the plant journal 59 rice encodes a pentatricopeptide...

Rice OGR1 encodes a pentatricopeptide repeat–DYWprotein and is essential for RNA editing in mitochondria

Sung-Ryul Kim, Jung-Il Yang, Sunok Moon, Choong-Hwan Ryu, Kyungsook An, Kyung-Me Kim, Jieun Yim and Gynheung An*

Department of Integrative Bioscience and Biotechnology, National Research Laboratory of Plant Functional Genomics and

Functional Genomic Center, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Republic of Korea

Received 11 March 2009; revised 9 April 2009; accepted 27 April 2009; published online 11 June 2009.*For correspondence (fax +82 54 279 0659; e-mail [email protected]).

SUMMARY

RNA editing is the alteration of RNA sequences via insertion, deletion and conversion of nucleotides. In

flowering plants, specific cytidine residues of RNA transcribed from organellar genomes are converted into

uridines. Approximately 35 editing sites are present in the chloroplasts of higher plants; six pentatricopeptide

repeat genes involved in RNA editing have been identified in Arabidopsis. However, although approximately

500 editing sites are found in mitochondrial RNAs of flowering plants, only one gene in Arabidopsis has been

reported to be involved in such editing. Here, we identified rice mutants that are defective in seven specific

RNA editing sites on five mitochondrial transcripts. Their various phenotypes include delayed seed

germination, retarded growth, dwarfism and sterility. Mutant seeds from heterozygous plants are opaque.

This mutation, named opaque and growth retardation 1 (ogr1), was generated by T-DNA insertion into a gene

that encodes a pentatricopeptide repeat protein containing the DYW motif. The OGR1–sGFP fusion protein is

localized to mitochondria. Ectopic expression of OGR1 in the mutant complements the altered phenotypes. We

conclude that OGR1 is essential for RNA editing in rice mitochondria and is required for normal growth and

development.

Keywords: DYW, mitochondria, Oryza sativa, pentatricopeptide repeat, RNA editing, seed.

INTRODUCTION

Mitochondria are semi-autonomously reproductive organ-

elles within eukaryotic cells that carry their own genetic

material (mtDNA) and protein-synthesizing machinery

(ribosomes, tRNAs and other components) (Taiz and Zeiger,

1998). Plant mitochondria primarily act in the respiratory

oxidation of organic acids and in transferring electrons to O2

via the electron transport chain coupled to ATP synthesis.

The plant mitochondrial proteome contains 2000–3000 gene

products; the overwhelming majority of mitochondrial pro-

teins are encoded in the nucleus and are actively transported

into mitochondria by complex protein machinery (Millar

et al., 2005). The 200–2400 kb mitochondrial genome

encodes 50–60 gene products, mainly various components

of the electron transport system, ribosomal proteins and

tRNA (Kubo and Newton, 2008).

RNA editing is the alteration of an RNA sequence from

that transcribed from the genome. Several types of editing,

such as nucleotide insertion, deletion and conversion, have

been reported in many organisms (Wedekind et al., 2003;

Shikanai, 2006). In the flowering plants examined so far,

these events occur at specific sites of organellar transcripts

(Shikanai, 2006; Takenaka et al., 2008). Plastid-type RNA

editing and plant mitochondria editing have striking simi-

larities. Both involve C fi U conversions and have com-

mon nearest-neighbor biases (5¢ pyrimidine and 3¢ purine)

and codon position preferences (Bock, 2000). The major

difference between these two organellar systems lies in their

editing frequency. Compared with fewer than 40 editing

sites on the chloroplast transcriptome (Tsudzuki et al., 2001;

Kahlau et al., 2006), RNA editing in flowering-plant mito-

chondria alters 350–500 sites (Giege and Brennicke, 1999;

Notsu et al., 2002; Handa, 2003; Mower and Palmer, 2006;

Bentolila et al., 2008; Zehrmann et al., 2008). Such editing of

plant organellar transcripts is essential for the synthesis of

functional proteins that, after editing, generally exhibit

closer sequence conservation across species. It may also

generate a translational start codon or stop codon (Shikanai,

2006; Takenaka et al., 2008). RNA editing is also required for

excision of tRNA(Phe) from precursors in plant mitochondria

(Marchfelder et al., 1996).

738 ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd

The Plant Journal (2009) 59, 738–749 doi: 10.1111/j.1365-313X.2009.03909.x

Although RNA editing was identified as occurring in

plant mitochondria in the late 1980s (Covello and Gray,

1989; Gualberto et al., 1989), it remains an enigmatic

process. Pentatricopeptide repeat (PPR) proteins have been

identified in Arabidopsis as specific nuclear factors for

plastid RNA editing. These proteins carry tandem repeats

of the PPR motif, a highly degenerate unit of 35 amino

acids. Three PPR proteins participate in the editing of

single sites (Kotera et al., 2005; Okuda et al., 2007; Zhou

et al., 2008), while another three have two or three specific

target sites in various transcripts (Chateigner-Boutin et al.,

2008; Okuda et al., 2009). MEF1, which encodes a PPR

protein, is required for editing three specific sites in

mitochondrial mRNAs of Arabidopsis (Zehrmann et al.,

2009).

PPR genes are highly numerous in plants (> 400 mem-

bers) compared with other eukaryotic organisms; many PPR

proteins are predicted to be targeted to either mitochondria

or chloroplasts (Lurin et al., 2004). Although several PPR

genes have other conserved domains, e.g. protein kinase

and Small MutS Related (SMR) domains (Saha et al., 2007),

most PPR proteins belong to two major sub-families: P and

PLS. The P sub-family has a typical PPR (P) motif and

contains no other conserved domains. The PLS sub-family

consists of the typical P motif plus longer (L) and shorter (S)

variant PPR motifs in turn. These can be further divided into

four sub-groups based on their C-terminal domain: PLS (no

additional C-terminal domain), E (with E), E+ (with E and E+)

and DYW (with E, E+ and DYW) (Lurin et al., 2004). Although

typical PPR motifs are found in the genomes of all eukary-

otes, these variant PPR motifs appear to be plant-specific.

