JXB Advance Access originally published online on November 29, 2005
Journal of Experimental Botany 2006 57(1):193-200; doi:10.1093/jxb/erj025
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RESEARCH PAPER |
Identification of a mitochondrial ATP synthase small subunit gene (RMtATP6) expressed in response to salts and osmotic stresses in rice (Oryza sativa L.)
1Alkali Soil Natural Environmental Science Center (ASNESC), Stress Molecular Biology Laboratory, Northeast Forestry University, Harbin 150040, PR China
2Asian Natural Environment Science Center (ANESC), The University of Tokyo, 1-1-1 Midori-cho, Nishi-tokyo, Tokyo 188-0002, Japan
* To whom correspondence should be addressed. E-mail: shenkuiliu{at}nefu.edu.cn
Received 14 August 2005; Accepted 21 October 2005
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Abstract |
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Large areas of northern China have alkaline soil due to the accumulation of sodium carbonates (NaHCO3, Na2CO3). To understand better how plants can tolerate alkaline soil, a cDNA library was prepared from rice (Oryza sativa L.) roots grown in the presence of NaHCO3 stress. A cDNA clone isolated from this library was identified by a homology search as a mitochondrial ATP synthase 6 kDa subunit gene (RMtATP6; GenBank accession nos AB055076, BAB21526). In transformed yeast and tobacco protoplasts, the RMtATP6 protein was localized in mitochondria using the green fluorescent protein (GFP) marker. Analysis of RMtATP6 mRNA levels suggested that the expression of this gene was induced by stress from sodium carbonates and other sodium salts. Transgenic tobacco overexpressing the RMtATP6 gene had greater tolerance to salt stress at the seedling stage than untransformed tobacco. Among the other genes for F1F0-ATPase of rice, some were found to be up-regulated by some environmental stresses and some were not. These data suggest that the RMtATP6 protein acts as a subunit of ATP synthase, and is expressed in response to stress from several salts, with the other genes coding for the subunits of the same ATP-synthase.
Key words: Carbonate stress, cloning, GFP, mitochondria, rice mitochondrial ATP synthase 6 kDa subunit, transgenic, yeast
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Alkaline soil is a type of soil that has high levels of carbonate salts. According to incomplete statistics of UNESCO and FAO, 950 million ha (6.4%) of the world's land area has saline-alkali soil, and about one-tenth of this area is found in China. The alkaline soils of China are formed by the accumulation of NaHCO3 and Na2CO3 (Wang et al., 1993
), the hydrolytic decomposition of which raises the pH to above 9.0. The alkalinity results in a hardened, poorly ventilated soil. Plants in severely alkaline soil can barely survive. Only a few species of plants grow in such soils and they are sparsely distributed. This limits the development of agriculture and livestock husbandry. Presently, an important policy in many parts of the world, including China, is to exploit existing alkaline-tolerant plants and to develop new ones. Therefore, there is interest in breeding transgenic plants that are tolerant of alkaline soil. In this study, some genes from a rice (Oryza sativa) root cDNA library that was prepared from plants grown under carbonate (NaHCO3) stress were isolated. One of the genes was found to be the gene for a mitochondrial ATP synthase 6 kDa subunit (RMtATP6; GenBank accession nos AB055076, BAB21526).
