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STRIPE2 Encodes a Putative dCMP Deaminase that Plays an Important Role in Chloroplast Development in Rice
Available online at www.sciencedirect.com
ScienceDirect Journal of Genetics and Genomics 41 (2014) 539e548
JGG ORIGINAL RESEARCH
STRIPE2 Encodes a Putative dCMP Deaminase that Plays an Important Role in Chloroplast Development in Rice Jing Xu a, Yiwen Deng a, Qun Li a, Xudong Zhu b,*, Zuhua He a,* a
National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China b China National Rice Research Institute, Hangzhou 31006, China Received 31 March 2014; revised 8 May 2014; accepted 9 May 2014 Available online 19 June 2014
ABSTRACT Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the g-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination. Additionally, exogenous application of dUMP could partially rescue the st2 phenotype. Therefore, our study identified a putative dCMP deaminase as a novel regulator in chloroplast development for the first time. KEYWORDS: stripe2; Chloroplast development; dCMP deaminase; Oryza sativa
INTRODUCTION The chloroplast is the crucial organelle for plant photosynthesis and essential for the production of hormones and metabolites (Pogson and Albrecht, 2011). About 3000 proteins in the chloroplast participate in transition from proplastids to mature chloroplasts, and this process is coordinated by both nuclear and plastid genome involved in synthesis of chloroplast DNA, the plastidic transcription/translation apparatus and the photosynthetic system (Sakamoto et al., 2008). Numerous chlorophyll-deficient or abnormal chloroplast mutants have been identified, and they provide ideal genetic * Corresponding authors. Tel: þ86 21 5492 4121, fax: þ86 21 5492 4123 (Z. He); Tel: þ86 571 6337 0327, fax: þ86 571 6337 0389 (X. Zhu). E-mail addresses: [email protected] (X. Zhu); [email protected] (Z. He).
materials to investigate regulation mechanisms of chlorophyll biosynthesis and chloroplast development in rice. Screening for chloroplast development mutants has identified most steps in these biological processes, such as yellowgreen leaf1 ( ygl1) and faded green leaf ( fgl ), which result from lesions of chlorophyll synthase that catalyzes esterification of chlorophyllide in the last step of chlorophyll biosynthesis (Wu et al., 2007) and NADPH:protochlorophyllide oxidoreductase that catalyzes the photoreduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) (Sakuraba et al., 2013). Both mutations led to reduced contents of chlorophyll and undeveloped chloroplasts. The magnesium chelatase, catalyzing the chelation of Mg2þ into proto IX to produce Mg-Proto IX, comprises three subunits including ChlH, ChlD and ChlI (Jung et al., 2003; Zhang et al., 2006). Mutations of the chlorina-1 and chlorina-9 led to deficiency in
http://dx.doi.org/10.1016/j.jgg.2014.05.008 1673-8527/Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
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chlorophyll content and incomplete development of chloroplasts, due to the disruption of ChlD and ChlI subunits, respectively. Moreover, an insert mutation in ygl2 ( yellowgreen leaf 2), encoding heme oxygenase (HO) that catalyzes the degradation of heme to synthesize phytochrome precursor, results in significantly reduced content of chlorophyll and tetrapyrrole intermediates (Chen et al., 2013). As a semiautonomous organelle, chloroplast genome only encodes about 100 genes (Delannoy et al., 2009). Most of the proteins essential for chloroplast development and function are nuclear-encoded (Chen et al., 2010). It is well documented that the coordination of nuclear and plastid genes is crucial for chloroplast biogenesis (Mullet, 1988). Under illumination, one-third of the nuclear genes change expression, including many transcription factors such as PIFs (phytochrome interacting factors). Either pif1 or pif3 mutant showed delayed development of chloroplast (Moon et al., 2008; Stephenson et al., 2009). Gene transcription, RNA maturation, and protein translation in the chloroplasts also have impact on chloroplast biogenesis and development. There are two types of plastid RNA polymerases: plastid-encoded RNA polymerase (PEP) and nucleus-encoded RNA polymerase (NEP) responsible for the transcription of the plastome. In Arabidopsis, mutations in SIG6 (sigma factor 6) cause a weakly virescent phenotype and transcripts of several PEP-dependent plastid genes are specifically reduced (Loschelder et al., 2006). Decreased expression of AtRpoTp, one of the NEP genes, leads to a typical virescent phenotype which can be recovered after two weeks of growth (Swiatecka-Hagenbruch et al., 2008). PPR proteins (pentatricopeptide repeat proteins), characterized by tandem arrays of a 35 amino acid motif, have been demonstrated to be critical for RNA processing, splicing, editing, stability, maturation and translation in the chloroplast (Takenaka et al., 2013). YSA (young seedling albino) encodes a PPR protein and the disruption of its function causes a seedling stage-specific albino phenotype in rice. The mutant plants can recover and develop normal green leaves after the four-leaf stage. Interestingly, the ysa mutant has been used as a marker for efficient identification and elimination of false hybrids in commercial hybrid rice production (Su et al., 2012). The Arabidopsis mutant sel1 (seedling lethal 1) exhibited a pigment-defective and seedling-lethal phenotype with a disrupted PPR gene. In the sel1 plants, RNA editing of acetylCoA carboxylase b subunit transcripts was disrupted (Pyo et al., 2013). The virescent rice mutants v1, v2 and v3 are temperature-conditional, which produce chlorotic leaves at a restrictive temperature (20 C) but develop nearly green leaves at a permissive temperature (30 C). V1 encodes a chloroplast-localized protein NUS1 which is involved in the regulation of chloroplast rRNA metabolism during early chloroplast development (Kusumi et al., 1997; Kusumi et al., 2011). V2 encodes a new type of guanylate kinase (pt/ mtGK) localized both to plastids and mitochondria. It has been proposed that V2 functions at an early stage of chloroplast differentiation particularly in the chloroplast translation machinery during early leaf development (Sugimoto et al., 2004, 2007). V3 and STRIPE1 encode the large and small subunits of
ribonucleotide reductase (RNR), respectively, which regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. Yoo et al. (2009) speculated that, upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves to sustain the continuous plant growth. In this study, we identified a stripe variegated mutant named st2 (stripe2). The st2 plants develop chlorotic leaves caused by low content of chlorophylls. Examination of the ultrastructure showed that the thylakoid membranes are extremely disturbed in the mutants. Map-based cloning and genetic complementation indicated that ST2 encodes a cytidine deaminase-like protein, which most likely functions as the dCMP deaminase. RESULTS Phenotype characterization of stripe2 mutant The rice (Oryza sativa L.) st2 mutant was isolated from g-rayinduced mutations of an indica cultivar (Longtepu, LTP). The mutant leaves were virescent with stripes (Fig. 1A and E). To determine its effect on chlorophyll formation, the leaves of 3-week seedling were analyzed for chlorophyll contents. Compared with the wild type, the chlorophyll contents in st2 were reduced by 30% (Fig. 1B), and autofluorescence in the st2 leaves also decreased (Fig. 1C and D). The ultrastructure of the wild-type chloroplasts was crescent-shaped and contained well-formed thylakoid structure including stroma thylakoids and grana thylakoids (Fig. 2A). In contrast, the mutant chloroplasts were small and thylakoid membrane was disturbed, some with less thylakoid structure (Fig. 2B and C). Some chloroplasts formed rudimentary thylakoids consisting of only grana lamellae without formation of stroma lamellae (Fig. 2D), while some chloroplasts displayed well-developed lamellar structures equipped with normally stacked grana but no starch grains in the st2 leaves (Fig. 2E and F). These results indicated the st2 phenotype is caused by the underdevelopment of the chloroplast. Fine mapping of the ST2 Gene For genetic analysis of the st2 mutant, we firstly crossed st2 mutant with a japonica cultivar Zhonghua11 (ZH11), and the F1 generation exhibited normal green leaves. Among the 762 F2 individuals, 183 were virescent and 579 were green. The segregation ratio of the F2 population accorded with 3:1 (c2 ¼ 0.39 < c20.05 ¼ 3.81; P > 0.05), suggesting that the st2 phenotype was controlled by a single recessive gene. Genetic mapping was performed using the same population. The locus was primarily mapped to a 6.5-CM region flanking by the makers of RM8139 and C7962 on the long arm of chromosome 1. To further narrow down the region, new markers including insertion/deletion (InDel) markers and CAPS (cleaved amplified polymorphic sequence) were designed; however, little polymorphism could be found between st2 and ZH11. Therefore, we developed a larger F2
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Fig. 1. Phenotypic characterization of the st2 mutant. A: Three-week-old seedlings of LTP (left) and st2 mutant (right). The st2 plants exhibited striped leaves. Scale bar ¼ 2.5 cm. B: The contents of chlorophyll a and b were greatly reduced in the three-week-old seedling of st2 mutant compared with those of LTP. Student’s t-test was performed on the raw data; asterisk indicates statistical significance at P < 0.01. C: Chlorophyll autofluorescence of LTP leaf sampled from three-week-old seedling. Scale bar ¼ 100 mm. D: Chlorophyll autofluorescence of st2 leaf sampled from three-week-old seedling. Scale bar ¼ 100 mm. E: The stripe phenotype was sustained in st2 adult plants. Scale bar ¼ 10 cm.
population from a cross of st2 and 9311 (indica) for further mapping. By genotyping 1723 homologous st2 individuals, the ST2 gene was finally confined to a 27-kb physical interval between S1614 and S9131 on BAC P0403C05. According to the rice genome annotation database (http://rice.plantbiology.
msu.edu/), seven genes were predicted in the region (Fig. 3A). The genomic DNA of the candidate genes was sequenced and compared with LTP. A 5-bp deletion (TAGGT) was detected on the ORF of Os01g0765000 in st2 mutant (Fig. 3B). This deletion leads to the abortion of Bln I
Fig. 2. Ultrastructure of chloroplasts in mesophyll cells of LTP (WT) and st2 mutant. The chloroplasts of LTP have well-ordered thylakoids and stacked membranes (A), while st2 shows various defects in chloroplasts, among which some form little thylakoid structure (B and C) or form thylakoid with only grana lamellae (D), and some could form normal thylakoid though without starch grains (E and F). Scale bars ¼ 1 mm. Cp, chloroplast; M, mitochondrion; SG, starch grain.
