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Plant Physiol. (1999) 120: 757-764
Periplasmic Carbonic Anhydrase Structural Gene (Cah1)
Mutant in Chlamydomonas reinhardtii 1
Kyujung Van and
Martin H. Spalding*
Interdepartmental Plant Physiology Major and Department of Botany,
353 Bessey Hall, Iowa State University, Ames, Iowa 50011
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ABSTRACT |
To survive in various conditions of
CO2 availability, Chlamydomonas reinhardtii
shows adaptive changes, such as induction of a
CO2-concentrating mechanism, changes in cell organization, and induction of several genes, including a periplasmic carbonic anhydrase (pCA1) encoded by Cah1. Among a collection of
insertionally generated mutants, a mutant has been isolated that showed
no pCA1 protein and no Cah1 mRNA. This mutant strain,
designated cah1-1, has been confirmed to
have a disruption in the Cah1 gene caused by a single
Arg7 insert. The most interesting feature of
cah1-1 is its lack of any significant
growth phenotype. There is no major difference in growth or
photosynthesis between the wild type and cah1-1 over a pH range from 5.0 to 9.0 even though this mutant apparently lacks Cah1 expression
in air. Although the presence of pCA1 apparently gives some minor
benefit at very low CO2 concentrations, the characteristics
of this Cah1 null mutant demonstrate that pCA1 is not
essential for function of the CO2-concentrating mechanism or for growth of C. reinhardtii at limiting
CO2 concentrations.
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INTRODUCTION |
Aquatic and soil-borne photosynthetic organisms, including
Chlamydomonas reinhardtii, live in quite variable conditions
of CO2 availability. To survive in limiting
CO2 conditions, C. reinhardtii and
other microalgae show adaptive changes, such as induction of a CCM
(Badger et al., 1980 ; for review, see Spalding, 1998), changes in cell
organization (Geraghty and Spalding, 1996), increased photorespiratory
enzyme activity (Marek and Spalding, 1991), induction of pCA1
(Cah1) (Fujiwara et al., 1990; Fukuzawa et al., 1990; Ishida
et al., 1993), mitochondrial CA (Mca1 and Mca2)
(Eriksson et al., 1996; Geraghty and Spalding, 1996), and Ccp
(Ccp1 and Ccp2) (Geraghty et al., 1990; Ramazanov
et al., 1993; Chen et al., 1997), and transient down-regulation in the
synthesis of Rubisco (Coleman and Grossman, 1984; Winder et al., 1992).
Of these the CCM is the most studied adaptive change (for review, see
Spalding, 1998), because it affects photosynthetic characteristics through increasing intracellular CO2
concentration. So far, at least Ci transport
(Badger et al., 1980; Spalding et al., 1983b) and a thylakoid CA
(Cah3) to supply CO2 for Rubisco by
dehydration of HCO3 (Spalding
et al., 1983a; Funke et al., 1997; Karlsson et al., 1998) have been
shown to be required for operation of the CCM. Characteristics
resulting from the CCM include a high apparent affinity for
CO2, a low CO2 compensation
point, and reduced photorespiration due to a high ratio of
CO2 to O2. It is not clear
yet whether the induced genes (Cah1, Mca1,
Mca2, Ccp1, and Ccp2) are required for
function of the CCM.
The Cah1 gene product, pCA1, is a Zn-containing
metalloenzyme that catalyzes the interconversion of
CO2 and
HCO3 in the cell wall space of
C. reinhardtii (Badger and Price, 1994). There are two
periplasmic CA isozymes: pCA1, which is induced by limiting
CO2, and pCA2, which is the Cah2 gene
product and is expressed only at very low abundance and only under
elevated CO2 (Fujiwara et al., 1990; Rawat and
Moroney, 1991). pCA1 has been considered a candidate for involvement in
the CCM because it is the most abundant gene product induced by
limiting CO2 in C. reinhardtii.
However, evidence for any function of pCA1 in the CCM has been
contradictory, with Moroney et al. (1985) arguing that pCA1 is required
to supply CO2 from
HCO3 for rapid photosynthesis
at low CO2 concentrations and alkaline pH, and
Williams and Turpin (1987) disagreeing. Among a collection of
insertionally generated mutants, we found a mutant, named
cah1-1, that showed no pCA1 protein and no
Cah1 mRNA. Here we report on the photosynthetic
characteristics of this Cah1 null mutant.
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MATERIALS AND METHODS |
Cell Strains, Culture Conditions, and Mating
All Chlamydomonas reinhardtii strains (Table
I) were grown as previously described
(Geraghty et al., 1990). Cells were cultured on an orbital shaker under
aeration with 5% CO2 in air
(CO2-enriched cells) or no aeration (air-adapted
cells). Cell cultures were switched from elevated
CO2 to limiting CO2 (no
aeration) for 1 or 2 d of induction. All matings were performed by
crossing PCA57-61 with CC1068 (Table I) according to the protocol of
Harris (1989).
