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Silencing of the Nuclear RPS10 Gene Encoding Mitochondrial
Ribosomal Protein Alters Translation in
Arabidopsis Mitochondria
W
Malgorzata Kwasniak,a Pawel Majewski,a,1 Renata Skibior,a Aleksandra Adamowicz,a Malgorzata Czarna,a
Elwira Sliwinska,b and Hanna Janskaa,2
a Department
b Department
of Biotechnology, University of Wroclaw, 51-148 Wroclaw, Poland
of Plant Genetics, Physiology, and Biotechnology, University of Technological and Life Sciences, 85-789 Bydgoszcz,
Poland
Hardly anything is known about translational control of plant mitochondrial gene expression. Here, we provide evidence for
differential translation of mitochondrial transcripts in Arabidopsis thaliana. We found that silencing of the nuclear RPS10 gene
encoding mitochondrial ribosomal protein S10 disturbs the ratio between the small and large subunits of mitoribosomes, with
an excess of the latter. Moreover, a portion of the small subunits are incomplete, lacking at least the S10 protein. rps10 cells
also have an increased mitochondrial DNA copy number per cell, causing an upregulation of all mitochondrial transcripts.
Mitochondrial translation is also altered so that it largely overrides the hyperaccumulation of transcripts, and as a consequence,
only ribosomal proteins are oversynthesized, whereas oxidative phosphorylation subunits are downregulated. Expression of
nuclear-encoded components of mitoribosomes and oxidative phosphorylation system (OXPHOS) complexes seems to be less
affected. The ultimate coordination of expression of the nuclear and mitochondrial genomes occurs at the complex assembly
level. These findings indicate that mitoribosomes can regulate gene expression by varying the efficiency of translation of mRNAs
for OXPHOS and ribosomal proteins.
INTRODUCTION
Bigenomic biogenesis is a characteristic feature of most mitochondrial complexes, such as those of the oxidative phosphorylation system (OXPHOS) and mitoribosomes. The stable
stoichiometry of these complexes implies that the synthesis of
their subunits must be tightly coordinated both within and between the two genomes. The coordination between the nuclear
and mitochondrial genomes could be achieved, in principle, at
various levels (i.e., transcriptional, RNA maturation and degradation, translational, and posttranslational).
The regulation of expression of plant mitochondrially encoded
genes is not fully understood. Some results suggest a correlation between the number of mitochondrial gene copies and the
steady state level of their transcripts (Janska et al., 1998; Hedtke
et al., 1999; Kühn et al., 2009; Shedge et al., 2010), but there are
also data supporting a lack of such a correlation (Woloszynska
et al., 2006; Preuten et al., 2010). Differences in the gene promoter strength were observed between plant mitochondrial
genes (Brennicke et al., 1999; Fey and Maréchal-Drouard, 1999)
1 Current
address: Department of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Krakow,
Poland.
2 Address correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Hanna Janska (janska@
ibmb.uni.wroc.pl).
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.111294
even for genes coding for components of the same complex
(Giegé et al., 2000). However, the transcriptional activity appears
to have only a small effect on transcript abundance because it is
largely counterbalanced by processes at different steps of RNA
maturation and degradation (Giegé et al., 2000; Leino et al.,
2005; Holec et al., 2008). Therefore, it is thought that modulation
of transcription is not essential for regulation of expression of
genes encoded in mitochondria and that posttranscriptional
processes are more important. It should be emphasized that
hardly anything is known about the regulation of plant mitochondrial gene expression at the translational level (Bonen,
2004). The importance of regulation at the posttranslational level
was highlighted by the finding of selective proteolysis of proteins synthesized from unedited mRNA (Binder and Brennicke,
2003) and by data demonstrating that the mitochondrial gene
expression remained more or less unaffected by sugar starvation at the transcriptional, posttranscriptional, and translational
levels, but was modulated at the posttranslational level (Giegé
et al., 2005).
Coordination of the expression of some OXPHOS genes between the nuclear and mitochondrial genomes at the transcript
level has been reported during, for example, flower development
in sunflower (Helianthus annuus; Smart et al., 1994; Ribichich
et al., 2001), wheat (Triticum aestivum) leaf development
(Topping and Leaver, 1990), and early steps of rice (Oryza sativa)
germination (Howell et al., 2009). On the other hand, an evident
lack of coordination between mitochondrial and nuclear transcripts of OXPHOS genes has been found in global analyses
during sugar starvation (Giegé et al., 2005) and germination in
Arabidopsis thaliana (Law et al., 2012). The sugar starvation
The Plant Cell Preview, www.aspb.org ã 2013 American Society of Plant Biologists. All rights reserved.
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study has revealed that such coordination is achieved at the
posttranslational level, possibly at the level of complex assembly (Giegé et al., 2005). Furthermore, that study suggested that
excess unassembled subunits of the complexes were rapidly
degraded by ATP-dependent proteases, which had earlier been
identified in plant mitochondria (Janska et al., 2010), and their
deficit was shown to compromise the assembly/stability of
OXPHOS complexes (Kolodziejczak et al., 2007).
Despite the controversy over the transcriptional coordination
of mitochondrial and nuclear genes during the biogenesis of
mitochondrial complexes, the coordination of expression of
nuclear genes themselves is well documented. This regulation
operates through the interaction of nuclear transcription factors
with specific sequence motifs in promoter regions of nuclear
genes encoding mitochondrial proteins. Numerous promoters
of nuclear genes encoding OXPHOS components from Arabidopsis and rice contain a sequence known as site II motif
(Welchen and Gonzalez, 2006), whose binding by TCP transcription factors ensures coordinated expression (Giraud et al.,
2010; Hammani et al., 2011). Furthermore, a global study has
shown that expression of mitochondrially encoded genes involved in energy production and gene expression in mitochondria is also highly coregulated at the transcript level, even more
tightly than are their nuclear counterparts (Leister et al., 2011).
In plants, all mitochondrial OXPHOS complexes, except
complex II, comprise subunits encoded in both the mitochondrial and the nuclear genome. In Arabidopsis, complex I contains
the largest proportion of mitochondrially encoded subunits (nine
out of the total 30) (Heazlewood et al., 2003), while complex III
contains the least (one of 10) (Braun et al., 1994). Mitoribosomes
also have a dual genetic origin; however, their exact composition
is unknown. In Arabidopsis, the mitochondrial genome encodes
four ribosomal proteins of the small subunit (SSU), three of the
large subunit (LSU), and all three rRNA species (Bonen, 2004). The
full repertoire of nuclear-encoded mitoribosomal proteins remains
to be established.
