Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice

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1 Journal of Integrative Plant Biology 2007, 49 (6): Structural and Expressional Variations of the Mitochondrial Genome Conferring the Wild Abortive Type of Cytoplasmic Male Sterility in Rice Zhen-Lan Liu *, Hong Xu *, Jing-Xin Guo and Yao-Guang Liu ** (Key Laboratory of Plant Functional Genomics and Biotechnology of Education Department, Guangdong Province, College of Life Sciences, South China Agricultural University, Guangzhou , China) Abstract The so-called wild abortive (WA) type of cytoplasmic male sterility (CMS) derived from a wild rice species Oryza rufipogon has been extensively used for hybrid rice breeding. However, extensive analysis of the structure of the related mitochondrial genome has not been reported, and the CMS-associated gene(s) remain unknown. In this study, we exploited a mitochondrial genome-wide strategy to examine the structural and expressional variations in the mitochondrial genome conferring the CMS. The entire mitochondrial genomes of a CMS-WA line and two normal fertile rice lines were amplified by Long-polymerase chain reaction into tilling fragments of up to 15.2 kb. Restriction and DNA blotting analyses of these fragments revealed that structural variations occurred in several regions in the WA mitochondrial genome, as compared to those of the fertile lines. All of the amplified fragments covering the entire mitochondrial genome were used as RNA blot probes to examine the mitochondrial expression profile among the CMS-WA and fertile lines. As a result, only two mrnas were found to be differentially expressed between the CMS-WA and the fertile lines, which were detected by a probe containing the nad5 and orf153 genes and the other having the ribosomal protein gene rpl5, respectively. These mrnas are proposed to be the candidates for further identification and functional studies of the CMS gene. Key words: cytoplasmic male sterility; mitochondrial genome; rice; transcription. Liu ZL, Xu H, Guo JX, Liu YG (2007). Structural and expressional variations of the mitochondrial genome conferring the wild abortive type of cytoplasmic male sterility in rice. J. Integr. Plant Biol. 49(6), Available online at Received 20 Dec Accepted 7 Mar Supported by Grants from the Ministry of Science and Technology of China (2005CB12080), the National Natural Science Foundation of China ( ), Ministry of Education of China, and China Postdoctoral Science Foundation to Liu Zhen-Lan ( ) and Xu Hong ( ). Publication of this paper is supported by the National Natural Science Foundation of China ( ). *Authors contributed equally. **Author for correspondence. Tel: +86 (0) ; Fax: +86 (0) ; <ygliu@scau.edu.cn>. Present address: College of Life Sciences, Northwest A&F University, Yangling , China Institute of Botany, the Chinese Academy of Sciences doi: /j x Mitochondria play important roles in a broad spectrum of physiological and developmental events in higher eukaryotes. Compared to other eukaryotes, the structure and size of plant mitochondrial genomes are highly variable (Wolstenholme and Fauron 1995). A number of studies show that recombination events frequently occur in the mitochondrial genomes of higher plant species, which play an important role in enlarging the genome size (Palmer and Shields 1984; Schardl et al. 1984; Mackenzie et al. 1988; Folkerts and Hanson 1989). Homologous recombination between repeated sequences can lead to a multipartite organization of mitochondrial genomes, which is indicative of a variety of different, highly redundant, subgenomic molecules (Ogihara et al. 2005; Sugiyama et al. 2005). On the other hand, illegitimate recombination between non-homologous sequences results in expanding of the size of plant mitochondrial genomes, or production of new chimeric genes. In plants, mitochondrial genes can directly influence male

