A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis

JOURNAL OF

GENETICS AND GENOMICS J. Genet. Genomics 37 (2010) 667−683

www.jgenetgenomics.org

A nuclear-encoded mitochondrial gene AtCIB22 is essential for plant development in Arabidopsis Lihua Han a, Genji Qin b, Dingming Kang a, Zhangliang Chen a, b, Hongya Gu b, c, Li-Jia Qu b, c, * b

a College of Agronomy and Biotechnology, China Agricultural University, Beijing 100094, China National Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China c The National Plant Gene Research Center (Beijing), Beijing 100101, China

Received for publication 28 February 2010; revised 18 July 2010; accepted 22 July 2010

Abstract Complex I (the NADH:ubiquinone oxidoreductase) of the mitochondrial respiratory chain is a complicated, multi-subunit, membranebound assembly and contains more than 40 different proteins in higher plants. In this paper, we characterize the Arabidopsis homologue (designated as AtCIB22) of the B22 subunit of eukaryotic mitochondrial Complex I. AtCIB22 is a single-copy gene and is highly conserved throughout eukaryotes. AtCIB22 protein is located in mitochondria and the AtCIB22 gene is widely expressed in different tissues. Mutant Arabidopsis plants with a disrupted AtCIB22 gene display pleiotropic phenotypes including shorter roots, smaller plants and delayed flowering. Stress analysis indicates that the AtCIB22 mutants’ seed germination and early seedling growth are severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis reveals that in moderate knockdown AtCIB22 mutants, genes including cell redox proteins and stress related proteins are significantly up-regulated, and that in severe knockdown AtCIB22 mutants, the alternative respiratory pathways including NDA1, NDB2, AOX1a and AtPUMP1 are remarkably elevated. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in Arabidopsis. Our findings also enhance our understanding about the physiological role of Complex I in plants. Keywords: Arabidopsis; mitochondria; Complex I; B22 subunit; ethanol treatment; alternative oxidase; uncoupling protein

Introduction The mitochondrial oxidative phosphorylation (OXPHOS) system consists of five multi-subunit enzyme complexes: Complex I, II, III, IV and V, among which Complex I (NADH:ubiquinone oxidoreductase; EC 1.6.5.3) is the first and most complicated protein complex. It catalyzes the transfer of two electrons from the mitochondrial matrix NADH generated by the tricarboxylic acid (TCA) * Corresponding author. Tel: +86-10-6275 3018, Fax: +86-10-6275 3339. E-mail address: [email protected] DOI: 10.1016/S1673-8527(09)60085-0

cycle to ubiquinone with the concurrent pumping of 4 protons across the inner mitochondrial membrane (IMM), generating the proton electrochemical gradient (ΔμH+) required for the ATP synthesis (Walker, 1992; Lambert and Brand, 2009). Complex I has a characteristic L-shape structure. One arm is embedded within the inner mitochondrial membrane (membrane arm) and the other protrudes into the mitochondrial matrix or the bacterial cytoplasm (peripheral arm) (Guenebaut et al., 1998; Hirst et al., 2003; Baranova et al., 2007). In plants, an additional peripheral domain is attached to the membrane arm at a central position on its matrix-exposed side, which mainly in-

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cludes carbonic anhydrase subunits (Sunderhaus et al., 2006; Klodmann et al., 2010). Currently 46 proteins have been isolated from the Complex I of bovine heart tissues and at least 49 from Arabidopsis suspension cell culture (Hirst et al., 2003; Klodmann et al., 2010). In plants, 9 subunits of Complex I are encoded by the mitochondrial DNA (mtDNA), i.e., nad1, nad2, nad3, nad4, nad4L, nad5, nad6, nad7 and nad9, while the other subunits are all encoded by the nuclear DNA (nuDNA) and translated in the cytosol and imported into the mitochondria (Lamattina et al., 1993; Rasmusson et al., 1998; Hirst et al., 2003). Disruption of Complex I subunits causes a variety of problems. In humans, it causes a range of neuromuscular diseases, i.e., Parkinson’s disease (Greenamyre et al., 2001; Khan, 2006), Leber’s heredity optic neuropathy (Chinnery et al., 2001; Fauser et al., 2002; Zhang et al., 2008) and leukodystrophy (Schuelke et al., 1999). As for higher plants, until now, only a few Complex I subunit mutants have been characterized and all exhibit various developmental defects (Marienfeld and Newton, 1994; Gutierres et al., 1997; Brangeon et al., 2000; Lee et al., 2002; Perales et al., 2005; de Longevialle et al., 2007; Meyer et al., 2009). Two sites of the mitochondria electron transport chain (ETC) have been widely hypothesized to be responsible for O2− generation in mitochondria Complex I and Complex III (Cadenas et al., 1977). During the electron transport, electrons can leak out from the respiratory chain and combine with oxygen, resulting in ROS production such as superoxide anion radical (O2−), hydrogen peroxide (H2O2), the hydroxyl radical (OH−) and peroxynitrite (ONOO−), which can damage cellular macromolecules including DNA, proteins and lipids (Cadenas et al., 1977; Grivennikova and Vinogradov, 2006; Cocheme and Murphy, 2008; Wang et al., 2008a; Lambert and Brand, 2009). In the IMM, plants present three non-phosphorylating bypasses of respiratory chains, which can reduce the cellular ROS levels and alter phosphorylation efficiency. These bypasses are NAD(P)H dehydrogenases (ND), alternative oxidases (AOX) and uncoupling proteins (UCP). ND, located on both sides of the IMM, delivers electrons from the matrix side or from the intermembrane space NADH pool directly to ubiquinone (Rasmusson et al., 2004). AOX directly accepts electrons from the ubiquinone pool of the ETC and reduces oxygen to water, thus bypassing the cytochrome oxidase (COX) pathway through complexes III