One of the known components of the SSU of Arabidopsis
mitoribosomes encoded in the nucleus is the S10 protein.
Transgenic lines with a decreased transcript level of ribosomal
protein S10 (RPS10) have been generated using the RNA interference method (Majewski et al., 2009). Homozygous Arabidopsis plants presented severe morphological abnormalities
and either died during vegetative growth or were rescued by
reversion. Among the hemizygous plants, several phenotype
categories were identified. These categories were correlated
with the timing of the onset of silencing but not with its efficiency. The role of mitochondrial S10 protein in plants is
unknown, but in bacteria its homolog, called as NusE, is
a multifunctional protein (Kaczanowska and Rydén-Aulin, 2007).
NusE is one of the proteins that complete the ribosomal assembly and is involved in transcriptional antitermination and in
tethering the ribosome to the RNA polymerase (Baumann et al.,
2010).
In this work, we analyzed the effects of the silencing of the
RPS10 gene encoding one of the SSU proteins on the expression of mitochondrial and nuclear genes encoding constituents
of OXPHOS complexes and of mitoribosomes. Our findings reveal that alterations in the mitoribosome divergently affect the
relative efficiency of translation of different types of mitochondrial mRNA. As a consequence, the amount of mitochondrially
encoded ribosomal proteins is higher in rps10 plants than in the
wild type, while the opposite is true for mitochondrially encoded
OXPHOS subunits. These findings establish that differential
regulation of translation in plant mitochondria can be caused by
alterations in mitoribosome status. We also confirmed using
plants rather than tissue culture the earlier suggestion of Giegé
et al. (2005) that the coordination of expression of the mitochondrial and nuclear genomes occurs at the complex assembly
level.
RESULTS
Two Phenotypes Are Generated by Different Timing of
RPS10 Silencing
Two phenotypes of the rps10 Arabidopsis hemizygous transformants, P2 and P3, differing by the onset of RPS10 silencing
were reported earlier (Majewski et al., 2009). In the P2 phenotype, the silencing occurs at the very early stage of vegetative
growth and in consequence the decreased level of RPS10
transcripts is observed in all rosette leaves. Since the silencing
is associated with leaf morphological abnormalities, all leaves
of P2 plants have an altered shape. By contrast, the developmentally late silencing characteristic of the P3 phenotype
begins in newly emerging leaves of 7-week-old plants and results in rosettes comprising leaves of different morphology; the
older ones are similar to the wild type, while the younger leaves
resemble those of P2. An analysis of the unaltered and altered
leaves of P3 plants showed a correlation between the reduced
RPS10 transcript level and leaf morphology at different developmental stages of Arabidopsis (Figure 1). Unless noted
otherwise, all analyses described below were performed using
the youngest leaves harvested from 9- to 10-week-old P2, P3,
and wild-type plants growing under short-day conditions. At that
Figure 1. Decreased Level of RPS10 Transcript Correlates with Altered
Leaf Morphology during Growth of P3 Phenotype of rps10.
The values obtained were averaged for three biological replicates, with
error bars representing SD. WT, the wild type.
Translation in Arabidopsis Mitochondria
age, the young leaves of both P2 and P3 plants exhibited abnormal morphology and decreased RPS10 transcript. It should
be emphasized that the efficiency of silencing is similar in the
altered leaves of P2 and P3 plants (Majewski et al., 2009).
Increased but Imbalanced Amounts of Mitoribosomal
Subunits Are Present in rps10 Plants
To check if the RPS10 silencing affects mitochondrial ribosome
biogenesis, the level of mitoribosomal subunits was quantified
based on rRNA abundance. As rRNAs are unstable when unassembled, the rRNA level can serve as a proxy for ribosomal
subunit accumulation (Walter et al., 2010). We determined the
abundance of cytosolic (cyt 18S and cyt 25S), mitochondrial (mt
18S and mt 26S), and chloroplast (chl 16S and chl 23S) rRNAs
(Figure 2). Since the S10 protein is a component of the SSU of
the mitoribosome, a deficit of mitochondrial SSU and, consequently, also of 18S rRNA was expected in P2 and P3 plants.
Surprisingly, we found an increased amount of mitochondrial
18S rRNA in the mutant compared with the wild type (Figure 2A;
the ratio mt 18S rRNA/cyt 18S rRNA was 1 for the wild type and
;1.5 for both rps10 phenotypes). Moreover, the abundance of
the mitochondrial rRNA of LSU, 26S rRNA, was even more increased (Figure 2A; the ratio mt 26S rRNA/cyt 25S rRNA was
;3.5). We then calculated the ratio of LSU to SSU for the cytosolic, mitochondrial, and chloroplast ribosomes based on the
respective rRNA levels (Figure 2B). A significant increase of that
ratio was only found for mitoribosomes in both the P2 and P3
phenotypes (Figure 2B; approximately twofold relative to the
;1:1 stoichiometry expected and found for wild-type plants),
Figure 2. Accumulation of rRNAs as a Proxy for Corresponding Ribosomal Subunits in P2 and P3 Phenotypes of rps10 Compared with WildType Plants.
(A) Ratio of mitochondrial to cytosolic rRNAs. WT, the wild type.
(B) Ratio of rRNAs of large and SSUs in cytosolic, mitochondrial, and
chloroplast ribosomes. The values obtained were averaged for four biological replicates, with error bars representing SD. Statistically significant
differences from the wild type are indicated by asterisks (Student’s t test;
P < 0.05).
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indicating an imbalance between mitoribosomal subunits. This
conclusion is additionally supported by an approximately threefold higher level of a polynucleotide phosphorylase-like protein,
an enzyme known to be involved in 18S rRNA degradation
(Perrin et al., 2004), in the P2 and P3 phenotypes (3.06 6 0.12
and 2.78 6 0.05, respectively) compared with wild-type plants
(1.00 6 0.06).
To quantitate the abundance of the mitoribosomal subunits in
rps10 in an independent manner, we estimated the amounts of
three mitoribosomal proteins, S4 and S10 of SSU and L16 representing LSU, using immunoblotting (Figure 3; see Supplemental
Figure 1 online). S4 and L16 are encoded mitochondrially,
whereas S10 is a nuclear-encoded protein. Both S4 and L16 were
more abundant in rps10 than in wild-type plants, but the increase
was substantially higher for L16 as calculated relative to total
mitochondrial protein. This result supports the above conclusion
based on rRNA quantity that the LSUs of mitoribosomes are
in excess over SSUs in the rps10 plants. However, the amount
of the S10 protein was comparable in the mitochondrial fraction of
rps10 and wild-type plants. This difference in the abundance of
the S10 and S4 proteins suggests that in rps10 plants, a fraction
of the mitochondrial SSUs were not fully assembled, lacking at
least the S10 protein.