2 Variations of the Mitochondrial Genome in CMS-WA Rice 909 development. The best known phenomenon is cytoplasmic male sterility (CMS), which has been observed in more than 150 plant species. CMS is a maternally inherited trait, by which plants fail to produce functional pollens. CMS is often associated with unusual open reading frames (ORFs) found in mitochondrial genomes. In many instances, male fertility can be restored specifically by nuclear-encoded, fertility restorer (Rf) gene(s). Therefore, CMS systems are ideal models for studying the genetic interaction and cooperative function of mitochondrial and nuclear genomes in plants (Schnable and Wise 1998; Hanson and Bentolila 2004). In addition, CMS systems have been exploited for the commercial production of F 1 hybrid seeds in a number of important crops. The sequencing of the mitochondrial genomes in plants provides critical information for understanding the mechanisms underlying these variations. The lower plant liverwort Marchantia polymorpha is the first plant of which the mitochondrial genome has been sequenced (Oda et al. 1992). To date, complete plant mitochondrial genome sequences have been determined for dicots of Arabidopsis thaliana (Unseld et al. 1997), sugar beet (Beta vulgalis L.)( Kubo et al. 1999; Satoh et al. 2006), rapeseed (Brassica napus L.) (Handa 2003), tobacco (Nicotiana tabacum L.) (Sugiyama et al. 2005) and monocots of rice (Oryza sativa L.) (Notsu et al. 2002; Tian et al. 2006), maize (Zea mays L.) (Clifton et al. 2005) and wheat (Triticum aestivum L.) (Ogihara et al. 2005). In rice, various types of CMS systems have been identified. Among them, the wild abortive system (CMS-WA) that was derived from a population of wild species O. rufipogon (Yuan 1977) is the most extensively used for hybrid rice production. The molecular basis of another CMS system CMS-BT (boro II) in rice has been elucidated (Wang et al. 2006), however, little is known about the mitochondrial genome conferring the WA type of CMS, and the related sterile gene(s) are unknown. In this study, we examined the structural and expressional variations of the mitochondrial genomes between a CMS-WA line and two normal fertile lines by exploiting a mitochondrial genome-wide strategy in combination with the use of the Long and Accurate polymerase chain reaction (LA-PCR) technique. A number of varied mitochondrial genomic regions were detected between the CMS-WA and fertile rice lines, and two mrnas that were differentially expressed between the CMS-WA and normal fertile lines were found. These results provide a basis for further identification and functional studies of the CMS gene. (490.5 kb) (Notsu et al. 2002), we first designed primer sets to amplify the entire mitochondrial genomes of a typical CMS-WA line Zhen-Shan 97A (ZS97A, indica) and two normal fertile cultivars Nipponbare (japonica) and (indica) into 43 tilling fragments (numbering as 1 43) of kb (Table 1). LA-PCR was used to amplify these relatively large fragments. Except for two (32, 41), the fragments from Nipponbare and were amplified efficiently. However, ten of the fragments in ZS97A could not be amplified efficiently (Figure 1). Therefore, new primers were prepared to amplify each of these regions into two sub-fragments. Finally, these regions, except for two (14-1 and 29), could be amplified from ZS97A. Due to the presence of large duplicated blocks within the rice mitochondrial genome (Notsu et al. 2002), some primer sets amplified identical fragments from two regions of the mitochondrial genome. To analyze the sequence variations in detail, the amplified fragments were subjected to restriction digestion with HindIII and DraI. Differences in the restriction pattern were observed between ZS97A and the two fertile lines in four regions (Table 1, Figure 2). By this restriction assay, only one region (25-2) of was found to be different from Niponbare (Figure 2). Structural variations revealed by DNA blotting analysis To further detect possible structural variations of the mitochondrial genomes among the rice lines, DNA blot analysis was carried out on total genomic DNAs digested by DraI and HindIII using nine fragments (14-2, 15, 17, 21-1, 28, 29-2, 30, 38, 41-1) amplified from Niponbare as the probes. Three (14-2, 28, and 38) of the probes detected identical hybridization patterns among the three rice lines. The remaining probes, including those showing monomorphism (15, 17, 30, 41-1) assayed by the restriction digestions, detected restriction fragment length polymorphisms (RFLPs) (Figure 3). Dramatically-different intensities of the hybridization signal were observed among the bands of each line detected by 30 (Figure 3), suggesting the presence of subgenomic circles at different copy numbers in the mitochondria. The 29 region (9.3 kb), in which an essential gene, coxi and predicted genes, orf284 and orf165 are present, Results PCR amplification and restriction analysis of the whole mitochondrial genomes of CMS and fertile lines On the basis of the rice mitochondrial genomic sequence Figure 1. Examples of amplification of mitochondrial genomic fragments by Long and Accurate polymerase chain reaction (LA-PCR) from Nipponbare (1), ZS97A (2), and 93-11(3), respectively.