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Fig. 3. Map-based cloning and candidate gene identification of ST2. A: The ST2 locus was initially mapped on chromosome 1 by flanking markers RM8139 and C7962 with 183 recessive individuals. A larger population consisting of 1723 recessive individuals was used for fine mapping, and finally the locus was confined to about 27 kb between markers S1614 and S9131. Seven ORFs were identified according to the genome annotation data. B: A 5-bp deletion (TAGGT in bold) located on the splicing site of exon 3 and intron 3 was found in ORF3 (Os01g0765000) and lead to the abortion of Bln I site (CCTAGG, underlined). C: The deletion was detected by primer C1377, and only st2 showed resistance to Bln I digestion. 1, st2; 2, LTP; 3, Nipponbare; 4, 9311; 5, Zhonghua 11; 6, Taipei309; 7, Zhejing 22; 8, Gumei 4; 9, Teqing; 10, F1 plants of st2 and 9311. D: Full length of cDNA of ORF3 was amplified from the st2 mutant. According to the genomic DNA (I) and cDNA (II) in the wild type, two new transcripts (III and IV) with parts of intron 3 were generated in the mutant. E: RT-PCR detection of the new transcripts in the st2 mutant, which were larger than that of LTP.
cutting site, and a primer (C1377) was designed to detect the deletion. To confirm that the deletion was exclusive for st2 mutant, several other varieties were also genotyped with C1377, and only st2 was resistant to the Bln I digestion (Fig. 3C). There were nine exons in Os01g0765000, and the deletion located on the conjunction of the third exon and
intron, which could affect mRNA splicing (Fig. 3D). RT-PCR showed that there are two larger bands in st2 instead of one band in LTP (Fig. 3E). Sequence alignment of the exceptional bands confirmed that the additional nucleotides came from the third intron (Fig. S1). The additional intron sequence led to the premature termination of the predicted
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which is a mitochondrial marker (Carrie et al., 2007), indicating that ST2 is localized to mitochondria. ST2 encodes a putative dCMP deaminase
Fig. 4. Functional complementation of st2 by genomic DNA. The st2 phenotype can be recovered by the external genomic DNA including the whole ORF3 and its promoter. Three independent transgenic lines (T1, T4, T6) showed normal leaf phenotype similar to the wild type (LTP). Scale bar ¼ 2 cm.
protein, therefore disturbing its normal function. The identification of the ST2 gene was subsequently confirmed by the genetic complementary experiments. More than 30 independent transgene-positive plants of T0 generation were obtained and most of them showed normal leaf color (Fig. 4). Moreover, 12 T1 lines were planted, which showed segregation of wild type and st2 phenotypes. Together, Os01g0765000 is the underlying gene responsible for the st2 phenotype.
Expression pattern and subcellular localization of ST2 To understand the roles of ST2 in plant growth, we detected the expression pattern of ST2 in different tissues including root, seedling, leaf, leaf sheath, stem, and panicle. RT-PCR result showed that the ST2 is expressed ubiquitously, suggesting that ST2 may function in most tissues (Fig. 5A). It was predicted that ST2 has a mitochondrial or chloroplast transit peptide comprising 40 amino acids at N-terminal (http://www. cbs.dtu.dk/services/TargetP/). To determine the subcellular localization of ST2, fusion proteins of ST2-YFP (yellow fluorescent protein) and truncated ST2 without the transit peptide (DST2-YFP) were transiently expressed in onion epidermal cells and rice protoplasts driven by the 35S promoter. The ST2-YFP showed a punctate pattern but did not coincide with the red chlorophyll auto-fluorescence. When the transit peptide is excluded (DST2-YFP), the punctate location pattern of ST2-YFP disappeared, confirming that the transit peptide guides its cellular location (Fig. 5B and C). Moreover, we observed that ST2-GFP was co-localized with AOX-RFP (mitochondrial alternative oxidase fused to RFP) (Fig. 5D),
According to the genome annotation and sequence similarity, we found that ST2 belongs to the cytidine deaminase-like family, which is involved in the nucleotide metabolism. There are 7 and 16 cytidine deaminase members in rice and Arabidopsis, respectively (Fig. 6A). Phylogenetic analysis showed that the ST2 shared 83% sequence similarity with Arabidopsis At3g48540. We examined T-DNA insertion mutants of At3g48540 available from the Arabidopsis community (http://www.arabidopsis.org/), but no phenotype like st2 was observed in the null mutant FLAG_475E06, probably due to the functional redundancy of the family in Arabidopsis. The members of cytidine deaminase-like family (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi? uid¼cd00786&seltype¼1) can be divided into four subfamilies, including riboflavin deaminase, cytidine deaminase, dCMP deaminase and nucleoside deaminase. In mammals, the cytidine deaminase such as APOBEC-1 is responsible for C-to-U editing of the apolipoprotein mRNA (Prohaska et al., 2014). In higher plants, RNA editing is a post-transcriptional process of altering a specific nucleotide C to U (and less frequently, from U to C) in organelle mRNAs. Most RNA editing events are necessary for expressing functional proteins, as demonstrated by examples of RPL2 (ribosomal protein L2) in maize (Hoch et al., 1991) and NDHD-1 (a subunit of the chloroplast NADH dehydrogenase-like complex (NDH), involved in cyclic electron flow) in Arabidopsis (Boussardon et al., 2012). However, enzyme(s) for the C-to-U conversion has not been identified yet and cytidine deaminase is most likely a candidate. To determine whether ST2 functions as a cytidine deaminase, we scanned the mitochondrial RNA editing sites by using 48 pairs of primers from the study of Kim et al. (2009). Compared with the wild type, all the editing sites still existed, suggesting that ST2 might not function as a cytidine deaminase to catalyze RNA nucleotides. We then compared ST2 with the human and yeast dCMP deaminases, and high homology was found especially in the catalytic site from the 80th to the 140th amino acid (Fig. 6B). Since dCMP deaminases catalyze the process from dCMP to dUMP by deamination, we considered that the st2 mutant might be deficient in the dUMP supply and the external application of dUMP should recover or attenuate the st2 phenotype. As showed in Fig. 7, the leaf color was recovered with 1 mmol/L dUMP feeding, albeit plant growth was inhibited at that concentration. Therefore, we proposed that ST2 most likely functions as a dCMP deaminase. DISCUSSION In this study, we isolated a new leaf-color mutant st2 in rice. The mutant exhibits the phenotype of underdeveloped chloroplast with inhibited chlorophyll accumulation. To recognized the ST2 gene functionally, we map-cloned the ST2 gene, and found that the gene encodes a putative mitochondrialocated dCMP deaminase. Our study for the first time identified a putative plant dCMP deaminase, and is also the first
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Fig. 5. Expression pattern and subcellular localization of ST2. A: ST2 can be detected in different tissues by RT-PCR. Ubiquitin (UB) was amplified as control. R, root; SL, seedling; LH, flag leaf during heading date; SH, sheath; ST, shoot; PH, panicle during heading stage; S, spikelets during heading stage. B: Transient expression of ST2-YFP, DST2-YFP and YFP in onion epidermal cells. The ST2-YFP showed a punctate YFP signal. When the N-terminal signal peptide of ST2 was excluded, it showed the same ubiquitous expression pattern as YFP control. The images showed the fluorescence both under dark field (YFP fluorescence) and bright field (Bright image). Scale bar ¼ 50 mm. C: The same constructions were transformed into rice protoplasts and exhibited similar localization patterns. The chlorophyll autofluorescence was also imaged and merged with the YFP fluorescence to detect the ST2 localization. Scale bar ¼ 20 mm. D: ST2-GFP was co-localized with AOX-RFP, a mitochondria maker, indicating that ST2 is localized to mitochondria in the rice protoplast. Scale bar ¼ 20 mm.
