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JGG

ORIGINAL RESEARCH

STRIPE2 Encodes a Putative dCMP Deaminase that Playsan Important Role in Chloroplast Development in Rice

Jing Xu a, Yiwen Deng a, Qun Li a, Xudong Zhu b,*, Zuhua He a,*

aNational 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, ChinabChina 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 andchlorophyll 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 mutantwas controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-basedcloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint ofexon 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 inmitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes theformation 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 photosyn-thesis and essential for the production of hormones and me-tabolites (Pogson and Albrecht, 2011). About 3000 proteins inthe chloroplast participate in transition from proplastids tomature chloroplasts, and this process is coordinated by bothnuclear and plastid genome involved in synthesis of chloro-plast DNA, the plastidic transcription/translation apparatusand the photosynthetic system (Sakamoto et al., 2008).Numerous chlorophyll-deficient or abnormal chloroplast mu-tants 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: ricezxd@126.com (X. Zhu); zhhe@sibs.ac.cn (Z. He).

http://dx.doi.org/10.1016/j.jgg.2014.05.008

1673-8527/Copyright� 2014, Institute of Genetics and Developmental Biology, Chin

Limited and Science Press. All rights reserved.

materials to investigate regulation mechanisms of chlorophyllbiosynthesis and chloroplast development in rice.

Screening for chloroplast development mutants has identi-fied most steps in these biological processes, such as yellow-green leaf1 ( ygl1) and faded green leaf ( fgl ), which resultfrom lesions of chlorophyll synthase that catalyzes esterifica-tion of chlorophyllide in the last step of chlorophyll biosyn-thesis (Wu et al., 2007) and NADPH:protochlorophyllideoxidoreductase that catalyzes the photoreduction of proto-chlorophyllide (pchlide) to chlorophyllide (chlide) (Sakurabaet al., 2013). Both mutations led to reduced contents ofchlorophyll and undeveloped chloroplasts. The magnesiumchelatase, catalyzing the chelation of Mg2þ into proto IX toproduce Mg-Proto IX, comprises three subunits includingChlH, ChlD and ChlI (Jung et al., 2003; Zhang et al., 2006).Mutations of the chlorina-1 and chlorina-9 led to deficiency in

ese Academy of Sciences, and Genetics Society of China. Published by Elsevier

540 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

chlorophyll content and incomplete development of chloro-plasts, due to the disruption of ChlD and ChlI subunits,respectively. Moreover, an insert mutation in ygl2 ( yellow-green leaf 2), encoding heme oxygenase (HO) that catalyzesthe degradation of heme to synthesize phytochrome precursor,results in significantly reduced content of chlorophyll andtetrapyrrole intermediates (Chen et al., 2013).

