The Arabidopsis BLAP75/Rmi1 Homologue Plays Crucial
Roles in Meiotic Double-Strand Break Repair
Liudmila Chelysheva, Daniel Vezon, Katia Belcram, Ghislaine Gendrot¤, Mathilde Grelon*
INRA de Versailles, Institut Jean-Pierre Bourgin, Station de Génétique et d’Amélioration des Plantes UR-254, Versailles, France
Abstract
In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIa/Top3 to maintain
genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double
Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic
DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of
the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination
progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome
fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is
observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in
meiotic double-strand break (DSB) repair—that it acts after the invasion step mediated by RAD51 and associated proteins
and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In
conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination
machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis
but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in
the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to
form bivalent structures, even when ZMM are absent.
Citation: Chelysheva L, Vezon D, Belcram K, Gendrot G, Grelon M (2008) The Arabidopsis BLAP75/Rmi1 Homologue Plays Crucial Roles in Meiotic Double-Strand
Break Repair. PLoS Genet 4(12): e1000309. doi:10.1371/journal.pgen.1000309
Editor: Michael Lichten, National Cancer Institute, United States of America
Received August 12, 2008; Accepted November 14, 2008; Published December 19, 2008
Copyright: ß 2008 Chelysheva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by INRA, Department of Genetics and Plant Breeding.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: grelon@versailles.inra.fr
¤ Current address: Laboratoire Reproduction et Développement des Plantes, Ecole Normale Supérieure de Lyon, ENS/CNRS UMR 5667/INRA, Université Lyon I,
Lyon, France
proceed through the capture of the second processed DNA end to
produce a double Holliday junction intermediate (dHJ). The dHJ
is then resolved by an unknown resolvase to generate COs
products. This CO pathway is under the control of a set of genes
which includes the ZMM family (for Zip1, Zip2, Zip3, Zip4, Mer3
and Msh4/Msh5). Another CO pathway, that does not proceed
through dHJ formation, also coexists in most species and is under
the control of the Mus81/Mms4 endonuclease [6].
In somatic cells, homologous recombination (HR) is also used to
repair DNA DSBs that arise either from damage or from stalled
replication forks. In this context, contrary to what happens during
meiotic HR, repair is mostly directed towards the sister chromatids
rather than the homologous chromosome. Furthermore, COs are
generally prevented in favour of NCO events, probably by
preferential involvement of the SDSA repair pathway and to
dissolve dHJ to generate NCO events.
The eukaryotic homologues of the highly conserved RecQ
helicase family are known to be particularly crucial components of
regulation mechanisms against CO formation. The Bloom protein
(BLM, one of the five human RecQ helicases) was shown to
disrupt D-loop intermediates in vitro [7], to dissolve dHJ [8,9] and
to disrupt the Rad51 presynaptic filament [9,10]. In vivo, the
antiCO effect of BLM/Sgs1 helicase was demonstrated by the fact
that yeast sgs1 mutants as well as Bloom’s syndrome patients or
Introduction
From a diploid mother cell, meiosis generates four haploid
products from which gametes differentiate. This ploidy reduction
is a direct consequence of two rounds of chromosomal segregation
(meiosis I and meiosis II) following a single S phase. The first
meiotic division separates homologous chromosomes from each
other while meiosis II separates sister chromatids.
Recombination is one of the key events in meiosis. It gives rise
to crossovers (COs), which are essential for the correct segregation
of homologous chromosomes during meiosis I, ensuring the
association of homologous chromosomes into bivalents. Meiotic
recombination can also lead to gene conversion not associated
with COs (NCOs), events that are probably much more frequent
than COs at least in plants and mammals [1].
The current model for meiotic recombination [2,3] proposes
that it is initiated by the programmed formation of DNA doublestrand breaks (DSBs), which are then resected to generate 39 single
stranded DNA molecules that drive DNA repair onto the
homologous chromosome by invading an intact homologous
chromosome. DNA strand exchange results in the formation of
joint molecules. These joint molecules either dissociate enabling
the broken chromosome to rejoin through synthesis-dependent
strand annealing (SDSA) [3–5], or form stable D-loops which
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Arabidopsis BLAP75 in Meiosis
the BLM protein was shown to colocalise with the recombination
proteins RPA, RAD51/DMC1 and MSH4 [32–34], but BLM
disruption in mouse has no effect on meiotic CO rates [14]. This is
not the case for the Drosophila melanogaster, Caenorhabditis elegans and
Schizosaccharomyces pombe BLM orthologues, for which depletion is
associated with a decrease in CO rates [35–37].
In this study we show that the Arabidopsis BLAP75/Rmi1
homologue is absolutely required for meiotic DSB repair onto
homologous chromosome or sister chromatid. We also provide
evidence that, in the absence of A. thaliana BLAP75, recombination
is initiated and progresses until the formation of recombination
intermediates that allow the formation of bivalents even in the
absence of ZMM.
Author Summary
Recombination is a process by which cells can repair DNA
damage. Such repair can either be crossovers (CO), in
which DNA molecules are submitted to major exchanges,
or non-crossover (NCO) events. Eukaryotic cells have
developed several mechanisms to maintain genome
stability during vegetative development by limiting the
occurrence of CO events in favour of NCO. BLAP75/Rmi1,
BLM/Sgs1, and TopoIIIa/Top3 act together in a complex
(BTB/RTR) known to be a crucial component of regulation
mechanisms against CO formation. However, CO/NCO
regulation is thought to be very different during meiosis
since homologous chromosomes (paternal and maternal)
overcome at least one CO/pair. In this study, we
investigate the role of the BTB/RTR complex during
meiotic recombination through the analysis of a function
of one of its members: the A. thaliana homologue of
BLAP75/Rmi1. We show for the first time that BLAP75/Rmi1
is also a key protein of the meiotic homologous
recombination machinery. In Arabidopsis, we found that
this protein is dispensable for homologous chromosome
recognition and synapsis, but necessary for the repair of
meiotic double-strand breaks. Furthermore, in the absence
of BLAP75, bivalent formation can happen even in the
absence of CO.
