Download DCW11, Down-Regulated Gene 11 in CW-Type

Plant Cell Physiol. 49(4): 633–640 (2008)
doi:10.1093/pcp/pcn036, available online at www.pcp.oxfordjournals.org
ß The Author 2008. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of
this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and the Japanese Society of Plant
Physiologists are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its
entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact [email protected]
DCW11, Down-Regulated Gene 11 in CW-Type Cytoplasmic Male Sterile Rice,
Encoding Mitochondrial Protein Phosphatase 2C is Related to Cytoplasmic
Male Sterility
Sota Fujii and Kinya Toriyama*
Laboratory of Environmental Biotechnology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutumidori-Amamiyamachi,
Aoba-ku, Sendai, 981-8555 Japan
produce functional pollen, and it is widely utilized for F1
hybrid breeding. Beside their commercial use, CMS studies
contribute to a better understanding of the plant nuclear–
mitochondrial intracellular genomic barrier. For instance,
many researchers have concluded that an aberrant chimeric
gene in mitochondria possibly induces CMS in various
plant species. On the contrary, nuclear fertility restorers
have been genetically identified to ameliorate the ectopic
protein derived from a chimeric gene (Hanson and Bentolila
2004). In addition to the above research, a recent review has
pointed out that the cause of CMS is thought to involve the
mitochondria to nucleus signaling pathway, the so-called
mitochondrial retrograde signaling pathway (Zubko 2004).
For example, wheat APETALA3 homolog WAP3 was
considerably down-regulated in alloplasmic wheat expressing the pistillody phenotype (Murai et al. 2002). Linke
et al. (2003) found that MADS box genes DcMADS2 and
DcMADS3, which are homologous to Antirrhinum
GLOBOSA and DEFICIENS, are down-regulated in CMS
carrot. The APETALA3 gene was found to be expressed
ectopically in CMS Brassica napus (Geddy et al. 2004).
Teixeira et al. (2005) found evidence of a retrograde
influence of the expression levels of various homeotic genes.
Retrograde responses derived from mitochondrial
stress have been widely investigated in yeast and mammalian cells (Butow and Avadhani 2004). The RTG pathway is
one of the most investigated systems of mitochondrial
retrograde signaling. This pathway is characterized by two
basic helix–loop–helix-leucine zipper (bHLH-Zip) transcription factors, Rtg1p and Rtg3p, that are thought to
function in activation of the retrograde genes. In addition,
the ATP-binding protein Rtg2g acts as the mediator of
mitochondrial signals to Rtg1p and Rtg3p (Sekito et al.
2000). Thus, genes that possess the function of signaling and
transcriptional regulation are likely to be involved in
retrograde signaling. It is likely that pathways similar to
the yeast or animal stress-derived mitochondrial retrograde
response exist in plants. Mitochondrial-mediated signaling
in wild-type plants is perhaps essential for normal male
sporophyte or gametophyte development. Such factors
related to retrograde signaling could be revealed as CMS
Causes of cytoplasmic male sterility (CMS) in plants have
been studied for two decades, and mitochondrial chimeric genes
have been predicted to induce CMS. However, it is unclear
what happens after CMS-associated proteins accumulate in
mitochondria. In our previous study of microarray analysis, we
found that 140 genes are aberrantly regulated in anthers of
CW-type CMS of rice (Oryza sativa L.). In the present study,
we investigated DCW11, one of the down-regulated genes
in CW-CMS encoding a protein phosphatase 2C (PP2C).
DCW11 mRNA was preferentially expressed in anthers, with
the highest expression in mature pollen. As predicted by the
N-terminal sequence, DCW11 signal peptide–green fluorescent protein (GFP) fusion protein was localized in mitochondria. Knockdown of DCW11 in wild-type rice by RNA
interference caused a major loss of seed-set fertility, without
visible defect in pollen development. Since this knockdown
phenotype resembled that of CW-CMS, we concluded that the
down-regulation of DCW11 is correlated with CW-CMS. This
idea was supported by the up-regulation of alternative oxidase
1a (AOX1a), which is known to be regulated by mitochondrial
retrograde signaling, in DCW11 knockdown lines. Downregulation of DCW11 and up-regulation of AOX1a were also
observed in two other types of rice CMS. Our result indicates
that DCW11 could play a role as a mitochondrial signal
transduction mediator in pollen germination.
