Growing Slowly 1 locus encodes a PLS-type PPR protein required for RNA editing and plant development in Arabidopsis
Journal of Experimental Botany
Growing Slowly 1 locus encodes a PLS-type PPR protein required for RNA editing and plant development in Arabidopsis
Tingting Xie 0 2
Dan Chen 2
Jian Wu 1
Xiaorong Huang 2
Yifan Wang 2
Keli Tang 2
Jiayang Li 1
Mengxiang Sun 2
Xiongbo Peng 2
Karl-Josef Dietz, Bielefeld University
0 Present address: College of Life Sciences, Huazhong Agricultural University , Wuhan 430070 , China
1 State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Genetics and Developmental Biology, Chinese Academy of Sciences , Beijing 100101 , China
2 State Key Laboratory for Hybrid Rice, College of Life Sciences, Wuhan University , Wuhan 430072 , China
Most pentatricopeptide repeat (PPR) proteins are involved in organelle post-transcriptional processes, including RNA editing. The PPR proteins include the PLS subfamily, containing characteristic triplets of P, L, and S motifs; however, their editing mechanisms and roles in developmental processes are not fully understood. In this study, we isolated the Arabidopsis thaliana Growing slowly 1 (AtGRS1) gene and showed that it functions in RNA editing and plant development. Arabidopsis null mutants of grs1 exhibit slow growth and sterility. Further analysis showed that cell division activity was reduced dramatically in the roots of grs1 plants. We determined that GRS1 is a nuclear-encoded mitochondria-localized PPR protein, and is a member of the PLS subfamily. GRS1 is responsible for the RNA editing at four specific sites of four mitochondrial mRNAs: nad1-265, nad4L-55, nad6-103, and rps4-377. The first three of these mRNAs encode for the subunits of complex I of the electron transport chain in mitochondria. Thus, the activity of complex I is strongly reduced in grs1. Changes in RPS4 editing in grs1 plants affect mitochondrial ribosome biogenesis. Expression of the alternative respiratory pathway and the abscisic acid response gene ABI5 were up-regulated in grs1 mutant plants. Genetic analysis revealed that ABI5 is involved in the short root phenotype of grs1. Taken together, our results indicate that AtGRS1 regulates plant development by controlling RNA editing in Arabidopsis.
ABI5; mitochondria; pentatricopeptide repeat proteins; RNA editing; root
Pentatricopeptide repeat (PPR) proteins are a class of RNA
binding proteins characterized by the presence of a
degenerate 35-amino-acid repeat, the PPR motif, which is arranged
in tandem 2–50 times (Small and Peeters, 2000). The PPR
motif (P motif) has another two variants, namely the S
(short) motif with a length of 31 amino acids and the L
(long) motif with a length of 35–36 amino acids. Based on
their motifs, PPR proteins are divided into two subfamilies:
the P subfamily has only P motifs, and the PLS subfamily
contains characteristic triplets of P, L, and S motifs. Most
members of the PLS subfamily contain extra conserved
domains at their C-terminus, and these are designated the
© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.
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E, E+, and DYW domains (Lurin et al., 2004; Cheng et al.,
PPR proteins are involved in many aspects of RNA
processing in mitochondria and chloroplasts, including RNA
cleavage, splicing, editing, and translation, and play crucial
roles in plant developmental processes and responses to
environmental stresses (Andrés et al., 2007; Zehrmann et al., 2009;
Liu et al., 2010; Murayama et al., 2012; Zhu et al., 2012; Haili
et al., 2013; Mei et al., 2014; Yang et al., 2014; Hsieh et al.,
2015). RNA editing is an important step in the
post-transcriptional control of organelle gene expression. Most RNA
editing in plants results in the conversion of cytidine (C) to
uridine (U) (Covello and Gray, 1989; Gualberto et al., 1989;
Hiesel et al., 1989; Shikanai, 2006; Chateigner-Boutin and
Small, 2010). In the mitochondria of Arabidopsis,
approximately 500 C-to-U editing sites had been uncovered (Giegé
and Brennicke, 1999; Bentolila et al., 2005, 2008). The
mechanism of the editing reaction puzzled researchers for many
years, until the first PPR protein, CHLORORESPIRATORY
REDUCTION 4, was found to be involved in chloroplast
RNA editing (Kotera et al., 2005). Since then, PPR proteins
have been found to be involved in RNA editing and all the
discovered trans-factors involved in RNA editing in plants
belong to the PLS subfamily (Takenaka et al., 2013; Shikanai,
2015). Although several PPR proteins target individual sites,
some are found to recognize more than two and even as many
as eight sites (Kim et al., 2009; Zehrmann et al., 2009, 2012;
Zhu et al., 2012; Glass et al., 2015). Although recently
bioinformatics, biochemical, and structural analyses have shown
that PPR proteins recognize RNA in one-motif to
one-nucleotide binding mode (Yagi et al., 2013; Yin et al., 2013; Barkan
and Small, 2014), the mechanism of how a single PPR
protein recognizes multiple target sequences still needs further
Mutations in many RNA-editing PPR proteins do not
result in any evident developmental defect (Zehrmann et al.,
2009; Verbitskiy et al., 2010; Härtel et al., 2013), although
some PPRs are important in development (Yu et al., 2009;
Koprivova et al., 2010; Liu et al., 2010; Murayama et al.,
2012; Haili et al., 2013; Yang et al., 2014). The
relationship between mutant phenotype and RNA editing has not
received much attention until recently. Mutations in PPR
proteins involved in chloroplast RNA editing have been shown
to impair chloroplast biogenesis (Yu et al., 2009). Several
reports have shown that an increase in reactive oxygen species
(ROS) is responsible for the developmental defects observed
in the mitochondrial RNA editing by those mutant PPRs
(Liu et al., 2010; Yang et al., 2014). The nature of other
signaling pathways linking PPRs involved in mitochondrial RNA
editing and plant development remains largely unknown.
In this study, we analyzed the Arabidopsis T-DNA
knockout mutant grs1-1, which displays a phenotype of slow
growth and sterility. Genetic and molecular analysis indicates
that the GRS1 gene encodes a PPR protein. Further studies
showed that GRS1 is required for the RNA editing of four
mitochondrial transcripts. The upstream sequences of these
editing sites share some conserved nucleotides. The lack of
RNA editing at these sites leads to reduced levels of functional
mitochondrial complex I and affects mitochondrial ribosome
biogenesis. Abscisic acid (ABA) response gene ABI5 but not
ROS is involved in the short root phenotype in grs1.
Materials and methods
Mutant library construction and selection of grs1-1
We generated an Arabidopsis mutant library with T-DNA encoding
LAT52::EGFP, a cell-autonomous pollen-specific reporter (Twell
et al., 1989; Sessions et al., 2002), and a hygromycin-resistance gene.