Results
Identification and Molecular Characterisation of A.
thaliana BLAP75/RMI1
In a screen for A. thaliana T-DNA (Agrobacterium tumefaciens
transferred DNA) insertions that generate meiotic mutants, we
isolated a mutant (line FCN288) disrupted in the A. thaliana
predicted open reading frame, At5g63540 (see Materials and
Methods), annotated as a protein of unknown function in TAIR
(http://arabidopsis.org/). Another insertion line in At5g63540
available in the public databases (http://signal.salk.edu/),
SALK_093589, was obtained (Figure 1A) and showed the same
meiotic phenotype as FCN288 (Figure 1A). Genetic tests
confirmed that these two mutations were allelic (see Materials &
Methods), demonstrating that disruption of At5g63540 is responsible for the mutant phenotype observed in both lines. Interestingly, other insertion lines which we investigated (Salk_005449,
Salk_054053, Salk_054062 and Salk_094387, Figure 1A) that
contained a T-DNA insertion in the 39 region of At5g63540 did
not show any detectable phenotype (not shown). According to the
T-DNA insertion sites, these mutant lines are expected to produce
a truncated protein, suggesting that the C-terminal part of the
protein is not necessary for its function.
The At5g63540 cDNA encodes a 644-amino acid (aa) protein
(Figure 1B). Database searches using the BLASTP program
(Blosum 45) for proteins similar to that encoded by At5g63540
revealed the existence of a conserved domain (from aa 101 to 194,
e value 6610220) [38] annotated as a domain of unknown
function (DUF1767, pfam08585) but found in the N-terminus of
the nucleic acid binding domain of several protein families,
represented mainly by the mammalian BLAP75 proteins and
showing weak homology with an OB-fold domain [19]. When
BLAST searches against the non-redundant database with the
At5g63540 protein sequence were carried out, the highest scores
(outside the plant kingdom) were obtained with several sequences
similar to the protein BLAP75/Rmi1, including similarities outside
the DUF1467 region (Figure 1B). Alignment of these proteins
revealed two conserved domains: one spanning from aa 101 to aa
294 (DUF1467, Figure 1B) and another one from aa 484 to aa
627. Recent biochemical studies performed on the human
BLAP75 protein showed that aa 151 to aa 211 (contained in
DUF1467, and corresponding to aa 219 to aa 279 on At5g63540)
are necessary for the interaction of BLAP75 with BLM and
TopoIIIa. One conserved lysine (K166, corresponding to K235 in
At5g63540, Figure 1B) is absolutely necessary not only for
interaction with BLM-TopoIIIa but also for enhancing dHJ
dissolution and HJ dissociation [25]. These authors also identified
a single strand DNA binding activity domain lying in the C
terminus of the human protein [25]. It corresponds to the second
BLM-deficient mice have elevated rates of mitotic recombination
(either reciprocal sister chromatid exchanges (SCE) or increased
frequency of exchange between homologous chromosomes) [11–
14]. In plants at least seven RECQ-like genes were identified [15]
and functional analyses showed that A. thaliana RECQ4A is likely to
be the functional homologue of BLM [16,17]. It was also shown to
partially suppress the embryo-lethality of A. thaliana top3a and to be
lethal in conjunction with the A. thaliana mus81A mutation [18].
The human protein BLAP75 (for Bloom Associated Protein of
75 kD) was recently identified [19,20] as a 75 kD protein which copurified in diverse Bloom (BLM)-containing complexes from HeLa
cells. It was proposed to form the structural core of all BLM
complexes with BLM and TopoIIIa (the human topoisomerase
3a) [19]. A BLAP75 homologue was described in yeast (Rmi1/
Nce4) and the conservation of the BTB complex (BLM-TopoIIIBLAP75) was demonstrated in S. cerevisiae, where it is known as the
RTR (RecQ helicase-Top3-Rmi1) complex [21,22]. Recent
biochemical studies showed that the BTB/RTR complex plays a
crucial role in the dissolution of dHJ to produce NCOs [9,23,24].
The proposed mode of action is that BLM/Sgs1 decatenates dHJs
to form a hemicatenane substrate for the topoisomerase 3.
BLAP75/Rmi1 would be necessary for the loading and stability
of the complex. It strongly enhances BLM-TopoIIIa dependant
dHJ dissolution in vitro [9,21,24–26]. It is also possible that this
complex works on other HR substrates such as Rad51 presynaptic
filaments or D loops [10,27]. Therefore, the BTB/RTR
complexes are proposed to act at different levels of the HR
process to limit CO formation in favour of NCO events.
Limited data is available on the involvement of BTB/RTR
complexes in meiotic DNA repair. In S. cerevisiae, sgs1D top3D, and
rmiD mutants show reduced sporulation and decreased spore
viability [11,12,22,28,29]. For sgs1, the phenotype was correlated
with meiosis I nondisjunction and precocious sister segregation
[11,28] but, unlike the situation in somatic cells, in most cases no
increase in meiotic recombination was detected [11,28,30,31].
Nevertheless, Sgs1 was shown to prevent CO maturation in zmm
mutants, and could suppress sister chromatid dHJ formation
during meiotic recombination [30,31]. In mouse spermatocytes,
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Figure 1. The A. thaliana BLAP75/RMI1 open reading frame. (A) Schematic representation of the A. thaliana BLAP75/RMI1 coding sequence.
Exons are represented as grey boxes and T-DNA insertions in the studied alleles are indicated. Arrows show the orientation of the sequenced T-DNA
left border. (B) Alignment of A. thaliana (At5g63540), H. sapiens (AL732446.4), Xenopus tropicalis (NM_001016296.1) and Oryza sativa (XM_472298.1)
BLAP75 homologues. Identical aminoacids (aa) are boxed in black whereas similar aa are boxed in grey. The amino acids underlined in yellow
represent the two conserved domains of the BLAP75 protein family. The region shown in H. sapiens BLAP75 (aa 151 to 211) to be necessary for
binding to BLM and TopoIIIa [25] is underlined in red. The asterisk indicates the conserved lysine necessary for interaction with TopoIIIa [25].
doi:10.1371/journal.pgen.1000309.g001
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Arabidopsis BLAP75 in Meiosis
conserved domain of this protein family which is found in plant
BLAP75 (Figure 1B).
