Research ArticleENVIRONMENTAL AND STRESS PHYSIOLOGY

Periplasmic Carbonic Anhydrase Structural Gene (Cah1) Mutant in Chlamydomonas reinhardtii

Kyujung Van, Martin H. Spalding

Published July 1999. DOI: https://doi.org/10.1104/pp.120.3.757

Abstract

To survive in various conditions of CO2 availability, Chlamydomonas reinhardtiishows 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 singleArg7 insert. The most interesting feature ofcah1-1 is its lack of any significant growth phenotype. There is no major difference in growth or photosynthesis between the wild type andcah1-1 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.

Aquatic and soil-borne photosynthetic organisms, includingChlamydomonas 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, seeSpalding, 1998), because it affects photosynthetic characteristics through increasing intracellular CO2concentration. 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 ofC. 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, andWilliams and Turpin (1987) disagreeing. Among a collection of insertionally generated mutants, we found a mutant, namedcah1-1, that showed no pCA1 protein and noCah1 mRNA. Here we report on the photosynthetic characteristics of this Cah1 null mutant.

MATERIALS AND METHODS

Cell Strains, Culture Conditions, and Mating

All Chlamydomonas reinhardtii strains (TableI) 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 ofHarris (1989).

View this table:
Table I.

Strains

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)2SO4overnight, 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 appropriate32P-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 mmMops-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 m−2 s−1) 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).

RESULTS

Generation and Isolation of Mutants

A pool of more than 7000 transformants was generated by complementation transformation of an Arg-requiring mutant with theArg7 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 Cah1mRNA (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 andMca2 genes) and Ccp (Ccp1 and Ccp2genes), apparently was not affected by the mutation (data not shown).

Fig. 1.
Fig. 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 andNcoI 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.

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-kbSalI 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 theCah1 structural gene. Cah1 and Cah2are 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 Cah2structural gene appeared undisturbed. The 5.7-kbBglII fragment containing the 5′ end of the Cah2gene 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 theBglII site in Cah1. The simplest interpretation of these results is that the Cah1 gene 5′ to theBglII site of Cah1 had been disrupted by an insertion, a deletion, or a rearrangement. Since both theArg7 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 theCah1 structural gene.

Fig. 2.
Fig. 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-lengthCah1 cDNA. Arrows indicate the band that hybridized both to the full-length Cah1 cDNA and the 1.3-kbSalI fragment of the Arg7 probe. B, Restriction map of the Cah1 and Cah2genomic 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.

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 theArg7 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.

View this table:
Table II.

Genetic analysis of PCA57-61 progeny from a cross with CC1068

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), aCah1 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 (TableI), 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.

Fig. 3.
Fig. 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.

Fig. 4.
Fig. 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.

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 K½(CO2) calculated from these data is higher for PCA57-61-612 (21 μm) than the wild type (11 μm). Since the O2evolution is measured in a closed system, these experiments used fairly low cell densities (9–11 μg chlorophyll mL−1) 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 mL−1) 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.

Fig. 5.
Fig. 5.

Photosynthetic response to NaHCO3concentration (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 mL−1; cia5, 10.12 μg mL−1; PCA57-61-612, 9.14 μg mL−1. 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.

Fig. 6.
Fig. 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 mL−1; PCA57-61-612, 9.14 μg mL−1. Three independent measurements are averaged from three different cultures. B, Chlorophyll concentrations were: CC125, 37.53 μg mL−1; PCA57-61-612, 35.11 μg mL−1. One sample was measured.

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 CO2from HCO3 − dehydration under the conditions used, but they do not exclude some minor benefit conferred by pCA1 at low Ci concentrations.

View this table:
Table III.

Effect of pH on the photosynthetic rate of wild-type CC125 and PCA57-61-612

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 CO2concentration, 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, seeSpalding, 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 CO2is 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 ofC. 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 theCah1 and Cah2 genes has disrupted theCah1 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 Cah1expression 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 K½(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 byMoroney 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. reinhardtiiwashed 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.

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.

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
  • Received December 14, 1998.
  • Accepted April 9, 1999.