T-DNA mutagenesis was carried out on qrt1 plants (Preuss et al.,
1994), where mature pollen grains maintain male meiotic products
in tetrads (Supplementary Fig. S1A, B at JXB online).
Hygromycinresistant plants, heterozygous for a single locus T-DNA insertion,
produced tetrads with two mutant pollen grains emitting green
fluorescent protein (GFP) fluorescence, and two wild-type grains that
did not display any GFP activity (Supplementary Fig. S1C, D). This
simplified the process of determining whether a T2 plant was
heterozygous (tetrads are two GFP+ to two GFP−, HYG resistant),
homozygous (all four tetrad members are GFP+, HYG resistant)
(Supplementary Fig. S1E, F) or wild-type (all four tetrads members
are GFP−) for a T-DNA induced mutation.
For grs1-1 selection, T1 seeds were obtained by self-pollination of
hygromycin-resistant grs1-1 plant and sown on 1/2 MS plates with
hygromycin to select grs1-1 seedlings. Thirty-two
hygromycin-resistant seedlings were grown on soil and the pollen grains of each plant
were visualized under a fluorescence microscope to determining
whether a T2 plant was heterozygotes, homozygotes, or wild-type.
T1 seeds were sown on 1/2 MS plates for germination.
Plant materials and growth conditions
Arabadopsis thaliana qrt1 (Preuss et al., 1994) was used as a
wildtype strain. The grs1-1 allele was isolated from our mutant library
with hygromycin resistance (Wu et al., 2012, Supplementary data).
The grs1-2 (CS428796) and gin1-3 lines were obtained from the
Arabidopsis Biological Resource Center (ABRC; Ohio, USA). The
mutant abi5-1 (Liu et al., 2012) was provided by Dr Lei Zhang
(College of Life Sciences, Wuhan University). The transgenic line
pCyclinB1;1:Dbox-GUS (Colon-Carmona et al., 1999) was provided
by Dr Jian Xu (Temasek Life Sciences Laboratory, Singapore).
Seeds were surface-sterilized with 20% bleach for 10 min, and
washed three times with sterile distilled water. Seeds were stratified
for 3 d at 4 °C and then sown on 1/2 MS plates with 1.0% (w/v)
sucrose. To decrease the ROS level in seedlings,
diphenyleneiodonium (DPI, 100 μM, Sigma) or reduced glutathione (GSH, 300 μM,
Sigma) was added to the culture media. Agar plates were placed in
a growth room with a photoperiod of 16 h light/8 h dark. For
kanamycin selection, 50 mg l–1 of kanamycin (Sigma) was supplemented
to the media. Similarly, 50 mg l–1 of hygromycin (Roche) and 10 mg
l–1L of sulfadiazin (Sigma) were added for hygromycin selection and
sulfadiazin selection, respectively. Plants were grown on soil in a
greenhouse under long-day conditions (16 h light/8 h dark) at 22 °C.
Cloning of the T-DNA flanking sequence and characterization of
the grs1-1 and grs1-2 alleles
TheT-DNAflankingsequenceinthegrs1-1mutantwasclonedbyTAILPCR (Liu et al., 1995). The authenticity of the cloned sequence was
confirmed by PCR using two pairs primers located around the T-DNA
left border (GRS1-T1, TGGAACAAGTTCATCACGGTTTC;
LB-S, CCAAAATCCAGTACTAAAATCCAG) and right
border (GRS1-T2, ATTCATGGTTTGTGCATAAAAAGAG;
RB-S, CGCGCGGTGTCATCTATG). For the grs1-2 allele, the
T-DNA site was confirmed by PCR using the following
primers: GRS1-RP, GTGAAAATGGGAGCAAAAGTG; and LB3,
Vector construction and plant transformation
Plasmids P092, P093, and P094 were produced as described
previously (Wu et al., 2012; Yan et al., 2016). To generate the
pGRS1::GRS1 complementation construct, a 3876-bp
wildtype genomic sequence containing the AT4G32430 gene,
1078bp upstream of the ATG codon and 506-bp downstream of the
TAG codon sequences, was PCR-amplified (primers: GRS1-F1,
from genomic DNA and was then cloned into the P092
plasmid with T-DNA encoding pLAT52::DsRED and a
kanamycin-resistance gene (Supplementary Fig. S2C). To examine the
subcellular location of GRS1, we amplified and cloned the 35S
promoters into P094 to generate the 35S::EGFP construct.
Then the GRS1 ORF was amplified (primers: GRS1-CDS1,
and GRS1-CDS2, NNNNCTCGAGAACTGCAACTTTCCCC
TCCAAATTCATC) from genomic DNA and cloned into the
35S::EGFP plasmid to generate a 35S::GRS1-EGFP construct. To
produce the mitochondrial marker line, we amplified the TagRFP-T (Shibata
et al., 2010) and put it under the control of 35S to generate 35S::RFP.
Then we amplified and cloned the 129-bp DNA fragment containing
the mitochondria-targeted pre-sequence of the located F1-ATPase
gene At5g13450 (Robison et al., 2009) (using primers MITO-1,
and MITO-2, NNNNCTGCAGTGTTTGAGCAGAAGCA
GTTGCATAAG) into 35S::RFP to generate the 35S::Mito-RFP
construct. To investigate the expression pattern of GRS1, the GRS1
promoter was amplified (primers: GRS1-F1 as above, and GRS1-R2:
and put upstream of GUS (β-glucuronidase) in P093 to
generate pGRS1::GUS. All the gene constructs were transferred into
Agrobacterium tumefaciens strain GV3101 and transformed into
Arabidopsis plants by the floral dip method (Clough and Bent, 1998).
Genotype analysis of the genomic complemented lines
To identify the genotype of the genomic complemented lines, the DNA
of these plants was extracted and PCR analysis was conducted using
three pairs of primers (S1+A1, S2+A1, S1+A2) (Supplementary
Fig. S2B, C): Primer S1, CATCTGTAGGCAACAGTTTCATCAC
located upstream of the T-DNA insertion site; Primer S2,
CCAAAATCCAGTACTAAAATCCAG located around the T-DNA
left border; Primer A1, CTCTTCTCTCGCTTTTTAAGTTGC
located downstream of the AT4G32430 gene and beyond the
genomic fragment used for complementation; and Primer A2,
TGACTTAGTTGATTTGGAGGGTG located downstream of the
genomic fragment used for complementation.