and IV (Vanlerberghe and McIntosh, 1997). UCPs, members of the anion-carrier superfamily in the IMM, catalyze a free fatty acid-mediated proton recycling and dissipate the ΔμH+ across the IMM, and hence modulate the coupling tightness between the mitochondrial respiration and ATP synthesis (Considine et al., 2003; Hourton-Cabassa et al., 2004; Smith et al., 2004; Echtay, 2007). ND, AOX and UCP proteins are critical for normal growth and development and are dramatically induced by various abiotic stresses, or in mutants with genetic lesions of redox proteins (Maxwell et al., 1999; Fernie et al., 2004; Borecky et al., 2006; Vidal et al., 2007). In this study, starting from a late flowering mutant, we characterize the Arabidopsis homologue (At4g34700, designated AtCIB22) of the B22 subunit of eukaryotic mitochondrial complex I. The B22 subunit was grouped into the LYR family and named after a highly conserved tripeptide LYR (LYK) motif close to the N-terminus of these proteins. Proteins in this family have been identified as a component of the higher eukaryotic complex I. Proteomic studies indicate that the complex I B22 subunit in Bos taurus is located in the membrane arm (Hirst et al., 2003), whereas studies of AtCIB22 regarding its localization give conflicting results. In 2006, AtCIB22 was first isolated from the mitochondrial complex I of Arabidopsis cell cultures by Sunderhaus et al. (2006), but not found by other researchers (Heazlewood et al., 2003; Cardol et al., 2004; Meyer et al., 2008; Klodmann et al., 2010). Moreover, the AtCIB22 protein was also identified from the tonoplast of Arabidopsis cell cultures (Jaquinod et al., 2007). Our AtCIB22 subcellular localization data indicated that AtCIB22 was located in mitochondria. Bioinformatic analysis showed that AtCIB22 was a single-copy gene and was highly conserved throughout eukaryotes. The AtCIB22 gene was widely expressed in different tissues of Arabidopsis plants, with the highest expression in the stems. Lack of AtCIB22 resulted in pleiotropic phenotypes including shorter roots, smaller plant size and later flowering. Stress analysis indicated that the AtCIB22 mutants’ seed germination and early seedling growth were severely inhibited by sucrose deprivation stress but more tolerant to ethanol stress. Molecular analysis revealed that genes involved in alternative respiratory chains were significantly up-regulated in the AtCIB22 severe knockdown mutants. These data demonstrate that AtCIB22 is essential for plant development and mitochondrial electron transport chains in plants.

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Materials and methods

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GGAGTTTCGACGGC-3′) and AtCIB22-2 (5′-TTATGG GTTGTCTTCTAGACCA-3′).

Materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) was used in all experiments. Seeds were germinated on half-strength Murashige and Skoog (1/2 MS) medium and synchronized for 3 days at 4°C, then grown under long-day conditions (16 h light/8 h dark) at 22 ± 2°C.

Subcellular protein localization analysis A Bgl II/Spe I fragment containing full length AtCIB22 cDNA was cloned into pCAMBIA-1302 (Cambia, Australia). The construct was transformed into Arabidopsis plants with Agrobacterium tumefaciens GV3101 by the floral dipping method (Clough and Bent, 1998). Harvested seeds were screened on 1/2 MS medium containing 50 μg/mL hygromycin (Sigma, USA). Drug-resistant seedlings were transferred to soil and grown under the same conditions as previously described (Guan et al., 2009). Primers used to clone AtCIB22 cDNA were as follows: GFP-1: 5′-GA AGATCTATGAGCGGAGTTTCGACGG-3′ (Bgl II site underlined); GFP-2: 5′-GGACTAGTTGGGTTGTCTTCT AGACCATA-3′ (Spe I site underlined). Roots of 5-dayold transgenic seedlings and the abaxial side of 3-week-old rosette leaves were visualized using a Leica SPE confocal microscope equipped with a 40 × 1.25 NA oil immersion objective lens. For mitochondrial-specific dyes, Arabidopsis roots were incubated for 15 min in the dark with 500 nmol/L MitoTracker Orange CMTMRos (Molecular Probes, M-7510, Sigma), and then washed three times in 0.2 mol/L phosphate buffered saline (PBS), pH 6.8. Green and MitoTracker Orange fluorescence signals were detected with 488 nm and 543 nm laser lines for excitation respectively. Chlorophyll autofluorescence was excited at 488 nm wavelength.