Generation and Isolation of Mutants
Using glass bead transformation (Kindle, 1990; Davies et al.,
1994), CC425 (Table I) was transformed with pArg7.8 (Debuchy et al.,
1989) to generate a pool of insertional mutants on
CO2 minimal medium that lacked Arg. This plasmid
has the structural gene (Arg7) for argininosuccinate lyase
to complement the arg2 mutation. Each of more than 7000 colonies was suspended with air minimal medium in a 1.5-mL
microcentrifuge tube and grown with shaking but no aeration for 2 d. After centrifugation the supernatant of each culture was screened
for a lack of pCA1 expression using a slot blot and immunodetection, as
described for western blots. Mutants identified in this primary screen
as having reduced pCA1 expression were screened again by western
immunoblots, following SDS-PAGE of extracellular protein from larger
scale cultures.
Protein Extracts, SDS-PAGE, and Western Immunoblots
Cell cultures exposed to limiting CO2 (no
aeration) for 2 d were harvested by centrifugation with a GSA
rotor (5,000 rpm, 15 min, Sorvall). The supernatants were precipitated
with
(NH4)2SO4 overnight, and the precipitated proteins were collected by
centrifugation with a GSA rotor (10,000 rpm, 30 min), dissolved in 1×
resolving gel buffer, and then desalted using spin columns (1-mL
disposable syringe, Becton Dickinson) of Sephadex G-25. Protein samples
were separated by SDS-PAGE with 12% polyacrylamide using the buffer system of Laemmli (1970). Separated proteins were electrophoretically transferred to nitrocellulose with a semi-dry blotting unit (Fisher Biotech, Itasca, IL) and immunodetected using affinity-purified anti-pCA1 polyclonal antiserum as the primary antibody (Roberts and
Spalding, 1995), horseradish peroxidase-linked second antibody, and
aminoethylcarbazole as the chromogenic substrate (Harlow and Lane,
1988).
Nucleic Acid Extractions and Analysis
Genomic DNA was isolated using Elu-Quik DNA purification kits
(Schleicher & Schuell) and digested with appropriate restriction enzymes. Total RNA was prepared (Chomczynski and Sacchi, 1987) from
induced cells exposed to limiting CO2 for 1 or
2 d. Nucleic acids (10 µg) were separated in 0.8% agarose gels
for Southern analysis or in 0.66 M formaldehyde-1.5%
agarose gels for northern analysis, and then blotted onto nylon
transfer membranes (Micron Separations Inc., Westborough, MA). After UV
cross-linking, membranes were hybridized with appropriate
32P-labeled probes (Random Primers DNA Labeling
System, Life Technologies). Southern and northern analyses were
performed according to the method of Ausubel et al. (1989).
Growth Tests and Photosynthetic O2 Exchange
For spot growth tests, active growing cells were suspended to
similar cell densities with minimal medium, spotted (10 µL) onto agar
plates with different pHs, and kept at an air level of
CO2 for 10 d (Harris, 1989). Each medium was
buffered with 25 mM Mes-KOH for pH 6.0, 25 mM Mops-KOH for pH 7.0, 25 mM Hepps (4-2 [-hydroxyethyl]-1-piperazine propane sulfonic acid)-KOH for pH 8.0 and 25 mM Ampso (3-(N-[1,1-dimethyl
hydroxyethyl] amino)-2-hydroxypropane sulfonic acid)-KOH for pH 9.0.
For growth-curve tests, active, fully air-adapted cells were inoculated
into different pH liquid minimal medium and buffered as described
above. The cell density was determined by using a hemacytometer
(Reichert Scientific Instruments, Buffalo, NY) (Harris, 1989).
For O2-exchange measurements, 1-d-induced cells
were harvested by centrifugation and resuspended in 25 mM
Mops-KOH (pH 7.0) for analysis of the response to
CO2 concentration or in 25 mM of the
appropriate buffer for analysis of the effects of pH. Suspended cells
(1 mL) were added to an O2 electrode (Rank
Brothers, Bottisham, Cambridge, UK) at 25°C. The measurements were
started by the addition of different concentrations of
NaHCO3 after confirmation by cessation of
O2 evolution of the depletion of
Ci in medium under illumination (500 µmol
photons m2 s1) provided
by a slide projector. O2 concentration data were
collected every second, averaged, recorded every 5 s by a 21×
datalogger (Campbell Scientific, Logan, UT), and transferred to an IBM
computer. Smoothed data (Savitzky and Golay, 1964) were used for
calculation of exchange rates. Maximum spontaneous
CO2 supply rates from uncatalyzed dehydration of
bicarbonate were calculated as described by Miller and Colman (1980).
