STRIPE2 Encodes a Putative Dcmp Deaminase That Plays an Important Role in Chloroplast Development in Rice

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Journal of Genetics and Genomics 41 (2014) 539e548

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 materials to investigate regulation mechanisms of chlorophyll biosynthesis and chloroplast development in rice. The chloroplast is the crucial organelle for plant photosyn- Screening for chloroplast development mutants has identi- thesis and essential for the production of hormones and me- fied most steps in these biological processes, such as yellow- tabolites (Pogson and Albrecht, 2011). About 3000 proteins in green leaf1 ( ygl1) and faded green leaf ( fgl ), which result the chloroplast participate in transition from proplastids to from lesions of chlorophyll synthase that catalyzes esterifica- mature chloroplasts, and this process is coordinated by both tion of chlorophyllide in the last step of chlorophyll biosyn- nuclear and plastid genome involved in synthesis of chloro- thesis (Wu et al., 2007) and NADPH:protochlorophyllide plast DNA, the plastidic transcription/translation apparatus oxidoreductase that catalyzes the photoreduction of proto- and the photosynthetic system (Sakamoto et al., 2008). chlorophyllide (pchlide) to chlorophyllide (chlide) (Sakuraba Numerous chlorophyll-deficient or abnormal chloroplast mu- et al., 2013). Both mutations led to reduced contents of tants have been identified, and they provide ideal genetic chlorophyll and undeveloped chloroplasts. The magnesium chelatase, catalyzing the chelation of Mg2þ into proto IX to produce Mg-Proto IX, comprises three subunits including * 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). ChlH, ChlD and ChlI (Jung et al., 2003; Zhang et al., 2006). E-mail addresses: [email protected] (X. Zhu); [email protected] (Z. He). 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. 540 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548 chlorophyll content and incomplete development of chloro- ribonucleotide reductase (RNR), respectively, which regulates plasts, due to the disruption of ChlD and ChlI subunits, the rate of deoxyribonucleotide production for DNA synthesis respectively. Moreover, an insert mutation in ygl2 ( yellow- and repair. Yoo et al. (2009) speculated that, upon insufficient green leaf 2), encoding heme oxygenase (HO) that catalyzes activity of RNR, plastid DNA synthesis is preferentially the degradation of heme to synthesize phytochrome precursor, arrested to allow nuclear genome replication in developing results in significantly reduced content of chlorophyll and leaves to sustain the continuous plant growth. tetrapyrrole intermediates (Chen et al., 2013). In this study, we identified a stripe variegated mutant As a semiautonomous organelle, chloroplast genome only named st2 (stripe2). The st2 plants develop chlorotic leaves encodes about 100 genes (Delannoy et al., 2009). Most of the caused by low content of chlorophylls. Examination of the proteins essential for chloroplast development and function are ultrastructure showed that the thylakoid membranes are nuclear-encoded (Chen et al., 2010). It is well documented that extremely disturbed in the mutants. Map-based cloning and the coordination of nuclear and plastid genes is crucial for genetic complementation indicated that ST2 encodes a cyti- chloroplast biogenesis (Mullet, 1988). Under illumination, dine deaminase-like protein, which most likely functions as one-third of the nuclear genes change expression, including the dCMP deaminase. many transcription factors such as PIFs (phytochrome interacting factors). Either pif1 or pif3 mutant showed delayed RESULTS development of chloroplast (Moon et al., 2008; Stephenson et al., 2009). Gene transcription, RNA maturation, and pro- Phenotype characterization of stripe2 mutant tein translation in the chloroplasts also have impact on chloroplast biogenesis and development. There are two types of The rice (Oryza sativa L.) st2 mutant was isolated from g-ray- plastid RNA polymerases: plastid-encoded RNA polymerase induced mutations of an indica cultivar (Longtepu, LTP). The (PEP) and nucleus-encoded RNA polymerase (NEP) respon- mutant leaves were virescent with stripes (Fig. 1A and E). To sible for the transcription of the plastome. In Arabidopsis, determine its effect on chlorophyll formation, the leaves of mutations in SIG6 (sigma factor 6) cause a weakly virescent 3-week seedling were analyzed for chlorophyll contents. phenotype and transcripts of several PEP-dependent plastid Compared with the wild type, the chlorophyll contents in st2 genes are specifically reduced (Loschelder et al., 2006). were reduced by 30% (Fig. 1B), and autofluorescence in the Decreased expression of AtRpoTp, one of the NEP genes, st2 leaves also decreased (Fig. 1C and D). The ultrastructure of leads to a typical virescent phenotype which can be recovered the wild-type chloroplasts was crescent-shaped and contained after two weeks of growth (Swiatecka-Hagenbruch et al., well-formed thylakoid structure including stroma thylakoids 2008). PPR proteins (pentatricopeptide repeat proteins), and grana thylakoids (Fig. 2A). In contrast, the mutant chlo- characterized by tandem arrays of a 35 amino acid motif, have roplasts were small and thylakoid membrane was disturbed, been demonstrated to be critical for RNA processing, splicing, some with less thylakoid structure (Fig. 2B and C). Some editing, stability, maturation and translation in the chloroplast chloroplasts formed rudimentary thylakoids consisting of only (Takenaka et al., 2013). YSA (young seedling albino) encodes grana lamellae without formation of stroma lamellae a PPR protein and the disruption of its function causes a (Fig. 2D), while some chloroplasts displayed well-developed seedling stage-specific albino phenotype in rice. The mutant lamellar structures equipped with normally stacked grana but plants can recover and develop normal green leaves after the no starch grains in the st2 leaves (Fig. 2E and F). These results four-leaf stage. Interestingly, the ysa mutant has been used as a indicated the st2 phenotype is caused by the underdevelop- marker for efficient identification and elimination of false ment of the chloroplast. hybrids in commercial hybrid rice production (Su et al., 2012). The Arabidopsis mutant sel1 (seedling lethal 1) exhibited a Fine mapping of the ST2 Gene pigment-defective and seedling-lethal phenotype with a disrupted PPR gene. In the sel1 plants, RNA editing of acetyl- For genetic analysis of the st2 mutant, we firstly crossed st2 CoA carboxylase b subunit transcripts was disrupted (Pyo mutant with a japonica cultivar Zhonghua11 (ZH11), and the et al., 2013). The virescent rice mutants v1, v2 and v3 are F1 generation exhibited normal green leaves. Among the 762 temperature-conditional, which produce chlorotic leaves at a F2 individuals, 183 were virescent and 579 were green. The restrictive temperature (20 C) but develop nearly green segregation ratio of the F2 population accorded with 3:1 2 2 leaves at a permissive temperature (30 C). V1 encodes a (c ¼ 0.39 < c0.05 ¼ 3.81; P > 0.05), suggesting that the st2 chloroplast-localized protein NUS1 which is involved in the phenotype was controlled by a single recessive gene. Genetic regulation of chloroplast rRNA metabolism during early mapping was performed using the same population. The chloroplast development (Kusumi et al., 1997; Kusumi et al., locus was primarily mapped to a 6.5-CM region flanking by 2011). V2 encodes a new type of guanylate kinase (pt/ the makers of RM8139 and C7962 on the long arm of mtGK) localized both to plastids and mitochondria. It has been chromosome 1. To further narrow down the region, new proposed that V2 functions at an early stage of chloroplast markers including insertion/deletion (InDel) markers and differentiation particularly in the chloroplast translation ma- CAPS (cleaved amplified polymorphic sequence) were chinery during early leaf development (Sugimoto et al., 2004, designed; however, little polymorphism could be found be- 2007). V3 and STRIPE1 encode the large and small subunits of tween st2 and ZH11. Therefore, we developed a larger F2 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548 541