Thus, researchers have proposed that the PLS sub-family is

involved in plant-specific post-transcriptional processes,

especially RNA editing (Lurin et al., 2004; Salone et al.,

2007). PPR proteins play crucial functions in plant organellar

gene expression associated with RNA cleavage (Hashimoto

et al., 2003; Kazama et al., 2008), RNA processing (Meierhoff

et al., 2003; Hattori et al., 2007), RNA splicing (Schmitz-

Linneweber et al., 2006; Falcon de Longevialle et al., 2007),

RNA editing (Kotera et al., 2005; Okuda et al., 2007, 2009;

Chateigner-Boutin et al., 2008; Zhou et al., 2008; Zehrmann

et al., 2009) and translational activation (Schmitz-Linnewe-

ber et al., 2005).

The minimal requirements for mammalian C fi U RNA

editing in the nucleus are the cis-acting element (known as a

mooring sequence), cytidine deaminase (APOBEC-1), and an

RNA binding protein (ACF) capable of binding to both the

mooring sequence and the cytidine deaminase (Smith et al.,

1997; Smith, 2007). A similar working model for plastid RNA

editing has been proposed by Bock (2000). The model states

that a site-specific trans-acting factor binds upstream (cis-

acting element) of the editing site, followed by recruitment

of an unknown editing enzyme (editase). Arabidopsis CRR4,

a PPR protein of the E sub-group, has been identified as a

site-recognition trans-acting factor (Okuda et al., 2006). This

protein specifically binds to the target RNA, and its C-

terminal domain (E/E+) is suspected to be an interaction

domain with an unidentified RNA editing enzyme. In plant

mitochondria, RNA editing may require other factors in

addition to the three components needed for chloroplast

RNA editing (cis-element, trans-acting factor and editing

enzyme). Non-specific RNA binding proteins, such as gluta-

mate dehydrogenase, are attached to the cis-acting element

and must be removed by RNA helicase to access the trans-

factor (Takenaka et al., 2008). Although six PPR proteins in

the chloroplasts and one PPR protein in the mitochondria

have been identified as being involved in this editing, only

CRR4 has been shown to exhibit specific RNA binding

activity. The roles of the other PPR proteins in the RNA

editing machinery have not been elucidated.

Here we report identification of the nuclear OGR1 gene,

which encodes a PPR protein of the DYW sub-group

localized to mitochondria and is essential for mitochondrial

RNA editing.

RESULTS

Mutant isolation and phenotypic analysis

We isolated three opaque-seed mutants from rice T-DNA

tagged pools. Their germination was late and their growth

was slow compared with the wild-type (WT). Although all

three exhibited similar phenotypes from seedling to adult

stages, the mutations were located at three loci on the rice

nuclear genome. We named them opaque and growth

retardation 1 (ogr1), ogr2 and ogr3.

The ogr1 seeds had opaque endosperm and were slightly

smaller in width and thickness than the WT (OGR1/OGR1)

(Figure 1a). Scanning electron microscopy revealed that

starch granules were round and loosely packed in the

mutant endosperm (Figure 1c). Mutant plants had pheno-

types of growth retardation and less tillering (Figure 1d).

They flowered late and did not produce seeds (data not

shown).

Heterozygous (OGR1/ogr1) plants harbored approxi-

mately 2.7–9.2% mutant seeds. This proportion was sig-

nificantly lower than 25%, the expected value based

on Mendelian segregation. Therefore, we stained pollen

grains with iodine to determine whether the mutation

affects male gametophyte viability. Approximately 28.2–

36.5% of the grains from anthers of heterozygous (OGR1/

ogr1) plants were weakly stained (Figure 1b). This propor-

tion of defective pollen grains was significantly higher than

in WT (OGR1/OGR1) (3.1%). We performed genetic analysis

by reciprocal crossing between heterozygous (OGR1/ogr1)

and WT (OGR1/OGR1) plants. When the former were used

as the female donor, nearly 50% of the F1 progeny were

OGR1/ogr1. However, when those heterozygous plants

were used as the male, only 23.5% were OGR1/ogr1

RNA editing in rice mitochondria 739

ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749

(Table 1). These results demonstrated that a large propor-

tion of ogr1 pollen is defective, and that female gameto-

phytes are normal.

OGR1 encodes a PPR protein containing the DYW motif

The phenotypes of the ogr1 mutant were caused by T-DNA

insertion in a short arm of chromosome 12, and four ESTs

have been reported at the insertion site. Based on this EST

information, we amplified a 1770 bp fragment of cDNA that

encodes an open reading frame of 589 amino acids. This

protein consists of six PPR-related motifs and E, E+, and

DYW motifs (Figure 2a,b). The gene contains a single exon,

as is found in many other PPR genes (Lurin et al., 2004;

O’Toole et al., 2008).

From our rice flanking sequence tag database (An et al.,

2003; Jeong et al., 2006) we isolated another T-DNA tagged

line, ogr1-2, in which T-DNA is inserted 686 bp upstream of

the first mutant ogr1-1 insertion site (Figure 2a). These ogr1-

2 mutant plants showed the same phenotype as ogr1-1. The

OGR1 transcript was not present in ogr1-1 and ogr1-2

mutants, demonstrating that both mutants are null alleles

(Figure 2c).

