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CENTRAL REGION COMPONENT1, a Novel Synaptonemal
Complex Component, Is Essential for Meiotic Recombination
Initiation in Rice
CW
Chunbo Miao,a,1 Ding Tang,a,1 Honggen Zhang,b Mo Wang,a Yafei Li,a Shuzhu Tang,b Hengxiu Yu,b Minghong Gu,b
and Zhukuan Chenga,2
a State
Key Laboratory of Plant Genomics and Center for Plant Gene Research, Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences, Beijing 100101, China
b Key Laboratory of Crop Genetics and Physiology of Jiangsu Province/Key Laboratory of Plant Functional Genomics of Ministry of
Education, Yangzhou University, Yangzhou 225009, China
ORCID ID: 0000-0003-3673-1879 (C.M.).
In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal
complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species,
their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component,
designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC
transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization,
CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene
checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved
SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with
HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to
promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous
chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The
roles of CRC1 identified here have not been reported for Pch2 or TRIP13.
INTRODUCTION
In meiosis, homologous recombination is initiated by programmed
DNA double-strand break (DSB) formation. Meiotic DSB formation has been most extensively studied in Saccharomyces
cerevisiae in which the DSBs are formed by a large complex,
with sporulation protein11 (Spo11) as the catalytic subunit
(Edlinger and Schlögelhofer, 2011). Spo11, a conserved type II
topoisomerase, is responsible for catalyzing meiotic DSB formation in various eukaryotes (Edlinger and Schlögelhofer, 2011).
In addition to Spo11, nine factors (radiation sensitive50, meiotic
recombination11 [Rec11], X ray-sensitive2, recombination102,
recombination104, recombination114, superkiller8, meiotic recombination2 [Mer4], and meiosis defective4 [Mei4]) were characterized
as being subunits of the complex (Edlinger and Schlögelhofer,
2011). In contrast with Spo11, the nine factors are poorly conserved at the sequence or functional level, and among them,
only the Mei4 orthologs (MEI4 in Mus musculus and PUTATIVE
1 These
authors contribute equally to this work.
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Zhukuan Cheng
([email protected]).
C
Some figures in this article are displayed in color online but in black and
white in the print edition.
W
Online version contains Web-only data.
www.plantcell.org/cgi/doi/10.1105/tpc.113.113175
2 Address
RECOMBINATION INITIATION DEFECT2 [PRD2] in Arabidopsis
thaliana) were found to be essential for meiotic DSB formation in
plants and mammals (De Muyt et al., 2009; Kumar et al., 2010).
MEI4 and PRD2 show enormous sequence divergence from
Mei4 and were identified as Mei4 orthologs by a sophisticated in
silico study (Kumar et al., 2010).
In plants, besides SPO11, four meiotic DSB-forming factors
(PRD1, PRD2, PRD3, and DSB FORMATION [DFO] in Arabidopsis) were identified (De Muyt et al., 2007, 2009; Zhang et al.,
2012), but the mechanisms underlying their functions remain
elusive. Among them, PRD1 is the ortholog of mouse MEIOSIS
DEFECTIVE1, and PRD3 and DFO were considered to be plantspecific proteins. In rice (Oryza sativa), the PRD3 ortholog
HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1)
is essential for homologous chromosome pairing (Nonomura et al.,
2004).
In addition to DSB formation, meiotic homologous recombination also requires synaptonemal complex (SC) assembly. The
SC mediates the intimate association of homologous chromosomes (homologs) and promotes the posthomology search
steps in meiotic recombination. SC assembly starts from early
prophase I when a longitudinal protein core, called the axial element (AE), is formed between the two sister chromatids of each
chromosome. When chromosomes become synapsed from
zygotene to pachytene, the AEs of each homologous pair are
physically connected by numerous transverse filaments (TFs)
and run parallel to each other. Thus, the SC is a zipper-like
structure in which the lateral elements (LEs; corresponding to
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the AEs formed before synapsis) constitute the two parallel
backbones and the TFs are similar to the teeth of the zipper
(Schmekel and Daneholt, 1995). The region where the TF teeth
meet and interdigitate is the central element, corresponding to
the electron-dense structure running along and between the two
parallel LEs in electron microscopy examination (Schmekel and
Daneholt, 1995).
Several TF proteins have been identified, including zipper1
(Zip1) in budding yeast (Dong and Roeder, 2000), SYNAPTONEMAL COMPLEX PROTEIN1 in mice (Meuwissen et al., 1992),
crossover suppressor on 3 of Gowen in Drosophila melanogaster (Anderson et al., 2005), SYP proteins in Caenorhabditis
elegans (Schild-Prüfert et al., 2011), and ZYP1 in Arabidopsis
(Higgins et al., 2005). Although they bear no apparent sequence
similarity, these TF proteins exhibit significantly similar structures. All TF proteins identified share a coiled-coil domain in the
central region with one globular domain at each end. In rice, a TF
protein termed ZEP1 was also identified (Wang et al., 2010).