3 910 Journal of Integrative Plant Biology Vol. 49 No Table 1. Polymerase chain reaction amplification and restriction analysis of the mitochondrial genomes of rice lines Niponbare, 93-11, and ZS97A. Fragment no. Fragment position (bp) Presence of genes Restriction pattern Hind III Dra I m m 2 (11) coxiii, orf25, orf152a, nad5-1 m m (12) nad5-2, ψtrnv, trnm m m (13) trnh, ψtrnp, trnw m m trnq, rps7 m m orfb, trnm, nad6, ccmc m m trni, trnd, trnn, trnk m m orfx m m trnp, nad1-1 m m nad1 m m trnq, orf288 N = 9, A = NP N = 9, A = NP nad4-4 m m 15 (42) m m m m rps3, rpl16, nad3, rps12, m m rps2, trnfm m m nad4 m m cox2, orf161 m N = 9 = A orf152b, orf187 m m atp6 N = 9 = A N = 9 = A nad5-4,5, orf153 N = A = 9 m nad1-2,3, rps13, rps4, nad1-4 m m orf176, trns, atp9 m m orf161 m m m N = 9 = A orf152b, orf187, rrn5, rrn18 N = 9 = A N = 9 = A N = A = 9 N = A = rpl2, rps19, nad4l, cob m m nad5-3,5, nad1-5, mat-r m m m m rps1, ccmfn m m ccmfc m m trns, orf165, orf284 N = 9, A = NP N = 9, A = NP cox1 N = 9, A = NP N = 9, A = NP rpl5 m m 30-2 (37-2) m m m m 31 (38) atp1, trns, trnf m m (39-1) nad9, trny, nad2-3,4 m m (39-2) orf173 m m trne, rrn26, orf162 m m trnc, ccmb m m nad2-2 m m nad2-1, trnr m m rpl5 m m rrn26 m m orf162 m m nad4-4 m m m m The primary fragments (1 43) were numbered in order of their locations along the mitochondrial genome sequence, and those numbered as 14-1 and 14-2 were sub-fragments of the primary fragments. The fragments listed in parentheses are duplicated and amplified with the same primer sets. Fragment positions and the presence of genes or predicted open reading frames are based on the mitochondrial genome of Niponbare (Notsu et al. 2002). m, monomorphic among the three lines; N = 9 = A, Niponbare was identical with 93-11, but different from ZS97A; N = A = 9, Niponbare was identical with ZS97A, but different from 93-11; NP, no amplified product.

4 Variations of the Mitochondrial Genome in CMS-WA Rice 911 Figure 2. Sequence variation of the amplified mitochondrial genomic fragments revealed by restriction analysis. 1, Nipponbare; 2, 93-11; 3, ZS97A. could not be amplified from ZS97A even new primers were used to amplify the region into sub-fragments. We then carried out DNA blot analysis using a fragment 29-2 of Niponbare covering the coxi sequence as the probe. The result showed that sequence variations were widely present in this region of ZS97A (Figure 3). Transcriptional variations among the mitochondrial genomes To examine the possible alterations in mitochondrial transcription patterns among the CMS and fertile lines, RNA blotting analysis was carried out by using all of the amplified fragments from Nipponbare and ZS97A (Table 1) as the probes. Six fragments (1, 14-2, 15, 16, 27-1, and 32-2) did not detect a hybridization signal. This indicated that no active genes exist in these regions, even though an ORF (orf173) was predicted within the 32-2 region (Notsu et al. 2002). The other probes detected transcripts, of which four (5, 6, 21-2, 30-1) showed different transcription profiles among the lines (Figure 4). Although the mrna patterns in ZS97A detected by the probes 5 containing rps7 and 6 containing orfb, nad6, and ccmc were different from those of Nipponbare, they were the same with those of This suggests that these mrnas are not correlated with the CMS. However, the probe 21-2 containing nad5 and orf153 and the one 30-1 having the ribosomal protein gene rpl5 detected a special mrna with relatively large size in ZS97A, respectively. The same mrna patterns were detected by these Figure 3. Restriction fragment length polymorphisms (RFLPs) revealed with the amplified mitochondrial genomic fragments as the probes. 1, Nipponbare; 2, 93-11; 3, ZS97A. two probes in other CMS-WA lines (data not shown). Although macro structural variation is shown in the 29-2 region, the coxi gene located in this region was transcribed into the same mrna pattern among the lines (Figure 4). Discussion Effective strategy for comparative studies of the whole mitochondrial genomes Structural variations between CMS-WA lines and normal fertile lines were studied using the RAPD method (Cai et al. 1998; Yang et al. 2002). However, this method can find only a small part of the variations. In this study, we amplified the entire mitochondrial genomes of the CMS-WA and normal fertile lines into tilling fragments of up to 15.2 kb using the LA-PCR technology, also called Long Range PCR or Long PCR (Barnes 1994; Cheng et al. 1994). These fragments were subjected to