The S. cerevisiae Rmi1 protein is much shorter than higher
eukaryotic BLAP75, containing only the N-terminal region [22]
and when it was used to query the A. thaliana non-redundant
accessions using PSI-BLAST (Blosum 45), no hits were obtained.
When the same search was carried out using Homo sapiens BLAP75,
however, we picked up At5g63540 after the first round of iteration
(e value = 8e-14, Identities = 54/187 (28%), Positives = 93/187
(49%)). We therefore called this new A. thaliana gene BLAP75/
RMI1. The insertional line FCN288 (accession Ws) was named
blap75-1, and the Salk_093589 line (Col-0 accession) blap75-2.
This BLAST search also resulted in a high score with the A.
thaliana At5g19950 gene (e value = 1e-11, Identities = 32/78 (41%),
Positives = 47/78 (60%)). However, the GABI-Kat Line 679A11
with an insertion in At5g19950 did not display a vegetative or
reproductive phenotype.
Reverse-transcriptase PCR (RT-PCR) studies showed that
BLAP75 was expressed at low levels in roots and flower buds but
not in leaves (see Figure S1).
Figure 3. A. thaliana blap75 mutants show defects in male
sporogenesis. Male meiocytes (A–C) as well as meiotic products (D–F)
are shown after anther clearing for wild type (A, D), blap75-1 (B, E) and
blap75-2 (C, F). Bar, 10 mm.
doi:10.1371/journal.pgen.1000309.g003
between wild-type and mutant plants when the early stages of
microsporogenesis were compared, with round pollen mother cells
(PMCs) found within the anther locules (Figure 3A–C). In wildtype anthers, these cells underwent two meiotic divisions to
produce a characteristic microspore tetrad (Figure 3D). Meiosis
products were also detected in mutant plants but these lacked the
regular tetrahedral structure, and either single, double or multiple
cell products were observed (Figure 3E, F). blap75 mutants
produced a majority of dyads (55% of the cells counted for
blap75-1 (n = 348), and 73% of the cells for blap75-2 (n = 316))
suggesting that the meiotic program is disrupted in blap75 mutants.
We therefore investigated male meiosis by staining PMC
chromosome spreads with 49,6-diamidino-2-phenylindole (DAPI).
Wild-type A. thaliana meiosis has been described in detail in [39],
and the major stages are summarised in Figure 4 (A–H). During
prophase I, meiotic chromosomes condense, recombine, and
undergo synapsis, resulting in the formation of five bivalents, each
consisting of two homologous chromosomes attached to each other
by sister chromatid cohesion and chiasmata, which become visible
at diakinesis (Figure 4C, arrow heads). Synapsis (the close
association of two chromosomes via the synaptonemal complex
(SC)) begins at zygotene and is complete by pachytene (Figure 4B),
by which point the SC has polymerised along the whole length of
the bivalents. At metaphase I, the five bivalents are easily
distinguishable (Figure 4D). During anaphase I, each chromosome
separates from its homologue, leading to the formation of dyads
corresponding to two pools of five chromosomes (Figure 4E–F).
The second meiotic division then separates the sister chromatids,
generating four pools of five chromosomes (Figure 4G–H), which
give rise to a tetrad of four microspores (Figure 3D).
In A. thaliana blap75 mutants, the early stages of meiosis could
not be distinguished from wild type: chromosomes condensed and
synapsis of the homologous chromosomes proceeded normally
(shown for blap75-1 allele in Figure 4 I–J). To confirm that no
synapsis defects could be detected in blap75 mutants we performed
immunolocalization studies by double-labelling wild-type and
mutant PMCs with anti-ZYP1 (a major component of the central
element of the SC, [40]) and anti-ASY1 (a protein associated with
the axial element of the SC, [41]) antibodies. We could not detect
any difference in mutant compared to wild-type cells (Figure 4Q–
T), either in the progression (Figure 4Q and 4S) or completion of
synapsis (Figure 4R and 4T). However, chromosomal abnormalities appeared later at prophase, when condensing bivalents could
be recognised (diakinesis, Figure 4K). At this stage in wild type, the
five bivalents can be identified, each of them composed of a pair of
homologous chromosomes connected one to the other where COs
have occurred (some of these chiasmata are shown by arrowheads
The A. thaliana blap75 Mutants Are Meiosis-Defective
The two blap75 mutants displayed the same phenotype: normal
vegetative growth but short siliques (Figure 2) suggesting fertility
defects. Indeed, the mean seed number per silique of both blap75
mutants was extremely low (0.03 for blap75-1 and 0.0006 for
blap75-2, counted on 1,000 siliques) whereas the average is 63 and
71 seeds per silique for Ws (blap75-1 accession) and Col-0 (blap75-2
accession), respectively (n = 50).
We examined the reproductive development of these mutants
and found that blap75 sterility is due to abortion of male and
female gametophytes (data not shown). No differences were seen
Figure 2. A. thaliana blap75 mutants are sterile. Comparison of
wild-type (Wt) and homozygous blap75-1 (blap75) mutant plants after
30 days in the greenhouse. Arrows show siliques that elongate in wild
type but not in mutant.
doi:10.1371/journal.pgen.1000309.g002
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Figure 4. Meiotic phenotype of blap75 mutants. (A–P) DAPI Staining of wild-type (Ws, A–H) or mutant (blap75-1, I–P) PMCs during meiosis. (A, I)
Leptotene, (B, J) pachytene, (C, K) diakinesis, (D, L–N) metaphase I, (E) telophase I, (F) metaphase II, (G) anaphase II, (H) telophase II, (P) anaphase I. (Q–
T) Co-immunolocalization of ASY1 (red) and ZYP1 (green) in wild-type (Ws, Q–R) and mutant (blap75-1, S–T) PMCs. Bar, 10 mm.
doi:10.1371/journal.pgen.1000309.g004
to the A. thaliana centromere repeat sequences (Figure 5A–D). This
probe allows the very clear positioning of the ten A. thaliana
centromeres, which were observed in wild-type and in most blap75
cells, as expected, grouped in two pools of five, pointing toward the
two spindle poles (Figure 5A–C). It also showed that contrary to
what occurs in wild type, the chromosome arms are floating on the
metaphase plate (Figure 5B–C, arrows), explaining the rattle-like
structures seen in Figure 4N. When probed with the centromeric
repeat, the ‘‘compact’’ blap75 bivalents shown in Figure 4O (here
in Figure 5D) appeared to have the same structure as in 5B and 5C
with two centromeres directed towards opposite directions and two
chromosome arms floating, except that the whole structure is more
condensed (compare Figure 5D to Figure 5B,C). In some cases
however, more than two centromere signals could be observed
(asterisks, Figure 5D), suggesting that these entities underwent
premature sister centromere uncoupling.
