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The Plant Cell, Vol. 16, 1008–1020, April 2004, www.plantcell.org ª 2004 American Society of Plant Biologists
The Novel Gene HOMOLOGOUS PAIRING ABERRATION
IN RICE MEIOSIS1 of Rice Encodes a Putative Coiled-Coil
Protein Required for Homologous Chromosome Pairing
in Meiosis
Ken-Ichi Nonomura,a,1 Mutsuko Nakano,a Toshiyuki Fukuda,a,b Mitsugu Eiguchi,a Akio Miyao,c
Hirohiko Hirochika,c and Nori Kurataa,b
a Experimental
Farm/Plant Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
of Life Science, Graduate University for Advanced Studies/Sokendai, Mishima, Shizuoka 411-8540, Japan
c Department of Molecular Genetics, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba,
Ibaraki 305-8602, Japan
b Department
We have identified and characterized a novel gene, PAIR1 (HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1),
required for homologous chromosome pairing and cytokinesis in male and female meiocytes of rice (Oryza sativa). The pair1
mutation, tagged by the endogenous retrotransposon Tos17, exhibited meiosis-specific defects and resulted in complete
sterility in male and female gametes. The PAIR1 gene encodes a 492–amino acid protein, which contains putative coiled-coil
motifs in the middle, two basic regions at both termini, and a potential nuclear localization signal at the C terminus.
Expression of the PAIR1 gene was detected in the early stages of flower development, in which the majority of the
sporocytes had not entered meiosis. During prophase I of the pair1 meiocyte, all the chromosomes became entangled to
form a compact sphere adhered to a nucleolus, and homologous pairing failed. At anaphase I and telophase I, chromosome
nondisjunction and degenerated spindle formation resulted in multiple uneven spore production. However, chromosomal
fragmentation frequent in plant meiotic mutants was never observed in all of the pair1 meiocytes. These observations clarify
that the PAIR1 protein plays an essential role in establishment of homologous chromosome pairing in rice meiosis.
INTRODUCTION
Sexual reproduction is required for generating gametes containing a haploid chromosome complement from diploid somatic
cells. Meiosis is a crucial event in this process, in which two
successive rounds of chromosome segregation follow a single
round of DNA replication. Meiosis has evolved to achieve two
contrary purposes: the maintenance of genome stability and the
creation of genetic diversity. To achieve these two purposes, the
most important processes are homologous chromosome pairing
and recombination.
Chromosome pairing becomes evident by the close parallel
association of homologous pairs during meiotic prophase, which
is divided into several substages according to the state of
synapsis and condensation. Homologous chromosomes have
begun to condense at leptotene and to become partially
synapsed at zygotene, fully synapsed at pachytene, and
desynapsed but held together by chiasmata at diplotene and
diakinesis. Synapsis guarantees a strong and close connection
1 To
whom correspondence should be addressed. E-mail knonomur@
lab.nig.ac.jp; fax 81-559-81-6879.
The author responsible for distribution of materials integral to this
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Ken-Ichi Nonomura
([email protected]).
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.020701.
of the homologous pairs facilitated by a network of longitudinal
and transversal protein fibers called the synaptonemal complex
(SC; Moens, 1969; Westergaard and von Wettstein, 1972; Gillies,
1975), whereas the SC function on early prophase has not yet
been elucidated.
On the other hand, presynaptic alignment of homologous
chromosomes is believed to be important in facilitating a homology search before SC formation in many organisms (Zickler and
Kleckner, 1998). Recent analyses that used the fluorescent in situ
hybridization (FISH) technique clearly revealed the meiosisspecific clustering of telomeres in the nucleus (the so-called
bouquet formation), in some cases of the centromeres as well,
contributes to presynaptic alignment (Chikashige et al., 1994;
Scherthan et al., 1996; Bass et al., 1997; Martinez-Perez et al.,
1999; Armstrong et al., 2001). In addition, chromosome
aggregation adhered to the nucleolus (the so-called synizetic
knot) is believed to occur as part of the homology search in early
meiotic prophase (Loidl, 1988). To elucidate the relationship
between presynaptic chromosome conformation and homologous pairing, genetic analyses using meiotic mutants are necessary. In Schizosaccharomyces pombe, loss of a telomere
binding protein TAZ1 (Cooper et al., 1998; Nimmo et al., 1998)
and a spindle pole body (SPB)–associated protein KMS1
(Shimanuki et al., 1997; Niwa et al., 2000) impair telomere
clustering and decrease the frequency of meiotic recombination.
In plants, PAM1 of maize (Zea mays) is reported that obviously
affects bouquet formation (Golubovskaya et al., 2002). In
Rice PAIR1 for Homolog Pairing in Meiosis
addition, several interesting genes and mutations affecting
homology search have been identified in plant meiosis. The
phs1 mutation of maize causes nonhomologous synapsis, delay
of DNA double-strand break (DSB) repair, and dramatically
reduced RAD51 foci on meiotic chromosomes, although it has
little effect on bouquet formation (Pawlowski et al., 2004). The
AHP2 of Arabidopsis (Arabidopsis thaliana) is homologous to
Saccharomyces cerevisiae HOP2 (Leu et al., 1998) and S. pombe
MEU13 (Nabeshima et al., 2001), which are known to act in
monitoring homology between pairing partner at meiosis, and its
mutation causes some early meiotic aberrations, such as
chromosome fragmentation and unbalanced chromosome
segregation (Schommer et al., 2003). The SYN1/DIF1 (Peirson
et al., 1997; Bai et al., 1999) and SWI1 (Mercier et al., 2001, 2003)
of Arabidopsis are required for precise reductional division. The
loss-of-function of both genes resulted in precocious separation
of sister chromatid cohesion and chromosome fragmentation
during early meiosis. However, genetic and biochemical interaction among these genes is still unclear. In addition, the
PHS1 and SWI1 encode unknown proteins, suggesting that
identification of key players in early plant meiosis is not completed yet.
Initiation and repair of DSBs are also known to affect
presynaptic chromosome alignment (Loidl et al., 1994; Weiner
and Kleckner, 1994), and in some species they influence
recombination and SC formation as well (Giroux et al., 1989;
Celerin et al., 2000; Grelon et al., 2001). In S. cerevisiae, meiotic
recombination is initiated by DSBs (Sun et al., 1989; Cao et al.,
1990) that are catalyzed by SPO11, a type II topoisomerase
(topoisomerase VI, subunit A; Bergerat et al., 1997). Although
meiotic DSB formation has only been proven experimentally in
yeast, this process is considered to be extensively conserved in
mammals and plants (Grelon et al., 2001). In the spo11 mutant of
Arabidopsis, stages typical of pachytene and SC formation are
seldom observed (Grelon et al., 2001), thereby indicating the
relationship between presynaptic events and DSB initiation also
exists in the plant kingdom. However, recent analysis using mei1/
spo11 double mutants revealed a SPO11-independent initiation/
repair pathway of DSBs in meiosis of Arabidopsis (Grelon et al.,
2003). To build a comprehensive understanding of the molecular
mechanism promoting early meiosis in plants, further studies
using meiotic mutants are necessary.