OGR1 is localized to the mitochondria

PPR genes are encoded in the nuclear genome and most are

predicted to be localized to mitochondria or plastids (Lurin

et al., 2004; O’Toole et al., 2008). The MitoProt, TargetP and

Predotar programs predict that OGR1 protein is mitochon-

drial (Claros and Vincens, 1996; Emanuelsson et al., 2000;

Small et al., 2004). To confirm these predictions, we

constructed a plasmid in which the OGR1 coding sequence

was ligated to green fluorescent protein (GFP) under the

control of the maize ubiquitin promoter (pUbi). This pUb-

i_OGR1::GFP construct was electroporated into protoplasts

derived from the rice Oc cell line. The transformed protop-

lasts were treated with MitoTracker Red for staining. As

expected, the GFP signal was detected in mitochondria

(Figure 3a–d). We also confirmed its location in mesophyll

protoplasts. The pUbi_OGR1::GFP construct was co-elec-

troporated with the mitochondrial reference molecule

p35S_F1-ATPase::RFP (Jin et al., 2003) to rice mesophyll

Table 1 Reciprocal crosses between ogr1-1 heterozygous (OGR1/ogr1) and wild-type (OGR1/OGR1) plants

Parent Progeny

Female ($) Male (#) OGR1/OGR1 OGR1/ogr1OGR1/ogr1 OGR1/OGR1 89 84OGR1/OGR1 OGR1/ogr1 208 64

(a)

(b)

(c)

(d)

Figure 1. Morphological abnormalities of the ogr1 mutant.

(a) Comparison of mature seeds between mutant and WT (left), and cross-

section images (right).

(b) Pollen grain staining of heterozygous anther with 1% iodine solution.

(c) SEM images at the endosperm core region of mature seeds.

(d) Phenotypes of WT (left), ogr1-1 (middle) and ogr1-2 (right) at 45 days after

germination.

(a)

(b)

(c)

Figure 2. OGR1 structure and T-DNA insertion.

(a) OGR1 (GenBank accession number FJ527826) is a single-exon gene on

chromosome 12. The protein coding region (1770 bp, 590 codons) is

indicated by a closed box and deduced motifs are shown below. The 143

amino acid N-terminal region was predicted to be a mitochondrial targeting

sequence by MitoProt analysis (Claros and Vincens, 1996). The locations of

T-DNA insertions and RT-PCR primers (F1, R1 and R’) are shown. The F1 and R’

primers are in the coding region, and the R1 primer is in the 3¢ UTR region.

(b) Alignment of six PPR-related motifs present in OGR1. Amino acids that are

conserved more than 60% are shaded in black; similar amino acids are shaded

in gray. Each PPR motif was suggested to form anti-parallel a-helices based on

their similarity to the tetratricopeptide motif (Small and Peeters, 2000).

(c) RT-PCR analysis of OGR1 gene expression in mutants (ogr1/ogr1) and

segregating WT (OGR1/OGR1).

740 Sung-Ryul Kim et al.


cells. This experiment showed that OGR1::GFP is localized to

mitochondria but not to chloroplasts (Figure 3e–h).

OGR1 is involved in RNA editing of mitochondrial

transcripts

Because PPR proteins are involved in a wide range of post-

transcriptional processes in plant organelles, we postulated

that the product of the OGR1 gene is probably involved in a

similar manner. OGR1 is a PPR protein of the DYW sub-

group (Figure 2a). Arabidopsis CRR2, another such protein,

is active in the inter-cistronic cleavage of the rps7–ndhB

transcript in plastids (Hashimoto et al., 2003). Four other PPR

proteins in the DYW sub-group – CRR22, CRR28, YS1 and

MEF1 – are essential for RNA editing in Arabidopsis orga-

nellar transcripts (Zhou et al., 2008; Okuda et al., 2009;

Zehrmann et al., 2009). OGR2 is a nuclear-encoded subunit

of mitochondrial respiratory complex I (NADH dehydroge-

nase). OGR3, which encodes a putative pyruvate decarbox-

ylase, is also indirectly associated with the mitochondrial

electron transfer chain (unpublished data). The phenotypes

of ogr1 mutants were very similar to those of ogr2 and ogr3

mutants. Therefore, we speculate that the ogr1 mutants are

defective in RNA cleavage or editing of the mRNAs of com-

plex I subunits that are transcribed from the mitochondrial

genome.

Mitochondrial respiratory complex I has more than 30

subunits (Heazlewood et al., 2003; Brandt, 2006). Among

them, nine (nad1–4, 4L, 5–7 and 9) are from the mitochon-

drial genome, and the others are encoded by the nuclear

genome. We performed RNA gel-blot analyses to examine

any change in RNA accumulation patterns of these nine nad

genes in the mutants (Figures 4 and S1). Although polycis-

(a) (b) (c) (d)

(e) (f) (g) (h)

Figure 3. Localization of OGR1::sGFP fusion proteins.

(a–d) Protoplasts were prepared from the rice Oc cell line and transfected with the pUbi_OGR1::sGFP construct. Cells were treated with MitoTracker Red to detect

mitochondria.

(e–h) Mesophyll cells were co-transfected with pUbi_OGR1::sGFP and p35S_F1-ATPase::RFP. Images were obtained by confocal scanning microscopy. Images are

GFP fusion proteins (a, e), MitoTracker Red (b), RFP (f), merged images (c, g) and bright-field (d, h).

Figure 4. RNA gel-blot analyses of mitochondrial transcripts from nine NADH

dehydrogenase genes and three other genes (ccmC, cox2 and cox3) with RNA

editing defects.