As a semiautonomous organelle, chloroplast genome onlyencodes about 100 genes (Delannoy et al., 2009). Most of theproteins essential for chloroplast development and function arenuclear-encoded (Chen et al., 2010). It is well documented thatthe coordination of nuclear and plastid genes is crucial forchloroplast biogenesis (Mullet, 1988). Under illumination,one-third of the nuclear genes change expression, includingmany transcription factors such as PIFs (phytochrome inter-acting factors). Either pif1 or pif3 mutant showed delayeddevelopment of chloroplast (Moon et al., 2008; Stephensonet al., 2009). Gene transcription, RNA maturation, and pro-tein translation in the chloroplasts also have impact on chlo-roplast biogenesis and development. There are two types ofplastid RNA polymerases: plastid-encoded RNA polymerase(PEP) and nucleus-encoded RNA polymerase (NEP) respon-sible for the transcription of the plastome. In Arabidopsis,mutations in SIG6 (sigma factor 6) cause a weakly virescentphenotype and transcripts of several PEP-dependent plastidgenes 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 recoveredafter two weeks of growth (Swiatecka-Hagenbruch et al.,2008). PPR proteins (pentatricopeptide repeat proteins),characterized by tandem arrays of a 35 amino acid motif, havebeen demonstrated to be critical for RNA processing, splicing,editing, stability, maturation and translation in the chloroplast(Takenaka et al., 2013). YSA (young seedling albino) encodesa PPR protein and the disruption of its function causes aseedling stage-specific albino phenotype in rice. The mutantplants can recover and develop normal green leaves after thefour-leaf stage. Interestingly, the ysa mutant has been used as amarker for efficient identification and elimination of falsehybrids in commercial hybrid rice production (Su et al., 2012).The Arabidopsis mutant sel1 (seedling lethal 1) exhibited apigment-defective and seedling-lethal phenotype with a dis-rupted PPR gene. In the sel1 plants, RNA editing of acetyl-CoA carboxylase b subunit transcripts was disrupted (Pyoet al., 2013). The virescent rice mutants v1, v2 and v3 aretemperature-conditional, which produce chlorotic leaves at arestrictive temperature (�20�C) but develop nearly greenleaves at a permissive temperature (�30�C). V1 encodes achloroplast-localized protein NUS1 which is involved in theregulation of chloroplast rRNA metabolism during earlychloroplast 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 beenproposed that V2 functions at an early stage of chloroplastdifferentiation particularly in the chloroplast translation ma-chinery during early leaf development (Sugimoto et al., 2004,2007). V3 and STRIPE1 encode the large and small subunits of

ribonucleotide reductase (RNR), respectively, which regulatesthe rate of deoxyribonucleotide production for DNA synthesisand repair. Yoo et al. (2009) speculated that, upon insufficientactivity of RNR, plastid DNA synthesis is preferentiallyarrested to allow nuclear genome replication in developingleaves to sustain the continuous plant growth.

In this study, we identified a stripe variegated mutantnamed st2 (stripe2). The st2 plants develop chlorotic leavescaused by low content of chlorophylls. Examination of theultrastructure showed that the thylakoid membranes areextremely disturbed in the mutants. Map-based cloning andgenetic complementation indicated that ST2 encodes a cyti-dine deaminase-like protein, which most likely functions asthe dCMP deaminase.

RESULTS

Phenotype characterization of stripe2 mutant

The rice (Oryza sativa L.) st2 mutant was isolated from g-ray-induced mutations of an indica cultivar (Longtepu, LTP). Themutant leaves were virescent with stripes (Fig. 1A and E). Todetermine its effect on chlorophyll formation, the leaves of3-week seedling were analyzed for chlorophyll contents.Compared with the wild type, the chlorophyll contents in st2were reduced by 30% (Fig. 1B), and autofluorescence in thest2 leaves also decreased (Fig. 1C and D). The ultrastructure ofthe wild-type chloroplasts was crescent-shaped and containedwell-formed thylakoid structure including stroma thylakoidsand grana thylakoids (Fig. 2A). In contrast, the mutant chlo-roplasts were small and thylakoid membrane was disturbed,some with less thylakoid structure (Fig. 2B and C). Somechloroplasts formed rudimentary thylakoids consisting of onlygrana lamellae without formation of stroma lamellae(Fig. 2D), while some chloroplasts displayed well-developedlamellar structures equipped with normally stacked grana butno starch grains in the st2 leaves (Fig. 2E and F). These resultsindicated the st2 phenotype is caused by the underdevelop-ment of the chloroplast.

Fine mapping of the ST2 Gene

For genetic analysis of the st2 mutant, we firstly crossed st2mutant with a japonica cultivar Zhonghua11 (ZH11), and theF1 generation exhibited normal green leaves. Among the 762F2 individuals, 183 were virescent and 579 were green. Thesegregation ratio of the F2 population accorded with 3:1(c2 ¼ 0.39 < c2

0.05 ¼ 3.81; P > 0.05), suggesting that the st2phenotype was controlled by a single recessive gene. Geneticmapping was performed using the same population. Thelocus was primarily mapped to a 6.5-CM region flanking bythe makers of RM8139 and C7962 on the long arm ofchromosome 1. To further narrow down the region, newmarkers including insertion/deletion (InDel) markers andCAPS (cleaved amplified polymorphic sequence) weredesigned; however, little polymorphism could be found be-tween st2 and ZH11. Therefore, we developed a larger F2

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.

