A CMS-HL line, YuetaiA (YtA), and its maintainer line of YuetaiB (YtB, Indica) and ShijinB (SB, japonica) were used in this study. T0 progeny transgenic plants were grown at 25°C under 16 h of daylight in a greenhouse. For physiological analysis, plants were grown under a controlled temperature of 23°C and at 70% relative humidity.
Plasmid construction and rice transformation
The sequence of the gene coding for the mitochondrial transit peptide was derived from RFo (Genbank accession EF472240.1) . To enhance the expression of orfH79 in the nuclear genome of rice, the fusion between this transit peptide sequence and orfH79 was synthesized using rice-preferred genetic codons based on the Codon Usage Database that provided by Kazusa DNA Research Institute http://www.kazusa.or.jp/codon/. The fusion was synthesized by overlap extension PCR . Four long primers with 10 bp of overlap were synthesized and PCR reactions were carried out as described by : s1, 5'-atgaccaacctgctccgctggctcttctccaccacccgcggcaccaacggtctcccatactt catcttcg-3';s2, 3'aagtagaagccacagc accacccgcc gcgggacaaaagcgaaacgatc atagtcggggagacatg-5'; S3, 5'-cctctgtacgaccccgctctactggacaagatcatagat cataacataaaggccggctaccctatagagg-3'; S4, 3'-gatatctccacctgaaccccgtga gataagcacaccagaagggattcac T-5'. The synthetic gene was ligated to the OsPMP41  to form an intermediated construct designated pGUS1. This was then digested with HindIII and BamHI and ligated to a strong constitutive promoter Pubi (the maize ubiquitin promoter). The entire expression cassette (Pubi:: synthetic gene:: Nos) was digested with HindIII and EcoRI and ligated into a binary vector pCAMBIA1301, that had been digested with the same enzymes. The resulting constructs were designated pCON. The japonica rice variety ShijinB was transformed using Agrobacterium tumefaciens EHA-105, following the published procedures by Hiei et al. .
Preparation of proteins
Total protein was extracted from etiolated shoots, spikelets and anthers of YtA and YtB. Tissues were ground into a fine powder in liquid nitrogen. 400-500 mg of tissue were extracted in 1 ml of extraction buffer (66 mM Tris-Cl, pH 6.8, 2% SDS, 2% v/v 2-mercaptoethanol) at room temperature followed by centrifugation for 20 min at 40000 × g to remove the insoluble fraction. Proteins were precipitated at -20°C for 1 h by the addition of 1 ml of an 8:1 mixture of ice-cold acetone and trichloroacetic acid with 0.1% v/v 2-mercaptoethanol. The supernatant was discarded after centrifugation at 18000×g for 15 min at 4°C, and the pellet was washed in 1 ml of ice-cold acetone. The remaining acetone was removed by air drying at room temperature. The resulting pellet was redissolved in the protein extraction solution. Purified mitochondrial proteins were extracted from the same tissues and transgenic callus mitochondria as described by Wan et al. . The YtA line mitochondria protein were separated into soluble and insoluble fractions according to a method described by Uyttewaal et al. 
Antibodies and western blotting analysis
A polyclonal antibody against ORFH79 was obtained from a commercial service (Alpha Diagnostic International, San Antonio, Tex., USA) by injection of a synthetic peptide, comprising residues 37 to 55 of ORFH79 into rabbits. The specificity of the antiserum was tested by western blot the ORFH79 protein expressed in E. coli (Additional file 4). Equal amounts of protein from each fraction were separated by 18% SDS-PAGE gel at 4°C, and then transferred onto an Immobilon-PSQ transfer membrane (PVDF type; Millipore) at 80 V for 40 min. The membrane was incubated in 5% w/v non-fat milk, 0.05% v/v Tween-20, in phosphate buffered saline (PBS) for 1 h, washed for 10 min three times in PBST (PBS, 0.05% Tween-20), and incubated in a 1:500 dilution of rabbit antiserum overnight at 4°C. After four washes with PBST, the membrane was incubated with goat anti-rabbit IgG conjugated with alkaline phosphatase (AP) in PBST solution for 2 h. After four 10-min washes in PBST, signal was detected according to a method described by Yue et al . Briefly, the membrane was washed with AP 7.5 buffer (0.1 M Tris-HCl pH 7.5, 0.1 M NaCl, 2 mM MgCl2) twice, and once with AP 9.5 buffer (0.1 M Tris-HCl pH 9.5, 0.1 M NaCl, 50 mM MgCl2) for 10 min each. Then the membrane was incubated with 2.5 mg nitroblue tetrazolium (NBT; Promega) and 1.25 mg 5-bromo-4-chloro-3-indolyl phosphate (BCIP; Promega) in 7.5 ml of AP 9.5 buffer at room temperature until the signal appeared. Finally, TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) was added to stop the reaction. As a negative control, a western blot was simultaneously processed in which only the secondary antibody was used.
Immunofluorescence analysis of the orfH79 product in anther and root tip
Five-day-old root tips and premeiotic anthers of YtA were dissected, fixed and sectioned, as described by Paciorek et al. . After dewaxing and rehydration, the sections were washed six times in PBST, 5 min each wash. They were then blocked in 0.5% (w/v) BSA and 5% (v/v) normal goat serum in PBST for 30 min at room temperature, and finally incubated overnight at 4°C in a 1:100 dilution of the primary antibody in PBS, 5% (w/v) non-fat milk, 0.05% (v/v) Tween-20. After six washes of 5 min each in PBST, the slides were incubated with goat anti-rabbit FITC-conjugated secondary antibody (1:50) at room temperature for 2 h, and subjected to five 10-min washes in PBST. Hybridization signals were visualized and captured by a cool CCD camera mounted on an Olympus BX51 microscope.