The results for five mitochondrial genes are shown here; the rest are shown in

Figure S1. In each autoradiograph, the right two lanes are from ogr1/ogr1 and

the left two are OGR1/OGR1. Putative processed transcripts are indicated by

an arrowhead. The deduced major bands are unspliced nad4 (1), ccmC-nad6

(2), orf25-cox3 (3) and orf152a-orf25-cox3 (4) transcripts.



tronic and unspliced transcripts were detected from most of

the genes, no significant difference was found between the

mutant and the segregating WT (OGR1/OGR1), which was

derived from OGR1/ogr1.

We then analyzed RNA editing. Rice has 171 reported

editing sites in its nine nad genes (Notsu et al., 2002). cDNAs

were synthesized from the mutant (ogr1/ogr1) and segre-

gant WT (OGR1/OGR1), and their sequences were deter-

mined. This analysis revealed that three C residues (C401,

C416 and C433) on nad4 and one C residue (C1457) on nad2

were not edited in the ogr1 mutant (Figure 5a,b), thereby

indicating that the OGR1 protein is involved in mitochondrial

RNA editing. To examine whether the protein also functions

in the editing of other mitochondrial RNAs, we extended our

survey to all 491 editing sites in the mitochondria (Notsu

et al., 2002). These analyses identified three additional

editing sites – C458 on ccmC (cytochrome c maturation C),

C167 on cox2 (cytochrome c oxidase subunit 2) and C572 on

cox3 (cytochrome c oxidase subunit 3) – that were not edited

in the mutant (Figure 5c–e). Sequencing the seven target

sites from the heterozygous (OGR1/ogr1) plants showed that

the sites were edited correctly, as in the WT (OGR1/OGR1).

This demonstrated that a half dosage of OGR1 is sufficient

for RNA editing (data not shown).

Interestingly, all of the seven editing sites were non-silent

(i.e. RNA editing altered the coded amino acid identity)

(Table 2). All except C433 of nad4 were found at the second

position of the codon. Except for C416 and C433 of nad4,

which were within codons for proline and leucine, respec-

tively, the codons of the other editing sites from the

pre-mRNA encoded serine. The transcript levels of ccmC,

cox2 and cox3 did not differ significantly between the ogr1

mutant and segregating WT (Figure 4). Therefore, we

concluded that OGR1 is involved in RNA editing of seven C

residues on five distinct mitochondrial transcripts. Our

observations suggest that RNA editing of plant mitochon-

(a)

(b)

(f)

(c) (d) (e)

Figure 5. Identification of unedited C residues in the ogr1 mutant.

Sequencing chromatograms were derived by direct sequencing of the RT-PCR products containing seven target sites.

(a–e) Green, black, red and blue represent A, G, T and C, respectively. Unedited sites are indicated by with arrows; normally edited sites are indicated by asterisks.

(f) Sequence alignment of cDNA starting from –40 to +10 of seven unedited C residues. Each bold upper-case T is generated by RNA editing. Nucleotides conserved

more than 60% are shaded. Numbers after C on the left are nucleotide positions from ATG start codon.



drial and chloroplast transcripts is performed by similar

machinery, in that the editing is site-specifically regulated by

PPR proteins.

RNA editing efficiency in ogr1 mutants

We estimated the editing efficiency at the C401, C416 and

C433 positions on nad4 for the ogr1-1 and ogr1-2 mutants

and their segregating WT. After synthesis of cDNAs, PCR

reactions were performed using Pfu DNA polymerase to

reduce PCR errors. The products were cloned into the

pBluescript SK vector digested with EcoRV. From each

library, we sequenced more than 50 clones, giving a total of

435 from eight libraries (Table S1). In WT plants, all three

target C residues were 100% edited. In mutants, however,

C401 was 0% edited, C416 was 4.4% edited, and C433 was

17.1% edited (Tables 2 and S1). Interestingly, all the clones

that carried an edited C416 also carried edited C433. We also

measured the editing efficiency of C1457 in nad2 (Table S1).

This site was completely edited in the WT, but only 1.8%

edited in the mutant (Table 2). Although the rate was low in

the mutant, the event was not due to a PCR error that oc-

curred randomly (not specifically C fi T) and at a much

lower frequency.

Our observations were different from those for chloro-

plast editing mutants, in which the specific target site(s)

were entirely unedited. One possibility is that the completely

unedited C401 in nad4 caused a mitochondrial ETC dysfunc-

tion that influenced the extent of RNA editing on the other

six sites. To find out whether this is true, we analyzed the

mitochondrial dysfunction mutants. In the ogr2 and ogr3

mutants, the seven sites were fully edited, indicating that the

defect in editing of the specific sites in the ogr1 mutant was

caused by the loss of OGR1.

Identification of low-efficiency editing sites

To evaluate whether the ogr1 mutation can affect other

editing sites, we selected a 350 bp region (from 118–467)

of nad4 and a 415 bp region (from 1053–1467) of nad2,

which together contain 19 known editing sites as well as

our four OGR1 target sites. Of these, 15 were fully edited

and four were nearly fully edited (98.2–99.6%). During

these analyses, we also found ten new editing sites: C124,

C156, C291, C303 and C385 in the nad4 transcript, and

C1057, C1080, C1212, C1336 and C1404 in the nad2

transcript. All were C fi U conversions at low editing

efficiency (1.1–36.2%, Table 3). Because this efficiency was

similar between WT and ogr1 mutants, we concluded that

the editing is not dependent on OGR1. Interestingly, all ten

were silent editing sites, i.e. did not change encoded

amino acids.

