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First published online February 27, 2008; 10.1104/pp.107.111260 Plant Physiology 146:1721-1737 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Arabidopsis PPR40 Connects Abiotic Stress Responses to Mitochondrial Electron Transport1,[W],[OA]Institute of Plant Biology (L.Z., G.R., G.S., K.Ö., C.K., L.S.), and Proteomics Research Group (Z.D., K.F.M.), Biological Research Centre, Hungarian Academy of Sciences, 6726–Szeged, Hungary; Department of Applied Biotechnology and Food Science, Laboratory of Biochemistry and Molecular Biology, Budapest University of Technology and Economics, 1111–Budapest, Hungary (A.S.); Pathobiochemistry Research Group, Hungarian Academy of Sciences and Semmelweis University, 1111–Budapest, Hungary (A.S.); Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143–0446 (K.F.M.); and Max-Planck-Institut für Züchtungsforschung, D–50829 Cologne, Germany (C.K., Z.K.)
Oxidative respiration produces adenosine triphosphate through the mitochondrial electron transport system controlling the energy supply of plant cells. Here we describe a mitochondrial pentatricopeptide repeat (PPR) domain protein, PPR40, which provides a signaling link between mitochondrial electron transport and regulation of stress and hormonal responses in Arabidopsis (Arabidopsis thaliana). Insertion mutations inactivating PPR40 result in semidwarf growth habit and enhanced sensitivity to salt, abscisic acid, and oxidative stress. Genetic complementation by overexpression of PPR40 complementary DNA restores the ppr40 mutant phenotype to wild type. The PPR40 protein is localized in the mitochondria and found in association with Complex III of the electron transport system. In the ppr40-1 mutant the electron transport through Complex III is strongly reduced, whereas Complex IV is functional, indicating that PPR40 is important for the ubiqinol-cytochrome c oxidoreductase activity of Complex III. Enhanced stress sensitivity of the ppr40-1 mutant is accompanied by accumulation of reactive oxygen species, enhanced lipid peroxidation, higher superoxide dismutase activity, and altered activation of several stress-responsive genes including the alternative oxidase AOX1d. These results suggest a close link between regulation of oxidative respiration and environmental adaptation in Arabidopsis.
Adaptation of plants to environmental stresses has important metabolic implications, including changes in photosynthesis, respiration, metabolite assimilation, and catabolism. Mitochondria are in the center of regulation of cellular energy homeostasis and redox balance, and integrate numerous metabolic pathways that are important in adaptive responses to extreme environmental conditions. Respiration and oxidative phosphorylation; metabolism of Pro, Cys, ascorbate, and folate; and the control of redox balance are examples of processes illustrating the importance of mitochondria in coordination of cellular metabolism during stress adaptation (Sweetlove et al., 2007  ).
Respiration is the core process of mitochondrial metabolism in which a large amount of free energy is released and used for ATP production. During respiration, controlled oxidation of reduced carbohydrates, such as malate and pyruvate, takes place through glycolysis and tricarboxylic acid cycle producing, respectively, reducing NADPH and FADH2. Electrons from the NADPH and FADH2 are transferred to O2 via the electron transport chain generating the energy carrier ATP and oxidized NADP+ and FAD+ (Siedow and Day, 2000
Mitochondrial electron transport is also important to neutralize the excess of reducing capacity of photosynthesis, preventing oxidative damage of thylakoid membranes and other cellular components (Møller, 2001
Mitochondrial proteins encoded in the nucleus are imported by a specialized organelle transport system. A particular nuclearly encoded organellar protein family is characterized by 9 to 15 tandem arrays of pentatricopeptide repeats (PPRs), which are composed of degenerate 35 amino acid units (Small and Peeters, 2000
Posttranscriptional regulation of gene expression is a dominant mode of controlling gene activity in mitochondria. PPR proteins are implicated in the regulation of organellar gene expression by controlling diverse aspects of RNA metabolism, such as RNA splicing, editing, processing, and translation (Meierhoff et al., 2003