report that mitochondria-located deaminase plays a critical role in chloroplast development as well as chlorophyll biosynthesis. According to the sequence alignment, ST2 belongs to the cytidine deaminase-like family with 7 and 16 members in rice and Arabidopsis, respectively. Only two members of the family, AtCDA1 (cytidine deaminase 1) (Faivre-Nitschke et al., 1999; Vincenzetti et al., 1999; Kafer and Thornburg, 2000) and AtTadA (Karcher and Bock, 2009) have been reported in Arabidopsis. AtCDA1 can utilize both cytidine and 20 -deoxycytidineas substrates but unable to deaminate cytosine, CMP or dCMP. AtTadA encodes a chloroplast tRNA adenosine deaminase which triggers A-to-I editing in the anticodon of the plastid tRNA-Arg (ACG), presumably regulating chloroplast translational efficiency. This cytidine deaminase-like family can be divided into four subfamilies performing functions in different bioprocesses. One possibility is that ST2 functions as a cytidine deaminase for RNA editing. However, no editing site was found disappeared in st2 compared with the wild type. We then found that ST2 shares
similarity with the human and yeast dCMP deaminases. According to reports in animal, T4 phage and yeast, dCMP deaminases catalyze the deamination of dCMP to form dUMP. dUMP is the precursor for dTMP that is subsequently phosphorylated to thymidine triphosphate for DNA synthesis and repair. The exogenous application of dUMP could recover the st2 phenotype, suggesting that ST2 most likely functions as a dCMP deaminase. Therefore, the mutation in the ST2 gene reduces the dUMP pool in plant, and further disturbs the balance of subsequent dTTP formation. It is intriguing how a mitochondria-located deaminase affects chloroplast development. As previously reported, in the rice virescent3 and strip1 mutants, plastid DNA synthesis for chloroplast biogenesis is supposed to be relatively less critical for plant survival and can sacrifice under insufficient dNTP levels (Yoo et al., 2009). Similarly, we speculate that chloroplast development is preferentially arrested to allow both nuclear and mitochondrial genome replication. However, the mechanism how the dNTP pool prioritizes nuclear and possible mitochondrial DNA (mtDNA) synthesis keeps elusive.
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Fig. 6. Sequence analysis of ST2 homologs. A: The phylogenetic tree represents alignment of ST2 protein with its homologs in rice and Arabidopsis. Seven proteins including ST2 and 16 proteins in Arabidopsis were aligned by ClustalX2 and the phylogram tree was constructed by TreeView. Scale represents percentage substitution per site. B: The ST2 protein was aligned with the human dCMP deaminase (HsDCD, NP_001012750.1) and yeast dCMP deaminase (ScDCD, NP_012014.1). Identical residues were boxed in black and similar residues were boxed in gray.
In T4 phage, the endogenous dTTP content was greatly reduced and the dCTP increased 30 times as high as the normal level when dCMP deaminase was inactive. Such imbalance led to the reduced fidelity of DNA replication with increased nucleotide mismatch (Sargent and Mathews, 1987). Correspondingly, we did detect up-regulated DNA repair genes from microarray data of st2 compared with LTP, including endonuclease, meiotic recombination protein DMC1, DNA helicase RecQ, and RAD51 homolog RAD51B (data not shown). The study of the maize ncs (nonchromosomal strip) mutant indicated that mitochondria are necessary for the chloroplast biogenesis, and the mtDNA mutations can result in strip phenotype (Jiao et al., 2005). We also suggest another possibility that the imbalance dNTP in the st2 mutants may also increase the mutation rate of mtDNA, thereby disturbing the chloroplast
development. Further investigation is necessary to dissect the mechanism of nucleotide distribution between chloroplasts, mitochondria and the nucleus. MATERIALS AND METHODS Plant materials and grow condition The rice st2 mutant was isolated from g-ray-induced mutations of an indica cultivar (LTP). A japonica variety, Zhonghua11 (ZH11), was crossed with st2 to construct a F2 population for genetic study and preliminary mapping. Additional 1723 recessive individuals from the F2 populations derived from the cross between st2 and 9311 (indica) were used for fine mapping. Plants were cultivated in the
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mixture was centrifuged, and the supernatant was used to measure the absorbance values under 663 nm and 645 nm, using a spectrophotometer (Beckman Coulter-DU800, USA), with the extraction buffer as control. Each sample was assayed with three biological repeats. Transmission electron microscopy (TEM) assay Seedlings of st2 and LTP of 20-day-old were sampled for TEM observation. All the leaf samples were cut into sections less than 2 mm2 and infiltrated for 30 min with fixation buffer [2.5% glutaraldehyde in phosphate buffer (pH 7.2)] under vacuum. After 3 days at 4 C, samples were post-fixed in 0.1 mol/L cacodylate (pH 7.4) with 2% OsO4 at 4 C, followed by washing with 0.1 mol/L PBS, then dehydrated with a gradient ethanol-acetone series and embedded in Polybed 812 (Sigma, USA) resin (Li et al., 2011). Ultrathin sections were obtained with an ultramicrotome, mounted on grids, and stained. The sections were viewed via an electron microscopy H7650 (Hitachi, Japan). Map-based cloning of ST2
Fig. 7. Recovery of st2 plants by dUMP feeding. A: The LTP seedlings treated with dUMP under concentration of 0, 105, 104 and 103 mol/L (from left to right). Two seedlings were imaged for each concentration. Scale bar ¼ 2 cm. Note that 103 mol/L dUMP slightly inhibited seedling growth. B: The st2 mutant plants were fed with dUMP under the same concentrations as in (A). Note that the leaf phenotype was recovered by 103 mol/L dUMP. Scale bar ¼ 2 cm.
experimental field during natural growth seasons. For seedlings, seeds were germinated in the dark for 2 days and then transferred to liquid medium in a growth chamber under growth conditions with 12-h day, 28 C, 80% relative humidity (RH) followed by 12-h night, 26 C, 60% RH. Measurement of chlorophyll a and b The contents of chlorophyll a and b (Chl a and Chl b) were measured according to the previously method with some modifications (Arnon, 1949). The fresh rice leaves were cut into small pieces with scissors and soaked in the extraction buffer (95% ethanol:acetone:water ¼ 5:4:1), and incubated at 4 C in the dark for 18 h with periodically inversion. The
For gene mapping, a total of 120 SSR (simple sequence repeats) markers distributing evenly on 12 chromosomes according to database information in the GRAMENE (http://www.gramene. org/bd/markers/) were used for preliminary mapping. To construct a high-density linkage map for fine mapping in the target region, new InDel markers and CAPS markers were developed according to the sequence differences between indica (9311) and japonica (Nipponbare) genomes. In case that markers did not exhibit polymorphism between st2 and 9311, we sequenced PCR products to search for SNPs to develop new CAPS markers. The primer sequences and enzymes used are listed in Table S1. All the primers were designed using the program Primer Premier 5.0 (http://www.premierbiosoft.com). Subsequently, we placed ST2 in a 27-kb region between the markers S1614 and S9131. Genomic DNA fragments of this region were amplified from st2 and wild-type (LTP) plants, sequenced, and compared using MegAlign (DNASTAR). Complementation test and transgenic expression The rice Nipponbare ( japonica) BAC P0403C05 bearing ST2 was digested to isolate a 9-kb genomic DNA fragment with the full ST2 region, which was inserted into the binary vector pCAMBIA1301 to generate the plasmid pST2-ST2. The plasmid was introduced into the st2 mutants by Agrobacterium-mediated transformation (Hiei et al., 1994). More than 10 independent transgenic lines were produced that could successfully complement the mutant phenotypes. Subcellular localization assay ST2-YFP, DST2-YFP and ST2-GFP fusions were obtained by in-frame fusing the cDNAs with YFP or GFP, which were amplified using the specific primers ST2-YF/ST2-YR, DST2-
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YF/ST2-YR, and ST2-YF/ST2-GR, respectively (Table S2). The resulted fusions were driven by the 35S promoter. Transient expression of the fusion proteins ST2-YFP, DST2-YFP and the controls YFP in onion epidermal cells was performed using a helium biolistic device (Bio-Rad PDS-1000, USA) as previously described (Collings et al., 2000). Rice mesophyll protoplasts isolated from leaf sheaths of 11-day-old seedlings were transfected with fusion constructs, or cotransfected with constructs of ST2-GFP and AOX-RFP to determine mitochondrial location, according to previously reported methods (Bart et al., 2006). Onion epidermal cells and protoplasts expressing the proteins were imaged under a laser scanning confocal microscope using a Plan-Apochromat 100/1.4 oil objective (LSM510 META NLO, Zeiss, Germany). RT-PCR analysis Total RNAs were extracted from the whole 2-week-old seedlings using TRIzol reagent (Invitrogen, USA) and treated with DNase RQ1 (Promega, USA). cDNAs were synthesized from 3 mg total RNAs using oligo (dT) primer and SuperScript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen). RT-PCR analysis and full-length cDNA amplification were performed using gene-specific primers (Table S3), under the following conditions: 5 min at 95 C, 28 cycles (30 s at 95 C, 30 s at specific Tm, 1 min/kb at 72 C), followed by 10 min at 72 C. Analysis of RNA editing Total RNAs extracted from seedling leaves were treated with RQ1 DNase (Promega). Then cDNAs were synthesized from 3 mg total RNAs using hexanucleotide oligomers primer and MMLV reverse transcriptase. These cDNAs were used as templates for PCR amplification of mitochondrial genes. The primers used for scanning mitochondrial RNA editing sites were the same as those reported by Kim et al. (2009). The RTPCR products were directly sequenced and manually compared between the wild type and the mutant. ACKNOWLEDGEMENTS We thank Jiqin Li and Xiaoshu Gao for technical assistance. We also acknowledge James Whelan (University of Western Australia) for providing AOX-RFP plasmid. This work was supported by the grant from the Ministry of Science and Technology of China (No. 2012AA10A302-2).