Keywords: Cytoplasmic male sterility (CMS) —
Mitochondrial retrograde signaling — Oryza sativa —
Protein phosphatase 2C.
Abbreviations: AOX, alternative oxidase; CaMV, cauliflower
mosaic virus; CMS, cytoplasmic male sterility; DCW, downregulated in CW-CMS; GFP, green fluorescent protein; PP2C,
protein phosphatase 2C; PPR, pentatricopeptide repeat; RNAi,
RNA interference; RT–PCR, reverse transcription–PCR; UTR,
untranslated region.
Introduction
Cytoplasmic male sterility (CMS) is a maternally
inherited trait that results in the inability of plants to
*Corresponding author: E-mail, [email protected]; Fax, þ81-22-717-8834.
633
634
CMS-related mitochondrial protein phosphatase 2C
Fig. 1 Alignment of the DCW11 amino acid sequence with the deduced At5g53140 sequence. The broken line indicates the region of
predicted mitochondrial targeting peptides. The bold line shows the fairly conserved PP2C catalytic domain.
downstream factors in a comprehensive transcriptomic
study of CMS.
Among several types of CMS in rice, we have been
studying three types of gametophytic CMS: BT-CMS,
LD-CMS and CW-CMS (for a review, see Fujii et al.
2008). BT-CMS originated from indica variety Chinsurah
boro II (Shinjyo 1975). Although BT-CMS has been studied
most extensively (Iwabuchi et al. 1993, Kazama and
Toriyama 2003, Wang et al. 2006), we thought it was
unsuitable for a comprehensive transcriptomic study
because the pollen of the BT-CMS line shows apparent
reduction of starch content and abnormal morphology.
Pollen grains of the LD-CMS line, which originated from
the Burnese cultivar Lead Rice, also show reduction of
starch content and abnormal morphology. In contrast,
pollen of the CW-CMS line, which is derived from Oryza
rufipogon Griff., W1 strain, appeared to be normal but lacks
pollen germination ability (Fujii and Toriyama 2005). Thus,
we considered that CW-CMS should be the model for a rice
CMS transcriptomic study using a microarray, so that the
effects of morphological abnormality are minimally
reflected. As a result, we have obtained 140 aberrantly
regulated genes in a CW-CMS plant (Fujii et al. 2007). The
genes differentially regulated in our CMS microarray were
designated as up-regulated gene in CW-CMS (UCW) or
down-regulated gene in CW-CMS (DCW). In order to
identify the core factors that mediate retrograde signaling in
CMS, or CMS downstream factors, we applied a reverse
genetic approach in UCW and DCW genes.
In this study, we analyzed the expressional and
functional characters of one of the DCW genes, DCW11,
which encodes a protein phosphatase 2C (PP2C) gene. The
possible role of DCW11 in CMS induction mechanism of
rice is discussed.
Results
DCW11 encodes a mitochondrial protein phosphatase 2C
protein
DCW11 (accession No. AK069289 in the rice fulllength cDNA database, KOME, http://cdna01.dna.affrc.
go.jp/cDNA/) was identified as a down-regulated gene
under the CW-CMS background by our microarray
analysis (Fujii et al. 2007). DCW11 is predicted to encode
a PP2C family protein. The most similar protein in
Arabidopsis is that encoded by At5g53140 with a similarity
of 46.9% (Fig. 1). The PP2C region is fairly conserved
between the two proteins.
CMS-related mitochondrial protein phosphatase 2C
GFP
Merged
635
Mito Tracker Red
Fig. 2 DCW11 signal peptides target mitochondria. A BY-2 cell expressing GFP fused to the DCW11 putative signal peptide (left),
counterstained with Mito Tracker Red (right), with a merged image (center).
In Arabidopsis, the PP2C family consists of 76 members
(Schweighofer et al. 2004). It has been known that this gene
family is involved in various biological processes such as the
ABA response, the mitogen-activated protein kinase
(MAPK) pathway, or even flower development
(Schweighofer et al. 2004). Seventy-six Arabidopsis PP2Cs
fall into 10 groups (A–J) (Kerk et al. 2002). At5g53140 is
assigned to group F, but the functions of genes in group F
remain unknown (Schweighofer et al. 2004). We searched
for PP2C genes in rice, and found that 79 genes are
predicted to contain a PP2C domain (data not shown).