Isolation of ppr40 Mutants
Screening of our T-DNA-tagged Arabidopsis collection (Szabados et al., 2002
The At3g16890 gene has a single exon with an open reading frame (ORF) of 1,980 bp and encodes a 74-kD protein composed of 659 amino acid residues (Fig. 1D). The At3g16890 protein belongs to the P subclass of the PPR protein family and was previously named PPR40 (PPR model, PPR_3_5768407; Lurin et al., 2004 ). The PPR40 protein carries a predicted mitochondrial targeting signal and 14 conserved PPR motifs arranged in two separate domains (Fig. 1, D and F). In different plant species, genome sequencing revealed a large number of PPR domain proteins (Lurin et al., 2004). PPR40 orthologs sharing highly conserved domain structure have been identified in Vitis and rice (Oryza sativa; Supplemental Fig. S1). Reverse transcription (RT)-PCR analysis indicated the lack of full-length At3g16890 transcript in the homozygous ppr40-1 and ppr40-2 mutants (Fig. 1C). However, 3'-truncated transcripts from both mutant alleles were detected with primers PPRF+PPRR1 positioned 5' upstream of the ppr40-1 T-DNA insertion site. Primers PPRF+PPRR2 positioned upstream of the ppr40-2 insertion detected truncated transcripts only in the ppr40-2 mutant (Fig. 1C). The RT-PCR analysis thus suggested that C-terminally truncated proteins may be produced in both ppr40-1 and ppr40-2 mutants. The ppr40-1 allele allows theoretically the synthesis of a truncated protein of 121 amino acids composed of 103 residues encoded by the PPR40 coding region and 18 C-terminal amino acids encoded by T-DNA sequences. If produced, this truncated PPR40-1 protein carries only the mitochondrial targeting signal without PPR sequences. On the other hand, the truncated PPR40-2 protein of 318 amino acids is predicted to contain 284 PPR40-encoded amino acids followed by 34 T-DNA encoded C-terminal amino acids (Fig. 1, D and E). Although so far no evidence supports the existence of these truncated proteins in the ppr40 mutants, the fact that the ppr40-2 mutant shows less severe growth retardation than ppr40-1 (Fig. 1B; see below) suggests possible synthesis of the truncated ppr40-2 protein resulting in a partial loss of function phenotype. Data deposited in microarray transcript profiling databases indicate that the PPR40 gene is constitutively transcribed at low levels in all tissue types throughout plant development and not regulated by any thus far recorded treatment (https://www.genevestigator.ethz.ch). To verify these data, we examined the PPR40 expression profile by quantitative RT-PCR (qRT-PCR) using RNA samples from different organs of wild-type plants. The PPR40 transcript levels were almost three orders of magnitude lower than the reference ACTIN2/8 mRNA in all tested tissues showing somewhat higher abundance in green siliques and seedlings (Supplemental Fig. S2). The level of PPR40 transcript was not changed significantly by hormones (auxin, cytokinin, ethylene, and salicylic acid) and stress (salt, osmotic, and cold) treatments (data not shown).
In comparison to wild type, the rosette size of the ppr40-1 mutant was 50% ± 10% (n = 50) smaller, whereas the ppr40-2 mutant showed only a 20% ± 5% (n = 50) reduction of rosette diameter during vegetative development (Fig. 1B). Both ppr40 mutants were fertile and produced comparable amounts of seed as wild-type plants. The ppr40 mutants displayed slightly delayed seed germination. Although germination of wild-type (ecotype Columbia of Arabidopsis [Col-0]) seed was 100% at day 4, the ppr40-1 and ppr40-2 mutants completed their germination with 2- and 1-d delays, respectively (Fig. 2B ). Both ppr40 mutants displayed enhanced, although different, degrees of sensitivity to inhibition of seed germination by ABA (Fig. 2A). In the presence of 0.5 µM ABA, only 20% ± 6% of the ppr40-1 seed germinated after 1 week compared to germination of 60% ± 3% of the ppr40-2 seed and 98% ± 2% of the wild-type seed (in each case n = 300; Fig. 2B). Phenotypic differences in rosette growth and ABA sensitivity indicated that ppr40-1 is likely a null allele, whereas ppr40-2 represents a leaky mutation. Therefore, we used the ppr40-1 allele in further characterization of PPR40 function. Except ABA, other hormone responses of the ppr40-1 mutant (e.g. auxin, cytokinin, ethylene, salicylic acid, GA, and brassinolide; Supplemental Table S1) were similar to the wild type.