541J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

population from a cross of st2 and 9311 (indica) for furthermapping. By genotyping 1723 homologous st2 individuals,the ST2 gene was finally confined to a 27-kb physical intervalbetween S1614 and S9131 on BAC P0403C05. According tothe rice genome annotation database (http://rice.plantbiology.

Fig. 2. Ultrastructure of chloroplasts in mesophyll cells of LTP (WT) and st2 mut

The chloroplasts of LTP have well-ordered thylakoids and stacked membranes (A),

thylakoid structure (B and C) or form thylakoid with only grana lamellae (D), and so

bars ¼ 1 mm. Cp, chloroplast; M, mitochondrion; SG, starch grain.

msu.edu/), seven genes were predicted in the region(Fig. 3A). The genomic DNA of the candidate genes wassequenced and compared with LTP. A 5-bp deletion(TAGGT) was detected on the ORF of Os01g0765000 in st2mutant (Fig. 3B). This deletion leads to the abortion of Bln I

ant.

while st2 shows various defects in chloroplasts, among which some form little

me could form normal thylakoid though without starch grains (E and F). Scale

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.

542 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

cutting site, and a primer (C1377) was designed to detect thedeletion. To confirm that the deletion was exclusive for st2mutant, several other varieties were also genotyped withC1377, and only st2 was resistant to the Bln I digestion(Fig. 3C). There were nine exons in Os01g0765000, and thedeletion located on the conjunction of the third exon and

intron, which could affect mRNA splicing (Fig. 3D). RT-PCRshowed that there are two larger bands in st2 instead of oneband in LTP (Fig. 3E). Sequence alignment of the excep-tional bands confirmed that the additional nucleotides camefrom the third intron (Fig. S1). The additional intronsequence led to the premature termination of the predicted

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.

543J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

protein, therefore disturbing its normal function. The iden-tification of the ST2 gene was subsequently confirmed by thegenetic complementary experiments. More than 30 inde-pendent transgene-positive plants of T0 generation were ob-tained and most of them showed normal leaf color (Fig. 4).Moreover, 12 T1 lines were planted, which showed segre-gation of wild type and st2 phenotypes. Together,Os01g0765000 is the underlying gene responsible for the st2phenotype.

Expression pattern and subcellular localization of ST2

To understand the roles of ST2 in plant growth, we detectedthe expression pattern of ST2 in different tissues includingroot, seedling, leaf, leaf sheath, stem, and panicle. RT-PCRresult showed that the ST2 is expressed ubiquitously, sug-gesting that ST2 may function in most tissues (Fig. 5A). It waspredicted that ST2 has a mitochondrial or chloroplast transitpeptide comprising 40 amino acids at N-terminal (http://www.cbs.dtu.dk/services/TargetP/). To determine the subcellularlocalization of ST2, fusion proteins of ST2-YFP (yellowfluorescent protein) and truncated ST2 without the transitpeptide (DST2-YFP) were transiently expressed in onionepidermal cells and rice protoplasts driven by the 35S pro-moter. The ST2-YFP showed a punctate pattern but did notcoincide with the red chlorophyll auto-fluorescence. When thetransit peptide is excluded (DST2-YFP), the punctate locationpattern of ST2-YFP disappeared, confirming that the transitpeptide 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),