Histochemical analysis of GUS activity
For pCYCB1;1:Dbox-GUS staining, we crossed the
pCYCB1;1:DboxGUS stable lines with grs1-1 mutant plants. F2 seeds were obtained
by self-pollination of F1 and sown on 1/2 MS plates with
hygromycin to select seedlings with the grs1-1 background. Individual F3
seeds were obtained by self-pollination of these seedlings and sown
on 1/2 MS plates for germination. GUS activity analysis was
performed with 8-d-old seedlings (with normal roots and short roots),
and the lines with all normal roots with GUS activity were regarded
as homozygous for pCYCB1;1:Dbox-GUS. The seedlings with short
roots were regarded as homozygoous for both
pCYCB1;1:DboxGUS and grs1-1.
The histochemical analysis of GUS activity was performed
according to Vielle-Calzada et al. (2000). Plant tissues were
incubated at 37 °C in GUS-staining solution [2 mM
5-bromo-4-chloro3-indolyl glucuronide (X-Gluc) in 50 mM sodium phosphate buffer,
pH 7.0] containing 0.1% Triton X-100, 2 mM K4Fe(CN)6 and 2 mM
K3Fe(CN)6. The stained tissues were then transferred to 70% (v/v)
ethanol solution. Samples were mounted with traditional clearing
solution and placed under a microscope (Olympus) fitted with
differential interference contrast optics for imaging.
Analysis of subcellular localization of GRS1
The iPSORT Prediction program (Bannai et al., 2002) predicted that
GRS1 is targeted to the mitochondria. To confirm its mitochondrial
localization, transgenic plants containing the 35S::GRS1-EGFP
construct were crossed with a transgenic mitochondrial marker
line expressing 35S::mito-RFP. The petal cells of the F1 progeny
were visualized using a FV1000 confocal laser-scanning microscope
(CLSM; Olympus). GFP fluorescence was detected with
excitation at 488 nm and emission at 510–530 nm; red fluorescent protein
(RFP) fluorescence was detected with excitation at 568 nm and
emission at 590–620 nm.
Analysis of RNA editing
The status of Arabidopsis mitochondrial RNA editing in grs1
plants was examined as described by Zehrmann et al. (2008). Total
RNA was extracted from 20-d-old grs1 and wild-type seedlings.
Complementary DNA fragments of all mitochondrial transcripts
containing RNA editing sites were amplified by RT-PCR. The
primers used in this experiment are given in Supplementary Table S3. The
amplified PCR products were directly sequenced and the results were
compared to the corresponding DNA sequence for each transcript.
For the determination of the root meristem size, root tips were
excised from seedlings 8 d after germination, and examined with a
differential interference contrast (DIC) microscope (Olympus).
Measurement of ROS in roots
For nitrobluetetrazolium (NBT) staining to detect superoxides,
seedlings were incubated in a reaction buffer containing 1 mM NBT
(Sigma-Aldrich) and 20 mM K-phosphate at pH 6.0 for 20 min. The
seedlings stained by NBT were washed three times with water and
then transferred to acetic acid:ethanol (1:3, v/v) solution. To enable
3, 3- diaminobenzidine (DAB) staining to detect H2O2, the seedlings
were incubated in 0.3 mg ml–1 DAB (Sigma-Aldrich) dissolved in
50 mM Tris-HCl (pH 5.0) for 12 h. The seedlings stained by DAB
were washed three times with water, and were then examined in 10%
glycerol with an Olympus microscope.
Total RNAs of seeds before germination and 7-d-old seedlings
were extracted using the RNAqueous Phenol-free total RNA
Isolation kit (Ambion)according to the manufacturer’s
protocol. After digestion with RNase-free DNase I (Promega), the first
strand of cDNA was synthesized using oligo-dT and M-MLV
reverse transcriptase (Invitrogen). Quantitative PCR analysis was
performed using FastStart Essential DNA Green Master (Roche)
on a CFX ConnectTM Real-Time System (BioRad). Each
experiment was repeated three times and samples were normalized using
UBQ10 expression. Data acquisition and analyses used Bio-Rad
CFX Manager software; the relative expression levels were
measured using the 2(–∆Ct) analysis method and the error bars in the
figures represent the variance of three replicates. The genes and the
primers used for detection of the mRNA expression are listed in
Supplementary Table S4.
Detection of enzyme activity of complex I
Analysis of the NADP dehydrogenase activity of mitochondrion
complex I was performed according to Wu et al. (015). Proteins of
crude organelle extract from young seedlings were solubilized with
was also found to be retarded and did not appear to be able
to attract wild-type pollen tubes into the ovules (Fig. 1C–F).
Cell division is impaired in grs1-1
After germination, the growth rate of the primary root was
dramatically reduced in grs1-1 plants compared to the
wildtype. To determine the cellular basis for the observed defects
in the root development of grs1-1 plants, we examined the
size of the root meristem in seedlings 8 d after germination.
It was observed that the size of root meristem in grs1-1 was
much shorter than that of the wild-type (Fig. 2A). To further
substantiate the role of GRS1 in controlling root cell
division, we crossed pCyclin B1;1:Dbox-GUS stable lines
(ColonCarmona et al., 1999) with grs1-1 mutant plants. The pCyclin
B1;1:Dbox-GUS reporter allows the visualization of cells at
the G2-M phase of the cell cycle, and thus to monitor mitotic
activity in the root meristem (Colon-Carmona et al., 1999).
In contrast to the wild-type, we found that there was almost
no GUS signal in grs1-1 roots (Fig. 2B). The results indicate
that the number of dividing cells was reduced dramatically in
grs1-1compared to wild-type roots.
Molecular characterization of grs1-1
Arabidopsis grs1-1 plants were generated by T-DNA
insertion with resistance to hygromycin. All the grs1-1/+
heterozygous plants were resistant to hygromycin, suggesting that the
mutant phenotype was caused by a T-DNA insertion. We
cloned the T-DNA flanking sequence by using the thermal
asymmetric interlaced polymerase chain reaction
(TAILPCR) technique (Liu et al., 1995). The grs1-1 mutant was
shown to carry a T-DNA insertion in the gene AT4G32430
located 1325 bp downstream of the ATG start codon
(Fig. 3A, Supplementary Fig. S2B). Another allele
containing a T-DNA insertion in the GRS1 gene, CS428796, was
obtained from the Arabidopsis Biological Resource Center.
We verified that the CS428796 mutant carries a T-DNA
insertion in the AT4G32430 gene at 850 bp downstream of the
ATG start codon (Fig. 3A). We then renamed the CS428796
allele grs1-2. Homozygous grs1-2 plants were found to
phenocopy grs1-1 homozygous plants (Fig. 1A, B).