Besides germination, ABA controls other physiological responses including stomatal opening, desiccation, plant growth, and expression of numerous genes. Stomatal closure is one of the most characteristic ABA-controlled responses during water deprivation. To compare ABA-induced stomatal closure of wild-type and ppr40-1 plants, we prepared leaf epidermal peels from plants treated with different concentrations of ABA and recorded changes in the stomatal pore diameter. Whereas untreated wild-type and ppr40-1 plants showed no significant difference, the ppr40-1 mutant compared to the wild type responded with enhanced stomatal closure to increasing ABA concentrations (Fig. 2, C and D). Because stomatal closure affects the evaporation rate during drought, we have examined the water loss property of the ppr40-1 mutant in desiccation assays. Water loss from isolated ppr40-1 leaves was significantly lower compared to the wild type (Fig. 2E), correlating with enhanced ABA sensitivity of stomatal closure of the ppr40-1 mutant. To assess whether enhanced ABA sensitivity of ppr40-1 correlates with either elevated ABA biosynthesis or alteration in signal transduction, we have first compared the free ABA concentrations in wild-type and ppr40-1 seedlings exposed to treatment with 150 mM NaCl for 0, 6, and 24 h. The ABA levels were comparable in wild-type and ppr40-1 seedlings cultured in medium without salt. Treatment for 6 h with 150 mM NaCl resulted in about a 50% increase of ABA content in both mutant and wild-type plants. Upon 24 h of salt treatment, however, the ABA concentration increased 3-fold in ppr40-1 and 2.5-fold in wild type compared to untreated controls, suggesting that some increase in endogeneous ABA levels during salt stress could contribute to the enhancement of stress sensitivity of the ppr40-1 mutant (Fig. 2F).
To determine whether enhanced ABA and salt sensitivity of ppr40-1 correlates with altered transcription of well-characterized stress-responsive genes acting in the parallel ABA-signaling pathways (Yamaguchi-Shinozaki and Shinozaki, 2005
To test possible implication of PPR40 in the regulation of plant responses to various environmental conditions and plant hormones, we performed seed germination and seedling growth assays (Supplemental Table S1). These indicated that in addition to ABA the ppr40-1 mutant also shows enhanced sensitivity to salinity, osmotics, sugars, and oxidative stress. Germination of ppr40-1 seeds was particularly inhibited by NaCl (Fig. 3, A and B ). Under standard conditions, the root growth rate of the ppr40-1 mutant measured on vertical agar plates was 40% lower compared to wild type, whereas in the presence of 100 and 150 mM NaCl the ppr40-1 root elongation rate of was, respectively, 65% to 75% lower than wild type. NaCl (200 mM) completely blocked root growth of the mutant, whereas wild-type roots continued to grow at a low rate at this salt concentration (Fig. 3C). Salt stress also caused lower fresh weight accumulation in the ppr40-1 mutant compared to wild type (data not shown). The ppr40-1 mutant showed enhanced sensitivity to oxidative stress. On media containing sublethal concentrations of either hydrogen peroxide or paraquat, the ppr40-1 mutant displayed faster bleaching and chlorophyll degradation compared to the wild type (Fig. 3, D–F). This prompted us to test the accumulation of ROS, in particular hydrogen peroxide, in the ppr40-1 mutant. Histochemical 3,3-diaminobenzidine (DAB) assays indicated 28% ± 12% higher level of H2O2 accumulation in ppr40-1 leaves (P < 0.0001; n = 50) suggesting that ROS damage could, at least partially, contribute to enhanced stress sensitivity of the mutant (Fig. 3G).