SUPPLEMENTARY DATA Fig. S1. Sequences representing the disturbed splicing shown in Fig. 3D (III and IV). Table S1. PCR-based markers linked to St2 locus. Table S2. Primers for RT-PCR. Table S3. Primers for plasmid construction.
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Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2014.05.008. REFERENCES Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta vulgaris. Plant Physiol. 24, 1e15. Bart, R., Chern, M., Park, C.J., Bartley, L., Ronald, P., 2006. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2, 13. Boussardon, C., Salone, V., Avon, A., Berthome, R., Hammani, K., Okuda, K., Shikanai, T., Small, I., Lurin, C., 2012. Two interacting proteins are necessary for the editing of the NdhD-1 site in Arabidopsis plastids. Plant Cell 24, 3684e3694. Carrie, C., Murcha, M.W., Millar, A.H., Smith, S.M., Whelan, J., 2007. Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria. Plant Mol. Biol. 63, 97e108. Chen, H., Cheng, Z., Ma, X., Wu, H., Liu, Y., Zhou, K., Chen, Y., Ma, W., Bi, J., Zhang, X., Guo, X., Wang, J., Lei, C., Wu, F., Lin, Q., Liu, L., Jiang, L., 2013. A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Rep. 32, 1855e1867. Chen, M., Galvao, R.M., Li, M., Burger, B., Bugea, J., Bolado, J., Chory, J., 2010. Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell 141, 1230e1240. Collings, D.A., Carter, C.N., Rink, J.C., Scott, A.C., Wyatt, S.E., Allen, N.S., 2000. Plant nuclei can contain extensive grooves and invaginations. Plant Cell 12, 2425e2440. Delannoy, E., Le Ret, M., Faivre-Nitschke, E., Estavillo, G.M., Bergdoll, M., Taylor, N.L., Pogson, B.J., Small, I., Imbault, P., Gualberto, J.M., 2009. Arabidopsis tRNA adenosine deaminase arginine edits the wobble nucleotide of chloroplast tRNAArg(ACG) and is essential for efficient chloroplast translation. Plant Cell 21, 2058e2071. Faivre-Nitschke, S.E., Grienenberger, J.M., Gualberto, J.M., 1999. A prokaryotic-type cytidine deaminase from Arabidopsis thaliana. Eur. J. Biochem. 263, 896e903. Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271e282. Hoch, B., Maier, R.M., Appel, K., Igloi, G.L., Kossel, H., 1991. Editing of a chloroplast mRNA by creation of an initiation codon. Nature 353, 178e180. Jiao, S., Thornsberry, J.M., Elthon, T.E., Newton, K.J., 2005. Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize. Plant Mol. Biol. 57, 303e313. Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., An, G., 2003. Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system. Plant Cell Physiol. 44, 463e472. Kafer, C., Thornburg, R., 2000. Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes. J. Plant Biol. 43, 162e170. Karcher, D., Bock, R., 2009. Identification of the chloroplast adenosine-toinosine tRNA editing enzyme. RNA 15, 1251e1257. Kim, S.R., Yang, J.I., Moon, S., Ryu, C.H., An, K., Kim, K.M., Yim, J., An, G., 2009. Rice OGR1 encodes a pentatricopeptide repeat-DYW protein and is essential for RNA editing in mitochondria. Plant J. 59, 738e749. Kusumi, K., Mizutani, A., Nishimura, M., Iba, K., 1997. A virescent gene V1 determines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice. Plant J. 12, 1241e1250. Kusumi, K., Sakata, C., Nakamura, T., Kawasaki, S., Yoshimura, A., Iba, K., 2011. A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions. Plant J. 68, 1039e1050. Li, W., Zhong, S., Li, G., Li, Q., Mao, B., Deng, Y., Zhang, H., Zeng, L., Song, F., He, Z., 2011. Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence. Cell Res. 21, 835e848.
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Loschelder, H., Schweer, J., Link, B., Link, G., 2006. Dual temporal role of plastid sigma factor 6 in Arabidopsis development. Plant Physiol. 142, 642e650. Moon, J., Zhu, L., Shen, H., Huq, E., 2008. PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis. Proc. Natl. Acad. Sci. USA 105, 9433e9438. Mullet, J.E., 1988. Chloroplast development and gene expression. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 475e502. Pogson, B.J., Albrecht, V., 2011. Genetic dissection of chloroplast biogenesis and development: an overview. Plant Physiol. 155, 1545e1551. Prohaska, K.M., Bennett, R.P., Salter, J.D., Smith, H.C., 2014. The multifaceted roles of RNA binding in APOBEC cytidine deaminase functions. Wiley Interdiscip. Rev. RNA http://dx.doi.org/10.1002/wrna.1226. Pyo, Y.J., Kwon, K.C., Kim, A., Cho, M.H., 2013. Seedling Lethal1, a pentatricopeptide repeat protein lacking an E/Eþ or DYW domain in Arabidopsis, is involved in plastid gene expression and early chloroplast development. Plant Physiol. 163, 1844e1858. Sakamoto, W., Miyagishima, S.Y., Jarvis, P., 2008. Chloroplast biogenesis: control of plastid development, protein import, division and inheritance. Arabidopsis Book 6, e0110. Sakuraba, Y., Rahman, M.L., Cho, S.H., Kim, Y.S., Koh, H.J., Yoo, S.C., Paek, N.C., 2013. The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions. Plant J. 74, 122e133. Sargent, R.G., Mathews, C.K., 1987. Imbalanced deoxyribonucleoside triphosphate pools and spontaneous mutation rates determined during dCMP deaminase-defective bacteriophage T4 infections. J. Biol. Chem. 262, 5546e5553. Stephenson, P.G., Fankhauser, C., Terry, M.J., 2009. PIF3 is a repressor of chloroplast development. Proc. Natl. Acad. Sci. USA 106, 7654e7659. Su, N., Hu, M.L., Wu, D.X., Wu, F.Q., Fei, G.L., Lan, Y., Chen, X.L., Shu, X.L., Zhang, X., Guo, X.P., Cheng, Z.J., Lei, C.L., Qi, C.K., Jiang, L., Wang, H.,
Wan, J.M., 2012. Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production. Plant Physiol. 159, 227e238. Sugimoto, H., Kusumi, K., Noguchi, K., Yano, M., Yoshimura, A., Iba, K., 2007. The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria. Plant J. 52, 512e527. Sugimoto, H., Kusumi, K., Tozawa, Y., Yazaki, J., Kishimoto, N., Kikuchi, S., Iba, K., 2004. The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation. Plant Cell Physiol. 45, 985e996. Swiatecka-Hagenbruch, M., Emanuel, C., Hedtke, B., Liere, K., Borner, T., 2008. Impaired function of the phage-type RNA polymerase RpoTp in transcription of chloroplast genes is compensated by a second phage-type RNA polymerase. Nucleic Acids Res. 36, 785e792. Takenaka, M., Zehrmann, A., Verbitskiy, D., Hartel, B., Brennicke, A., 2013. RNA editing in plants and its evolution. Annu. Rev. Genet. 47, 335e352. Vincenzetti, S., Cambi, A., Neuhard, J., Schnorr, K., Grelloni, M., Vita, A., 1999. Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana. Protein Expr. Purif. 15, 8e15. Wu, Z., Zhang, X., He, B., Diao, L., Sheng, S., Wang, J., Guo, X., Su, N., Wang, L., Jiang, L., Wang, C., Zhai, H., Wan, J., 2007. A chlorophylldeficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol. 145, 29e40. Yoo, S.C., Cho, S.H., Sugimoto, H., Li, J., Kusumi, K., Koh, H.J., Iba, K., Paek, N.C., 2009. Rice virescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development. Plant Physiol. 150, 388e401. Zhang, H., Li, J., Yoo, J.H., Yoo, S.C., Cho, S.H., Koh, H.J., Seo, H.S., Paek, N.C., 2006. Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Mol. Biol. 62, 325e337.