Generation of AtCIB22 RNAi plants To create an RNAi construct, a 354 bp fragment of AtCIB22 full length cDNA was amplified by RT-PCR. The AtCIB22 RNAi fragment cloning, Arabidopsis transformation and transgenic plants screening were performed as described previously (Qin et al., 2005). Primers used in the RNAi construct were as follows: AtCIB22-1 (5′-ATGAGC

Identification of T-DNA insertion in atcib22-1 and atcib22-2 mutants Three primers, P1 (5′-TTGGTCATTCTTGGGCTTTA -3′), P2 (5′-TTGTCTTACTGGAACGAAAC-3′) and LS3 (5′-GCTTTCGCCTATAAATACGACGG-3′) were designed for co-segregation analysis of the atcib22-1 mutant. A 766 bp fragment would be amplified from the wild type and 538 bp from the homozygous atcib22-1 mutants. The T-DNA insertion line SALK_097732 (designated atcib22-2) was requested from the SALK mutant collection (Alonso et al., 2003). Three primers P3 (5′-CGTGAGAAG TTCAATGTCAACC-3′), P4 (5′-GCTTCACCATGAGC AATCAGTT-3′) and LBa1 (5′-TGGTTCACGTAGTGG GCCATCG-3′) were designed for genotyping analysis of atcib22-2 plants. An 864 bp fragment would be amplified from the wild type and 408-bp from the homozygous atcib22-2 mutants.

Quantitative real-time PCR analysis For tissue-specific analysis, total RNA was extracted from roots, stems, leaves, flowers and siliques of 45-day-old Arabidopsis plants. For other quantitative real-time PCR (qRT-PCR) analysis mentioned in this paper, all the RNA was extracted by Trizol reagent (Invitrogen, USA) from 10-day-old seedlings grown on plates under long-day conditions at 24°C. The reverse transcription and qRT-PCR analysis were performed as previously described (Livak and Schmittgen, 2001; Qin et al., 2007). Primers used to check AtCIB22 transcripts are AtCIB22-1 and AtCIB22-2. Previous descriptions of primers used have been given for At4g34710 (Urano et al., 2005), for ORA47 and WRKY40 (Wang et al., 2008b), and for NDA1, NDA2, NDB1, NDB2, AOX1a and AtPUMP1 (Umbach et al., 2005). Primers for checking transcripts of genes located in the 10 kb region flanking the T-DNA insert in atcib22-1 mutants and AtPUMP3 have been included in Supplemental Table 1.

Root length and flowering time assays For root length assay, wild type and atcib22-2 seeds

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were harvested from plants grown under the same conditions and stored dry for more than 2 months. Triplicates of 60 seeds for each treatment were surface-sterilized, plated on 1/2 MS medium containing 1% sucrose, and chilled at 4oC in the dark for 3 days, then moved to long-day conditions at 24oC. For the sucrose deprivation stress treatment, no sucrose was added into the 1/2 MS medium. For ethanol stress treatment, 0.5% (v/v) ethanol was added into the 1/2 MS medium. The roots were photographed and the lengths were measured by the SPOT software. All values are given as mean ± S.E. (n = 20). The flowering time was determined by the day when the bolting is 1 cm in height under long-day conditions, and then the total leaf number was counted. All values were given as mean ± S.E. (n = 30).

Affymetrix microarray analysis Wild type and atcib22-2 seedlings were grown on the same plate for 10 days under long-day conditions at 24°C. The whole seedlings of wild type and atcib22-2 mutants were pooled and homogenized in liquid nitrogen. Total RNA was isolated from 400 mg seedlings by Trizol reagent (Invitrogen). RNA was purified using RNeasy mini kits (Qiagen) and then was used for hybridization of microarray chips (ATH1 22K, Affymetrix) according to the manufacturer’s instructions. The data image analysis was performed by Affymetrix Microarray Suit (version 5.0) software. After normalization, the adjusted log2 ratio analysis was carried out and genes showing a significant difference (P<0.05) between wild type and atcib22-2 plants were selected (Qin et al., 2007).

Results Identification of the AtCIB22 locus A late flowering mutant was isolated from ~25,000 T1 transgenic plants that were transformed with the activation-tagging vector pSKI015 and later designated atcib22-1 (Fig. 1A) (Weigel et al., 2000). The F1 generation of a cross between wild type (female) and atcib22-1 (male) plants all phenocopied the wild type (Table 1). In the F2 generation, plants segregated for wild type and late flowering mutant phenotype at a ratio of ~3:1 (Table 1), suggesting that atcib22-1 is caused by a single recessive nu-