OXPHOS Complexes and Their Subunits Are Less Abundant
in rps10 Mitochondria
The effect of the increased but altered biogenesis of mitoribosomes on the abundance and activity of OXPHOS complexes
was investigated by blue-native gel electrophoresis (BN-PAGE;
Figure 4). The intensity of separated bands stained with Coomassie blue (Figures 4A and 4C; protein level) or after specific
histochemical staining (Figures 4B and 4C; activity level) was
quantified. A significantly lower abundance of OXPHOS complexes was observed in rps10 compared with the wild type, both
as protein amount and activity level. The exception was complex
III that was reduced to a much lesser extent, while complex I
was the most affected. This suggests an interesting correlation
as complex III has only one and complex I the highest number
of mitochondrially encoded subunits among all OXPHOS
complexes.
Subsequently, immunoblotting was performed using antibodies
against one mitochondrially encoded and one nuclear-encoded
subunit of complexes I, III, IV, and V (Figure 5; see Supplemental
Figure 1 online). We consistently observed an approximately
similar reduction of the amount of mitochondrially and nuclear–
encoded subunits for complex I (NAD9 and 49 kD; ;40% of wildtype level), III (COB and CYTC1; ;60% of wild-type level), and IV
(COX2 and COXVC; ;25% of wild-type level) but not for complex
V. There, the level of a nuclear-encoded subunit was markedly
less affected (ATP2; ;50% of wild-type level) compared with a
mitochondrially encoded one (ATP6; ;15% of wild-type level).
The evident imbalance between the above mitochondrially
and nuclear-encoded subunits of complex V prompted us to
check the overall stoichiometry of the subunits of this complex
using two-dimensional BN-PAGE in the P2 phenotype of the
rps10 mutant. In contrast with the imbalance described above,
the relative proportion of protein subunits of complex V was
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to fourfold). Although a similar trend was observed for both
phenotypes, the upregulation of the mitochondrially encoded
genes was more pronounced in P3 than in P2. Furthermore, the
abundance of the majority of the nuclear-encoded transcripts
studied tended to be unchanged in P3, but slightly decreased in
Figure 3. Quantification of Ribosomal Proteins in Mitochondrial Fractions from P2 and P3 Phenotypes of rps10 and Wild-Type Plants.
Protein gel blots of equal amounts (20, 10, and 5 mg) of total mitochondrial proteins were probed with antibodies against S4, S10, and
L16. Representative immunoblots are shown in Supplemental Figure 1
online. Intensities of stained bands were analyzed with Image Quant
Software and averaged for the three protein loadings. The values obtained were averaged for three independent experiments, with error bars
representing SD. Statistically significant differences from the wild type
(WT) are indicated by asterisks (Student’s t test; P < 0.05).
nearly the same. The ratio of the four analyzed subunits of
complex V was as follows: ATP1 and ATP2/ATP g/FAd was
1/0.19/0.10 in wild-type plants and 1/0.15/0.08 in P2 phenotype
of the rps10 mutant.
Mitochondrial DNA Content and Abundance of
Mitochondrial mRNAs Are Upregulated in rps10 Plants
Since the biogenesis of OXPHOS complexes and mitoribosomes
is bigenomic, we analyzed the consecutive steps of the expression of the mitochondrial and nuclear genomes to understand the
effect of RPS10 silencing on the biogenesis of the mitochondrial
complexes.
First, we investigated the abundance of mRNAs encoding
representative components of mitoribosomes and OXPHOS
complexes, both mitochondrially and nuclear encoded, using
quantitative RT-PCR (Figure 6). The abundance of mitochondrially encoded transcripts was increased, while that of nuclearencoded ones was unaltered or slightly decreased for all the
complexes surveyed in rps10. The level of mitochondrial transcripts for most of the ribosomal proteins analyzed showed
a somewhat stronger upregulation (twofold up to even eightfold)
than did transcripts encoding the OXPHOS subunits (1.2-fold up
Figure 4. Mitochondrial Respiratory Complexes Separated by BN-PAGE
from P2 Phenotype of rps10 and Wild-Type Plants.
(A) Coomassie blue staining. WT, the wild type.
(B) In-gel activity staining.
(C) Quantification of the protein amount (top panel) and activity (bottom
panel) of individual OXPHOS complexes in the P2 phenotype of rps10
compared with wild-type plants. Intensities of stained bands were analyzed with Image Quant Software. The values obtained were averaged for
three independent experiments, with error bars representing SD. Statistically significant differences from the wild type are indicated by asterisks
(Student’s t test; P < 0.05).
Translation in Arabidopsis Mitochondria
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Figure 5. Mitochondrially and Nuclear-Encoded OXPHOS Proteins in Mitochondrial Fractions from P2 and P3 Phenotypes of rps10 Compared with
Wild-Type Plants.
Protein gel blots of equal amounts (20, 10, and 5 mg) of total mitochondrial proteins were probed with antibodies against the following: I, NAD9, 49 kD;
III, COB, CYTC1; IV, COX2, COXVC; V, ATP6, ATP2. Representative immunoblots are shown in Supplemental Figure 1 online. Intensities of stained
bands were analyzed with Image Quant Software and averaged for the three protein loadings. The values obtained were averaged for three independent
experiments, with error bars representing SD. Statistically significant differences between mitochondrially and nuclear-encoded subunits within the same
complex are indicated by asterisks (Student’s t test; P < 0.05). WT, the wild type.
P2. We also noticed that the mitochondrial mRNAs encoding
subunits of complexes I and IV were more elevated (twofold up
to fourfold) than those encoding subunits of complex III and V
(2.5-fold or less) in the both phenotypes. This observation is
interesting in the light of the earlier finding that RPOTm, one
of the RNA polymerases present in Arabidopsis mitochondria,
more efficiently transcribes genes encoding subunits of complexes I and IV (Kühn et al., 2009).
The fact that all of the mitochondrially encoded transcripts
tested were elevated in rps10 prompted us to check if that increase could be a consequence of mitochondrial genome amplification. To quantify the mitochondrial genome relative to the
nuclear DNA content, we first established the ploidy of the nuclear genome in leaf cells using flow cytometry. 2C, 4C, 8C, and
16C nuclei were detected (see Supplemental Figure 2 online).