Complementation of mutant phenotypes and RNA editing

by ectopic expression of the wild-type OGR1 gene

To verify that the mutant phenotypes and RNA editing

defect are indeed due to a lack of functional OGR1, we

Table 2 RNA editing efficiency

Editing site

Codon (amino acid)Editing efficiency(%)

WT Mutant WT Mutant

cox2-C167 TTA (Leu) TCA (Ser) NT NTcox3-C572 TTC (Phe) TCC (Ser) NT NTccmC-C458 TTA (Leu) TCA (Ser) NT NTnad2-C1457 TTA (Leu) TCA (Ser) 100 1.8nad4-C401 TTC (Phe) TCC (Ser) 100 0nad4-C416 CTT (Leu) CCT (Pro) 100 4.4nad4-C433 TTT (Phe) CTT (Leu) 100 17.1

RNA editing efficiency was estimated as described in Table S1. NT,not tested.

Table 3 Sites with low editing efficiency discovered in 765 bp regions of nad4 and nad2

Sample

nad4 nad2

Clone number C124 C156 C291 C303 C385 Clone number C1057 C1080 C1212 C1336 C1404

OGR1-1 #1 51 1 0 5 1 1 28 8 3 3 1 4#2 54 0 0 2 1 0 29 14 1 6 0 1

ogr1-1 #1 61 1 1 7 5 1 29 11 0 4 1 2#2 56 2 1 3 1 3 26 8 0 2 0 0

OGR1-2 #1 52 0 1 4 0 0 29 9 1 3 0 7#2 51 0 0 2 0 0 30 8 0 7 1 3

ogr1-2 #1 57 1 2 5 0 2 29 13 0 2 1 2#2 53 1 0 6 2 1 29 12 0 2 0 2

Total 435 6 5 34 10 8 229 83 5 29 4 21Edited(%) 1.4 1.1 7.8 2.3 1.8 36.2 2.2 12.7 1.7 9.2Codona CTG tCC ACC ATC CTA CtA GTC TTC CTA TtC

The number of edited clones is shown for each editing site. aAn underlined C indicates a new editing site. A bold lower-case t indicates a knownediting site. We regarded a nucleotide as a new editing site when RNA editing events were observed from at least three libraries at the samenucleotide position.



introduced into ogr1-1 mutants a construct harboring the

wild-type OGR1 coding sequence under the control of

the maize ubiquitin promoter. Transgenic plants expressed

the exogenous gene at a high level and grew normally,

indicating that the dwarf mutant phenotype was rescued

(Figure 6a,b). The seven sites that were unedited in the

mutant were correctly processed in these transgenic plants

(Figure 6c). We concluded that the defect of RNA editing

observed in the ogr1 mutants is solely due to the absence

of functional OGR1, and that abnormal growth derives from

a defect in RNA editing.

DISCUSSION

ogr1 mutants show a malfunction in the mitochondrial ETC

due to defective RNA editing

Our ogr1 mutants showed pleiotropic effects with slow

growth and late flowering that resulted in dwarf and sterile

phenotypes. A large proportion of the pollen grains from

heterozygous (OGR1/ogr1) plants were abnormal. These

phenotypes were due to mutations in the nuclear-encoded

OGR1 gene, which caused a defect in RNA editing at seven

specific sites on five mitochondrial transcripts: nad2, nad4,

cox2, cox3 and ccmC. Rice mitochondria have 491 editing

sites on 34 transcripts (Notsu et al., 2002). All OGR1 target

transcripts are involved in the mitochondrial electron

transport chain (ETC) coupled to ATP generation. The nad2

and nad4 transcripts encode a subunit of respiratory com-

plex I (proton-pumping NADH dehydrogenase). Transcripts

of cox2 and cox3 encode a subunit of respiratory complex IV

(cytochrome c oxidase), and ccmC is involved in the bio-

genesis of cytochrome c, which transfers electrons from

complex III to complex IV. Growth retardation and male

sterility have previously been observed in mitochondrial

mutants that are defective in the respiratory complex, e.g.

maize NCS5 and NCS6 (deletions of the 5¢ end of cox2),

maize NCS2 (truncated nad4) and Nicotiana sylvestris CMS I

and CMS II (mtDNA deletions in nad7) (Lauer et al., 1990;

Newton et al., 1990; Marienfeld and Newton, 1994; Gutierres

et al., 1997). Mutants that are defective in nuclear genes

associated with the mitochondrial ETC have similar pheno-

types. The Arabidopsis mutant fro1, which is deficient in a

subunit of complex I, shows growth retardation and dwarf-

ism (Lee et al., 2002). Our ogr2 mutant, which is also defi-

cient in a subunit of complex I, had a phenotype very similar

to that of ogr1 (unpublished data). Moreover, OTP43, a

member of the Arabidopsis PPR family, is involved in trans-

splicing of nad1 intron 1. Its T-DNA insertion mutation

exhibits delayed development and flowering (Falcon de

Longevialle et al., 2007). Our ogr1 mutant phenotypes

coincided with those of these mitochondrial ETC-defective

mutants.

Generally, RNA editing in protein coding sequences in

plant organellar transcripts is required to generate amino

acids that are conserved with respect to the protein homo-

logs in other systems (Gualberto et al., 1989; Maier et al.,

1992; Yura and Go, 2008). However, editing is not always

essential for the synthesis of functional proteins (Freyer

et al., 1997; Fiebig et al., 2004). For example, the Arabidopsis

mef1 mutant, which is defective in mitochondrial RNA

editing of rps4, shows a normal growth phenotype under

standard conditions (Zehrmann et al., 2009). We compared

cDNA sequences containing seven editing sites from three

dicots (Arabidopsis thaliana, Brassica napus and Beta vul-

garis) and two monocots (Triticum aestivum and Oryza

sativa). All these organisms have T nucleotides at seven

(a)

(b)

(c)

Figure 6. Complementation of ogr1-1.