The TF protein is not the only component in the SC central
region. Studies in mice and Drosophila have identified several nonTF proteins (SYNAPTONEMAL COMPLEX CENTRAL ELEMENT1
[SYCE1], SYCE2, SYCE3, and TESTIS EXPRESSED SEQUENCE12
in mice; Corona in Drosophila) localizing to the SC central element (Costa et al., 2005; Hamer et al., 2006; Schramm et al.,
2011; Lake and Hawley, 2012). These non-TF central region
proteins are essential for TF assembly within SCs, but their
orthologs could not be identified in plants by BLAST searches
in the database.
Pachytene checkpoint2 (Pch2), a conserved AAA-ATPase,
was first identified as a pachytene checkpoint factor in budding
yeast (San-Segundo and Roeder, 1999). Subsequently, the essential role of Pch2 in pachytene checkpoint control was also
uncovered in C. elegans and Drosophila (Bhalla and Dernburg,
2005; Joyce and McKim, 2009). In mice, the Pch2 ortholog
THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) promotes the normal localization of RAD51 on meiotic chromosomes
(Roig et al., 2010). In addition, the establishment of normal crossover distribution also requires Pch2/TRIP13 in budding yeast and
mice (Zanders and Alani, 2009; Roig et al., 2010).
Here, we identified the rice ortholog of budding yeast Pch2
and mouse TRIP13. To our surprise, we found that rice Pch2/
TRIP13 is an SC component and localizes to the central region
of SCs. Hence, this rice protein was named CENTRAL REGION
COMPONENT1 (CRC1). In addition, we provide evidence that
CRC1 is essential for the initiation of meiotic recombination and
the recruitment of PAIR2 onto meiotic chromosomes. The roles of
CRC1 identified here have not been reported for Pch2 or TRIP13.
wild-type pollen did not result in seed production, indicating that
the mutant is both male and female sterile.
To isolate the target gene, we crossed the heterozygotes with
the indica rice variety Guangluai 4 and used a total of 1487 F2
and F3 segregates showing the sterile phenotype to map the
target gene. The gene was preliminarily mapped between the
sequence-tagged site (STS) markers S2 and S3 on the long arm
of chromosome 4 using 411 F2 segregates, and then further
linkage analysis using F3 segregates mapped the gene to a 16kb segment of the BAC clone AL606595 (see Supplemental
Figure 1A online). In this region, there was only one predicted
gene (gene ID: 4336167), and a 2-bp deletion at the position 830
to 831 in the gene’s coding sequence was identified in the
mutant by sequencing. This mutant allele was named crc1-1.
Additionally, three other mutant alleles of CRC1, named crc1-2,
crc1-3, and crc1-4, were also identified through map-based
cloning (see Supplemental Figure 1B online). Both crc1-2 and
crc1-3 were isolated from Yandao 8 (japonica). In crc1-2, a G-toA transition induces an amino acid substitution of Gly-264 to Arg
in the AAA-ATPase domain of CRC1. The crc1-3 mutant contains a nonsense mutation (G-to-A substitution) at position 488
in the gene’s coding sequence. The crc1-4 mutant was isolated
from Zhongxian 3037 (indica), and a G-to-A substitution at the
last nucleotide site of the third intron, which results in transcripts
lacking the fourth exon, was identified in this mutant. All of the
mutants grew normally at the vegetative stage but were completely sterile.
Real-time RT-PCR showed that CRC1 was expressed most
highly in young panicles (5 to 7 cm), at lower levels in internodes,
roots, and flowering panicles, but not in leaves (see Supplemental
Figure 2 online).
An RNA interference (RNAi) experiment of CRC1 generated 74
transgenic lines, 67 of which grew normally at the vegetative
stage, but exhibited severely reduced fertility, with seed setting
rates lower than 10%.
Full-Length cDNA Cloning and Deduced Protein Sequence
of CRC1
RESULTS
By performing rapid amplification of cDNA ends (RACE) and RTPCR, we obtained a 1785-bp full-length cDNA of CRC1, which
differs remarkably from the cDNA sequence AK106344 provided
by the Rice Full-Length cDNA Project. The deduced CRC1
protein, which contains 475 amino acid residues with a central
AAA-ATPase domain, is 43.8% identical and 58.7% similar to
mouse TRIP13 and 23.1% identical and 34.6% similar to budding yeast Pch2. Multiple sequence alignment of CRC1 with its
orthologs revealed that the CRC1/Pch2 proteins were highly
conserved within the AAA-ATPase domain (see Supplemental
Figure 3 online).
