Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas
1997; Springer Nature; Volume: 16; Issue: 22 Linguagem: Inglês
10.1093/emboj/16.22.6713
ISSN1460-2075
Autores Tópico(s)Protist diversity and phylogeny
ResumoArticle15 November 1997free access Disruption of the plastid ycf10 open reading frame affects uptake of inorganic carbon in the chloroplast of Chlamydomonas Norbert Rolland Norbert Rolland Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France Search for more papers by this author Albert-Jean Dorne Albert-Jean Dorne Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France Search for more papers by this author Gabi Amoroso Gabi Amoroso Department of Biology, University of Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany Search for more papers by this author Dieter F. Sültemeyer Dieter F. Sültemeyer Department of Biology, University of Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany Search for more papers by this author Jacques Joyard Jacques Joyard Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France Search for more papers by this author Jean-David Rochaix Corresponding Author Jean-David Rochaix Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Norbert Rolland Norbert Rolland Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France Search for more papers by this author Albert-Jean Dorne Albert-Jean Dorne Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France Search for more papers by this author Gabi Amoroso Gabi Amoroso Department of Biology, University of Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany Search for more papers by this author Dieter F. Sültemeyer Dieter F. Sültemeyer Department of Biology, University of Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany Search for more papers by this author Jacques Joyard Jacques Joyard Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France Search for more papers by this author Jean-David Rochaix Corresponding Author Jean-David Rochaix Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland Search for more papers by this author Author Information Norbert Rolland1,2, Albert-Jean Dorne2, Gabi Amoroso3, Dieter F. Sültemeyer3, Jacques Joyard2 and Jean-David Rochaix 1 1Departments of Molecular Biology and Plant Biology, University of Geneva, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland 2Laboratoire de Physiologie Cellulaire Végétale, URA 576 (CEA/CNRS/Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, F-38054 Grenoble-cedex 9, France 3Department of Biology, University of Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany The EMBO Journal (1997)16:6713-6726https://doi.org/10.1093/emboj/16.22.6713 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The product of the chloroplast ycf10 gene has been localized in the inner chloroplast envelope membrane (Sasaki et al., 1993) and found to display sequence homology with the cyanobacterial CotA product which is altered in mutants defective in CO2 transport and proton extrusion (Katoh et al., 1996a,b). In Chlamydomonas reinhardtii, ycf10, located between the psbI and atpH genes, encodes a putative hydrophobic protein of 500 residues, which is considerably larger than its higher plant homologue because of a long insertion that separates the conserved N and C termini. Using biolistic transformation, we have disrupted ycf10 with the chloroplast aadA expression cassette and examined the phenotype of the homoplasmic transformants. These were found to grow both photoheterotrophically and photoautotrophically under low light, thereby revealing that the Ycf10 product is not essential for the photosynthetic reactions. However, under high light these transformants did not grow photoautotrophically and barely photoheterotrophically. The increased light sensitivity of the transformants appears to result from a limitation in photochemical energy utilization and/or dissipation which correlates with a greatly diminished photosynthetic response to exogenous (CO2 + HCO3−), especially under conditions where the chloroplast