(a) Seedling phenotypes of WT (left), the ogr1-1 mutant (right), and a

transgenic plant expressing a wild-type OGR1 gene in the ogr1-1 background

(middle).

(b) RT-PCR analysis of the OGR1 transcript. The positions of primers are

shown in Figure 2a.

(c) Sequencing chromatograms from direct sequencing of RT-PCR products.

Seven target editing sites were fully edited in transgenic plants (ogr1-

1 + OGR1). Only the results for two target sites (C1457 of nad2 and C167 of

cox2) are presented here.



positions on the cDNA sequences. These nucleotides either

originate from genomic DNA or are generated by RNA

editing (Figure S2). As a result, the five organisms encode

the same amino acid residues at those positions, indicating

that editing the seven target sites of OGR1 is required for

production of the conserved amino acids and for normal

growth.

Molecular function of the OGR1 protein

OGR1 is a member of the DYW sub-group of PPR proteins and

is localized to mitochondria (Figures 2a and 3). RNA editing

at seven sites on five mitochondrial transcripts was nearly

abolished in our ogr1 mutants (Figure 5). This defect was

fully recovered by introducing exogenous OGR1 (Figure 6).

RNA accumulation patterns for 12 mitochondrial genes in-

volved in the respiratory complex were similar between the

ogr1 mutant and WT RNAs (Figures 4 and S1). Therefore, we

conclude that OGR1 is active in RNA editing rather than RNA

cleavage in rice mitochondria. Although seven Arabidopsis

PPR genes reportedly play roles in RNA editing, six of them

are involved in editing chloroplast transcripts; only one has

been identified for mitochondrial RNA editing. Our mutant

will be a valuable resource for further understanding the RNA

editing mechanism in plant mitochondria.

A cis-element is required for recognition of an editing site

by a trans-acting factor in both plastids and mitochondria.

In chloroplasts, a cis-element has been analyzed using

plastid transformation. Sequences that are shorter than 22

nucleotides upstream of the editing site or shorter than five 5

nucleotides downstream are sufficient for editing (Bock

et al., 1996, 1997; Chaudhuri and Maliga, 1996). Likewise, the

distance between the editing site and the upstream cis-

acting element plays a critical role (Hermann and Bock,

1999). Similarly, in mitochondria, analysis of an in organello

RNA editing system from purified wheat mitochondria has

indicated that both the 16-nucleotide upstream and 6-

nucleotide downstream regions are essential for editing

cox2 mRNA (Farre et al., 2001). Takenaka et al. (2004) have

reported that efficient editing of pea atp9 transcript requires

a longer sequence than for cox2. Two functional regions are

located in the 5¢ upstream sequence of the C20 editing site of

atp9: one from )40 to )35 that is required for efficient

editing, and a second, from )15 to )5, that is essential for the

reaction with an editing enzyme.

Some chloroplast RNA editing sites have sequence sim-

ilarity between their upstream sequences, and may recruit

the same trans-acting factor (Karcher et al., 2008; Kobayashi

et al., 2008). This phenomenon can be explained as recog-

nition of several targets by a common trans-acting factor

(Choury and Araya, 2006). We therefore speculated that a

consensus cis-acting element exists near the OGR1 editing

sites (region from )40 to +10). However, we did not find any

consensus sequences (Figure 5f). Similar results have been

obtained for the editing sites of clb19, crr22 and mef1

(Chateigner-Boutin et al., 2008; Okuda et al., 2009; Zehr-

mann et al., 2009).

Most PPR proteins function in post-transcriptional pro-

cesses by specifically binding to RNA (Delannoy et al., 2007).

For example, CRR4 and Rf1 show such activity in vitro

without the aid of other factors (Okuda et al., 2006; Kazama

et al., 2008). However, preliminary analyses using in vitro

binding and immunoprecipitation of protein–RNA com-

plexes failed to demonstrate that CLB19 binds specifically

to its target RNAs (Chateigner-Boutin et al., 2008). Because

no obvious conserved sequences surround the editing sites,

OGR1 may require another trans-factor to achieve target

specificity. Alternatively, the RNA-binding sites may contain

a specific secondary structure that is formed during tran-

script maturation.

The protein structures of the PPR motif and the tetratric-

opeptide motif (Small and Peeters, 2000) are similar. The

latter consists of a pair of anti-parallel a-helices; tandem

arrays of tetratricopeptide motifs are expected to form a

superhelix enclosing a groove, which is likely to be a protein-

binding site. Some PPR proteins have been identified from

protein complexes (Williams and Barkan, 2003; Uyttewaal

et al., 2008), including HCF152, which forms a homodimer

(Nakamura et al., 2003). Therefore, it is possible that the PPR

motif of OGR1 interacts with other proteins for site-specific

binding.

The DYW domain has been proposed to possess editing

activity based on evolutionary considerations and sequence

similarity with cytidine deaminase (Salone et al., 2007).

Recently, that hypothesis was tested via enzymatic assay

using the recombinant DYW protein At2g02980 (Nakamura

and Sugita, 2008). The recombinant domain possesses

novel endoribonuclease activity instead of editing activity.

However, a target RNA of At2g02980 has not been identified,

and the T-DNA insertional mutant does not show aberrant

transcripts. Arabidopsis CRR22 and CRR28 are essential for

RNA editing of chloroplast transcripts. Interestingly, trun-

cated proteins that lack the DYW motifs can completely

restore RNA editing in vivo (Okuda et al., 2009). In contrast,

the DYW motif of CRR2 is essential for RNA cleavage in vivo.