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

ST2 encodes a putative dCMP deaminase

According to the genome annotation and sequence similarity, wefound that ST2 belongs to the cytidine deaminase-like family,which is involved in the nucleotide metabolism. There are 7 and16 cytidine deaminase members in rice and Arabidopsis,respectively (Fig. 6A). Phylogenetic analysis showed that theST2shared 83% sequence similarity with Arabidopsis At3g48540.We examined T-DNA insertion mutants of At3g48540 availablefrom the Arabidopsis community (http://www.arabidopsis.org/),but no phenotype like st2 was observed in the null mutantFLAG_475E06, probably due to the functional redundancy of thefamily in Arabidopsis. The members of cytidine deaminase-likefamily (http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid¼cd00786&seltype¼1) can be divided into four sub-families, including riboflavin deaminase, cytidine deaminase,dCMP deaminase and nucleoside deaminase. In mammals, thecytidine deaminase such asAPOBEC-1 is responsible for C-to-Uediting of the apolipoprotein mRNA (Prohaska et al., 2014). Inhigher plants, RNA editing is a post-transcriptional process ofaltering a specific nucleotide C to U (and less frequently, from Uto C) in organelle mRNAs. Most RNA editing events are neces-sary for expressing functional proteins, as demonstrated by ex-amples of RPL2 (ribosomal protein L2) in maize (Hoch et al.,1991) and NDHD-1 (a subunit of the chloroplast NADHdehydrogenase-like complex (NDH), involved in cyclic electronflow) in Arabidopsis (Boussardon et al., 2012). However, en-zyme(s) for the C-to-U conversion has not been identified yet andcytidine deaminase is most likely a candidate. To determinewhether ST2 functions as a cytidine deaminase, we scanned themitochondrial RNA editing sites by using 48 pairs of primersfrom the study ofKim et al. (2009). Comparedwith thewild type,all the editing sites still existed, suggesting that ST2 might notfunction as a cytidine deaminase to catalyze RNA nucleotides.We then compared ST2 with the human and yeast dCMP de-aminases, and high homology was found especially in the cata-lytic site from the 80th to the 140th amino acid (Fig. 6B). SincedCMP deaminases catalyze the process from dCMP to dUMP bydeamination,we considered that the st2mutantmight be deficientin the dUMP supply and the external application of dUMP shouldrecover or attenuate the st2 phenotype. As showed in Fig. 7, theleaf color was recovered with 1 mmol/L dUMP feeding, albeitplant growth was inhibited at that concentration. Therefore, weproposed 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 chlo-roplast with inhibited chlorophyll accumulation. To recog-nized the ST2 gene functionally, we map-cloned the ST2 gene,and found that the gene encodes a putative mitochondria-located dCMP deaminase. Our study for the first time identi-fied a putative plant dCMP deaminase, and is also the first

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.

544 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

report that mitochondria-located deaminase plays a criticalrole in chloroplast development as well as chlorophyllbiosynthesis.

According to the sequence alignment, ST2 belongs to thecytidine deaminase-like family with 7 and 16 members in riceand Arabidopsis, respectively. Only two members of thefamily, AtCDA1 (cytidine deaminase 1) (Faivre-Nitschkeet al., 1999; Vincenzetti et al., 1999; Kafer and Thornburg,2000) and AtTadA (Karcher and Bock, 2009) have been re-ported in Arabidopsis. AtCDA1 can utilize both cytidine and20-deoxycytidineas substrates but unable to deaminate cyto-sine, CMP or dCMP. AtTadA encodes a chloroplast tRNAadenosine deaminase which triggers A-to-I editing in theanticodon of the plastid tRNA-Arg (ACG), presumably regu-lating chloroplast translational efficiency.

