyanobacteria Synechococcus and Synechocystis in a screen of high CO2-requiring mutants. Clearly, mutants having defective or missing carboxysomal CA can no longer grow on limiting CO2. Paradoxically, these cells can still accumulate Ci to high levels. These observations support the notion that the carboxysomal CA converts accumulated HCO3 to CO2 for Rubisco in the carboxysome. Cells without the carboxysomal CA still accumulate HCO3 but cannot rapidly convert the HCO3 to CO2 for fixation.

The location of the carboxysomal CA is considered essential to its function. Price & Badger (1989b) postulated that cytosolic CA activity might short-circuit the Ci accumulation step allowing CO2 to leak from the cell before being fixed. This was tested by Price & Badger (1989a) who expressed a human CA in the cytosol of Synechococcus and found that it indeed short-circuited CCM by increasing leakage of CO2 out of the cells.

The function of the periplasmic α-CAs identified in Anabaena and Synechococcus (Soltes-Rak et al. 1997) is still unclear but it is likely that its function resembles that of micro-algal periplasmic CAs which is to facilitate the diffusion of CO2 across the plasma membrane. The physiological role of the γ-CA analog CcmM is not known. However, cells deleted in CcmM require high CO2 for optimal growth and no longer make functional carboxysomes. In fact CcmM cells have empty carboxysomes. From these results it is clear that CcmM is required for correct carboxysome assembly and for optimal growth on low levels of CO2. However, it is not clear whether CcmM has carbonic anhydrase activity or whether its enzymatic activity is required for correct carboxysome assembly.


There are multiple isoforms of CA even in unicellular micro-algae. In micro-algae, the expression of CA is influenced by the environmental level of CO2 and in some cases the presence of alternate carbon sources (Villarejo, Orus & Martinez 1997). The eukaryotic alga in which the function of CAs has been most extensively studied is the green micro-alga C. reinhardtii. C. reinhardtii contains at least five genes coding for CAs, 3 α-CAs and two β-CAs. Two of the α-CA proteins are located in the periplasmic space (Fujiwara et al. 1990; Rawat & Moroney 1991), and one α-CA is located in the thylakoid lumen (Karlsson et al. 1998). The two β-CA proteins are almost identical and are located in the mitochondrial matrix (Eriksson et al. 1996; Geraghty & Spalding 1996).

It has been proposed that the function of the two periplasmic α-CAs is to facilitate the diffusion of CO2 across the plasma membrane. One of them, the periplasmic CA CAH1, is strongly induced by air growth conditions and it has therefore been postulated to be part of the CCM in C. reinhardtii. It is thought that this CA speeds up the equilibrium between HCO3 and CO2 so that CO2 at the cell surface can diffuse across the plasma membrane. The role of the periplasmic CA is to replenish the external CO2 from the HCO3 supply as CO2 enters the cell. In support of this idea membrane impermeant carbonic anhydrase inhibitors have a strong inhibitory effect on CO2 fixation at high external pH but a less pronounced effect at low external pH (Moroney et al. 1985). There is also a strong correlation between the presence of the CCM and the expression of CAH1. In C. reinhardtii there is a second periplasmic CA, CAH2. The function of CAH2 is obscure. CAH2 is never expressed at high levels and is not expressed when the CCM is functional.

The α-CA of the thylakoid lumen, CAH3, is required for growth of C reinhardtii in air levels of CO2 (Funke, Kovar & Weeks 1997; Karlsson et al. 1998). Complementation of a mutant (Cia3) that cannot grow under low CO2 with the wild-type gene restores the ability to grow with air as carbon source. The function of this PSII-associated CA (Park et al. 1999) was analysed by Pronina & Semenenko (1990) and Pronina & Borodin (1993) and later by Raven (1997). The model presented by Raven (1997) proposes that CAH3 speeds up formation of CO2 from HCO3 in the acidic lumen and that this CO2 diffuses through the thylakoid membrane to the pyrenoid, partly surrounding the thylakoid membranes, where it is carboxylated by Rubisco. The model is based on the assumption that HCO3 is actively pumped into the lumen from the stroma. The results by Park et al. (1999) support this model although it cannot be excluded that CAH3 has other functions more directly involved in PSII photochemistry.