ScienceDirect Journal of Genetics and Genomics 41 (2014) 539e548
JGG ORIGINAL RESEARCH
STRIPE2 Encodes a Putative dCMP Deaminase that Plays an Important Role in Chloroplast Development in Rice Jing Xu a, Yiwen Deng a, Qun Li a, Xudong Zhu b,*, Zuhua He a,* a
National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China b China National Rice Research Institute, Hangzhou 31006, China Received 31 March 2014; revised 8 May 2014; accepted 9 May 2014 Available online 19 June 2014
ABSTRACT Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the g-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination. Additionally, exogenous application of dUMP could partially rescue the st2 phenotype. Therefore, our study identified a putative dCMP deaminase as a novel regulator in chloroplast development for the first time. KEYWORDS: stripe2; Chloroplast development; dCMP deaminase; Oryza sativa
INTRODUCTION The chloroplast is the crucial organelle for plant photosynthesis and essential for the production of hormones and metabolites (Pogson and Albrecht, 2011). About 3000 proteins in the chloroplast participate in transition from proplastids to mature chloroplasts, and this process is coordinated by both nuclear and plastid genome involved in synthesis of chloroplast DNA, the plastidic transcription/translation apparatus and the photosynthetic system (Sakamoto et al., 2008). Numerous chlorophyll-deficient or abnormal chloroplast mutants have been identified, and they provide ideal genetic * Corresponding authors. Tel: þ86 21 5492 4121, fax: þ86 21 5492 4123 (Z. He); Tel: þ86 571 6337 0327, fax: þ86 571 6337 0389 (X. Zhu). E-mail addresses: [email protected] (X. Zhu); [email protected] (Z. He).
materials to investigate regulation mechanisms of chlorophyll biosynthesis and chloroplast development in rice. Screening for chloroplast development mutants has identified most steps in these biological processes, such as yellowgreen leaf1 ( ygl1) and faded green leaf ( fgl ), which result from lesions of chlorophyll synthase that catalyzes esterification of chlorophyllide in the last step of chlorophyll biosynthesis (Wu et al., 2007) and NADPH:protochlorophyllide oxidoreductase that catalyzes the photoreduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) (Sakuraba et al., 2013). Both mutations led to reduced contents of chlorophyll and undeveloped chloroplasts. The magnesium chelatase, catalyzing the chelation of Mg2þ into proto IX to produce Mg-Proto IX, comprises three subunits including ChlH, ChlD and ChlI (Jung et al., 2003; Zhang et al., 2006). Mutations of the chlorina-1 and chlorina-9 led to deficiency in
http://dx.doi.org/10.1016/j.jgg.2014.05.008 1673-8527/Copyright Ó 2014, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved.
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chlorophyll content and incomplete development of chloroplasts, due to the disruption of ChlD and ChlI subunits, respectively. Moreover, an insert mutation in ygl2 ( yellowgreen leaf 2), encoding heme oxygenase (HO) that catalyzes the degradation of heme to synthesize phytochrome precursor, results in significantly reduced content of chlorophyll and tetrapyrrole intermediates (Chen et al., 2013). As a semiautonomous organelle, chloroplast genome only encodes about 100 genes (Delannoy et al., 2009). Most of the proteins essential for chloroplast development and function are nuclear-encoded (Chen et al., 2010). It is well documented that the coordination of nuclear and plastid genes is crucial for chloroplast biogenesis (Mullet, 1988). Under illumination, one-third of the nuclear genes change expression, including many transcription factors such as PIFs (phytochrome interacting factors). Either pif1 or pif3 mutant showed delayed development of chloroplast (Moon et al., 2008; Stephenson et al., 2009). Gene transcription, RNA maturation, and protein translation in the chloroplasts also have impact on chloroplast biogenesis and development. There are two types of plastid RNA polymerases: plastid-encoded RNA polymerase (PEP) and nucleus-encoded RNA polymerase (NEP) responsible for the transcription of the plastome. In Arabidopsis, mutations in SIG6 (sigma factor 6) cause a weakly virescent phenotype and transcripts of several PEP-dependent plastid genes are specifically reduced (Loschelder et al., 2006). Decreased expression of AtRpoTp, one of the NEP genes, leads to a typical virescent phenotype which can be recovered after two weeks of growth (Swiatecka-Hagenbruch et al., 2008). PPR proteins (pentatricopeptide repeat proteins), characterized by tandem arrays of a 35 amino acid motif, have been demonstrated to be critical for RNA processing, splicing, editing, stability, maturation and translation in the chloroplast (Takenaka et al., 2013). YSA (young seedling albino) encodes a PPR protein and the disruption of its function causes a seedling stage-specific albino phenotype in rice. The mutant plants can recover and develop normal green leaves after the four-leaf stage. Interestingly, the ysa mutant has been used as a marker for efficient identification and elimination of false hybrids in commercial hybrid rice production (Su et al., 2012). The Arabidopsis mutant sel1 (seedling lethal 1) exhibited a pigment-defective and seedling-lethal phenotype with a disrupted PPR gene. In the sel1 plants, RNA editing of acetylCoA carboxylase b subunit transcripts was disrupted (Pyo et al., 2013). The virescent rice mutants v1, v2 and v3 are temperature-conditional, which produce chlorotic leaves at a restrictive temperature (20 C) but develop nearly green leaves at a permissive temperature (30 C). V1 encodes a chloroplast-localized protein NUS1 which is involved in the regulation of chloroplast rRNA metabolism during early chloroplast development (Kusumi et al., 1997; Kusumi et al., 2011). V2 encodes a new type of guanylate kinase (pt/ mtGK) localized both to plastids and mitochondria. It has been proposed that V2 functions at an early stage of chloroplast differentiation particularly in the chloroplast translation machinery during early leaf development (Sugimoto et al., 2004, 2007). V3 and STRIPE1 encode the large and small subunits of
ribonucleotide reductase (RNR), respectively, which regulates the rate of deoxyribonucleotide production for DNA synthesis and repair. Yoo et al. (2009) speculated that, upon insufficient activity of RNR, plastid DNA synthesis is preferentially arrested to allow nuclear genome replication in developing leaves to sustain the continuous plant growth. In this study, we identified a stripe variegated mutant named st2 (stripe2). The st2 plants develop chlorotic leaves caused by low content of chlorophylls. Examination of the ultrastructure showed that the thylakoid membranes are extremely disturbed in the mutants. Map-based cloning and genetic complementation indicated that ST2 encodes a cytidine deaminase-like protein, which most likely functions as the dCMP deaminase. RESULTS Phenotype characterization of stripe2 mutant The rice (Oryza sativa L.) st2 mutant was isolated from g-rayinduced mutations of an indica cultivar (Longtepu, LTP). The mutant leaves were virescent with stripes (Fig. 1A and E). To determine its effect on chlorophyll formation, the leaves of 3-week seedling were analyzed for chlorophyll contents. Compared with the wild type, the chlorophyll contents in st2 were reduced by 30% (Fig. 1B), and autofluorescence in the st2 leaves also decreased (Fig. 1C and D). The ultrastructure of the wild-type chloroplasts was crescent-shaped and contained well-formed thylakoid structure including stroma thylakoids and grana thylakoids (Fig. 2A). In contrast, the mutant chloroplasts were small and thylakoid membrane was disturbed, some with less thylakoid structure (Fig. 2B and C). Some chloroplasts formed rudimentary thylakoids consisting of only grana lamellae without formation of stroma lamellae (Fig. 2D), while some chloroplasts displayed well-developed lamellar structures equipped with normally stacked grana but no starch grains in the st2 leaves (Fig. 2E and F). These results indicated the st2 phenotype is caused by the underdevelopment of the chloroplast. Fine mapping of the ST2 Gene For genetic analysis of the st2 mutant, we firstly crossed st2 mutant with a japonica cultivar Zhonghua11 (ZH11), and the F1 generation exhibited normal green leaves. Among the 762 F2 individuals, 183 were virescent and 579 were green. The segregation ratio of the F2 population accorded with 3:1 (c2 ¼ 0.39 < c20.05 ¼ 3.81; P > 0.05), suggesting that the st2 phenotype was controlled by a single recessive gene. Genetic mapping was performed using the same population. The locus was primarily mapped to a 6.5-CM region flanking by the makers of RM8139 and C7962 on the long arm of chromosome 1. To further narrow down the region, new markers including insertion/deletion (InDel) markers and CAPS (cleaved amplified polymorphic sequence) were designed; however, little polymorphism could be found between st2 and ZH11. Therefore, we developed a larger F2
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Fig. 1. Phenotypic characterization of the st2 mutant. A: Three-week-old seedlings of LTP (left) and st2 mutant (right). The st2 plants exhibited striped leaves. Scale bar ¼ 2.5 cm. B: The contents of chlorophyll a and b were greatly reduced in the three-week-old seedling of st2 mutant compared with those of LTP. Student’s t-test was performed on the raw data; asterisk indicates statistical significance at P < 0.01. C: Chlorophyll autofluorescence of LTP leaf sampled from three-week-old seedling. Scale bar ¼ 100 mm. D: Chlorophyll autofluorescence of st2 leaf sampled from three-week-old seedling. Scale bar ¼ 100 mm. E: The stripe phenotype was sustained in st2 adult plants. Scale bar ¼ 10 cm.
population from a cross of st2 and 9311 (indica) for further mapping. By genotyping 1723 homologous st2 individuals, the ST2 gene was finally confined to a 27-kb physical interval between S1614 and S9131 on BAC P0403C05. According to the rice genome annotation database (http://rice.plantbiology.
msu.edu/), seven genes were predicted in the region (Fig. 3A). The genomic DNA of the candidate genes was sequenced and compared with LTP. A 5-bp deletion (TAGGT) was detected on the ORF of Os01g0765000 in st2 mutant (Fig. 3B). This deletion leads to the abortion of Bln I
Fig. 2. Ultrastructure of chloroplasts in mesophyll cells of LTP (WT) and st2 mutant. The chloroplasts of LTP have well-ordered thylakoids and stacked membranes (A), while st2 shows various defects in chloroplasts, among which some form little thylakoid structure (B and C) or form thylakoid with only grana lamellae (D), and some could form normal thylakoid though without starch grains (E and F). Scale bars ¼ 1 mm. Cp, chloroplast; M, mitochondrion; SG, starch grain.