Root length and the number of lateral root assay
T1 seeds of the transgenic and non-transgenic plants were sown on 1/2 MS medium containing 0.3% (w/v) phytagel (Sigma, St Louis, MO, USA). Images of 5-day-old and 7-day-old roots were collected, and length and lateral root numbers were recorded. Eight to ten roots were measured in each of three to five replicates.
Detection of ROS
T1 seeds of three independent transgenic and non-transgenic plants and seeds of YtA and YtB were de-husked, sterilized and then germinated on 1/2 MS medium containing 1.0% (w/v) phytagel gel. Five-day-old seedling root tips were harvested. ROS detection was performed as previously described by Umbach et al. with slight modifications. Briefly, root tips were excised and placed in 4 ml of 1 mM Tris-Cl, pH 8.0, and gently shaked at 23°C in dark for 30 min. Then the roots were incubated with 10 μM 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA[Sigma]) in 1 mM Tris-Cl buffer (pH 8.0) for 10 min. After a briefly rinsing with the Tris-Cl buffer to remove the dye, the roots were imaged with a confocal microscope. Optical filters were set to the maximum absorption wavelength of 488 nm and the emission wavelength of 530 nm. The experiment was performed in triplicate. For anther ROS detection, the anthers were punctured by a dissecting needle so that the H2DCFDA could penetrate easily. Image analysis and quantification of the fluorescence intensity were performed by the Leica LCS Lite software.
ATP and ADP measurements
0.5 g fresh weight (FW) of one week old root tip from YtA and YtB were collected respectively. Three-week old T1 progeny transgenic seedlings, as well as the negative plants that had been segregated from T1 progeny as a negative control were etiolated for three days. Root tips and etiolated plants (0.5 g FW) were ground in liquid nitrogen. ATP and ADP were extracted using 4.5% HCLO4 according to Botrel and Kaiser  and measured by the luciferin-luciferase method [38, 39] following the protocol of the ATP detection kit (Beyotime, China). ADP was converted to ATP by pyruvate kinase (EC18.104.22.168) (Sigma). The ATP content was assayed by luminescence in a Multifunctional Microplate Reader (SpectraMax M2, MDC). Three independent transgenic lines were used in this experiment, and each measurement was repeated three times.
Measurement of mitochondria membrane potential (ΔΨm)
T1 seeds of transgenic and non-transgenic plants were de-husked, sterilized, and germinated on 1/2 MS medium at 28°C in the dark. Five-day-old etiolating seedling were harvested for protoplast isolation. About 1 g of young seedlings was sliced into 0.5 mm pieces and placed into a 10 M CPW solution containing 0.7 M mannitol for 2 h for preplasmolysis. The pieces were then incubated in digesting medium (1.5% cellulose enzyme [Sigma], 1% Hemicellulase [Sigma], 0.5% pectolyaser [Onazuk, Japan] in 10 M CPW) for 4 h at 30°C in the dark. Protoplast were purified as described by Parys et al. . The integrity and activity of the protoplasts were checked by staining them with neutral red and observing them with a microscope. JC-1 was used a measure the mitochondrial potential (ΔΨm) as described by Simeonova et al. . The protoplasts (106 ml-1 ) were suspended in JC-1 buffer supplied by the JC-1 kit (Beyotime, China). Briefly, the protoplasts were treated with 10 μg ml-1 of JC-1 in the dark for 20 min, washed twice with JC-1 buffer and analyzed on a Multifunctional Microplate Reader (SpectraMax M2, MDC) with excitation at 490 nm and emission at 530 and 590 nm, respectively. ΔΨm was determined by the ratio of fluorescence intensity at 590 nm to 530 nm in triplicate. With the same kit, mitochondria isolated from the root of YtA and YtB were suspended in JC-1 buffer. Then, 10 μg ml-1 of JC-1 was added. After incubation in the dark for 10 to 15 min, then the mitochondria membrane potential of YtA and YtB was assayed by calculating the ratio of fluorescence intensity at 590 nm to 530 nm in triplicate.
Root respiration measurements
Roots from five-day old YtA and YtB, and one-week-old transgenic and wild type seedlings were prepared respectively. The roots were briefly rinsed in a buffer (10 mM MES-KOH pH 6.5) to remove dead cells and then immediately transferred to the temperature-controlled chamber of a chlorolab 2 electrode (Hansatech, UK) containing 2 ml of buffer. Respiration rate was measured by detection of oxygen consumption at 25°C.
Vitamin C (VC) treatment
Seeds of YtA and YtB were de-husked, sterilized, and germinated in culture solution  containing 100 nM vitamin C at 28°C for five days. The length of the primary roots and the development of lateral roots were measured. Five independent plants for each sample were measured respectively.
Assays for mitochondrial distribution in root tip
Five-day-old roots tips were harvested, then the root tips were stained with 10 μM MitoTracker red (Molecular Probes) for 40 min and then washed in water for 20 min. Image analysis was performed using the Leica LCS Lite software.
P-values were calculated using Microsoft Excel (2003 version). All error bars in the graphs indicate standard errors of the mean. The statistical significance of the experimental data was determined using the two-sided Student's t-test.