The PSORT computer program (http://psort.nibb.
ac.jp/) and the Mitoprot web site (http://ihg.gsf.de/ihg/
mitoprot.html) predicted that DCW11 would be imported
into mitochondria, and that the first 59 amino acids are
possibly mitochondrial targeting signal peptides (Fig. 1,
broken line). In order to verify that DCW11 localizes in
mitochondria, we fused the fragment for putative signal
peptides to a gene for green fluorescent protein (GFP)
under the control of the cauliflower mosaic virus (CaMV)
35S promoter, and introduced it into tobacco BY-2 cells by
Agrobacterium-mediated transformation. As expected from
the sequence, DCW11 signal peptide–GFP fusion protein
was shown to co-localize with the Mito Tracker Red signal
(Fig. 2). DCW11, therefore, was confirmed to localize in
mitochondria.
DCW11 is preferentially expressed in anthers, with the
highest accumulation in mature pollen
The expression specificity of DCW11 in wild-type rice
was monitored in anthers at the tetrad stage, uninucleate
microspore stage, bicellular pollen stage and tricellular
pollen stage, as well as in isolated tricellular pollen, stigmas,
seeds, seedlings and roots using quantitative reverse
transcription–PCR (RT–PCR) (Fig. 3). Expression of
DCW11 increased during pollen development in anthers
from the tetrad stage to the tricellular pollen stage. The
highest accumulation of DCW11 mRNA was observed in
tricellular pollen. The expression of DCW11 was not
10000
9000
8000
7000
6000
5000
4000
3000
2000
1000
0
d
tra
Te
i-
Un
-
Bi
i-
ic
Tr
lle
o
rp
la
lu
el
Anthers
n
Tr
as
m
ig
St
gs
s
ed
lin
Se
d
ee
s
ot
Ro
S
Fig. 3 DCW11 mRNA is preferentially expressed in tricellular
pollen. The figure shows the relative DCW11 expression level
compared with that of the tubulin alpha-1 chain (AK069140)
detected by real-time quantitative RT–PCR. Transcript levels were
measured in three biological replicates from cDNA prepared from
respective plant organs. Uni-, Bi- and Tri- indicate anthers at the
uninucleate microspore stage, bicellular pollen stage and tricellular
pollen stage, respectively.
detected in stigmas, seeds, seedlings or roots. This expression profile indicates that DCW11 plays a role in pollen
development.
Knockdown of DCW11 in Taichung 65 results in reduced seed
sets and AOX1a up-regulation
To gain insight into the nature of DCW11 involvement
in CMS, we produced DCW11 knockdown plants by the
RNA interference (RNAi) method. The DCW11 cDNAspecific region in the 30 untranslated region (UTR) of the
gene was cloned for utilization as the trigger for RNAi. We
obtained seven independent RNAi transgenic lines. In five
transgenic plants, expression of DCW11 was shown to be
636
A
CMS-related mitochondrial protein phosphatase 2C
120%
100%
80%
60%
40%
20%
0%
B
120%
100%
80%
60%
40%
20%
0%
C
120%
100%
80%
60%
40%
20%
0%
D
100
80
60
40
20
0
6
4
3
3
1
7
2
5
2
1
1
3
2 3
5 6
-3
US 11- 11- 11- 11- 11- 11- 11US -1- -3- -4- -5- -7- -1- -3- -4- -5-7
i::G CW CW CW CW CW CW CW
i::G 11 11 11 11 11 W1 W1 W1 W1 W1
b
b
U
U CW CW CW CW CW DC DC DC DC DC
D
D D
D
D
D
D
D
D
D
D
D D
KD KD KD KD KD KD KD
KD KD KD KD
KD KD KD KD KD K
T0 generation
T1 generation
with transgenes
S
S
S 65
CM -CM -CM ng
D
W
L ichu
C
Ta
BT
T1 generation
w/o transgenes
Fig. 4 Characterization of DCW11 knockdown plants and three types of CMS. (A) Relative DCW11 expression level in anthers at the
tricellular pollen stage monitored as described in Fig. 3. (B) Seed set and (C) pollen stainability. (D) Relative AOX1a expression level
investigated using the same method as that for DCW11. Scores in each plant are shown as percentages for (A–C) (untransformed Taichung
65 ¼ 100%), and relative expression values in (D) (untransformed Taichung 65 ¼ 1).
knocked down to 4–38% in comparison with that of the
wild-type in the T0 generation (Fig. 4A, KDDCW11-1,
KDDCW11-3, KDDCW11-4, KDDCW11-5, KDDCW11-7).