This cytidine deaminase-like family can be divided into foursubfamilies performing functions in different bioprocesses. Onepossibility is that ST2 functions as a cytidine deaminase forRNA editing. However, no editing sitewas found disappeared inst2 compared with the wild type. We then found that ST2 shares

similarity with the human and yeast dCMP deaminases. Ac-cording to reports in animal, T4 phage and yeast, dCMP de-aminases catalyze the deamination of dCMP to form dUMP.dUMP is the precursor for dTMP that is subsequently phos-phorylated to thymidine triphosphate for DNA synthesis andrepair. The exogenous application of dUMP could recover thest2 phenotype, suggesting that ST2 most likely functions as adCMP deaminase. Therefore, the mutation in the ST2 gene re-duces the dUMP pool in plant, and further disturbs the balanceof subsequent dTTP formation. It is intriguing how amitochondria-located deaminase affects chloroplast develop-ment. As previously reported, in the rice virescent3 and strip1mutants, plastid DNA synthesis for chloroplast biogenesis issupposed to be relatively less critical for plant survival and cansacrifice under insufficient dNTP levels (Yoo et al., 2009).Similarly, we speculate that chloroplast development is pref-erentially arrested to allow both nuclear and mitochondrialgenome replication. However, the mechanism how the dNTPpool prioritizes nuclear and possible mitochondrial DNA(mtDNA) synthesis keeps elusive.

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.

545J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

In T4 phage, the endogenous dTTP content was greatlyreduced and the dCTP increased 30 times as high as the normallevel when dCMP deaminase was inactive. Such imbalance ledto the reduced fidelity of DNA replication with increasednucleotide mismatch (Sargent and Mathews, 1987). Corre-spondingly, we did detect up-regulated DNA repair genes frommicroarray data of st2 compared with LTP, including endonu-clease, meiotic recombination protein DMC1, DNA helicaseRecQ, and RAD51 homolog RAD51B (data not shown). Thestudy of themaize ncs (nonchromosomal strip) mutant indicatedthat mitochondria are necessary for the chloroplast biogenesis,and the mtDNA mutations can result in strip phenotype (Jiaoet al., 2005). We also suggest another possibility that theimbalance dNTP in the st2 mutants may also increase the mu-tation rate of mtDNA, thereby disturbing the chloroplast

development. Further investigation is necessary to dissect themechanism 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 muta-tions of an indica cultivar (LTP). A japonica variety, Zhong-hua11 (ZH11), was crossed with st2 to construct a F2population for genetic study and preliminary mapping. Addi-tional 1723 recessive individuals from the F2 populationsderived from the cross between st2 and 9311 (indica) wereused for fine mapping. Plants were cultivated in the

Fig. 7. Recovery of st2 plants by dUMP feeding.

A: The LTP seedlings treated with dUMP under concentration of 0, 10�5, 10�4

and 10�3 mol/L (from left to right). Two seedlings were imaged for each

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

under the same concentrations as in (A). Note that the leaf phenotype was

recovered by 10�3 mol/L dUMP. Scale bar ¼ 2 cm.

546 J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

experimental field during natural growth seasons. For seed-lings, seeds were germinated in the dark for 2 days and thentransferred to liquid medium in a growth chamber undergrowth 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) weremeasured according to the previously method with somemodifications (Arnon, 1949). The fresh rice leaves were cutinto small pieces with scissors and soaked in the extractionbuffer (95% ethanol:acetone:water ¼ 5:4:1), and incubated at4�C in the dark for 18 h with periodically inversion. The

mixture was centrifuged, and the supernatant was used tomeasure 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 assayedwith three biological repeats.

Transmission electron microscopy (TEM) assay

Seedlings of st2 and LTP of 20-day-old were sampled forTEM observation. All the leaf samples were cut into sectionsless than 2 mm2 and infiltrated for 30 min with fixation buffer[2.5% glutaraldehyde in phosphate buffer (pH 7.2)] undervacuum. After 3 days at 4�C, samples were post-fixed in0.1 mol/L cacodylate (pH 7.4) with 2% OsO4 at 4

�C, followedby washing with 0.1 mol/L PBS, then dehydrated with agradient ethanol-acetone series and embedded in Polybed 812(Sigma, USA) resin (Li et al., 2011). Ultrathin sections wereobtained with an ultramicrotome, mounted on grids, andstained. The sections were viewed via an electron microscopyH7650 (Hitachi, Japan).