Four amino acid residues in the DYW motif differ between

RNA editing factors and RNA cleavage factors (Okuda et al.,

2009). However, when we aligned the DYW motif of two

mitochondrial editing factors (OGR1 and MEF1) with the

chloroplast proteins, we did not find significant differences

between the plastid and mitochondrial editing factors. We

also did not find any major differences between RNA editing

factors and an RNA cleavage factor (CRR2), in contrast to the

results obtained by Okuda et al. (2009) (Figure S3).

OGR1 is involved in multiple editing sites on mitochondrial

transcripts

All plant organellar editing factors belong to the E, E+ and

DYW sub-groups of PPR proteins. Three of six chloroplast



RNA editing factors participate in the editing of single sites

(Kotera et al., 2005; Okuda et al., 2007; Zhou et al., 2008),

while the others have two or three specific target sites in

various transcripts (Chateigner-Boutin et al., 2008; Okuda

et al., 2009). The mitochondrial editing factor MEF1 has

three target sites in three mitochondrial transcripts (Zehr-

mann et al., 2009). The OGR1 protein is responsible for

seven specific editing sites on five distinct mitochondrial

transcripts. The number of target sites is greater than pre-

viously reported for chloroplast editing. Higher plants have a

large number of PPR genes, including 450 members in

Arabidopsis and 477 in rice. Approximately half belong to

the P sub-family; the others belong to the PLS sub-family

(O’Toole et al., 2008). Computational analysis has predicted

that 19 and 54% of the PPR proteins are targeted to plastids

and mitochondria, respectively (Lurin et al., 2004). Whereas

chloroplasts contain approximately 35 editing sites in higher

plants, rice mitochondria have 491 editing sites (Notsu et al.,

2002). Because PPR proteins are involved not only in editing

but in other post-transcriptional roles, some should

have multiple target sites in the mitochondria, as found

for OGR1.

Although the C401 site of nad4 was entirely unedited in

our mutant, other target sites (C416 and C433 of nad4, and

C1457 of nad2) were not completely edited (Tables 2 and

S1). One possible explanation is that some editing sites

recruit one or more trans-factors that have various RNA

binding activities. For example, editing of the C401 site of

nad4 is completely dependent on OGR1, which has no close

homologs in the rice genome. However, the other sites are

edited by an additional factor with low binding activity.

Another possibility is that efficient editing of some sites

might require prior editing of the cis-element that forms the

target for the editing factor. For example, editing of C416 of

nad4 might require prior editing of C401 of nad4, which lies

within the region that is likely to form the binding site for the

C416 editing factor.

Sites with very low editing efficiency in mitochondria

By analysis of the 765 bp region of nad RNAs, we discovered

ten C fi U editing sites. This suggests that numerous sites

are edited at low efficiency in mitochondrial RNAs. However,

unlike the major sites, this type of editing does not alter the

coding of amino acids. In plant organellar RNA editing,

some sites show variable efficiency that depends on tissue

type, developmental stage or ecotype (Grosskopf and Mul-

ligan, 1996; Peeters and Hanson, 2002; Zehrmann et al.,

2008). Therefore, it is not unexpected that our examination

of ten new sites from four tissues (leaf, root, anther and

callus) revealed no significant differences. We compared

cDNA sequences containing the ten sites among five higher

plants, and observed that six were either edited or contained

genomic T (Figure S4). For example, the 1057 nucleotide of

nad2, which was edited at low efficiency in rice, is fully

edited from C fi U in Triticum aestivum, and there is a T

residue at this position in the genomic sequence of Arabid-

opsis thaliana, Brassica napus and Beta vulgaris. These data

support the authenticity of the new editing sites found in our

study.

Another interesting feature is that two adjacent C

residues in one codon may be edited with various

efficiencies. For example, C1403 of nad2 was 99.6% edited,

compared with 9.2% for C1404 of nad2 (Table 3). Similar

results were observed for C1057/C1058 of nad2 and C154/

C156 of nad4. This indicates that RNA editing occurs at a

specific nucleotide, and each site requires its own editing

machinery.

EXPERIMENTAL PROCEDURES

Plant materials and growing conditions

Two rice mutants – ogr1-1 (PFG_3A-10853, ‘Dongjin’ background)and ogr1-2 (PFG_1D-02332, ‘Hwayoung’ background) – were iden-tified from T-DNA-tagging lines generated from Oryza sativa var.japonica cv. Dongjin or Hwayoung (Jeon et al., 2000; Jeong et al.,2002; Ryu et al., 2004). Seeds were germinated on half-strengthMurashige and Skoog medium. Ten-day-old plants were trans-planted to soil and grown in the greenhouse.

Pollen grain staining and scanning electron microscope

analysis

Pollen grains from dehiscing anthers were stained with 1% iodinesolution and observed under an optical microscope. Fully driedbrown grains were bisected with a razor blade, mounted on SEMstubs, and coated with gold. The specimens were observed under ascanning electron microscope (LEO, 1450 vp; LEO ElectronMicroscopy Inc., Carl Zeiss, http://www.zeiss.com/).

RT-PCR analysis

Seedlings were grown on Murashige and Skoog medium undercontinuous light at 27�C. Total RNA was extracted from leaf tissueusing RNAiso (Takara, http://www.takara-bio.com/), and first-strandcDNA was synthesized using MMLV reverse transcriptase (Pro-mega, http://www.promega.com/) and oligo(dT)15 primer. RT-PCRwas performed using the following primers: F1 (5¢-CGTGA-TACCATGCGAAGCAA-3¢), R1 (5¢-GAAGTGATATGCATGGTTCAAG-3¢) and R’ (5¢-TTACCAGTAATCCCTGCAGG-3¢). Actin1 was used fornormalization of the cDNA quantity. PCR reactions included an ini-tial 5 min of denaturation at 95�C, followed by 95�C for 30 sec, 56�Cfor 40 sec and 72�C for 45 sec (33 cycles for OGR1, 25 cycles forActin1).