Surprisingly, the mean C-values in P2 and P3 plants (5.02 6
0.42 and 4.34 6 0.47, respectively) were higher than in the wild
type (2.92 6 0.08; P = 0.05, Duncan’s test). This finding implies
that the silencing of the RPS10 gene stimulated endoreduplication. Then, using quantitative PCR, we estimated DNA
level for several mitochondrial genes encoding OXPHOS and
ribosomal proteins. These values were corrected for the nuclear
genome endopolyploidization to give the absolute number of
mitochondrial gene copies per cell. Figure 7 presents copy
numbers of individual mitochondrial genes in wild-type and
rps10 cells expressed as log2 ratios. All of the mitochondrial
genes quantitated gave similar results: They were present in
;4.5- to 6.5-fold more copies in the rps10 cells than in the wildtype ones. Given the above results (Figure 7), we reanalyzed the
data presented in Figure 6 to establish the relationship between
the copy number of mitochondrial genes and their RNA abundance. For several of the ribosomal proteins, the increase of
their mRNA abundance reflected closely the increase in the
gene copy number, implying that the accumulation of those
transcripts was simply due the elevated mitochondrial DNA level.
For the remaining ribosomal proteins, and all of the OXPHOS
subunits, the increase in abundance of their transcripts was not
as high and more variable. Thus, the level of those mitochondrial
transcripts is most probably determined by a combined effect of
the elevated cellular mtDNA level and the variable selectivity of
RNA polymerases.
Number of Mitochondria Is Unaffected in rps10 Mutants
The elevated number of mitochondrial gene copies per cell
could result from an increased DNA content per mitochondrion
and/or an increased number of mitochondria per cell. To assess
the overall number of mitochondria per cell, we prepared protoplasts from P2, P3, and wild-type plants and counted the
mitochondria using MitoTracker GreenFM staining and confocal
scanning microscopy. The mean number of mitochondria was
slightly higher in P2 and P3 compared with wild-type protoplasts
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Figure 6. Steady State Transcript Levels of Mitochondrial and Nuclear Genes Encoding Subunits of OXPHOS and Mitoribosomes in P2 and P3
Phenotypes of rps10 Compared with Wild-Type Plants.
The abundance of transcripts is expressed as a log2 ratio, in the following order: nad2, nad4, nad6, 76 kD, B14 SU, PSST SU, cob, CYTC1, 11 kD,
MPPa, cox1, cox2, cox3, COXVC, COXVB, COXVIB, atp6, atp8, atp9, ATP2-1, ATP2-3, ATPd, rpl2, rpl5, rpl16, rps3, rps4, rps7, rps12, RPS14, and
RPS24. The values obtained were averaged for four biological replicates, with error bars representing SD. WT, the wild type.
(see Supplemental Table 1 online), but that difference was not
statistically significant. Given the similar numbers of mitochondria in all three types of cells and the markedly different numbers
of mitochondrial gene copies per cell, one must conclude that
most if not all the rps10 mitochondria contain significantly more
mitochondrial DNA copies than do wild-type mitochondria.
with the wild type (Figure 8A). For the nad9 transcript, the shift
was observed only for P3. These shifts indicate that the number of
ribosomes present on the examined transcripts is lower in the
rps10 mutant, suggesting a lower efficiency of translation. The
second parameter describing translatability also implies a decrease in mitochondrial translation for most of the OXPHOS
The Translation Status of OXPHOS and Mitoribosomal
mRNAs Is Differentiated in rps10 Plants
The perturbation of the assembly/stability of mitoribosomes in
rps10 seemed likely to affect mitochondrial translation. To check
for this, we determined the translational activity of individual
mRNAs encoding OXPHOS and mitoribosomal proteins in rps10
and wild-type plants. mRNA attached to ribosomes (polysomal
fraction) was separated from unbound mRNA (nonpolysomal
fraction) by Suc gradient centrifugation of total RNA extracts
from P2, P3, and wild-type leaves, followed by quantitative
analysis of specific transcripts by RT-PCR. Two parameters
related to translatability of mRNA were analyzed: ribosomal
density (the number of ribosomes present on a transcript visualized in the mRNA distribution across the polysome fraction)
and ribosomal loading (the proportion of ribosome-bound
mRNA expressed as the percentage of total mRNA). Representative data are shown in Figure 8 and Supplemental Figure 3
online.
For the mitochondrially encoded cob, cox2, and atp6 transcripts, the mRNA distribution in the density gradient was shifted
toward lighter fractions in both phenotypes of rps10 compared
Figure 7. Mitochondrial Gene Copy Numbers in P2 and P3 Phenotypes
of rps10 Compared with Wild-Type Plants.
The values were calculated per nucleus by multiplying the ratios of mitochondrial genes to nuclear DNA (ACT2 gene) by the mean C-value and
are expressed as log2 ratios. The data were averaged for four biological
replicates, with error bars representing SD. Genes are presented in the
following order: I, nad9; III, cob; IV, cox2; V, atp6; mitoribosomes: rpl2,
rpl5, rpl16, rps3, rps4, rps7, and rps12. WT, the wild type.
Translation in Arabidopsis Mitochondria
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Figure 8. Distribution of mRNAs across Polysome Profiles for OXPHOS and Mitoribosomal Mitochondrial and Nuclear Genes.
(A) Transcripts of mitochondrially encoded genes (nad9, cob, cox2, atp6, rpl2, rpl5, rpl16, rps3, rps4, rps7, and rps12).
(B) Transcripts of nuclear-encoded genes (76 kD, CYTC1, COXVC, ATP2-1, RPL29, RPS14, and RPS24). Specific mRNA was analyzed quantitatively
over the entire profile (fractions 1 to 10) by real-time PCR. Graphs represent percentage of total mRNA present in each fraction for wild-type (WT), P2,
and P3 plants. The distribution of mRNA in the gradients was compared for P2 and P3 phenotypes of rps10 and wild-type plants, and shifts of their
maxima to lighter or heavier gradient fractions relative to the wild-type maximum are indicated by arrows.
transcripts, although the decrease in ribosomal loading was
considerably variable (Figure 8A; see Supplemental Figure 3A
online). The only exception was the cob mRNA, showing a higher
proportion in the polysomal fraction. The increased polysome
loading with the reduced ribosomal density found for the cob
transcript explains why the decrease of the COB protein amount
in rps10 was less drastic than that observed for the other mitochondrially encoded OXPHOS subunits (Figure 5). Conversely,
enhanced translation was found for the majority of the mitochondrially encoded ribosomal transcripts as judged by both
a shift to heavier polysomes in rps10 compared with the wild
type (rpl2, rpl5, rps4, and rps7; Figure 8A) and strongly enhanced
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loading onto polysomes (for all the mitochondrially encoded ribosomal mRNAs; Figure 8A; see Supplemental Figure 3A online).