We also carried out FISH experiments using probe mixes
designed to specifically label pairs of chromosomes: a 45S rDNA
probe together with a cocktail of chromosome 4 BACs, shown in
Figure 5E to L; a mixture of 45S and 5S rDNA repeats, shown in
Figure 5 M to 5Q; and a mixture of chromosome 1 BACs shown
in Figure 5R to U. These combinations allowed the clear
identification of either chromosomes 2 and 4 (Figure 5E–L),
chromosomes 2, 4 and 5 (Figure 5M–Q) or chromosomes 1
(Figure 5R–U).
Labelling of a blap75-1 pachytene cell (Figure 5G, 5T) showed
that the multiple BAC probes were correctly positioned along the
chromosome arms, demonstrating that in blap75, synapsis is
occurring between homologous chromosomes. When metaphase I
PMCs were probed, we found that homologous chromosomes
were associated together in bivalent-like structures (Figure 5H, J,
O, P, Q) as in wild-type cells (Figure 5E, M or R). We also
in Figure 4C). In blap75 mutants, chromosome arms are visible but
it was impossible to distinguish a chromosome arm from its
homologue as if the two were intimately linked (compare 3K to
3C). At metaphase I, abnormalities were even more obvious, with
a range of phenotypes illustrated in Figure 4L to 4O. In the
majority of the metaphase I cells (86% n = 76 for blap75-1 and
89% n = 107 for blap75-2) chromosomes did appear to be
associated in bivalent-like structures because five entities can be
recognised (Figure 4M–O). Nevertheless, their shape is very
unusual, often showing (52% of the metaphase for each allele)
bubble-like extensions (Figure 4L, M arrows) that sometimes seem
to connect the bivalents together, leading to the whole set of
chromosomes having a rattle-like structure (Figure 4N). In the
remaining cells, the bivalent-like entities displayed a very unusual
compact appearance (Figure 4O), and we never observed the five
typical bivalents observed during wild-type metaphase (Figure 4D).
Next, anaphase I proceeded and led to dramatic chromosome
fragmentation (Figure 4P). Nevertheless, chromosome migration
occurred and was followed by de-condensation of the various
DNA pools produced after anaphase I. Typical telophase I could
be recognised (data not shown) but meiotic division appeared to
stop at this stage, and we could never identify a second meiotic
division in any of the two blap75 mutants.
When we analysed female meiosis in blap75 mutants we
observed the same defects as for male meiosis (Figure S2).
A. thaliana BLAP75 Is Not Necessary for Homologous
Bivalent Formation
In order to understand the nature of the metaphase structures
observed in A. thaliana blap75 mutants at metaphase I, we
performed fluorescent in situ hybridization (FISH) analyses on
PMCs with diverse probes. Firstly, we used a probe corresponding
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Figure 5. Homologous bivalents are formed in A. thaliana blap75 mutants. (A–D) FISH on wild-type (Wt, A) or mutant (blap75-1, B–D)
metaphase I cells with a probe directed against centromeric repeats (CEN, red). Arrows indicate the floating chromosome arms. Asterisks indicate the
bivalents with more than two signals. (E–L) FISH with a 45S rDNA probe (red) and chromosome 4 long arm BACs (green) on wild-type (Wt, E) or
mutant cells (blap75-1, F–L). (E, H, J) metaphase I, (G) pachytene stage, (K–L) anaphase I. Double asterisks indicate chromosome 4s that can be
recognised with its short arm labelled in red and its long arm labelled in green, as shown on the schema in I, and in a somatic cell (F). (M–Q) FISH with
probes directed against 45S (red) and 5S (green) rDNA on metaphase I cells. Double asterisks indicate chromosome 4s that can be identified by
double labelling of its short arm: at the pericentromeric region in green and at the subtelomeric region in red, as shown on the schema in N. (R–U)
FISH with probes directed against chromosome 1 BACs on metaphase chromsome (R), pachytene (T) or anaphase I (U) cells. All FISH preparations
were DAPI-stained. Arrowheads indicate chromosome fragmentation. Bar, 10 mm.
doi:10.1371/journal.pgen.1000309.g005
observed that in many cases chromosome arms are much less
compact than in wild type (compare Figures 5H, J, Q to Figure 5E,
M), float around, and sometimes appeared connected to each
other (Figure 5H). Furthermore we observed very frequent
evidences of chromosomal fragmentation (Figure 5 arrowheads).
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Therefore, we can conclude from these results that the
structures observed at metaphase I in blap75 mutants are
bivalent-like in the sense that they connect homologous chromosomes from pachytene to anaphase I. Nevertheless, the architecture is highly perturbed with chromosome arms floating on the
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metaphase plate and numerous evidence of chromosome breakages as early as metaphase I/anaphase I transition.
trace of synapsis was observed during prophase (not shown) as is
the case in dmc1 (Figure 6M). Then, at metaphase/anaphase I,
chromosomes with very altered morphology, showing fragmentation and chromosome bridges, were observed (Figure 6P,Q). This
fragmentation was even more spectacular while anaphase I
proceeded, but second division figures were observed (Figure 6R),
contrary to the situation in the A. thaliana blap75 single mutant.