Chlorophyll content was estimated after extraction with 96% (v/v)
ethanol (Wintermans and De Mots, 1965).
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RESULTS |
Generation and Isolation of Mutants
A pool of more than 7000 transformants was generated by
complementation transformation of an Arg-requiring mutant with the Arg7 gene. In an attempt to identify mutants failing to
adapt to limiting CO2, this pool of potential
insertional mutants was screened immunochemically for the absence of
pCA1, which was being used as a reporter for induction of genes
involved in adaptation to limiting CO2.
Sixty-eight putative mutants were identified in the primary screen, and
18 were selected for further characterization based on the absence of
or reduction in pCA1 expression in western blots. Among this collection
of insertionally generated mutants, we found a mutant (PCA57-61) that
showed no detectable pCA1 protein (Fig.
1A) and no detectable Cah1
mRNA (Fig. 1B). Since the Cah1 cDNA probe used (Spalding et
al., 1991) also detects the Cah2 mRNA (Fujiwara et al.,
1990) and the antibody used should cross react with pCA2, these blots
also demonstrate that Cah2 mRNA and pCA2 were undetectable
as well. Expression of other proteins normally induced by limiting
CO2, mitochondrial CA (Mca1 and
Mca2 genes) and Ccp (Ccp1 and Ccp2
genes), apparently was not affected by the mutation (data not shown).
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| Figure 1.
Western and northern analysis for wall-less
"wild-type" C. reinhardtii (CC400 or CC425) and
PCA57-61. Extracellular protein and total RNA (10 µg per lane) were
isolated after adaptation of cells for 2 d in air. A, pCA1 protein
detected with affinity-purified anti-pCA1 polyclonal antiserum. B,
Cah1 mRNA probed with 1.4-kb BglII and
NcoI fragment of Cah1 cDNA. The total RNA
was probed with 25S and 5.8S rRNA. Northern analysis was performed on
the same membrane, but CC425 and PCA57-61 are selectively shown.
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Genomic Analysis of PCA57-61
PCA57-61 genomic DNA was compared with that of CC425 (Table I) by
Southern analysis (Fig. 2). The presence
of one insert in PCA57-61 was confirmed by probing with the 1.3-kb
SalI fragment of Arg7 (no data shown). When the
same blot was probed with the entire Cah1 cDNA, different
patterns were observed for CC425 and PCA57-61 (Fig. 2A), indicating
that the Cah1 structural gene had been disrupted. Some of
the restriction fragments (indicated by arrows) apparently hybridized
to both the Cah1 and the Arg7 probes (data not
shown), indicating that the Arg7 insert is located near the
Cah1 structural gene. Cah1 and Cah2
are arranged in tandem, separated by approximately 0.8 kb (Fig. 2B).
The Cah1 cDNA probe hybridizes with both genes (Fujiwara et
al., 1990), so bands from both Cah1 and Cah2 can
be seen on the Southern analysis in Figure 2A. The Cah2
structural gene appeared undisturbed. The 5.7-kb BglII fragment containing the 5 end of the Cah2
gene and the 3 end of the Cah1 gene appeared intact, but
the 6.8-kb KpnI fragment covering about the same area had
been disrupted, along with all restriction fragments 5 to the
BglII site in Cah1. The simplest interpretation
of these results is that the Cah1 gene 5 to the BglII site of Cah1 had been disrupted by an
insertion, a deletion, or a rearrangement. Since both the
Arg7 probe and the Cah1 probe hybridized to the
new EcoRI and KpnI fragments of PCA57-61
(indicated by arrows in Fig. 2A), it appears that this disruption had
been caused by insertion of the Arg7 gene in or near the
Cah1 structural gene.
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| Figure 2.
Southern analysis of wild-type CC425 and PCA57-61.
A, Genomic DNA (10 µg per lane) was isolated, digested with different
restriction enzymes, and hybridized with the full-length
Cah1 cDNA. Arrows indicate the band that hybridized both
to the full-length Cah1 cDNA and the 1.3-kb
SalI fragment of the Arg7 probe. B,
Restriction map of the Cah1 and Cah2
genomic region (adapted from Fujiwara et al., 1990). Restriction enzyme
sites: B, BglII; E, EcoRI; H,
HindIII; Hc, HincII; K,
KpnI. The disrupted region is indicated by a bold arrow.
Fragment sizes for each enzyme are indicated below the restriction map.
Exons are indicated by filled boxes.
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Phenotypes and Genetic Analysis of PCA57-61
The most interesting feature of PCA57-61 was its lack of any
significant growth phenotype even though this mutant apparently lacked
any Cah1 expression in air. After PCA57-61 was crossed with
CC1068 (Table I), progeny showed Mendalian 2:2 segregation of wild-type
and Arg-requiring phenotypes (Table II).