A similar analysis of the nuclear-encoded transcripts revealed
almost unaffected cytoplasmic translation in the P2 phenotype
(Figure 8B; see Supplemental Figure 3B online). In P3, a small
decrease in ribosomal loading was accompanied by a higher ribosome density on translating mRNAs. Considering the contrasting
changes of ribosomal density and loading, we predict no substantial changes in the translation efficiency of the nuclear-encoded
OXPHOS and mitoribosomal transcripts in the P3 phenotype either.
A clear exception was the COXVC mRNA showing a significant
decrease in polysome loading in both P2 and P3 phenotypes.
The Pattern of Proteins Synthesized in rps10 Mitochondria
Differs from That in the Wild Type
To provide an independent evidence for aberrant mitochondrial
translation in rps10, we conducted in organello protein synthesis
using isolated mitochondria from the P2 and P3 rps10 mutants
and wild-type plants. Radioactively labeled mitochondrial proteins were separated by SDS-PAGE electrophoresis and visualized by autoradiography (Figure 9). As we expected from the
affected association of mRNAs with polysomes, the pattern of
labeled proteins synthesized by mitochondria of P2 and P3
plants differed notably from the wild type. The profile of proteins
synthesized in mitochondria from wild-type plants resembled
that shown by Giegé et al. (2005) and Kühn et al. (2009). By
contrast, several of those bands were hardly visible in P2 and P3
mitochondria (Figure 9, open arrowhead), while some bands
barely detectable in wild-type mitochondria were very strong in
P2 and P3 (Figure 9, closed arrowhead). These results confirm
that mitochondrial translation is misregulated in the rps10 mutant with enhanced synthesis of certain proteins and repression
of others. Based on the polysome analysis results, we postulate
that at least some of the proteins observed almost exclusively
in P2 and P3 after the in organello translation are mitoribosomal
proteins, while some of those hardly visible are OXPHOS subunits. This prediction is supported by the data presented in
Supplemental Figure 4 online. The apparent molecular masses
of some proteins synthesized preferentially in mitochondria of
rps10 mutants or wild-type plants determined from their
electrophoretic mobility matched well the theoretical sizes
of several mitochondrially encoded ribosomal or OXPHOS
proteins, respectively.
Mitochondrial ATP-Dependent Proteases Are Upregulated
in rps10 Plants
The data reported here indicate that only the efficiency of synthesis of mitochondrially encoded proteins is significantly altered
during biogenesis of OXPHOS complexes in rps10 mutants, but
nevertheless at the steady state protein level the ratio of both
mitochondrially and nuclear-encoded components is similar, at
least for the representative subunits of complexes I, III, and IV.
This discrepancy suggests that the excess of synthesized proteins unassembled into the dual-origin complexes should be
removed. To test that prediction, we estimated the abundance
of mitochondrial ATP-dependent proteases known to digest
unassembled/damaged proteins (Janska et al., 2010, 2013). An
increased level of transcripts was detected only for the Lon
proteases, LON1 and LON4 (Figure 10A), which degrade mainly
soluble proteins. Furthermore, despite unaltered transcript levels, the protein abundance of FtsH4 and FtsH10 proteases was
also significantly increased in rps10 compared with wild-type
mitochondria (Figure 10B). These proteases have been shown to
degrade membrane proteins like OXPHOS subunits in yeast.
DISCUSSION
Figure 9. In Organello Protein Synthesis.
Autoradiogram of proteins synthesized for 30 and 60 min by mitochondria isolated from P2 and P3 phenotypes of rps10 and wild-type (WT)
plants. After synthesis, 25 µg of mitochondria was fractionated by 12%
(w/v) SDS-PAGE. The lane marked C. bact. (P3) is a specific control
performed in mitochondria isolated from P3 in the presence of a substrate for bacterial translation. Open arrowheads, protein bands less
abundant in P2 and P3 than in wild-type plants; closed arrowheads,
protein bands undetectable or less abundant in wild-type plants.
In this study, we have shown that a deficit in the ribosomal
S10 protein leads to an imbalance between ribosome subunits
in Arabidopsis mitochondria. Furthermore, a portion of SSUs are
incomplete, lacking at least the S10 protein. Next, we found that
the expression of mitochondrially encoded components of mitoribosomes and OXPHOS complexes is markedly modulated in
response to the ensuing perturbations. As a consequence, the
ribosomal proteins are overexpressed, whereas the oxidative
phosphorylation system subunits are downregulated. The expression of nuclear-encoded components of mitoribosomes and
OXPHOS complexes seems to be less affected. The final coordination of expression of the mitochondrial and nuclear genomes
occurs at the level of protein complex assembly, probably with an
involvement of mitochondrial ATP-dependent proteases.
To explain how the RPS10 silencing affects mitoribosome
biogenesis, we propose the following scenario. The silencing
provokes a compensatory response increasing the abundance
Translation in Arabidopsis Mitochondria
Figure 10. Abundance of Mitochondrial ATP-Dependent Proteases in
P2 and P3 Phenotypes of rps10 and Wild-Type Plants.
(A) Steady state transcript level. The values obtained were averaged for
four biological replicates, with error bars representing SD. WT, the wild
type.
(B) Steady state protein level. Protein gel blots of equal amounts (20, 10,
and 5 mg) of total mitochondrial proteins were probed with antibodies
against FtsH4 and FtsH10. Quantification of intensities of stained bands
was done with Image Quant Software and averaged for the three protein
loadings. Values obtained were averaged for three independent experiments, with error bars representing SD. Statistically significant differences
from the wild type are indicated by asterisks (Student’s t test; P < 0.05).
of the components of both LSU and SSU of mitoribosomes.
However, the upregulation is imbalanced since the S10-deficient
SSU are unstable and therefore prone to degradation, resulting in
an excess of LSU. It should be emphasized that owing to the
feedback response, the steady state level of the S10 protein is
nearly the same in the mutant and wild-type mitochondria. Thus,
the rps10 mutant does not suffer from an absolute insufficiency
of functional mitoribosomes but rather from the excess of LSU
and/or the presence of a portion of partially assembled SSU. That
such defective complexes are formed is indicated by an excess of
another SSU component, S4, over S10. The goal of this study
was to evaluate how these changes in the mitoribosome status
affect the multiple levels of the bigenomic biogenesis of mitoribosomes themselves and oxidative phosphorylation complexes.