Mitochondrial F1F0-ATP synthase is a multimeric enzyme, in which F1 is a hydrophilic sector carrying the catalytic sites for ATP synthesis/hydrolysis and F0 includes hydrophobic subunits embedded in the membrane so as to constitute a proton channel (Fillingame, 1999; Pedersen et al., 2000). Prokaryotes and eukaryotes have the same F1 structures which consist of five subunits designated 
, ß, 
, 
, and 
(Amzel and Pedersen, 1983), but somewhat different F0 structures (Boyer, 1997; Jones et al., 1998; Kinosita et al., 1998; Mueller, 2000; Velours and Arselin, 2000). A major challenge is to understand how the F0 sector functions (Kinosita et al., 1998). Jansch et al. (1996) resolved the mitochondrial F1F0-ATP synthase from potato (Solanum tuberosum) by blue native (BN)-PAGE into two forms: the intact F1F0 complex (580 kDa) comprising 13 subunits and the separated F1 part (350 kDa). The F1 section is composed of six polypeptides, and the F0 section is composed of at least seven polypeptides with apparent molecular masses of 27, 23, 20, 20, 15, 6, and 6 kDa. One of the two 6 kDa proteins is the F0 part of complex V. The deduced amino acid sequence of RMtATP6 is homologous to the 6 kDa protein from potato (Jansch et al., 1996). However, the function of this small protein is not clear and its gene has not been identified. Recently, Heazlewood et al. (2003b) purified rice mitochondrial proteins using IEF/SDS–PAGE and BN/SDS–PAGE. Among the isolated proteins is a 6 kDa protein (spot 23 in BN-PAGE). This protein is part of F0 of the mitochondrial ATP synthase, and its gene corresponds to the RMtATP6 gene. In this study, the RMtATP6 gene was identified and its gene expression was characterized. Green fluorescent protein (GFP) was used as a marker to study RMtATP6 protein localization in yeast and plant cells. Overexpression of the gene in tobacco was found to increase stress tolerance at the seedling stage.
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Materials and methods |
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Plant materials
Rice (Oryza sativa L., cv. Nipponbare) seeds were surface-sterilized with 70% ethanol for 1 min with constant agitation, washed at least three times with autoclaved water, immersed in autoclaved water at 30 °C for 24 h, and germinated in a culture room maintained at 25 °C with 80% humidity under a 12/12 h light/dark cycle. The roots of 7-d-old seedlings were immersed in 30 mM NaHCO3 for 24 h and then harvested for cDNA library construction. In addition, the 7-d-old seedlings were divided into five groups. The roots of each group were immersed in one of five solutions (H2O, 80 mM NaCl, 30 mM NaHCO3, 15 mM Na2CO3, 10% PEG 6000) for 24 h, and then the leaves and roots were harvested for northern and semi-quantitative RT-PCR analyses.
Construction and screening of the rice root cDNA library
Poly(A)+ RNA extracted from rice roots was used to construct a cDNA library in the E. coli expression vector 
TriplEx2 using a SMART cDNA Library Construction Kit (Clontech) according to the manufacturer's instructions. The rice root expression cDNA library was plated on LB medium containing 120 mM NaHCO3. About 3000 E. coli cells were inoculated on each plate (
=90 mm). After incubation overnight at 37 °C, a few carbonate-resistant E. coli clones were transferred to grow on LB medium containing 125 mM NaHCO3 at 37 °C overnight. Faster growing colonies were picked and incubated with LB liquid medium. Plasmid DNA was prepared by the alkali method and sequenced. The GenBank database was searched for homologous sequences with the BLAST algorithm.
DNA sequence analysis
The sequences of the RMtATP6 clone and other clones were determined from DNA cycle sequencing reactions, which were performed with fluorescent dye terminators followed by product analysis on an automated model 377 DNA sequencer.
Northern blot analyses
For northern blot analysis, 10 µg total RNA was electrophoresed on a 1% formaldehyde agarose gel and blotted onto a nylon membrane. RNA blot analyses were performed using a digoxigenin (DIG)-labelled RMtATP6 cDNA probe. The membrane was hybridized and washed according to Sambrook et al. (1989). Hybridization signals were detected with CDP-Star (Tropix) using the ImageMaster VDS-CL system (Amersham Pharmacia).
