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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 genean@postech.ac.kr).
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.
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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.
RNA editing in rice mitochondria 741
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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.
742 Sung-Ryul Kim et al.
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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.
RNA editing in rice mitochondria 743
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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.
744 Sung-Ryul Kim et al.
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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 in rice mitochondria 745
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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).
746 Sung-Ryul Kim et al.
ª 2009 The AuthorsJournal compilation ª 2009 Blackwell Publishing Ltd, The Plant Journal, (2009), 59, 738–749
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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