inorganic carbon transport system is not induced. Mass spectrometric measurements with either whole cells or isolated chloroplasts from the transformants revealed that the CO2 and HCO3− uptake systems have a reduced affinity for their substrates. The results suggest the existence of a ycf10-dependent system within the plastid envelope which promotes efficient inorganic carbon (Ci) uptake into chloroplasts. Introduction The complete DNA sequences of a dozen plastid genomes have been determined and they have revealed the existence of many open reading frames of unknown function (for review, see Reardon and Price, 1995). Those that are conserved in algae and in higher and lower plants have been designated ycf for ‘hypothetical chloroplast open reading frame’. The ycf10 gene codes for a putative polypeptide of 229–231 amino acids. This ORF was located upstream of petA (encoding apo-cytochrome f) in tobacco (Shinozaki et al., 1986), rice (Hiratsuka et al., 1989) and Marchantia (Ohyama et al., 1986). In pea, ycf10 has been shown to be cotranscribed with petA (Willey and Gray, 1990; Nagano et al., 1991). The putative ycf10 product was first called HBP (for Heme-Binding Protein) because of a short region of homology, around a conserved histidine residue, to the heme-binding domain of two cytochrome b polypeptides (Willey and Gray, 1990). The Ycf10 product was immuno-localized in the inner membrane of the pea chloroplast envelope (Sasaki et al., 1993; Gray, 1996). Accordingly, ycf10 was renamed cemA (chloroplast envelope membrane). Cyanobacterial mutants affected in CO2 transport have been shown to contain mutations within the cotA gene which displays significant sequence identity to ycf10 (Katoh et al., 1996a). More recently a role for the cotA product in light-induced proton extrusion has been proposed (Katoh et al., 1996b). This raises the possibility that Ycf10 may have a role in CO2 assimilation. To overcome the low affinity of ribulose 1,5-bisphosphate carboxylase (Rubisco) for its substrate, CO2, green algae like Chlamydomonas reinhardtii have developed a carbon concentrating mechanism (CCM) which elevates the CO2 concentration available to Rubisco in order to favour the carboxylation reaction at the expense of the oxygenase reaction (Badger and Price, 1994). Perhaps the most important components of the CCM are carbonic anhydrase and the transport systems for inorganic carbon (Ci). It has been shown that wild-type cells of C.reinhardtii have the capacity to utilize both CO2 and HCO3− for photosynthesis (Moroney et al., 1985; Williams and Turpin, 1987; Sültemeyer et al., 1991; Palmqvist et al., 1994). Periplasmic carbonic anhydrase facilitates CO2 aquisition as it rapidly converts HCO3− to CO2 which in turn is then taken up by the cells (Badger and Price, 1994). Besides the presence of external carbonic anhydrase, the presence of different intracellular isozymes has been demonstrated in mitochondria and chloroplasts (Badger and Price, 1994; Husic and Marcus, 1994; Amoroso et al., 1996; Eriksson et al., 1996). These enzymes are induced 4- to 10-fold when cells are cultivated under limiting CO2 concentrations. However, their exact physiological role is unknown. A cytosolic carbonic anhydrase has been proposed (Sültemeyer et al., 1991; Badger and Price, 1994) but its presence has not yet been conclusively demonstrated. In addition, a Ci transporter in the chloroplast envelope is also induced in cells grown in high-salt-minimum (HSM) medium with air, but not in cells grown in Tris-acetate-phosphate (TAP) medium or in HSM medium with CO2-enriched air (Moroney et al., 1987; Sültemeyer et al., 1988). However, it is not known which Ci species is transported into the plastid. To gain further insights into the function of ycf10, we have disrupted this chloroplast gene in C.reinhardtii, using biolistic transformation. Here we show that the Ycf10 product is not essential for cell viability and photosynthetic function, provided cells are grown under low light. However, the