A number of spontaneous or induced synapsis mutants have
been reported in rice (Oryza sativa; Kitada and Omura, 1983;
Kitada et al., 1983; Nonomura et al., 2004). However, the PAIR2
gene, which is the ortholog of S. cerevisiae HOP1 (Hollingsworth
et al., 1990) and Arabidopsis ASY1 (Caryl et al., 2000; Armstrong
et al., 2002), is the only reported case of cloning rice meiotic
genes (Nonomura et al., 2004). Herein, we report the second
case of the isolation and characterization of a novel meiotic gene
PAIR1 (HOMOLOGOUS PAIRING ABERRATION IN RICE
MEIOSIS1), encoding a putative coiled-coil protein in O. sativa
ssp japonica (2n ¼ 24). A tagged pair1 mutation by the retrotransposon Tos17 exhibited complete male and female sterility
and a complex phenotype during rice meiosis I, in which an unusual entangled configuration of chromosomes at early prophase
resulted in a defect of bivalent formation. Spindle formation and
sporulation were also affected. Such a broad phenotype from
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early to late stages of meiosis is usual in plant synaptic mutants.
Here, we clarify that the PAIR1 protein of rice plays an essential
role in establishment of homologous chromosome pairing in
early meiotic prophase.
RESULTS
Isolation of the pair1 Mutant
We screened meiotic mutants from Tos17 insertion lines induced
by somatic culture and regeneration (Hirochika et al., 1996;
Yamazaki et al., 2001). In the ND0016 of 600 sterile lines,
complete male- and female-sterile plants segregated but with
normal plant morphology (Figure 1A). The sterile phenotype
segregated as a single recessive mutation (fertile:sterile ¼
241:76, x2 ¼ 0.178 for 3:1). The mutant showed complete pollen
sterility (Figure 1C) and no obvious construction of female spores
in the ovule (Figure 1G), in contrast with normal spores in wildtype siblings (Figures 1B and 1F). Pollination of the mutant
flowers with wild-type pollen did not result in seed production
(data not shown), thus supporting the notion that the female
gamete was not functional in the mutant. No noticeable abnormalities were observed in the morphology of anther walls
and ovules of the homozygous pair1 mutant (Figures 1D to 1G).
These results clearly indicate that the pair1 mutation only
affected male and female sporogenesis.
Tos17 Insertion Caused pair1 Mutant Phenotype
Segregants (n ¼ 317) of the third generation of regenerated
plants (R3) of the ND0016 line were screened for the presence of
a Tos17 insertion tag of the PAIR1 gene. Approximately 10
copies of transposed Tos17 were detected in those plants,
whereas the original Nipponbare cultivar contained two Tos17
copies (data not shown). Of these transposed copies, a 4.5-kb
XbaI fragment was completely linked to the pair1 sterile
phenotype. A genomic 2.0-kb fragment flanking the Tos17 copy
was cloned from the 4.5-kb fragment, and the same blot was
reprobed with this fragment. This probe led to the detection of
two allelic bands of 6.5 kb and 4.5 kb (part of this result is shown
in Figure 2A); the former was linked to the wild-type phenotype,
whereas the homozygous latter bands were linked to the sterile
phenotype. The sterile phenotype was reproduced in the next
generation of fertile R3 plants carrying heterozygous bands (H in
Figure 2A). Thus, we concluded that segregation mode of
tagged site was completely consistent with that of sterile
phenotype in ND0016. An identical sequence to the tagged site
was included in the bacterial artificial chromosome clone
OSJNBa0009C08, which mapped to the short-arm end of
chromosome 3 (0.0-centimorgan position; http://rgp.dna.affrc.
go.jp/).
We were unsuccessful in our attempt to isolate the cDNA clone
against the tagged sequence, despite screening >70,000 clones
derived from young panicles. On the other hand, three independent trials of rapid amplification of 59 cDNA ends (59-RACE) and
39-RACE reactions (see Methods) successfully amplified a 1.7kb cDNA. In all 59-RACE trials, the cDNA extension stopped at
the same position (underlined in Figure 2B). A TATA box,
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ND4557 and pair1-3 in NC0012 (Figure 2C). All insertions
appeared in the sixth exon except for pair1-3, which appeared
in the fourth exon. The pair1-2 and pair1-3 mutants also showed
complete sterility and a defect in bivalent formation in meiosis
(data not shown). An in-frame stop codon appeared within all
allelic insertions to interrupt PAIR1 translation, and a 5-bp target
sequence duplicated at both ends of all the insertions as
described by Hirochika et al. (1996).
To confirm that the Tos17 insertion in this locus resulted in the
mutant phenotype, a 8.2-kb XhoI-BamHI fragment including the
entire coding region and a 3.4-kb upstream sequence (Figure 2C)
Figure 1. Defects of the pair1 Mutation Observed Only in Male and
Female Sporocytes.
(A) The plant morphology of the homozygous pair1-1 mutant (/) was
normal, whereas complete sterility resulted in erected panicles and
prolonged greening of leaves compared with the heterozygous wild-type
sibling (1/).
(B) Fertile pollens in a heterozygous sibling, stained by iodium potassium
iodide solution.
(C) Completely sterile pollen in the homozygous pair1-1 mutant.
(D) and (E) In anther wall development, no discernible difference was
observed between the wild type (D) and the pair1-1 mutant (E),
respectively. Ep, epidermis; En, endothecium; Ml, middle layer; Ta,
tapetum cells; PMC, pollen mother cell. Bar ¼ 20 mm.
(F) and (G) In the wild-type ovule, three of four female spores were
degenerated (arrows and bottom left illustration, smaller arrows for
degenerated spores; [F]), whereas no obvious structure of spores was
observed in the mutant ovules (G). Bar ¼ 20 mm.
a potential binding site of transcription factors (White and
Jackson, 1992), was found 37 bp upstream of the 59-RACE
end (Figure 2B). In addition, a translation initiation codon of the
gene was predicted 100 bp downstream of the 59 end by the
RiceGAAS gene annotation software (http://ricegaas.dna.affrc.
go.jp/). From these results, we concluded that the isolated
cDNAs would cover the entire mRNA of the tagged gene.
Comparison of the genomic sequence with the cDNA enabled
the prediction of the 11 exons and 10 introns in this locus (Figure
2C). The Tos17 was inserted into the sixth exon in the ND0016
line, designated as pair1-1. A search for the coding sequence in
the Tos17-flanking database (http://pc7080.abr.affrc.go.jp/) revealed two allelic lines in addition to pair1-1: pair1-2 found in
Figure 2. Identification and Characterization of the PAIR1 Gene.