Another striking difference between both genotypes was the
absence in the double mutant of any bivalent-like structures at
metaphase I. Therefore it appears that in the absence of BLAP75,
the repair of meiotic DSBs onto sister chromatids is altered.
Furthermore, bivalent-like structures formed in blap75 mutants are
dependant upon DMC1 function.
A. thaliana BLAP75 Is Necessary for Meiotic DSB Repair
using Homologous Chromosomes or Sister Chromatids
as Templates
Meiotic recombination is initiated by the formation of DNA
DSBs that are catalysed by Spo11 in budding yeast and in all other
eukaryotes studied to date [42]. In A. thaliana, the disruption of
SPO11-1 or SPO11-2 induces a typical asynaptic phenotype
(Figure 6A–C) associated with a dramatic decrease in meiotic
recombination, leading to the formation of achiasmatic univalents,
which is correlated with an absence of meiotic DSBs [43,44]. In
order to understand if the meiotic chromosomal defects observed
in blap75 mutants were dependent upon DSB formation, we
generated spo11-1blap75 double mutants (Figure S3). These plants
showed a typical spo11-1 phenotype: synapsis failed to engage
(Figure 6D), there was an absence of bivalents (Figure 6E) and lack
of chromosome fragmentation at anaphase I (Figure 6F) or II (not
shown). Therefore, blap75 bivalent-like structures as well as blap75
fragmentation are dependent upon meiotic DSB formation.
Next, we analysed the nuclear distribution of the DMC1
protein, which is an essential component of the recombination
machinery (Figure S3). Its appearance on meiotic chromosomes
during prophase is thought to mark the sites of recombination
repair. To follow DMC1 focus formation throughout meiosis, coimmunolocalisation was performed with antibodies that recognise
the meiotic protein ASY1. Detailed analysis of DMC1 progression
in wild-type Arabidopsis meiotic prophase was described in [45].
DMC1 foci appear at late leptotene/early zygotene reaching an
average of 240 foci per nucleus (239 +/2 74 n = 49) and disappear
by pachytene [45]. DMC1 foci had similar characteristics in
blap75-1 male meiocytes, with an average of 235+/268 per
zygotene nuclei (n = 60) (Figure 7). Therefore, early DSB repair
events do not appear to be disrupted in blap75 mutants.
In order to obtain more precise information concerning the
function and position of A. thaliana BLAP75 in the DSB repair
steps, we also generated the rad51blap75 and mnd1blap75 double
mutants. The Rad51 protein is a recombinase that is loaded on
single-stranded DNA generated after DSB processing and
mediates the search for homology and invasion of an intact
homologous DNA molecule [46]. The Mnd1 protein is another
key actor of the strand invasion step, stimulating the activity of
Dmc1 and/or Rad51 [47]. In A. thaliana the two mutants, rad51
and mnd1, show drastic meiotic defects that can be summarised by
an absence of synapsis, the formation at metaphase I of a mass of
entangled chromosomes linked together by chromosomes bridges
and prominent chromosome fragmentation at anaphase I [48–
51](shown on Figure 6G–I for rad51). Nevertheless, these
abnormalities do not prevent meiosis II from occurring, and a
second round of chromosomal segregation is observed, leading to
the formation of very abnormal meiotic products (not shown). The
phenotype of rad51blap75 and mnd1blap75 double mutants could
not be distinguished from that of the rad51 or mnd1 single mutants
(shown for rad51blap75 in Figure 6J–L), suggesting that A. thaliana
BLAP75 acts after RAD51 and MND1 in the DSB repair cascade.
The situation in the A. thaliana dmc1 mutant is very different
because even if meiotic DSBs are formed in this background, they
are completely repaired, probably using the sister chromatid as a
template [52–55] leading to a typical asynaptic phenotype
(Figure 6M–O). Therefore, we wondered whether A. thaliana
BLAP75 is involved in this repair pathway and we analysed the
phenotype of the blap75dmc1 double mutant. In this background
we observed a cumulative effect of the two mutations. Firstly, no
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Bivalent-Like Structures in A. thaliana blap75
Backgrounds Are Independent of ZMM Proteins
In order to understand the nature of the association between
homologous chromosomes existing in blap75 mutants, we analysed
the involvement of two ZMM proteins: MSH5 and MER3 (Figure
S3). Both were previously shown to be involved in the maturation
of class I COs, which represent 85% of the total CO number in A.
thaliana. Their mutation has no effect on early meiosis events but
results in a highly pronounced decrease in CO formation (85% of
the wild-type level for A. thaliana msh5 and 76% for A. thaliana mer3)
leading to a large number of achiasmatic univalents at metaphase I
([56,57], shown for msh5 in Figure 8A–C). When we analysed
blap75msh5 and blap75mer3 double mutants, we could not detect
any difference between them and the single blap75 (Compare
Figure 8 D–I to Figure 4J, M and P).
Therefore, we can conclude that the formation of stable
associations between homologous chromosomes observed in
blap75 mutants does not require ZMM proteins.
Discussion
Is the BTB/RTR Complex Conserved in Plants?
In human cells and S. cerevisiae, BLAP75/Rmi1, BLM/Sgs1 and
TopoIIIãTop3 were demonstrated to interact [19–22], to form
one or several complexes involved in maintaining genome stability
[27]. In yeast, Rmi1 and Top3 appear to act in the same pathway
downstream of Sgs1 since most of the defects exhibited by top3 and
rmi1 mutants are suppressed by mutation of SGS1 [22,58,59].
These data led to the hypothesis that the yeast RecQ helicase
activity (Sgs1) produces toxic DNA structures that are removed by
the combined action of Top3 and Rmi1. In A. thaliana, the
existence of this complex has not yet been shown, but the results of
several recent studies provide evidence for its conservation. Firstly,
the A. thaliana recq4A-4 mutation suppresses (at least partially) the
lethality of the A. thaliana top3a-1 mutation [16]. Secondly, A.
thaliana blap75/rmi1 mutants as well as recq4A-4 and the leaky top3a
-2 show hypersensitivity to the same DNA damaging agents as well
as increased rates of somatic homologous recombination [60].