ycf10-deficient mutants are very sensitive to high light, a property which cannot be related to altered chlorophyll, carotenoid or quinone metabolism. Instead, it appears to be a consequence of a limitation in photochemical energy utilization and/or dissipation which correlates with a decrease in CO2-dependent photosynthesis and a reduced affinity of the CO2 and HCO3− uptake systems for their substrates. Results Localization and unusual structure of the ycf10 gene of C.reinhardtii Using a ycf10 tobacco probe, we mapped ycf10 by Southern hybridization on the adjacent chloroplast EcoRI fragments R7 and R8 of C.reinhardtii between rbcL-atpA-psbI and atpH (cf. Figure 3). While still associated with other photosynthetic genes, the ycf10 gene of C.reinhardtii is found in a different region of the chloroplast genome as compared with higher plants. This result is not surprising, due to the considerable variation in chloroplast genome organization in C.reinhardtii compared with land plants (see Boudreau et al., 1994). The chloroplast DNA region containing ycf10 was sequenced and the deduced amino acid sequence of the Ycf10 product is shown in Figure 1. The ycf10 open reading frame is oriented in the same direction as the upstream atpA and psbI genes and the downstream atpH gene (cf. Figure 3). The putative ycf10 ATG initiation codon is preceded by a 40 nucleotide sequence which is highly AT rich (93%) and which lacks any apparent ribosome binding site. However, only 40% of chloroplast genes examined contain a potential Shine–Dalgarno sequence within the 15 nucleotides upstream of the initiation codon (Bonham-Smith and Bourque, 1989). Figure 1.Nucleotide sequence of the ycf10 region of the chloroplast DNA of C.reinhardtii. The ycf10 and atpH DNA and the deduced amino acid sequences are shown. For Ycf10, the amino acids are numbered from the first putative initiating methionine. The two additional in-frame sequences IS1 and IS2 are indicated as well as the more conserved domains CRO, CR1 and CR2. A 16 nucleotide inverted repeat in the ycf10-atpH intergenic region is marked with arrows. The accession number of this sequence in the DDBJ/EMBL/GenBank nucleotide sequence database is X90559. Download figure Download PowerPoint The C.reinhardtii ycf10 open reading frame encodes a putative polypeptide of 500 amino acids with a molecular weight of 57 818, which greatly exceeds the size of its higher plant homologues usually consisting of ∼230 residues. The size of Ycf10 of Marchantia polymorpha (ORF 434) is closer to that of C.reinhardtii. The Ycf10 protein can be divided into several domains based on its sequence conservation in C.reinhardtii, M.polymorpha and higher plants. The N-terminal 80 amino acid region (IS1 in Figure 2) is very basic in C.reinhardtii and followed by a 45 amino acid region, CR0, partially conserved between C.reinhardtii and M.polymorpha (45% sequence identity and 60% sequence similarity). This N-terminal domain is absent from Ycf10 in higher plants. The two conserved regions, CR1 and CR2 are separated by a poorly conserved domain, IS2, which is considerably larger in C.reinhardtii (150 residues, Figure 2). Regions IS1, CR1 and CR2 together contain five hydrophobic domains of which domains 2, 3 and 5 (indicated by black boxes in Figure 2A) are predicted to be transmembrane segments (Persson and Argos, 1994; Rost et al., 1995). In C.reinhardtii and M.polymorpha the CR1 regions (68 amino acids) are 50% identical and 54% similar and the CR2 regions (138 amino acids) are 44% identical and 60% similar. Comparison of 10 known Ycf10 protein sequences reveals poor conservation: 3% identity (11% similarity) for CR1 and 14% identity (22% similarity) for CR2 (Figure 2B). Figure 2.Comparative analysis of the structure of Ycf10 of C.reinhardtii, cyanobacteria and plants. Cr, C.reinhardtii; S1 (slr1596) CotA, Synechocystis sp. PCC 6803 (Katoh et al., 1996a); S2 (sll1168) Synechocystis sp. PCC 6803; Mp, M.polymorpha (Ohyama et al., 1986); Pp, Porphyra purpurea (Reith and Munholland, 