(A) DNA gel blot analysis of a sample of the 317 progenies of the pair1-1
heterozygote probed with the PAIR1 cDNA. A Tos17 insertion induced
a 4.5-kb polymorphic band (arrow), whereas the original Nipponbare
showed a 6.5-kb band (lane P). Presence of the 4.5-kb bands was
completely linked with the sterile phenotype (S). Homozygosity (W) or
heterozygosity (H) of fertile plants was determined in their progeny.
(B) Genomic sequence 150 bp upstream of the PAIR1 coding region.
Underlining indicates the 59 end predicted by three independent
reactions of RACE. The predicted TATA box 35 bp upstream of the 59
end and the translation initial codon ATG are boxed.
(C) PAIR1 coding region and Tos17-inserted sites. Shaded bars indicate
allelic Tos17 insertions, in which arrowheads represent long terminal
repeats and their orientation. A small bar under the gene structure
indicates the position of the DNA probe used in (A) and (D). Arrowheads
indicate the position and orientation of the PCR primers.
(D) RT-PCR for the PAIR1 mRNA. The panicles were classified into five
groups (P1 to P5) according to their development stage (see the text). In
lane 9, the P3 total RNA was templated without RTase as a negative
control. In lanes 10 and 11, total DNA from Nipponbare and the PAIR1
cDNA clone were templated as negative and positive controls,
respectively.
Rice PAIR1 for Homolog Pairing in Meiosis
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was inserted into the pPZP2H-lac binary vector (Fuse et al.,
2001) and transformed into pair1-1 homozygous callus. Seed
fertility subsequently recovered in 18 of 24 regenerated plants,
although it ranged from 10% to 60%. On the other hand, no
recovery was observed in all four transformants that contained
the empty vector as a negative control. From these results, we
concluded that this locus encoded functional the PAIR1 gene.
The expression profile of PAIR1 was examined by RT-PCR
using total RNAs extracted from vegetative and reproductive
organs. Accumulation of PAIR1 transcripts was observed in
young panicles but not in vegetative organs (Figure 2D). This
result corresponds well to the mutant aberration that was
observed only in reproductive cells but not in vegetative organs
(Figure 1). In the reproductive phase, panicles were classified
into five groups according to their development: 5 mm
panicles as a group of P1; 10 mm as P2; 30 mm as P3;
60 mm as P4; and 100 mm as P5. Although stage
progression varied among individual plants or panicles, P1
panicles usually had developed a flower primordia, P2 and P3
panicles had developed anthers not entering meiosis, P4 had
some meiocytes entering meiosis, and P5 had all meiocytes
entering meiosis (Nonomura et al., 2003, 2004). PAIR1 expression was first detected in P1 panicle initiates, reached a peak in
P3 panicles, and rapidly decreased in P4 panicles (Figure 2D).
This result suggested that the peak of PAIR1 expression
occurred in the early stages of meiocyte development.
PAIR1 Encoded a Novel Unknown Protein
The PAIR1 gene encoded a peptide sequence of 492 predicted
amino acids with a molecular mass of 54 kD (Figure 3A). A
database search revealed significant similarity only to the
hypothetical protein T1N6.6 (832 amino acids) of Arabidopsis,
which is 32.6% identical to PAIR1 in a 507–amino acid overlap
(data not shown). The fact that few proteins share the similarity
with PAIR1 suggests that the peptide sequence of PAIR1 might
be less conserved among organisms.
a-Helical coiled-coil motifs containing 13 heptad repeats were
predicted in the middle of the peptide (166 to 207 amino acids
[cc1], 229 to 249 amino acids [cc2], and 274 to 301 amino acids
[cc3]) by the online network protein sequence analysis (Combet
et al., 2000). Three clusters of heptad repeats were rich in
hydrophobic residues at the first and fourth heptad (highlighted
in Figure 3A). Generally, the first and fourth hydrophobic residues
of the heptad are located on the same side of the a-helix and
contribute to protein dimerization (Landschulz et al., 1988; Moitra
et al., 1997). In the cc1 sequence, a helix-turn-helix structure was
also predicted according to Chou and Fasman (1978) (dotted in
Figure 3A). All three alleles contained Tos17 insertions in the
coiled-coil region (arrows in Figure 3A). The pI value was
estimated at 10.32 for the whole PAIR1 peptide, and three basic
regions with a higher pI value than the average were predicted (0
to 60 amino acids, 330 to 430 amino acids, and 460 to 492 amino
acids; Figure 3A). The basic region at the C terminus contained
a KRRRR peptide alignment (Figure 3A), which was predicted as
a potential nuclear localization signal (Chelsky et al., 1989). A
transient assay by bombardment of a 35S-GFP (green fluorescent protein)-PAIR1 fusion construct into Allium cepa (onion)
Figure 3. Primary and Secondary Structure Prediction of PAIR1.
(A) The vertical arrows indicate the position of Tos17 insertion in the
respective three alleles. The a-helical regions were predicted according
to Chou and Fasman (1978). The pI values of 20 amino acids were
estimated in each 10–amino acid interval along the PAIR1 peptide and
plotted on the graph. The coiled-coil structure with three heptad clusters
(cc1, cc2, and cc3), separated by two nonhelical sequences, was
predicted at the middle of the peptide (shaded bars and wavy lines). The
coiled-coil regions contained 13 heptad repeats in total, whose first and
fourth residues were frequently hydrophobic (highlighted). The cc1
sequence was predicted to make a helix-turn-helix structure (turned at
the residues with dots). Three basic amino acid clusters were detected
(hatched bars and underlines). The C-terminal basic region including
a KRRRR sequence was predicted to act as a nuclear localization signal
(NLS; closed box and double underline). The S/T-P-X-X or S/T-S/T-X-X
motifs, characteristic of DNA binding proteins (Suzuki 1989), are boxed.
(B) Nuclear localization of GFP-PAIR1 fusion protein during transient
expression in A. cepa epidermal cells.
epidermal cells revealed that the fusion protein was localized in
the nucleus in contrast with 35S-GFP alone (Figure 3B),
suggesting that PAIR1 also acts in the nucleus of the rice
meiocytes. The N terminus including a basic region was rich in S/
T-P-X-X or the conformational mimic S/T-S/T-X-X motif (boxed
in Figure 3A), which are abundant in many DNA binding proteins
(Suzuki, 1989).
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Aberrant Kinetics of Homologous Chromosomes in
pair1 Meiocytes
The chromosome behavior in the meiocytes of wild-type siblings
and pair1-1 mutants was characterized. In the wild type, the 24
chromosomes appeared as very thin threads at leptotene (Figure
4A) and underwent synapsis at zygotene (Figure 4B). During
zygotene, chromosomes formed a synizetic knot (Figure 4B),
followed by a relaxed formation of fully synapsed bivalents in
pachytene (Figure 4C). After synapsis was relieved at diplotene
(Figure 4D), 12 bivalents became tightly condensed at diakinesis
(Figure 4E) and arranged at the metaphase plate subsequent to
nuclear envelope breakdown (Figure 4F). Reductional division of
bivalents occurred at anaphase I (Figure 4G), and subsequent
equational division produced tetrad spores (Figure 4H).
