全 文 ：Received 29 May 2003 Accepted 28 Aug. 2003
Supported by the National Natural Science Foundation of China (30070475), “948” Fund in Ministry of Agronomy of China (201010) and the
State Key Basic Research and Development Plan of China (2001CB108).
* Author for correspondence. E-mail: .
http://www.chineseplantscience.com
R Region of S Type of Cytoplasmic Male Sterility in Maize
Mitochondrial DNA is Transcribed in Both Directions
and May Be Associated with Male Sterility
ZHANG Sai-Qun, ZHANG Fang-Dong, XIAO Hai-Lin, ZHENG Yong-Lian*
(State Key Laboratory of Crop Genetic Improvement and National Center of Crop Molecular Breeding,
Huazhong Agricultural University, Wuhan 430070, China)
Abstract: It is well accepted that S type of cytoplasmic male sterility (CMS) in maize (Zea mays L.) is
associated with R region in mitochondrial genome. R region includes two open reading frames — orf355
and orf77 and it is speculated that orf77 is an important candidate gene of CMS. RT-PCR showed that both
DNA strands of R region are transcribed. Nuclear background or developing stages of plant do not influence
the transcription of R region in the strand which also acts as template strand of coxⅠ/coxⅡ locating just
upstream of R region, but they do influence the transcription of R region in the other strand (template
strand of orf355-orf77). In tassel with nuclear background of rf3rf3, transcription of the template strand of
orf355-orf77 is different from that in etiolated shoots with nuclear background of Rf3- or rf3rf3 and tassel
with nuclear background of Rf3-. Compared to the same other three, it is truncated at about 238 base from
the 5 end of R region. Rabbit antiserum against putative ORF77 expressed in Escherichia coli was prepared
but Western blot did not detect ORF77 in plant materials. It seems that double-strand transcription of R
region inhibits the translation of orf77 and the transcribing mode of R region may be associated with male
sterility.
Key words: maize; S type of CMS; mitochondrial DNA; R region; double-strand transcription
Cytoplasmic male sterility (CMS), a common phenomenon
in higher plants, is maternally inherited and has been demon-
strated to be related to the unique arrangements of the mito-
chondrial genome in different plants studied (Hanson, 1991).
CMS in maize was originally classified into three groups, S
(USDA), T (Texas) and C (Charrua) based on the nuclear genes
that can restore them to fertility (Beckett, 1971). Mitochon-
drial genome of CMS-S maize consists of one circular “mas-
ter” DNA molecule and two linear double-stranded DNA
episomes, named S1 and S2. Both episomes have identical
terminal inverted repeats (IR). Similar IR sequences are part of
a recombinant repeat designated R, present in the “master”
DNA at the junction of region σ-σ and Y-Y. Recombination
between the IRs of episomes and R results in the rearrange-
ment of mitochondrial genome and several polymorphism of
R region (Schardl et al., 1984; Schardl et al., 1985; Zabala et
al., 1997). It is well accepted that CMS-S in maize is associated
with the recombinational region R. Northern blotting analysis
showed that mRNA transcription in microspore of CMS-S
sterile plants changed in Rf3- nuclear background (Wen and
Christin, 1999a; 1999b; Zabala et al., 1997). Sequence analy-
sis showed that R region includes two open reading frames
(orf355 and orf77) (Zabala et al., 1997). Both coxⅠ and coxⅡ
are reported locating just upstream of R region (Schardl et al.,
1984; Zhang and Zheng, 2000). Figure 1 shows the structure of
R region.
1 Materials and Methods
1.1 Plant materials
The characteristics of the maize (Zea mays L.) lines used
in this study are listed in Table 1.
1.2 Preparation of mitochondrial protein, antiserum
against ORF77 and Western blot
Mitochondria were isolated according to the method of
Singh and Brown (1991). Then suspended mitochondria in
the solution including 8 mol/L urea, 4% CHAPS, 40 mmol/L
Tris-HCl; bathed in ice, centrifuged, prepared the superna-
tant for SDS-PAGE electrophoresis. At the same time, puta-
tive ORF77 was expressed in Escherichia coli using fusion
protein expression vector PGEK-KG and purified according
to Guan and Jack (1991). Immunization was then programmed
in rabbits and the antiserum was used in Western blot.
1.3 Primers for revert-transformation and PCR
Primers for revert-transformation including: oligo(dT)18;
P1 (5 -CGTCGGCGATCCTCTACTATT-3); P2 (5 -
C C A A C G AT T C C T G T C T A C T T G C - 3 ) ; P 3 ( 5 -
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 337-341
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004338
CGTCTAGAGAGCACGATTCGATATT-3); P4(5 -
GCTCATGACCTAATCAATCCACTCATCG-3). The target
sites of the primers are shown in Fig.1.