The first step of the feedback response in rps10 occurs at the
mitochondrial DNA level. A 4.5- to 6.5-fold increase in the mitochondrial gene copy number was detected in the P2 and P3
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phenotypes. This increase was similar for all the genes tested,
suggesting amplification of the whole mitochondrial genome.
The observed upregulation was not accompanied by a proportional increase in the number/mass of mitochondria but
was reflected in enhanced steady state levels of mitochondrial
transcripts. The best correlation between the gene copy number
and the transcript level was found for several ribosomal proteins.
Enhanced transcript levels as a consequence of elevated mitochondrial gene copy numbers have been reported earlier
(Hedtke et al., 1999; Kühn et al., 2009; Shedge et al., 2010) and
suggested to reflect a feedback response to energy constraints
caused by defects in mitochondria (Kühn et al., 2009). In contrast with the upregulated mitochondrial transcripts, the abundance of nuclear-encoded mRNAs of both mitoribosomes and
OXPHOS complexes was unchanged or only slightly decreased.
Thus, an evident lack of coordination between nuclear and mitochondrial gene expression at the transcript level was detected
in the rps10 mutant.
The data regarding polysomes revealed substantial alterations
in mitochondrial translation in the rps10 plants. In contrast with
the transcript level, where the same trend was noted for both
mitoribosome and OXPHOS proteins, a divergent translational
response was observed. Generally, within mitochondria, the
majority of transcripts of OXPHOS subunits are less actively
translated, whereas transcripts of most mitoribosome proteins
are preferentially synthesized. This contrasting response was
preserved at the protein steady state level, where the OXPHOS
subunits were present at reduced levels, while ribosomal proteins overaccumulated. The translation efficiency of nuclearencoded mRNAs for mitoribosome and OXPHOS components
was much less affected. However, there was a significant reduction in the steady state protein level of the nuclear-encoded
OXPHOS subunits of complexes I, III, and IV. Moreover, this
decrease was comparable to that found for the mitochondrially
encoded components of the same complexes. Thus, it seems
that the nuclear-encoded OXPHOS proteins, after almost unchanged synthesis, are degraded to bring their abundance
down to the level of the mitochondrially encoded components.
No similar analysis could be performed for mitoribosomes due
to a lack of antibodies against their nuclear-encoded constituents,
except for S10.
The conclusion that the coordination of expression of mitochondrial and nuclear genes coding for subunits of OXPHOS
complexes occurs posttranslationally was reached earlier by
Giegé et al. (2005) in their study of the biogenesis of Arabidopsis
OXPHOS complexes under sugar starvation conditions. They
postulated that the coordination takes place at the level of
protein complex assembly. Our results further confirmed that
suggestion. The nuclear-encoded subunit of complex V was
reduced in rps10 to a markedly lesser extent than its mitochondrially encoded one, but the stoichiometry of the complex
V subunits was virtually identical to that in wild-type mitochondria. Apart from this similarity, there are also differences in the
feedback responses initiated by sugar starvation and by RPS10
silencing. The response to the RPS10 silencing is mainly associated with changes in mitochondrial genome expression,
whereas under sugar starvation the biogenesis of OXPHOS
complexes was regulated mostly by changes in nuclear genome
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The Plant Cell
expression. As a consequence, the limiting factor in the biogenesis of OXPHOS complexes in response to sugar deprivation
is the availability of nuclear-encoded subunits, while in the case
of RPS10 silencing it is the availability of mitochondrially encoded ones. Our results argue that the excess unassembled
mitochondrial proteins are degraded by ATP-dependent proteases. We found transcriptional activation of the Lon1 and Lon4
proteases in both phenotypes of rps10 and upregulation of
FtsH4 and FtsH10 proteases at the posttranscriptional level. The
possibility of translational regulation of FtsH10 expression had
been reported earlier (Piechota et al., 2010).
Although the molecular details remain to be determined, our
data indicate that the altered translation found in rps10 mitochondria is caused by changes of at least two factors: ribosomal
loading and density. The reduced ribosomal densities found for
all the mitochondrial OXPHOS transcripts tested explains at
least partially the reduced steady state levels of the corresponding OXPHOS subunits. It seems that the diverse decrease
in the steady state levels of individual OXPHOS proteins reflects
the differences in the ribosomal loading of their mRNAs. Conversely, the highly increased steady state levels of mitochondrially
encoded ribosomal proteins result from enhanced ribosomal
loading and for majority of proteins also from increased ribosomal density. Taken together, the perturbation in mitoribosomes resulting from RPS10 silencing leads to extensive
and diverse changes in mitochondrial translation reflected in the
steady state protein level. The mitoribosomal proteins are overaccumulated, while the OXPHOS subunits are present at reduced
levels in rps10. This demonstrates that differential changes of the
efficiency of translation in response to compromised ribosomal
assembly/stability occur in plant mitochondria. Autoregulation of
ribosome biosynthesis by translational control has been reported
for a fission yeast mutant depleted of Arg-methylated ribosomal
protein S2 (Bachand et al., 2006). This mutant, like the Arabidopsis rps10, shows an imbalanced level of small and large ribosomal subunits. However, in the yeast mutant, an enhanced
translation of several mRNAs encoding proteins of SSU was
observed with little variation in the overall mRNA abundance.
Translational regulation of ribosome biogenesis has also been
documented in bacteria (Nomura, 1999), mammalian cells
(Meyuhas and Hornstein, 2000), and chloroplasts (Fleischmann
et al., 2011).
Coupling of the rate of synthesis of organellar proteins to the
rate of their assembly into complexes is a common feature of
several related mechanisms known as control by epistasy of
synthesis (CES) (Choquet and Vallon, 2000). In this context, it
is feasible that a CES-like regulation triggered by partially assembled mitoribosome may operate to activate translation of
ribosomal proteins in rps10 mitochondria. However, the regulation of translation by the assembly status of the mitoribosome
observed in the rps10 mutant is not limited to the components
of the mitoribosome itself, as would be postulated by the CES
model, but also concerns the OXPHOS subunits, being therefore
more general. The effect of the accumulation of free LSU and the
presence of a portion of defective SSU on the efficiency of
protein synthesis in plant mitochondria could be based on the
formation of atypical interactions between those subunits and
other components of the translation apparatus. The ribosome
filter hypothesis posits that ribosomes are not simply translation
machines but also function as regulatory elements that differentially affect or filter the translation of particular mRNAs (Mauro
and Edelman, 2007). Our findings support the hypothesis in that
the misregulation of the mitoribosome subunits in rps10 differentially affects the translational status of particular mitochondrial
transcripts. We further postulate that similar translational regulation could occur in wild-type plants. Variation in the interactions between the ribosome subunits could be triggered by
posttranslational modifications of ribosomal proteins. In this
respect, it is interesting to note that methylation of the S10
protein in bacteria affects the assembly of ribosomes and protein synthesis (Ren et al., 2010).