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 msu.edu/), seven genes were predicted in the region mapping. By genotyping 1723 homologous st2 individuals, (Fig. 3A). The genomic DNA of the candidate genes was the ST2 gene was finally confined to a 27-kb physical interval sequenced and compared with LTP. A 5-bp deletion between S1614 and S9131 on BAC P0403C05. According to (TAGGT) was detected on the ORF of Os01g0765000 in st2 the rice genome annotation database (http://rice.plantbiology. 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. 542 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

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 intron, which could affect mRNA splicing (Fig. 3D). RT-PCR deletion. To confirm that the deletion was exclusive for st2 showed that there are two larger bands in st2 instead of one mutant, several other varieties were also genotyped with band in LTP (Fig. 3E). Sequence alignment of the excep- C1377, and only st2 was resistant to the Bln I digestion tional bands confirmed that the additional nucleotides came (Fig. 3C). There were nine exons in Os01g0765000,andthe from the third intron (Fig. S1). The additional intron deletion located on the conjunction of the third exon and sequence led to the premature termination of the predicted J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548 543

which is a mitochondrial marker (Carrie et al., 2007), indicating that ST2 is localized to mitochondria.

ST2 encodes a putative dCMP deaminase

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 Fig. 4. Functional complementation of st2 by genomic DNA. cytidine deaminase such as APOBEC-1 is responsible for C-to-U The st2 phenotype can be recovered by the external genomic DNA including editing of the apolipoprotein mRNA (Prohaska et al., 2014). In the whole ORF3 and its promoter. Three independent transgenic lines (T1, T4, higher plants, RNA editing is a post-transcriptional process of T6) showed normal leaf phenotype similar to the wild type (LTP). Scale altering a specific nucleotide C to U (and less frequently, from U ¼ bar 2 cm. to C) in organelle mRNAs. Most RNA editing events are neces- protein, therefore disturbing its normal function. The iden- sary for expressing functional proteins, as demonstrated by ex- tification of the ST2 gene was subsequently confirmed by the amples of RPL2 (ribosomal protein L2) in maize (Hoch et al., genetic complementary experiments. More than 30 inde- 1991) and NDHD-1 (a subunit of the chloroplast NADH pendent transgene-positive plants of T0 generation were ob- dehydrogenase-like complex (NDH), involved in cyclic electron tained and most of them showed normal leaf color (Fig. 4). flow) in Arabidopsis (Boussardon et al., 2012). However, en- Moreover, 12 T1 lines were planted, which showed segre- zyme(s) for the C-to-U conversion has not been identified yet and gation of wild type and st2 phenotypes. Together, cytidine deaminase is most likely a candidate. To determine Os01g0765000 is the underlying gene responsible for the st2 whether ST2 functions as a cytidine deaminase, we scanned the phenotype. 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 Expression pattern and subcellular localization of ST2 function as a cytidine deaminase to catalyze RNA nucleotides. We then compared ST2 with the human and yeast dCMP de- To understand the roles of ST2 in plant growth, we detected aminases, and high homology was found especially in the cata- the expression pattern of ST2 in different tissues including lytic site from the 80th to the 140th amino acid (Fig. 6B). Since root, seedling, leaf, leaf sheath, stem, and panicle. RT-PCR dCMP deaminases catalyze the process from dCMP to dUMP by result showed that the ST2 is expressed ubiquitously, sug- deamination, we considered that the st2 mutant might be deficient gesting that ST2 may function in most tissues (Fig. 5A). It was in the dUMP supply and the external application of dUMP should predicted that ST2 has a mitochondrial or chloroplast transit recover or attenuate the st2 phenotype. As showed in Fig. 7,the peptide comprising 40 amino acids at N-terminal (http://www. leaf color was recovered with 1 mmol/L dUMP feeding, albeit cbs.dtu.dk/services/TargetP/). To determine the subcellular plant growth was inhibited at that concentration. Therefore, we localization of ST2, fusion proteins of ST2-YFP (yellow proposed that ST2 most likely functions as a dCMP deaminase. fluorescent protein) and truncated ST2 without the transit peptide (DST2-YFP) were transiently expressed in onion DISCUSSION epidermal cells and rice protoplasts driven by the 35S promoter. The ST2-YFP showed a punctate pattern but did not In this study, we isolated a new leaf-color mutant st2 in rice. coincide with the red chlorophyll auto-fluorescence. When the The mutant exhibits the phenotype of underdeveloped chlo- transit peptide is excluded (DST2-YFP), the punctate location roplast with inhibited chlorophyll accumulation. To recog- pattern of ST2-YFP disappeared, confirming that the transit nized the ST2 gene functionally, we map-cloned the ST2 gene, peptide guides its cellular location (Fig. 5B and C). Moreover, and found that the gene encodes a putative mitochondria- we observed that ST2-GFP was co-localized with AOX-RFP located dCMP deaminase. Our study for the first time identi- (mitochondrial alternative oxidase fused to RFP) (Fig. 5D), fied a putative plant dCMP deaminase, and is also the first 544 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