clear mutation. In order to identify the mutated gene in this mutant, the genomic sequence flanking the T-DNA was recovered by thermal asymmetric interlaced PCR (Fig. 1B) (Liu et al., 1995). An inverted T-DNA repeat was identified to insert into At4g34700, which encodes the Arabidopsis homologue of the B22 subunit of eukaryotic mitochondrial complex I, and is later designated AtCIB22. One insert site is located 13 bp away from the end of the fourth intron, the other is about 250 bp upstream of the first insert site. To examine whether the T-DNA insertion corresponds to the late flowering phenotype observed, we genotyped the wild type, heterozygous and homozygous plants, and found that the T-DNA insertion closely co-segregated with the observed phenotypes (Fig. 1C). To determine whether the AtCIB22 locus is responsible for the mutated phenotype, we examined the expression level of AtCIB22 and the adjacent genes located in the 10 kb region flanking the T-DNA insertion using qRT-PCR analysis. The results showed that AtCIB22 expression was completely knocked out whereas the expression of At4g34710 (encoding arginine decarboxylase 2, ADC2) was remarkably up-regulated in the homozygous atcib22-1 mutants (Fig. 1D). Interestingly we accidentally discovered that atcib22-1 mutants were more resistant to a high concentration of ethanol during germination (Fig. 1E). Since the transcript levels of both AtCIB22 and ADC2 were changed in the atcib22-1 mutants, we carried on the functional dissection of AtCIB22 by reverse genetics.

Reduction of AtCIB22 expression results in pleiotropic developmental defects in Arabidopsis To reveal the function of AtCIB22 in plant development, we isolated a T-DNA insertion mutant SALK_097732 in which the T-DNA insertion was located at 1 bp upstream of the third exon of AtCIB22 (Fig. 2A) (Alonso et al., 2003). qRT-PCR analysis showed that the expression level of AtCIB22 in homozygous SALK_097732 mutants was reduced to 18% of that in the wild type, suggesting that SALK_097732 is a knockdown mutant (Fig. 2, B and D), which we thus designated this mutant as atcib22-2. The atcib22-2 mutants displayed obvious developmental phenotypes such as late flowering and short roots. The phenotypes were co-segregated with the T-DNA insertion, suggesting that the deficiency of AtCIB22 possibly caused the developmental defects found in atcib22-2 mutants.

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Fig. 1. Isolation and characterization of a late flowering mutant atcib22-1. A: wild type (WT) and atcib22-1 plants grown for 40 days in long-day conditions. The atcib22-1 mutants displayed a later flowering phenotype compared to wild type plants. B: scheme of the genomic region flanking the T-DNA insertion site in atcib22-1 mutants. Genes are represented by black arrows, intergenic regions by lines. The arrow direction represents the transcriptional orientation of the genes. The four black arrowheads represent the four 35S enhancers from pSKI015. LB, T-DNA left border; bar, Basta resistance gene; 4Enhancer, CaMV 35S enhancer tetrad; RB, T-DNA right border; P1 and P2, primers for the T-DNA linkage analysis. C: linkage analysis of the T-DNA insertion and phenotypes. P1 and P2 will amplify a 766 bp fragment from the wild type, and P2 and LS3 will amplify a 538 bp fragment from the homozygous atcib22-1 mutant. M, λDNA EcoR I/Hind III marker. D: transcript analysis of At4g34700 and At4g34710 in atcib22-1 plants by quantitative real-time PCR. E: 3-day-old wild type and atcib22-1 mutants germinated on 1/2 MS control and 1/2 MS + 0.5% ethanol medium. Red arrows point to the germinated seeds.

Table 1 Genetic analysis of the atcib22-1 mutant Generation

Plants tested

WT

atcib22-1

F1 *

020

020

00

F2

240

178

62

* Female is wild type and male is atcib22-1.

χ2

P

0.089

0.765

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Fig. 2. Identification of AtCIB22-2 mutants. A: T-DNA insertion in the SALK_097732 (atcib22-2) line. P3 and P4 are the primers used in co-segregation analysis. B: molecular identification of homozygous atcib22-2 mutants. An 864 bp fragment was amplified from the wild type and 408 bp from the homozygous SALK mutants. M, 1 kb DNA ladder marker. C: scheme of the AtCIB22 RNAi construct. LB, T-DNA left border; 35S-P, CaMV 35S promoter; AtCIB22, CDS of the AtCIB22 gene; GUSF, 1 kb fragment of GUS; Ter, nopaline synthase terminator; Kan R, kanamycin resistance gene NPTII; RB, T-DNA right border. D: transcript analysis of AtCIB22 in the wild type, atcib22-2 and R14 mutants.

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To further confirm that AtCIB22 is responsible for the plant developmental defects, we knocked down the expression of AtCIB22 in Arabidopsis using RNA interference (RNAi) (Fig. 2C). Among the 20 transgenic lines we obtained, 4 lines showed delayed flowering phenotypes. We designated the 4 lines as R3, R9, R14 and R20. R14 was chosen for further study because of its most severe phenoltype. qRT-PCR analysis showed that AtCIB22 transcripts in homozygous R14 plants decreased to 8% of that in the wild type, even lower than that in atcib22-2 mutants (Fig. 2D). Statistical analysis showed that both atcib22-2 and R14 plants displayed shorter roots (Fig. 3, A and B), smaller size (Fig. 3C) and later flowering phenotypes (Fig. 3, D and E) compared to the wild type plants. At the same growth stage, atcib22-2 and R14 plants produced smaller and less leaves than the wild type, but there was no obvious difference in the number of leaves at bolting stage (Fig. 3E). The adult atcib22-2 plants had similar height with the wild type, but R14 plants displayed a shorter stature compared to the wild type (Fig. 3, F and G). It is interesting to note that the severity of the phenotypes (i.e., flowering time and plant size) is well correlated with the knock-down levels of the AtCIB22 gene. These results suggest that AtCIB22 is responsible for the mutant phenotypes and is essentially required in plant growth and development.