In the pair1-1 mutant, chromosome configuration appeared
normal at leptotene (Figure 4J), and the first visible abnormality
was detected at zygotene. Throughout this stage, very thin
threads aggregated around the nucleolus in the mutant (Figure
4K). The normal appearance of thick threads was not observed at
pachytene and diplotene (Figures 4L and 4M). In addition, the
chromosome configuration with a synizetic knot was maintained
up to late diplotene or early diakinesis (Figure 4M), whereas the
knot was released at pachytene in the wild type (Figure 4C).
There were 24 completely unpaired univalents observed at
diakinesis (Figure 4N). Even after synizetic configuration eventually diffused, we observed several univalents connected by
a chromatin bridge (arrowheads in Figure 4N). Despite a complete absence of homologous pairing, all the univalents
frequently arranged at the metaphase plate (Figure 4O). The
univalents were divided unequally at anaphase I, and several
lagging univalents were observed (Figure 4P). Though considerable synchronous segregation of sister chromatids was observed at anaphase II (Figure 4Q), several chromatids often
moved independently (Figure 4Q, arrowheads). Throughout
meioses I and II, precocious sister chromatid separation and
Figure 4. Homologous Chromosome Pairing Is Defective in Male and
Female Meiocytes of the Wild Type and pair1-1 Mutant.
(A) to (H) Chromosomal spreads from wild-type male meiocytes in
various stages: leptotene (A), zygotene (B), pachytene (C), diplotene (D),
diakinesis (E), metaphase I (F), metaphase II (G), and anaphase II (H).
(I) Homologous chromosomes at diakinesis in wild-type female
meiocytes.
(J) to (Q) Chromosomal spreads from pair1-1 male meiocytes in various
stages. No noticeable aberration was observed at leptotene (J) and
synizetic zygotene (K). However, synizetic conformation still remained at
pachytene and diplotene in the mutant ([L] and [M]). At late diakinesis, 24
complete univalents were observed in the mutant (N), in which several
univalents were frequently connected by a thin-chromatin thread
(arrowheads). Though all univalents aligned on the division plane (O),
nondisjunction and delayed univalents (arrows) were observed in the
mutant (P). Most of the chromatids were synchronously divided to the
opposite poles, whereas several chromatids independently moved
(arrowheads; [Q]).
(R) Approximately 24 univalents were observed in the mutant female
meiocytes at diakinesis. Bar ¼ 20 mm.
Rice PAIR1 for Homolog Pairing in Meiosis
detectable fragmentation of chromosomes were not observed in
pair1-1 meiocytes (Figure 4P). In female meiocytes of pair1-1,
unpaired univalents were also observed at diakinesis (Figure 4R).
Ratios of characteristic meiocytes in 0.6- to 0.9-mm anthers
were compared between the wild type and the mutant (Figure 5).
In the wild type, 80% of the meiocytes entered into zygotene
(Figure 5, light blue bars) in 0.6-mm anthers and completely
shifted to pachytene or later stages in 0.9-mm anthers. Of the 86
mutant meiocytes observed, however, zygotene cells with synizetic configuration were constantly observed, even in 0.9-mm
anthers. A normal appearance of pachytene and diplotene was
seldom observed. The FISH foci of the 25S rDNA locus were
counted on male meiocytes with 0.6- to 0.8-mm anthers to
assess homologous chromosome pairing at early prophase
(Figure 6). In the wild type, separated foci at leptotene (1.55 foci
averaged) or zygotene (1.30 foci) converged into a single focus
(1.21 foci) at pachytene, whereas foci in the mutant did not
converge (1.52 foci, even in 0.8-mm anther), indicating synapsis
was completely defective in the mutant (Table 2). Thus, we
concluded that the crucial events for meiosis, such as the timing
of the release the synizetic knot and subsequent homolog
synapsis at zygotene and pachytene, were defective both in male
and female meiocytes of the pair1 mutant.
Abnormal Spore Formation in the pair1 Mutant
The pair1 mutation was abnormal not only in chromosome
division but also in cytokinesis of meiocytes. In the male
meiocytes at dyad stage, all of the wild-type dyads shared an
equal volume of nuclei and cell mass (Figure 7A). By contrast,
46.7% of the pair1-1 dyads shared an unequal nuclei and/or
cell mass (Figures 7B and 7C). In addition to various sizes of
nuclei, multiple micronuclei were frequently observed. Unusual
Figure 5. Frequency of Each Meiotic Stage Observed in Anthers of the
Wild Type and the pair1-1 Mutant.
Staging was performed using anthers of 0.6, 0.7, 0.8, and 0.9 mm in
length. Meiotic stages were classified into leptotene (red), zygotene with
synizetic conformation (light blue), pachytene (yellow), diplotene (green),
diakinesis (orange), and metaphase I or later stages (dark blue). In the
mutant anthers, typical features of pachytene and diplotene chromosomes were seldom observed. Then, the meiocytes were classified
according to the extent of chromosome condensation.
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Figure 6. Chromosome Pairing in Male Meiocytes of the 0.8-mm
Anthers.
The pictures, a part of the experiments shown in Table 2, are
superimposed images of DAPI-stained chromatin (blue) and FISH (green)
with a probe for the 25S rDNA. Most of the wild-type meiocytes showed
a single focus (left), whereas most of the mutant meiocytes showed two
unpaired foci (right).
triad formation was observed in 8.3% of the pair1-1 dyads
(Figure 7D).
At tetrad stage, 70% of the spore sets of the pair1-1 mutant
showed abnormal morphology (Table 1). Only 1.9% of the
tetrads observed had wild-type morphology. Most of the mutant
tetrads exhibited various sizes of nuclei and/or cell mass, such as
unequal nuclei distribution with multiple micronuclei (Figure 7F)
and spores without a nucleus (Figure 7G). Polyads, tetrads
with more than four spores, were also observed in 5.1% of the
mutant spore sets (Figures 7H and 7I; Table 1). These results
indicate that the loss-of-function mutation in PAIR1 gene also
affects correct chromosome disjunction and cytokinesis during
sporulation.
Abnormal Spindle Formation at Meiosis I in pair1 Mutants
Spindle morphogenesis was observed at meioses I and II by
indirect immunofluorescent staining using anti-a-tubulin antibody. The negative control without the primary antibody gave no
signal (data not shown). In the wild type, the network of
cytoplasmic microtubules appeared to surround the nuclear
envelope at diakinesis (Figure 8A). After nuclear envelope
breakdown, a bipolar short array of microtubules associated to
bivalents arranged at the metaphase plate (Figure 8B) and
formed an obvious structure of bipolar spindles at metaphase I
(Figure 8C). Each set of 12 univalents synchronously segregated
toward opposite poles by the anaphase I spindles (Figure 8D). At
late telophase I, the phragmoplast formed between the daughter
pronuclei (Figure 8E). The phragmoplast is a cytoskeletal
structure held by two arrays of microtubule bundles and is
required for determining the position of the cytokinetic plane
(Verma, 2001). As the cell plate expanded, the phragmoplast
depolymerized in the center and repolymerized along the edge of
the growing cell plate (Figure 8F). In meiosis II, the shape and
density of the spindles were almost the same as that of meiosis I
(Figures 8G and 8H).