Both the absence of Cah1 mRNA accumulation and the
restriction polymorphisms of genomic DNA co-segregated with the
Arg7 insert, confirming that this insert was responsible for
the absence of Cah1 mRNA in PCA57-61 (Table II). Progeny
57-61-612 was chosen for further physiological analysis because it had
the same biochemical phenotype as PCA57-61 but had a normal cell wall.
Effects of pH on Growth
Because Moroney et al. (1985) and Williams and Turpin (1987)
reported contradictory results for photosynthesis at alkaline pH using
either CA inhibitors (Moroney et al., 1985) or washed, wall-less cells
to eliminate pCA1 activity (Williams and Turpin, 1987), a
Cah1 null mutant, such as PCA57-61, should help in resolving the function of pCA1 in the CCM. In spot tests growth of PCA57-61 and
its walled progeny, PCA57-61-612, was similar to that of two wild
types, one wall-less (CC400, Table I) and one walled (CC125, Table I), at all pHs in air (Fig. 3).
However, other known mutants with defects in the CCM and related
pathways, cia5, ca1-1,
pmp1-1, and pgp1-1 (Table
I), showed slow or no growth in air within the tested pH range. All
cells grew poorly at pH 5.0 (data not shown). Both wild types, PCA57-61
and PCA57-61-612, showed variable growth at pH 9.0, as illustrated by
the poor growth of CC400 at pH 9.0 in Figure 3. However, the slow
growth usually caught up with that on the other pH plates. In liquid,
the growth rate of PCA57-61-612 was similar to that of the CC125 wild
type at pH 8.0 in air (Fig. 4) and at
other pHs from 6.0 to 9.0 (data not shown). These growth tests
demonstrated that there was no major difference in growth between the
wild type and PCA57-61 over a pH range from 6.0 to 9.0, indicating that
the mutant grows normally under low CO2 in this
pH range even without detectable pCA1.
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| Figure 3.
Spot test for growth response to different pH
conditions of PCA57-61, wild-type strains (CC125 and CC400) and four
high-CO2-requiring mutants (cia5,
ca1-1,
pmp1-1, and
pgp1-1). All plates (except the high
CO2 plate) were kept at air level of CO2 for
10 d.
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| Figure 4.
Cell growth curve of CC125 ( ) and PCA57-61-612
( ) at pH 8.0 in air levels of CO2. The growth curves
shown are from a single experiment but are representative of three
independent growth experiments.
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Photosynthetic O2 Exchange
Photosynthetic O2 evolution of PCA57-61-612
was investigated at different concentrations of
NaHCO3 and at different pHs. PCA57-61-612 showed
a similar pattern of photosynthetic response to
NaHCO3 concentration as the CC125 wild type (Fig.
5A), and there was no difference of
photosynthesis rate between these strains at air levels of
CO2. At very low CO2,
however, PCA57-61-612 showed slightly lower photosynthesis rates than
the wild type (Fig. 5B). The
K1/2(CO2) calculated from
these data is higher for PCA57-61-612 (21 µM) than the
wild type (11 µM). Since the O2
evolution is measured in a closed system, these experiments used fairly
low cell densities (9-11 µg chlorophyll mL1)
to minimize problems with rapid Ci depletion at
low Ci concentrations. At these relatively low
cell densities, the rate of photosynthetic consumption in PCA57-61-612
was similar to the spontaneous CO2 supply rate
from uncatalyzed bicarbonate dehydration (Fig.
6A). However, this similarity is
coincidental because at higher cell densities (35-38 µg chlorophyll
mL1) CO2 response curves
for both strains were similar to those obtained at lower cell densities
(data not shown), and the photosynthesis rates of both strains clearly
exceeded the spontaneous CO2 supply rate at all
bicarbonate concentrations up to 100 µM (Fig. 6B). The
apparently large difference between the calculated photosynthetic CO2 consumption rates of PCA57-61-612 and CC125
in Figure 5A results only from the slightly lower cell density for the
mutant because the rates are expressed as micromoles of
CO2 per liter per hour to match the units used
for the spontaneous CO2 supply rate. The measured
rates of O2 evolution were more variable at the
higher cell density, but PCA57-61-612 maintained a rate similar to that of the wild type even though the calculated maximum spontaneous CO2 supply rate was only one-half that of the
photosynthetic rate.
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| Figure 5.
