Inactivation of the Chloroplast ATP Synthase γ Subunit Results in High Non-photochemical Fluorescence Quenching and Altered Nuclear Gene Expression in Arabidopsis thaliana
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m308435200
ISSN1083-351X
AutoresCristina Dal Bosco, Lina Lezhneva, Alexander Biehl, Dario Leister, Heinrich Strotmann, G. Wanner, Jörg Meurer,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe nuclear atpC1 gene encoding the γ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the γ subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase α and β subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1. The nuclear atpC1 gene encoding the γ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the γ subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase α and β subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1. In oxygenic photosynthesis light-dependent electron transport from water to NADP+ is coupled to synthesis of ATP (photophosphorylation) and energetically mediated by a transmembrane electrochemical proton gradient. Photophosphorylation, which essentially resembles oxidative phosphorylation, is carried out at the thylakoid membrane of photosynthetic eubacteria and chloroplasts and is catalyzed by a proton-translocating reversible ATPase (“ATP synthase”). The basic organization, structure, and composition of this protein complex have been extensively investigated on the eubacterial level as well as in mitochondria and plastids and were found to be vastly conserved (1.Strotmann H. Shavit N. Leu S. The Molecular Biology of Chloroplast and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Norwell, MA1998: 477-500Google Scholar, 2.Groth G. Pohl E. J. Biol. Chem. 2001; 276: 1345-1352Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The plastid ATP synthase complex consists of nine different subunits, four of them are localized in the membrane integral CF0 subcomplex (a, b, b′, and c14) which is responsible for proton translocation, and five subunits constitute the extrinsic CF1 subcomplex (α3, β3, γ, δ, and ϵ) which forms the catalytic entity (3.Groth G. Strotmann H. Physiol. Plant. 1999; 106: 142-148Crossref Scopus (30) Google Scholar, 4.Nelson N. Curr. Opin. Cell Biol. 1992; 4: 654-660Crossref PubMed Scopus (69) Google Scholar). Three independent studies proved the rotation of the γ subunit relative to (αβ)3 during ATP hydrolysis in the isolated F1 (5.Ducan T.M. Bulygin V.V. Zhou Y. Hutcheon M.L. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10964-10968Crossref PubMed Scopus (461) Google Scholar, 6.Sabbert D. Engelbrecht S. Junge W. Nature. 1996; 381: 623-625Crossref PubMed Scopus (465) Google Scholar, 7.Noji H. Yasuda R. Yoshida M. Kinosita K. 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The seven amino acids Cys199–Cys205 (numbering in Arabidopsis) are present only in land plants and green algae as in Chlamydomonas reinhardtii (10.Ketcham S.R. Davenport J.W. Warncke K. McCarty R.E. J. Biol. Chem. 1984; 259: 7286-7293Abstract Full Text PDF PubMed Google Scholar, 12.Ross S.A. Zhang M.X. Selman B.R. J. Biol. Chem. 1995; 270: 9813-9818Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Nevertheless, differences in the redox regulation mechanism have been pointed out between algae and higher plants (13.Ort D.R. Oxborough K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 269-291Crossref Scopus (82) Google Scholar). Whereas in higher plants the re-oxidation of CF1, following a light-dark transition, takes over 1 h, in the alga Dunaliella salina CF1 is several times faster re-oxidized by a specific endogenous oxidant (14.Selman-Reimer S. Duke R.J. Stockman B.J. Selman B.R. J. Biol. Chem. 1991; 266: 182-188Abstract Full Text PDF PubMed Google Scholar). Only recently redox modulation of the γ rotation has also been reported (15.Bald D. Noji H. Yoshida M. Hirono-Hara Y. Hisabori T. J. Biol. Chem. 2001; 276: 39505-39507Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In bacteria the genes for the two subcomplexes are organized in separate operons suggesting that the enzyme evolutionarily derived from a proton channel (F0) and an ATPase (F1) (16.Falk G. Walker J.E. Biochem. J. 1988; 254: 109-122Crossref PubMed Scopus (71) Google Scholar). The organization of the genes reflects also a possible mechanism of assembly. The bacterial F0 and F1 accumulate independently and associate then to the membrane to assemble a functional complex (17.Klionsky D.J. Simoni R.D. J. Biol. Chem. 1985; 260: 11207-11215Abstract Full Text PDF PubMed Google Scholar). However, only little is known about the assembly of the chloroplast enzyme (1.Strotmann H. Shavit N. Leu S. The Molecular Biology of Chloroplast and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Norwell, MA1998: 477-500Google Scholar). In Chlamydomonas neither CF0 nor CF1 is assembled, although the unimpaired polypeptides are synthesized, if one of the nine subunits is missing. Several mutants in Chlamydomonas affected in different chloroplast-encoded subunits of the ATP synthase have been used to characterize the assembly process (18.Wollman F.A. Minai L. Nechushtai R. Biochim. Biophys. Acta. 1999; 1411: 21-85Crossref PubMed Scopus (218) Google Scholar). In Arabidopsis two genes, atpC1 and atpC2 (accession numbers M61741 and J05761, respectively), located on chromosomes 4 and 1, respectively, encode for the plastid γ subunit (19.Inohara N. Iwamoto A. Moriyama Y. Shimomura S. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 7333-7338Abstract Full Text PDF PubMed Google Scholar, 20.Legen J. Misera S. Herrmann R.G. Meurer J. DNA Res. 2001; 8: 53-60Crossref PubMed Scopus (14) Google Scholar). In plants grown under continuous illumination, atpC1 is much higher expressed than atpC2 (19.Inohara N. Iwamoto A. Moriyama Y. Shimomura S. Maeda M. Futai M. J. Biol. Chem. 1991; 266: 7333-7338Abstract Full Text PDF PubMed Google Scholar). The two AtpC proteins in Arabidopsis share 73% sequence homology, whereas the homology of the AtpC subunits between other plants and AtpC1 in Arabidopsis is about 88 ± 4%. This raises the intriguing question about the role(s) of the two atpC gene copies in Arabidopsis, and whether the two genes possess distinct functions and under which physiological conditions they become operative. Proton efflux through the plastid ATP synthase, associated with ATP synthesis, results in an accelerated relaxation of the electric field-associated absorption change at 518 nm (21.Witt H.T. Biochim. Biophys. Acta. 1978; 505: 355-427Crossref Scopus (430) Google Scholar). This property has been used to identify coupling factor reduction mutants in Arabidopsis which grow poorly in dim light and showed slowed ΔA518 decay after light-dark transition (22.Gabrys H. Kramer D.M. Crofts A.R. Ort D.R Plant Physiol. 1994; 104: 769-776Crossref PubMed Scopus (18) Google Scholar). One coupling factor quick recovery mutant, cfq, contains an E244D point mutation in the γ subunit leading to a decreased acidification of the lumen compared with wild type in the initial few minutes of the induction period under subsaturating light conditions. This mutant has been used to correlate non-photochemical chlorophyll fluorescence quenching (NPQ) 1The abbreviations used are: NPQ, non-photochemical quenching; chl, chlorophyll; PAM, pulse amplitude modulation; PSI and PSII, photosystem I and II, respectively; QA, primary electron acceptor quinones of PSII; qE, high energy quenching; WT, wild type; PVDF, polyvinylidene difluoride; T-DNA, transfer DNA; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. with ΔpH (23.Govindjee Spilotro P. Funct. Plant Biol. 2002; 29: 425-434Crossref PubMed Scopus (15) Google Scholar). In the present study we have identified the Arabidopsis thaliana mutant dpa1 in which the atpC1 gene has been inactivated by T-DNA insertion. The mutant has been characterized on the level of accumulation, activity, and assembly of ATP synthase. Deficiency of the γ subunit leads to loss of ATP synthesis and unusual NPQ which is much below the dark Fo level due to proton accumulation in the lumen. The pattern of mRNA expression of nuclear genes coding for chloroplast proteins in dpa1 is interpreted as a compensatory effect. Plant Material, Growth Conditions, and Mutant Selection—Seed sterilization and growth conditions of dpa1 and wild type were as described (24.Meurer J. Meierhoff K. Westhoff P. Planta. 