Construction of expression vectors and transformation
The RMtATP6 fragment was ligated to the pBI121 binary vector (Clontech) that had been digested with SmaI and SacI to construct the plasmid pBI121-RMtATP6. The GFP gene was digested from the plasmid pEGFP (Clontech) by BamHI and NotI, and the GFP fragment was ligated into BamHI/NotI sites in pYES2 (Clontech) to construct the plasmid pYES2-GFP. The RMtATP6 gene was amplified by PCR with sense primer 5'-CGTAAGCTTAGATCGAGGATGGTT-3' (HindIII site underlined) and antisense primer 5'-CGGGATCCCGGGCTTCTTGTG-3'(BamHI site underlined), and the RMtATP6 fragment was ligated into the HindIII/BamHI sites in pYES2-GFP to construct the plasmid pYES2-RMtATP6-GFP. The GFP and RMtATP6-GFP genes were amplified by PCR with sense primer 5'-TCCCCCGGGAGATCGA GGATGGTT-3' (SmaI site underlined) and antisense primer 5'-CCGGAGCTC TTACTTGTACAGCTC-3' (SacI site underlined). The GFP fragment was cloned into the SmaI/SacI sites in pBI121 vector to construct the plasmid pBI121-GFP. The RMtATP6-GFP fragment was blunt ended with a Klenow fragment, and was cut with SacI and ligated into pBI121 to construct the plasmid pBI121-RMtATP6-GFP. The plasmids pYES2-GFP and pYES2-RMtATP6-GFP were transformed into competent yeast strain INVSc1 using the electric impulse method. The plasmids pBI121-RMtATP6, pBI121-GFP, and pBI121-RMtATP6-GFP were transformed into Agrobacterium tumefaciens strain EHA105.
Staining of mitochondria and analysis by confocal microscopy
Yeast cells in the mid-log phase were incubated without fixation in YPGR medium (1% yeast extract and 2% peptone+2% galactose and 1% raffinose) containing 100 nM MitoTracker Red CmxRos (Molecular Probes) for 45 min at 30 °C with gentle shaking and washed three times before confocal analysis. Protoplasts were incubated in culture medium with 400 nM MitoTracker for 40 min at 28 °C, and washed three times before confocal analysis. Confocal microscopy was performed with a FV500 laser-scanning confocal imaging system (Olympus, Japan). GFP fluorescence was detected between 505 nm and 550 nm with excitation at 488 nm. MitoTracker staining was detected between 585 nm and 615 nm with excitation at 568 nm.
Transformation of tobacco
The RMtATP6, GFP genes and the RMtATP6-GFP fusion gene were separately cloned in the plant transformation binary vector pBI121. Each gene was placed under the control of the CaMV 35S promoter with nptII as the selectable marker. To transform tobacco (Nicotiana tabacum) leaf discs, the recombinant plasmids were introduced into Agrobacterium tumefaciens strain EHA105. Agrobacterium-mediated transformation of tobacco was performed per standard protocol (Rogers et al., 1986). Integration of the transgene in different lines was confirmed by PCR and Southern blot analysis. Expression of the introduced gene was evaluated with an RNA blot analysis using RNA from young leaves and roots. Transgenic tobacco protoplasts were prepared from calli and roots as described (Carneiro et al., 1993).
Transgenic plants and salinity stress tolerance
To assess the relative salinity tolerance of various plants, wild-type (WT) and T1 generation transgenic seeds over-expressing the RMtATP6 gene were germinated in 1/2 strength MS medium containing 0 and 50 mg l–1 kanamycin, respectively. The surviving seedlings (30-d-old) were transferred onto fresh 1/2 MS medium with one of four salt solutions (150 mM NaCl, 200 mM NaCl, 5 mM NaHCO3, and 7.5 mM NaHCO3) to impose salinity stress or onto plain 1/2 MS medium as a control. The seedlings were maintained under culture room conditions, and their growth under stress was monitored for 25 d. Root length was measured 25 d after the salt treatments.
Semi-quantitative RT-PCR analysis
Total RNA (50 ng) was added to a 20 µl reverse transcription mixture for first-strand cDNA synthesis, using the RT-PCR kit (ReverTra DashTM) (Toyobo, Japan) according to the manufacturer's instructions. After a 30-fold dilution, 1 µl of the reverse transcripts was added to a 50 µl PCR mixture. Annealing temperature was calculated according to the nucleotide composition of the primers and the general program was 30 cycles of 94 °C (30 s), 57.8 °C (1 min) and 72 °C (1 min), followed by an extended incubation at 72 °C for 10 min. The PCR products (15 µl) were then electrophoresed in a 1.2% ethidium bromide (EB)-agarose gel and viewed under UV. Rice actin1 transcripts were used as internal standards. The primers used are listed in Table 1 (sense and antisense, respectively). The ImageMaster TotalLab software (Amersham Pharmacia) was used to measure the intensity of each band. The ratio intensity of the bands relative to the normal condition (CV) was shown.