1.4 Preparation of mitochondrial RNA and RT-PCR
analysis
Mitochondria were isolated as above, then Trizol (Gibco,
USA) was applied to extract RNA. Prior to the synthesis of
first-strand cDNA, total RNA was treated with DNaseⅠ.
First-strand cDNA is synthesized with RevertaidTM H Mi-
nus First Strand cDNA Synthesis kit #K1631 (MBI,
Fermentas). PCR was performed after digesting the first-
strand cDNA with RNase H and RNase A.
1.5 Sequence analysis of products from RT-PCR
Amplified fragments were reclaimed and constructed to
pGEM®-T Easy Vector (Promega, USA). Sequence analy-
sis was then finished in Shangon Biotechnology Company
(Shanghai, China).
2 Results
2.1 Western blotting analysis of plant mitochondrial
protein
To investigate the expression of orf77 at translation level
under different nuclear backgrounds, rabbit antiserum
against putative ORF77 was prepared. The specificity of
the antiserum was tested in Western blot on protein ex-
tracted from E. coli expressing the 26 kD glutathione S-
transferase (GST) fused to 9 kD putative ORF77 and a 35
kD protein was detected. However, when 150 mg of mito-
chondrial proteins were analyzed in Western blot, no spe-
cific bands could be identified.
2.2 RT-PCR analysis of the transcription of R region
RT-PCR experiments with primer oligo(dT)18 on all four
samples showed the same amplified cDNA fragment of 1.6
kb with P1 and P2 (Fig.2A, lanes 5-8 ). When using P2
which is located in the 3 terminal of R region for RT, differ-
ent PCR results with P1 and P2 appeared, only sample 3 had
an amplified cDNA fragment of 1.6 kb, but the other three
had none (Fig.2A, lanes 1-4). Two pairs of primers P3 and
P4, P3 and P2 were then used as PCR primers respectively
and the same amplified fragments of 1.1 kb (Fig.2C, lanes
1-4), 1.4 kb (Fig.2C, lanes 5-7) were obtained accordingly
in all four samples. When using P1 anchored in the IR se-
quence of R region for RT and then P1 and P2 for PCR, the
two same fragments of 1.6 kb, 1.0 kb appeared in all samples
(Fig.2B, lanes 9-12). All the results are shown in Table 2.
2.3 Sequence analysis result of RT-PCR products
Sequence analysis showed that all the amplified frag-
ments from RT-PCR of 1.6 kb, 1.4 kb and 1.1 kb above were
almost the same as the DNA sequence of R region. But the
fragments of all 1.0 kb amplified by P1 and P2 were totally
different from the sequence in R region and sequence
BLAST showed that they were almost the same as the DNA
sequence in episome S1.
Because there is no similar sequence to P2 in the ampli-
fied product of 1.0 kb and a pair of invert repeat sequence
of P1 locates on its 5 terminal and 3 terminals, it can be
deduced that the 1.0 kb fragment actually amplified from
mRNA of episome S1 only by P1. Furthermore PCR by P1
confirmed this speculation (Fig.2D).
2.4 Based on all the data above, several conclusions can
be made as follows
2.4.1 Reverse transcription succeeded with both specific
primers P1 and P2 showed that there are two opposite tran-
scription directions of R. One is the same as that of coxⅡ
(defined as positive direction), the other is opposite (defined
as negative direction).
Table 1 Names, characteristics and genotypes of materials
Sample Name Fertility Restorer genotype Source part for mtRNA
1 Tangxu 77 Sterile rf3rf3 Etiolated shoots
2 Tangxu HZ85× S7913 Fertile Rf3rf3 Etiolated shoots
3 Tangxu HZ85 Sterile rf3rf3 Tassels
4 Tangxu 77×HZ129 Fertile Rf3rf3 Tassels
Fig.1. Structure of R region. Total R region comprised 1 727 bp. IR sequence of 208 bp is at the upstream of R region. P1, P2, P3 and
P4 are primers for RT-PCR. P1 is in the IR sequence, from +68 to +88 of R region. P2 is from +1 703 to +1 723 of R region. P3 and P4
are in orf355 from +238 to +1 395 of R region. Short dark arrows show the directions of primers. Long gray arrows show the transcription
directions of coxⅠ/coxⅡ and orf355, orf77 in R region.
339ZHANG Sai-Qun et al.: R Region is Transcribed in Both Directions in Maize S-CMS
2.4.2 Reverse transcription with oligo(dT)18 is in the same
direction as that of P1 and the mRNA template is in positive
direction.
2.4.3 Nuclear background or developing period of plant
do not influence the transcription of R region in positive
direction, but they do influence the transcription of R re-
gion in the negative direction.
2.4.4 The transcription product of R region in negative
direction in tassel of nuclear background of rf3fr3 is differ-
ent from that in etiolated shoots with nuclear background
of Rf3- or rf3fr3 and that in tassel with nuclear background
of Rf3-. RT-PCR result implied that the difference is limited
to the sequence between P1 and P3 (from IR sequence to
+238) of negative mRNA strand.