Map-Based Cloning of CRC1
Defects in Meiosis Result in the Sterility of crc1
The first crc1 mutant was characterized from the japonica rice
variety C418. The mutant grew normally at the vegetative stage
but was completely sterile (Figures 1A and 1B). I2-KI staining
showed that the pollen grains were completely nonviable in the
mutant (Figures 1C and 1D). Pollinating the mutant flowers with
To explore the reason for the sterility in crc1 mutants, we investigated the meiotic chromosome behavior of pollen mother
cells from wild-type and mutant plants. In the wild type, meiotic
chromosomes condensed as thin threads at leptotene (Figure 2A).
At subsequent stages, the chromosomes continued to condense,
CRC1 in Rice Meiosis
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Figure 1. Phenotypic Characterization of the crc1-1 Mutant.
(A) and (B) Comparison of wild-type (left) and crc1-1 (right) plants and panicles.
(C) I2-KI staining of wild-type pollen grains.
(D) I2-KI staining of crc1-1 pollen grains.
Bars = 50 mm.
[See online article for color version of this figure.]
and the thin threads began to synapse at zygotene (Figure 2B). At
pachytene, the chromosomes were fully synapsed and present as
thick threads (Figure 2C). At diplotene, the synapsis was relieved
and chiasmata, which correspond to the crossovers formed at
pachytene, were visible. Twelve bivalents could be clearly observed at diakinesis (Figure 2D), and the further condensed bivalents aligned on the equatorial plate at metaphase I (Figure 2E).
Reductional segregation of the chromosomes occurred at anaphase I (Figures 2F and 2G), and then the equational segregation
of sister chromatids at the second meiotic division produced
tetrad spores (Figure 2I).
In crc1-1, the chromosome behavior at leptotene was similar
to that in the wild type (Figure 3A). However, abnormal chromosome behavior was observed thereafter. Although the chromosomes condensed normally during prophase I, obvious features
of homologous chromosome pairing were not observed from zygotene to pachytene (Figures 3B and 3C). Subsequently, 24 chromosome univalents were clearly observed at diakinesis (Figure 3D),
and the univalents were often scattered throughout the entire nucleus at metaphase I (Figure 3E). At anaphase I, the chromosomes
were unequally separated, and lagging chromosomes were always
observed (Figures 3F and 3G). At the end of meiosis, different
numbers of chromosomes and micronuclei were observed in the
daughter cells (Figures 3H and 3I). The meiotic chromosomes in
sterile CRC1RNAi plants and other crc1 mutants behaved similarly
to those in crc1-1 (see Supplemental Figures 4A to 4D online).
CRC1 Is a Prerequisite for Abnormal Chromosome
Entanglement and Fragmentation in rec8 Meiocytes
REC8, a conserved meiosis-specific component of the cohesin
complex, is essential for meiotic DSB repair in various organisms. In rice rec8 meiocytes, chromosome entanglements were
observed from diplotene to metaphase I, and chromosome
fragments appeared at anaphase I (Figure 4A) (Shao et al.,
2011). To investigate the role of CRC1 in meiotic pairing and
recombination, the meiosis in the rec8 crc1 double mutant was
investigated. In rec8 crc1, the chromosomes during prophase
I also exhibited the fluffy appearance observed in the rec8 single
mutant. However, the abnormal chromosome entanglement and
fragmentation observed in rec8 were not observed in the double
mutant (Figure 4A). In rec8 crc1, the chromosomes were clearly
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Figure 2. Meiosis in the Wild Type.
(A) Leptotene.
(B) Zygotene.
(C) Pachytene.
(D) Diakinesis.
(E) Metaphase I.
(F) Anaphase I.
(G) Late anaphase I.
(H) Telophase I.
(I) Telophase II.
Bars = 5 mm.
observed as univalents at the end of prophase I; thereafter,
equational segregation of the chromosomes occurred at anaphase I (Figure 4A).
CRC1 Is Essential for Meiotic DSB Formation
DSB formation is essential for meiotic chromosome pairing in
most eukaryotes, including plants. The elimination of rec8
chromosome entanglement and fragmentation by the crc1 mutation, plus the lack of meiotic pairing in crc1, indicates that
CRC1 is very likely a DSB-forming factor. To confirm this
speculation, immunostaining was conducted in wild-type and
crc1-1 microsporocytes using antibodies against several meiotic proteins of rice, including COM1, DMC1, ZIP4, and MER3.