Interestingly C. reinhardtii also contains a mitochondrial CA encoded by two genes with only one amino acid difference in the coding region (Eriksson et al. 1996). The polypeptide denoted LIP 21 (Geraghty & Spalding 1996) is not synthesized under high CO2 but is strongly induced when the CO2 concentration is decreased to air levels. For the same reason as for the periplasmic CAs it has been assumed that the mitochondrial CA is important to the acclimation of algae cells to low CO2 conditions. Eriksson et al. (1998) suggested that one function of the mitochondrial CAs could be to buffer the mitochondrial matrix by increasing the rate at which photorespiratory NH3 is converted to NH4+. Alternatively, Raven recently postulated that the function of the mitochondrial CA is to decrease leakage of inorganic carbon back into the medium. His quantitative analyses show that a 10% increase in CO2 supply to Rubisco occurs at a cost of less than 1% of total mitochondrial and plastid generated ATP, granted that his other assumptions are correct (J. A. Raven personal communication).

C3 plants

The function of the chloroplast β-CA in higher plants has been the subject of several studies over the years. Potential roles range from modulation of pH of the stroma to facilitating diffusion of CO2 across the chloroplast envelope. Another proposed role of the β-CA is to replenish the CO2 supply in the chloroplast stroma from HCO3, which is present in almost 100 times the concentration of CO2 in the alkaline stromal compartment. To study the function of the chloroplast β-CA, two groups have made transgenic plants containing antisense CA constructs. The leaves of tobacco plants containing the antisense β-CA gene have only about 10% or less of the CA activity of wild-type plants. One group reported no deleterious effects of the reduction in CA activity (Price et al. 1994) and a second group reported that the antisense plants compensated for the decrease in CA with an increase in stomatal conductance leading to an increase in water loss (Majeau, Arnoldo & Coleman 1994). The reasons for the increased drought sensitivity are unclear although it is possible that a reduction in photosynthesis due to a decrease in the rate of delivery of CO2 to Rubisco caused the leaf stomata to remain open. Another possibility is that lowering the chloroplast CA activity somehow results in a disruption of the signal for the plant to close its stomata under certain conditions. Finally, it is possible that the chloroplast β-CA played a more critical role in photosynthesis during the early expansion of land plants in the Cretaceous era when CO2 concentrations were lower than they are today. The increase of CO2 in the atmosphere may have rendered the chloroplast β-CA expendable except during times when CO2 is limiting.

C4 plants

In C4 plants, the most abundant β-CA is localized in the cytosol of mesophyll cells (Ku, Kano-Murakami & Matsuoka 1996). The mesophyll CA is thought to play a crucial role in C4 photosynthesis by providing HCO3 for PEP carboxylase (Hatch & Burnell 1990). In fact, the presence of CA in C4 plants has been suggested to accelerate the rate of photosynthesis in C4 plants 104-fold over what it would be if this enzyme were absent (Badger & Price 1994). Furthermore, it was postulated that CA must be excluded from the bundle sheath cells to avoid loss of accumulated CO2 due to its conversion to HCO3 and leakage of the latter back to mesophyll cells through plasmodesmata (Burnell & Hatch 1988). However, results of later modelling studies indicated that effects of CA in the bundle sheath would be less severe (Jenkins, Furbank & Hatch 1989).