1996; 198: 385-396Crossref PubMed Scopus (176) Google Scholar) with the exception that only 1.4% (w/v) sucrose has been supplemented to the medium. Seedlings were grown under continuous light at a photon flux density of 20 μmol photons m–2 s–1 and at a constant temperature of 21 °C if not otherwise indicated. Prior to illumination, plates were placed for 2 days at 4 °C to synchronize germination. Propagation of the dpa1 mutant occurred via heterozygous offsprings. All comparisons between mutant and wild type were carried out with leaf material of the same developmental stage. Individual mutant plants of segregants grown in Petri dishes have been identified by chlorophyll (chl) fluorescence video imaging (FluorCam 690M; Photon Systems Instruments, Brno, Czech Republic). The FluorCam software package (protocol for quenching analysis) has been used to follow the chl fluorescence induction and to determine fluorescence parameters and PSII yield. The dpa1 mutant could easily be distinguished from wild type plants due to a strong NPQ (see below). Molecular Mapping and Complementation Studies—F1 populations were produced by pollinating emasculated flowers of the accession Landsberg erecta with Wassilewskija plants heterozygous for the dpa1 mutation. F2 families selected for the mutant offsprings were grown on medium, and individual mutant plants were chosen for genetic mapping of the mutation with molecular markers. The oligonucleotides 5′-CAGTCGAATCTTGATGACCGTCGATGATG-3′ and 5′-GTTCGTCGAGAATCAGAGTGGCTC-3′ were used as a simple sequence length polymorphism marker Cer452226 (25.Jander G. Norris S.R. Rounsley S.D. Bush D.F. Levin I.M. Last R.L. Plant Physiol. 2002; 129: 440-450Crossref PubMed Scopus (538) Google Scholar) located 850 kb upstream of the atpC1 gene on the bacterial artificial chromosome F4C21. This marker is polymorphic between Landsberg and Wassilewskija producing 203- and 285-bp PCR products, respectively. The full-length coding sequence of the intron-less atpC1 gene was amplified by Pfu polymerase from wild type genomic DNA using the forward primer 5′-AACAAAAAAATGGCTTGCTCTAATCTAACA-3′ and the reverse primer 5′-AAGAGGGTTCTAGACAAATCAAACCTGTGC-3′, which inserts an XbaI restriction site at the 3′-end of the gene. The XbaI-digested and -purified PCR product was ligated into the SmaI/XbaI sites of the binary vector pS001-VS under control of the cauliflower mosaic virus 35S rRNA promoter (26.Reiss B. Klemm M. Kosak H. Schell J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3094-3098Crossref PubMed Scopus (69) Google Scholar). Successful cloning was verified by sequencing. The construct was introduced into progenies of plants that segregate the mutation via Agrobacterium using the floral dip method (27.Clough S.J. Bent A.F. Plant J. 1998; 16: 735-743Crossref PubMed Google Scholar). Transformant plants were efficiently selected on rock wool (Grodan, Hobro, Denmark) immersed in 10 mg/liter sulfadiazine prior to spreading the seeds (28.Hadi M.Z. Kemper E. Wendeler E. Reiss B. Plant Cell Rep. 2002; 21: 130-135Crossref Scopus (24) Google Scholar). For PCR analysis of transformed lines (see Fig. 1) the following primers have been used: Atactr (5′-GCTCATTCTGTCGGCGATTCCAGG-3′) and Atactf (5′-TCCTAGTATTGTGGGTCGTCCTCG-3′) of the actin3 gene as a control; primer 1 (5′-CGAACCATCCACTAATACCCAGCC-3′) of the atpC1 promoter; primer 2 (5′-CGTCTCTTCGTGAGCTCAGAGACCGTATCG-3′) of the 5′ atpC1 coding region; primer 3 (5′-GCATAGATGCACTCGAAATCAGCC-3′) of the left border of the T-DNA; primer 4 (5′-GGTGATAAAGGCAGTAGCGTGTGGATCACG-3′) of the 3′ atpC1 coding region; and primer Pv (5′-GCCATCGTTGAAGATGCCTCTGCCG-3′) of the 35S promoter. Northern and Southern Analyses—Total RNA and DNA were isolated and subjected to Northern and Southern analysis, respectively, as described (24.Meurer J. Meierhoff K. Westhoff P. Planta. 1996; 198: 385-396Crossref PubMed Scopus (176) Google Scholar, 29.Steiner J.J. Poklema C.J. Fjellstrom R.G. Elliott L.F. Nucleic Acid Res. 1995; 23: 2569-2570Crossref PubMed Scopus (145) Google Scholar, 30.Dellaporta S.L. Wood J. Hicks J.B. 