1995); Pt, Pinus thunbergii (Wakasugi et al., 1994); Zm, Zea mays (Maier et al., 1995); Sb, soybean (N.Nielsen, 1995, Swissprot P49160); Nt, Nicotiana tabacum (Shinozaki et al., 1986); Os, Oryza sativum (Hiratsuka et al., 1989) and Ps, Pisum sativum (Willey and Gray, 1990). (A) Diagram displaying the regions of homology between the C.reinhardtii and plant proteins. Gray regions (CRO, CRI and CR2) represent conserved domains. White regions are less conserved (IS1 and IS2). Black boxes correspond to hypothetical membrane-associated helices (Persson and Argos, 1994; Rost et al., 1995), open boxes correspond to hydrophobic domains. Hydropathy plots (Kyte and Doolittle, 1982) are shown for pea (Willey and Gray, 1990) and C.reinhardtii (span-length of nine amino acids). (B) Sequence comparison of the Ycf10 proteins. Letters or asterisks on the consensus line (CO) indicate identical or functionally similar amino acids with one possible mismatch according to the following grouping: ILMV; ASPTG; NQ; DE; KRH; FYW and C. Dashes represent gaps introduced to maximize similarity. Numbers refer to the last amino acid of the regions shown. The putative membrane associated helices are underlined. The open wedge indicates the non-conserved his residue proposed to be involved in heme binding (Willey and Gray, 1990). The position of the initiator met of CotA (S1) proposed by Katoh et al. (1996a) is indicated by a closed wedge. Download figure Download PowerPoint Figure 3.Disruptions of the ycf10 gene of C.reinhardtii. The map of the region of the chloroplast genome containing atpH, ycf10, psbI, atpA and rbcL is shown. Except for rbcL transcription of the other genes proceeds from right to left. The two aadA cassettes used for ycf10 disruption, 5′ atpA-aadA- 3′rbcL and 5′ psbD-aadA, were inserted in the orientations shown. The transformants obtained with the corresponding constructs are indicated in the legend of Figure 4. Download figure Download PowerPoint Besides the reported homology between Ycf10 and the CotA gene product from Synechocystis sp. 6803 (37% sequence identity, 59% sequence similarity; Katoh et al., 1996a), we found that another gene from Synechocystis (accession number sll1685) encodes a protein related to Ycf10 (23% sequence identity, 48% sequence similarity, Figure 2B). The chloroplast ycf10 gene is not required for cell viability To investigate the function of ycf10 in C.reinhardtii, this ORF was disrupted independently at two unique sites (NdeI within the CR1 domain and EcoRI within the CR2 domain, cf. Figures 1 and 3) with the aadA expression cassette (Goldschmidt-Clermont, 1991). Since this cassette contains the rbcL 3′ end which is known to exert a polar effect on transcription and/or RNA accumulation of the downstream region, it was inserted in the antisense direction at the two sites indicated relative to the transcription of ycf10. The corresponding constructs were introduced into a wild-type strain of C.reinhardtii, using biolistic transformation and selecting for spectinomycin resistance. In transformants 2D and 13A the aadA cassette was inserted at the EcoRI and NdeI sites of ycf10, respectively. Hybridization of PstI-digested DNA of transformant 2D with an aadA probe revealed as expected a 6.8 kbp fragment (Figure 4A). However, further analysis of this transformant revealed loss of atpA, psbI and part of ycf10 (Figure 4A and data not shown). This deletion was created through homologous recombination between the 3′ rbcL end of the aadA cassette and the authentic 3′ rbcL end which are oriented in the same direction 6 kbp apart (Figure 3, data not shown). A similar deletion also occurred in transformant 13A in which additional DNA rearrangements were observed (Figure 4A). Since the homoplasmic transformant 2D was still able to grow on a medium containing acetate (cf. Figure 5), it can be concluded that the entire chloroplast DNA region between rbcL and ycf10 is not essential for cell viability. Figure 4.Southern analysis of the ycf10::aadA transformants. (A) Transformants 