Seed sets of knockdown lines were reduced to 4–56%,
whereas the control plants with -glucuronidase (gus)
genes were reduced to 75%. Since reduced seed fertility
is often observed in the transgenic T0 generation, we
planted eight plants per line for the T1 generation, of which
DCW11 mRNA was successfully down-regulated in the T0
generation. The seed sets in the T1 generation were reduced
to 15–49%, which was almost the same as that in the
T0 generation (Fig. 4B). T1 plants that lost the RNAi
transgenes showed normal seed-set fertility and normal
DCW11 expression, which proved that reduction of seed
set in knockdown plants was caused by DCW11 mRNA
down-regulation. Since DCW11 mRNA was barely detected
in stigmas or seeds (Fig. 2), we considered that downregulation of DCW11 caused male sterility but did not cause
dysfunction in female organs or seed maturation. This
was supported by the fact that we obtained F1 seeds by
crossing wild-type pollen onto DCW11 knockdown
plants (data not shown). We could not find any morphological defects in the pollen of DCW11 knockdown plants
under light microscopy (data not shown). The values of
pollen stainability were 485% in the T1 generation of
DCW11 knockdown plants, which was the same as that
observed in the CW-CMS line (Fig. 4C). We suspected that
DCW11 functions in pollen germination ability. This
phenotype resembled CW-CMS, in which the pollen
looks normal until it fails to germinate on the stigma.
CMS-related mitochondrial protein phosphatase 2C
8000
637
Discussion
7000
6000
5000
4000
3000
2000
1000
0
S
S
S
65
CM -CM -CM ng
u
D
W
L
C
ich
Ta
BT
Uninucleate
microspores
S
S
S
65
CM -CM -CM ng
u
D
W
L
C
ich
Ta
S
S
S
65
CM -CM -CM ng
u
D
W
L
C
ich
Ta
BT
BT
Bicellular pollen
Tricellular pollen
Fig. 5 mRNA expression of DCW11 in uninucleate microspores, and bicellular and tricellular pollen. Transcript levels
were measured in three biological replicates. Relative transcript abundance was normalized to the levels of tubulin alpha-1
chain.
These results led us to believe that DCW11 could be related
to CW-CMS.
In order to obtain further evidence, we investigate the
expression levels of AOX1a mRNA in DCW11 knockout
lines. AOX1a is known to be up-regulated in the mitochondrial complex I mutant of tobacco (Sabar et al. 2000). Our
previous study detected ectopic overdrive of AOX1a in
CW-CMS mature anthers (Fujii et al. 2007). To the best of
our knowledge, AOX1a is the most common nucleusencoded marker gene for CMS. As expected, the AOX1a
mRNA level was highly overexpressed in anthers at the
tricellular pollen stage in T0 and T1 knockdown lines
(Fig. 4D). These data indicate that repression of DCW11
causes a CW-CMS-like effect, and DCW11 could be
involved in a pathway stimulating AOX1a expression in
rice CMS.
DCW11 is down-regulated in other CMS mature anthers
To determine if DCW11 is involved in other CMS
systems in rice, we checked the expression level of DCW11
in mature anthers of BT-CMS and LD-CMS lines, both of
which exhibit the pollen abortion phenotype (Fig. 4A). We
also isolated total RNA from uninucleate microspores,
bicellular pollen and tricellular pollen of BT-CMS and
LD-CMS lines, as well as CW-CMS and Taichung 65. The
resulting total RNA was subjected to quantitative real-time
RT–PCR. As a result, down-regulation of DCW11 was
evident in pollen at the tricellular stage in all three CMS
cytoplasms, but not in uninucleate microspores or bicellular
pollen (Fig. 5). From these results, we expect that
transcriptional regulation of DCW11 is common in all
these CMS systems.
DCW11 is involved in pollen germination
DCW11 encodes PP2C targeted to mitochondria. The
function of DCW11 or an ortholog of Arabidopsis has not
yet been elucidated. We have analyzed the expressional and
functional characters of DCW11 in wild-type rice.
Knockdown of DCW11 resulted in reduced seed set
although pollen grains of the knockdown lines were visibly
normal.