Molecular characterization of the AtCIB22 gene The AtCIB22 gene contains five exons in the coding region and encodes a protein of 117 amino acid residues. It is a single copy gene in the Arabidopsis genome and highly conserved in plants and animals (Fig. 4A). In the amino acid sequence AtCIB22 has 31% identity with the B22 subunit of Complex I from Bos taurus and 71%, 73%, 77%, 82% and 83% identity with that from Zea mays, Oryza sativa, Populus trichocarpa, Vitis vinifera and Ricinus communis respectively (Fig. 4B). There is no transmembrane domain predicted by transmembrane helices analysis in AtCIB22 (http://www.cbs.dtu.dk/services/ TMHMM/) (Hirst et al., 2003).

AtCIB22 is mitochondria-located and the AtCIB22 gene is widely expressed in Arabidopsis A putative mitochondrial targeting signal peptide was predicted in AtCIB22 by the MitoProt software, which suggested that AtCIB22 was possibly a mitochondria-

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located protein (http://ihg2.helmholtz-muenchen.de/ihg/ mitoprot.html). To test whether AtCIB22 is localized to mitochondria in vivo, a construct in which AtCIB22 fused with a GFP gene driven by the CaMV 35S promoter was generated and transformed into Arabidopsis plants by Agrobacterium-mediated transformation using the floral dip method. Stable transgenic plants from T2 generation were obtained and used for AtCIB22-GFP localization analysis with a confocal microscope. As shown in Fig. 5A, the GFP signal is co-localized with the MitoTracker Orange marker in a single root hair from an AtCIB22-GFP transgenic line, indicating that AtCIB22-GFP was localized in mitochondria. AtCIB22-GFP fluorescence was also detected in the root tips (Fig. 5B), mature roots (Fig. 5C) and mesophyll cells (Fig. 5D). To evaluate the expression pattern, we examined the transcript of the AtCIB22 gene in different tissues by qRT-PCR analysis. The results showed that AtCIB22 was expressed in roots, stems, flowers, rosette leaves, cauline leaves and siliques, with the highest expression in the stems, suggesting that the mitochondrial content in stems is possibly the highest (Fig. 5E). These data suggest that AtCIB22 is mitochondria-located and the AtCIB22 gene is widely expressed in Arabidopsis.

The seedling growth of AtCIB22 mutants is highly sensitive to sucrose deprivation but tolerant to ethanol stress It was reported that the ETC dysfunction could lead to a metabolism adaption from the TCA to the fermentative glycolysis pathway to synthesize carbon skeletons and ATP. During the fermentative glycolysis, more carbohydrate substrates are consumed to keep the metabolic flux, and typical examples of fermentation products are ethanol and lactic acid. In our mutant and transgenic plant, we noticed that the root length of atcib22-2 and R14 seedlings was severely reduced after 8 days growth on sucrose-deprived MS medium as compared to that of the wild type (Fig. 6, A and B), suggesting that carbon source supply is critical for the growth of AtCIB22 mutants. More interestingly, the root and hypocotyls length of atcib22-2 and R14 mutants was obviously longer than that of wild types on MS + 0.5% ethanol medium (Fig. 6, A and C), suggesting that AtCIB22-deficient mutants are more tolerant to ethanol stress. These results demonstrated that the deficiency of AtCIB22 impairs the balance of carbohydrate metabolism in Arabidopsis.

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Fig. 3. Phenotypic analysis of AtCIB22 mutants. A: root length of 10-day-old wild type, atcib22-2 and R14 seedlings, including views of the leaves from above in the bottom three images. The roots of atcib22-2 and R14 mutants all displayed shorter roots and smaller cotyledons than that of wild type. Bar = 1 cm. B: root length analysis of 10-day-old wild type, atcib22-2 and R14 seedlings. Data represent mean ± S.E. (n = 20). Student’s t-test, ** P < 0.01. C: leaves from of 3-week-old wild type, atcib22-2 and R14 plants. D: AtCIB22 mutants grown for 5 weeks in long-day conditions, including detail of the R14 sample. The mutants displayed retarded growth compared to wild type plants. Bar = 1 cm. E: flowering time and leaf number analyses at bolting time of wild type, atcib22-2 and R14 plants. The mutants exhibited delayed flowering as compared to the wild type, whereas the leaf number of the mutants is similar to that of the wild type. Data represent mean ± S.E. (n = 30). Student’s t-test, ** P < 0.01. F: 8-week-old wild type, atcib22-2 and R14 plants. G: Plant height analysis of 8-week-old wild type, atcib22-2 and R14 plants. The atcib22-2 adult plants had similar height with the wild type, but R14 plants displayed a shorter stature compared to the wild type. Data represent mean ± S.E. (n = 30). Student's t-test, ** P < 0.01.