Therefore, the existence of a plant BTB/RTR complex composed
of A. thaliana RECQ4A, TOP3a and BLAP75/RMI1 that would
be involved in vegetative cell cycle surveillance, is very likely.
However, its function during meiosis is less clear. Our data
together with the those of [60] clearly show that two members of
the plant BTB/RTR complex (BLAP75/RMI1 and TOP3a) are
involved in meiotic recombination where they are likely to act in
the same pathway. In A. thaliana, the topoisomerase 3a protein is
essential for somatic development [16] making its function during
meiosis difficult to investigate. Nevertheless, partial suppression of
the top3a -1 somatic phenotype by the recq4a mutation, together
with analysis of a leaky top3a-2 allele made it possible to show that
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Arabidopsis BLAP75 in Meiosis
Figure 6. The blap75 meiotic phenotype depends on SPO11-1, RAD51 and DMC1. DAPI staining of male meiocytes of spo11-1-2 (A–C), blap751spo11-1-2 (D–F), rad51 (G–I), blap75-1rad51 (J–L), dmc1 (M–O), blap75-1dmc1 (P–R) at prophase I (A, D, G, J, M), or metaphase I/anaphase I transition
(B, E, H, K, N, P, Q), end of anaphase I (C, F, I, L, O) or second meiotic division (R). Bar, 10 mm.
doi:10.1371/journal.pgen.1000309.g006
blap75/rmi1, top3a-2 and recq4A-4top3a-1 have the same meiotic
defects [60] suggesting that TOP3a and BLAP75/RMI1 act
together during meiosis. Several findings, however, suggest that
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the last member of the complex, RECQ4A, may not be involved in
meiosis. Firstly, its disruption does not impair meiosis [16,17,60].
Secondly, recq4A-4 suppresses (at least partially) the somatic
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Arabidopsis BLAP75 in Meiosis
The Arabidopsis BLAP75/RMI1 Homologue
By characterising the A. thaliana homologue of BLAP75/RMI1,
we could report one of the first studies of the role played by a
member of the BTB/RTR complex in meiosis. The BLAP75/
Rmi1 proteins share a N-terminal domain containing a putative
OB (oligonucleotide/oligosaccharide binding)-fold which is responsible for the single stranded DNA binding activities of proteins
such as RPA or BRCA2 [61]. Nevertheless, to date, a DNA
binding capacity was not associated with this region in the
BLAP75/Rmi1 protein family. However it was recently shown, in
vitro, to be necessary for H. sapiens BLAP75 to form a complex with
BLM and hTopoIIIa and to activate the dissolution activity of this
complex. The C-terminal region of BLAP75 proteins is specific to
higher eukaryotes: it is not found in S. cerevisiae Rmi1 which is
much shorter than the vertebrate or plant BLAP75. We found that
mutations that disrupt this C terminal region of the A. thaliana
BLAP75 do not lead to any detectable phenotype, at least at the
reproductive level. Since in the H. sapiens BLAP75, the C-terminal
domain was recently shown to bind to single stranded DNA in vitro
[25], it suggests that, in A. thaliana, the single strand DNA binding
Figure 7. Early recombination events are not altered in blap75.
Co-immunolocalization of ASY1 (Red) and DMC1 (green) in wild-type
(Ws, A) and mutant (blap75-1, B) PMCs. Bar, 10 mm.
doi:10.1371/journal.pgen.1000309.g007
phenotype of the top3a-1 mutation [16], but not the meiotic
phenotype [60]. This suggests that if Arabidopsis BLAP75 and
TOP3a do act together with a meiotic helicase, it is probably not
RECQ4A.
Figure 8. Bivalent-like structures are formed in the absence of MSH5 or MER3. DAPI staining of male meiocytes of msh5 (A–C), blap75-1msh5
(D–F), blap75-1mer3 (G–I) at pachytene (A, D, G), metaphase I (B, E, H) and anaphase I (C, F, I). Bar, 10 mm.
doi:10.1371/journal.pgen.1000309.g008
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Arabidopsis BLAP75 in Meiosis
capacity of BLAP75 is dispensable for its meiotic function. In vitro
studies comparing the activity of a truncated BLAP75 protein
containing only the N-terminal region and the full length protein
might help understand the function of this conserved higher
eukaryote C-terminus extension.
complex activity (blap75 mutants), did not restore CO formation
(since normal bivalents were not observed), but recombination
nevertheless progressed, allowing the formation of stable bivalentlike structures.
What Role Does BLAP75 Play in Meiotic Recombination?
A. thaliana BLAP75 Is a Key Protein of the Meiotic
Homologous Recombination Machinery Dispensable for
Homologous Chromosome Recognition and Synapsis
and Whose Disruption Allows Bivalent Formation in the
Absence of ZMM Proteins
Mutations affecting the BTB/RTR complexes lead to genomic
instability at mitosis and meiosis. During mitosis, these mutations
provoke a characteristic ‘‘hyper-rec’’ phenotype showing that this
complex acts to suppress CO formation, likely acting at different
steps of the recombination process, one of the most documented
steps being the dissolution of dHJ intermediates toward NCO
events [9,23,24,26,65]. This complex could also be involved in
repressing recombination by acting on earlier intermediates [27],
by destabilising the Rad51 filament (as was shown in vitro for BLM
protein [10]), or by disrupting D-loop intermediates (shown also
for the BLM protein, [7]). Biochemical studies have shown that
BLAP75/Rmi1 has affinity for a number of DNA structures, with
a preference for HJ [21]. The role of BLAP75/Rmi1 in the BTB/
RTR complex would be to promote BLM-dependent dissolution
of homologous recombination intermediates by recruiting TopoIIIa [9,23,24,26].
