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BIOENERGETICS AND PHOTOSYNTHESIS

Rubisco Activase Is Required for Optimal Photosynthesis in the Green Alga Chlamydomonas reinhardtii in a Low-CO₂ Atmosphere¹

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

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
This report describes a Chlamydomonas reinhardtii mutant that lacks Rubisco activase (Rca). Using the Ble^R (bleomycin resistance) gene as a positive selectable marker for nuclear transformation, an insertional mutagenesis screen was performed to select for cells that required a high-CO₂ atmosphere for optimal growth. The DNA flanking the Ble^R insert of one of the high-CO₂-requiring strains was cloned using thermal asymmetric interlaced-polymerase chain reaction and inverse polymerase chain reaction and sequenced. The flanking sequence matched the C. reinhardtii Rca cDNA sequence previously deposited in the National Center for Biotechnology Information database. The loss of a functional Rca in the strain was confirmed by the absence of Rca mRNA and protein. The open reading frame for Rca was cloned and expressed in pSL18, a C. reinhardtii expression vector conferring paromomycin resistance. This construct partially complemented the mutant phenotype, supporting the hypothesis that the loss of Rca was the reason the mutant grew poorly in a low-CO₂ atmosphere. Sequencing of the C. reinhardtii Rca gene revealed that it contains 10 exons ranging in size from 18 to 470 bp. Low-CO₂-grown rca1 cultures had a growth rate and maximum rate of photosynthesis 60% of wild-type cells. Results obtained from experiments on a cia5 rca1 double mutant also suggest that the CO₂-concentrating mechanism partially compensates for the absence of an active Rca in the green alga C. reinhardtii.

Rca catalyzes the activation of Rubisco in vivo by removing inhibitory sugar phosphates (Robinson and Portis, 1988; Portis, 1992, 2003). This enzyme is required to maintain Rubisco activity in higher plants grown at ambient CO₂ concentrations. A substantial reduction (Mate et al., 1993, 1996; Jiang et al., 1994; Hammond et al., 1998) or absence of this protein (Portis, 1992) drastically impairs the photosynthetic process in higher plants. It has been postulated independently by different research groups that the activities of Rubisco and Rca are important key regulation points for photosynthesis under different environmental stress conditions (Hammond et al., 1998; Crafts-Brandner and Salvucci, 2000). Rubisco is activated by the binding of CO₂ to form a carbamate on a Lys residue in the active site and by the binding of Mg²⁺ (Portis, 1992). Once activated, Rubisco catalyzes the carboxylation of RuBP to form two molecules of 3-phosphoglycerate or the oxygenation of RuBP to form one molecule of 3-phosphoglycerate and one molecule of phosphoglycolate. Thus, the concentration of CO₂ at the active site of Rubisco is important both to the activation of Rubisco and the carboxylation reaction. C. reinhardtii cells that are grown in air (350 ppm CO₂) are capable of concentrating CO₂ at the active site of Rubisco. This localized increase in CO₂ concentration allows the algal cell to photosynthesize efficiently at very low levels of external dissolved inorganic carbon (DIC; HCO₃^- and CO₂). This phenomenon has been termed the CO₂-concentrating mechanism (CCM; for review, see Kaplan and Reinhold, 1999; Moroney and Somanchi, 1999). The CCM is repressed in cells that are grown in a high-CO₂ atmosphere (1%-5% [v/v] CO₂). Cyanobacteria also possess a CCM, but it is not clear whether Rca is even present in cyanobacteria. There have been reports of Rca-like activity, yet the cyanobacterial genomes sequenced to date do not appear to have an Rca gene. Therefore, although it is clear that Rca is required for growth in higher plants, it is not clear whether Rca is required for optimal photosynthetic activity in organisms with a CCM.