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Fig. 4. AtCIB22 protein and its phylogenetic homologs in plants and animals. A: alignment of AtCIB22 homologs from 11 species, which are Arabidopsis thaliana (A. thaliana), Ricinus communis (R. communis), Vitis vinifera (V. vinifera), Populus trichocarpa (P. trichocarpa), Oryza sativa (O. sativa), Zea mays (Z. mays), Picea sitchensis (P. sitchensis), Physcomitrella patens (P. patens), Homo sapiens (H. sapiens), Bos Taurus (B. Taurus), Xenopus tropicalis (X. tropicalis). Identical amino acid residues are shaded in black. Similar amino acid residues are shaded in gray. Conserved residues are indicated by asterisks. B: phylogenetic analysis of AtCIB22 homologs. The Neighbor-Joining tree was built using MEGA version 4.1 (Tamura et al., 2007) with a bootstrap of 1,000 replicates.

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Fig. 5. Subcellular protein localization and tissue-specific analysis of AtCIB22. A: mitochondrial localization analysis of AtCIB22. An Arabidopsis root hair cell from a plant transformed with AtCIB22-GFP and stained with MitoTracker Orange. GFP fluorescence is false-colored green. MitoTracker Orange CMTMRos (Molecular Probes) fluorescence is false-colored red. Yellow color indicates the overlap of green and red fluorescence. Bar = 10 µm. B–D: fluorescence in the root tip zone (B), mature root zone (C) and mesophyll cells (D) of the AtCIB22-GFP transgenic plants. The red false color is chlorophyll autofluorescence. Bar = 10 µm. E: transcript levels of AtCIB22 in different tissues.

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Fig. 6. Sucrose deprivation and ethanol treatments on AtCIB22 mutants. A: 8-day-old wild type, atcib22-2 and R14 seedlings on MS control, MS sucrose deprivation medium and MS + 0.5% ethanol medium. Bar = 0.5 cm. B: root length analysis of 8-day-old wild type, atcib22-2 and R14 seedlings on MS control and MS sucrose deprivation medium. Data are expressed as the ratio between the decreased root length by sucrose deprivation and the root length on MS control medium. Data represent mean ± S.E. (n = 20). Student’s t-test, ** P<0.01. C: root length analysis of 8-day-old wild type, atcib22-2 and R14 seedlings on MS control and MS + 0.5% ethanol medium. Data represent mean ± S.E. (n = 20).

Redox and stress related genes were significantly up-regulated in atcib22-2 mutant plants To investigate the molecular mechanism causing the phenotypes, we carried out the microarray analysis to reveal the gene expression changes in atcib22-2 mutants. If 2-fold change was taken as the cutting threshold, there were 47 genes up-regulated and 7 genes down-regulated in the mutants. As shown in Table 2, there were several groups of genes whose expression levels were altered in the atcib22-2 mutants. The first group of genes includes those involved in cell redox proteins, e.g., thioredoxin family protein (At1g52990), FAD-binding domain-containing protein (At2g46740) and cytochrome P450 (CYP71B22). The second group contains proteins participating in various defense responses, including salt, cold and pathogen, such as ZAT10 (Mittler et al., 2006), BAP1(Yang et al., 2007), ORA47 (Wang et al., 2008b), PCC1 (Sauerbrunn and Schlaich, 2004), WRKY36 and WRKY40 (Dong et al., 2003), and cell wall proteins including pectin esterase

(At3g10710) and jacalin lectin family protein (At5g49870). The third group involves signal proteins, calcium binding protein (At4g27280), two protease inhibitors (At4g08530, At5g55450), ABA response protein (At3g02480), cell wall proteins encoding lectin protein kinase (At5g59260) and two WAK1 cell wall-associated kinases (At1g21250, At1g69730). We also found that some genes participating in the carbohydrate metabolism were up-regulated, such as the genes encoding DIC2 (Palmieri et al., 2008), fucosyltransferase 6 (FUT6), qua-quine starch (QQS) (Li et al., 2009), glycosyltransferase (At3g18170) and beta-galactosidase (At4g29200). Our microarray data showed that the AtCIB22 transcript level was similar to the results obtained by qRT-PCR, suggesting that the microarray data is reliable. We also confirmed WRKY40 and ORA47 transcript levels by qRT-PCR analysis and a similar change was observed (Fig. 7). These data suggest that knockdown of AtCIB22 has affected the gene transcript pool, and that transcripts of some redox, stress, signal and metabolism related genes are significantly up-regulated.