Mutations affecting any member of the BTB/RTR complex also
affect meiosis, but are not accompanied by a ‘‘hyper-rec’’ phenotype
(see Introduction). Studies we performed on the A. thaliana BLAP75
homologue showed that abnormalities in blap75 mutant meiosis
appear at diakinesis. Homologous bivalents were formed but
showed very abnormal structures, with no visible chiasmata but
intimately linked homologous chromosomes arms. The recombinases Rad51 and Dmc1 act very early during meiotic recombination (Figure S3). They are loaded onto 39 single strand DNA
generated at DSB sites and are thought to play a crucial role in the
search for homologous intact DNA duplexes. Mnd1, together with
its partner Hop2, was shown to stabilise the Rad51 presynaptic
filament and to promote D-loop formation [66]. We analysed the
phenotypes of the rad51blap75 and mnd1blap75 double mutants and
found that we could not distinguished them from single rad51 or
mnd1, showing that the blap75 phenotype depends not only on
SPO11-1 (as discussed earlier), but also RAD51 and MND1. We
also found that DMC1 focus number was identical in blap75 and in
wild type. The Dmc1 protein is a meiotic specific RecA homologue
that plays a crucial role in driving meiotic DNA repair towards the
homologous chromosome instead of the sister chromatid. If
BLAP75 was involved in destabilizing early recombination
intermediates, one might expect to see a difference in DMC1 focus
formation. Since this difference was not observed, it suggests that
BLAP75 is not involved in destabilising early recombination
intermediates. Therefore, A. thaliana BLAP75/RMI1 is a protein
necessary for the repair of meiotic DSBs that acts after the invasion
step mediated by RAD51 and associated proteins. We also showed
that BLAP75 is necessary for repair onto sister chromatids, since
DSB repair in the dmc1 background is perturbed in the absence of
BLAP75. Therefore, taken together all these data suggest that
BLAP75, probably along with TOP3a, fulfils a key function in
meiotic recombination by processing (dissolving) recombination
intermediates, which are dependent upon RAD51, MND1 and
DMC1 and generated during meiotic DSB repair on either
homologous chromosomes or sister chromatids.
Our study of A. thaliana blap75/rmi1 insertional mutants showed
that this gene is crucial for meiosis. Disruption of A. thaliana
BLAP75 led to drastic chromosome fragmentation at anaphase I
and to an absence of the second meiotic division. Inhibition of
meiosis II is either indirect and due to the strong chromosomal
fragmentation observed at meiosis I or A. thaliana BLAP75 is
directly involved in meiosis II induction, but further studies are
needed to decipher the precise explanation. Our study revealed
that A. thaliana BLAP75 is involved in meiotic recombination.
Firstly, we showed that the A. thaliana blap75 meiotic phenotype
depends on SPO11-1 and therefore on DSB formation. Thus it is
likely that the DNA fragmentation observed in blap75 mutants
reflects DSB repair defects. Secondly, we showed that A. thaliana
BLAP75 is not necessary for homologue recognition and synapsis.
Even if we cannot exclude subtle effects (timing differences for
example), major perturbations to homologous recognition, association and synapsis can be ruled out: all pachytene stages
appeared perfectly normal in terms of synapsis (observed by DAPI
spreads as well as ZYP1 immunolabelling) and homology
(according to FISH results). Therefore, homologue recognition
and synapsis occur normally in the absence of A. thaliana BLAP75.
Even in the absence of A. thaliana BLAP75, homologous
bivalents are formed and can be observed from diakinesis to
metaphase I. We observed that these bivalents are formed
independently of two A. thaliana ZMM proteins (MSH5 and
MER3), that are known to be necessary for all (MSH5) or most
(MER3) Class I CO formation (see Introduction, Figure S3, and
[56,57,62]). The biochemical function of ZMM is still poorly
understood, but data obtained in yeast support the idea that these
proteins allow the formation of stable SEI intermediates,
committing these to the Class I pathway [63,64]. Thus these
proteins act just after the invasion step by the Rad51-Dmc1-coated
single stranded DNA, to stabilise the newly formed heteroduplex
in order to direct repair toward dHJ intermediates and CO
formation. In the absence of these proteins, a stable heteroduplex
cannot occur. Since homologous bivalents are formed in
blap75mer3 or blap75msh5, we conclude that in blap75 mutants,
recombination intermediates exist that are stable enough to form
bivalent structures, even when ZMM are absent. Such intermediates could be, for example, the complex joint molecules
corresponding to several interconnected DNA duplexes that have
been observed in yeast sgs1 mutants [30]. Our findings are also in
agreement with data from [30,31] who detected an anti-CO effect
of Sgs1 in zmm mutants. It was suggested that yeast Zmm protect
recombination intermediates from dissolution by Sgs1. When
Zmm are removed, the anti-CO activity of Sgs1 occurs and
recombination intermediates do not produce COs but are repaired
onto the sister chromatid, explaining the decrease in COs
observed in these backgrounds. However, when both Zmm
and Sgs1 are removed, CO recombination intermediates are
formed and the CO level is close to that of wild type [30,31]. In
the case of A. thaliana, the removal of both ZMM and BTB/RTR
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Materials and Methods
Plant Material
The blap75-1 mutant (FCN288 line) was obtained from the
Versailles Arabidopsis T-DNA transformant collection [67]. Mutant
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Arabidopsis BLAP75 in Meiosis
blap75-1 allele was amplified with primers P2 and P7
(GCTGGTCCGTTTGTTCTGCAG).
blap75-2 T-DNA left border was amplified by PCR with primers
P6 and primer LbSalk2 (GCTTTCTTCCCTTCCTTTCTC).
The wild-type allele of blap75-2 was PCR amplified with primers
P6 and P1R.
screening was performed as described in [68]. The blap75-2
mutant, line Salk_093689, was obtained from the collection of TDNA mutants at the Salk Institute Genomic Analysis Laboratory
(SIGnAL, http://signal.salk.edu/cgi-bin/tdnaexpress) [69] and
provided by the Nottingham Arabidopsis Stock Centre (NASC)
(http://nasc.nott.ac.uk) as well as lines Salk_005449,
Salk_054062, Salk_054053 and Salk_094387.
The spo11-1 allele used is spo11-1-2 described in [44,54]. The
dmc1, rad51-1, msh5, and mer3 mutants were described in
[48,52,56,57,70].