COM1, which is involved in DSB end resection, is essential for
rice meiotic recombination (Ji et al., 2012). DMC1 mediates the
single-strand invasion in meiotic recombination and is required
for rice meiotic pairing (Deng and Wang, 2007). ZIP4 and MER3,
two components of the ZMM complex (a protein complex required for crossover formation), are essential for interferencesensitive crossover formation in rice (Wang et al., 2009; Shen
et al., 2012). Therefore, the antibodies against COM1, DMC1,
ZIP4, and MER3 can be used as markers to monitor the process
of meiotic recombination. In immunostaining assays, COM1,
DMC1, ZIP4, and MER3 were observed as punctuate foci on
wild-type chromosomes during early meiosis, and these foci
persisted until pachytene (Figures 5A to 5C and 5G) (Wang et al.,
2009; Ji et al., 2012; Shen et al., 2012). By contrast, no signals
of COM1, DMC1, ZIP4, or MER3 were observed on the crc1-1
meiotic chromosomes (Figures 5D to 5F and 5J), suggesting
that CRC1 is essential for meiotic DSB formation.
Meiotic DSBs trigger the phosphorylation of histone H2AX
from leptotene to early zygotene in various eukaryotes (Dickey
et al., 2009). Therefore, to further confirm that CRC1 is essential
CRC1 in Rice Meiosis
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Figure 3. Meiosis in the crc1-1 Mutant.
(A) Leptotene.
(B) Zygotene.
(C) Pachytene.
(D) Diakinesis.
(E) Metaphase I.
(F) Anaphase I.
(G) Late anaphase I.
(H) Telophase I.
(I) Telophase II.
Bars = 5 mm.
for meiotic DSB formation, an antibody specifically recognizing
the rice phospho-histone H2AX (gH2AX) was raised in rabbits,
and immunostaining with the antibody was conducted in microsporocytes. The results revealed numerous chromatinassociated gH2AX dot and patchy signals during early prophase
I in the wild type (Figure 4B). In crc1-1, however, no gH2AX
signals were observed in meiocytes at the corresponding stages
(Figure 4B). Collectively, these results indicate that CRC1 is
essential for meiotic DSB formation.
CRC1 Is Essential for the Recruitment of PAIR2 onto
Meiotic Chromosomes
PAIR2, the rice ortholog of budding yeast Hop1 and mouse
HORMAD1 and -2, is essential for homologous chromosome
pairing (Nonomura et al., 2006). In budding yeast and mice, the
normal chromosome localization of Hop1, HORMAD1, and
HORMAD2 requires Pch2/TRIP13 (Börner et al., 2008; Wojtasz
et al., 2009). Therefore, immunostaining for PAIR2 was conducted in wild-type and crc1-1 microsporocytes. The result
showed that PAIR2 preferentially associated presynaptic chromosome axes in the wild type (Figure 5H) (Nonomura et al.,
2006; Wang et al., 2009). In crc1-1, however, no PAIR2 signals
were observed (Figure 5K), indicating that CRC1 is essential for
the chromosome recruitment of PAIR2.
Immunostaining for another chromosome axis-associated
protein, PAIR3, was also conducted. Similar to the wild type, the
antibody against PAIR3 labeled the chromosome axes during
prophase I in crc1-1 (Figures 5I and 5L).
CRC1 Interacts with PAIR1 in Vitro
We then tried to identify CRC1-interacting partners to help
elucidate the roles of CRC1. Therefore, yeast two-hybrid (Y2H)
assays between CRC1 and proteins including PAIR1, PAIR2,
PAIR3, SPO11-1, and SPO11-4 were conducted. The results
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Figure 4. CRC1 Is Essential for Meiotic DSB Formation.
(A) Comparison of meiotic chromosome behavior in rec8 and rec8 crc1. Bars = 5 mm.
(B) Immunostaining for gH2AX (red) in the wild type and crc1-1 at zygotene. Chromosomes were stained with 4,6-diamidino-2-phenylindole (DAPI;
blue). REC8 signals (green) were used to indicate the meiocytes. Bars = 5 mm.
(C) CRC1 interacts with itself and PAIR1 in Y2H assays. The interactions were verified by the growth of yeast on selective medium (SD-Leu-Trp-Ura-His
with 5 mM 3-AT) and the blue color of yeast in X-Gal assays. BD, bait vector; AD, prey vector; 3-AT, 3-amino-1,2,4-triazole.
(D) Pull-down assays. Lane 1, purified maltose-binding protein (MBP) tag alone; lane 2, purified MBP-CRC1 alone; lane 3, MBP-CRC1 incubated with
resin-bound GST tag; lane 4, MBP-CRC1 incubated with resin-bound GST-PAIR1; lane 5, MBP tag incubated with resin-bound GST-PAIR1; lane 6,
MBP tag incubated with resin-bound GST-CRC1; lane 7, MBP-CRC1 incubated with resin-bound GST-CRC1; lane 8, MBP tag incubated with resinbound GST tag.
revealed an interaction between CRC1 and PAIR1 (Figure 4C).
The self-interaction of CRC1 was also detected in this system
(Figure 4C).
To further validate these interactions, we conducted in vitro
glutathione S-transferase (GST) pull-down assays. In the assays, MBP-CRC1 showed specific affinity to GST-PAIR1 and
GST-CRC1 (Figure 4D), consistent with the results of the Y2H
assays. The interactions suggest that CRC1 and PAIR1 act as
a complex to promote meiotic DSB formation.
