Download The DUET gene is necessary for chromosome

Research article
5975
The DUET gene is necessary for chromosome organization and
progression during male meiosis in Arabidopsis and encodes a
PHD finger protein
Thamalampudi Venkata Reddy1,*, Jagreet Kaur1,*, Bhavna Agashe1,*, Venkatesan Sundaresan2 and
Imran Siddiqi1,†
1Centre for Cellular and Molecular Biology, Uppal
2Department of Plant Biology and Agronomy, Life
Road, Hyderabad – 500007, India
Sciences Addition 1002, University of California, Davis, CA95616, USA
*These authors contributed equally to this work
†Author for correspondence (e-mail: [email protected])
Accepted 27 August 2003
Development 130, 5975-5987
© 2003 The Company of Biologists Ltd
doi:10.1242/dev.00827
Summary
Progression through the meiotic cell cycle is an essential
part of the developmental program of sporogenesis in
plants. The duet mutant of Arabidopsis was identified as a
male sterile mutant that lacked pollen and underwent an
aberrant male meiosis. Male meiocyte division resulted in
the formation of two cells instead of a normal tetrad. In
wild type, male meiosis extends across two successive bud
positions in an inflorescence whereas in duet, meiotic stages
covered three to five bud positions indicating defective
progression. Normal microspores were absent in the
mutant and the products of the aberrant meiosis were unito tri-nucleate cells that later degenerated, resulting in
anthers containing largely empty locules. Defects in male
meiotic chromosome organization were observed starting
from diplotene and extending to subsequent stages of
meiosis. There was an accumulation of meiotic structures
at metaphase 1, suggesting an arrest in cell cycle
progression. Double mutant analysis revealed interaction
with dyad, a mutation causing chromosome cohesion
during female meiosis. Cloning and molecular analysis of
DUET indicated that it potentially encodes a PHD-finger
protein and shows specific expression in male meiocytes.
Taken together these data suggest that DUET is required
for male meiotic chromosome organization and
progression.
Introduction
al., 1999; Bai et al., 1999; Yang et al., 1999a; Grelon et al.,
2001; Armstrong et al., 2002; Chen et al., 2002). Others appear
to be unique to plants and do not have obvious homologues in
yeast or animals (Byzova et al., 1999; Mercier et al., 2001;
Azumi et al., 2002). The pathway leading to sporocyte
specification is found only in plants and the two genes that have
been identified in this pathway, SPOROCYTELESS/NOZZLE
which encodes a nuclear protein related to MADS box
transcription factors and is required for the initiation of
sporogenesis (Yang et al., 1999b; Schiefthaler et al., 1999) and
EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS
CELLS, which encodes a putative LRR receptor kinase
required for tapetum formation and control of male meiocyte
number (Canales et al., 2002; Zhao et al., 2002), do not have
corresponding homologues in yeast or animals.
The control of meiotic cell cycle progression in yeast is
dependent upon checkpoints that monitor morphogenesis of
the chromosomes during meiosis. Mutations that affect
synapsis and recombination lead to arrest of meiotic
progression at the pachytene stage. The identification and
analysis of extragenic suppressors of pachytene arrest has led
to an understanding of the pachytene checkpoint (Roeder and
Bailis, 2000). The pachytene checkpoint has also been found
in animals (Edelmann et al., 1996), but remains to be identified
Meiosis in plants is the transition between the diploid
sporophyte and haploid gametophyte generations which in
lower plants exist as distinct free-living organisms. During the
reproductive phase of development in flowering plants,
specialized meiotic cells, the sporocytes, are formed in the
anthers and ovules. In plants there is no separate germline as
in animals, and the sporocytes are derived from the L2 layer
of the meristem (Dawe and Freeling, 1990). The understanding
of sporocyte specification and meiosis in plants has advanced
significantly in recent years (reviewed by Yang and
Sundaresan, 2000; Bhatt et al., 2001). Genetic and molecular
approaches in Arabidopsis and maize have led to the isolation
of mutants that are altered in sporocyte development or meiosis
(Curtis and Doyle, 1991; McCormick, 1993; Chaudhury et al.,
1994; Sheridan et al., 1996; Golubovskaya et al., 1997; Ross
et al., 1997; Taylor et al., 1998; Sanders et al., 1999) as well
as to the identification of several genes that are required for
development of the sporocyte or for meiosis. Many of the plant
genes that are responsible for basic components of the meiotic
machinery that is common to all eukaryotes such as
chromosome cohesion, synapsis, recombination and
chromosome segregation show conservation with genes in
yeast and other organisms (Klimyuk et al., 1997; Couteau et
Key words: Chromatin, Male sterility, Checkpoint, Cohesion,
Synapsis
5976 Development 130 (24)
in plants (Couteau et al., 1999; Garcia et al., 2003). In addition
to the pachytene checkpoint, which is specific to meiosis,
chromosomal checkpoints that act during mitosis have also
been shown to function in meiosis (Lydall et al., 1996). This
is consistent with the view that meiosis is a specialized cell
cycle based on the mitotic cell cycle (reviewed by Lee and
Amon, 2001).