5 912 Journal of Integrative Plant Biology Vol. 49 No Figure 4. RNA blot analysis with the amplified mitochondrial genomic fragments as the probes, showing differential or identical transcripts among the fertile and cytoplasmic male sterility (CMS) lines. 1, Nipponbare; 2, 93-11; 3, ZS97A. analyses on structural and expressional variations by restriction analysis, DNA blot and RNA blot. This PCR-based genomewide strategy is effective for comparative analyses of the genomes of mitochondrion, chloroplast, or focused chromosomal regions in organisms with reference sequence information available. Structural variations among the mitochondrial genomes of CMS-WA, indica, and japonica cultivars It has been demonstrated that mitochondrial genomes of higher plants are organized in a more complex way than those of other eukaryotes (Knoop 2004). Divergent arrangements in mitochondrial genomes have been widely detected in higher plants, even within species (Ullrich et al. 1997). The multipartite structure is a widely accepted model for interpreting the complexity and arrangements of the mitochondrial genome in higher plants, which involves a single circular master chromosome generating multiple subgenomic circles by recombination events between repeated sequences residing in the mitochondrial genome. This is documented in sugar beet (Beta vulgaris), in which multiple structural differences were found between the mitochondrial genomes of normal and CMS lines (Kubo et al. 1999; Satoh et al. 2006). By restriction analysis of the amplified fragments, we identified several varied regions of the mitochondrial genome in ZS97A as compared to the indica and japonica fertile lines. In addition, two regions (14-1, 29) in ZS97A could not be amplified, indicating the presence of macro structural variation. Furthermore, by using DNA blotting analysis, more varied regions were detected, including those that exhibited monomorphic restriction patterns among the examined lines. This demonstrates that DNA blotting analysis is more sensitive for detection of structural variation among mitochondrial genomes than the restriction digestion of PCR products. This different ability is likely to be due to the multipartite structure of the mitochondrial genome: the PCR method amplifies fragments only from part of the multiple subgenomic circles in which the both primer sites are present, whereas DNA blot hybridization detects all homologous sequences existing in different subgenomic molecules. Subgenomic circles can exist at different copy numbers within the mitochondrial genome (Janska et al. 1998). The hybridization profiles detected by 30 suggest that substoichinometric forms of genomic configurations at lower copy numbers might exist in the rice mitochondrial genome. These configurations may account for an underlying mechanism by which genetic diversity has arisen in the mitochondrial genome (Small et al. 1987, 1989). Our results demonstrated that, in the macro scale, considerable structural variations occurred in the mitochondrial genome of the WA cytoplasm, while the variation between the indica and japonica fertile cytoplasm was relatively small. Recent phylogenetic studies have proposed that the indica and japonica subspecies were independently originated from different O. rufipogon populations (Zhu and Ge 2005; Londo et al. 2006). The WA cytoplasm also was derived from a population of O. rufipogon. Therefore, the different degrees of the structural variation between these three types of mitochondrial genomes suggest that the divergence of the ancient O. rufipogon lineage with the WA cytoplasm was earlier than those of the ancestors of indica and japonica, respectively. Differentially expressed mrnas as the CMS-associated candidates Although we found a number of varied mitochondrial genomic regions in the CMS-WA line, it is still difficult to link these variations to the CMS locus. It has been documented that an altered transcription profile is one of the main features observed in CMS lines of various plants (Hanson and Bentolila 2004, Wang et al. 2006). In this study, we examined the transcription patterns between the CMS and normal rice lines at the whole mitochondrial genome level by using all of the PCR products, regardless of them being polymorphic or monomorphic, as the RNA blot probes. This strategy can avoid missing any differentially expressed transcripts. By examination of all of the transcripts of the whole mitochondrial genome, only two mrnas specific in the CMS-WA were found, which were detected by a probe containing the nad5 and orf153 genes and the another having the ribosomal protein gene rpl5, respectively. CMS genes often originate from rearrangements of the mitochondrial genomes through recombination events, and in many cases, they