CRC1 signals were situated in the center between and along the
REC8 signals (Figure 6D), indicating that CRC1 localizes to the
SC central region. The CRC1 protein was rapidly removed
from the chromosomes at diplotene, and at diakinesis, no CRC1
signals were observed on the chromosomes (Figure 6E).
Similar immunostaining signals were observed when we used
polyclonal antibodies against the N-terminal 171 residues of
CRC1 or against the CRC1 C-terminal 175 residues. The CRC1
immunostaining signals were not detected in mitotic cells or in
crc1 mutants.
CRC1 Localizes to the Central Region of SCs
To investigate the spatial and temporal localization of CRC1,
a polyclonal antibody was raised against the full-length sequence of CRC1, and immunostaining with the antibody was
conducted in rice microsporocytes. In this assay, CRC1 first
appeared as punctuate foci on chromosomes at leptotene (Figure
6A). At zygotene, the cells began to show linear CRC1 signals along
the chromosome axes (Figure 6B). At early pachytene, CRC1 was
observed as linear signals along the entire chromosome axes, and
these linear signals were slightly narrower than the REC8 signals at
this stage (Figure 6C). At late pachytene or early diplotene, when
REC8 was present as double thread signals on each chromosome,
CRC1 and ZEP1 Colocalize to SC Central Region in
a Mutually Dependent Fashion
The pattern of CRC1 localization was similar to that of ZEP1.
Therefore, dual immunostaining for CRC1 and ZEP1 was conducted in the wild type. The result revealed complete colocalization of CRC1 and ZEP1 from leptotene to pachytene (Figure
7A). Immunostaining for CRC1 and ZEP1 was also conducted in
zep1 and crc1-1, respectively. CRC1 was not detected on zep1
meiotic chromosomes, nor was ZEP1 detected in crc1-1 (Figure
7B), indicating that CRC1 and ZEP1 colocalize to the SC central
region in a mutually dependent fashion.
CRC1 in Rice Meiosis
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Figure 5. Immunostaining Detection of Several Meiotic Proteins in the Wild Type and crc1-1.
(A) to (C), (G), and (H) Staining for COM1, DMC1, ZIP4, MER3, and PAIR2 (green) at early zygotene in the wild type.
(D) to (F) and (J) to (K) Staining for COM1, DMC1, ZIP4, MER3, and PAIR2 (green) at early zygotene in crc1-1.
(I) and (L) PAIR3 signals (green) at late zygotene in the wild type (I) and crc1-1 (L).
REC8 signals (red) were used to indicate the meiotic chromosome axes. Bars = 5 mm.
Here, the meiosis in zep1 crc1 double mutant was also investigated. In zep1, the homologous chromosomes can pair, but cannot
synapse, with each other (Wang et al., 2010). In zep1 crc1, however,
no homologous chromosome pairing was observed (Figure 7C),
consistent with the essential role of CRC1 in meiotic recombination
initiation.
CRC1 Interacts with ZEP1 in Yeast
Subsequently, Y2H assays were conducted between CRC1 and
ZEP1. The result revealed an interaction between CRC1 and
ZEP1 (Figure 7D). The TF proteins were reported to be aligned in
head-to-head dimers, with the C terminus positioned in the LE
and the N terminus in the central element, to bridge the parallel
LEs (de Boer and Heyting, 2006). Thus, the domain of ZEP1
responsible for interacting with CRC1 may define the ultrastructural position of CRC1 within SCs. Therefore, Y2H assays
between CRC1 and the three ZEP1 domains were conducted.
The results showed that CRC1 only interacted with the
N-terminal domain (residues 1 to 70) of ZEP1 (see Supplemental
Figure 5 online), consistent with the SC central region localization
of CRC1.
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Figure 6. Dual Immunostaining for REC8 (Red) and CRC1 (Green) in Rice Microsporocytes.
(A) Leptotene.
(B) Zygotene.
(C) Early pachytene.
(D) Late pachytene or early diplotene. Only two chromosomes in a cell are shown.
(E) Diakinesis.
Bars = 5 mm.
DISCUSSION
CRC1 May Be a Conserved SC Component
Although SCs are highly conserved at the ultrastructural level,
their components identified are poorly conserved at the
sequence level. Here, we identified CRC1, a novel SC central
region component. CRC1 is highly orthologous to budding yeast
Pch2 and mouse TRIP13. Thus, it may be a conserved SC
component.
Pch2 is a protein that was thought to have close relationships
with synapsis (Wu and Burgess, 2006). Its orthologs exist
CRC1 in Rice Meiosis
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Figure 7. CRC1 Is a Novel SC Component.