Recently, several mutants have been isolated that could be defective in one or more components of the CCM (Colombo et al., 2002). These mutants grow more slowly in a low-CO₂ atmosphere but grow normally, in relation to wild-type (WT) cells, in a high-CO₂ atmosphere. In contrast, photosynthesis-deficient mutants exhibit an acetate-requiring phenotype, and many of these mutants must be grown in the dark to avoid chlorophyll (Chl) bleaching caused by elevated levels of photosynthetically generated reactive oxygen species (Rochaix, 1995). This report describes an Rca mutant, rca1, in the green alga C. reinhardtii that grows normally in a high-CO₂ atmosphere but grows poorly in a low-CO₂ atmosphere. This report also presents data that suggest that although the presence of the CCM partially obviates the need for Rca, in a low-CO₂ atmosphere, C. reinhardtii requires both an active CCM and Rca to obtain maximal rates of photosynthesis and growth.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

rca1 Has an Insertion in the Gene Encoding Rca

C. reinhardtii strain D66 (nit2^-, cw15, and mt⁺; Schnell and Lefebvre, 1993) was transformed with linearized pSP124s, which contains the modified Ble^R gene (Lumbreras et al., 1998). D66 is a cell wall-deficient strain that exhibits normal photosynthetic characteristics but requires ammonia as a source of nitrogen for growth. Over 42,000 Ble^R insertional mutants were generated and screened for a high-CO₂-requiring phenotype (Colombo et al., 2002). Figure 1 shows the growth characteristics on minimal media plates in high CO₂ (5% [v/v]; Fig. 1B), air levels (350 ppm CO₂; Fig. 1C), and low (35 ppm CO₂; Fig. 1D) CO₂ atmospheres of the WT parent D66, the rca1 mutant, a CCM-deficient mutant cia5 (Moroney et al., 1989), and the double mutant cia5 rca1. The cia5 and rca1 mutants clearly show a reduction in photoautotrophic growth under low-CO₂ conditions compared with the parental strain D66. The cia5 rca1 double mutant's growth is severely retarded when the concentration of CO₂ in the environment is at air levels or below.

View larger version (140K): [in this window] [in a new window]   Figure 1. Photoautotrophic growth in high CO2 (5% [v/v]; B), air levels of CO2 (350 ppm CO2; C), and low CO2 (35 ppm CO2; D), for D66, rca1, cia5, and cia5 rca1 on solid media (see key in A).

Southern-blot analysis revealed that a single insertion of the Ble^R cassette was integrated into the genome (Fig. 2). The probe was amplified using PCR and contained the second and third exons of the Ble^R and an intron and 3'-UTR arising from the C. reinhardtii RbcS2 gene (Fig. 2A). The more diffuse bands indicate hybridization of the probe to the endogenous RbcS2 intron and 3'-UTR that are present in the parent D66 strain and rca1 (Fig. 2B).

View larger version (68K): [in this window] [in a new window]   Figure 2. Southern-blot analysis of D66 and rca1 probed with a 588-bp PCR fragment of the BleR gene containing two BleR exons and part of the RbcS2 gene. A, Map of the BleR portion of the pSP124s plasmid. Bars, RbcS2-derived portions of the gene; arrows, coding regions for BleR. RbcS2-P, RbcS2 promoter; RbcS2-T, RbcS2 terminator; arrows above and below the map indicate the primers used to make the probe. B, D66 (odd lanes) and rca1 (even lanes) genomic DNA was digested with the indicated restriction enzymes. The higher intensity bands correspond to the BleR insertion, whereas the less intense bands correspond to the endogenous RbcS2 intron and portion of the RbcS2 3'-untranslated region (UTR).

Genetic crosses were performed to determine if the resistance to zeocin was genetically linked to the rca mutation localized in rca1. In the cross between rca1 (mt⁺) and the strain cc124 (mt^-), which grows normally on low-CO₂ concentrations, the segregation of Ble^R resistance showed a ratio of 1:1 in all tetrads examined, and, in all cases, the Ble^R cells required a high-CO₂ atmosphere for optimal growth. In parallel crosses using random spore analysis, all of the Ble^R resistant segregants (approximately 200) exhibited the growth phenotype of rca1, further supporting that the pSP124s insertion is the cause of the high-CO₂-requiring phenotype.