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Results |
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Sequence analysis and identification of the RMtATP6 gene in rice
The RMtATP6 gene was isolated as described in the Materials and methods. The length of the RMtATP6 cDNA is 505 bp. Sequencing revealed that RMtATP6 cDNA contains a major GC-rich (62%) open reading frame (ORF) of 174 nucleotides (85–258) encoding a protein of 58 amino acids. The open reading frame encodes a predicted 58 amino acid protein with a calculated molecular mass 6578 Da. The TMpred method predicted the RMtATP6 protein has a strong transmembrane domain (Fig. 1). The amino acids at the boundary of the mitochondrial targeting sequence are SRFDPW which are shown in a rectangle in Fig. 2.
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The BLAST algorithm identified two proteins with similarity to RMtATP6 (Fig. 2). One is from Arabidopsis thaliana (At3g46430; 76% amino acid identity) and the other is from potato (Solanum tuberosum) (ID: P80497 [GenBank] ; 73% amino acid identity). RMtATP6 corresponds to a 6 kDa protein from the F0 part of rice mitochondrial ATP synthase (Heazlewood et al., 2003b
) and thus appears to be the mitochondrial ATP synthase 6 kDa subunit. According to the computer program PSORT, the intracellular localizations of RMtATP6 with the highest certainties were the mitochondrial intermembrane space (0.827), microbody peroxisome (0.640), mitochondrial matrix space (0.605), and mitochondrial inner membrane (0.310). These results further suggest that RMtATP6 is located in rice mitochondria.
Intracellular localization of RMtATP6 protein using a GFP fusion in yeast and tobacco cells
To determine whether RMtATP6 is localized to mitochondria, a fusion gene was constructed which encodes a protein with the GFP at the C terminus of the RMtATP6 protein and it was introduced into yeast and plant cells. The fusion protein was expressed in the yeast strain INVSc1, and its localization was analysed with a laser confocal microscope. The mitochondria were visualized with MitoTracker, a mitochondria-specific dye for living cells. As a control, a construct encoding GFP was used to study its targeting in yeast cells (Fig. 3d–f). The green fluorescence of GFP was almost evenly distributed throughout the yeast cells (Fig. 3d). The localization signal was not consistent with the mitochondrial localization signal (red fluorescence area) (Fig. 3e, f). This result indicates the GFP is not targeted to a specific site in yeast cells. The RMtATP6-GFP fusion protein is evenly distributed throughout the mitochondria, as can be observed by RMtATP6-GFP/MitoTracker double staining (Fig. 3a–c).
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The RMtATP6-GFP gene was cloned into the plant expression vector pBI121 to study the localization of RMtATP6 in a tobacco plant (Nicotiana tabacum). The fusion gene was placed under the control of the CaMV 35S promoter. In the confocal microscopy analysis of the transgenic plants, GFP fluorescence was visible throughout the cytosol (Fig. 4a, b). In root protoplasts transformed with the RMtATP6-GFP construct, microscopic analysis of GFP expression revealed a punctate pattern of fluorescence (Fig. 4d), which was also labelled by the fluorescence of MitoTracker (Fig. 4e). Colocalization is shown by yellow in the merged image in Fig. 4f, and shows that the RMtATP6-GFP fusion protein is in mitochondria. Callus protoplasts transformed with the RMtATP6-GFP construct showed fluorescence in mitochondria (Fig. 4g–i). These results show that RMtATP6 is localized in the mitochondria of protoplasts of tobacco roots and calli.