6 Variations of the Mitochondrial Genome in CMS-WA Rice 913 are co-transcribed with adjacent essential genes to produce longer, dicistronic mrnas (Schnable and Wise 1998), such as the rice CMS locus B-atp6/orf79 in the BT type of cytoplasm (Wang et al. 2006). Therefore, either of these two transcripts having this feature is the possible CMS-associated candidate. Using the rpl5 gene as a probe, we detected the same transcription profile as we did with 30-1 (data not shown), indicating that this specific mrna contained rpl5. The downstream of rpl5 (ca. 0.7 kb) is linked to the 29-2 region, which was found in this study to be structurally different from the normal lines. Therefore, it is most likely that the rearrangement in the 29-2 region resulted in a CMS-related locus and altered the transcription pattern of rpl5. Cloning and sequencing analysis of these focused regions are underway. Materials and Methods Rice lines and DNA preparation A CMS-WA line ZS97A (indica) and two normal fertile rice lines Nipponbare (japonica) and (indica) were used for this study. Total cellular DNA was isolated from leaves by the CTAB (Cetyl trimethylammonium bromide) method. LA-PCR amplification and restriction digestion Primers for the LA-PCR were designed as mer in length, and the sequence information is available on request. PCR amplification was carried out in a 25-μL reaction mixture with LA-Taq DNA polymerase in 1 LA or 1 GC-I buffer (TaKaRa, Dalian, China) according to the protocol supplied by the manufacturer. The LA-PCR products (2-5 μl) were subjected to digestion reactions (20 μl) with DraI or HindIII. Digested PCR products were run on 0.8% agarose gels. DNA blotting analysis Genomic DNAs were digested with restriction enzymes, run on 0.8% agarose gels, and transferred onto Hybond N + nylon membranes (Amersham Bioscience, Bucks, UK) by the alkaline transfer recommended by the supplier. PCR products were labeled with [α- 32 P]dCTP by random priming reaction (TaKaRa) and used as hybridization probes. Hybridization was carried out at 65 C in hybridization buffer (5 SSC (0.75 mmol/l NaCl/ mmol/l sodium citrate ph 7.0), 0.1% sodium dodecyl sulfate (SDS), 0.1% Sarkosyl, and 0.5% blocking reagent). The membranes were washed successively in 2 SSC, and 0.1% SDS at 65 C. Hybridization signals were detected and analyzed by using an FX Molecular Imaging System (Bio-Rad, Hercules, CA, USA). RNA blot analysis Total RNAs were isolated from leaves using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the protocol supplied by the manufacturer. For RNA blotting analysis, about 20 μg of total RNA was denatured and separated on 1.2% denaturing formaldehyde agarose gels and transferred to Hybond- NX nylon membranes. PCR products were labeled with [α- 32 P] dctp by random priming reaction kit (TaKaRa, Dalian, China) and used as hybridization probes. Hybridization was carried out at 42 C in hybridization buffer (5 SSC, 0.5% SDS, 0.5% Sarkosyl, 50 mmol/l Tris-HCl, ph 7.5, 50% formamide, and 1% blocking reagent). The membranes were washed successively in 2 SSC, and 0.1% SDS at 42 C. Hybridization signals were detected and analyzed by using an FX Molecular Imaging System (Bio-Rad, Hercules, CA, USA). References Barnes WM (1994). PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc. Natl. Acad. Sci. USA 91, Cai CL, Yang Z, Zhu YG (1998). Analysis of rice mitochondrial DNA of sporophyte male sterility lines by RAPD. Yi Chuan Xue Bao 25, Cheng S, Fockler C, Barnes WM, Higuchi R (1994). Effective amplification of long targets from cloned inserts and human genomic DNA. Proc. Natl. Acad. Sci. USA 91, Clifton SW, Minx P, Fauron CM, Gibson M, Allen JO, Sun H et al. (2004). Sequence and comparative analysis of the maize NB mitochondrial genome. Plant Physiol. 136, Folkerts O, Hanson MR (1989). Three copies of a single recombination repeat occur on the 443 kb master circle of the Petunia hybrida 3704 mitochondrial genome. Nucleic Acids Res. 17, Handa H (2003). The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): Comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana. Nucleic Acids Res. 31, Hanson MR, Bentolila S (2004). Interactions of mitochondrial and nuclear genes that affect male gametophyte development. Plant Cell 16, Janska H, Sarria R, Woloszynska M, Arrieta-Montiel M, Mackenzie SA (1998). Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility. Plant Cell 10, Knoop V (2004). The mitochondrial DNA of land plants: Peculiarities in phylogenetic Perspective. Curr. Genet. 46, Kubo T, Nishizaw S, Mikami T (1999). Alterations in organization and transcription of the mitochondrial genome of cytoplasmic

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