Inverse-PCR and thermal asymmetric interlaced-PCR were used to clone the DNA flanking the Ble^R insert. The sequences of the PCR fragments were identical to the Rca cDNA sequence (Roesler and Ogren, 1990) deposited at the National Center for Biotechnology Information database. The opposite end of the insert was amplified using primers designed to complement the known sequences of the Ble^R insert and the Rca cDNA. Eighty-nine base pairs containing the 3' end of exon 8 and the 5' end of intron 8 were deleted upon insertion of the linearized pSP124s. Figure 3 shows the structure of the Rca locus and the location of the Ble^R insert in rca1. The WT gene was isolated from an indexed cosmid library using a PCR-based technique. The Rca gene was completely sequenced and shown to contain 10 exons and nine introns spanning 3 kb (Fig. 3; Rca). The deduced structure of the Rca gene agrees with the distribution of expressed sequence tags in this region of the C. reinhardtii genome (http://genome-.jgi-psf.org/chlre1/chlre1.home.html; scaffold 1895). In rca1, the Ble^R insert was found to be located at the 3' end of exon 8 (Fig. 3; rca1).

View larger version (8K): [in this window] [in a new window]   Figure 3. Map of the C. reinhardtii Rca and rca1 loci. Black arrows, Exons; white arrow, pSP124s insertion.

Rca Expression in rca1

Northern-blot analysis showed that a null mutant for Rca had been selected (Fig. 4). In WT cells, a slight increase in the Rca transcript abundance was observed when cells were switched from a high- to low-CO₂ atmosphere (Fig. 4A). No message for Rca was detected in the rca1 mutant (Fig. 4A). Western-blot analysis using polyclonal antibodies raised against Rca from C. reinhardtii failed to detect the Rca protein in the mutant rca1 cultures (Fig. 4B) and, consistent with the mRNA hybridization results, that the abundance of the Rca peptide also increased in the WT cells upon transfer to a low-CO₂ atmosphere. This increase in Rca in WT cells under low-CO₂ conditions is in agreement with earlier reports (Rawat and Moroney, 1995; Im et al., 2003).

View larger version (45K): [in this window] [in a new window]   Figure 4. Expression of Rca in D66 and rca1cells. A, Northern-blot analysis of high-CO2- and air-grown D66 (WT) cells and rca1 cells using 10 µg of total RNA per lane and a 1.2-kb Rca cDNA PCR product as the probe using a random primer protocol. B, Western-blot analysis of high-CO2- and air-grown D66 (WT) cells and rca1 cells. Cell extracts (50 µg of protein) were probed with a polyclonal antibody raised against C. reinhardtii Rca. Cells were grown in minimal medium with an atmosphere of 5% (v/v) CO2 in air (high CO2) or 350 ppm CO2 (air).

Complementation of rca1

Attempts to complement the rca1 strain with the cosmid containing the WT Rca gene were unsuccessful. Partial complementation of rca1 was observed by expressing the Rca cDNA under the control of the constitutive PsaD promoter and terminator in the vector pSL18 (Fig. 5A; Depege et al., 2003). For this experiment, rca1 cells were transformed with the Rca ORF under the control of the C. reinhardtii PsaD promoter and terminator linked to a paromomycin resistance cassette. Paromomycin resistance colonies were selected, and a number of these showed improved growth under low CO₂ as compared with the rca1 strain (Fig. 5B). Partial complementation was verified by selection on minimal media in 35 ppm CO₂ in air (Fig. 5B) and confirmed by the reappearance of the 42-kD peptide after transformation (Fig. 6A). In addition, the strains expressing Rca showed rates of photosynthesis that were higher than rca1, although not as high as the WT strain (Fig. 6A). The improved growth of the transformants expressing the WT Rca protein provides additional support that the poor growth observed in rca1 is due to the loss of Rca (Fig. 6B).