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RMtATP6 gene expression responds to environmental stresses in rice
RMtATP6 gene expression was induced by both salts (80 mM NaCl, 30 mM NaHCO3, 15 mM Na2CO3) and osmotic stress (exposure to 10% PEG 6000) in both leaves (Fig. 5A) and roots (Fig. 5B). In roots, expression was strongly induced by NaCl, Na2CO3, and PEG. In the presence of 30 mM NaHCO3, transcript levels of the RMtATP6 gene in leaves peaked at 12 h, and remained high until 24 h (Fig. 5C), and those in roots increased from 6 h to 24 h (Fig. 5D).
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Salt stress tolerance of transgenic tobacco seedlings harbouring RMtATP6
Three independent transgenic lines (T31, T32, T40) of tobacco were first confirmed to harbour pBI121-RMtATP6 by PCR using genomic DNA as templates (Fig. 6A). Northern analyses further showed that each of these lines constitutively expressed RMtATP6 in leaves (Fig. 6B) and roots (Fig. 6C). In order to investigate the biological role of RMtATP6 in salinity tolerance, three lines of the T1 generation transgenic seedlings over-expressing the RMtATP6 gene were selected and compared with WT plants in salt-tolerance tests. WT and the three independent transgenic lines (T31, T32, T40) showed comparable growth in the absence of salts (Fig. 6D). However, in the presence of NaCl and NaHCO3, the growth of WT plants was reduced compared with that of the transgenic lines. The root length of stressed transgenic plants was longer compared with that of WT, as reflected on visual inspection (Fig. 6E–H) and quantitative estimation (Fig. 7). When plants were grown on medium containing 150 mM NaCl, 200 mM NaCl, 5 mM NaHCO3 and 7.5 mM NaHCO3, the root lengths of the three transgenic plants were about 4.6
6, 3.74.1, 4.65.7, and 3.24.1-fold, respectively, of that in the WT plants (Fig. 7).
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Semi-quantitative RT-PCR analysis
The stresses had different effects on the transcript levels of the genes encoding the other mitochondrial F1F0-ATPase subunits of rice (Fig. 8). The expressions of RMtATP
and RMtATPß in leaves and roots were hardly affected by the various stress conditions. The expression of RMtATP9 in leaves was little affected under several stresses, but the expression of RMtATP9 in roots clearly decreased by treatment with Na2CO3 and PEG. Stress treatments with NaCl and NaHCO3 induced the expressions of RMtATP1 and RMtATP2 in leaves, but the expression of these two genes in roots was clearly decreased by treatment with Na2CO3. Expression of RMtATP6 was increased in leaves by treatment with NaCl, NaHCO3, Na2CO3, and PEG, and was increased in roots by treatment with NaCl and PEG.
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Discussion |
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The compositions of the yeast and mammalian mitochondrial ATP synthase complex subunits have been extensively studied, while much less is known about the plant complex. Several studies have purified and partially sequenced components of F1F0-ATP synthase from spinach (Hamasur and Glaser, 1992
), potato (Jansch et al., 1996), and Arabidopsis (Kruft et al., 2001; Millar et al., 2001). These previous works have mainly identified F1 subunits, and some of the corresponding genes from these species have been sequenced. However, the compositions of the plant mitochondria F0, in contrast, are largely unknown. Recently, four subunits in the F0 part of Arabidopsis mitochondrial ATP synthase were identified, which were subunit 9, d subunit, orfB and orf25, but the function is not clear (Heazlewood et al., 2003a). So it is important to study the structures of the plant mitochondria F0 subunit, which will help in understanding its role in ATP synthase. In this study, the RMtATP6 gene was identified as a rice mitochondrial ATP synthase 6 kDa subunit gene. This subunit was separately purified from the F0 part of mitochondrial F1F0-ATP synthase of potato (Solanum tuberosum) and rice (Oryza sativa L.) by blue native (BN)-PAGE (Jansch et al., 1996; Heazlewood et al., 2003b). An Arabidopsis protein (At3g46430) was found which had 76% amino acid identity with RMtATP6, although the protein was not identified in the F0 component of Arabidopsis (Heazlewood et al., 2003a). Use of GFP as a marker also showed that RMtATP6 is targeted to mitochondria (Figs 3, 4). A BAC clone of rice chromosome 3 was also found which contained the nucleotide sequence of RMtATP6 (BAC OSJNBa0091E13; GenBank accession no. AC133860). Genomic Southern hybridization showed that there is only one RMtATP6-like gene in rice (data not shown). Therefore, RMtATP6 is encoded by the nuclear genome and functions in mitochondrial F1F0-ATP synthase. The transcript level of the RMtATP6 gene showed that it was induced by several abiotic stresses (Fig. 5). Moreover, transgenic tobacco over-expressing RMtATP6 displayed enhanced tolerance to salt stresses from NaCl and NaHCO3 (Figs 6, 7). These results suggest that the RMtATP6 gene plays an important role in the response to salt and osmotic stress.