To confirm that the grs1-1 mutant phenotypes were indeed
caused by knockout of the AT4G32430 gene, we performed
a complementation test with the genomic sequence of
AT4G32430. Fifty-nine T1 transgenic plants were screened
on double-resistance plates with hygromycin and
kanamycin (for the transformed genomic sequence). Among them,
eleven plants were homozygous for grs1-1. All these grs1-1
homozygous plants carrying the fragments of the
exogenous genomic sequence (resistance to kanamycin) showed
no obvious differences compared to the wild-type, and were
named the genomic complemented lines (homozygous for
grs1-1, heterozygous for exogenous genomic fragment)
(Supplementary Fig. S2A). Genotype analysis confirmed the
genomic complemented lines contained both the mutated
grs1-1 version and expression of the wild-type version
(Supplementary Fig. S2D). These results indicate that the
AT4G32430 gene can successfully complement the grs1-1
phenotype. The AT4G32430 gene was therefore renamed as
GRS1 encodes a mitochondria-targeted
pentatricopeptide repeat protein
To investigate the expression pattern of GRS1, we fused
the GRS1 promoter sequence to a GUS reporter gene, and
transformed this construct into the wild-type. In seedlings,
GRS1::GUS was preferentially expressed in the meristematic
region of both roots and stems. In flowers, GUS activity
was detected in the sepal, stigma, stamen, and pollen grains
Fig. 2. The activity of the root meristem division is reduced in grs1-1. (A) The root meristematic zone of 8-d-old wild-type (WT) plants is much longer
than that of grs1-1 plants. (B) Expression of pCyclinB1;1:Dbox-GUS in the meristematic zone of 8-d-old WT and grs1-1 seedlings. Arrows indicate the
boundary between the root meristematic and elongation zone. Scale bars = 100 µm.
Fig. 3. Structural features, expression patterns, and subcellular localization
of GRS1. (A) Diagram showing the relative position of the T-DNA insertion
in the GRS1 gene and the structural features of the GRS1 protein. Various
protein domains are indicated below the diagram. (B) GUS expression
patterns in different plant parts of transgenic proGRS1::GUS lines. Top row:
7-d-old seedling. GUS signal is observed in root and shoot meristems. Scale
bar = 5mm. Bottom row, left: an inflorescence, scale bar = 3 mm. right: an
anther with pollen grains, scale bar = 50 µm. (C) Localization of GRS1-GFP
protein in the mitochondria. Petal cells of plant co-expressing GRS1-GFP
and the mitochondrial marker Mito-RFP were examined with confocal laser
scanning microscopy. From left to right: green fluorescent signal from
GRS1GFP; red fluorescent signal from the mitochondrial marker Mito-RFP; merged
picture with green and red signals showing co-localization. Scale bar = 20µm.
BLAST analysis identified GRS1 as a member of the PPR
family, more specifically belonging to the PLS subfamily.
Thus, GRS1 encodes a PLS-type pentatricopeptide repeat
protein, as proposed by Lurin et al. (2004). It consists of six
PPR-like S, six PPR-like L, and five P motifs with E1, E2, and
DYW C-terminal extensions (Lurin et al., 2004; Barkan and
Small, 2014; Cheng et al., 2016) (Fig. 3A, Supplementary Fig.
S3, and Table S1). The iPSORT Prediction program (Bannai
et al., 2002) predicted that GRS1 is targeted to mitochondria
and, indeed, GRS1-GFP was found to co-localize with the
mitochondria-localized Mito-RFP (Fig. 3C), indicating that
GRS1 is a nuclear-encoded mitochondrial protein.
GRS1 is required for mitochondrial RNA editing
Since GRS1 encodes a DYW-type PPR protein, we tested its
involvement in mitochondrial RNA editing. We identified
several unedited sites in the mitochondrial RNA in the grs1-1
mutants. Our results revealed that C-to-U editing at the positions
of nad1-265, nad4L-55, nad6-103, and rps4-377 was specifically
blocked in the grs1-1 plants. Editing of these four sites is also
inhibited in grs1-2 mutants (Fig. 4). The C-to-U editing in the
nad1 mRNA results in an arginine-to-tryptophane amino acid
change (R89W) in the NAD1 protein. The C-to-U editing in
the nad4L mRNA results in an arginine-to-tryptophane amino
acid change (R19W) in the NAD4L protein. The C-to-U
editing in the nad6 mRNA results in an arginine-to-cystine amino
acid change (R35C) in the NAD6 protein. The C-to-U
editing in the rps4 mRNA results in a proline-to-leucine amino
acid change (P126L) in the RPS4 protein. Editing of the four
mRNAs at these four editing sites was highly efficient in the
wild-type, as shown by the detection of a single peak equivalent
to the T nucleotide at these positions, whereas editing of these
sites was totally abolished in grs1-1 and grs1-2 mutants (Fig. 4).
Editing deficiencies of the mutant alleles were restored in the
grs1-1 complemented lines (Fig. 4). These results confirmed
that mutation in the GRS1 gene was responsible for the defect
of mitochondrial RNA editing in the grs1-1 mutants.
Fig. 4. GRS1 is responsible for RNA editing of four sites in Arabidopsis mitochondria. Wild-type plants show that RNA editing of the mitochondrial
editing sites nad1-265, nad4L-55, nad6-103, and rps4-377 is efficient, while in grs1-1 and grs1-2 these sites are not edited. Editing deficiencies were
restored in the grs1-1 complemented lines.
AtGRS1 is required for RNA editing and plant development | 5693
Complex I function and mitoribosomal biogenesis are
impaired in grs1-1 mutants
An alternative respiratory pathway is activated in
The proteins NAD1, NAD4L, and NAD6 are components
of the mitochondrial electron transport chain complex
I (NADH dehydrogenase). Having observed that RNA
editing of these genes was altered in grs1-1 mutants and resulted
in amino acid changes, we hypothesized that RNA editing
defects of these transcripts may lead to complex
I malfunction in grs1-1 mutants. To test this hypothesis, we isolated
crude mitochondria from seedlings of wild-type, grs1-1
mutants, and grs1-1 complemented lines. Separation of
mitochondrial complexes by blue-native PAGE and NADH
dehydrogenase activity staining showed that both protein
levels and activity of complex I could barely be detected in
grs1-1 mutants (Fig. 5A, B).
Since the RPS4 protein is a component of the small
subunit (SSU) of the mitoribosome, we tested whether the
change in RPS4 editing in the grs1 mutants affects
mitochondrial ribosome biogenesis. As rRNAs are unstable
when unassembled, rRNA levels can serve as a marker for
the accumulation of ribosomal subunits (Walter et al., 2010;
Kwasniak et al., 2013). We determined the abundance of
mitochondrial (mt 18S and mt 26S), chloroplast (chl 16S
and chl 23S) and cytosolic (cyt 18S and cyt 25S) rRNAs.