Genetic Complementation of the ppr40-1 Mutation
The coding region of intronless At3g16890 gene was PCR amplified and cloned into the pPILY intron-tagged hemagglutinine (HA)-epitope fusion vector (Ferrando et al., 2000
Association of PPR40 with Mitochondrial Electron Transport
So far, all characterized PPR domain proteins have been localized in chloroplasts and mitochondria, and many of them are suggested to participate in the control of organellar gene expression (Andrés et al., 2007
To search for possible function of PPR40, we have tested its RNA-binding capability using combined UV-formaldehyde cross-linking of mitochondrial protein-RNA complexes and RNA-binding gel shift assays with purified PPR40 and in vitro translated mitochondrial RNAs. However, these assays lead to negative results (data not shown). Therefore, we have investigated whether PPR40 is associated with protein complexes localized in mitochondrial membranes. Mitochondrial extracts purified from PPR40-HA-expressing cell cultures were subjected to fractionation of membrane proteins by Suc gradient centrifugation and blue-native PAGE (BN-PAGE). Suc gradient fractions were analyzed by BN-PAGE and western blotting using anti-HA antibody. Immunoblotting detected PPR40-HA in a protein complex of about 500 kD in the Suc gradient fractions 7 to 9 (Fig. 6A ). The size of this complex corresponded to that of Complex III of mitochondrial electron transport system (Dudkina et al., 2005 ). To confirm association of PPR40-HA with Complex III, preparative two-dimensional BN-PAGE and SDS-PAGE was performed. Using this alternative separation method, PPR40-HA was also detected in Complex III by western blotting of preparative BN gels (Fig. 6B; western blotting of first dimension BN-PAGE). To analyze more precisely the composition of PPR40-HA-associated protein complex, the anti-HA cross-reacting protein complex was excised from the BN gel and size fractionated by SDS-PAGE. Subsequent western analysis confirmed that the excised complex indeed carried PPR40-HA of predicted molecular mass of 74 kD (Fig. 6B) in association with five major subunits of Complex III system that were identified by mass spectrometry (MS; Table I
; Heazlewood et al., 2004).
To test whether the lack of PPR40 protein caused any alteration in the stoechiometry of core subunits of electron transport complexes, mitochondria were isolated from wild-type and ppr40-1 mutant cell suspension cultures and membrane proteins were separated in BN gels. The respiratory complexes from wild-type and mutant mitochondria showed similar BN gel resolution patterns (Fig. 7A ; Supplemental Fig. S4A), and the stoechiometry of Complex III subunits analyzed by SDS-PAGE also appeared to be unaffected by the ppr40-1 mutation (Fig. 7B; Supplemental Fig. S4B). Although PPR40 showed clear cofractionation with Complex III, these data indicated that PPR40 probably does not affect the composition and stability of core subunits of Complex III. Furthermore, the ppr40-1 mutation did not appear to influence the transcript levels of genes coding for subunits of Complex III. qRT-PCR analysis of mRNA levels of these genes revealed no more than a 50% difference between the wild type and the ppr40-1 mutant (Fig. 7C).