2D and 13A were obtained with the 5′ atpA -aadA-3′ rbcL cassette inserted at the EcoRI and NdeI sites, respectively. Ct is a control transformant in which the aadA cassette was inserted near atpB. Total DNA was digested with PstI and hybridized with the probes aadA (0.7 kbp NcoI–SphI fragment, corresponding to the coding sequences of aadA), ycf10 (0.7 kbp XbaI–EcoRI fragment of R7 corresponding to part of the ycf10 coding regions, residues 226–453) and atpA (1 kbp PstI–EcoRI fragment of R7 corresponding to an internal portion of atpA). (B) Transformants T1, T2 and T3 were obtained with the 5′ psbD-aadA cassette. Total DNA was digested with HindIII and hybridized with the ycf10 probe. The 1.7 kbp band corresponds to the wild-type DNA and the 2.5 and 2.6 kbp bands correspond to the transformant DNA after integration of the aadA cassette in the EcoRI (T1 same orientation as ycf10) and NdeI site (T2, T3, same and opposite orientation as ycf10, respectively). Download figure Download PowerPoint Figure 5.Growth patterns of the ycf10-deficient transformants. Equal number of cells were plated either on TAP medium (photoheterotrophic growth) or on HSM medium (photoautotrophic growth in the presence or absence of 150 μg/ml spectinomycin; spec). Plates were photographed after 10 days of growth under strong light (SL; 90 μE/m2/s) or dim light (DL; 3 μE/m2/s). HSM plates exposed to dim light were photographed after 18 days. Controls included cells from wild type (WT); Ct;T FuD50 lacking ATP synthase and transformant 2D. Download figure Download PowerPoint To avoid chloroplast DNA rearrangements, another cassette in which aadA is driven by the promoter and 5′ untranslated region of psbD, but lacking a chloroplast termination or processing site, was used for transformation. Transformants T1 and N3-1 were obtained with the cassette inserted at the EcoRI site in the same orientation as ycf10 (Figure 3). Transformants T2 and T3 had the cassette inserted at the NdeI site in the same and opposite orientation as ycf10, respectively (Figure 3). As shown in Figure 4B hybridization of the ycf10 probe to HindIII digested DNA yields a 1.72 kbp fragment in the wild type. The corresponding fragments of the transformants are 2.45 (T1) and 2.62 kbp (T2 and T3) as expected from a homologous recombination event at the EcoRI and NdeI sites (including the 0.17 kbp EcoRI–HindIII deletion in T1), respectively. Although the ycf10 and atpH genes are oriented in the same direction and separated by only 223 bp, disruption of ycf10 did not significantly affect the level of atpH mRNA (data not shown). The chloroplast ycf10 gene is not essential for the photosynthetic reactions Fluorescence transients of six independently generated ycf10::aadA transformants were undistinguishable from those of wild type, thus indicating that photosystem I and II and the cytochrome b6f complex are not significantly affected in these mutants (data not shown). The growth pattern of the ycf10 transformants was compared with several control strains including wild type, Fud50, a mutant lacking atpB, a control strain (Ct) containing the aadA cassette in a region of the chloroplast genome which does not encode any essential or photosynthetic gene and the transformant 2D with a deletion of ycf10, psbI and atpA (see above). These strains were tested under photoheterotrophic conditions (TAP medium), and photoautotrophic conditions (HSM medium) under high (90 μE/m2/s) or dim light (3 μE/m2/s) in the presence or absence of spectinomycin (Figure 5). As expected, the control transformant (Ct) grew under all conditions tested, and the wild-type and Fud50 strains did not grow on spectinomycin plates. Fud50 and strain 2D were unable to grow on HSM medium and their growth was impaired in high light on TAP medium because of their photosensitivity (Figure 5), due to the lack of ATP synthase (Woessner et al., 1982). The transformants T1, T2 and T3 grew as fast as wild type and the control strain on TAP medium in dim light. On HSM