Results of recent experiments support the view of Jenkins et al. (1989). Ludwig et al. (1998) transformed the NADP-ME-type C4 dicot Flaveria bidentis with a construct encoding the tobacco chloroplast CA lacking a transit sequence. Expression of the construct was driven by the β-glucuronidase (GUS) promoter so that CA would be expressed in the cytosol of both mesophyll and bundle sheath cells. One line of transgenic plants had 50% more CA activity in whole leaves and five times more CA activity in bundle sheath cells than control plants when CA activity was measured on a Rubisco-site basis. The transgenic plants also had increased C isotope discrimination and decreased photosynthesis at high concentrations of oxygen compared with control plants, consistent with the notion that HCO3 was leaking from the bundle sheath cells. However, in spite of these differences, the plants were phenotypically normal. It is possible that transgenic plants expressing higher levels of the tobacco CA in the bundle sheath cells died during selection. On the other hand, it is also possible that it is the abundance of CA in the cytosol of mesophyll cells, rather than its absence from bundle sheath cells that is crucial for C4 photosynthesis.

Unresolved questions

Chloroplast stroma β-CA

To date antisense constructs developed in tobacco suggest that the chloroplast stroma β-CA in higher plants plays a minimal role, if any, in photosynthesis (Majeau et al. 1994; Price et al. 1994). Observations using Arabidopsis suggest that this question needs to be investigated further. The antisense approach was also used to reduce CA activity in Arabidopsis leaves (H.-J. Kim and S.G. Bartlett, in preparation). As was observed with the antisense tobacco plants, no phenotype was evident when seedlings were grown in soil, supporting the notion that the chloroplast CA has little effect on photosynthesis in C3 plants. However, when the transgenic Arabidopsis seedlings were grown on agar, the antisense plants died whereas control plants were normal. These results suggest that the chloroplast β-CA is required at least when levels of carbon dioxide are limiting.

So far the antisense approach has resulted in plants with somewhat less than 10% of the wild-type level of CA activity. What would happen if all of the stroma CA activity were eliminated? Experimentally it may be difficult to reduce the activity completely if there is more than one isoform of CA in the chloroplast stroma, particularly if the genes encoding CAs are regulated in such a way that other isoforms are up-regulated when expression of the major CA is suppressed. It also may be difficult to select for transgenic plants with very low CA activity if these low activity plants germinate poorly or are very sickly.

Thylakoid CA

Although the evidence for a thylakoid-associated CA in C. reinhardtii is strong, the question whether there is a thylakoid CA in higher plants remains controversial. A number of groups have argued for the presence of a thylakoid CA. The evidence for a CA associated with the thylakoid membranes in higher plants is based on sensitive assays of CA activity in a variety of thylakoid membrane preparations. Groups from Russia and Bulgaria detected CA activity in thylakoid membranes more than 20 years ago (for a review see Stemler 1997; and references therein). The recent discovery of a lumenal α-CA in C. reinhardtii by Karlsson et al. (1998) has renewed interest in the search for a corresponding CA in higher plant thylakoids. Recently, groups in both Russia and USA (see Stemler 1997) have been able to measure CA activity in both maize and pea thylakoids and in PSII preparations. One problem with this type of measurement is that the CA activity measured could originate from contamination by the abundant stromal CA. However, experiments by Shutova (personal communication) showed that polypeptides from pea PSII membrane preparations did not cross-react with antibodies raised against the pea stromal CA in Western blots indicating that there is a CA activity from a novel CA associated with thylakoid membranes.

It has also been postulated that a thylakoid CA associated with PSII might play a role in the primary photosynthetic reactions. Swader & Jacobson (1972) reported that high concentrations of AZ inhibited electron transport in thylakoids. Several more recent publications have indicated an involvement of HCO3 in donor side reactions of PSII (Klimov et al. 1995; Allakhverdiev et al. 1997). There are further indications that this thylakoid CA may be regu-lated in some unknown way by light and redox levels (Moubarak-Milad & Stemler 1994; Moskvin, Ovchinnikova & Ivanov 1996). If it can be finally proved that a novel CA is involved in the PSII reactions it reasonable to suggest that it either is involved in supplying HCO3 as a ligand in the oxygen-evolving complex, or that it is involved in the proton-buffering capacity around PSII.