Apocytochrome B (cob, ATMG00220) is the only Complex III subunit that is encoded by the mitochondrial genome and is expressed as a 5-kb transcript (Brandt et al., 1993 ). All other Complex III subunits are encoded in nuclear genome and proteins are imported into mitochondria. To test whether PPR40 controls splicing of the apocytochrome B mRNA, northern hybridization of total mitochondrial RNA isolated from the wild type, the ppr40-1 mutant, and the complemented mutant plants was performed. We observed no difference in cob transcript size between mutant, wild-type, and complemented plants (Fig. 7D). In addition, splicing of mitochondrially encoded intron-containing subunits of Complex I (nad1, nad4, and nad7) and Complex IV (cox2 and ccb452) was tested by RT-PCR analysis and revealed no difference between wild-type, mutant, and complemented plants (data not shown). RNA editing of mitochondrial transcripts can influence gene activity. To test whether editing of apocytochrome B mRNA is different in the ppr40-1 mutant, full-length cob complementary DNA (cDNA) was amplified and sequenced from RNA samples of wild-type, mutant, and complemented mutant plants. Sequence analysis could identify all seven cob C/U editing sites in each ORF as described by Giegé and Brennicke (1999), and confirmed that RNA editing did not change in the ppr40-1 mutant (Supplemental Fig. S5).
Because the ppr40-1 mutation did not appear to affect the subunit composition of respiratory complexes, we have asked the question whether PPR40 is required for proper control of respiration-associated mitochondrial functions, such as consumption of oxygen with different respiratory substrates and generation of ROS. Respiration was measured by oxygen consumption in mitochondria isolated from wild-type and ppr40-1 mutant cell suspension cultures. Using NADH as the electron donor for Complex I, which is a major electron source in the respiration system, we observed 50% reduction of oxygen consumption in ppr40-1 mitochondria compared to wild type (Fig. 8A ). Similarly, application of succinate as electron donor for Complex II that transfers electrons to Complex III via ubiquinon, revealed 40% lower oxygen consumption in ppr40-1 mitochondria (Fig. 8A). These data indicated that electron transport through Complexes I and II, which act upstream of Complex III, was greatly reduced in the mutant.
During oxidative respiration Complex III transfers electrons from ubiquitin to cytochrome c (ubiquinol cytochrome c reductase activity) toward Complex IV, which has COX and mediates electron transfer to oxygen. Using ascorbate as respiratory substrate for Complex IV to measure direct electron transport from this substrate to oxygen, we detected 2.5- to 3.0-fold higher oxygen consumption in ppr40-1 mutant mitochondria compared to wild type (Fig. 8A). In contrast to reduced activities of Complexes I and II, this result indicated that Complex IV was fully functional and that ascorbate could at least partially bypass the defect of electron transport through Complex III in the ppr40-1 mutant. Furthermore, we observed that COX activity was about twice as high in the ppr40-1 mutant than in wild type (Fig. 8B), and the ascorbate consumption was 30% higher in roots and 80% higher in cell culture of the ppr40-1 mutant compared to wild type (Fig. 8C). These data indicated that Complex IV worked at a higher rate in the ppr40-1 mutant than in wild-type mitochondria. Thus, despite remarkable reduction of electron transport through Complex III, the activity of Complex IV via ascorbate maintained a high level of oxygen consumption in the ppr40-1 mutant.
Decreased ubiquinol cytochrome c reductase activity of Complex III results in hindered electron transport and accumulating electron pool, which can generate ROS. AOXs capture the excess electrons from ubiquinon, producing water and preventing accumulation of ROS during stress when electron transport through Complex III is reduced (Navrot et al., 2007
Complexes I and III are considered to be main sources for generation of ROS in mitochondria during oxidative respiration (Chen et al., 2003
Lipid peroxidation is a direct consequence of ROS damage and is therefore considered as major indication for ROS accumulation. In correlation with the higher level of hydrogen peroxide accumulation, we observed that the ratio of oxidized lipids was 20% to 25% higher in leaves of the ppr40-1 mutant compared to wild-type and genetically complemented mutant plants (Fig. 9B). Exposure to 150 mM NaCl salt stress for 24 h has slightly increased the level of lipid peroxidation, but the difference between ppr40-1 and controls remained approximately the same as in nonstressed plants.