LITERATURE  CITED

  1. ↵
    1. Aizawa K,
    2. Miyachi S
    (1986) Carbonic anhydrase and CO2 concentrating mechanisms in microalgae and cyanobacteria. FEMS Microbiol Rev 39:215–233.
    OpenUrlCrossRef
  2. ↵
    1. Ausubel FM,
    2. Brent R,
    3. Kingston RE,
    4. Moore DD,
    5. Seidman JG,
    6. Smith JA,
    7. Struhl K
    (1989) Current Protocols in Molecular Biology, Vol 2. (John Wiley & Sons, New York).
  3. ↵
    1. Badger MR,
    2. Kaplan A,
    3. Berry JA
    (1980) Internal inorganic carbon pool of Chlamydomonas reinhardtii. Evidence for a CO2 concentrating mechanism. Plant Physiol 66:407–413.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Badger MR,
    2. Price GD
    (1994) The role of carbonic anhydrase in photosynthesis. Annu Rev Plant Physiol Plant Mol Biol 45:369–392.
    OpenUrlCrossRef
  5. ↵
    1. Chen Z-Y,
    2. Lavigne MD,
    3. Mason CB,
    4. 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.
    OpenUrlAbstract
  6. ↵
    1. Chomczynski P,
    2. Sacchi N
    (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Coleman JR,
    2. Grossman AR
    (1984) Biosynthesis of carbonic anhydrase in Chlamydomonas reinhardtii during adaptation to low CO2. Proc Natl Acad Sci USA 81:6049–6053.
    OpenUrlAbstract/FREE Full Text
  8. ↵
    1. Davies JP,
    2. Yildiz F,
    3. Grossman AR
    (1994) Mutants of Chlamydomonas with aberrant responses to sulfur deprivation. Plant Cell 6:53–63.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Debuchy R,
    2. Purton S,
    3. 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.
    OpenUrlPubMed
  10. ↵
    1. Eriksson M,
    2. Karlsson J,
    3. Ramazanov Z,
    4. Garderstrom P,
    5. Samuelsson G
    (1996) Proc Natl Acad Sci USA 93:12031–12034.
    OpenUrlAbstract/FREE Full Text
  11. ↵
    1. Fujiwara S,
    2. Fukuzawa H,
    3. Tachiki A,
    4. Miyachi S
    (1990) Structure and differential expression of two genes encoding carbonic anhydrase in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87:9779–9783.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Fukuzawa H,
    2. Fujiwara S,
    3. Yamamoto Y,
    4. Dionisio-Sere ML,
    5. 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.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Funke RP,
    2. Kovar JL,
    3. Weeks DP
    (1997) Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Plant Physiol 114:237–244.
    OpenUrlAbstract
  14. ↵
    1. Geraghty AM,
    2. Anderson JC,
    3. 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.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Geraghty AM,
    2. Spalding MH
    (1996) Molecular and structural changes in Chlamydomonas under limiting CO2. A possible mitochondrial role in adaptation Plant Physiol 111:1339–1347.
  16. ↵
    1. Harlow E,
    2. Lane D
    (1988) Antibodies: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  17. ↵
    1. Harris EH
    (1989) The Chlamydomonas Source Book: A Comprehensive Guide to Biology and Laboratory Use. (Academic Press, San Diego, CA).
  18. ↵
    1. Ishida S,
    2. Muto S,
    3. Miyachi S
    (1993) Structural analysis of periplasmic carbonic anhydrase 1 of Chlamydomonas reinhardtii. Eur J Biochem 214:9–16.
    OpenUrlPubMed
  19. ↵
    1. Karlsson J,
    2. Clarke AK,
    3. Chen ZY,
    4. Hugghins SY,
    5. Park YI,
    6. Husic HD,
    7. Moroney JV,
    8. 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.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Kindle KL
    (1990) Proc Natl Acad Sci USA 87:1228–1232.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Laemmli UK
    (1970) Cleavage of structural proteins during the assembly of the bacteriophage T4. Nature 227:680–685.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Marek LF,
    2. Spalding MH
    (1991) Changes in photorespiratory enzyme activity in response to limiting CO2 in Chlamydomonas reinhardtii. Plant Physiol 97:420–425.
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Miller AG,
    2. Colman B