medium in dim light the growth patterns of the transformants were the same as for wild type, although growth was considerably slower than on TAP medium. Hence no essential photosynthetic function is affected in the transformants lacking functional ycf10. Loss of ycf10 leads to increased light sensitivity On TAP medium in high light (90 μE/m2/s) growth of the ycf10 mutants was considerably impaired as compared with wild type and the control strain Ct in the presence of spectinomycin (Figure 5). These mutants appeared to be even more photosensitive than the ATP synthase lacking mutant Fud50. Growth of the ycf10 mutants was undetectable on HSM medium in high light (Figure 5). Interestingly, the ability of the ycf10 mutants to grow in HSM medium in high light was partially restored in the presence of spectinomycin. Since carotenoid deficiency is known to confer light sensitivity, the carotenoid and chlorophyll content of wild-type and the ycf10-deficient mutants were determined and compared. No significant difference was detected for wild-type cells grown in dim light or grown in dim light and transferred to high light for 24 h (Table I). Furthermore, we observed no significant difference in carotenoid composition between wild-type and mutant cells grown in dim light. This observation appears to rule out a direct role of the ycf10 gene product in the carotenoid biosynthetic pathway. Table 1. Pigment composition of wild-type and ycf10-deficient mutant cells Wild-type DL Mutants DL Wild-type SL 24 h Mutants SL 24 h Chla/Chlb 2.44 2.59 2.42 2.31 Car/Chla+b 0.27 0.26 0.20 0.22 Carotenoid composition (% of total carotenoids): α- and β-carotene 40.5 42.1 36.5 20.4 Lutein 18.6 15.8 20.0 35.6 Violaxanthin 12.6 11.0 10.0 8.7 Loroxanthin 15.3 13.6 8.4 4.8 Neoxanthin 8.9 10.1 21.0 13.0 Zeaxanthin/Cryptoxant. n.d. n.d. n.d. 10.0 Others 4.6 4.5 4.2 6.9 Chla/b, chlorophyll a, b; Car, carotenoids. The values presented for the mutants correspond to the average obtained from the three ycf10::aadA transformants (T1, T2 and T3). The cultures (400 ml) were grown in dim light (DL; 2 μE/m2/s) until the mid-log phase (2×106 cells/ml). Half of the culture was kept in dim light and the other half was transferred to high light (SL: 90 μE/m2/s) for 24 h. n.d., not detected. Chlorophyll content per 106 cells was 4.13 μg for wild type and 3.7 ± 0.3 μg for the three mutants. Carotenoid content per 106 cells was 1.01 μg for wild type and 1.03 ± 0.1 μg for the mutants. Light treatment did not affect these values significantly. However, upon transfer to high light, the carotenoid composition was markedly modified in the ycf10 mutants (Table I). α- and β-carotene were reduced two-fold and lutein was increased >2-fold in the mutants. Other pigments were observed in mutant cells that were undetectable in wild-type cells grown under the same conditions. In particular one of the major pigments accumulating in high light was identified as zeaxanthin, a pigment directly involved in energy dissipation. Evidence has been presented that one major pathway for zeaxanthin synthesis occurs through hydroxylation of β-carotene via β-cryptoxanthin (Demmig-Adams, 1990). The 50% reduction of β–carotene in the mutant cells (Table I) could be directly correlated to the accumulation of zeaxanthin. The latter was not detectable in wild-type cells grown under the same conditions. It is an excess of light, and not high light per se, that induces the increased accumulation of zeaxanthin (for review see Demmig-Adams, 1990). These results suggest that disruption of ycf10 lowers the threshold level of light perceived as excessive. However, similar changes in carotenoid composition upon exposure to high light as observed for the ycf10-deficient strains were also found in FuD50, a mutant strain that is light-sensitive because it lacks ATP synthase (data not shown). The quinone (phylloquinone, plastoquinone and α-tocopherol) content was not found to be affected in the mutants when compared with wild-type cells (not shown), thus excluding