There is also evidence against a higher plant thylakoid CA. First it can be argued that in higher C3 plants lacking an active transport of inorganic carbon, there is no need for such a thylakoid associated CA. There is also no evidence for an α-CA in Synechocystis 6803, a cyanobacteria with functional PSII. In addition, others have found no CA activity in PSII core preparations from spinach and CA inhibitors have no effect on O2 evolution in PSII preparations (Bricker and Moroney, unpublished observations). However the discovery of six α-CAs in the Arabidopsis genome leaves open the possibility that one of those genes encodes a thylakoid CA required for activation or maintenance of PSII. Despite the somewhat scattered information available today it seem worthwhile to continue to work on this subject until a clearer picture emerges.

Periplasmic CA

The role of the periplasmic CA of green algae has long been thought to aid in the diffusion of CO2 across the plasma membrane (Raven 1997). Presumably, as CO2 enters the cell the external CO2 pool is replenished from HCO3 outside the cell through the action of the periplasmic CA. This role was thought to be especially important in alkaline aquatic environments where HCO3 predominates. Support for this role comes mainly from inhibitor studies contrasting the effects of acetazolamide (AZ) and ethoxyzolamide (EZ). In these studies AZ was utilized as an impermeant CA inhibitor and EZ as the membrane permeable inhibitor. Moroney et al. (1985) demonstrated that AZ caused a strong inhibition of photosynthesis in C. reinhardtii at high external pH, but did not inhibit photosynthesis at acidic external pH. In contrast, EZ inhibited photosynthesis at both alkaline and acidic external pH. A similar pattern has been observed in a number of micro-algae and macro-algae. The interpretation of these studies was that AZ only inhibited photosynthesis at high external pH where the conversion of HCO3 to CO2 is limiting the entry of CO2 to the cell. In contrast, EZ was thought to inhibit under all conditions because it enters the cell and inhibits an internal CA. It appears from mutant studies that the internal CA inhibited by EZ is the thylakoid CAH3 (Karlsson et al. 1998).

Recent studies have cast some doubt on these earlier conclusions. Van & Spalding (1999) reported that C. reinhardtii cells with a defective CAH1 gene grew as well as wild-type cells on low CO2 concentrations. Their data would imply that the periplasmic CA is not required for optimal diffusion of CO2 across the plasma membrane. There are a number of explanations for the different results obtained with inhibitors and mutants. The first is that most of Van and Spalding's growth and photosynthesis data were obtained at external pH close to neutral (Van & Spalding 1999). Perhaps the growth of the mutant would be impaired under more alkaline conditions. The presence of the CAH2 protein also confounds a clean interpretation of the mutant data. Although the expression of Cah2 is low, there may still be enough periplasmic CA to fulfill the physiological role. In this case the CAH2 protein would be functionally complimenting the Cah1 defect. The inhibitor AZ would inhibit both CA isoforms and the physiological effect would be more obvious. Another possibility is that the Chlamydomonas can adjust its CO2 and HCO3 uptake systems to compensate for the reduced periplasmic CA. Additional studies will be needed to clarify the role of the periplasmic CA.

Future directions

There has been a resurgence of interest in CAs from plants and algae over the past decade. This interest began with the discovery of a second major class of CA in plants in 1990, the β-CAs, and has continued with the finding of multiple α- and β-CAs in C. reinhardtii and A. thaliana and the determination of the critical physiological roles CAs have in cyanobacteria and macro-algae. These CAs have been found in many intracellular locations and tissues. With the completion of the Arabidopsis genome project we will soon know exactly how many isoforms of CA there are in a higher plant. The challenge for future CA researchers will be to determine the expression patterns, intracellular location and physiological roles for each of these isoforms. As there appears to be a large number of isoforms in plants and algae, CA researchers should be busy during the next few years.


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