Map-based cloning of ST2

For genemapping, a total of 120 SSR (simple sequence repeats)markers distributing evenly on 12 chromosomes according todatabase information in the GRAMENE (http://www.gramene.org/bd/markers/) were used for preliminary mapping. Toconstruct a high-density linkage map for fine mapping in thetarget region, new InDel markers and CAPS markers weredeveloped according to the sequence differences between indica(9311) and japonica (Nipponbare) genomes. In case thatmarkers did not exhibit polymorphismbetween st2 and 9311,wesequenced PCR products to search for SNPs to develop newCAPS markers. The primer sequences and enzymes used arelisted in Table S1. All the primers were designed using theprogram Primer Premier 5.0 (http://www.premierbiosoft.com).Subsequently, we placed ST2 in a 27-kb region between themarkers S1614 and S9131. Genomic DNA fragments of thisregion 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 ST2was digested to isolate a 9-kb genomic DNA fragment with thefull ST2 region, which was inserted into the binary vectorpCAMBIA1301 to generate the plasmid pST2-ST2. Theplasmid was introduced into the st2 mutants by Agro-bacterium-mediated transformation (Hiei et al., 1994). Morethan 10 independent transgenic lines were produced that couldsuccessfully complement the mutant phenotypes.

Subcellular localization assay

ST2-YFP, DST2-YFP and ST2-GFP fusions were obtained byin-frame fusing the cDNAs with YFP or GFP, which wereamplified using the specific primers ST2-YF/ST2-YR, DST2-

547J. Xu et al. / Journal of Genetics and Genomics 41 (2014) 539e548

YF/ST2-YR, and ST2-YF/ST2-GR, respectively (Table S2).The resulted fusions were driven by the 35S promoter. Tran-sient expression of the fusion proteins ST2-YFP, DST2-YFPand the controls YFP in onion epidermal cells was per-formed using a helium biolistic device (Bio-Rad PDS-1000,USA) as previously described (Collings et al., 2000). Ricemesophyll protoplasts isolated from leaf sheaths of 11-day-oldseedlings were transfected with fusion constructs, or co-transfected with constructs of ST2-GFP and AOX-RFP todetermine mitochondrial location, according to previouslyreported methods (Bart et al., 2006). Onion epidermal cellsand protoplasts expressing the proteins were imaged under alaser 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-oldseedlings using TRIzol reagent (Invitrogen, USA) and treatedwith DNase RQ1 (Promega, USA). cDNAs were synthesizedfrom 3 mg total RNAs using oligo (dT) primer and SuperScriptIII reverse transcriptase according to the manufacturer’s in-structions (Invitrogen). RT-PCR analysis and full-lengthcDNA amplification were performed using gene-specificprimers (Table S3), under the following conditions: 5 min at95�C, 28 cycles (30 s at 95�C, 30 s at specific Tm, 1 min/kb at72�C), followed by 10 min at 72�C.

Analysis of RNA editing

Total RNAs extracted from seedling leaves were treated withRQ1 DNase (Promega). Then cDNAs were synthesized from3 mg total RNAs using hexanucleotide oligomers primer andMMLV reverse transcriptase. These cDNAs were used astemplates for PCR amplification of mitochondrial genes. Theprimers used for scanning mitochondrial RNA editing siteswere the same as those reported by Kim et al. (2009). The RT-PCR products were directly sequenced and manuallycompared 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 WesternAustralia) for providing AOX-RFP plasmid. This work wassupported by the grant from the Ministry of Science andTechnology of China (No. 2012AA10A302-2).

SUPPLEMENTARY DATA

Fig. S1. Sequences representing the disturbed splicing shownin 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.

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.jgg.2014.05.008.

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