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The full-length cDNAs obtained from the ABRC (Arabidopsis Biological Resource Center at Ohio State) were amplified using the vectorial (pZL1, Invitrogen) forward primer 5′-TAATACGACTCACTATAGGG-3′ and the reverse primer 5′-ATTTAGGTGACACTATAG-3′. Real Time PCR Technique—The LightCycler Thermal Cycler System (Roche Applied Science) was used to perform quantitative two-step reverse transcriptase-PCR for atpC1, atpC2 mRNA, and 18S rRNA applying the SYBR Green protocol (33.Wittwer C.T. Ririe K.M. Andrew R.V. David D.A. Gundry R.A. Balis U.J. BioTechniques. 1997; 22: 176-181Crossref PubMed Scopus (826) Google Scholar). cDNA synthesis was carried out with total RNA using SuperScript II RNase H– Reverse Transcriptase (Invitrogen) and hexanucleotides according to the manufacturer's instructions (Roche Applied Science). Primer combinations specific for the 5′-ends of atpC1 (primer 2 and 5′-GGTTAAGGGAACATCGACATCATCGG-3′), atpC2 (5′-GCTTCGAGCTCAGAGTCCTACTCTT-3′ and 5′-CCAGTAACAACAACCAAAGCAACTCTC-3′), and the 18S rRNA (5′-GCTCAAAGCAAGCCTACGCTCTGG-3′ and 5′-GGACGGTATCTGATCGTCTTCGAGCC-3′) were chosen. After addition of MgCl2 (4 mm) and template cDNA to the master mix, an initial denaturation step followed by 45 cycles of denaturation (95 °C for 15 s), annealing (58 °C, atpC1 and atpC2, or 63 °C, 18S rRNA for 5 s) and extension (72 °C for 1 s/20 bp) were performed. All ramp rates were set to 20 °C per s. Serially diluted samples of Arabidopsis genomic DNA, corresponding to 15 ng to 1.5 pg, were used for calibration. Specific amplification has been confirmed by melting curve analysis and agarose gel electrophoresis. Photophosphorylation—Chloroplast thylakoids from Arabidopsis leaves were prepared as described for spinach (34.Strotmann H. Bickel-Sandkötter S. Biochim. Biophys. Acta. 1977; 460: 126-135Crossref PubMed Scopus (111) Google Scholar). The reactions were conducted in a ΔpH clamp instrument as described (35.Strotmann H. Thelen R. Müller W. Baum W. Eur. J. Biochem. 1990; 193: 879-886Crossref PubMed Scopus (23) Google Scholar). The reaction cell of 2.5-ml volume contained a medium consisting of 25 mm Tricine buffer, pH 8.0, 5 mm dithiothreitol, 5 mm MgCl2, 5 mm32P-labeled Na2HPO4, 50 mm KCl, 50 μm phenazine methosulfate, and thylakoids corresponding to a chl concentration of 25 μg/ml. The experiments were conducted at pre-chosen ΔpH values (35.Strotmann H. Thelen R. Müller W. Baum W. Eur. J. Biochem. 1990; 193: 879-886Crossref PubMed Scopus (23) Google Scholar, 36.Kothen G. Schwarz O. Strotmann H. Biochim. Biophys. Acta. 1995; 1229: 208-214Crossref Scopus (13) Google Scholar) which were kept constant throughout the experiment by the employed clamp device. ΔpH was continuously controlled by the fluorescence quenching of 9-aminoacridine. The fluorescence signal was calibrated as described (37.Schwarz O. Strotmann H. Photosynth. Res. 1998; 57: 287-295Crossref Scopus (5) Google Scholar). The thylakoids were pre-illuminated for 2 min to obtain the prechosen proton gradient. Then 0.5 mm ADP was added in the light. After 10, 20, and 30 s, 0.2-ml samples were taken and deproteinized by HClO4 (final concentration 0.6 m). The formed 32P-labeled organic phosphate was determined as described (38.Avron M. Biochim. Biophys. Acta. 1960; 40: 257-272Crossref PubMed Scopus (505) Google Scholar). Immmunological and Translation Analyses—Thylakoid membrane proteins of 3-week-old plants were isolated as described (39.Meurer J. Berger A. Westhoff P. Plant Cell. 1996; 8: 1193-1207Crossref PubMed Scopus (73) Google Scholar). Proteins were quantified (40.Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), and samples were heated for 5 min at 80 °C in 2% SDS mixed with 1/10 volume of glycerol/dye solution and immediately applied onto SDS-PAGE at 30 mA for 12–15 h at room temperature. The protein pattern was visualized in silver-stained gels (41.Blum H. Beier H. Gross H.J. Electrophoresis. 1987; 8: 93-99Crossref Scopus (3742) Google Scholar). For immuno-detection, proteins were transferred to PVDF membranes (Pall Biodyne, Dreieich, Germany) by semi-dry electroblotting (Peqlab, Erlangen, Germany). The membranes were incubated with antisera raised against thylakoid membrane proteins, and signals were identified by the enhanced chemiluminescence technique (Amersham Biosciences). Most of the antibodies used in this study were raised in rabbits against Chlamydomonas or spinach polypeptides (24.Meurer J. Meierhoff K. Westhoff P. Planta. 