a role for the ycf10 product in the biosynthesis or regulation of these compounds. Loss of ycf10 strongly limits the apparent photosynthetic affinity for Ci when mutant cells have reached the CO2 compensation point A significant sequence homology has been noticed between Ycf10 and the cyanobacterial CotA protein which has been shown to be required for CO2 uptake and proton extrusion (Katoh et al., 1996a,b) thus raising the question whether Ycf10 may may be involved in CO2 assimilation. Several mutants of C.reinhardtii affected in this process have been shown to be light-sensitive (Spreitzer and Mets, 1981; Spreitzer and Ogren, 1983a). To test whether the apparent photosynthetic affinity for CO2 is affected in the ycf10-deficient mutants, cells from wild type and the T1 mutant were grown either in HSM medium in dim light or in TAP medium in the dark. Cells were then collected and resuspended in 10 mM HEPES pH 7.15 in an illuminated O2 electrode chamber (872 μE/m2/s) for 1.5 h to deplete their internal Ci pool (‘low Ci’ cells). At this stage cells had reached their CO2 compensation point (i.e. the concentration of CO2 at which the CO2 fixed by photosynthesis is equal to the CO2 released by respiration) which is near zero for C.reinhardtii (Spalding et al., 1983b and Figure 6, as deduced from the O2 evolution curves). CO2-dependent photosynthetic O2 evolution of the cells was measured after addition of increasing amounts of Ci. To test for photoinhibition under these conditions, Fv/Fm fluorescence measurements were performed. The T1 mutant was found to be slightly more photoinhibited than wild type as the Fv/Fm value decreased from 0.77 ± 0.02 before, and to 0.37 ± 0.02 after the light treatment whereas the corresponding wild-type values were 0.78 ± 0.03 and 0.56 ± 0.05, respectively. Figure 6.Upper part: scheme of inorganic carbon fluxes in the wild-type and the ycf10-deficient T1 mutant of C.reinhardtii grown on HSM or TAP medium. Under the former growth conditions, a bicarbonate transporter is induced in the chloroplast envelope. The periplasmic, putative cytosolic and chloroplast carbonic anhydrases (CA) are indicated (Moroney et al., 1987; Husic et al., 1989). Chloro, chloroplast; Mito, mitochondria; Cyto, cytosol. Lower part: response of photosynthesis to external [CO2 + HCO−3] in the wild-type (WT) and ycf10-deficient mutant (T1) cells pregrown in HSM in dim light (3 μE/m2/s, left panels) or TAP in the dark (right panels). Prior to all measurements, cells were transferred into 10 mM HEPES pH 7.15 for 90 min in high light (872 μE/m2/s) until cessation of O2 evolution. In panels A–D photosynthetic oxygen evolution was measured continuously with increasing amounts of Ci which was added in the form of NaHCO3 (a, 0.25 nmol; b, 2.5 nmol; c, 12.5 nmol; d, 25 nmol; e, 62.5 nmol; f, 187.5 nmol; g′, 187.5 nmol; g″, 1.25 μmol; h, 2.5 μmol). (A) Cells pregrown in HSM medium. The chlorophyll concentration for wild-type and T1 cells was 4.5 and 4 μg/ml, respectively. DCMU was added to a final concentration of 5 μM. (B) Same as (A), except that the cells were pregrown in TAP medium. The chlorophyll concentration for the wild-type and T1 cells was 5.7 and 5.4 μg/ml, respectively. (C) Effect of EZ: cells pregrown in HSM medium. After cessation of O2 evolution, the cells were transferred to the dark and later to the light as indicated. EZ (at a final concentration of 100 μM), HCO−3 (2.5 mM) and DCMU (5 μM) were added as shown. The chlorophyll concentration for the wild-type and T1 cells was 5 and 4.4 μg/ml, respectively. (D) Same as in C for cells pregrown in TAP medium. The chlorophyll concentration for the wild-type and T1 cells was 5 and 4 μg/ml, respectively. The vertical scale represents 20 nmol O2. (E and F) Estimation of the affinities (K1/2) for HCO3− and CO2 from mass-spectrometric measurements of HCO3− and CO2 uptake in cells grown in HSM (E) or TAP (F) medium. The K1/2 values were determined from response curves similar to those shown in Figure 7. Downlo
Referência(s)