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 similarity with the human and yeast dCMP deaminases. Ac- role in chloroplast development as well as chlorophyll cording to reports in animal, T4 phage and yeast, dCMP de- biosynthesis. aminases catalyze the deamination of dCMP to form dUMP. According to the sequence alignment, ST2 belongs to the dUMP is the precursor for dTMP that is subsequently phos- cytidine deaminase-like family with 7 and 16 members in rice phorylated to thymidine triphosphate for DNA synthesis and and Arabidopsis, respectively. Only two members of the repair. The exogenous application of dUMP could recover the family, AtCDA1 (cytidine deaminase 1) (Faivre-Nitschke st2 phenotype, suggesting that ST2 most likely functions as a et al., 1999; Vincenzetti et al., 1999; Kafer and Thornburg, dCMP deaminase. Therefore, the mutation in the ST2 gene re- 2000) and AtTadA (Karcher and Bock, 2009) have been re- duces the dUMP pool in plant, and further disturbs the balance ported in Arabidopsis. AtCDA1 can utilize both cytidine and of subsequent dTTP formation. It is intriguing how a 20-deoxycytidineas substrates but unable to deaminate cyto- mitochondria-located deaminase affects chloroplast develop- sine, CMP or dCMP. AtTadA encodes a chloroplast tRNA ment. As previously reported, in the rice virescent3 and strip1 adenosine deaminase which triggers A-to-I editing in the mutants, plastid DNA synthesis for chloroplast biogenesis is anticodon of the plastid tRNA-Arg (ACG), presumably regu- supposed to be relatively less critical for plant survival and can lating chloroplast translational efficiency. sacrifice under insufficient dNTP levels (Yoo et al., 2009). This cytidine deaminase-like family can be divided into four Similarly, we speculate that chloroplast development is pref- subfamilies performing functions in different bioprocesses. One erentially arrested to allow both nuclear and mitochondrial possibility is that ST2 functions as a cytidine deaminase for genome replication. However, the mechanism how the dNTP RNA editing. However, no editing site was found disappeared in pool prioritizes nuclear and possible mitochondrial DNA st2 compared with the wild type. We then found that ST2 shares (mtDNA) synthesis keeps elusive. J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548 545

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 development. Further investigation is necessary to dissect the reduced and the dCTP increased 30 times as high as the normal mechanism of nucleotide distribution between chloroplasts, level when dCMP deaminase was inactive. Such imbalance led mitochondria and the nucleus. to the reduced ﬁdelity of DNA replication with increased nucleotide mismatch (Sargent and Mathews, 1987). Corre- MATERIALS AND METHODS spondingly, we did detect up-regulated DNA repair genes from microarray data of st2 compared with LTP, including endonu- Plant materials and grow condition clease, meiotic recombination protein DMC1, DNA helicase RecQ, and RAD51 homolog RAD51B (data not shown). The The rice st2 mutant was isolated from g-ray-induced muta- study of the maize ncs (nonchromosomal strip) mutant indicated tions of an indica cultivar (LTP). A japonica variety, Zhong- that mitochondria are necessary for the chloroplast biogenesis, hua11 (ZH11), was crossed with st2 to construct a F2 and the mtDNA mutations can result in strip phenotype (Jiao population for genetic study and preliminary mapping. Addi- et al., 2005). We also suggest another possibility that the tional 1723 recessive individuals from the F2 populations imbalance dNTP in the st2 mutants may also increase the mu- derived from the cross between st2 and 9311 (indica) were tation rate of mtDNA, thereby disturbing the chloroplast used for ﬁne mapping. Plants were cultivated in the 546 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

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 4C, 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

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). Fig. 7. Recovery of st2 plants by dUMP feeding. Subsequently, we placed ST2 in a 27-kb region between the A: The LTP seedlings treated with dUMP under concentration of 0, 10 5,10 4 markers S1614 and S9131. Genomic DNA fragments of this and 103 mol/L (from left to right). Two seedlings were imaged for each region were amplified from st2 and wild-type (LTP) plants, concentration. Scale bar ¼ 2 cm. Note that 10 3 mol/L dUMP slightly inhibited seedling growth. B: The st2 mutant plants were fed with dUMP sequenced, and compared using MegAlign (DNASTAR). under the same concentrations as in (A). Note that the leaf phenotype was recovered by 103 mol/L dUMP. Scale bar ¼ 2 cm. Complementation test and transgenic expression experimental field during natural growth seasons. For seed- The rice Nipponbare ( japonica) BAC P0403C05 bearing ST2 lings, seeds were germinated in the dark for 2 days and then was digested to isolate a 9-kb genomic DNA fragment with the transferred to liquid medium in a growth chamber under full ST2 region, which was inserted into the binary vector growth conditions with 12-h day, 28C, 80% relative humidity pCAMBIA1301 to generate the plasmid pST2-ST2. The (RH) followed by 12-h night, 26C, 60% RH. plasmid was introduced into the st2 mutants by Agro- bacterium-mediated transformation (Hiei et al., 1994). More Measurement of chlorophyll a and b than 10 independent transgenic lines were produced that could successfully complement the mutant phenotypes. The contents of chlorophyll a and b (Chl a and Chl b) were measured according to the previously method with some Subcellular localization assay modifications (Arnon, 1949). The fresh rice leaves were cut into small pieces with scissors and soaked in the extraction ST2-YFP, DST2-YFP and ST2-GFP fusions were obtained by buffer (95% ethanol:acetone:water ¼ 5:4:1), and incubated at in-frame fusing the cDNAs with YFP or GFP, which were 4C in the dark for 18 h with periodically inversion. The amplified using the specific primers ST2-YF/ST2-YR, DST2- J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548 547