The mt 18S showed no evident difference between grs1-1
and the wild-type, while a significant increase was observed
for mt 26S rRNA in grs1-1 plants compared to the wild-type
(Fig. 5C), with the increased ratio of mt 26S to mt 18S
indicating an imbalance between mitoribosomal subunits. The
chl 16S, chl 23S, cyt 18S, and cyt 25S showed no obvious
differences between grs1-1 and the wild-type (Fig. 5C),
suggesting the grs1 mutation only affects mitochondrial
Lack of complex I activities is known to result in elevated
levels of an alternative respiratory pathway in Arabidopsis
(Yuan and Liu, 2012). The components of this alternative
respiratory pathway include several alternative NAD(P)H
dehydrogenases (NDs) and alternative oxidases (AOXs). To
determine whether grs1-1 mutants had the same phenotype,
we performed quantitative RT-PCR assays for the transcripts
levels of six ND genes and three AOX genes in wild-type and
grs1-1 plants. As shown in Fig. 6, the expression levels of the
nine examined genes in grs1-1 increased significantly relative
to the wild-type. These results indicate that the alternative
respiratory pathway is activated in grs1-1. grs1-2 mutants
had a similar phenotype with up-regulation of transcripts
for alternative respiration compared with the wild-type.
(Supplementary Fig. S4).
The grs1-1 mutant does not accumulate higher
amounts of ROS than the wild-type
Reports have shown that impaired activity of the
mitochondrial electron transport chain of complex I can cause a
redox imbalance and increases in ROS accumulation,
leading to the accumulation of more ROS in mutants than in
the wild-type (Liu et al., 2010; Yang et al., 2014). We
analyzed the ROS levels in grs1-1 mutants and wild-type plants
and showed that grs1-1 mutants do not accumulate higher
amounts of ROS than the wild-type (Fig. 7A, B). Consistent
with these results, addition of the reducing agent glutathione
(GSH) or diphenyleneiodonium chloride (DPI) was not able
to complement the root growth defects of grs1-1 mutant
plants (Fig. 7C).
Fig. 6. The alternative respiratory pathway is activated in grs1-1. The expression levels of alternative respiratory pathway genes in grs1-1 increased
significantly relative to the wild-type. These genes include three alternative oxidases (AOXs) and six alternative NAD(P)H dehydrogenases (NDs). The
values obtained were averaged for three independent experiments, with error bars representing SD. Statistically significant differences between grs1-1
and the wild-type are indicated: **P<0.01 (Student’s t-test;).
abi5 partially rescues the post-germination growth
arrest of grs1-1
Since grs1 mutant display defects in seed germination and
post-germination growth, it is possible that the ABA
signaling pathway is activated in these mutants. Given that the
transcription factors ABI3 and ABI5 are key proteins in
the ABA signaling pathway (Finkelstein and Lynch, 2000;
Lopez-Molina et al., 2001), expression of ABI3 and ABI5
was analyzed in grs1-1 mutant and wild-type seedling plants 8
d after germination. Expression of ABI5 was found to be
significantly up-regulated in grs1-1 mutants, whereas expression
levels of ABI3 were not significantly altered (Fig. 8A),
implying that ABI5, but not ABI3, is activated in grs1-1 mutants
and is involved in the short-root phenotype. To test this
hypothesis, the grs1-1 abi5-1 double-mutant was generated,
and it showed longer roots than those of the grs1-1 mutants
(Fig. 8B, Supplementary Table S2). While only about 10 cells
could be observed in the meristems of in grs1-1 mutants,
approximately 20 cells were established in the meristem of
grs1-1 abi5-1 double-mutant plants (Fig. 8C). These results
indicate that abi5-1 partially rescues the post-germination
growth arrest of the grs1-1 mutants.
We then tested whether a decrease in the ABA content in
grs1-1 mutants can rescue the post-germination growth arrest
of these plants. The gin1-3 mutant line is a knockout allele
of the ABA2 gene, one of the key genes involved in ABA
synthesis However, the grs1-1 gin1-3 double-mutant did not
show any evident differences compared with the grs1-1
singlemutant plants in post-germination growth (Fig. 8B, C).
Putative cis-acting elements recognized by GRS1
Recently bioinformatics, biochemical, and structural analyses
have shown that PPR proteins recognize RNA in one-motif
to one-nucleotide binding mode (Kim et al., 2009; Yagi et al.,
2013; Yin et al., 2013; Barkan and Small, 2014). The major
determinant is the amino acid at position 5 of the motif (Yagi
et al., 2013; Yin et al., 2013; Barkan and Small, 2014; Cheng
et al., 2016). The second major determinant is at position 2
of the motif and position 35 of the following motif (Yagi
et al., 2013; Yin et al., 2013; Barkan and Small, 2014; Cheng
et al., 2016). The site-specific RNA editing factors PPR and
the RNA target sequences show optimal correlations when
the PPR domains are aligned with the nucleotide sequences
upstream of RNA editing sites up to the fourth nucleotide
(nucleotide −4). The last S motif of GRS1 is accordingly
positioned at the −4 nucleotides site of all the editing sites
(Supplementary Fig. S3 and Table S1). In this way, the
conserved A nucleotide at position −12 and G nucleotide at
position −6 are consistent with the predictions of bioinformatics
(Kim et al., 2009; Yagi et al., 2013; Yin et al., 2013; Barkan
and Small, 2014).
Cis-elements located between 20 to 25 nucleotides upstream
and one to three nucleotides downstream of the edited C are
known to be important in the context of RNA editing in
mitochondria and plastids (Zehrmann et al., 2009; Barkan
and Small, 2014). When comparing the context of the four
RNA sites edited by GRS1, five nucleotides are identical in
addition to the edited C (Supplementary Fig. S3), suggesting
that these positions are important for guiding editing through
GRS1 in the mitochondria. These five nucleotides, however,
are not sufficient to specify a unique site in the plant
mitochondrial transcriptome. An in silico screen identified
NAD4403, another editing site with the same RNA context in the
mitochondrial genome (Supplementary Fig. S3). NAD4-403
is edited normally in the wild-type and in the grs1 mutant,
confirming that the five shared nucleotide positions are not
sufficient to guide editing through GRS1. More information
may be provided by other nucleotides inside the context of
RNA editing of the four sites to ensure GRS1 specifically
binds to them. It was reported that PPR proteins distinguish
purines from pyrimidines much better than they distinguish
between C/U or A/G (Yagi et al., 2014; Kindgren et al.,
2015). The conservation between these four sequences is
better than shown when this is taken into account, with several
other nucleotide positions, such as −4, −7, −9, −14, and −15,
showing expected matches to the protein sequence in
addition to the ones that have been indicated. The correlations
of the amino acid codes in GRS1 and the diversity of its
targeted RNA bases can offer more information for predicting
whether a PPR protein can bind a particular RNA.