Little is known on the response of expression of F1F0-ATPase subunit genes against environmental stresses. Sweetlove et al. (2002) exposed Arabidopsis cells to oxidative stresses, and found that the ATP synthase -subunit and ß-subunit was significantly decreased by the stress treatments. The authors speculated that the ATP synthase subunits are the greatest casualties of oxidative stress. Christie et al. (2001) reported that exposure of wheat (Triticum aestivum) to aluminium increased mitochondrial F1F0-ATPase activity only in the aluminum-tolerant wheat variety, although the level of transcript encoding the -subunit of F1F0-ATPase remained constant. They considered that the up-regulation of ATP synthase activity was an adaptive response involved in aluminium tolerance. In this study, to understand better the function of RMtATP6 as a subunit of mitochondrial F1F0-ATPase under environmental stresses, the transcript levels of the genes encoding the other subunits of rice mitochondrial F1F0-ATPase were examined under various stress conditions by semi-quantitative RT-PCR analyses. In this study, there was little or no effect of salt stress on the expression of the -subunit, which was the same as the response to Al stress (Christie et al., 2001); the ß-subunit and 9 subunit of F1F0-ATPase were observed, respectively (Fig. 8). On the other hand, the level of transcript encoding the 6, 1-, 2-subunits of F1F0-ATPases increased with salt stress. The -subunit, ß-subunit, 1-subunit, and 2-subunit are the parts of the F1-complex, which is a catalytic portion of ATP synthase, whereas the 6 subunit and 9 subunit are parts of the F0-complex which performs the proton translocation needed for ATP synthase activity. Although the change in F1F0-ATPase activity has not been analysed in this study, the up-regulation of the level of transcript encoding the subunits of F1F0-ATPases may indicate a response of rice to maintain the activity of F1F0-ATPases under damage suffered from environmental stresses. Because the over-expression of the RMtATP6 gene greatly improved the salt-tolerance of transgenic tobacco plants, increased RMtATP6 must have a role to keep or intensify the activity of the F1F0-ATPase under stress conditions, although the function of RMtATP6 in the complex of the F1F0-ATPase is not understood.
14-3-3 proteins are known as regulators of the H+-ATPase. They are the highly conserved hydrophilic proteins and widely present in animal and plant cells (Aitken, 1996). Recently, Bunney et al. (2001) demonstrated that 14-3-3 proteins are present in the inner mitochondrial-membrane compartment and regulate the activity of mitochondrial and chloroplast F1F0-ATPases via an interaction with the F1 ß-subunit. The activity of ATP synthases in both organellas is drastically reduced by 14-3-3 proteins. The authors suggested that 14-3-3 proteins provide a mechanism to regulate the activity of ATP synthase in response to environmental stresses. It will be very interesting to know whether the 14-3-3 proteins also regulate the activity of mitochondrial ATPase under salt and osmotic stress conditions or not.
In the near future, the yeast two or three-hybridization system will be applied to study the interaction between RMtATP6 and other ATP synthase subunits, and to analyse the ATP synthase activity of transgenic plant with the RMtATP6 gene under environmental stresses, which will help us to discover how the mitochondrial ATP synthase activity is regulated and to understand the function of RMtATP6 in the regulation of ATP synthase activity in response to environmental stresses.
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Acknowledgements |
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This work was supported by the National High Technology Research and Development Program (863 Program) from the People's Republic of China.
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