Photosynthetic response to NaHCO3
concentration (pH 7.0) of wild-type CC125 (), cia5
( ), and PCA57-61-612 (). A, Air-adapted cells (1 d) were used for
all measurements. Chlorophyll concentrations were: CC125, 11.09 µg
mL1; cia5, 10.12 µg mL1; PCA57-61-612,
9.14 µg mL1. B, Expansion of the data in A at
NaHCO3 concentrations up to 50 µM. Three
independent measurements were averaged from three different cultures.
SD is indicated by error bars.
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| Figure 6.
Photosynthetic CO2 consumption rate of
wild-type CC125 () and PCA57-61-612 () calculated from
O2 evolution assuming 1:1 O2:CO2.
The calculated maximum spontaneous CO2 supply rate is
indicated by the diagonal line. A, Chlorophyll concentrations were:
CC125, 11.09 µg mL1; PCA57-61-612, 9.14 µg
mL1. Three independent measurements are averaged from
three different cultures. B, Chlorophyll concentrations were: CC125,
37.53 µg mL1; PCA57-61-612, 35.11 µg
mL1. One sample was measured.
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Because it had been suggested that pCA1 was needed to supply
CO2 at alkaline pH (Moroney et al., 1985),
photosynthetic O2 evolution at different pHs was
also investigated. Photosynthetic rates at two bicarbonate
concentrations of wild type, CC125 and PCA57-61-612, decreased with
increasing pH, but both showed approximately the same rate of
photosynthetic O2 evolution at the given
concentration and pH (Table III).
Although there was no significant difference between the wild type and
PCA57-61-612 in photosynthetic O2 evolution at
any NaHCO3 concentration or pH, small effects
might be masked by the variation in the rate measurement. These results
indicate that pCA1 is not required to supply CO2
from HCO3 dehydration under
the conditions used, but they do not exclude some minor benefit
conferred by pCA1 at low Ci concentrations.
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Table III.
Effect of pH on the photosynthetic rate of
wild-type CC125 and PCA57-61-612
One-day air-adapted cells were resuspended in either 25 mM
citrate-KOH buffer (pH 5.0), 25 mM Mes-KOH buffer (pH 6.0),
25 mM Mops-KOH buffer (pH 7.0), 25 mM HEPPS-KOH
buffer (pH 8.0), or 25 mM AMPSO-KOH buffer (pH 9.0).
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DISCUSSION |
Like C4 plants, C. reinhardtii,
as well as many other microalgae and cyanobacteria, have an active CCM
that allows cells to assimilate Ci efficiently
when they grow under limiting CO2 conditions. The
CCM results in increased internal CO2
concentration, which increases the substrate for Rubisco carboxylation
and favors the carboxylation reaction of Rubisco over the oxygenation
reaction (Badger et al., 1980; Aizawa and Miyachi, 1986; Spalding,
1998). This mechanism is inducible after transfer from high (5%
CO2 in air) to low CO2
(0.03% CO2 in air). As previously described,
C. reinhardtii shows adaptive changes to limiting
CO2 conditions other than the CCM, including the
induction of several major genes (Cah1, Mca1,
Mca2, Ccp1, Ccp2) (for review, see
Spalding, 1998). However, it is not clear yet whether any of these
induced genes are required for function of the CCM.
pCA1 has been considered a potential candidate for an essential CCM
component because large amounts of this polypeptide accumulate under
low CO2 conditions, and affinity for
Ci is also increased, coincident with the induction of
pCA1. For pCA1 to be essential, conversion between
CO2 and
HCO3 would have to be a
critical step for C. reinhardtii in terms of adapting to low
CO2, but many studies have shown that C. reinhardtii can use both CO2 and
HCO3 (Williams and Turpin,
1987; Sültemeyer et al., 1989; Palmqvist et al., 1994). On the
other hand, the evidence does indicate that CO2
is the preferred substrate (Moroney et al., 1985; Aizawa and Miyachi,
1986).
The function of pCA1 in the CCM has been controversial. Moroney et al.
(1985) and Williams and Turpin (1987) arrived at contradictory conclusions regarding the need of pCA1 to supply
CO2 at alkaline pH. The photosynthetic rate of
C. reinhardtii was significantly decreased in limiting
CO2 when pCA1 was inhibited by nominally nonpermeant CA inhibitors, but only at alkaline pH (Moroney et al.,
1985). The authors interpreted this to mean pCA1 was required to supply
CO2 from
HCO3 for rapid photosynthesis
at low CO2 concentrations and alkaline pH,
because the HCO3 concentration
is higher than that of CO2 under these
conditions. However, Williams and Turpin (1987) were unable to
demonstrate decreased photosynthetic rates at alkaline pH using washed
wall-less cells that lack pCA1 activity rather than using CA
inhibitors. These authors concluded that the pCA1 activity is not
absolutely required for utilizing Ci under
alkaline pH and suggested that, in the work of Mononey et al. (1985),
nominally nonpermeant CA inhibitors like AZA may have penetrated the
cells and partially inhibited internal CA, which is essential for
photosynthesis. Although Moroney and coworkers tried to control for
secondary effects of the CA inhibitors based on the work reported here
and by Williams and Turpin, it appears that they did have effects other
than inhibition of extracellular CA.