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Table 2 Expression profiles of genes in wild type and atcib22-2 plants by microarray analysis Locus ID

Annotation

Log2 value

Thioredoxin family protein

+1.1

Redox proteins At1g52990 At2g46740

FAD-binding domain-containing protein

+1.1

At3g26200

CYP71B22, electron carrier/oxygen binding/monooxygenase

−2.3

At1g27730

Zat10, salt-tolerant finger protein

+1.4

At3g61190

BON1-associated protein 1 (BAP1)

+1.3

At1g74930

ORA47, response to biotic stress and wounding

+1.1

At1g72920

Disease resistance protein (TIR-NBS class), putative

+1.2

At1g02530

PGP12, multi-drug resistance protein

+1.1

At1g07730

Disease resistance-responsive family protein

+1.0

Stress response

At2g19970

Pathogenesis-related protein, putative

+1.0

At1g69810

WRKY36, transcription factor

+1.0

At1g80840

WRKY40, transcription factor

+1.0

At4g33450

Myb domain protein 69 (Myb69), transcription factor

+1.5

At5g51190

AP2 domain-containing transcription factor, putative

+1.0

At5g58890

AGL82, DNA binding / transcription factor

+1.0

At3g22231

Pathogen and circadian controlled 1 (PCC1)

−1.8

At3g10710

Pectinesterase family protein

+1.1

At5g49870

Jacalin lectin family protein

+1.4

Signal transduction At5g59260

Lectin protein kinase, putative

+1.6

At1g21250

Cell wall-associated kinase (WAK1)

−1.1

At1g69730

Wall-associated kinase

−1.0

At4g27280

Calcium-binding EF hand family protein

+1.0

At4g08530

Protease inhibitor/ seed storage/lipid transfer protein

+3.1

At5g55450

Protease inhibitor/seed storage/lipid transfer protein

−1.0

At3g02480

ABA response protein-related

+1.2

At1g70450

Protein kinase family protein

+1.6

Others (metabolism, development ) At4g24570

DIC2, dicarboxylate carrier 2

+1.8

At3g18170

Transferase, transferring glycosyl groups

+2.3

At1g14080

Fucosyltransferase 6 (FUT6)

+1.2

At2g46860

Arabidopsis thaliana pyrophosphorylase 3(AtPPa3)

+2.1

At3g30720

QQS(QUA-QUINE STARCH), starch metabolism

+1.0

At5g44120

CRA1, nutrient reservoir

−2.8

At4g13240

Rho guanyl-nucleotide exchange factor (ROPGEF9)

+3.8

At1g01750

Actin depolymerizing factor 11 (ADF11)

+1.0

At3g16320

Cell division cycle family protein (CDC27a)

+1.3

At2g33880

HB-3, positive regulation of cell division

+1.1

At1g58450

Peptidyl-prolyl cis-trans isomerase FKBP-type family protein

+2.5

At5g13150

Exocyst subunit EXO70 family protein C1 (ATEXO70C1)

+1.0

AT4g14730

Transmembrane protein-related

+1.5

At2g04680

DC1 domain-containing protein

+1.1

At4g01760

DC1 domain-containing protein

+3.6

At1g14220

Ribonuclease T2 family protein

+1.0

Lihua Han et al. / Journal of Genetics and Genomics 37 (2010) 667−683

Transcripts of alternative respiratory pathway were remarkably elevated in AtCIB22 severe knockdown plants To investigate whether there is any change in the ROS response signaling in R14 plants, we examined the transcript change of those genes in non-phosphorylating respiratory chains including alternative NADH dehydrogenases (NDA1, NDA2, NDB1 and NDB2), alternative oxidase (AOX1a) and uncoupling proteins (AtPUMP1, AtPUMP3) by qRT-PCR analysis. The result showed that NDA1, NDB2, AOX1a and AtPUMP1 transcripts were significantly

Fig. 7. Confirmation of up-regulation of WRKY40 and ORA47 genes in atcib22-2 plants by quantitative real-time PCR. Data represent mean ± S.E. of three independent experiments.

Fig. 8. Transcript analysis of NDA1, NDB2, AOX1a and AtPUMP1 genes in R14 plants by quantitative real-time PCR. Data represent mean ± S.E. of three independent experiments.

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up-regulated in R14 plants (Fig. 8). These data suggest that severe deficiency of AtCIB22 will lead to the activation of non-phosphorylating respiratory chains in plants.

Discussion In this study, we found that the disruption of a nuclear gene, encoding the homologue of the B22 subunit of eukaryotic mitochondrial Complex I, results in abnormal development and a modified respiratory pathway in Arabidopsis. Mutants concerning mitochondrial Complex I in plants are associated with different developmental abnormalities. The maize (Zea mays L.) non-chromosomal stripe (NCS) 2 mutant, possessing a fused nad4-nad7 mitochondrial gene, displays striped sectors of pale-green tissue on the leaves and retarded growth (Newton and Coe, 1986; Marienfeld and Newton, 1994). The Nicotiana sylvestris cytoplasmic male sterile (CMS) I and II, NMS1 mutants and Arabidopsis fro1, otp43, css1 and ndufs4 mutants, exhibit a slower growth rate, and the fertility of tobacco CMSI, CMSII and NMS1 mutants is reduced (Gutierres et al., 1997; Brangeon et al., 2000; Lee et al., 2002; Nakagawa and Sakurai, 2006; de Longevialle et al., 2007; Meyer et al., 2009). However, the developmental defiency in ca2 mutants devoid of the plant-specific Complex I subunit gamma carbonic anhydrase 2 was much less apparent. The mutants developed normally under standard growth conditions, but their suspension cell culture displayed reduced growth rates and respiration (Perales et al., 2005). Further studies reveal that the activity or assembly of complex I was impaired in isolated mitochondria of NCS2, CMSII, NMS2, otp43 and ca2 mutants, and an increased respiration in the dark and a decreased photosynthesis were observed in CMSII mature leaves (Brangeon et al., 2000; Sabar et al., 2000). Our data show that the severe knockdown AtCIB22 mutants exhibit shorter roots, a smaller plant size, late flowering and a reduced plant height at the end of growth stage, whereas their fertility was normal. The previous report shows that the mitochondrial matrix protein Isd11 in Saccharomyces cerevisiae, belonging to the LYR family, has a limited sequence similarity with the eukaryotic Complex I subunits B22 and B14, and plays an important role in Fe/S cluster biogenesis in mitochondria (Wiedemann et al., 2006). But whether the function of AtCIB22 protein is to participate the Fe/S