Apparent abnormalities were not observed at diakinesis
(Figure 8I) and early metaphase I (Figure 8J). The first noticeable
abnormality appeared at metaphase I in the pair1-1 mutant. In all
of the 10 meiocytes observed, the spindles at metaphase I and
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(Figures 8Q and 8R). These observations suggest that the effects
of the pair1 mutation might be restricted to meiosis I.
DISCUSSION
Figure 7. Abnormal Male Sporulation in the pair1-1 Mutant.
(A) to (D) DAPI-stained male sporocytes in dyad stage. All wild-type
anthers carried normal dyads and tetrads. The mutant carried abnormal
dyads that had one or more of the following: nuclei of different sizes (A),
multiple nuclei (B), unequal cytoplasm (C), and micronuclei (D).
(E) to (I) DAPI-stained male spores in the tetrad stage. The mutant carried
abnormal tetrads with an unequal volume or misdivided nuclei ([F] and
[G]) with several micronuclei. Abnormal pentayads (H) and hexayads (I)
were also observed.
anaphase I were composed of wavy and thin bundles of
microtubules (Figure 8K), whereas all the chromosomes were
captured by the spindle microtubules at anaphase I (Figure 8L).
Of the 18 meiocytes observed at early telophase I, a single
meiocyte exhibited an extra extension of microtubules at
a different direction from the poleward axis (Figure 8M). In
another meiocyte, ectopic small bundles of microtubules formed
along the phragmoplast at late telophase I (Figure 8N). In the
remaining 16 meiocytes, striking abnormalities were seldom
observed except for chromosome nondisjunction. At cytokinesis, multiple micronuclei were observed in half of 20 meiocytes
(Figures 8O and 8P). In Figure 8P, two micronuclei are clearly
localized outside the microtubule corona surrounding the
nuclear envelop. On the other hand, the morphology of the
spindle appeared to be normal in the meiosis II of the mutants
(Figures 8Q to 8S). No spindle formation was observed around
the micronuclei observed at metaphase II and anaphase II
We identified a rice pair1 mutant with complete male and female
sterility that exhibited a complex phenotype during meiosis I. The
mutant phenotype was limited to germ cells (Figure 1), and the
PAIR1 gene was expressed mainly at the premeiotic or early
meiotic stages (Figure 2D). The mutant phenotype was first
apparent at zygotene, the stage of synizetic knot formation
(Figures 4J and 5). In the mutant meiocytes, no pairing of 25S
rDNA foci was detected through zygotene to pachytene (Figure
6; Table 2). The rDNA locus in japonica rice is close to the shortarm end of chromosome 9. Because synapsis is generally
initiated subtelomerically in many organisms (von Wettstein et al.,
1984), unpairing of rDNA foci may represent asynapsis of whole
extent of homologous chromosomes. Thus, we conclude that the
normal PAIR1 product has an essential role in establishment of
homologous chromosome pairing in early meiosis.
We previously identified and characterized the mutation in rice
PAIR2 gene (Nonomura et al., 2004), the ortholog of Arabidopsis
ASY1 that is an important factor for synapsis and functions by
localizing along lateral elements of the SC (Caryl et al., 2000;
Armstrong et al., 2002). The pair2 mutant, as well as the pair1,
completely lacked chromosome pairing in early meiosis.
However, no tangled chromosomes appeared in the pair2,
indicating that tangled chromosomes observed in the pair1
mutant are not simply the result of the failure of SC formation.
Rather, premeiotic expression suggests that the PAIR1 is
involved in or before presynaptic homolog alignment.
The newly identified PAIR1 gene encoded a putative nuclear
protein of 492 amino acids containing an a-helical coiled-coil
structure (Figure 3). The S/T-P-X-X or S/T-S/T-X-X motifs, which
are present in many DNA binding proteins (Suzuki, 1989), were
abundant in the N terminus of PAIR1. In addition, a nonhelical
basic region was also identified at the N terminus. A basic amino
acid cluster juxtaposed with Leu-zipper motifs is called a bZIP
domain and is often shared in transcription factors binding to
sequence-specific DNA (Fassler et al., 2002). Thus, we speculate
that PAIR1 forms a coiled-coil dimer and acts on the chromosomes during meiosis I. Indeed, the SMC (structure maintenance
of chromosomes) family of proteins contains a coiled-coil
structure, and their heterodimers function directly in sister
chromatid cohesion or in the condensation pathway during
mitosis and meiosis (Hirano, 2000). In addition, SC is known to be
composed of many coiled-coil proteins (Meuwissen et al., 1992;
Offenberg et al., 1998; Tung and Roeder, 1998; Yuan et al., 1998).
Table 1. Frequency of Appearance of Abnormal Tetrad Spores
Tetrad (%)
PAIR1-1 Genotype
Dyad (%)
Triad (%)
Normal
Aberranta
Pentayad (%)
Hexayad (%)
1/
/
1 (0.9)
47 (12.7)
0
36 (9.7)
109 (98.2)
7 (1.9)
1 (0.9)
262 (70.6)
0
16 (4.3)
0
3 (0.8)
a Contains
an uneven volume of cytoplasm, various sizes of nuclei, and/or several micronuclei.
Rice PAIR1 for Homolog Pairing in Meiosis
1015
Figure 8. Immunostaining of Spindles in Wild-Type and pair1-1 Male Meiocytes.
(A) to (H) Male meiocytes of the wild type.
(I) to (T) Male meiocytes of pair1-1 mutant.
(A) Tubulin fibers were present throughout the cytoplasm at diakinesis. Chromosomes were stained by PI (red), and microtubules were immunologically
stained by Alexa488 (green).
(B) Bipolar-oriented microtubules had developed at early metaphase I.
(C) Mature spindles were formed at metaphase I.
(D) Bivalents were separated by the spindle into two sets of 12 univalents at anaphase I.
(E) The phragmoplast had begun to form between daughter pronuclei at telophase I, in which diminished staining in the midzone indicated the
cytokinetic plane (arrow).
(F) A division plate with a phragmoplast ring had grown to separate the daughter cells.
(G) Two sets of univalents aligned at metaphase-II plates.
(H) Two sets of univalents were separated to the opposite poles by spindles.
(I) and (J) No obvious abnormality was observed in the microtubule array in pair1-1 mutant at diakinesis (I) and early metaphase I (J).
(K) The mutant spindle is composed of wavy, thin bundles of microtubules.
(L) Several delayed univalents (arrowheads) were always observed at anaphase I.