2.4.5 Perhaps R region can not be translated to proteins
because its mRNA exists in double strand. Western blot
result of ORF77 just coincide with this hypothesis.
3 Discussion
Novel open reading frames (ORFs), which are often chi-
meric in structure, have been found to be associated with
CMS in different plants (Hanson, 1991). Fertility restorer
Table 2 RT-PCR results
Primers for
Primers for PCR
Size of amplified fragment of RT-PCR (kb)
RT Sample 1 Sample 2 Sample 3 Sample 4
Oligo(dT)18 P1, P2 1.6 1.6 1.6 1.6
P 1 No amplified band No amplified band No amplified band No amplified band
P 1 P1, P2 1.6, 1.0 1.6, 1.0 1.6, 1.0 1.6, 1.0
P 1 1.0 1.0 1.0 1.0
P 2 P1, P2 No amplified band No amplified band 1.6 No amplified band
P3, P4 1.1 1.1 1.1 1.1
P3, P2 1.4 1.4 - 1.4
Fig.2. RT-PCR results. A. Lanes 1-4, P2 is as revert transcription primer, P1 and P2 are PCR primers; lanes 5-8, oligo(dT)18 is as revert
transcription primer, P1 and P2 are PCR primers. Lane 1, sample 4; lane 2, sample 3; lane 3, sample 2; lane 4, sample 1; lanes 5-8,
samples 1-4. B. P1 is as revert transcription primer, P1 and P2 are PCR primers. Lanes 9-12, samples 1-4. C. P2 is as revert
transcription primer. Lanes 1-4, P3 and P4 are as PCR primers; lanes 5-8, P3 and P2 are as PCR primers. Lanes 1-4, samples 1-4; lane
5, sample 1; lane 6, sample 2; lane 7, sample 4. D. Oligo(dT)18 is as revert transcription primer for lanes 1 to 4 and 9, P1 is as revert
transcription primer for lanes 5 to 8 and 10. P1 is as PCR primer for lanes 1 to 8. P1 and P2 are as PCR primers for lanes 9 and 10. Lanes
1-4, samples 1-4; lane 5-8, samples 1-4; lane 9, sample 1; lane 10, sample 1. M1, 200 bp ladder; M2, 100 bp ladder.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004340
gene (Rf) can restore pollen fertility by regulating the
transcription, post-transcription editing, translation or post-
translation of these ORFs (Wise et al., 1987; Abad et al.,
1995; Singh et al., 1996; Tang et al., 1996; Bergman et al.,
2000; Bentolila et al., 2002).
In this experiment, RT-PCR revealed the double-strand
transcribing mode and the lengths of transcriptions of R
region (including orf77). It was found that mRNA of R re-
gion in tassel with sterile nuclear background is different
from that in tassel with fertile nuclear background or eti-
olated shoot with either of the two types of the nuclear
background. It can be concluded that this transcriptional
difference of R region is associated with the sterility of
tassel.
Among the sequence of orf77, there are three stretches
similar to atp9 of mitochondria, so it is presumed that S-
type male sterility could result from a partial impairment of
the mitochondrial function as a result of the expression of
orf77. The ORF77 antiserum did react with ORF77 expressed
in E. coli in Western analysis, but we did not detect ORF77
in plant mitochondrial proteins under the same condition
for Western blot. So we can deduce that orf77 does not
translate in sterile or fertile plant in different developing
stages. Perhaps double strand orf77 mRNA inhibits the
translation of orf77 and the sterility of S type CMS of maize
is only associated with the transcription of R region.
Both coxⅠ and coxⅡ are reported locating just up-
stream of R region. From RT-PCR results in this experiment
and bioinformation analysis, we can deduce that polyA
belongs to mRNA of coxⅠ or coxⅡ and one strand in R
region co-transcribe with coxⅠ or coxⅡ. Although RT-
PCR analysis in this experiment can not show the initiation
site of transcription in the DNA strand including orf355-
orf77 in sterile tassel, it does show that the transcribed
product is longer in sterile tassel than that in fertility re-
store tassel, so perhaps coxⅠ or coxⅡ has part or whole
double-strand mRNA in sterile tassel and results in abnor-
mally transcription of itself and sterile phenotype; but in
fertile tassel or etiolated shoots, mRNA of coxⅠ or coxⅡ
is in single-strand and can translate normally.
Double-strand mRNA of CMS-related segment is also
found in Vicia faba (Pfeiffer, 1998) and the analysis of orf274
in mtDNA associated with male-sterile tobacco showed a
similar Western blot result (Bergman et al., 2000) as that of
orf77 in this experiment. These results may mean some-
thing about the same mechanism of plant CMS.
Northern blotting analysis with single-strand cDNA
probe of R region and construction of S type CMS maize
mitochondrial cDNA library progressing in our laboratory
will contribute to test the hypothesis above and explain the
CMS mechanism to further degree.
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