RNA gel-blot analyses

Total RNAs were extracted from calli prepared from the ogr1mutant or segregant WT (OGR1/OGR1); 15 lg samples werefractionated in denaturing formaldehyde gels. RNA size markerswere obtained from Promega. After transfer to Hybond N+ nylonmembranes (Amersham Biosciences, http://www5.amershambio-sciences.com/), hybridization was performed with 32P-radiolabeledprobes and a Rediprime II random prime DNA labeling system(Amersham Biosciences). The DNA fragments used as probeswere obtained by PCR using gene-specific primers (see sequencesin Table S2). Procedures were as described previously (Kanget al., 1998).



Analysis of RNA editing

Total RNAs extracted from seedling leaves and calli were treatedwith RQ1 DNase (Promega). Then cDNAs were synthesized usingMMLV reverse transcriptase, hexanucleotide oligomers and totalRNA. These cDNAs were used as templates for PCR amplication ofmitochondrial genes. Information for sequences and editing siteswas obtained from the RNA Editing Database (REDIdb; http://bio-logia.unical.it/py_script/search.html) (Picardi et al., 2007). Primerswere designed to cover all 491 mitochondrial editing sites (Ta-ble S3), and PCR was performed using Taq polymerase (SolGent,http://www.solgent.co.kr/), with an initial 5 min denaturation at95�C, followed by 35 cycles of 94�C for 30 sec 55�C for 40 sec and72�C for 50 sec, with a final 7 min at 72�C. The RT-PCR productswere directly sequenced using the Applied Biosystems Big DyeTerminator 3.0 method and processed on an Applied Biosystems3730 DNA sequencer (http://www.appliedbiosystems.com/).Sequencing chromatograms were manually compared betweenwild-type and mutant.

Analysis of RNA editing efficiency and survey of

new editing sites

PCR was performed with Pfu DNA polymerase (SolGent), using twoprimer sets (nad2-F2 + nad2-newR2 for nad2 and nad4-F1 + nad4-R1 for nad4, see Table S3). The product was eluted from the agarosegel and ligated into a pBluescript SK vector digested with EcoRV.After transformation into Escherichia coli strain TOP10, plasmidDNAs were prepared from white colonies and sequenced. TheseDNA sequences were aligned using BioEdit software (Hall, 1999).

Subcellular localization

The OGR1 coding region was amplified without its stop codon,using primers 334start (5¢-ggaTCCATGTCGGTGTCGGC-3¢) and334R (5¢-actagtCCAGTAATCCCTGCAGGAA-3¢) (restriction sites forcloning are underlined). The fragment was inserted into the multiplecloning site (MCS) of the maize ubiquitin promoter–MCS–sGFPcoding sequence–NOS terminator cassette of a pGA3452 vector(Kim et al., 2009). This construct generated OGR1::sGFP fusionproteins. To label the mitochondria, we used MitoTracker Red(Invitrogen, http://www.invitrogen.com/), a mitochondrion-specificdye, and the CaMV 35S promoter-F1-ATPase::RFP-NOS terminatorplasmid (Jin et al., 2003). Protoplast preparation and transformationprocedures were as previously described (Han et al., 2006; Wooet al., 2007).

Complementation

A cDNA clone containing the complete OGR1 open reading framewas amplified using primers 334start (see above) and endR (5¢-ac-tagtTTACCAGTAATCCCTGCAGG-3¢) (restriction site underlined),then cloned between the ubiquitin promoter and the NOS termi-nator in binary vector pGA3426, which has a hygromycin B selec-tion marker (Kim et al., 2009). Embryonic calli that developed fromogr1-1 mutant seeds were co-cultured with Agrobacterium tum-efaciens strain LBA4404 harboring the above construct. Transgenicplants were obtained as described previously (Lee et al., 1999).

ACKNOWLEDGEMENTS

We thank In-Soon Park for rice transformation, Hyun-Woo Cho forsubcellular localization experiments, Hee-Jung Choi for confocalimaging, and Priscilla Licht for critical proofreading of the manu-script. This work was supported, in part, by grants from the CropFunctional Genomic Center, 21st Century Frontier Program (grant

number CG1111), the Biogreen 21 Program (grant number 034-001-007-03-00) of the Rural Development Administration, the KoreaScience and Engineering Foundation (KOSEF) through the NationalResearch Laboratory Program funded by the Ministry of Scienceand Technology (grant number M10600000270-06J0000-27010), andthe Basic Research Promotion Fund through a Korea ResearchFoundation grant (KRF-2007-341-C00028).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. RNA gel-blot analyses of mitochondrial NADH dehydro-genase genes.Figure S2. Comparison of cDNA sequences containing seven RNAediting sites from five higher plant species.Figure S3. Comparison of the E, E+ and DYW motifs among nine PPRproteins.Figure S4. Alignments of cDNA sequences containing ten newediting sites from five higher plants.Table S1. RNA editing efficiency between wild-type and mutant.Table S2. PCR primers for probes used in RNA gel-blot analyses.Table S3. PCR primers for amplification of transcripts from themitochondrial genome for RNA editing analyses.Please note: Wiley-Blackwell are not responsible for the content orfunctionality of any supporting materials supplied by the authors.Any queries (other than missing material) should be directed to thecorresponding author for the article.

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