Download Carbonic anhydrases in plants and algae

Plant, Cell and Environment (2001) 24, 141–153
INVITED REVIEW
Carbonic anhydrases in plants and algae
J. V. MORONEY,1 S. G. BARTLETT1 & G. SAMUELSSON2
1
Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803 USA and 2Umeå Plant Science
Centre, Department of Plant Physiology, Umeå University, S-901 87, Umeå, Sweden
ABSTRACT
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
a-, b-and g-CA. Animals have only the a-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.
Key-words: Arabidopsis; Chlamydomonas; cyanobacteria;
macro-algae; photosynthesis.
INTRODUCTION
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 (HewettEmmett & 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
Correspondence: Göran Samuelsson. Fax: 090 786 66 76; e-mail:
[email protected]
© 2001 Blackwell Science Ltd
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 a-CA, b-CA, and g-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
a-CA and b-CA and the CcmM protein that bears strong
similarity to g-CAs. Examples of a-CA and b-CA are
known in Chlamydomonas reinhardtii (Hewett-Emmett &
Tashian 1996; Samuelsson & Karlsson, 2000). However, in
animals only the a-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.
CO2 + H2O ´ HCO3 - + H + ´ CO3 -2 + 2H +
(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 ª 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
141
142 J. V. Moroney et al.
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).
STRUCTURAL COMPARISON OF
CARBONIC ANHYDRASE GENE FAMILIES
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 a-, b-and g-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.
a-Carbonic anhydrases
The a-type is the most studied CA and is very widely distributed. a-CAs have been found in animals, plants, eubacteria and viruses. Although a-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 a-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
a-CAs. The a-CA structure is dominated by antiparallel bsheets forming a spherical molecule with two halves. The
active site is a funnel-shaped crater with the zinc atom
located near the bottom. Most a-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 a-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 a-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 a-CAs have also been found in eubacteria, algae and
higher plants. In photosynthetic organisms only a few a-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 a-CAs. In C. reinhardtii, two of the a-CAs
are localized to the periplasmic space and one to the thylakoid membrane. Furthermore, the sequence of a novel
a-CA cDNA from A. thaliana was recently reported (accession no. U73462, Susanne Larsson, personal communication). All of the a-CAs observed to date in plants appear
to be soluble proteins although they are localized to distinct
cellular compartments.
b -Carbonic anhydrases
The distribution of b-CAs does not appear to be as broad
as the a-CAs at this time. b-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 b-CAs have been found and both appear to be
localized to mitochondria (Eriksson et al. 1996). In C3
plants the abundant b-CA localized to the chloroplast
stroma is usually the CA isoform with the highest total
activity in the plant. b-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 b-CAs are quite different to those of
a-CAs. X-ray absorption spectroscopy (EXAFS) studies on
spinach b-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
b-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
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
Carbonic anhydrases in plants and algae 143
CO2 residing in a hydrophobic pocket is required for activity is underscored by the recent comparison between the aCA and b-CA crystal structures (Kimber & Pai, 2000). It
turns out that amino acids in the active site of the b-CA are
a mirror image of the amino acids in the active site of the
a-CA hydrophobic pocket (Kimber & Pai, 2000).
g -Carbonic anhydrases
A third type of CA, the g-CA, was discovered in the archaebacterium Methanosarcina thermophila (Alber & Ferry
1994). Genes encoding putative proteins with sequences
similar to g-CA have been found in eubacteria and plants
(Newman et al. 1994). The g-CA from M. thermophila was
crystallized and its structure solved (Kisker et al. 1996). The
structure of g-CA is strikingly different from either the aCA or b-CA. The g-CA functions as a trimer of identical
subunits. The structure of each monomer is dominated by
a left-handed b-helix (Kisker et al. 1996). The trimer contains three Zn atoms, one each at the three subunit interfaces. As in the a-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 g-CA is similar to that of
a-CA (Kisker et al. 1996).
A g-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 g-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
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
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 d-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 Znlimiting 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.
NUMBER AND DISTRIBUTION OF GENES
a-Carbonic anhydrases
The first a-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. reinhardtii a-CA was discovered in 1995
(Karlsson et al. 1995). Later Karlsson et al. (1998) published
the sequence of the cDNA encoding this intracellular aCA, 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 a-CA has been found in any given
cyanobacterium. An a-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 a-CAs.
It is clear that a number of a-CAs remain to be identified in higher plants. As of September 2000, the Arabidopsis database contained sequences representing six different
genes that align with a-CAs (Table 1). In contrast, no
sequences in the Synechocystis 6803 genome align strongly
with a-CAs. To our knowledge only one a-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
144 J. V. Moroney et al.
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
family
Gene
number
cDNA
accession
EST accession
Genomic accession
Chromosome
location
References
a
1
U73462
AV442219
Z18493
AV441118
AL353912
III
Larsson et al. 1997
a
2
T45411
A1992753
AC006202
II
a
3,4
AL161554
IV
a
5,6
AC026875
I
b
1
X65541
AV440467
AV442587
AV441815
T42977
AC009325
III
Raines et al. 1992
b
2
L18901
AV442315
N95942
N96451
AL391149
V
Fett and Coleman 1994
b
3
AA597643
T45390
T43963
AA597734
AC003671
I
b
4
Z235745
AC005990
I
b
5
AA598084
AL031394
IV
g
1
T04294
AC024609
I
g
2
AV564979
BE524908
AC074226
?
g
3
AA72011
AI992681
AB013389
V
the Arabidopsis database do align with some of the other
a-CAs identified by the genome project, implying that
these are functional genes.
b-Carbonic anhydrases
b-CAs were first recognized to be carbonic anhydrases in
photosynthetic organisms (Burnell, Gibbs & Mason 1990;
Fawcett et al. 1990). Once recognized as a CA, b-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 b-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 b-CA
leads to a high CO2-requiring phenotype (Price & Badger
1989b). The Synechocystis 6803 genome contains no other
b-CA, indicating that the carboxysomal b-CA is the only bCA in at least this cyanobacterium. Two b-CAs have been
identified in the micro-alga, C. reinhardtii (Eriksson et al.