View larger version (42K): [in this window] [in a new window]   Figure 5. Partial complementation by the WT Rca open reading frame (ORF). A, rca1 was transformed with a linearized plasmid (pSL18) carrying the paromomycin resistance gene (AphVIII) driven by an RbcS2 and Hsp70 promoter fusion and the WT Rca ORF driven by the C. reinhardtii PsaD promoter. Colonies resistant to paromomycin were selected, and these transformants were screened on high (5% [v/v]) and low (35ppm CO2) CO2. Six of 250 (2.5%) of the paromomycin colonies grew on low CO2 better than the original rca1 strain but not as well as the WT D66. B, Growth characteristics of D66, rca1, two paromomycin-resistant colonies that grew better in low CO2 (pc1 and pc2), and two colonies that did not show complementation (p1 and p2) on minimal plates in high CO2 or low CO2 (35 ppm).

View larger version (26K): [in this window] [in a new window]   Figure 6. A, Western-blot analysis and maximum rates of photosynthesis of the rca1 mutant, the WT (D66), and one complemented (rca1-pc1) strain. The complemented strain was selected from those colonies shown in Figure 5. An additional paromomycin-resistant strain that did not exhibit complementation (rca1-p1) was also tested. Cells were bubbled with air (350 ppm CO2) in minimal medium at a constant light intensity of 80 µmol photons m-2 s-1 Error bars = SE of two independent experiments. B, Growth curves of the four strains in air levels of CO2.

Pyrenoid Structure

Rca is localized to the pyrenoid in C. reinhardtii (McKay et al., 1991). The pyrenoid of rca1 grown overnight at air levels of CO₂ was observed using transmission electron microscopy. No obvious changes with respect to the D66 WT cells were observed in the pyrenoid in mutants cells adapted to a low-CO₂ atmosphere for 12 h (data not shown).

Characteristics of a cia5 rca1 Double Mutant

In liquid media bubbled with air levels of CO₂ (0.035% [v/v] CO₂ in air), rca1 exhibited slower growth rates than the WT D66 (Fig. 7). In higher plants that lack a CCM, an active Rca is absolutely required for autotrophic growth in air levels of CO₂ (Somerville et al., 1982). To determine to what extent, if any, the presence of an active CCM compensates for the loss of Rca, the CCM-deficient double mutant cia5 rca1 was constructed. The growth and photosynthetic characteristics of the double mutant were severely reduced as compared with WT or either single mutant (Figs. 1, 7, and 8). Thus, the cia5 rca1 double mutant phenotype was similar to the Arabidopsis rca mutant, suggesting that the CCM does partially compensate for the loss of an active Rca in C. reinhardtii.

View larger version (18K): [in this window] [in a new window]   Figure 7. Representative experiment measuring growth of D66, rca1, cia5, and cia5 rca1 in liquid medium bubbled with air. The cell density is plotted versus time at a constant light intensity of 80 µmol photons m-2 s-1.

View larger version (24K): [in this window] [in a new window]   Figure 8. Rates of photosynthesis as a function of the DIC concentration for air grown D66 (), cia5 (), rca1 (), and cia5 rca1 (). Error bars = SE of two or three independent experiments.