Surprisingly, we found that whereas most nuclei in wild-type
plants were diploid (mean C-value ;3), the mutants nuclei exhibited a higher ploidy (mean C-value ;4 to 5). However, this
change is not dramatic in the light of data showing that the mean
C-value of nuclear DNA varies from 4 up to 13 during maturation
and aging of Arabidopsis leaves (Zoschke et al., 2007). Recently,
it was shown that the mammalian homolog of the S10 protein,
like many other ribosomal proteins, has a secondary function as
a cell proliferation regulator (Ren et al., 2010; Abbas et al., 2012).
Thus, it is highly likely that the plant S10 also has an extraribosomal
function connected with cell proliferation and a deficiency of this
protein leads to endoreduplication. Further studies will be needed
to test this hypothesis.
METHODS
Plant Material and Growth Conditions
Wild-type and mutant Arabidopsis thaliana plants were of the Columbia-0
ecotype. The transgenic lines of rps10 were generated using the RNA
interference method (Majewski et al., 2009). Seeds of wild-type plants
were germinated on half-strength Murashige and Skoog, supplemented
with kanamycin (50 mg L21) for the rps10 mutant. Seedlings were grown
for 3 d at 4°C in the dark followed by 11 d in an 8-h-light/16-h-dark (short
day) photoperiod at 22°C with light intensity of 100 mmol m22 s21. After
that time, the wild-type and the kanamycin-resistant rps10 plants were
transferred to the soil and grown in a growth chamber (short day, 22°C,
100 mmol m22 s21).
Nucleic Acid Isolation and cDNA Synthesis
Nucleic acids were extracted from fresh plant leaves and stored at –20°C.
Genomic DNA was isolated using a GeneMATRIX Plant and Fungi DNA
purification kit (EURx). Total RNA was isolated using a GeneMATRIX
Universal RNA purification kit (EURx). The reverse transcription reaction
was performed using up to 2 mg of total RNA and a reverse transcription
kit (Applied Biosystems). The resulting cDNA was used as a template for
quantitative real-time PCR.
Protoplast Isolation, Confocal Imaging, and Determination of the
Number of Mitochondria per Cell
Protoplasts were isolated from leaves using the method described by Yoo
et al. (2007). Isolated protoplasts were pelleted at 1000g for 1 min at room
temperature and resuspended in 50 mL MMg solution (0.4 M mannitol,
15 mM MgCl2, and 4 mM MES, pH 5.7) containing 200 nM MitoTracker
Green FM (Molecular Probes), prewarmed at 37°C. Images of a half-
Translation in Arabidopsis Mitochondria
protoplast were acquired as a z-series with 0.39-µm intervals using
a Zeiss LSM510 confocal laser scanning microscope equipped with
a 360 water immersion objective. The MitoTracker GreenFM stain was
visualized with a 488-nm argon laser and a BP505-530 filter. The number
of mitochondria was estimated using an approach similar to that of
Preuten et al. (2010). Twenty randomly chosen protoplasts from each
of three separate isolations were analyzed for P2 and P3 phenotypes of
rps10 and for wild-type plants. The number of mitochondria per protoplast
was determined using Imaris Software 7.6.1 (Bitplane).
Flow Cytometric Analysis of Nuclear DNA Content
For flow cytometry, samples were prepared as described previously (Sliwinska
et al., 2012). Single leaves were chopped in 1 mL of nuclei isolation buffer
supplemented with propidium iodide (50 mg mL21) and RNase A (50 mg
mL21) and analyzed using a CyFlow SL Green flow cytometer (Partec)
equipped with a high-grade solid-state laser with green light emission at
532 nm, long-pass filter RG 590 E, DM 560 A, and side and forward scatters.
For each sample, the DNA content of 3000 to 7000 nuclei was measured.
Analyses were performed on at least six replicates using a logarithmic
amplification. Histograms were evaluated using the FloMax program
(Partec), and the percentage of nuclei with particular DNA contents and
the mean C-value (Lemontey et al., 2000) were calculated before applying
a one-way analysis of variance and the Duncan’s test.
Isolation of Polysomal RNA
Polysomes were fractionated from homogenized leaf tissue as described
previously by Kahlau and Bock (2008) with slight modifications. Leaves
(200 mg) were ground in 1 mL of freshly prepared extraction buffer (200 mM
Tris-HCl, pH 9.0, 200 mM KCl, 35 mM MgCl 2 , 25 mM EGTA,
200 mM Suc, 100 mM b-mercapthoethanol, 1% [v/v] Triton X-100, 2% [v/v]
polyoxyethylene-10-tridecyl ether, 6% [w/v] digitonin, 1 mg/mL heparin,
100 mg/mL chloramphenicol, and 25 mg/mL cycloheximide) and centrifuged at 4°C and 13,200g for 5 min to remove cell debris. The cleared
extract was supplemented with 1/20 volume of 10% (w/v) sodium deoxycholate and fractionated in a Suc density gradient (56:40:30:15% [w/v]
Suc in 40 mM Tris-HCl, pH 8.5, 20 mM KCl, 10 mM MgCl2, 100 mg/mL
chloramphenicol, and 500 mg/mL heparin) by ultracentrifugation (4°C,
200,000g, 80 min). Ten aliquots of ;410 mL each obtained by fractionation were subjected to RNA extraction using phenol/chloroform/isoamyl
alcohol (25:24:1). Control gradients supplemented with puromycin were
used to identify fractions containing polysomes (fractions 6 to 10) and free
mRNA and ribosomes (fractions 1 to 5). RNA isolated from all of these
fractions was subjected to 2.5 M LiCl treatment to remove the heparin.
Reverse transcription was performed as described above and cDNA used
for quantitative real-time PCR.
11 of 13
single data acquisition; cooling, 40°C for 30 s. The specificity of the
amplification products was verified by analysis of the melting curve. The
primers used are listed in Supplemental Table 2 online.