(A) CRC1 (green) colocalizes with ZEP1 (red) during SC assembly. Chromosomes were stained with DAPI (blue). Bars = 5 mm.
(B) Immunostaining for CRC1 (green) and ZEP1 (green) in crc1-1 and zep1 microsporocytes. REC8 signals (red) were used to indicate the chromosome
axes. Bars = 5 mm.
(C) Comparison of meiotic chromosome behavior in zep1 and zep1 crc1. Bars = 5 mm.
(D) CRC1 interacts with ZEP1 and itself in Y2H assays. The interactions were verified by the growth of yeast on selective medium (SD-Leu-Trp-Ura-His
with 5 mM 3-AT) and the blue color of yeast in X-Gal assays. BD, bait vector; AD, prey vector.
extensively in organisms known to undergo synaptic meiosis but
are absent from asynaptic organisms (e.g., Schizosaccharomyces
pombe, Aspergillus nidulans, and Tetrahymena thermophila)
(Wu and Burgess, 2006), suggesting that Pch2 is highly associated with synapsis during evolution. In budding yeast,
Pch2 functions in a pachytene checkpoint pathway that
specifically monitors aberrant SC intermediates (Wu and Burgess,
2006; Ho and Burgess, 2011), and the mechanism underlying this
role remains elusive. The essential role of Pch2 in the synapsis
checkpoint control was also characterized in C. elegans (Bhalla
and Dernburg, 2005). The identification of CRC1 as an SC component provides a valuable insight into the role of CRC1 in the
synapsis-monitoring checkpoint control. Pch2 also may be an SC
component and thus functions as a primary sensor to signal the
state of SC assembly, thereby promoting progress through or
arrest at the pachytene stage.
CRC1 Is Essential for Meiotic Recombination Initiation and
PAIR2 Recruitment onto Chromosomes
In meiosis, DSB formation is catalyzed by the conserved Spo11
protein. In addition to Spo11, a suite of regulatory and accessory factors are also essential for the DSB formation. In budding
yeast, nine factors form a large complex with Spo11 to promote the Spo11-mediated DSB formation (Edlinger and
Schlögelhofer, 2011). Here, we provide evidence that CRC1 is
essential for meiotic DSB formation. CRC1 interacts with PAIR1,
a plant-specific DSB-forming factor, in vitro, suggesting that
CRC1 and PAIR1 act as a complex to promote the DSB formation. In Y2H assays, we did not detect the interactions between SPO11 (including SPO11-1 and SPO11-4) and PAIR1 or
between SPO11 and CRC1, and the exact functional relationship between SPO11 and the PAIR1-CRC1 complex remains to
be determined.
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PAIR2, the rice ortholog of budding yeast Hop1 and Arabidopsis ASYNAPTIC1 (ASY1), is essential for the chromosome
pairing in meiosis (Nonomura et al., 2006). The budding yeast
Hop1 functions in meiotic DSB formation, and significantly reduced
DSB level was observed in the absence of Hop1 (Mao-Draayer
et al., 1996). In addition, Hop1 also plays an essential role in establishing the interhomolog rather than intersister chromatid recombination (Niu et al., 2005). In Arabidopsis, ASY1 also plays
a key role in establishing the interhomolog recombination, but
meiotic DSBs are formed independently of ASY1 (Sanchez-Moran
et al., 2007). In this study, we found that CRC1 is essential for the
recruitment of PAIR2 onto meiotic chromosomes. This result
suggests that CRC1 is also involved in the early steps of meiotic
recombination and perhaps promotes the interhomolog recombination through the recruitment of PAIR2.
CRC1 Has Different Functions from Pch2 and TRIP13
The budding yeast Pch2 and mouse TRIP13 are highly orthologous
to rice CRC1, but the roles of CRC1 identified here have not been
reported for Pch2 or TRIP13.
CRC1 is essential for meiotic recombination initiation. However, it is known that Pch2 and TRIP13 are not essential for
meiotic recombination initiation. In budding yeast pch2 mutant
strains, meiotic recombination is slightly delayed, but meiotic
pairing appears to be normal, and recombination repair can be
completed (Wu and Burgess, 2006; Börner et al., 2008). Therefore, the mutant strains exhibit normal spore viability. In Trip13
knockout mice, meiotic recombination is initiated normally but
cannot be completed (Roig et al., 2010).
In rice, CRC1 is essential for the recruitment of PAIR2 onto
meiotic chromosomes. However, this is not the case for Pch2 or
TRIP13. In budding yeast and mice, the chromosome axis-associated
HORMA-domain proteins (budding yeast Hop1 and mouse
HORMAD1 and HORMAD2) preferentially localize to presynaptic
chromosome axes, and with the SC assembly, they are significantly depleted from the synapsed regions of chromosomes
(Börner et al., 2008; Wojtasz et al., 2009). Pch2 and TRIP13 are
essential for the depletion of the HORMA-domain proteins, but
not for the recruitment of these proteins (Börner et al., 2008;
Wojtasz et al., 2009; Roig et al., 2010). The distribution of Hop1,
HORMAD1, and HORMAD2 along the presynaptic chromosome
axes is normal in the pch2/Trip13 mutants.