Photosynthetic Kinetics of rca1

Figure 8 shows the rates of photosynthesis versus the DIC concentration for D66, rca1, cia5, and cia5 rca1. When grown on air levels of CO₂, rca1 exhibited a reduction in the maximum rate of photosynthesis (PS_max) when compared with D66 and cia5. After a 6-h acclimation period to low CO₂, the apparent affinity of rca1 for DIC was the same as D66, but the maximum rate of photosynthesis was reduced to 60% of the WT (Fig. 8). In a low-CO₂ atmosphere, the WT cells had a PS_max of 140 µmol O₂ mg Chl^-1 h^-1, rca1 had a PS_max of 88 µmol O₂ mg Chl^-1 h^-1, cia5 had a PS_max of 133 µmol O₂ mg Chl^-1 h^-1, and cia5 rca1 had a PS_max of 17 µmol O₂ mg Chl^-1 h^-1. D66 and rca1 had similar apparent affinities for DIC, as estimated by the DIC concentration required for half maximal rates of photosynthesis (k_0.5 [DIC]) suggesting that the CCM was operational in both cell lines. cia5 and cia5 rca1 had much lower apparent affinities for DIC, indicating that the CCM was not functioning in these cell lines, consistent with earlier reports (Moroney et al., 1989; Fukuzawa et al., 2001; Xiang et al., 2001).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
This report characterizes a C. reinhardtii mutant that lacks an active Rca. This novel mutant, designated rca1, requires a high-CO₂ atmosphere to grow at an optimum rate. This mutant was generated by insertional mutagenesis using the Ble^R gene. Using PCR techniques, the DNA flanking the pSP124s insert was cloned and sequenced. The genomic sequence of the Rca gene in C. reinhardtii was determined, and this sequence was confirmed by comparison with the first draft of the C. reinhardtii genome (http://genome.jgi-psf.org/chlre1/chlre1. home.html; scaffold 1895). This sequence also agrees with the Rca cDNA sequence (Roesler and Ogren, 1990) except that the 3'-UTR is 512 bp longer than the previously reported Rca cDNA. However, the predicted amino acid sequence is identical to that reported previously (Roesler and Ogren, 1990). The Rca gene is composed of 10 exons, one of which is only 17 bp long. Conserved splicing sites were located at all of the intron/exon junctions. Another characteristic of the Rca locus, shared with several C. reinhardtii genes, is the distribution of the largest exons toward the 3'-UTR and the shortest exons toward the 5'-UTR. The C. reinhardtii Rca was compared with two loci of Rca deposited in GenBank from spinach (Spinacia oleracea; S45033) and Arabidopsis (M86720). The Rca genes in Arabidopsis and spinach have very similar arrangements of their exons and introns. In contrast, the arrangement of exons was completely different in the Rca locus of C. reinhardtii, suggesting a relatively long evolutionary distance exists between the Rca locus of C. reinhardtii and the Rca loci of higher plants.

Northern and western analyses indicate that rca1 is a null mutant of Rca because no Rca mRNA or Rca protein was detected in the mutant. Genetic analysis and molecular complementation also support the conclusion that the loss of Rca causes a slow growth phenotype in a low-CO₂ atmosphere. If rca1 was grown on elevated CO₂ (5% [v/v] CO₂), it had a growth rate indistinguishable from WT cells (Fig. 1) and photosynthesis rates similar to WT cells. O₂ evolution assays also showed that rca1 has a WT affinity for DIC. However, a considerable reduction in the maximum capacity to perform photosynthesis is observed when the mutant is grown in air levels of CO₂ (Fig. 8). Because air-grown rca1 cultures undergo bleaching of their Chls during long-term growth, it is likely that photoreactive oxygen species are being formed by the inefficient use of energy from excited Chls.

The results in this report suggest that when the CO₂ level is low and Rca is absent, there is a reduction in the amount of active Rubisco and, thus, a reduction in the maximum attainable rates of photosynthesis. Rubisco can be the limiting step of photosynthesis, and the reduction of the total activity of this protein (physically, by destruction or by inhibition) can cause a reduction in the photosynthetic process. Researchers have reduced the amount of Rca in higher plants using antisense silencing. In plants with reduced Rca, a reduction in active Rubisco and photosynthetic capacity has been reported (Mate et al., 1993, 1996; Eckardt et al., 1997; Hammond et al., 1998). The results obtained from rca1 grown under low-CO₂ conditions are consistent with these earlier reports.

Although rca1 grown under low CO₂ has reduced photosynthesis, it is still able to grow at air levels of CO₂ in contrast to the original Arabidopsis mutant. An explanation for this difference comes from the observation that a high-CO₂ atmosphere compensates for the loss of Rca in both C. reinhardtii (this report) and Arabidopsis (Somerville et al., 1982). rca1 grown in high CO₂ has rates of photosynthesis and growth that are comparable with those of WT cells (data not shown). These observations suggest that more Rubisco is in its activated form in a high-CO₂ atmosphere even in the absence of Rca. If this were the case, a functional CCM should compensate for the loss of Rca in a low-CO₂ atmosphere.