To date, there have been large numbers of Arabidopsis
male gametophyte mutants identified, with morphologically
unaffected pollen but deficient in male transmission of the
mutant allele. A calmodulin-binding protein mutant no
pollen germination1 exhibited complete loss of T-DNA
transmission through male gametophytes, although no
obvious defects were observed in pollen development
(Golovkin and Reddy 2003). Johnson et al. (2004) screened
a large number of Arabidopsis T-DNA tag-lines and
identified in total 32 hapless mutants, which displayed
distorted genotype segregation. Twenty-nine of them were
deficient in steps later than pollen germination rather than
pollen development. SETH1 and SETH2 are genes encoding glycosylphosphatidylinositol-anchoring proteins, and
essential for maintenance of polarized pollen tube growth
(Lalanne et al. 2004). To the best of our knowledge, there
have been no reports demonstrating that a PP2C-encoding
gene is required for the pollen germination process.
Although we have not yet determined whether DCW11
possesses PP2C activity or not, our results demonstrated
that DCW11 mRNA is accumulated in mature pollen and
functions in pollen germination. Additional information
about the pollen tube growth mechanism could be obtained
by searching the signaling transduction components that
DCW11 is involved in.
Repression of DCW11 results in AOX1a up-regulation
Expression of AOX1a is well known to be regulated by
mitochondrial retrograde signaling (Djajanegara et al.
2002). Zarkovic et al. (2005) designed a forward genetic
screening to isolate the mitochondrial retrograde regulation
factor using mutagenized Arabidopsis carrying the firefly
luciferase gene fused to the AOX1a promoter, resulting in
the Arabidopsis plants with mutations in the genes related to
mitochondrial retrograde regulation failing to activate the
AOX1a promoter under antimycin A treatment. In our case,
such a forward genetic approach using a reporter gene was
difficult due to the nature of CMS. We therefore applied a
reverse genetic approach in DCW genes. Since knockdown
of DCW11 resulted in AOX1a up-regulation in this study
(Fig. 4D), DCW11 could be participating in suppression
of AOX1a mRNA expression under normal conditions,
or the knockdown of DCW11 was causative of the
638
CMS-related mitochondrial protein phosphatase 2C
CMS-like effect. This is also supported by the fact that
AOX1a is up-regulated in BT-CMS and LD-CMS, in which
DCW11 was down-regulated (Fig. 4D). To the best of our
knowledge, there have been no reports on AOX1a negative
regulators, although Zarkovic (2005) has indicated the
existence of a candidate positive regulator. Understanding
the molecular functions of DCW11 would help us interpret
the regulatory network of AOX1a and retrograde signaling
in plants.
In mammals, a recent study on novel mitochondrial
matrix-localized PP2C, PP2Cm, revealed that knockdown
resulted in cell death associated with loss of mitochondrial
membrane potential (Lu et al. 2007). Although DCW11 had
no significant similarities to human PP2Cm (data not
shown), we are not able to rule out the possibility of
DCW11 participating in a part of the cell death program.
Considering that DCW11 homologous At5g53140 in
Arabidopsis belongs to group F of PP2C division and
its function was previously unidentified, some of the
members of group F could be related in mitochondrial
stress signaling.
required for pollen development (for a review, see Hanson
and Bentolila 2004). Since this knockdown phenotype of a
defect in pollen germination ability resembled that of
CW-CMS, we have concluded that the down-regulation of
DCW11 is strongly correlated with CW-CMS. DCW11
could be involved in mitochondrial signal transduction in
pollen development, especially in acquisition of germination
ability.
DCW11 could be the downstream factor of CMS
Although CMS is caused by genetic incompatibility
between nuclei and mitochondria within male reproductive
organs, only a few hints are provided for the essentiality of
mitochondria in pollen development from mutant studies.
Recently, the Arabidopsis mitochondrial complex II mutant
sdh1-1 was isolated and was shown to have deficiency in
both male and female gametophyte development (Leon
et al. 2007). Pollen of the mutants develops normally until
the vacuolated microspore stage, but fails to undergo
mitosis I. The result indicates that mitochondrial complex
II activity may be indispensable for generating a vegetative
nucleus. One of the hapless mutants, hap11, was shown
to encode a mitochondrial ATP synthase subunit (Johnson
et al. 2004). In the case of hap11, the pollen tube growth
path appears normal, but the tube fails to enter the
micropyle. Since mitochondria are ubiquitous organelles
and provide energy to all kinds of cells, mitochondrial mutants should exhibit embryo lethality or early
developmental dysfunctions as they do in the case of the
maize empty pericarp4 (EMP4) mutant (GutierrezMarcos et al. 2007). These authors showed that EMP4
encodes a mitochondrial pentatricopeptide repeat (PPR)
protein. Many mitochondrial PPR mutants are embryonic
lethal in Arabidopsis, indicating that proper mitochondrial
function is critical for early plant development (Lurin
et al. 2004).