(M) Extra microtubule fibers (arrows) extended to a different direction from the poleward axis at telophase I.
(N) The ectopic extension of microtubule fibers (arrow) also appeared at late telophase I.
(O) Several micronuclei (arrowheads) were observed in the mutant dyad.
(P) Daughter nuclei were enclosed by the microtubule corona, in which micronuclei were clearly detected outside the corona at the dyad stage.
(Q) to (T) During meiosis II, the spindles exhibited normal appearance but not around the micronuclei (arrowheads). Bar ¼ 10 mm.
1016
The Plant Cell
Table 2. Distribution of the FISH Foci for the 25S rDNAs in Male Meiocytes
No. of Meiocytes Observeda
PAIR1-1 Genotype
Anther Length (mm)
Singlet Focus
Doublet Foci
Rate of Pachytene
Average No. of Foci
Standard Error
1/
0.6
0.7
0.8
0.6
0.7
0.8
143
272
255
153
141
110
176
115
67
148
118
119
4.1%
45.2%
64.9%
NDb
ND
ND
1.55
1.30
1.21
1.49
1.46
1.52
0.055
0.050
0.040
0.057
0.060
0.060
/
This result represents the experiments using three independent plants.
the meiocytes at leptotene, zygotene, and pachytene were examined.
b ND indicates not determined because no typical pachytene is observed in the mutant.
a Only
However, some coiled-coil members are known to act on the
SPB (Ikemoto et al., 2000) or microtubule fibers (Yamashita et al.,
1997) in yeast meiosis. An antibody against PAIR1 protein, which
is now under construction, would permit clarification of its
localization in rice meiocytes.
Recently, the PHS1 gene has been known to act in early
meiosis of maize (Pawlowski et al., 2004). The phs1 mutation
exhibits interesting meiotic defects of nonhomologous synapsis,
delay of DSB repair, and dramatically reduced RAD51 foci.
These defects result in tangled chromosomes compacted into
a small sphere in pachytene, unpaired univalents in diakinesis,
and abnormal polyads, closely resembling the pair1 phenotype.
The PHS1 is also conserved in rice genome (Pawlowski et al.,
2004). Although the status of DSB repair and RAD51 recombination machineries has not yet been examined in this study,
phenotypic similarity between maize and rice mutants suggests
the relation of both meiotic proteins.
The pair1-like defects in early meiosis, such as tangled
chromosomes and homolog unpairing, were also observed in
syn1/dif1 (Peirson et al., 1997; Bai et al., 1999) and ahp2 mutants
(Schommer et al., 2003) of Arabidopsis. The SYN1 is a homolog
of a meiotic cohesin REC8 in S. pombe and is required for sister
chromatid cohesion during meiosis I (Cai et al., 2003). AHP2 is
a homolog of S. cerevisiae HOP2 (Leu et al., 1998) and S. pombe
MEU13 (Nabeshima et al., 2001) and is proposed to function in
monitoring sequence homology to either promote homolog
pairing or destabilize the pairing between nonhomologs.
However, a significant difference lay between these Arabidopsis
mutants (syn1 and ahp2) and monocot mutants (phs1 and pair1).
In the Arabidopsis mutants, fragmented chromosomes were
detected from diakinesis to anaphase I (Bai et al., 1999;
Schommer et al., 2003) but never in both monocot mutants. In
this study, a complete set of 24 univalents was clearly visible in all
meiocytes at anaphase I (Figure 4P). Chromosome fragmentation is thought to result from the accumulation of unrepaired
DSBs of DNA (Schommer et al., 2003). These facts lead to
a possibility that the PAIR1 promotes homolog pairing independent of DSB initiation/repair pathway. Indeed, in the maize
phs1 mutant, the DSBs are initiated and repaired, even though
delayed (Pawlowski et al., 2004).
On the other hand, the At-spo11 mutant also exhibits pair1like phenotype in early meiosis. This indicates that the homolog pairing should be coupled with DSB formation. Thus, as
another possibility, we favor that the PAIR1 might act in
presynaptic homolog alignment before loading DSB initiation
machinery on meiotic chromosomes, although it is possible
that the PAIR1 is a component of SC or involved in DSB
initiation step.
The pair1 mutation also affected spindle and phragmoplast
formation at anaphase I and telophase I (Figures 8M and 8N).
There was further indirect evidence in support of this observation by the appearance of polyads and several micronuclei at dyad and tetrad stages (Figures 7 and 8P). Abnormal
spindle formation has also been reported in the maize
desynaptic mutants dys1 and dys2 (Chan and Cande, 1998).
Although there are few reports of striking aberrations in spindle
formation, most of the Arabidopsis meiotic mutants described
so far produce polyads; the spo11 and mei1 in DSB initiation
(Grelon et al., 2001, 2003), the dmc1 in recombination steps
(Couteau et al., 1999), the syn1/dif1 in sister chromatid
cohesion (Bai et al., 1999; Bhatt et al., 1999), the asy1 in
chromosome synapsis (Caryl et al., 2000), and ms5 in chromosome division (Glover et al., 1998). Abnormal formation of
nonbipolar spindles or polyads in those synaptic mutants
assumes the involvement of microtubule organization in early
meiotic events, such as homolog alignment, synapsis, and
recombination, whereas the abnormal phenotype may simply
be the result of unpaired and missegregated univalents.
Interestingly, in many organisms, treatment of meiotic tissues
with microtubule depolymerizing agents inhibited cells in spindle
formation and in homologous chromosome pairing (Shepard
et al., 1974; Loidl, 1988). Induction of chromosome intertwining
was also observed in colchicine-treated Triticum aestivum
(wheat) meiocytes (Thomas and Kaltsikes, 1977). At meiotic
prophase of S. pombe, it is known that all telomeres cluster near
the SPB moving along an oscillatory path, and this is probably for
efficient chromosome pairing (Chikashige et al., 1994; Hiraoka,
1998; Niwa et al., 2000). This movement is principally led by the
pull of microtubules that connect the SPB to microtubuleanchoring sites on the cell cortex (Yamamoto and Hiraoka,
2001). Although the yeast cell system is critically different from
plant cells, these findings strongly suggest that microtubule
organization is important for early meiotic events common in
eukaryotes. Analysis of PAIR1 function will contribute toward
elucidating the relationship between meiotic chromosome
behavior and microtubule organization in plant cells.
Rice PAIR1 for Homolog Pairing in Meiosis
METHODS
1017
tion, and the cultures were regenerated as described by Hiei et al.
(1994).
Plant Materials
Callus derived from the japonica cultivar Nipponbare was employed for
regeneration after 5 months of suspension culture, as described by
Hirochika et al. (1996). From a total of 27,998 regenerated plants,
reduction of seed fertility was observed in 4032 lines, both in the R1
generation and its segregant progenies (R2 or R3 generation). From those
sterile lines, 600 lines segregating sterile plants in the R2 were randomly
selected for meiotic chromosome observation. Crossing of the wild-type
pollens to the pair1 homozygous plants was performed according to
method described by Nonomura et al. (2003). All materials were grown in
a field in the city of Mishima, Shizuoka, Japan, or in a greenhouse at 308C
during the day and 248C at night.