To test the hypothesis that the CCM partially compensates for the loss of Rca, the cia5 rca1 double mutant was constructed and analyzed. Growth of the double mutant at air levels of CO₂ was compared with that of WT and the single mutants cia5 and rca1. Both rca1 and cia5 can grow on air levels of CO₂ although not WT cells (Fig. 7). In support of the hypothesis that the CCM partially compensates for the loss of Rca, the double mutant did not grow at air levels of CO₂ (Fig. 7) and had a greatly reduced maximal rate of photosynthesis (Fig. 8). Therefore, although an active CCM and a functional Rca are required for maximum rates of photosynthesis, the CCM does compensate partially for the loss of Rca presumably by raising the concentration of CO₂ in the vicinity of Rubisco. Thus, compared with the Arabidopsis rca mutant that grows very poorly at air levels of CO₂, the C. reinhardtii mutant is able to sustain appreciable rates of growth in air levels of CO₂ due to the presence of an active CCM. Because the CCM partially compensates for the loss of Rca in an alga with a CCM, a C4 plant deficient in Rca might also be less affected than a C3 plant missing Rca.

The action of Rca should be physically associated with Rubisco. Recently, Borkhsenious et al. (1998) and Morita et al. (1997) have demonstrated that almost 95% of Rubisco is located in the pyrenoid, a characteristic and well-defined structure observed in C. reinhardtii chloroplasts when the cells are acclimated to low CO₂. In addition, McKay et al. (1991) demonstrated that Rca is localized to the pyrenoid in green algae. Therefore, the structure of the pyrenoid in rca1 was examined to determine whether the pyrenoid structure was disrupted by the loss of Rca. Ultramicroscopic analysis revealed that the pyrenoid structure was unaltered in the rca1 mutant (data not shown), indicating that Rca is not essential to the assembly and/or maintenance of the pyrenoid structure.

The rca1 mutant may be useful in understanding the interaction of Rca with Rubisco. With this mutant, it may be possible to begin to dissect Rca's in vivo function in C. reinhardtii. Using site-directed mutagenesis and expression vectors, it may be feasible to manipulate the activity of Rubisco via a genetically modified Rca. In addition, a genetic screen for suppressors and modifiers of the rca1 mutant phenotype could discover yet uncharacterized genes that may play a central role in the carboxylation reaction of Rubisco.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Strains and Media

Chlamydomonas reinhardtii strain D66 (nit2^-, cw15, and mt⁺) was obtained from Rogene Schnell (University of Arkansas, Little Rock; Schnell and Lefebvre, 1993) and CC124 (nit1^-, nit2^-, and mt^-) from the C. reinhardtii Stock Center (Duke University, Durham, NC). The CCM mutant cia5 (Cia5 is a putative transcription factor; Fukuzawa et al., 2001; Xiang et al., 2001) was also used in this study because it does not induce the CCM (Moroney et al., 1989). Before transformation, D66 cells were grown in Tris-acetate phosphate (TAP) medium (Sueoka, 1960) under illumination to a density of between 1 and 3 10⁶ cells mL^-1. Transformants were selected on TAP plates supplemented with 7.5 µg of zeocin mL^-1 (Invitrogen, Carlsbad, CA). The screening for mutants with phenotype slc (sick in low CO₂) was performed on minimal media (Sueoka, 1960). To determine if the presence of an active CCM could compensate for the loss of Rca, a double mutant was constructed by crossing rca1 to cia5 and selecting for the double-mutant progeny. The atmospheric CO₂ levels used in these studies were: high CO₂ (5% [v/v] CO₂ in air), ordinary air (350 ppm CO₂), and low CO₂ (35 ppm CO₂). In all cases, the O₂ concentration was 21% (v/v). All strains were maintained axenically on TAP plates.