METHODS
Plant Materials
All crc1 lines were isolated from mutants induced by 60Co g-ray irradiation. To obtain the rec8 crc1 and zep1 crc1 double mutants, the crc1-1+/2
plants were crossed with rec8+/2 and zep1+/2; subsequently, genotyping of
the F1 and F2 progeny was conducted to select the double mutants. The
rec8 and zep1 mutants used in this study were named Osrec8-1 and zep1-1,
respectively, in previous reports (Wang et al., 2010; Shao et al., 2011). All of
the plants were grown in paddy fields.
Map-Based Cloning of the Target Gene
To map the target gene, STS and cleaved-amplified polymorphic sequence
markers were developed based on the genomic sequence difference
between Nipponbare (japonica) and 9311 (indica) according to the data
published at http://www.ncbi.nlm.nih.gov/. Among them, R1 and R3 were
cleaved-amplified polymorphic sequence markers, and the others were
STS markers (see Supplemental Table 1 online). The PCR products of R1
and R3 primers were digested with NheI and NcoI, respectively, to determine the polymorphism. The crc1-2+/2, crc1-3+/2, and crc1-4+/2 plants
were crossed with Longtepu (indica), Guangluai 4 (indica), and Nipponbare (japonica), respectively, to produce the mapping populations.
Full-Length cDNA Cloning of CRC1
RNA extraction was conducted using the TRIzol reagent (Invitrogen).
Reverse transcription and RACE were performed using the SMARTer
RACE cDNA amplification kit (Clontech) (see Supplemental Table 2 online
for the gene-specific primers used in this experiment). Gene-specific
primers R5-1 and R5-2, combined with the universal primers provided in
the kit, were used to perform 59 RACE PCR. For 39 RACE PCR, genespecific primers R3-1 and R3-2 were used along with the universal primers. PCR using primers ORF-F and ORF-R was performed to amplify the
open reading frame. The PCR products were cloned into the PMD18-T
vector (TaKaRa) and sequenced. The sequences were then spliced together to obtain the full-length cDNA sequence.
Real-Time RT-PCR for Transcript Expression Assay
Total RNA was extracted individually from the roots, internodes, leaves,
young panicles (5 to 7 cm), and flowering panicles of C418. Reverse
transcription was performed using the SuperScript III reverse transcriptase (Invitrogen). Real-time RT-PCR analysis was performed using the
Bio-Rad CFX96 real-time PCR instrument and EvaGreen (Biotium). The
RT-PCR was conducted with gene-specific primers CRC1RT-F and
CRC1RT-R for CRC1, and Actin-F and Actin-R for ACTIN (see Supplemental Table 2 online for the primer sequences).
RNAi Knockdown of CRC1
A fragment of the CRC1 coding sequence was amplified using the primers
CRC1i-1F and CRC1i-1R (see Supplemental Table 2 online for the primer
sequences). The RNAi vector (pCam23A) construction and transformation
were performed as previously described (Wang et al., 2009; Huang et al.,
2010).
Antibody Production
The anti-CRC1 antibodies were raised in mice against GST-fused proteins. The DMC1 polyclonal antibody was generated in mice against GSTfused DMC1 protein. The antibodies against REC8, ZEP1, PAIR2, PAIR3,
COM1, MER3, and ZIP4 were previously described (Wang et al., 2009,
2011; Wang et al., 2010; Ji et al., 2012; Shen et al., 2012). To produce the
rice (Oryza sativa) gH2AX antibody, a rabbit polyclonal antibody was
raised against a KLH conjugated peptide (DIGSA[pS]QEF, designated
H2AX-C-pS134) of the rice H2AX (gene ID: 4333939) and then the antibody was affinity purified first with H2AX-C (DIGSASQEF) and then with
H2AX-C-pS134 to obtain the antibody that specifically recognizes the
phosphorylated Ser-134 of rice H2AX.
Meiotic Chromosome Preparation
Fresh young panicles were fixed in Carnoy’s solution (ethanol:glacial
acetic acid, 3:1), and anthers at the proper developmental stage were then
squashed in an acetocarmine solution. After being frozen in liquid nitrogen
for 2 min, the slides with squashed meiocytes were dehydrated through
an ethanol series (70, 90, and 100%). Chromosomes on the slides were then
counterstained with DAPI in an antifade solution (Vector Laboratories).
CRC1 in Rice Meiosis
11 of 12
Chromosome images were captured under the Zeiss A2 fluorescence
microscope with a micro-charge-coupled device camera.