Thus, why male-specific defects are observed in CMS is
quite interesting. There might be a special function of
mitochondria in male organ development rather than
energy production, such as regulation of nuclear genes
Plasmid construction and plant transformation
DCW11 RNAi constructs driven by the maize ubiquitin
promoter were produced using the pANDA vector provided by
Dr. Miki and Dr. Shimamoto, following their protocols (Miki and
Shimamoto 2004). A gene-specific region of the 400 bp 30 UTR of
DCW11 (accession No. AK069289 in the rice full-length cDNA
database, KOME http://cdna01.dna.affrc.go.jp/cDNA/) was PCR
cloned by the primer set 50 -CACCCCCTTGCGAAAACA
and
50 -CCGTAATGGCAGGTAATTAG-30 .
GAAGAC-30
Nucleotides CACC were added to the original DCW11 sequence
for cloning into TOPO vector (Invitrogen, Carlsbad, CA, USA).
The resulting RNAi construct was introduced into Agrobacterium
tumefaciens EHA105, and further introduced into plants by the
method described by Yokoi et al. (1997). We also developed rice
lines with the gus gene driven by the maize ubiquitin promoter as a
negative control.
Materials and Methods
Plant materials
Oryza sativa L. japonica cultivar Taichung 65 was used as the
wild type throughout the study. A BT-CMS line was obtained from
a successive backcross of Chinsurah Boro II and Taichung 65
(Shinjyo 1975). A CW-CMS line was derived from four backcrosses of japonica Reimei into O. rufipogon Griff., W1 strain
(Toriyama and Hinata 1987), and five subsequent backcrosses of
Taichung 65. An LD-CMS line was derived from nine backcrosses
of japonica Akihikari into Lead Rice, followed by three successive
crosses of Taichung 65. All of the CMS lines were confirmed to be
occupied mainly by the Taichung 65 nuclear genome by markerassisted selection.
Pollen stainability
Spikelets 1 d before anthesis were harvested, and pollen
grains were stained with 1% (w/v) iodine–potassium iodide
solution. The numbers of darkly stained pollen grains were
counted for 4200 pollen grains per spikelet. Pollen stainability
was calculated as the average of the values of six spikelets. The seed
set percentage was calculated as the average of that of the five
panicles for each plant.
Quantitative RT–PCR analysis
Total RNA from various stages of anthers, pollen, stigmas,
seeds, seedlings and roots was extracted using RNeasy (Qiagen,
Tokyo, Japan) according to the manufacturer’s instructions. DNA
contamination was eliminated by treatment of RNase-free DNase I
(TAKARA-BIO INC., Ohtsu, Japan). cDNA synthesis was
accomplished using the ReverTraAce (TOYOBO, Osaka, Japan).
PCR was performed for three biological replicates, using SYBR
Premix Ex Taq (TAKARA BIO INC.) and Thermal Cycler Dice
Real Time System TP800 (TAKARA BIO INC.).
CMS-related mitochondrial protein phosphatase 2C
RT–PCR was performed using the specific set of primers for
each amplification reaction as follows: 50 -ATGAAGACTT
GGAATGCCTC-30 and 50 -CCGTAATGGCAGGTAATTAG-30
for DCW11; 50 -GTCTACTGCCGAGGATTTGCATCAC-30 and
50 -CCGATACAGCTAAAGAGCTCTC-30 for AOX1a (accession
No. AK064040 in the rice full-length cDNA database, KOME,
http://cdna01.dna.affrc.go.jp/cDNA/); 50 -TACAACGGTTGGC
GTCGCAC-30 and 50 AACTTGCGCACACGGTCCAG-30 for
tubulin alpha-1 chain (accession No. AK069140 in the rice fulllength cDNA database, KOME, http://cdna01.dna.affrc.go.jp/
cDNA/).