We have identified a mutant (PCA57-61) that shows no pCA1 protein and
no Cah1 mRNA (Fig. 1) but apparently normal Mca1,
Mca2, Ccp1, and Ccp2 mRNAs and normal
levels of the corresponding proteins. This insertional mutant was
isolated after transformation of strain CC425 with a plasmid containing
the argininosuccinate lyase gene (Arg7). Southern and
genetic analyses have established the presence of 1 insert in this
mutant that co-segregates with the lack of Cah1 mRNA and
with polymorphisms in the 5 region of Cah1. It is clear
that insertion of this Arg7 insert in the region of the Cah1 and Cah2 genes has disrupted the
Cah1 structural gene and that this disruption is responsible
for lack of Cah1 expression in this mutant. Thus, PCA57-61
is a Cah1 structural gene mutant apparently null in pCA1
expression, which we have named cah1-1.
The most interesting feature of PCA57-61 is that it does not have a
high CO2-requiring phenotype even though it has
no detectable pCA1. One might expect that the total absence of pCA1
would have a more significant effect on the growth rate of PCA57-61 at
air level of CO2, perhaps intermediate between
wild type and cia5, a mutant that lacks Cah1
expression along with all other induced genes (Moroney et al., 1989).
Both in spot tests and in liquid culture, cell growth rates for
PCA57-61 and PCA57-61-612 without detectable pCA1 were similar to those
of the wild type over a pH range from 6.0 to 9.0, which argues against
any essential role of pCA1 even at alkaline pH.
Consistent with the lack of any effect on growth, the measured
photosynthetic rates of the mutant and the wild type were very similar
over a wide range of pH values, as well (Table III). In addition, the
Ci response curve of PCA57-61-612 for
photosynthetic O2 evolution at pH 7.0 was very
similar to that of wild-type CC125, indicating that the lack of pCA1
had no major impact on photosynthetic rates over a range of
Ci concentrations or a range of pH conditions. However, even though the photosynthetic rates of the two strains were
indistinguishable at air levels of CO2 (60 µM Ci), the mutant was found to
have a slightly elevated
K1/2(Ci) relative to wild-type
(21 µM versus 11 µM) and slightly reduced
photosynthetic O2 evolution at
Ci concentrations lower than 50 µM.
Although not nearly as extreme as the differences reported by Moroney
et al. (1985), it appears that the abundant pCA1 activity may be of
some benefit under very low Ci concentrations.
The observations made here appear contradictory to the report by
Moroney et al. (1985), based on inhibitor studies, that pCA1 was
required to supply CO2 from bicarbonate for rapid
photosynthesis at low CO2 concentrations,
especially at alkaline pH. If pCA1 is essential to supply
CO2 through dehydration of bicarbonate, this
should be most evident under conditions where the photosynthetic rate
of the wild type clearly exceeds the calculated maximum spontaneous CO2 supply rate. However, as demonstrated in
Figure 5B, the Cah1 null mutant showed a photosynthetic rate
similar to that of wild-type CC125 even though the spontaneous
CO2 supply rate was only one-half the rate of
photosynthesis. This could possibly be explained by residual CA
activity from pCA2, but Cah2 expression should be repressed
under these low CO2 growth conditions (Fujiwara
et al., 1990; Rawat and Moroney, 1991), as confirmed by our inability to detect either the Cah2 mRNA or the pCA2 protein. It seems
more likely that, although C. reinhardtii apparently prefers
CO2 (Moroney et al., 1985; Aizawa and Miyachi,
1986), PCA57-61-612 must be using bicarbonate directly from the medium.
These results confirm those of others (Sültemeyer et al., 1989;
Palmqvist et al., 1994) including Williams and Turpin (1987), who
reported similar findings using wall-less C. reinhardtii
washed free of pCA1.
We conclude that, although some benefit may be derived from the
presence of pCA1 at very low Ci concentrations,
the benefit appears less substantial than that reported by Moroney et
al. (1985), and this protein certainly does not appear to be essential either for function of the CCM or for growth of C. reinhardtii at limiting CO2 concentrations.
These conclusions beg the question, therefore, of why the expression
level of pCA1 is so high if this protein provides only minimal benefit
for photosynthesis and growth. It is possible that further work with
this Cah1 null mutant will allow the identification of
conditions under which pCA1 provides more substantial benefits.