1996). Both b-CAs in C. reinhardtii are localized to the
mitochondria. In addition, expression of the b-CAs in C.
reinhardtii is strongly influenced by the CO2 concentration
(Eriksson et al. 1998). Under elevated CO2 conditions very
little b-CA mRNA is made but under low CO2 the mRNA
is quite abundant. b-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 b-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
b-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 b-CA. In A. thaliana cDNAs encoding cytosolic and
chloroplastic forms of b-CA have been described (Fett &
Coleman 1994). At this time sequences encoding at least
five b-CA genes from A. thaliana are in the database
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
Carbonic anhydrases in plants and algae 145
(Table 1), and it is possible that other b-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 b-CAs. Interestingly,
conceptual translation of the two CA cDNAs for maize bCA 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 bCAs 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).
g -Carbonic anhydrases
Several Arabidopsis ESTs in the databases have homology
with the g-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 g carbonic anhydrase homologues (Table1). The
Chlamydomonas EST database also has an EST that aligns
well with the M. thermophila g-CA. Cyanobacteria have the
CcmM gene that encodes a protein with an N-terminus that
has homology with M. thermophila g-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 bac© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
teria 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 a-CAs and two
b-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 b-CA and g-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.
LOCALIZATION
General
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 a-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.
146 J. V. Moroney et al.
Established locations of CA in photosynthetic
organisms
Cyanobacteria
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 b-CA family. Later a homologue
was described from another cyanobacterium, the closely
related Synechocystis (So & Espie 1998). A member of the
a-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 a-type and
immunogold localization studies revealed that these CAs
were located in the periplasmic space. Cyanobacteria also
contain the g-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 a-CA being periplasmic, the b-CA found in the
carboxysome, and the g-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 b-CA migrates as a hexamer in sieving gels, the
X-ray structure of the pea chloroplast b-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 b-CA,
appears to be a monomer or dimer when subjected to gel
filtration (Atkins, Patterson & Graham 1972). The chloroplast b-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 b-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 b-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 b-CA will be found in chloroplasts of all higher
C3-type plants.
Until recently it was assumed that the b-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 a-CAs from other organisms. This clone
(Z18493) denoted ACAH1, represents the first a-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
a-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
a-CA was located inside the thylakoid lumen (Karlsson
et al. 1998). In addition, uptake of this a-CA into the thylakoid lumen was powered by the DpH across the thylakoid
membrane as outlined by Robinson & Mant (1997). This aCA, 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 localiza© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
Carbonic anhydrases in plants and algae 147
tion for CA in any other higher plant or alga (see also below
under unresolved questions).
Mitochondria
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 b-CA.Two virtually identical genes encode
this mitochondrial CA and the gene product is approximately 21 kDa in molecular size. The mitochondrial b-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 b-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 aCAs (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 a-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
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
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 HCO3consumption (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 a-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 a-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 macroalgae including green, red and brown algae (Larsson &
148 J. V. Moroney et al.
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 a-CA and PSII in C. reinhardtii (Park et al.
1999) have led to a renewed discussion of existence of a
similar a-CA in higher plants.
PHYSIOLOGICAL ROLES OF CARBONIC
ANHYDRASES
Cyanobacteria
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 b-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 a-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 g-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.
Micro-algae
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 a-CAs and two b-CAs. Two of the
a-CA proteins are located in the periplasmic space
(Fujiwara et al. 1990; Rawat & Moroney 1991), and one aCA is located in the thylakoid lumen (Karlsson et al. 1998).
The two b-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 a-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 equi© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
Carbonic anhydrases in plants and algae 149
librium 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 a-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 b-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.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
Another proposed role of the b-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 b-CA, two groups have made transgenic plants
containing antisense CA constructs. The leaves of tobacco
plants containing the antisense b-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 b-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 b-CA
expendable except during times when CO2 is limiting.
C4 plants
In C4 plants, the most abundant b-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 bglucuronidase (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
150 J. V. Moroney et al.
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.
To date antisense constructs developed in tobacco suggest
that the chloroplast stroma b-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 b-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.
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 regulated 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 a-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 a-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.
Thylakoid CA
Periplasmic 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 a-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
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 HCO3outside 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
Unresolved questions
Chloroplast stroma b-CA
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
Carbonic anhydrases in plants and algae 151
photosynthesis at both alkaline and acidic external pH. A
similar pattern has been observed in a number of microalgae 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 b-CAs, and has continued with the finding of multiple
a- and b-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.
ACKNOWLEDGMENTS
This work was funded in part by National Science
Foundation grant IBN-9904425 to J.V.M.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 141–153
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Received 6 July 2000; received in revised form 25 September 2000;
accepted for publication 25 September 2000