Transformation of tobacco BY-2 cells
To analyze the DCW11 cellular localization, a 198 bp
fragment encoding predicted signal peptides was PCR cloned
by primers FORWARD: GGATCCATGGTATGCTTCGCT
AGCCT and REVERSE: GGATCCGGAGTCGAGCATCAT
CCTGG. A BamHI site was added to the original DCW11
sequence for cloning into the CaMV 35S- and GFP-containing
binary vector. Transformation of BY-2 cells was performed as
described by Shaul et al. (1996). Transformed cells were selected by
kanamycin treatment, and counterstained by Mito Tracker Red
(Invitrogen). Cells were observed under fluorescent microscopy.
Funding
The Ministry of Agriculture, Forestry and Fisheries of
Japan (Integrated research project for plant, insect and
animal using genome technology QT-2007); the Ministry of
Education, Science, Sports and Culture, Japan Grant-inAid for Special Research on Priority Areas (No. 18075002).
S.F. is the recipient of Research Fellowships of the Japan
Society for the promotion of Science for Young Scientists.
Acknowledgments
We are grateful to Dr. Kiyoyuki Miura at National
Agricultural Research Center for providing seeds of LD-CMS
lines. pANDA vector was kindly provided by Dr. Miki and
Dr. Shimamoto from the Nara Institute of Science and
Technology.
References
Butow, R.A. and Avadhani, N.G. (2004) Mitochondrial signaling: the
retrograde response. Mol. Cell 14: 1–15.
Djajanegara, I., Finnegan, P.M., Mathieu, C., McCabe, T., Whelan, J. and
Day, D.A. (2002) Regulation of alternative oxidase gene expression in
soybean. Plant Mol. Biol. 50: 735–742.
Fujii, S., Kazama, T. and Toriyama, K. (2008) Molecular studies on
cytoplasmic male sterility-related genes and restorer genes in rice. In Rice
Biology in the Genomics Era. Edited by Hirano, H.Y., Hirai, A.,
Sano, Y. and Sasaki, T. pp. 205–216. Spriger-Verlag, Berlin.
Fujii, S., Komatsu, S. and Toriyama, K. (2007) Retrograde regulation of
nuclear gene expression in CW-CMS of rice. Plant Mol. Biol. 63:
405–417.
Fujii, S. and Toriyama, K. (2005) Molecular mapping of the fertility
restorer gene for rice ms-CW-type cytoplasmic male sterility of rice.
Theor. Appl. Genet. 111: 696–701.
Geddy, R., Mahe, L. and Brown, G.G. (2004) Cell-specific regulation
of a Brassica napus CMS-associated gene by a nuclear restorer
639
with related effects on a floral homeotic gene promoter. Plant J. 41:
333–345.
Golovkin, M. and Reddy, A.S. (2003) A calmodulin-binding protein from
Arabidopsis has an essential role in pollen germination. Proc. Natl Acad.
Sci. USA 100: 10558–10563.
Gutiérrez-Marcos, J.F., Dal Prà, M., Giulini, A., Costa, L.M., Gavazzi, G.,
et al. (2007) empty pericarp4 encodes a mitochondrion-targeted pentatricopeptide repeat protein necessary for seed development and plant
growth in maize. Plant Cell 19: 196–210.
Hanson, M.R. and Bentolila, S. (2004) Interactions of mitochondrial and
nuclear genes that affect male gametophyte development. Plant Cell 16:
S154–S169.
Iwabuchi, M., Kyozuka, J. and Shimamoto, K. (1993) Processing followed
by complete editing of an altered mitochondrial atp6 RNA restores
fertility of cytoplasmic male sterile rice. EMBO J. 12: 1437–1446.
Johnson, M.A., von Besser, K., Zhou, Q., Smith, E., Aux, G., Patton, D.,
Levin, J.Z. and Preuss, D. (2004) Arabidopsis hapless mutations define
essential gametophytic functions. Genetics 168: 971–982.
Kazama, T. and Toriyama, K. (2003) A pentatricopeptide repeat-containing
gene that promotes the processing of abberant atp6 RNA of cytoplasmic
male-sterile rice. FEBS Lett. 544: 99–102.
Kerk, D., Bulgrien, J., Smith, D.W., Barsam, B., Veretnik, S. and
Gribskov, M. (2002) The complement of protein phosphatase catalytic
subunits encoded in the genome of Arabidopsis. Plant Physiol. 129:
908–925.