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cluster formation, or regulate Complex I enzymatic activity, assembly and structural stability, or protect the complex against oxidative stress, still remains unclear (Hirst et al., 2003). Complex I is the major NADH sink and electron entry point for the classical COX respiratory chain under normal conditions in mitochondria. During its dysfunction, ND can oxidize NADH from the cytosol and matrix side, supplying the respiratory substrate NAD+, and introduce electrons into the ubiquinone pool. Moreover, Complex II and the electron transfer flavoprotein can deliver electrons from succinate and branched chain amino acids respectively, directly to ubiquinone, and then to oxygen through AOX (Sun et al., 2005; Ishizaki et al., 2006). Mitochondria isolated from tobacco CMS leaves showed a decreased Complex I activity, but increased external ND activity and AOX protein level (Gutierres et al., 1997; Sabar et al., 2000). In previously described maize NCS2, Arabidopsis ndufs4 and otp43 mutants, the elevated AOX protein level is also observed. Additionally, in Arabidopsis suspension cell culture, treated with the classic Complex I inhibitor rotenone for 12 h, transcripts of NDA2, NDB2, AOX1a and AtPUMP1 were strongly up-regulated (Garmier et al., 2008). We found in this study that in AtCIB22 partial knockdown atcib22-2 mutants, transcripts of alternative respiratory pathways did not change, as shown in the microarray data. Different from the situation in atcib22-2 mutants, R14 plants in which AtCIB22 was severely knocked down showed a significant transcriptional induction of NDA1, NDB2, AOX1a and AtPUMP1, suggesting the important role of AtCIB22 in maintaining the normal mitochondrial ETC in plants. Mitochondria play essential roles in aerobic cellular respiration which mainly involves three stages: glycolysis, the TCA cycle and ETC (Giege et al., 2003; Fernie et al., 2004). During the ETC dysfunction, aerobic respiration is impaired and the anaerobic respiration is activated. The end-product of glycolysis, i.e., pyruvate, is not imported into the mitochondria, but is reduced to ethanol and lactate by alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) respectively, or is transaminated to alanine by the alanine aminotransferase (AlaAT) (Ismond et al., 2003; Bailey-Serres and Chang, 2005; Kato-Noguchi and Morokuma, 2007; Garmier et al., 2008). In ndufs4 mutants without the 18 kDa subunit of Complex I, amino acids derived from the convergence of glycolysis, the pentose phosphate pathway and TCA cycle intermediates were

found to accumulate, suggesting that glycolysis may operate at a higher metabolic rate (Meyer et al., 2009). In css1 mutants, defective in the functional nad4 subunit, various fundamental metabolic pathways including amino acid metabolism, triacylglycerol degradation and polysaccharide synthesis (cellulose and starch) were modified during the early stage of plant growth, and the content of alanine was significantly increased from 3 to 33% (Nakagawa and Sakurai, 2006). In addition, in Arabidopsis suspension cells treated with rotenone, glycolysis metabolic flux was increased and fermentation products, i.e., lactate and alanine, were induced (Garmier et al., 2008). Fermentation is an inefficient energy source with less ATP generation and more consumption of carbohydrate substrates. Germination of fro1 mutants and the seedling growth of ndufs4 and css1 mutants were all inhibited by sugar treatments, possibly due to severe osmotic stress. Moreover, the growth of css1 seedlings was also severely suppressed by sugar deprivation stress. Consistent with css1 mutants, the atcib22 seedling growth was also significantly inhibited in the absence of sugar, suggesting that a disturbance in cellular sugar homeostasis occurred. Interestingly atcib22 mutants also displayed a remarkably ethanol tolerant phenotype. Previous investigations have shown the enhanced cellular anti-oxidative system in certain complex I mutants, as shown in our microarray data in atcib22 mutants (Dutilleul et al., 2003; Meyer et al., 2009), whereas it is also known that, in plants, ethanol can be metabolized to acetaldehyde and then to acetate, and then into general metabolism (MacDonald and Kimmerer, 1993). Currently it is not yet clear whether the ethanol tolerance is caused by the strengthened antioxidant defense system, and/or the accelerated ethanol metabolism pathway, which will be investigated in the future.

Acknowledgement This study was supported by the National Basic Research Program of China (No. 2009CB941503).

Supplemental data Supplemental Table 1 associated with this article can be found in the online version at www.jgenetgenomics.org.

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