Isolation of Mitochondria and Gel Electrophoresis of Proteins
Isolation of mitochondria for SDS-PAGE and BN-PAGE experiments was
performed as described by Urantowka et al. (2005), whereas functional
mitochondria for in organello protein synthesis were isolated as described
by Lister et al. (2007). SDS-PAGE electrophoresis as well as protein and
catalytic staining of BN-PAGE gels were performed as described by
Kolodziejczak et al. (2007).
Protein Gel Blot Analysis
Equal amounts of mitochondrial proteins (20, 10, and 5 mg) were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride
membrane (Bio-Rad). Blots were probed with antibodies against the following OXPHOS subunits: NAD9 (obtained from P. Giegé, France), 49 kD
(obtained from L. Sweetlove, UK), COB (obtained from L. Sweetlove),
CYTC1 (obtained from P. Giegé), COX2 (purchased from Agrisera), COXVC
(obtained from P. Giegé), ATP6 (obtained from Christiane D. Chase, USA),
and ATP2 (obtained from C. Knorpp, Sweden). Furthermore, blots were also
probed with antibodies purchased from Agrisera, against Arabidopsis mitoribosomal proteins, anti-S10, anti-S4, and anti-L16, as well as against
mitochondrial proteases, anti-FtsH4 and anti-FtsH10. Anti-rabbit antibodies
conjugated with horseradish peroxidase were used as secondary antibodies and visualized with enhanced chemiluminescent ECL reagent.
Chemiluminescence was recorded using a chemiluminescence imager
(BioDoc-It Imaging System; UVP). Bands were quantified using QuantityOne software (Bio-Rad).
In Organello Protein Synthesis
Mitochondrial proteins (150 mg) were resuspended in a solution containing 5 mM KH2PO4, pH 7.0, 400 mM mannitol, 60 mM KCl, 50 mM
HEPES, 10 mM MgCl2, 10 mM malic acid, 1 mM pyruvate, 2 mM GTP,
2 mM DTT, 4 mM ADP, 0.1% (w/v) BSA, 25 mM unlabeled 19–amino acid
solution, and 30 mCi [35S]Met (>1000 Ci/mmol). Reactions were performed
in 100 mL for 30 and 60 min at 25°C on an orbital shaker and stopped by
addition of puromycin (50 mg/mL) and 350 mL mitochondria wash buffer
containing 10 mM unlabeled Met. In control experiments, 25 mM
Na-acetate was used instead of malic acid and pyruvate. Radiolabeled proteins were separated by SDS-PAGE and visualized by
autoradiography.
Accession Numbers
Real-Time PCR Analysis
The real-time PCR analyses were performed using DNA or cDNA on
a LightCycler 2.0 instrument (Roche Applied Science). The Real-Time
2x PCR Master Mix SYBR version B (A&A Biotechnology) was used.
Reactions were performed in a total volume of 15 mL with a final concentration of 0.5 mM primers. Relative quantification analysis using the
second derivative maximum method of the LightCycler 4.0 software was
used. The wild-type plants served as the calibrator, and the ACT2 gene
(At3g18780) was used as a reference. The values of amplification efficiency of the analyzed amplicons were calculated on a standard curve.
The standard curves were generated using six serial twofold dilutions of
the cDNA or DNA samples obtained from wild-type plants. The amplification protocol consisted of the following: denaturation, 95°C for 1 min;
amplification, 45 cycles at 95°C for 10 s, 50 to 65°C (the annealing
temperature was specific for used primers) for 10 s, 72°C for 20 s with
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: AtMg00090 (rps3), AtMg00290 (rps4), AtMg01270 (rps7),
At3g22300 (RPS10), AtMg00980 (rps12), At2g34520 (RPS14), At5g28060
(RPS24), AtMg00560 (rpl2), AtMg00210 (rpl5), AtMg00080 (rpl16),
At1g07830 (RPL29), AtMg00285 (nad2), AtMg00580 (nad4), AtMg00270
(nad6), AtMg00070 (nad9), At5g37510 (76 kD), At3g12260 (B14 SU),
At5g11770 (PSST SU), AtMg00220 (cob), At5g40810 (CYTc1), At1g15120
(11 kD SU), At1g51980 (MPP a), AtMg01360 (cox1), AtMg00160 (cox2),
AtMg00730 (cox3), At1g80230 (COXVb), At2g47380 (COXVc), At4g28060
(COXVIb), AtMg01170 (atp6), AtMg00480 (atp8), AtMg01080 (atp9),
At5g08670 (ATP2-1), At5g08690 (ATP2-3), At2g33040 (ATPg), At5g26860
(LON1), At3g05790 (LON4), At5g23140 (CLPP2), At5g53350 (CLPX ),
At2g29080 (FTSH3), At2g26140 (FTSH4), At1g07510 (FTSH10), and
At3g18780 (ACT2).
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The Plant Cell
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Representative Immunoblots of Total Mitochondrial Protein Isolated from Wild-Type, P2, and P3 Phenotypes of
rps10 Plants.
Supplemental Figure 2. Selected DNA Histograms of Nuclear
Preparations from Wild-Type Plants and P2 and P3 Phenotypes of
rps10.
Supplemental Figure 3. Ribosome Loading for Mitochondrially and
Nuclear-Encoded Gene Transcripts in P2 and P3 Phenotypes of rps10
and Wild-Type Plants.
Supplemental Figure 4. Identification of Mitochondrially Encoded
Proteins Whose Expression Is Affected by the Mitoribosome Defect.
Supplemental Table 1. Number of Mitochondria per Cell.
Supplemental Table 2. Primers Used for qRT-PCR.
ACKNOWLEDGMENTS
We thank Christine Chase, Carina Knorpp, Philippe Giegé, and Lee
Sweetlove for supplying the antibodies for OXPHOS complexes. This
work was supported by Grant N N301 784940 from the Ministry of
Science and Higher Education, Poland.
AUTHOR CONTRIBUTIONS
H.J. designed the study. M.K., P.M., R.S., A.A., M.C., and E.S. performed
experiments. H.J., M.K., R.S., A.A., M.C., and E.S. analyzed data. H.J.
wrote the article with significant input from M.K.
Received March 7, 2013; revised May 3, 2013; accepted May 10, 2013;
published May 30, 2013.
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Silencing of the Nuclear RPS10 Gene Encoding Mitochondrial Ribosomal Protein Alters
Translation in Arabidopsis Mitochondria
Malgorzata Kwasniak, Pawel Majewski, Renata Skibior, Aleksandra Adamowicz, Malgorzata Czarna,
Elwira Sliwinska and Hanna Janska
Plant Cell; originally published online May 30, 2013;
DOI 10.1105/tpc.113.111294
This information is current as of June 17, 2017
Supplemental Data
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