C. reinhardtii Mutagenesis and Transformation

Strain D66 was mutagenized by introducing the pSP124s plasmid (a gift from Saul Purton, University of London; Lumbreras et al., 1998) linearized with the restriction endonuclease KpnI according to the electroporation procedure described by Shimogawara et al. (1998) with modifications described by Colombo et al. (2002).

Identification of Flanking Regions

Thermal asymmetric interlaced PCR and inverse-PCR were used to obtain the DNA flanking the pSP124s insertion (Liu et al., 1995; Colombo et al., 2002). Homology searches were performed using the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST; Altschul et al., 1997), and at the Joint Genome Institute site (http://aluminum.jgi-psf.org/prod/bin/runBlast.pl?db=chlre1).

Nucleic Acid Isolation and Analysis

Total DNA was isolated from mutant cells grown as patches on TAP plates according to Newman et al. (1990; http://www.biology.duke.edu/chlamy/methods/dna.html). After digestion with restriction enzymes, DNA (2 µg in each lane) was loaded and separated on a 0.8% (w/v) agarose gel and blotted onto a nylon (Schleicher & Schull, Keene, NH) as described by Sambrook et al. (1989). ³²P-dCTP-labeled probes were prepared using a random primer procedure (Sambrook et al., 1989). Total RNA was isolated from mutant cells grown on TAP liquid media according to the Web site http://biosci.cbs.umn.edu/~amundsen/chlamy/methods/RNA_prep.html. Extraction of total RNA and RNA gel-blot analysis were performed by standard procedures (Sambrook et al., 1989). A cosmid containing the full copy of the gene was isolated from an indexed genomic library using a PCR-based technique.

Complementation

Partial complementation of rca1 was observed by expressing the Rca ORF under the control of the PsaD promoter and terminator in the vector pSL18 (Fig. 6A; Depege et al., 2003; gift from Stephane Lemaire, Université Paris-Sud). pSL18 contains the paromomycin resistance gene under the control of the C. reinhardtii Hsp70 and RbcS2 promoters (Sizova et al., 2001) linked to the PsaD promoter and terminator that can be used to express ORFs in C. reinhardtii (Depege et al., 2003). rca1 was transformed with 1 µg of the linearized plasmid using electroporation (Colombo et al., 2002). Transformants were selected on TAP plates containing 10 µg mL^-1 paromomycin (Sigma, St. Louis). Partial complementation was verified by the reappearance of the 42-kD peptide recognized by the Rca antibody after transformation and selection on minimal media in 35 ppm CO₂ in air (Fig. 6B).

Other Methods

Genetic crosses and tetrad analysis were performed as previously described (Sears et al., 1980; Moroney et al., 1986; Harris, 1989). Immunoblots were performed as described earlier (Rawat and Moroney, 1991). Protein extracts were fractionated by SDS-PAGE, transferred to a nitrocellulose membrane (Schleicher & Schull, Keene, NH), and probed with mouse anti-C. reinhardtii Rca polyclonal antibodies provided by Archie Portis (University of Illinois, Urbana). The specific protein bands were visualized using a secondary antibody conjugated to horseradish peroxidase (Bio-Rad Laboratories, Hercules, CA). Photosynthesis rates were measured using a Clark-type O₂ electrode as described earlier (Pollock and Colman, 2001). The light intensity used for the photosynthesis measurements was 1,000 µmol photons m^-2 s^-1. Cell density values were determined by direct counting in a hemacytometer chamber. Chl concentrations were estimated using the equations of Holden (1976).

Received August 20, 2003; returned for revision September 2, 2003; accepted September 9, 2003.

	FOOTNOTES

 
¹ This work was supported by the National Science Foundation (grant nos. IBN-9904425 and IBN-0212093 to J.V.M.) and by the Fund Antorchas (fellowship to S.L.C.).

² These authors contributed equally to the paper.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.032078.

^* Corresponding author; e-mail btmoro{at}lsu.edu; fax 225-578-2597.

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