1996; 198: 385-396Crossref PubMed Scopus (176) Google Scholar). For in vivo labeling analysis intact leaves of 3-week-old plants were immersed in a ½× MS solution containing 50 μCi of [35S]methionine for 20 min (42.Meurer J. Plücken H. Kowallik K.V. Westhoff P. EMBO J. 1998; 17: 5286-5297Crossref PubMed Scopus (181) Google Scholar). Subsequently, thylakoid membrane proteins were isolated, subjected to SDS gel electrophoreses, and transferred to PVDF membranes. Incorporation was detected by fluorography (43.Laskey R.A. Methods Enzymol. 1980; 65: 363-371Crossref PubMed Scopus (238) Google Scholar). Electron Microscopy—Sample preparations for ultrastructural analysis and electron microscopy were performed as described (44.Swiatek M. Regel R.E. Meurer J. Wanner G. Pakrasi H.B. Ohad I. Herrmann R.G. Mol. Genet. Genomics. 2003; 268: 699-710Crossref PubMed Scopus (59) Google Scholar). Fluorometric and Absorption Studies—chl a fluorescence measurements were performed with 3-week-old plants using a commercial pulse amplitude modulated fluorometer PAM 101 interphased with the PAM data acquisition system PDA-100 (Walz, Effeltrich, Germany). The fiber optic was held 2 mm distant from the upper side of plants grown under sterile conditions in Petri dishes. Leaves were dark-adapted for 5 min prior to the induction fluorescence measurements. The minimal (Fo), steady state (Fs), and maximal (Fm) fluorescence yield, and the variable fluorescence (Fv), calculated as (Fm – Fo), as well as the ratio Fv/Fm, which reflects the potential yield of the photochemical reaction of PSII (45.Krause G.H. Weis E. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1991; 42: 313-349Crossref Scopus (3695) Google Scholar), were recorded at 20 °C, unless noted otherwise. Photochemical quenching and NPQ was determined as described (46.van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar). The intensity of the saturating light flash (800 ms) used for detection of Fm and the maximal fluorescence during induction, Fm′, was 4000 μmol photons m–2 s–1. For nigericin studies, leaves of 3-week-old plants were harvested and cut into small pieces with a sharp razor blade at 4 °C in 50 mm HEPES (pH 7.6), 330 mm sorbitol, 1 mm MgCl2, 1 mm MnCl2, 2 mm EDTA, and 0.2% (w/v) defatted bovine serum albumin. The resuspension buffer was supplied with 0.1 mm methylviologen as an electron acceptor in all samples and, when indicated, with 2 μm nigericin as a ionophore (47.Nishio J.N. Whitmarsh J. Plant Physiol. 1993; 101: 89-96Crossref PubMed Scopus (76) Google Scholar). Expression Profiling Using Nuclear Arrays—The 3292 gene sequence tags array representing genes known or predicted to code for proteins having a chloroplast transit peptide has been described previously (48.Kurth J. Varotto C. Pesaresi P. Biehl A. Richly E. Salamini F. Leister D. Planta. 2002; 215: 101-109Crossref PubMed Scopus (33) Google Scholar, 49.Richly E. Dietzmann A. Biehl A. Kurth J. Laloi C. Apel K. Salamini F. Leister D. EMBO Rep. 2003; 4: 491-498Crossref PubMed Scopus (100) Google Scholar). At least three experiments with different filters and independent cDNA probes derived from plant material corresponding to pools of at least 50 individuals were performed for each condition or genotype tested, thus minimizing variations between individual plants, filters, or probes. cDNA probes were synthesized by using a mixture of oligonucleotides matching the 3292 genes in antisense orientation as primer, and hybridized to the gene sequence tags array as described previously (48.Kurth J. Varotto C. Pesaresi P. Biehl A. Richly E. Salamini F. Leister D. Planta. 2002; 215: 101-109Crossref PubMed Scopus (33) Google Scholar, 49.Richly E. Dietzmann A. Biehl A. Kurth J. Laloi C. Apel K. Salamini F. Leister D. EMBO Rep. 2003; 4: 491-498Crossref PubMed Scopus (100) Google Scholar). Images were read using the Storm PhosphorImager (Amersham Biosciences). Hybridization images were imported into the ArrayVision program (version 6; Imaging Research Inc., Ontario, Canada) and statistically evaluated (Bonferroni-adjusted significance test corresponding to a confidence interval of 0.9999), using the Array-Stat program (version 1.0 Rev. 2.0; Imaging Research). Normalization of data was performed with reference to all spots on the array as described previously (48.Kurth J. Varotto C. Pesaresi P. Biehl A. Richly E. Salamini F. Leister D. Planta. 