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FOOTNOTES |
1
This work was supported by the U.S. Department
of Agriculture National Research Initiative (grant no. 97-35100-4210 to
M.H.S.). This is journal paper no. J-18217 of project no. 3479 of the
Iowa Agriculture and Home Economics Experiment Station, Ames, and was supported by the Hatch Act and State of Iowa funds.
*
Corresponding author; e-mail mspaldin{at}iastate.edu; fax
1-515-294-1377.
Received December 14, 1998;
accepted April 9, 1999.
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ABBREVIATIONS |
Abbreviations:
AZA, acetazolamide.
CA, carbonic anhydrase.
CCM, CO2-concentrating mechanism.
Ccp, chloroplast carrier
protein.
Ci, inorganic carbon.
pCA1, periplasmic CA 1.
pCA2, periplasmic CA 2.
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LITERATURE CITED |
Aizawa K,
Miyachi S
(1986)
Carbonic anhydrase and CO2 concentrating mechanisms in microalgae and cyanobacteria.
FEMS Microbiol Rev
39:
215-233
[CrossRef]
Ausubel FM,
Brent R,
Kingston RE,
Moore DD,
Seidman JG,
Smith JA,
Struhl K
(1989)
Current Protocols in Molecular Biology, Vol 2.
John Wiley & Sons, New York
Badger MR,
Kaplan A,
Berry JA
(1980)
Internal inorganic carbon pool of Chlamydomonas reinhardtii. Evidence for a CO2 concentrating mechanism.
Plant Physiol
66:
407-413
[Abstract/Free Full Text]
Badger MR,
Price GD
(1994)
The role of carbonic anhydrase in photosynthesis.
Annu Rev Plant Physiol Plant Mol Biol
45:
369-392
[CrossRef][Web of Science]
Chen Z-Y,
Lavigne MD,
Mason CB,
Moroney JV
(1997)
Cloning and overexpressing of two cDNAs encoding the low-CO2-inducible chloroplast envelope protein LIP-36 from Chlamydomonas reinhardtii.
Plant Physiol
114:
265-273
[Abstract]
Chomczynski P,
Sacchi N
(1987)
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159
[Web of Science][Medline]
Coleman JR,
Grossman AR
(1984)
Biosynthesis of carbonic anhydrase in Chlamydomonas reinhardtii during adaptation to low CO2.
Proc Natl Acad Sci USA
81:
6049-6053
[Abstract/Free Full Text]
Davies JP,
Yildiz F,
Grossman AR
(1994)
Mutants of Chlamydomonas with aberrant responses to sulfur deprivation.
Plant Cell
6:
53-63
[Abstract]
Debuchy R,
Purton S,
Rochaix JD
(1989)
The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the ARG7 locus.
EMBO J
8:
2803-2809
[Web of Science][Medline]
Eriksson M,
Karlsson J,
Ramazanov Z,
Garderstrom P,
Samuelsson G
(1996)
Proc Natl Acad Sci USA
93:
12031-12034
[Abstract/Free Full Text]
Fujiwara S,
Fukuzawa H,
Tachiki A,
Miyachi S
(1990)
Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii.
Proc Natl Acad Sci USA
87:
9779-9783
[Abstract/Free Full Text]
Fukuzawa H,
Fujiwara S,
Yamamoto Y,
Dionisio-Sere ML,
Miyachi S
(1990)
cDNA cloning, sequence, and expression of carbonic anhydrase in Chlamydomonas reinhardtii: regulation by environmental CO2 concentration.
Proc Natl Acad Sci USA
87:
4383-4387
[Abstract/Free Full Text]
Funke RP,
Kovar JL,
Weeks DP
(1997)
Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2.
Plant Physiol
114:
237-244
[Abstract]
Geraghty AM,
Anderson JC,
Spalding MH
(1990)
A 36-kilodalton limiting-CO2 induced polypeptide of Chlamydomonas is distinct from the 37-kilodalton periplasmic carbonic anhydrase.
Plant Physiol
93:
116-121
[Abstract/Free Full Text]
Geraghty AM,
Spalding MH
(1996)
Molecular and structural changes in Chlamydomonas under limiting CO2.
A possible mitochondrial role in adaptation Plant Physiol
111:
1339-1347
Harlow E,
Lane D
(1988)
Antibodies: A Laboratory Manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Harris EH
(1989)
The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use.
Academic Press, San Diego, CA
Ishida S,
Muto S,
Miyachi S
(1993)
Structural analysis of periplasmic carbonic anhydrase 1 of Chlamydomonas reinhardtii.
Eur J Biochem
214:
9-16
[Medline]
Karlsson J,
Clarke AK,
Chen ZY,
Hugghins SY,
Park YI,
Husic HD,
Moroney JV,
Samuelsson G
(1998)
A novel alpha-type carbonic anhydrase associated with the thylakoid membrane in Chlamydomonas reinhardtii is required for growth at ambient CO2.