Superoxide radicals are known to be generated by Complex III misfunction during stress and represent the most damaging ROS species. Superoxide radicals are converted to H2O2 by mitochondrial manganese-containing superoxide dismutase (MnSOD; Navrot et al., 2007
Effects of the ppr40-1 Mutation on Complex III in Mitochondrial Electron Transport
Our study documents that the function of PPR domain protein PPR40 is important for the ubiquinol-cytochrome c reductase activity of Complex III in the mitochondrial electron transport chain, which has a significant influence on plant growth and responses to ABA and environmental stresses. This result adds a new aspect to the functional analysis of PPR domain proteins. PPRs located in chloroplasts and mitochondria are thought to interact with RNA and function in protein complexes as adaptors controlling either organellar RNA splicing and processing, or the stability of complex organelle structures (Williams and Barkan, 2003
The ppr40-1 mutation was originally identified in a genetic screen for mutants displaying enhanced sensitivity to ABA, but found later to confer delayed germination, semidwarf growth habit, and enhanced sensitivity to salt stress. Pleiotropic phenotype of the ppr40-1 mutant thus indicates that inactivation of PPR40, which leads to altered mitochondrial electron transport, affects a wide range of cellular functions and stress responses. Other mutations influencing mitochondrial electron transport have been reported to cause growth defects (Newton and Coe, 1986 In our experiments Suc gradient fractionation followed by BN and SDS electrophoresis in combination with proteomic analysis showed that PPR40 is associated with Complex III of the mitochondrial electron transport chain. Separation of Complex III by two independent methods, namely by Suc density gradient centrifugation and BN gel electrophoresis, did not disrupt stable association of PPR40-HA with Complex III. However, electroelution of Complex III followed by immunoprecipitation with a monoclonal anti-HA antibody did not pull down PPR40-HA (data not show), indicating that the C-terminal HA tag of PPR40-HA protein is hidden in Complex III. We have also tried complementing the ppr40-1 mutant with a HA-PPR40 construct, which carries an N-terminal HA-tag. However, this protein proved to be unstable and was never detected in the mitochondrial fraction, probably because the N-terminal HA tag interferes with mitochondrial import of PPR40. In conclusion, our data demonstrate that PPR40-HA is firmly associated with Complex III in nonstoechiometric amount. This observation is supported by the data showing that the ppr40 mutations lead to reduction but not to complete loss of Complex III activity. This suggests that PPR40 is an important regulator of cytochrome c reductase activity of Complex III, which is essential for mitochondrial electron transport and oxidative phosphorylation, but does not represent a core Complex III subunit. How PPR40 regulates Complex III by molecular interactions with its core subunits remains to be resolved by further crystallization and structural studies. Although several PPR proteins were reported to control organellar RNA processing, we could not find significant alterations in splicing and abundance of mitochondrial and nuclear transcripts encoding subunits of electron transport complexes I, III, and IV. However, comparative analysis of mitochondrial electron transport complexes clearly showed that subunit stoechiometry and abundance of Complex III in mutant and wild-type mitochondria was not significantly different suggesting that the ppr40 mutation influences the activity and not the composition of Complex III. The PPR40 protein carries two separate domains of five and nine tandem PPR repeats. We have isolated two ppr40 mutant alleles that differently affect leaf rosette development and ABA sensitivity of plants correlating with the position of T-DNA insertions in the PPR40 gene. The T-DNA insertion in the ppr40-1 allele is located upstream of the PPR-repeat coding domains and allows the synthesis of a 3'-truncated transcript, which is predicted to encode a protein lacking PPR repeats. In the ppr40-2 mutant that displays less severe alterations of developmental and stress responses, the T-DNA insertion permits the synthesis of a longer truncated transcript that encodes a C-terminally truncated protein retaining the first domain of five PPR repeats. Leaky phenotype of the ppr40-2 mutant suggests that the predicted PPR40-2 protein is likely produced and partially functional, whereas ppr40-1 represents a genuine null mutation causing a complete loss of PPR40 function.