Comparison of grs1-1 plants with other Arabidopsis
complex I mutant lines
Loss of GRS1 directly affects the editing of three components
of complex I: nad1-265, nad4L-55, and nad6-103, which
in turn impair the function of complex I. Most complex
I mutants show a retarded growth phenotype, such as ahg11
(Murayama et al., 2012), abo5 (Liu et al., 2010), abo8 (Yang
et al., 2014), bir6 (Koprivova et al., 2010), css1 (Nakagawa
and Sakurai, 2006), indh (Wydro et al., 2013), mtsf1 (Haili
et al., 2013), nMat1 (Keren et al., 2012), nMat2 (Keren et al.,
2009), nMat4 (Cohen et al., 2014), otp43 (de Longevialle
et al., 2007), otp439 and tang2 (Colas des Francs-Small et al.,
2014), slg1 (Sung et al., 2010), slo2 (Zhu et al., 2012), slo3
(Hsieh et al., 2015), and also assmk1 (small kernel 1), which
has been shown to be responsible for loss of editing of
NAD7448 transcripts in maize and rice (Li et al., 2014).
The phenotype of grs1-1 plants, however, cannot be fully
explained by the loss of function of complex I. The defects
observed in grs1-1 plants are much stronger than those of
mutants defective in complex I activity such as the slo2 (Zhu
et al., 2012), opt43 (de Longevialle et al., 2007), nMat1 (Keren
et al., 2012) and ndufs4 mutants (Meyer et al., 2009). Impaired
activity of the mitochondrial electron transport chain of
complex I can cause a redox imbalance and increases in ROS
accumulation, leading to the accumulation of more ROS in
mutants than in the wild-type (Liu et al., 2010; Yang et al.,
2014); however, the grs1-1 mutants do not accumulate more
ROS than the wild-type. Consistent with these results,
addition of GSH or DPI could not restore the root growth defects
of grs1-1 mutant plants. The results indicate that other
signals must be responsible for the retarded growth phenotype
observed in grs1-1 plants.
ABA is a well-established key player in seed germination
and post-germination growth. Furthermore, some reports
have shown that mutations of PPR proteins result in mutant
plants that are more sensitive to ABA than wild-type plants
(Liu et al., 2010; Murayama et al., 2012; Yang et al., 2014).
Expression of ABI5 was found to be significantly up-regulated
in the grs1-1 mutants compared to the wild-type plants, while
expression of ABI3 was not up-regulated in grs1-1 mutant
plants compared to the wild-type. The results indicate that
the up-regulated expression of ABI5 is independent of the
ABA signal. The grs1-1 abi5-1 double-mutant displayed
higher root meristem cell numbers than the
grs1-1singlemutant plants. The results indicate that abi5-1 partially
rescued the post-germination growth arrest of grs1-1 mutant
plants. The grs1-1 gin1-3 double-mutant, however, could not
partially rescue the post-germination growth arrest of
grs11 mutant plants. These findings suggest that ABI5, but not
ABA, is involved in the post-germination growth arrest of
grs1-1 mutant plants. The mechanism through which grs1-1
mutant plants activate ABI5 remains an interesting question
for future investigation.
Other factors must be involved in the root growth defects
of the grs1-1 mutant plants, since abi5-1 only partially
rescued their post-germination growth arrest. One possibility
is that the mutation of GRS1 also impairs the function of
mitoribosomes, leading to a dysfunction of mitochondria in
addition to the loss of function of complex I. This scenario
is found in mcsf1 mutants, where the activity of complexes
I and IV are both reduced, leading to severe defects in embryo
development, which is arrested at the early globular stage
(Zmudjak et al., 2013).
Supplementary data are available at JXB online.
Figure S1. Rapid identification of heterozygous and
homozygous mutants through pollen fluorescence.
Figure S2. Genomic complement fragment of At4g32430
rescues the phenotype of grs1-1.
Figure S3. Putative coordination of PPR motifs of GRS1
and RNA nucleotides around the editing sites targeted
Figure S4. Relative expression of alternative pathway genes
in wild-type and grs1-2.
Table S1. PLS repeat structure of At4g32430.
Table S2. The root length of 8-d-old seedlings.
Table S3. Primers used for RNA editing analysis.
Table S4. Primers used for quantitative RT-PCR.
We thank Dr Jian Xu for providing the transgenic line
pCYCB1;1:DboxGUS and Dr Lei Zhang for providing abi5-1 mutant seeds. This work was
supported by project number 2013CB945100 of the National Natural
Science Foundation (grant No. 31570317, 31270362).
Andrés C, Lurin C, Small ID. 2007. The multifarious roles of PPR
proteins in plant mitochondrial gene expression. Physiologia Plantarum
Bannai H, Tamada Y, Maruyama O, Nakai K, Miyano S. 2002.
Extensive feature detection of N-terminal protein sorting signals.
Bioinformatics 18, 298–305.
Barkan A, Small I. 2014. Pentatricopeptide repeat proteins in plants.
Annual Review of Plant Biology 65, 415–442.
Bentolila S, Chateigner-Boutin A-L, Hanson MR. 2005. Ecotype allelic
variation in C-to-U editing extent of a mitochondrial transcript identifies
RNA-editing quantitative trait loci in Arabidopsis. Plant Physiology 139,
Bentolila S, Elliott LE, Hanson MR. 2008. Genetic architecture of
mitochondrial editing in Arabidopsis thaliana. Genetics 178, 1693–1708.
Chateigner-Boutin A-L, Small I. 2010. Plant RNA editing. RNA Biology
Cheng S, Gutmann B, Zhong X, et al. 2016. Redefining the structural
motifs that determine RNA binding and RNA editing by pentatricopeptide
repeat proteins in land plants. The Plant Journal 85, 532–547.
Clough SJ, Bent AF. 1998. Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant
Journal 16, 735–743.
Cohen S, Zmudjak M, Colas des Francs-Small C, et al. 2014. nMAT4,
a maturase factor required for nad1 pre-mRNA processing and maturation,
is essential for holocomplex I biogenesis in Arabidopsis mitochondria. The
Plant Journal 78, 253–268.
Colas des Francs-Small C, Falcon de Longevialle A, Li Y,
Lowe E, Tanz SK, Smith C, Bevan MW, Small I. 2014. The
pentatricopeptide repeat proteins TANG2 and ORGANELLE TRANSCRIPT
PROCESSING439 are involved in the splicing of the multipartite nad5
transcript encoding a subunit of mitochondrial complex I. Plant Physiology
Colon-Carmona A, You R, Haimovitch-Gal T, Doerner P. 1999.
Technical advance: spatio-temporal analysis of mitotic activity with a labile
cyclin-GUS fusion protein. The Plant Journal 20, 503–508.