2002; 215: 101-109Crossref PubMed Scopus (33) Google Scholar, 49.Richly E. Dietzmann A. Biehl A. Kurth J. Laloi C. Apel K. Salamini F. Leister D. EMBO Rep. 2003; 4: 491-498Crossref PubMed Scopus (100) Google Scholar). Miscellaneous—Basic molecular biology methods were performed as described (31.Sambrook J. Ftitsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 7.1-9.62Google Scholar). Nucleotide sequences were determined by the dideoxy chain termination method (50.Sanger F. Nicklen S. Coulson A.R. Bio/Technology. 1992; 24: 104-108PubMed Google Scholar). Energy transfer fluorochrome dideoxynucleotide labeling (51.Rosenblum B.B. Lee L.G. Spurgeon S.L. Khan S.H. Menchen S.M. Heiner C.R. Chen S.M. Nucleic Acids Res. 1997; 25: 4500-4504Crossref PubMed Scopus (196) Google Scholar) was used for detection of sequencing products using the ABI 377 system (Applied Biosystems). Identification and Phenotype of the Dpa1 Mutant—The F2 progeny of 1100 EMS-treated seeds and 75 preselected pale mutants from T-DNA collections (53.Felmann K.A. Plant J. 1991; 1: 71-82Crossref Scopus (448) Google Scholar) obtained from the Arabidopsis Biological Resource Centre (Ohio State University, Columbus, OH) have been used for screening. 87 mutant plants were selected by their non-photoautotrophic growth on soil. They developed pale green cotyledons but no primary leaves. Cultivation on sucrose-supplemented MS medium (52.Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (54339) Google Scholar) often rescued the mutant seedlings, leading to a nearly normal pigmentation and development. Under these conditions the mutants could often hardly be distinguished from wild type plants, although growth rates were slightly retarded. Seven plants showing a high dark level of fluorescence (Fo) but a lower level during induction were selected from the collection by imaging analysis (Fig. 1A). In six plants the lowest fluorescence level became apparent after about 1–2 min, and the fluorescence again slowly increased close to the Fo level during induction. This unusual fluorescence behavior we have already observed previously in several high chlorophyll fluorescence (hcf) mutant plants (24.Meurer J. Meierhoff K. Westhoff P. Planta. 1996; 198: 385-396Crossref PubMed Scopus (176) Google Scholar). In one mutant, dpa1, light-dependent quenching of Fo was stable during induction. Therefore, we have chosen dpa1 for the present study. This increased NPQ was indicative of photosynthetic electron transport activity in dpa1 in contrast to a high chlorophyll fluorescence phenotype of photosystem I mutants such as hcf101 (Fig. 1A) (24.Meurer J. Meierhoff K. Westhoff P. Planta. 1996; 198: 385-396Crossref PubMed Scopus (176) Google Scholar). The screened seedling lethal dpa1 mutant of A. thaliana, accession Wassilewskija, originated from the T-DNA insertion collection. Inactivation of the atpC1 Gene—Initially, we have tried to isolate flanking genomic regions of the right border of the T-DNA by inverse PCR. Each attempt to get a specific product failed. We backcrossed the mutation in order to confirm the Mendelian segregation of dpa1, to remove possible background mutations and to generate a mapping population. The dpa1 mutation was mapped to the upper part of chromosome 4 by the use of the molecular Cer452226 marker closely located to atpC1 (see “Experimental Procedures”). Forty-eight meiotic chromosomes of individual F2 mutant plants derived from backcrosses to the accession Landsberg have been used in this study. No recombinations have been identified, indicating that the dpa1 mutation is closely linked to the atpC1 gene. Southern analyses with genomic DNA from dpa1, wild type, and heterozygous plants using probes of the T-DNA left border and the atpC1 gene resulted in restriction fragment length polymorphisms showing that there is a T-DNA insertion in the region of atpC1 (data not shown). Due to the close location of the mutation to atp
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