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Plant cDNA amplification were performed using gene-specific Cell 12, 2425e2440. primers (Table S3), under the following conditions: 5 min at Delannoy, E., Le Ret, M., Faivre-Nitschke, E., Estavillo, G.M., Bergdoll, M., 95 C, 28 cycles (30 s at 95 C, 30 s at specific Tm, 1 min/kb at Taylor, N.L., Pogson, B.J., Small, I., Imbault, P., Gualberto, J.M., 2009. 72C), followed by 10 min at 72C. Arabidopsis tRNA adenosine deaminase arginine edits the wobble nucleotide of chloroplast tRNAArg(ACG) and is essential for efficient chloroplast translation. Plant Cell 21, 2058e2071. Analysis of RNA editing Faivre-Nitschke, S.E., Grienenberger, J.M., Gualberto, J.M., 1999. A prokaryotic-type cytidine deaminase from Arabidopsis thaliana. Eur. J. Total RNAs extracted from seedling leaves were treated with Biochem. 263, 896e903. RQ1 DNase (Promega). Then cDNAs were synthesized from Hiei, Y., Ohta, S., Komari, T., Kumashiro, T., 1994. Efficient transformation of 3 mg total RNAs using hexanucleotide oligomers primer and rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6, 271e282. MMLV reverse transcriptase. These cDNAs were used as Hoch, B., Maier, R.M., Appel, K., Igloi, G.L., Kossel, H., 1991. Editing of a templates for PCR amplification of mitochondrial genes. The chloroplast mRNA by creation of an initiation codon. Nature 353, 178e180. primers used for scanning mitochondrial RNA editing sites Jiao, S., Thornsberry, J.M., Elthon, T.E., Newton, K.J., 2005. Biochemical and were the same as those reported by Kim et al. (2009).TheRT- molecular characterization of photosystem I deficiency in the NCS6 e PCR products were directly sequenced and manually mitochondrial mutant of maize. Plant Mol. Biol. 57, 303 313. Jung, K.-H., Hur, J., Ryu, C.-H., Choi, Y., Chung, Y.-Y., Miyao, A., compared between the wild type and the mutant. Hirochika, H., An, G., 2003. Characterization of a rice chlorophyll-deficient mutant using the T-DNA gene-trap system. Plant Cell Physiol. 44, 463e472. ACKNOWLEDGEMENTS Kafer, C., Thornburg, R., 2000. Arabidopsis thaliana cytidine deaminase 1 shows more similarity to prokaryotic enzymes than to eukaryotic enzymes. e We thank Jiqin Li and Xiaoshu Gao for technical assistance. J. Plant Biol. 43, 162 170. Karcher, D., Bock, R., 2009. Identification of the chloroplast adenosine-to- We also acknowledge James Whelan (University of Western inosine tRNA editing enzyme. RNA 15, 1251e1257. Australia) for providing AOX-RFP plasmid. This work was Kim, S.R., Yang, J.I., Moon, S., Ryu, C.H., An, K., Kim, K.M., Yim, J., supported by the grant from the Ministry of Science and An, G., 2009. Rice OGR1 encodes a pentatricopeptide repeat-DYW protein Technology of China (No. 2012AA10A302-2). 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 de- termines the expression timing of plastid genes for transcription/translation apparatus during early leaf development in rice. Plant J. 12, 1241e1250. SUPPLEMENTARY DATA 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 Fig. S1. Sequences representing the disturbed splicing shown system for early chloroplast development under cold stress conditions. e in Fig. 3D (III and IV). Plant J. 68, 1039 1050. Li, W., Zhong, S., Li, G., Li, Q., Mao, B., Deng, Y., Zhang, H., Zeng, L., Table S1. PCR-based markers linked to St2 locus. Song, F., He, Z., 2011. Rice RING protein OsBBI1 with E3 ligase activity Table S2. Primers for RT-PCR. confers broad-spectrum resistance against Magnaporthe oryzae by modi- Table S3. Primers for plasmid construction. fying the cell wall defence. Cell Res. 21, 835e848. 548 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

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