    (1980) Evidence for HCO3− transport by the blue-green alga (Cyanobacterium) Coccochloris peniocystis. Plant Physiol 65:397–402.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Moroney JV,
    2. Husic HD,
    3. Tolbert NE
    (1985) Effects of carbonic anhydrase inhibitors on inorganic carbon accumulation by Chlamydomonas reinhardtii. Plant Physiol 79:177–183.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Moroney JV,
    2. Husic HD,
    3. Tolbert NE,
    4. Kitayama M,
    5. Manuel LJ,
    6. Togasaki RK
    (1989) Isolation and characterization of a mutant of Chlamydomonas reinhardtii deficient in the CO2 concentration mechanism. Plant Physiol 89:897–903.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Palmqvist K,
    2. Yu J-W,
    3. 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.
    OpenUrlCrossRef
  27. ↵
    1. Ramazanov Z,
    2. Mason CB,
    3. Geraghty AM,
    4. Spalding MH,
    5. Moroney JV
    (1993) The low CO2-inducible 36-kD protein is localized to the chloroplast envelope of Chlamydomonas reinhardtii. Plant Physiol 101:1195–1199.
    OpenUrlAbstract
  28. ↵
    1. Rawat M,
    2. Moroney JV
    (1991) Partial characterization of a new isozyme of carbonic anhydrase isolated from Chlamydomonas reinhardtii. J Biol Chem 266:9719–9723.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Roberts CS,
    2. 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.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Savitzky A,
    2. Golay MJE
    (1964) Smoothing and differentiation of data by simplified least squares procedures. Anal Chem 36:1627–1639.
    OpenUrlCrossRef
  31. ↵
    1. Rochaix J-D,
    2. Goldschmidt-Clermont M,
    3. Merchant S
    1. Spalding MH
    (1998) CO2 acquisition: acclimation to changing carbon availability. in The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. eds Rochaix J-D, Goldschmidt-Clermont M, Merchant S (Kluwer Academic Publishers, Dordrecht, The Netherlands), pp 529–547.
  32. ↵
    1. Spalding MH,
    2. Spreitzer RJ,
    3. Ogren WL
    (1983a) Carbonic anhydrase deficient mutant of Chlamydomonas requires elevated carbon dioxide concentration for photoautotrophic growth. Plant Physiol 73:268–272.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Spalding MH,
    2. Spreitzer RJ,
    3. Ogren WL
    (1983b) Reduced inorganic carbonic transport in a CO2-requiring mutant of Chlamydomonas reinhardtii. Plant Physiol 73:273–276.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Spalding MH,
    2. Winder TL,
    3. Anderson JC,
    4. Geraghty AM,
    5. 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.
    OpenUrlCrossRef
  35. ↵
    1. Sültemeyer DF,
    2. Miller AG,
    3. Espie GS,
    4. Fock HP,
    5. Canvin DT
    (1989) Active CO2 transport by the green algae Chlamydomonas reinhardtii. Plant Physiol 89:1213–1219.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Suzuki K,
    2. Marek LF,
    3. Spalding MH
    (1990) A photorespiratory mutant of Chlamydomonas reinhardtii. Plant Physiol 93:231–237.
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Williams TG,
    2. Turpin DH
    (1987) The role of external carbonic anhydrase in inorganic carbon acquisition by Chlamydomonas reinhardtii at alkaline pH. Plant Physiol 83:92–96.
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Winder TL,
    2. Anderson JC,
    3. 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.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Wintermans JFGM,
    2. De Mots A
    (1965) Spectrophotometric characteristics of chlorophyll and their pheophytins in ethanol. Biochem Biophys Acta 109:448–453.
    OpenUrlPubMed
View Abstract
Back to top

Table of Contents

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Request Permissions
Periplasmic Carbonic Anhydrase Structural Gene (Cah1) Mutant in Chlamydomonas reinhardtii
Kyujung Van, Martin H. Spalding
Plant Physiology Jul 1999, 120 (3) 757-764; DOI: 10.1104/pp.120.3.757
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

In this issue

View this article with LENS