Covello PS, Gray MW. 1989. RNA editing in plant mitochondria. FASEB
Journal 7, 64–71.
de Longevialle AF, Meyer EH, Andres C, Taylor NL, Lurin C, Millar
AH, Small ID. 2007. The pentatricopeptide repeat gene OTP43 is
required for trans-splicing of the mitochondrial nad1 Intron 1 in Arabidopsis
thaliana. The Plant Cell 19, 3256–3265.
Finkelstein RR, Lynch TJ. 2000. The Arabidopsis abscisic acid response
gene ABI5 encodes a basic leucine zipper transcription factor. The Plant
Cell 12, 599–609.
Giegé P, Brennicke A. 1999. RNA editing in Arabidopsis mitochondria
effects 441 C to U changes in ORFs. Proceedings of the National
Academy of Sciences, USA 96, 15324–15329.
Glass F, Härtel B, Zehrmann A, Verbitskiy D, Takenaka M. 2015.
MEF13 requires MORF3 and MORF8 for RNA editing at eight targets
in mitochondrial mRNAs in Arabidopsis thaliana. Molecular Plant 8,
Gualberto JM, Lamattina L, Bonnard G, Weil J-H, Grienenberger
J-M. 1989. RNA editing in wheat mitochondria results in the conservation
of protein sequences. Nature 341, 660–662.
Haili N, Arnal N, Quadrado M, Amiar S, Tcherkez G, Dahan J,
Briozzo P, Colas des Francs-Small C, Vrielynck N, Mireau H.
2013. The pentatricopeptide repeat MTSF1 protein stabilizes the
nad4 mRNA in Arabidopsis mitochondria. Nucleic Acids Research 41,
Härtel B, Zehrmann A, Verbitskiy D, van der Merwe JA, Brennicke
A, Takenaka M. 2013. MEF10 is required for RNA editing at nad2-842
in mitochondria of Arabidopsis thaliana and interacts with MORF8. Plant
Molecular Biology 81, 337–346.
Hiesel R, Wissinger B, Schuster W, Brennicke A. 1989. RNA editing in
plant mitochondria. Science 246, 1632–1634.
Hsieh WY, Liao JC, Chang C, Harrison T, Boucher C, Hsieh MH.
2015. The SLOW GROWTH 3 pentatricopeptide repeat protein is required
for the splicing of mitochondrial nad7 intron 2 in Arabidopsis. Plant
Physiology 168, 490–501.
Keren I, Bezawork-Geleta A, Kolton M, Maayan I, Belausov E, Levy
M, Mett A, Gidoni D, Shaya F, Ostersetzer-Biran O. 2009. AtnMat2,
a nuclear-encoded maturase required for splicing of group-II introns in
Arabidopsis mitochondria. RNA 15, 2299–2311.
Keren I, Tal L, des Francs-Small CC, Araujo WL, Shevtsov S, Shaya
F, Fernie AR, Small I, Ostersetzer-Biran O. 2012. nMAT1, a
nuclearencoded maturase involved in the trans-splicing of nad1 intron 1, is
essential for mitochondrial complex I assembly and function. The Plant
Journal 71, 413–426.
Kim SR, Yang JI, Moon S, Ryu CH, An K, Kim KM, Yim J, An G.
2009. Rice OGR1 encodes a pentatricopeptide repeat-DYW protein and is
essential for RNA editing in mitochondria. The Plant Journal 59, 738–749.
Kindgren P, Yap A, Bond CS, Small I. 2015. Predictable alteration of
sequence recognition by RNA editing factors from Arabidopsis. The Plant
Cell 27, 403–416.
Koprivova A, des Francs-Small CC, Calder G, Mugford ST, Tanz
S, Lee BR, Zechmann B, Small I, Kopriva S. 2010. Identification of
a pentatricopeptide repeat protein implicated in splicing of intron 1 of
mitochondrial nad7 transcripts. The Journal of Biological Chemistry 285,
Kotera E, Tasaka M, Shikanai T. 2005. A pentatricopeptide repeat
protein is essential for RNA editing in chloroplasts. Nature 433, 326–330.
Kwasniak M, Majewski P, Skibior R, Adamowicz A, Czarna M,
Sliwinska E, Janska H. 2013. Silencing of the nuclear RPS10 gene
encoding mitochondrial ribosomal protein alters translation in arabidopsis
mitochondria. The Plant Cell 25, 1855–1867.
Li XJ, Zhang YF, Hou M, et al. 2014. Small kernel 1 encodes a
pentatricopeptide repeat protein required for mitochondrial nad7 transcript
editing and seed development in maize (Zea mays) and rice (Oryza sativa).
The Plant Journal 79, 797–809.
Liu Y, He J, Chen Z, Ren X, Hong X, Gong Z. 2010. ABA
overlysensitive 5 (ABO5), encoding a pentatricopeptide repeat protein required
for cis-splicing of mitochondrial nad2 intron 3, is involved in the abscisic
acid response in Arabidopsis. The Plant Journal 63, 749–765.
Liu YG, Mitsukawa N, Oosumi T, Whittier RF. 1995. Efficient isolation
and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal
asymmetric interlaced PCR. The Plant Journal 8, 457–463.
Liu ZQ, Yan L, Wu Z, Mei C, Lu K, Yu YT, Liang S, Zhang XF, Wang
XF, Zhang DP. 2012. Cooperation of three WRKY-domain transcription
factors WRKY18, WRKY40, and WRKY60 in repressing two
ABAresponsive genes ABI4 and ABI5 in Arabidopsis. Journal of Experimental
Botany 63, 6371–6392.
Lopez-Molina L, Mongrand S, Chua NH. 2001. A postgermination
developmental arrest checkpoint is mediated by abscisic acid and requires
the ABI5 transcription factor in Arabidopsis. Proceedings of the National
Academy of Sciences, USA 98, 4782–4787.
Lurin C, Andrés C, Aubourg S, et al. 2004. Genome-wide analysis of
Arabidopsis pentatricopeptide repeat proteins reveals their essential role in
organelle biogenesis. The Plant Cell 16, 2089–2103.
Mei C, Jiang SC, Lu YF, et al. 2014. Arabidopsis pentatricopeptide
repeat protein SOAR1 plays a critical role in abscisic acid signalling.
Journal of Experimental Botany 65, 5317–5330.
Meyer E, Tomaz T, Carroll A, Estavillo G, Delannoy E, Tanz SK,
Small ID, Pogson BJ, Millar AH. 2009. Remodeled respiration in ndufs4
with low phosphorylation efficiency suppresses Arabidopsis germination
and growth and alters control of metabolism at night. Plant Physiology
Murayama M, Hayashi S, Nishimura N, Ishide M, Kobayashi K, Yagi
Y, Asami T, Nakamura T, Shinozaki K, Hirayama T. 2012. Isolation of
Arabidopsis ahg11, a weak ABA hypersensitive mutant defective in nad4
RNA editing. Journal of Experimental Botany 63, 5301–5310.