Supplemental Figure 3. Alignment of CRC1 with Its Orthologs from
M. musculus, D. melanogaster, C. elegans, and S. cerevisiae.
Immunofluorescence
Supplemental Figure 4. Meiotic Chromosomes at Diakinesis in the
RNAi and Mutant Lines.
After being fixed in 4% (w/v) paraformaldehyde for 30 min at room
temperature, fresh young panicles were soaked in PBS solution. Anthers
at the proper developmental stage were then squashed on slides. After
soaking in liquid nitrogen for 2 min, the slides were dehydrated through
an ethanol series (70, 90, and 100%). Then, the slides with squashed
meiocytes were used in immunostaining with antibody combinations
diluted 1:500 in TNB buffer (0.1 M Tris-HCl, pH 7.5, 0.15 M NaCl, and
0.5% blocking reagent). After incubation in a humid chamber at 37°C for 4
h, the slides were washed three times with PBS solution. Texas red–
conjugated goat anti-rabbit antibody and fluorescein isothiocyanate–
conjugated sheep anti-mouse antibody (1:100) were then added to the
slides. Finally, after incubation in the humid chamber at 37°C for 30 min
and then three rounds of washes with PBS, the slides were counterstained
with DAPI in the antifade solution.
Supplemental Figure 5. Y2H Assays between CRC1 and Three ZEP1
Domains.
Supplemental Table 1. Markers Designed for Map-Based Cloning.
Supplemental Table 2. Primers for RACE, RT-PCR, and Plasmid
Construction.
ACKNOWLEDGMENTS
This work was supported by grants from the Ministry of Sciences and
Technology of China (2011CB944602 and 2012AA10A301) and the
National Natural Science Foundation of China (31230038).
AUTHOR CONTRIBUTIONS
Y2H Assays
The Y2H assays were performed using the ProQuest two-hybrid system
with Gateway Technology (Invitrogen). Plasmids pDEST32 and pDEST22
were used as the bait and prey vectors, respectively. The yeast strain used
here was MaV203.
Pull-Down Assays
The PAIR1 coding sequence was cloned into PGEX4T-2 (Amersham), and
the CRC1 coding sequence was cloned into PGEX4T-2 and PMAL-C5X
(NEB). Escherichia coli BL21 (DE3) cells harboring the empty or recombinant
vectors were cultured in Luria-Bertani medium at 37°C to reach the exponential phase, and the protein expression was then induced by adding
0.1 mM isopropyl b-D-1-thiogalactopyranoside to the cultures that would be
further cultured at 16°C for about 10 h. Then, the cell lysates containing ;15
to 50 mg soluble GST-fused proteins or GST tag were incubated with 120 mL
50% Glutathione Sepharose 4B (GE Healthcare) at 4°C for 1 h. Thereafter,
the Glutathione Sepharose 4B was washed three times with PBS solution.
Then, the cell lysates containing ;15 to 50 mg soluble MBP-fused proteins or
MBP tag were incubated with the Glutathione Sepharose 4B at 4°C for 1 h.
After washing the Glutathione Sepharose 4B five times with PBS solution, the
proteins were eluted with 100 mL elution buffer (50 mM Tris-HCl and 10 mM
reduced glutathione, pH 8.0). Then, the proteins were subjected to protein
gel blotting analysis using an anti-MBP antibody (EarthOx).
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: CRC1, KF245924;
COM1, AK119314; DMC1, AB079874; ZIP4, BAD82387; MER3, FJ008126;
H2AX, NP_001051106; PAIR1, AB158462; PAIR2, AB109238; PAIR3,
FJ449712; SPO11-1, GU170363; SPO11-4, GU177866; REC8, AY371049;
ZEP1, GU479042; TRIP13, NP_081458; PCH2 (Drosophila melanogaster),
NP_524282; PCH-2 (Caenorhabditis elegans), NP_495711; and Pch2
(Saccharomyces cerevisiae), NP_009745.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Molecular Identification of CRC1.
Supplemental Figure 2. Real-Time RT-PCR Expression Analysis of
CRC1.
C.M. and Z.C. designed the research and wrote the article. C.M., D.T.,
H.Z., M.W., and Y.L. performed the research and analyzed the data. S.T.,
H.Y., and M.G. provided technical assistance.
Received April 26, 2013; revised June 24, 2013; accepted July 25, 2013;
published August 13, 2013.
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CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential
for Meiotic Recombination Initiation in Rice
Chunbo Miao, Ding Tang, Honggen Zhang, Mo Wang, Yafei Li, Shuzhu Tang, Hengxiu Yu, Minghong
Gu and Zhukuan Cheng
Plant Cell; originally published online August 13, 2013;
DOI 10.1105/tpc.113.113175
This information is current as of June 17, 2017
Supplemental Data
/content/suppl/2013/07/30/tpc.113.113175.DC1.html
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