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Fig. 3. Map-based cloning and candidate gene identification of ST2. A: The ST2 locus was initially mapped on chromosome 1 by flanking markers RM8139 and C7962 with 183 recessive individuals. A larger population consisting of 1723 recessive individuals was used for fine mapping, and finally the locus was confined to about 27 kb between markers S1614 and S9131. Seven ORFs were identified according to the genome annotation data. B: A 5-bp deletion (TAGGT in bold) located on the splicing site of exon 3 and intron 3 was found in ORF3 (Os01g0765000) and lead to the abortion of Bln I site (CCTAGG, underlined). C: The deletion was detected by primer C1377, and only st2 showed resistance to Bln I digestion. 1, st2; 2, LTP; 3, Nipponbare; 4, 9311; 5, Zhonghua 11; 6, Taipei309; 7, Zhejing 22; 8, Gumei 4; 9, Teqing; 10, F1 plants of st2 and 9311. D: Full length of cDNA of ORF3 was amplified from the st2 mutant. According to the genomic DNA (I) and cDNA (II) in the wild type, two new transcripts (III and IV) with parts of intron 3 were generated in the mutant. E: RT-PCR detection of the new transcripts in the st2 mutant, which were larger than that of LTP.
cutting site, and a primer (C1377) was designed to detect the deletion. To confirm that the deletion was exclusive for st2 mutant, several other varieties were also genotyped with C1377, and only st2 was resistant to the Bln I digestion (Fig. 3C). There were nine exons in Os01g0765000, and the deletion located on the conjunction of the third exon and
intron, which could affect mRNA splicing (Fig. 3D). RT-PCR showed that there are two larger bands in st2 instead of one band in LTP (Fig. 3E). Sequence alignment of the exceptional bands confirmed that the additional nucleotides came from the third intron (Fig. S1). The additional intron sequence led to the premature termination of the predicted
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which is a mitochondrial marker (Carrie et al., 2007), indicating that ST2 is localized to mitochondria. ST2 encodes a putative dCMP deaminase
Fig. 4. Functional complementation of st2 by genomic DNA. The st2 phenotype can be recovered by the external genomic DNA including the whole ORF3 and its promoter. Three independent transgenic lines (T1, T4, T6) showed normal leaf phenotype similar to the wild type (LTP). Scale bar ¼ 2 cm.
protein, therefore disturbing its normal function. The identification of the ST2 gene was subsequently confirmed by the genetic complementary experiments. More than 30 independent transgene-positive plants of T0 generation were obtained and most of them showed normal leaf color (Fig. 4). Moreover, 12 T1 lines were planted, which showed segregation of wild type and st2 phenotypes. Together, Os01g0765000 is the underlying gene responsible for the st2 phenotype.
Expression pattern and subcellular localization of ST2 To understand the roles of ST2 in plant growth, we detected the expression pattern of ST2 in different tissues including root, seedling, leaf, leaf sheath, stem, and panicle. RT-PCR result showed that the ST2 is expressed ubiquitously, suggesting that ST2 may function in most tissues (Fig. 5A). It was predicted that ST2 has a mitochondrial or chloroplast transit peptide comprising 40 amino acids at N-terminal (http://www. cbs.dtu.dk/services/TargetP/). To determine the subcellular localization of ST2, fusion proteins of ST2-YFP (yellow fluorescent protein) and truncated ST2 without the transit peptide (DST2-YFP) were transiently expressed in onion epidermal cells and rice protoplasts driven by the 35S promoter. The ST2-YFP showed a punctate pattern but did not coincide with the red chlorophyll auto-fluorescence. When the transit peptide is excluded (DST2-YFP), the punctate location pattern of ST2-YFP disappeared, confirming that the transit peptide guides its cellular location (Fig. 5B and C). Moreover, we observed that ST2-GFP was co-localized with AOX-RFP (mitochondrial alternative oxidase fused to RFP) (Fig. 5D),
According to the genome annotation and sequence similarity, we found that ST2 belongs to the cytidine deaminase-like family, which is involved in the nucleotide metabolism. There are 7 and 16 cytidine deaminase members in rice and Arabidopsis, respectively (Fig. 6A). Phylogenetic analysis showed that the ST2 shared 83% sequence similarity with Arabidopsis At3g48540. We examined T-DNA insertion mutants of At3g48540 available from the Arabidopsis community (http://www.arabidopsis.org/), but no phenotype like st2 was observed in the null mutant FLAG_475E06, probably due to the functional redundancy of the family in Arabidopsis. The members of cytidine deaminase-like family (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi? uid¼cd00786&seltype¼1) can be divided into four subfamilies, including riboflavin deaminase, cytidine deaminase, dCMP deaminase and nucleoside deaminase. In mammals, the cytidine deaminase such as APOBEC-1 is responsible for C-to-U editing of the apolipoprotein mRNA (Prohaska et al., 2014). In higher plants, RNA editing is a post-transcriptional process of altering a specific nucleotide C to U (and less frequently, from U to C) in organelle mRNAs. Most RNA editing events are necessary for expressing functional proteins, as demonstrated by examples of RPL2 (ribosomal protein L2) in maize (Hoch et al., 1991) and NDHD-1 (a subunit of the chloroplast NADH dehydrogenase-like complex (NDH), involved in cyclic electron flow) in Arabidopsis (Boussardon et al., 2012). However, enzyme(s) for the C-to-U conversion has not been identified yet and cytidine deaminase is most likely a candidate. To determine whether ST2 functions as a cytidine deaminase, we scanned the mitochondrial RNA editing sites by using 48 pairs of primers from the study of Kim et al. (2009). Compared with the wild type, all the editing sites still existed, suggesting that ST2 might not function as a cytidine deaminase to catalyze RNA nucleotides. We then compared ST2 with the human and yeast dCMP deaminases, and high homology was found especially in the catalytic site from the 80th to the 140th amino acid (Fig. 6B). Since dCMP deaminases catalyze the process from dCMP to dUMP by deamination, we considered that the st2 mutant might be deficient in the dUMP supply and the external application of dUMP should recover or attenuate the st2 phenotype. As showed in Fig. 7, the leaf color was recovered with 1 mmol/L dUMP feeding, albeit plant growth was inhibited at that concentration. Therefore, we proposed that ST2 most likely functions as a dCMP deaminase. DISCUSSION In this study, we isolated a new leaf-color mutant st2 in rice. The mutant exhibits the phenotype of underdeveloped chloroplast with inhibited chlorophyll accumulation. To recognized the ST2 gene functionally, we map-cloned the ST2 gene, and found that the gene encodes a putative mitochondrialocated dCMP deaminase. Our study for the first time identified a putative plant dCMP deaminase, and is also the first
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Fig. 5. Expression pattern and subcellular localization of ST2. A: ST2 can be detected in different tissues by RT-PCR. Ubiquitin (UB) was amplified as control. R, root; SL, seedling; LH, flag leaf during heading date; SH, sheath; ST, shoot; PH, panicle during heading stage; S, spikelets during heading stage. B: Transient expression of ST2-YFP, DST2-YFP and YFP in onion epidermal cells. The ST2-YFP showed a punctate YFP signal. When the N-terminal signal peptide of ST2 was excluded, it showed the same ubiquitous expression pattern as YFP control. The images showed the fluorescence both under dark field (YFP fluorescence) and bright field (Bright image). Scale bar ¼ 50 mm. C: The same constructions were transformed into rice protoplasts and exhibited similar localization patterns. The chlorophyll autofluorescence was also imaged and merged with the YFP fluorescence to detect the ST2 localization. Scale bar ¼ 20 mm. D: ST2-GFP was co-localized with AOX-RFP, a mitochondria maker, indicating that ST2 is localized to mitochondria in the rice protoplast. Scale bar ¼ 20 mm.