Nakagawa N, Sakurai N. 2006. A mutation in At-nMat1a, which encodes
a nuclear gene having high similarity to group II intron maturase, causes
impaired splicing of mitochondrial NAD4 transcript and altered carbon
metabolism in Arabidopsis thaliana. Plant & Cell Physiology 47, 772–783.
Preuss D, Rhee SY, Davis RW. 1994. Tetrad analysis possible in
Arabidopsis with mutation of the QUARTET (QRT) genes. Science 264,
Robison MM, Ling X, Smid MP, Zarei A, Wolyn DJ. 2009. Antisense
expression of mitochondrial ATP synthase subunits OSCP (ATP5) and
γ (ATP3) alters leaf morphology, metabolism and gene expression in
Arabidopsis. Plant & Cell Physiology 50, 1840–1850.
Sessions A, Burke E, Presting G, et al. 2002. A high-throughput
Arabidopsis reverse genetics system. The Plant Cell 14, 2985–2994.
Shibata H, Inuzuka T, Yoshida H, Sugiura H, Wada I, Maki M. 2010.
The ALG-2 binding site in Sec31A influences the retention kinetics of
Sec31A at the endoplasmic reticulum exit sites as revealed by live-cell
time-lapse imaging . Bioscience, Biotechnology, & Biochemistry 74 , 1819 - 1826 .
Shikanai T. 2006 . RNA editing in plant organelles: machinery, physiological function and evolution . Cellular and Molecular Life Sciences 63 , 698 - 708 .
Shikanai T. 2015 . RNA editing in plants: machinery and flexibility of site recognition . Biochimica et Biophysica Acta 1847 , 779 - 785 .
Small ID , Peeters N. 2000 . The PPR motif - a TPR-related motif prevalent in plant organellar proteins . Trends in Biochemical Sciences 25 , 45 - 47 .
Sung TY , Tseng CC , Hsieh MH . 2010 . The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria . The Plant Journal 63 , 499 - 511 .
2013. RNA editing in plants and its evolution . Annual Review of Genetics 47 , 335 - 352 .
Twell D , Wing R , Yamaguchi J , McCormick S. 1989 . Isolation and expression of an anther-specific gene from tomato . Molecular and General Genetics 217 , 240 - 245 .
Verbitskiy D , Zehrmann A , van der Merwe JA , Brennicke A , Takenaka M. 2010 . The PPR protein encoded by the LOVASTATIN INSENSITIVE 1 gene is involved in RNA editing at three sites in mitochondria of Arabidopsis thaliana . The Plant Journal 61 , 446 - 455 .
Vielle-Calzada JP , Baskar R , Grossniklaus U. 2000 . Delayed activation of the paternal genome during seed development . Nature 404 , 91 - 94 .
Walter M , Piepenburg K , Schottler MA , Petersen K , Kahlau S , Tiller N , Drechsel O , Weingartner M , Kudla J , Bock R . 2010 . Knockout of the plastid RNase E leads to defective RNA processing and chloroplast ribosome deficiency . The Plant Journal 64 , 851 - 863 .
Wu J , Sun Y , Zhao Y , et al. 2015 . Deficient plastidic fatty acid synthesis triggers cell death by modulating mitochondrial reactive oxygen species .
Cell Research 25 , 621 - 633 .
Wu JJ , Peng XB , Li WW , He R , Xin HP , Sun MX . 2012 . Mitochondrial GCD1 dysfunction reveals reciprocal cell-to-cell signaling during the maturation of Arabidopsis female gametes . Developmental Cell 23 , 1043 - 1058 .
2013 . The evolutionarily conserved iron-sulfur protein INDH is required for complex I assembly and mitochondrial translation in Arabidopsis [corrected] . The Plant Cell 25 , 4014 - 4027 .
Yagi Y , Hayashi S , Kobayashi K , Hirayama T , Nakamura T. 2013 .
Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants . PLoS ONE 8 , e57286 .
Yagi Y , Nakamura T , Small I. 2014 . The potential for manipulating RNA with pentatricopeptide repeat proteins . The Plant Journal 78 , 772 - 782 .
Yan HL , Chen D , Wang YF , Huang J , Sun MX , Peng XB . 2016 .
Ribosomal protein L18aB is required for both male gametophyte function and embryo development in Arabidopsis . Scientific Reports 6 , 31195 .
Yang L , Zhang J , He J , Qin Y , Hua D , Duan Y , Chen Z , Gong Z. 2014 .
ABA-mediated ROS in mitochondria regulate root meristem activity by controlling PLETHORA expression in Arabidopsis . PLoS Genetics 10 , e1004791 .
Yin P , Li Q , Yan C , et al. 2013 . Structural basis for the modular recognition of single-stranded RNA by PPR proteins . Nature 504 , 168 - 171 .
Yu QB , Jiang Y , Chong K , Yang ZN . 2009 . AtECB2, a pentatricopeptide repeat protein, is required for chloroplast transcript accD RNA editing and early chloroplast biogenesis in Arabidopsis thaliana . The Plant Journal 59 , 1011 - 1023 .
Yuan H , Liu D. 2012 . Functional disruption of the pentatricopeptide protein SLG1 affects mitochondrial RNA editing, plant development, and responses to abiotic stresses in Arabidopsis . The Plant Journal 70 , 432 - 444 .
Zehrmann A , van der Merwe J , Verbitskiy D , Härtel B , Brennicke A , Takenaka M. 2012 . The DYW-class PPR protein MEF7 is required for RNA editing at four sites in mitochondria of Arabidopsis thaliana . RNA Biology 9 , 155 - 161 .
Zehrmann A , van der Merwe JA , Verbitskiy D , Brennicke A , Takenaka M. 2008 . Seven large variations in the extent of RNA editing in plant mitochondria between three ecotypes of Arabidopsis thaliana .
Mitochondrion 8 , 319 - 327 .
Zehrmann A , Verbitskiy D , van der Merwe JA , Brennicke A , Takenaka M. 2009 . A DYW domain-containing pentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana . The Plant Cell 21 , 558 - 567 .
Zhu Q , Dugardeyn J , Zhang C , et al. 2012 . SLO2, a mitochondrial pentatricopeptide repeat protein affecting several RNA editing sites, is required for energy metabolism . The Plant Journal 71 , 836 - 849 .
Zmudjak M , Colas des Francs-Small C , Keren I , Shaya F , Belausov E , Small I , Ostersetzer-Biran O. 2013 . mCSF1, a nucleus-encoded CRM protein required for the processing of many mitochondrial introns, is involved in the biogenesis of respiratory complexes I and IV in Arabidopsis.
The New Phytologist 199, 379 - 394 .