The mutant line described in this article will be made available at the
Rice Genome Resource Center of the National Institute of Agrobiological
Sciences, Japan (http://www.nias.affrc.go.jp/).
RT-PCR
Total RNA was isolated from roots, shoots, adult leaves, and young
panicles according to Sambrook et al. (1989), with a slight modification.
One microgram of total RNA was reverse transcribed by SuperScript II
RNaseH RT (Invitrogen, Carlsbad, CA) with the oligo(dT)20 primer. In the
PCR step, two specific primers within the PAIR1 cDNA were used, a sense
496 primer in the sixth exon and an antisense F09 primer, 59-GTTACCATTAATTAGCTAGGATGCGAGC-39, in the 11th exon (Figure 2C).
The PCR products were loaded on an agarose gel, and a DNA gel blot
analysis was performed using a nested PCR probe to confirm the precise
amplification from PAIR1 mRNA. As a control, the same RT products were
provided for PCR using primers in the rice actin (RAc): 59-AACTGGGATGATATGGAGAA-39 and 59-CCTCCAATCCAGACACTGTA-39.
Transient Assay of GFP-PAIR1 Localization
Genetic Analyses
The linkage relationship between the sterile phenotype and transposed
Tos17 fragments was analyzed by DNA gel blot hybridization or PCR (see
below) in the R3 population, which also included 317 pair1 heterozygous
R2 plants. The DNA extraction, DNA gel blot analysis, and cloning of
the Tos17-tagged sequence steps were performed as described by
Nonomura et al. (2003). A rice genomic sequence corresponding to the
Tos17 flanking sequence was identified using BLASTN (Altschul et al.,
1997) on the DNA Data Bank of Japan (DDBJ) and National Center for
Biotechnology Information Web sites.
To determine the cDNA sequence, total RNAs were extracted from
young (30- to 40-mm) panicles using the RNeasy plant mini kit (QIAGEN,
Hilden, Germany). Poly(A)1 RNAs were isolated using Oligotex-dT30
Super (Takara Bio, Shiga, Japan). The cDNA synthesis was performed by
59-RACE and 39-RACE using the Marathon cDNA amplification kit (BD
Biosciences, Franklin Lakes, NJ). In the 59- and 39-RACE analysis, a
specific antisense 497 primer, 59-CACCGCAGTTGTCTTATATCTGC-39,
and a sense 496 primer, 59-CCTCGACCCTTGCAACCTTGCAGAC-39,
were used, respectively (Figure 2C). Both RACE fragments were ligated at
the EcoRI site, and the resultant full-length cDNA was inserted into the
pGEM plasmid (Promega, Madison, WI). The cDNA sequence was
analyzed using GENETYX-MAC 12.0 software (Genetyx, Tokyo, Japan).
PCR genotyping for the pair1 segregated population was performed
using a set of primers: 465, 59-TTCAGCATTTTCTCCACTTTCCC-39, and
471, 59-TACCAGAAAAAAAATCACATGCTC-39, for the pair1-1, pair1-2,
and pair1-3 alleles, and of F05, 59-CAGAATAGCAGCATTTCACCTGAG-39, and F06, 59-AGGATTGTGTTTTGTGCTGC-39, for the pair1-4
allele (Figure 2C). The PCR conditions were an initial step of 948C incubation for 2 min followed by 30 cycles of 948C for 1 min, 558C for 1 min, and
728C for 2 min.
The PAIR1 cDNA was amplified by PCR with two primers tagged with
an NcoI site, 59-CATGCCATGGCATGAAGCTTAAGATGAAC-39 and
59-CATGCCATGGCATGGGATGCGAGCACGATG-39, and inserted to an
NcoI site of CaMV35S-sGFP(S65T)-NOS39 plasmid (Chiu et al., 1996) in
frame with the C terminus of a PAIR1 peptide. The plasmid was amplified
in DH10B cells and isolated by QIAGEN plasmid maxi kit. The
bombardment of plasmid DNAs was performed as described by Chiu
et al. (1996), using a PDS-1000/He biolistic particle delivery system (BioRad, Hercules, CA). The cells were stained with 1 mg/mL 49,6-diamidino2-phenylindole (DAPI), and signals were observed at 6 h after the
bombardment using an Olympus FLUOVIEW confocal laser-scanning
microscopy (CLSM) system (Tokyo, Japan) for GFP and an Axioscop20
(Zeiss, Jena, Germany) and Penguin 600CL camera system (Pixera, Los
Gatos, CA) for DAPI.
Meiotic Chromosome Observation
The young (40- to 60-mm) panicles, including flowers in meiosis, were
fixed with 3:1 ethanol:acetic acid (EAA) and stored at 48C until
observation. Chromosome spreads of pollen mother cells were prepared
according to Nonomura et al. (2003). They were stained with 4% Giemsa
solution (Sigma, St. Louis, MO) diluted in 33 mM phosphate buffer, pH
6.8, for 30 min and photographed using a BX50 light microscope with
a DP50 system (Olympus).
To observe female meiosis, flowers fixed in EAA were stained with
1.0 mg/mL of propidium iodide (PI) in 0.1 M L-Arg, pH 12.4, according to
Nonomura et al. (2003). Stained pistils in meiosis were dissected from
flowers, whole-mounted on the slide with a drop of Vectashield (Vector
Laboratories, Burlingame, CA), and viewed using a CLSM system
(Olympus). Images were merged and enhanced using Photoshop 7.0
(Adobe, Mountain View, CA) software.
Complementation of pair1 Phenotype
The genomic library constructed using a lEMBL3/BamHI vector
(Stratagene, La Jolla, CA) was screened by plaque hybridization probed
with PAIR1 cDNA according to Sambrook et al. (1989). An 8.2-kb XhoIBamHI fragment (Figure 2C) was inserted into the pPZP2H-lac binary
vector (Fuse et al., 2001) and transformed into pair1-1 homozygous
callus culture. We obtained 24 pair1-1 homozygous callus cultures from
the 96 R3 seeds, which were harvested from heterozygous R2 plants
and genotyped by PCR using the primers 465 and 471. A sample of
callus transformed with the empty vector was used as a negative
control. The PAIR1-1 plasmid and the empty vector were introduced into
the callus culture by Agrobacterium tumefaciens–mediated transforma-
Optical Tissue Sectioning
To observe the morphology of anther walls and ovules, Kasten’s
fluorescent periodic acid–Shiff methods were employed according to
Vollbrecht and Hake (1995). Flowers in meiosis were fixed with EAA. After
hydrolyzation in 50% and then 30% ethanol for 20 min, flowers were
treated in 0.5% periodic acid in 70% ethanol/20 mM NaOAc at room
temperature for 25 min and then rinsed twice for 5 min in water. They were
then stained in 0.25% acriflavine in 37.5 mM K2S2O5/0.017 N HCl at room
temperature for 20 min. Stained tissue was rinsed copiously with water
until very little acriflavine leeched out (1 to 2 h) and then dehydrated
through a series of ethanol solutions for 20 min each. Dehydrated tissue
1018
The Plant Cell
was treated by methyl salicylate series, 3:1, 1:1, and 1:3 of ethanol:methyl
salicylate, for at least 1 h each step and in 100% methyl salicylate twice
for 1 h each step. The anthers and pistils were dissected, whole-mounted
in methyl salicylate under a coverslip, and viewed using a CLSM system
(Olympus).