report that mitochondria-located deaminase plays a critical role in chloroplast development as well as chlorophyll biosynthesis. According to the sequence alignment, ST2 belongs to the cytidine deaminase-like family with 7 and 16 members in rice and Arabidopsis, respectively. Only two members of the family, AtCDA1 (cytidine deaminase 1) (Faivre-Nitschke et al., 1999; Vincenzetti et al., 1999; Kafer and Thornburg, 2000) and AtTadA (Karcher and Bock, 2009) have been reported in Arabidopsis. AtCDA1 can utilize both cytidine and 20 -deoxycytidineas substrates but unable to deaminate cytosine, CMP or dCMP. AtTadA encodes a chloroplast tRNA adenosine deaminase which triggers A-to-I editing in the anticodon of the plastid tRNA-Arg (ACG), presumably regulating chloroplast translational efficiency. This cytidine deaminase-like family can be divided into four subfamilies performing functions in different bioprocesses. One possibility is that ST2 functions as a cytidine deaminase for RNA editing. However, no editing site was found disappeared in st2 compared with the wild type. We then found that ST2 shares
similarity with the human and yeast dCMP deaminases. According to reports in animal, T4 phage and yeast, dCMP deaminases catalyze the deamination of dCMP to form dUMP. dUMP is the precursor for dTMP that is subsequently phosphorylated to thymidine triphosphate for DNA synthesis and repair. The exogenous application of dUMP could recover the st2 phenotype, suggesting that ST2 most likely functions as a dCMP deaminase. Therefore, the mutation in the ST2 gene reduces the dUMP pool in plant, and further disturbs the balance of subsequent dTTP formation. It is intriguing how a mitochondria-located deaminase affects chloroplast development. As previously reported, in the rice virescent3 and strip1 mutants, plastid DNA synthesis for chloroplast biogenesis is supposed to be relatively less critical for plant survival and can sacrifice under insufficient dNTP levels (Yoo et al., 2009). Similarly, we speculate that chloroplast development is preferentially arrested to allow both nuclear and mitochondrial genome replication. However, the mechanism how the dNTP pool prioritizes nuclear and possible mitochondrial DNA (mtDNA) synthesis keeps elusive.
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Fig. 6. Sequence analysis of ST2 homologs. A: The phylogenetic tree represents alignment of ST2 protein with its homologs in rice and Arabidopsis. Seven proteins including ST2 and 16 proteins in Arabidopsis were aligned by ClustalX2 and the phylogram tree was constructed by TreeView. Scale represents percentage substitution per site. B: The ST2 protein was aligned with the human dCMP deaminase (HsDCD, NP_001012750.1) and yeast dCMP deaminase (ScDCD, NP_012014.1). Identical residues were boxed in black and similar residues were boxed in gray.
In T4 phage, the endogenous dTTP content was greatly reduced and the dCTP increased 30 times as high as the normal level when dCMP deaminase was inactive. Such imbalance led to the reduced fidelity of DNA replication with increased nucleotide mismatch (Sargent and Mathews, 1987). Correspondingly, we did detect up-regulated DNA repair genes from microarray data of st2 compared with LTP, including endonuclease, meiotic recombination protein DMC1, DNA helicase RecQ, and RAD51 homolog RAD51B (data not shown). The study of the maize ncs (nonchromosomal strip) mutant indicated that mitochondria are necessary for the chloroplast biogenesis, and the mtDNA mutations can result in strip phenotype (Jiao et al., 2005). We also suggest another possibility that the imbalance dNTP in the st2 mutants may also increase the mutation rate of mtDNA, thereby disturbing the chloroplast
development. Further investigation is necessary to dissect the mechanism of nucleotide distribution between chloroplasts, mitochondria and the nucleus. MATERIALS AND METHODS Plant materials and grow condition The rice st2 mutant was isolated from g-ray-induced mutations of an indica cultivar (LTP). A japonica variety, Zhonghua11 (ZH11), was crossed with st2 to construct a F2 population for genetic study and preliminary mapping. Additional 1723 recessive individuals from the F2 populations derived from the cross between st2 and 9311 (indica) were used for fine mapping. Plants were cultivated in the
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mixture was centrifuged, and the supernatant was used to measure the absorbance values under 663 nm and 645 nm, using a spectrophotometer (Beckman Coulter-DU800, USA), with the extraction buffer as control. Each sample was assayed with three biological repeats. Transmission electron microscopy (TEM) assay Seedlings of st2 and LTP of 20-day-old were sampled for TEM observation. All the leaf samples were cut into sections less than 2 mm2 and infiltrated for 30 min with fixation buffer [2.5% glutaraldehyde in phosphate buffer (pH 7.2)] under vacuum. After 3 days at 4 C, samples were post-fixed in 0.1 mol/L cacodylate (pH 7.4) with 2% OsO4 at 4 C, followed by washing with 0.1 mol/L PBS, then dehydrated with a gradient ethanol-acetone series and embedded in Polybed 812 (Sigma, USA) resin (Li et al., 2011). Ultrathin sections were obtained with an ultramicrotome, mounted on grids, and stained. The sections were viewed via an electron microscopy H7650 (Hitachi, Japan). Map-based cloning of ST2
Fig. 7. Recovery of st2 plants by dUMP feeding. A: The LTP seedlings treated with dUMP under concentration of 0, 105, 104 and 103 mol/L (from left to right). Two seedlings were imaged for each concentration. Scale bar ¼ 2 cm. Note that 103 mol/L dUMP slightly inhibited seedling growth. B: The st2 mutant plants were fed with dUMP under the same concentrations as in (A). Note that the leaf phenotype was recovered by 103 mol/L dUMP. Scale bar ¼ 2 cm.
experimental field during natural growth seasons. For seedlings, seeds were germinated in the dark for 2 days and then transferred to liquid medium in a growth chamber under growth conditions with 12-h day, 28 C, 80% relative humidity (RH) followed by 12-h night, 26 C, 60% RH. Measurement of chlorophyll a and b The contents of chlorophyll a and b (Chl a and Chl b) were measured according to the previously method with some modifications (Arnon, 1949). The fresh rice leaves were cut into small pieces with scissors and soaked in the extraction buffer (95% ethanol:acetone:water ¼ 5:4:1), and incubated at 4 C in the dark for 18 h with periodically inversion. The
For gene mapping, a total of 120 SSR (simple sequence repeats) markers distributing evenly on 12 chromosomes according to database information in the GRAMENE (http://www.gramene. org/bd/markers/) were used for preliminary mapping. To construct a high-density linkage map for fine mapping in the target region, new InDel markers and CAPS markers were developed according to the sequence differences between indica (9311) and japonica (Nipponbare) genomes. In case that markers did not exhibit polymorphism between st2 and 9311, we sequenced PCR products to search for SNPs to develop new CAPS markers. The primer sequences and enzymes used are listed in Table S1. All the primers were designed using the program Primer Premier 5.0 (http://www.premierbiosoft.com). Subsequently, we placed ST2 in a 27-kb region between the markers S1614 and S9131. Genomic DNA fragments of this region were amplified from st2 and wild-type (LTP) plants, sequenced, and compared using MegAlign (DNASTAR). Complementation test and transgenic expression The rice Nipponbare ( japonica) BAC P0403C05 bearing ST2 was digested to isolate a 9-kb genomic DNA fragment with the full ST2 region, which was inserted into the binary vector pCAMBIA1301 to generate the plasmid pST2-ST2. The plasmid was introduced into the st2 mutants by Agrobacterium-mediated transformation (Hiei et al., 1994). More than 10 independent transgenic lines were produced that could successfully complement the mutant phenotypes. Subcellular localization assay ST2-YFP, DST2-YFP and ST2-GFP fusions were obtained by in-frame fusing the cDNAs with YFP or GFP, which were amplified using the specific primers ST2-YF/ST2-YR, DST2-
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YF/ST2-YR, and ST2-YF/ST2-GR, respectively (Table S2). The resulted fusions were driven by the 35S promoter. Transient expression of the fusion proteins ST2-YFP, DST2-YFP and the controls YFP in onion epidermal cells was performed using a helium biolistic device (Bio-Rad PDS-1000, USA) as previously described (Collings et al., 2000). Rice mesophyll protoplasts isolated from leaf sheaths of 11-day-old seedlings were transfected with fusion constructs, or cotransfected with constructs of ST2-GFP and AOX-RFP to determine mitochondrial location, according to previously reported methods (Bart et al., 2006). Onion epidermal cells and protoplasts expressing the proteins were imaged under a laser scanning confocal microscope using a Plan-Apochromat 100/1.4 oil objective (LSM510 META NLO, Zeiss, Germany). RT-PCR analysis Total RNAs were extracted from the whole 2-week-old seedlings using TRIzol reagent (Invitrogen, USA) and treated with DNase RQ1 (Promega, USA). cDNAs were synthesized from 3 mg total RNAs using oligo (dT) primer and SuperScript III reverse transcriptase according to the manufacturer’s instructions (Invitrogen). RT-PCR analysis and full-length cDNA amplification were performed using gene-specific primers (Table S3), under the following conditions: 5 min at 95 C, 28 cycles (30 s at 95 C, 30 s at specific Tm, 1 min/kb at 72 C), followed by 10 min at 72 C. Analysis of RNA editing Total RNAs extracted from seedling leaves were treated with RQ1 DNase (Promega). Then cDNAs were synthesized from 3 mg total RNAs using hexanucleotide oligomers primer and MMLV reverse transcriptase. These cDNAs were used as templates for PCR amplification of mitochondrial genes. The primers used for scanning mitochondrial RNA editing sites were the same as those reported by Kim et al. (2009). The RTPCR products were directly sequenced and manually compared between the wild type and the mutant. ACKNOWLEDGEMENTS We thank Jiqin Li and Xiaoshu Gao for technical assistance. We also acknowledge James Whelan (University of Western Australia) for providing AOX-RFP plasmid. This work was supported by the grant from the Ministry of Science and Technology of China (No. 2012AA10A302-2).