Sequence Analyses
Protein sequence similarity searches were performed at the
National Centre for Biotechnology Information (http://www.ncbi.
nlm.nih.gov/BLAST) and the Arabidopsis Information Resource
(TAIR, http://www.arabidopsis.org/Blast), using BLOSUM45
matrix and default parameters. Sequence analyses were performed
with BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
Genetic Analyses
Isolation of blap75-1: the FCN288 line segregated 3:1 for the
meiotic mutation (revealing the presence of a single recessive
mutation) and 3:1 for kanamycin resistance (one of the T-DNA
markers). After crossing to wild type, linkage between the T-DNA
insert and the meiotic phenotype was confirmed as described in
[44].
We tested for allelism between the blap75-1 and blap75-2
mutations by crossing two heterozygous plants blap75-1+/2 and
blap75-2+/2. Among the F1 plants, one fourth was sterile and
carried each of the mutant alleles.
Double mutants were obtained by crossing plants heterozygous
for each mutation. The resulting hybrids were self-pollinated. We
used PCR screening to select the sterile plants in the F2 progeny
homozygous for both mutations.
Antibodies
The anti-ASY1 polyclonal antibody has been described in [41].
It was used at a dilution of 1:500. The anti-ZYP1 polyclonal
antibody was described by [72]. It was used at a dilution of 1:500.
The anti-DMC1 antibody was described in [45] and the purified
serum was used at 1:20.
Microscopy
Comparison of early stages of microsporogenesis and the
development of PMCs was carried out as described in [44].
Preparation of prophase stage spreads for immunocytology was
performed according to [41] with the modifications described in
[73]. The fluorescence in situ hybridization (FISH) was performed
according to [74]. The A. thaliana 180 bp pericentromeric tandem
repeat (pAL1, [75]) and pTa71, a 9 kb clone containing 18S-5, 8S25S Triticum aestivum rDNA [76] were labelled by biotin nick
translation mix according the manufacturer’s instruction (Roche)
and detected by Avidin-Texas Red and goat anti-avidin-biotin
antibodies (Vector laboratories). A 3.5 kb fragment of 5S A. thaliana
rDNA (pCT4.2, [77]) and seven BACs (F25I24, F25E4, F28A21,
F17L22, F10M23, F4I10, F22I13) from the long arm of
chromosome 4 (http://www.arabidopsis.org/servlets/mapper)
were labelled by digoxigenin nick translation mix according the
manufacturer’s instruction (Roche) and were detected by mouse
anti-digoxigenin antibodies (Roche), rabbit anti-mouse FITC and
goat anti-rabbit Alexa-488 antibodies (Molecular Probes). To label
the chromosome 1 arm, ten BACs (F6F3, F24B9, F14N23,
F13K23, F19K19, F14010, F26F24, F17L21, F12K21, F26G16)
were labelled alternately with digoxigenin-dUTP and biotindUTP and detected as described above.
All observations were made using a Leica DMRXA2 microscope; photographs were taken using a CoolSNAP HQ camera
driven by Open-LAB 4.0.4 software; all images were further
processed with OpenLAB4.0.4 or AdobePhotoshop 7.0.
cDNA Studies
The full length cDNA sequence for At5g63540 was obtained
from NCBI (http://www.ncbi.nlm.nih.gov/entrez/) with the accession number AY954880 and checked by RT-PCR amplification.
Isolation of Plant T-DNA Flanking Sequences
The left border genomic sequence flanking the blap75-1 T-DNA
insert was amplified by thermal asymmetric interlaced PCR (TAIL
PCR) according to [71], with the modifications described in [44].
Subsequent sequencing revealed that the insertion was at nt 696 in
the AY954880 sequence. The right border could not be amplified
because of a complex insertion of two T-DNAs in tandem. The TDNA insertion led to a deletion of the 59 region of At5g63540
(since no amplification product could be detected with primers
P6 (GGAGCCCGTCTAGAAGTCGACAACGA) and P10
(GCTCACTGACTCCGACGGAT) or P3 (ACGAAGAAGAAGAAGATGAAACTGG) and P1R (TGAGTGGGCAGCCAATGTTAAC), however we checked that the gene located 59
to At5g63540 (At5g63550) was not affected.
The left border of blap75-2 was amplified with primers LbSalk2
and P3 and subsequently sequenced, showing that the T-DNA was
inserted in At5g63540 (nt 272 of sequence AY954880). The right
border could not be amplified, and we observed that the T-DNA
insertion induced a deletion of AY954880 39 region (at least from
nt 272 to nt 900 of AY954880).
Supporting Information
Figure S1 A. thaliana BLAP75 mRNA expression in different
plant tissues.
Found at: doi:10.1371/journal.pgen.1000309.s001 (0.13 MB
DOC)
Oligonucleotides for PCR Genotyping
The spo11-1-2 mutation was identified using a CAPS
marker. PCR amplification was performed with primers
MG52 (GGATCGGGCCTAAAAGCCAACG) and MG96
(CTTTGAATGCTGATGGATGCATGTAGTAG) and subsequently cleaved with AseI. The digestion generates two
500 bp fragments for the mutant allele only. The blap75-1
T-DNA left border was amplified with primers P2
(GCAGCTAGAGTTGCTCTGGTTG)
and
LbBAR2
(CGTGTGCCAGGTGCCCACGGAATAG). The wild-type
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Figure S2 A. thaliana blap75 mutants show defects in female
meiosis.
Found at: doi:10.1371/journal.pgen.1000309.s002 (0.40 MB
DOC)
Figure S3 Schematic representation of the different steps of
meiotic recombination investigated in this study.
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Arabidopsis BLAP75 in Meiosis
Found at: doi:10.1371/journal.pgen.1000309.s003 (0.06 MB
DOC)
We also wish to thank all the Arabidopsis group members for screening the
Versailles T-DNA transformant collection.
Acknowledgments
Author Contributions
We are grateful to Christine Mézard, Arnaud De Muyt, and Raphaël
Mercier for helpful discussions and constructive reading of the manuscript.
We wish to thank C. Franklin for providing ASY1 and ZYP1 antibodies.
Conceived and designed the experiments: LC DV KB GG MG. Performed
the experiments: LC DV KB GG MG. Analyzed the data: LC DV KB GG
MG. Wrote the paper: MG.
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