Reduction of respiration rate in the ppr40-1 mutant suggests that the PPR40 protein is important for proper function of Complex III, which has a cytochrome c reductase activity and catalyzes electron transfer from ubiquinon to cytochrome c in oxidative phosphorylation (Fig. 10
). Complex III is a 500-kD multiprotein complex, which is partially embedded in the inner mitochondrial membrane (Berry et al., 2000
Changes of ROS Regulation in the ppr40-1 Mutant
Complex III is a principal source of ROS and inhibition of cytochrome c reductase activity increases ROS generation and oxidative damage (Chen et al., 2003
In plants, mitochondrial ROS production is effectively reduced by nonphosphorylating respiratory pathways, which include AOXs. AOX diverts the electron flow from the ubiquinon pool to oxygen and produces water without ATP production (Vanlerberghe and Ordog, 2002 Mitochondria with reduced respiration may generate a permanent stress condition by producing ROS as a constitutive stress signal for activation of cellular defense responses. This appears to be the case in the ppr40-1 mutant, which is more sensitive to salinity, osmotic, and oxidative stress. The ppr40-1 mutation causes reduced electron transport through Complex III and we found that this leads to increased use of alternative electron donors (downstream Complex III), such as ascorbate, by Complex IV (Fig. 10).
It is notable that the ppr40-1 mutant displays acceleration of ABA-stimulated stomatal closure correlating with its enhanced sensitivity to ABA. Nonetheless, our efforts to associate the PPR40 function with correlative changes in the expression of key transcription factors of ABA-regulated stress response pathways did not provide a clear result. This, together with the above-discussed evidences, supports our conclusion that hypersensitivity of the ppr40-1 mutant to ABA and salt is caused by a mitochondrial defect resulting in enhancement of ROS generation and possible limitation of ATP production. ROS signaling is not only prominently linked to the control of programmed cell death (Gechev et al., 2006
Plant Materials and Growth Conditions
Arabidopsis (Arabidopsis thaliana) growth conditions in sterile culture and controlled growth chambers were as described earlier (Koncz et al., 1994
The T-DNA tagged ppr40-1 and ppr40-2 insertion mutants were identified in the Szeged (http://www.szbk.u-szeged.hu/
Total RNA was isolated from 3-week-old seedlings using the Tri-reagent extraction method (Chomczynski and Sacchi, 1987
Searches for putative protein targeting signals were performed using the TargetP (http://www.cbs.dtu.dk/services/TargetP), Predotar (http://urgi.infobiogen.fr/predotar/), and iPSORT (http://psort.nibb.ac.jp/) algorithms. PCR primers were designed with the Primer3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). Multiple sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/clustalw/index.html, http://align.genome.jp). Protein domain analyses were performed with the SMART service (http://smart.embl-heidelberg.de/).
Intact mitochondria were isolated from either 1-week-old cell suspension cultures or 3-week-old in vitro grown seedlings, or 4-week-old liquid root cultures using the method described by Werhahn et al. (2001)
Separation of mitochondrial protein complexes by Suc gradient ultracentrifugation was performed according to Dudkina et al. (2005)
The analysis of mitochondrial complexes was performed as described (Wittig et al., 2006
In-gel digestion was performed as described in http://donatello.ucsf.edu/ingel.html. For liquid chromatography-tandem MS (LC-MS/MS) analysis, samples were analyzed on an Agilent 1100 nanoLC system on-line coupled to an XCT Plus ion trap mass spectrometer in information-dependent acquisition mode: MS acquisitions were followed by three collision-induced dissociation analyses on computer-selected multiply charged ions. For the database search, raw data were converted into a Mascot generic file with the DataAnalysis for LC/MSD Trap v3.2 software. The resulting MS and MS/MS data were searched using the Mascot v2.1 software (www.matrixscience.com) against the SwissProt 51.7 nonredundant database without species restriction (259,034 sequences) and the Protein Prospector Batchtag (v.5.0.0.beta1) search engine against the Uniprot (2006.10.21) database with Arabidopsis species restriction (49,487 entries); subsequently the data were also manually inspected.