EMBO J
17:
1208-1216
[CrossRef][Web of Science][Medline]
Kindle KL
(1990)
Proc Natl Acad Sci USA
87:
1228-1232
[Abstract/Free Full Text]
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Marek LF,
Spalding MH
(1991)
Changes in photorespiratory enzyme activity in response to limiting CO2 in Chlamydomonas reinhardtii.
Plant Physiol
97:
420-425
[Abstract/Free Full Text]
Miller AG,
Colman B
(1980)
Evidence for HCO3 transport by the blue-green alga (Cyanobacterium) Coccochloris peniocystis.
Plant Physiol
65:
397-402
[Abstract/Free Full Text]
Moroney JV,
Husic HD,
Tolbert NE
(1985)
Effects of carbonic anhydrase inhibitors on inorganic carbon accumulation by Chlamydomonas reinhardtii.
Plant Physiol
79:
177-183
[Abstract/Free Full Text]
Moroney JV,
Husic HD,
Tolbert NE,
Kitayama M,
Manuel LJ,
Togasaki RK
(1989)
Isolation and characterization of a mutant of Chlamydomonas reinhardtii deficient in the CO2 concentration mechanism.
Plant Physiol
89:
897-903
[Abstract/Free Full Text]
Palmqvist K,
Yu J-W,
Badger MR
(1994)
Carbonic anhydrase activity and inorganic carbon fluxes in low- and high-Ci cells of Chlamydomonas reinhardtii and Scenedesmus obliquus.
Physiol Plant
90:
537-547
[CrossRef]
Ramazanov Z,
Mason CB,
Geraghty AM,
Spalding MH,
Moroney JV
(1993)
The low CO2-inducible 36-kD protein is localized to the chloroplast envelope of Chlamydomonas reinhardtii.
Plant Physiol
101:
1195-1199
[Abstract]
Rawat M,
Moroney JV
(1991)
Partial characterization of a new isozyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii.
J Biol Chem
266:
9719-9723
[Abstract/Free Full Text]
Roberts CS,
Spalding MH
(1995)
Post-translational processing of the highly processed, secreted periplasmic carbonic anhydrase of Chlamydomonas is largely conserved in transgenic tobacco.
Plant Mol Biol
29:
303-315
[CrossRef][Web of Science][Medline]
Savitzky A,
Golay MJE
(1964)
Smoothing and differentiation of data by simplified least squares procedures.
Anal Chem
36:
1627-1639
[CrossRef]
Spalding MH
(1998)
CO2 acquisition: acclimation to changing carbon availability.
In
J-D Rochaix,
M Goldschmidt-Clermont,
S Merchant,
eds, The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 529-547
Spalding MH,
Spreitzer RJ,
Ogren WL
(1983a)
Carbonic anhydrase deficient mutant of Chlamydomonas requires elevated carbon dioxide concentration for photoautotrophic growth.
Plant Physiol
73:
268-272
[Abstract/Free Full Text]
Spalding MH,
Spreitzer RJ,
Ogren WL
(1983b)
Reduced inorganic carbonic transport in a CO2-requiring mutant of Chlamydomonas reinhardtii.
Plant Physiol
73:
273-276
[Abstract/Free Full Text]
Spalding MH,
Winder TL,
Anderson JC,
Geraghty AM,
Marek LF
(1991)
Changes in protein and gene expression during induction of the CO2-concentrating mechanism in wild-type and mutant Chlamydomonas.
Can J Bot
69:
1008-1016
Sültemeyer DF,
Miller AG,
Espie GS,
Fock HP,
Canvin DT
(1989)
Active CO2 transport by the green algae Chlamydomonas reinhardtii.
Plant Physiol
89:
1213-1219
[Abstract/Free Full Text]
Suzuki K,
Marek LF,
Spalding MH
(1990)
A photorespiratory mutant of Chlamydomonas reinhardtii.
Plant Physiol
93:
231-237
[Abstract/Free Full Text]
Williams TG,
Turpin DH
(1987)
The role of external carbonic anhydrase in inorganic carbon acquisition by Chlamydomonas reinhardtii at alkaline pH.
Plant Physiol
83:
92-96
[Abstract/Free Full Text]
Winder TL,
Anderson JC,
Spalding MH
(1992)
Translational regulation of the large and small subunits of ribulose bisphosphate carboxylase/oxygenase during induction of the CO2-concentration mechanism in Chlamydomonas reinhardtii.
Plant Physiol
98:
1409-1414
[Abstract/Free Full Text]
Wintermans JFGM,
De Mots A
(1965)
Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol.
Biochem Biophys Acta
109:
448-453
[Medline]
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Abstract
Introduction