Lalanne, E., Honys, D., Johnson, A., Borner, G.H., Lilley, K.S.,
Dupree, P., Grossniklaus, U. and Twell, D. (2004) SETH1 and
SETH2, two components of the glycosylphosphatidylinositol anchor
biosynthetic pathway, are required for pollen germination and tube
growth in Arabidopsis. Plant Cell 16: 229–240.
León, G., Holuigue, L. and Jordana, X. (2007) Mitochondrial complex II is
essential for gametophyte development in Arabidopsis. Plant Physiol.
143: 1534–1546.
Linke, B., Nothnagel, T. and Borner, T. (2003) Flower development in
carrot CMS plants: mitochondria affect the expression of
MADS box genes homologous to GLOBOSA and DEFICIENS.
Plant J. 34: 27–37.
Lu, G., Ren, S., Korge, P., Choi, J., Dong, Y., Weiss, J., Koehler, C.,
Chen, J. and Wang, J. (2007) A novel mitochondrial matrix serine/
threonine protein phosphatase regulates the mitochondria permeability
transition pore and is essential for cellular survival and development.
Genes Dev. 21: 784–796.
Lurin, C., Andres, C., Aubourg, S., Bellaoui, M., Bitton, F., et al. (2004)
Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins
reveals their essential role in organelle biogenesis. Plant Cell 16:
2089–2103.
Miki, D. and Shimamoto, K. (2004) Simple RNAi vectors for stable and
transient suppression of gene function in rice. Plant Physiol. 45: 490–495.
Murai, K., Takumi, S., Koga, H. and Ogihara, Y. (2002) Pistillody,
homeotic transformation of stamens into pistil-like structures, caused by
nuclear–cytoplasm interaction in wheat. Plant J. 29: 169–181.
Sabar, M., De Paepe, R. and de Kouchkovsky, Y. (2000) Complex I
inpairment, respiratory compensations and photosynthetic decrease in
nuclear and mitochondrial male sterile mutants of Nicotiana sylvestris.
Plant Physiol. 124: 1239–1249.
Schweighofer, A., Hirt, H. and Meskiene, I. (2004) Plant PP2C
phosphatases: emerging functions in stress signaling. Trends Plant Sci.
9: 236–243.
Sekito, T., Thomson, J. and Butow, R.A. (2000) Mitochondria-to-nuclear
signaling is regulated by the subcellular localization of the transcription
factors Rtg1p and Rtg3p. Mol. Biol. Cell 11: 2103–2115.
Shaul, O., Nironov, V., Burssens, S., Montague, M.V. and Inze, D. (1996)
Two Arabidopsis cyclin promoters mediate distinct transcriptional
oscillation in synchronized tobacco BY-2 cells. Proc. Natl Acad. Sci.
USA 93: 4868–4872.
Shinjyo, C. (1975) Genetical studies of cytoplasmic male sterility and
fertility restoration in rice, Oryza sativa L. Sci. Bull. Coll. Agr. Univ.
Ryukyus 22: 1–57.
Teixeira, R.T., Farbos, I. and Glimelius, K. (2005) Expression levels
of meristem identity and homeotic genes are modified by
640
CMS-related mitochondrial protein phosphatase 2C
nuclear–mitochondrial interactions in alloplasmic male-sterile lines of
Brassica napus. Plant J. 42: 731–742.
Toriyama, K. and Hinata, K. (1987) Anther culture application to breeding
of a restorer for male-sterile cytoplasm of wild rice [ms-CW]. Jpn. J.
Breed. 37: 469–473.
Wang, Z., Zou, Y., Li, X., Zhang, Q., Chen, L., et al. (2006) Cytoplasmic
male sterility of rice with boro II cytoplasm is caused by a cytotoxic
peptide and is restored by two related PPR motif genes via distinct modes
of mRNA silencing. Plant Cell 18: 676–687.
Yokoi, S., Tsuchiya, T., Toriyama, K. and Hinata, K. (1997) Tapetumspecific expression of the Osg6B promoter–-glucuronidase gene in
transgenic rice. Plant Cell Rep. 16: 363–367.
Zarkovic, J., Anderson, S.L. and Rhoads, D.M. (2005) A reporter gene
system used to study developmental expression of alternative oxidase and
isolate mitochondrial retrograde regulation mutants in Arabidopsis.
Plant Mol. Biol. 57: 871–888.
Zubko, M.K. (2004) Mitochondrial tuning fork in nuclear homeotic
functions. Trends Plant Sci. 9: 61–64.
(Received December 10, 2007; Accepted February 25, 2008)