SUPPLEMENTARY DATA Fig. S1. Sequences representing the disturbed splicing shown in Fig. 3D (III and IV). Table S1. PCR-based markers linked to St2 locus. Table S2. Primers for RT-PCR. Table S3. Primers for plasmid construction.
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Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2014.05.008. REFERENCES Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in beta vulgaris. Plant Physiol. 24, 1e15. Bart, R., Chern, M., Park, C.J., Bartley, L., Ronald, P., 2006. A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts. Plant Methods 2, 13. Boussardon, C., Salone, V., Avon, A., Berthome, R., Hammani, K., Okuda, K., Shikanai, T., Small, I., Lurin, C., 2012. Two interacting proteins are necessary for the editing of the NdhD-1 site in Arabidopsis plastids. Plant Cell 24, 3684e3694. Carrie, C., Murcha, M.W., Millar, A.H., Smith, S.M., Whelan, J., 2007. Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria. Plant Mol. Biol. 63, 97e108. Chen, H., Cheng, Z., Ma, X., Wu, H., Liu, Y., Zhou, K., Chen, Y., Ma, W., Bi, J., Zhang, X., Guo, X., Wang, J., Lei, C., Wu, F., Lin, Q., Liu, L., Jiang, L., 2013. A knockdown mutation of YELLOW-GREEN LEAF2 blocks chlorophyll biosynthesis in rice. Plant Cell Rep. 32, 1855e1867. Chen, M., Galvao, R.M., Li, M., Burger, B., Bugea, J., Bolado, J., Chory, J., 2010. Arabidopsis HEMERA/pTAC12 initiates photomorphogenesis by phytochromes. Cell 141, 1230e1240. Collings, D.A., Carter, C.N., Rink, J.C., Scott, A.C., Wyatt, S.E., Allen, N.S., 2000. Plant nuclei can contain extensive grooves and invaginations. Plant Cell 12, 2425e2440. Delannoy, E., Le Ret, M., Faivre-Nitschke, E., Estavillo, G.M., Bergdoll, M., Taylor, N.L., Pogson, B.J., Small, I., Imbault, P., Gualberto, J.M., 2009. Arabidopsis tRNA adenosine deaminase arginine edits the wobble nucleotide of chloroplast tRNAArg(ACG) and is essential for efficient chloroplast translation. Plant Cell 21, 2058e2071. Faivre-Nitschke, S.E., Grienenberger, J.M., Gualberto, J.M., 1999. A prokaryotic-type cytidine deaminase from Arabidopsis thaliana. Eur. J. Biochem. 263, 896e903. Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271e282. Hoch, B., Maier, R.M., Appel, K., Igloi, G.L., Kossel, H., 1991. Editing of a chloroplast mRNA by creation of an initiation codon. Nature 353, 178e180. Jiao, S., Thornsberry, J.M., Elthon, T.E., Newton, K.J., 2005. Biochemical and molecular characterization of photosystem I deficiency in the NCS6 mitochondrial mutant of maize. Plant Mol. Biol. 57, 303e313. Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., Hirochika, H., An, G., 2003. Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system. Plant Cell Physiol. 44, 463e472. Kafer, C., Thornburg, R., 2000. Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes. J. Plant Biol. 43, 162e170. Karcher, D., Bock, R., 2009. Identification of the chloroplast adenosine-toinosine tRNA editing enzyme. RNA 15, 1251e1257. Kim, S.R., Yang, J.I., Moon, S., Ryu, C.H., An, K., Kim, K.M., Yim, J., An, G., 2009. Rice OGR1 encodes a pentatricopeptide repeat-DYW protein and is essential for RNA editing in mitochondria. Plant J. 59, 738e749. Kusumi, K., Mizutani, A., Nishimura, M., Iba, K., 1997. A virescent gene V1 determines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice. Plant J. 12, 1241e1250. Kusumi, K., Sakata, C., Nakamura, T., Kawasaki, S., Yoshimura, A., Iba, K., 2011. A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions. Plant J. 68, 1039e1050. Li, W., Zhong, S., Li, G., Li, Q., Mao, B., Deng, Y., Zhang, H., Zeng, L., Song, F., He, Z., 2011. Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence. Cell Res. 21, 835e848.
548
J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548
Loschelder, H., Schweer, J., Link, B., Link, G., 2006. Dual temporal role of plastid sigma factor 6 in Arabidopsis development. Plant Physiol. 142, 642e650. Moon, J., Zhu, L., Shen, H., Huq, E., 2008. PIF1 directly and indirectly regulates chlorophyll biosynthesis to optimize the greening process in Arabidopsis. Proc. Natl. Acad. Sci. USA 105, 9433e9438. Mullet, J.E., 1988. Chloroplast development and gene expression. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39, 475e502. Pogson, B.J., Albrecht, V., 2011. Genetic dissection of chloroplast biogenesis and development: an overview. Plant Physiol. 155, 1545e1551. Prohaska, K.M., Bennett, R.P., Salter, J.D., Smith, H.C., 2014. The multifaceted roles of RNA binding in APOBEC cytidine deaminase functions. Wiley Interdiscip. Rev. RNA http://dx.doi.org/10.1002/wrna.1226. Pyo, Y.J., Kwon, K.C., Kim, A., Cho, M.H., 2013. Seedling Lethal1, a pentatricopeptide repeat protein lacking an E/Eþ or DYW domain in Arabidopsis, is involved in plastid gene expression and early chloroplast development. Plant Physiol. 163, 1844e1858. Sakamoto, W., Miyagishima, S.Y., Jarvis, P., 2008. Chloroplast biogenesis: control of plastid development, protein import, division and inheritance. Arabidopsis Book 6, e0110. Sakuraba, Y., Rahman, M.L., Cho, S.H., Kim, Y.S., Koh, H.J., Yoo, S.C., Paek, N.C., 2013. The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions. Plant J. 74, 122e133. Sargent, R.G., Mathews, C.K., 1987. Imbalanced deoxyribonucleoside triphosphate pools and spontaneous mutation rates determined during dCMP deaminase-defective bacteriophage T4 infections. J. Biol. Chem. 262, 5546e5553. Stephenson, P.G., Fankhauser, C., Terry, M.J., 2009. PIF3 is a repressor of chloroplast development. Proc. Natl. Acad. Sci. USA 106, 7654e7659. Su, N., Hu, M.L., Wu, D.X., Wu, F.Q., Fei, G.L., Lan, Y., Chen, X.L., Shu, X.L., Zhang, X., Guo, X.P., Cheng, Z.J., Lei, C.L., Qi, C.K., Jiang, L., Wang, H.,
Wan, J.M., 2012. Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production. Plant Physiol. 159, 227e238. Sugimoto, H., Kusumi, K., Noguchi, K., Yano, M., Yoshimura, A., Iba, K., 2007. The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria. Plant J. 52, 512e527. Sugimoto, H., Kusumi, K., Tozawa, Y., Yazaki, J., Kishimoto, N., Kikuchi, S., Iba, K., 2004. The virescent-2 mutation inhibits translation of plastid transcripts for the plastid genetic system at an early stage of chloroplast differentiation. Plant Cell Physiol. 45, 985e996. Swiatecka-Hagenbruch, M., Emanuel, C., Hedtke, B., Liere, K., Borner, T., 2008. Impaired function of the phage-type RNA polymerase RpoTp in transcription of chloroplast genes is compensated by a second phage-type RNA polymerase. Nucleic Acids Res. 36, 785e792. Takenaka, M., Zehrmann, A., Verbitskiy, D., Hartel, B., Brennicke, A., 2013. RNA editing in plants and its evolution. Annu. Rev. Genet. 47, 335e352. Vincenzetti, S., Cambi, A., Neuhard, J., Schnorr, K., Grelloni, M., Vita, A., 1999. Cloning, expression, and purification of cytidine deaminase from Arabidopsis thaliana. Protein Expr. Purif. 15, 8e15. Wu, Z., Zhang, X., He, B., Diao, L., Sheng, S., Wang, J., Guo, X., Su, N., Wang, L., Jiang, L., Wang, C., Zhai, H., Wan, J., 2007. A chlorophylldeficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis. Plant Physiol. 145, 29e40. Yoo, S.C., Cho, S.H., Sugimoto, H., Li, J., Kusumi, K., Koh, H.J., Iba, K., Paek, N.C., 2009. Rice virescent3 and stripe1 encoding the large and small subunits of ribonucleotide reductase are required for chloroplast biogenesis during early leaf development. Plant Physiol. 150, 388e401. Zhang, H., Li, J., Yoo, J.H., Yoo, S.C., Cho, S.H., Koh, H.J., Seo, H.S., Paek, N.C., 2006. Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development. Plant Mol. Biol. 62, 325e337.