Cultured cells transformed with the PPR40-HA construct were digested for 4 h with an enzyme mixture containing 1% (w/v) cellulase Onozuka R-10 (Yakult), 0.5% (w/v) Macerozyme R-10 (Yakult), and 0.16% (w/v) Driselase (Sigma) in B5 media supplemented with 0.4 M mannitol. Before the fixation procedure, cells were treated with 100 nM MitoTracker Orange (Molecular Probes). Cells were fixed with 3.7% (w/v) formalin in microtubule stabilizing buffer (50 mM PIPES [pH 6.9], 5 mM MgSO4, 5 mM EGTA) for 1 h at room temperature (Ferrando et al., 2000
The full-length cDNA of At3g16890 gene was isolated by PCR amplification with ppr40F and ppr40R3 primers (Supplemental Table S2) and the PCR product was cloned as a NcoI-BglII fragment in the pPILY HA-epitope fusion vector (Ferrando et al., 2000
Samples (0.05 to 0.1 g) of 3-week-old in vitro grown control and treated seedlings were homogenized in liquid nitrogen and extracted with 80% (v/v) acetone for 2 h. The homogenate was centrifuged at 15,000g for 10 min. Absorption of the extracts was measured at 663 and 645 nm and the concentration of extracted chlorophylls was calculated according to Lichtenthaler (1987)
SOD activity was assayed by the inhibition of photochemical reduction of nitroblue tetrazolium as described (Dhindsa et al., 1981
Stomata opening assays were performed with epidermal peels from rosette leaves of 4- to 6-week-old plants (Leymarie et al., 1998
Ascorbate consumption was measured by reverse-phase HPLC as described (Szarka et al., 2002
Hydrogen peroxide generation was measured according to Dlasková et al. (2006)
For determination of ABA content, we used an ELISA (Phytodetek-ABA; Sigma-Aldrich) assay. (100 mg). Grinded plant samples (100 mg) were extracted with 5 mL of cold mixture of 100 mM NaHCO3:methanol (80:20, v/v) containing 1 mg of butylated hydroxytoluene in a volume of 100 mL. The extraction procedure was performed twice at 4°C for 24 h each, and then the solvent was evaporated. This assay utilizes monoclonal antibody for ABA, and the determination of (+)-cis-ABA in the plant extract is based on the competitive binding of ABA and the tracer (alkaline phosphatase-labeled ABA) to the antibody-coated microwells.
The following materials are available in the online version of this article.
We thank Annamária Király and Mónika Kispéterné Gál for their technical assistance, Mihály Dobó for growing the plants, Dr. Péter Doró and Miklós Mocsonoky for their help in bioinformatic analyses, and Dr. Irma Tari for her help in the ABA measurements. Received October 20, 2007; accepted February 20, 2008; published February 27, 2008.
1 This work was supported by the European Union FP5 (grant no. QLRT–2001–00841), NKFP (grant no. 4–038–04), OTKA (grant no. T–046552), and the joint research project (DFG 436UNG 13/172/01) between the Deutsche Forschungsgemeinschaft and the Hungarian Academy of Sciences. The Proteomics Research Group was supported by the Hungarian National Office for Research and Technology (RET–08/2004) and OTKA (grant no. K–60283). The Semmelweis University research group was supported by OTKA (grant no. 69187).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: László Szabados (szabados{at}brc.hu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.111260 * Corresponding author; e-mail szabados{at}brc.hu.
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kinase AKIN10 reached lower levels in ppr40-1, but mRNA levels of the seed-specific ABI5 transcription factor did not reveal any significant difference. Despite detecting some increase in ABA accumulation during salt stress and enhancement of ABA and salt sensitivity, these data indicated that the PPR40 function was not implicated in the primary control of either ABA synthesis or signaling, although the ppr40-1 mutation had some influence on these processes. Furthermore, transcription of PPR40 was not altered by externally added ABA (
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ek T, 
ek P (2006) Certain aspects of uncoupling due to mitochondrial uncoupling proteins in vitro and in vivo. Biochim Biophys Acta 1757: 467–473