FISH Analysis for Nucleolar Organizing Region
A probe for 25S rDNA locus (Takaiwa et al., 1985) was amplified by PCR
using the primers 59-CTGTGAAGGGTTCGAGTTGG-39 and 59-TTGCTACTACCACCAAGATCTG-39, in which the usual reaction buffer
contained a nucleotide mixture with 50% digoxigenin-labeled dUTP
(Roche, Indianapolis, IN) of unlabeled thymidine-59-triphosphate as an
addition. The young (40- to 60-mm) panicles were fixed with 4% (w/v)
paraformaldehyde in PMEG buffer (25 mM Pipes, 5 mM EGTA, 2.5 mM
MgSO4, 4% glycerol, and 0.2% DMSO, pH 6.8) in a 50-mL tube. After
vacuum infiltration, they were shaken at a medium speed at room
temperature for 3 h. The panicles were washed six times in PMEG at
medium shaking for 20 min each. They could be stored in PMEG at 48C at
least for 2 months. A single anther from the six in a flower was provided to
determine its meiotic stage by 1 mg/mL of DAPI in Vectashield (Vector
Laboratories) and viewed by fluorescent microscopy Axioscop 20 (Zeiss).
The anthers at appropriate stages were provided for cell-wall digestion in
an enzyme mixture at 378C for 20 min and at 48C for 10 min, in which 1.5
volumes of 50 mg/mL cytohelicase (Sigma) was mixed with 20 volumes of
2% cellurase Onozuka RS (Yakult Honsha, Tokyo, Japan), 0.3%
pectolyase Y-23 (Kikkoman, Chiba, Japan), and 1.5% macerozyme
R200 (Yakult Honsha) in PMEG. The anthers were squashed in 20%
acetic acid on the poly-L-Lys–coated slide and collapsed by a coverslip.
The coverslip was removed on dry ice. After air drying, the slide was
treated with 100 mg/mL RNaseA at 378C for 1 h, rinsed twice with 23 SSC
for 5 min and once with 50 mM Tris-HCl, pH 7.5, for 5 min, treated with 5
mg/mL of Proteinase K in 50 mM Tris-HCl at 378C for 30 min, and rinsed
twice with water at 48C for 10 min for each step. The male meiocytes on
the slides were refixed with EAA for 10 min, rinsed twice with 95% ethanol
for 10 min, and air dried. The hybridization procedure was performed
according to Schwarzacher and Heslop-Harrison (2000). The labeled
probe was detected by FITC-conjugated anti-digoxigenin antibody
(Roche). Chromosomes were counterstained with 1 mg/mL of DAPI,
and photos were taken using an Axioscop20 (Zeiss) and Penguin 600CL
camera system (Pixera).
Indirect Immunofluorescence
To observe the spindle morphology in male meiocytes, we adopted the
method described by Chan and Cande (1998), with slight modifications.
Fixation and digestion steps of the cells and cell walls were performed in
an identical manner to the FISH method. After fixed anthers were rinsed
five times with PMEG for 5 min each wash and squashed in 10 mL of
PMEG, the cell suspension was transferred to a 1.5-mL microtube. An
equal volume of prewarmed (208C) 3% SeaPrep agarose (BioWhittaker
Molecular Applications, Rockland, ME) in PMEG was added to the
suspension. After gently mixing the solution completely, it was then
cooled to 48C for 30 min. The agarose block was then rinsed with 500 mL
of PBS (137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM
KH2PO4, pH 7.4) twice at room temperature for 5 min each wash. The
agarose block was then incubated with 50 mL of monoclonal antibody
against the rat-a-tubulin subunit, OBT0614S (Oxford Biotechnology,
Oxfordshire, UK), diluted with PBS (1:100) at room temperature overnight.
It was rinsed with PBS twice for 1 h and then incubated in 50 mL of PBSdiluted (1:200) Alexa488 conjugated antibody against goat anti-rat IgG
(Invitrogen) at room temperature overnight. The agarose block was rinsed
with PBS twice for 1 h, incubated in 50 mL of 30 mg/mL of PI in PBS at
room temperature for 2 h, and washed with PBS twice for 5 min each
wash. The agarose block was placed with a drop of Vectashield (Vector
Laboratories) to the inside of a 18 3 18–mm square drawn by rubber
cement on the slide, heated to 558C for 3 min, and overlaid with
a coverslip. The male meiocytes were observed using the FLUOVIEW
CLSM system (Olympus). Five or six photos of optical sections from
a meiocyte were merged and enhanced using Photoshop 7.0 (Adobe).
Accession Numbers
The PAIR1 cDNA sequence described in this article has been deposited in
DDBJ under accession number AB158462. Accession numbers for the
other sequences mentioned are GenBank AC107224 (bacterial artificial
chromosome clone OSJNBa0009C08) and GenBank AC009273 (hypothetical protein T1N6.6).
Sequence data from this article have been deposited with the EMBL/
GenBank data libraries under accession numbers AB158462, AC107224,
and AC009273.
ACKNOWLEDGMENTS
We thank A. Morohoshi (National Institute of Genetics, Mishima, Japan)
for technical assistance. We also thank M. Yano (National Institute of
Agrobiological Sciences, Tsukuba, Japan) for providing binary vectors
for rice transformation and Y. Niwa (Shizuoka University, Shizuoka,
Japan) for providing 35S-sGFP plasmid. This research was supported
by the Program for Promotion of Basic Research Activities for Innovative
Biosciences (ProBRAIN), Japan, and in part by a Grant-in-Aid from the
Ministry of Agriculture, Forestry and Fisheries of Japan (Rice Genome
Project MP-2133).
Received January 5, 2004; accepted February 1, 2004.
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The Novel Gene HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 of Rice Encodes a
Putative Coiled-Coil Protein Required for Homologous Chromosome Pairing in Meiosis
Ken-Ichi Nonomura, Mutsuko Nakano, Toshiyuki Fukuda, Mitsugu Eiguchi, Akio Miyao, Hirohiko
Hirochika and Nori Kurata
Plant Cell 2004;16;1008-1020; originally published online March 18, 2004;
DOI 10.1105/tpc.020701
This information is current as of June 17, 2017
References
This article cites 73 articles, 29 of which can be accessed free at:
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