There is limited information on the control of meiotic
progression in plants. Arabidopsis mutants that affect meiotic
progression have been described (Siddiqi et al., 2000; Magnard
et al., 2001) and several genes have also been characterized at
the molecular level. The ASK1 gene is required for homologue
separation during male meiosis (Yang et al., 1999a). The
DYAD/SWI1 gene is required for meiotic chromosome
organization and meiotic progression (Mercier et al., 2001;
Agashe et al., 2002). The Swi1 protein has been shown to be
expressed in G1 and S phase of meiosis, and required for axial
element formation and initiation of recombination (Mercier et
al., 2003). The SOLO DANCERS gene encodes a cyclin-like
protein that is required for synapsis during meiosis (Azumi et
al., 2002). Several of these genes appear to be associated with
changes in chromosome organization and dynamics, however
the mechanism by which these changes are related to the
progression of meiosis in plants remains unknown.
We describe the isolation and characterization of the duet
mutant of Arabidopsis and molecular analysis of the DUET
gene. We show that the duet mutant is defective in chromosome
organization and progression during male meiosis. duet also
shows a synergistic genetic interaction with the dyad mutant.
The DUET gene encodes a plant homeo domain (PHD)-finger
protein that is expressed in male meiocytes.
Materials and methods
Plant material and growth conditions
Arabidopsis growth conditions were as described earlier (Siddiqi et
al., 2000).
Light microscopy
Developmental analysis of whole-mount anthers and ovules was done
after clearing inflorescences in methyl benzoate as described
previously (Siddiqi et al., 2000). The anthers were dissected on a slide
under a stereo dissecting microscope, mounted with a coverslip and
observed on a Zeiss Axioplan imaging 2 microscope under DIC optics
using ×40 and ×100 oil immersion objectives. Photographs were
captured on a CCD camera (Axiocam, Carl Zeiss) using the
Axiovision program (version 3.1). For a stage-wise comparison of
pollen development in the wild type and the duet mutant, the ovules
from the corresponding pistils were staged and used as a reference for
pollen developmental stage. Scoring of ovule stages was based on
examination of all the ovules in a pistil. The pistil was mounted either
intact or after separating the two carpels, and ovules were viewed
through the carpel wall. At the early stages of ovule development
corresponding to pollen meiosis and gametogenesis, all ovules in a
pistil were at the same stage. Anthers were also bissected and
developing pollen was observed in optical sections taken through the
anther wall. Pollen development in anthers was also found to be
synchronous. The correspondence between ovule and pollen stages
across different inflorescences was consistent. For plastic sections the
inflorescences were fixed in 2% paraformaldehyde, 0.5%
glutaraldehyde in 1× PBS overnight at 4°C. The inflorescences were
then washed with 1× PBS, treated with osmium tetroxide for 3 hours,
followed by dehydration in a graded ethanol series (30%, 50%, 70%,
Research article
90%, 100% ×5) for 15 minutes each. Ethanol was replaced by
propylene oxide and the samples infiltrated with Araldite resin
followed by embedding and curing for 3 days at 60°C. 2 µm sections
were cut using a Reichert Ultracut E microtome. Sections were stained
with 1% Toluidine Blue in 1% borax for 2-5 minutes and mounted in
the Araldite resin. Bright-field photographs of anther cross sections
were taken using a Zeiss Axioplan imaging 2 microscope.
Photographs were taken on Kodak Supra 100 ISO film using a blue
filter. All the photographs were edited using Adobe Photoshop 5.
cDNA isolation and expression analysis
Poly(A)+ mRNA was isolated from young flower buds and leaves
using the PolyA tract mRNA isolation kit (Promega) according to the
manufacturer’s protocol with an inclusion of RQ1 DNAase (Promega)
treatment before mRNA precipitation. Pistils were dissected and
stored in RNA Later (Ambion) and total RNA was isolated using
Trizol (Gibco BRL Life Sciences). The cDNA synthesis was carried
out with 150 ng of poly(A)+ RNA or 5 µg total RNA in the case of
dissected pistils, using the Superscript choice system for cDNA
synthesis (Gibco-BRL Life Sciences). Amplification of DUET cDNA
was carried out by using the gene-specific primers SETAF (5′CGTCTCCATCGAAGCTAAAATC-3′) and SETR1 (5′-ATCTACAAAGTTTGATCCAAAAACTGAC-3′). The amplified cDNA was
cloned into a pMOSBlue Vector (Amersham Pharmacia Biotech) and
sequenced. DUET expression was examined by PCR using cDNA
prepared from poly(A)+ mRNA as template and the primers SETF1
(5′-CCAATCATCGAAACGTGTCGTAAGAG-3′) and SETR13 (5′TCCGAGACTATTACAAAGCCGATCC-3′). GAPC expression was
detected using the primers GAPC1 (5′-CTTGAAGGGTGGTGCCAAGAAGG-3′) and GAPC2 (5′-CCTGTTGTCGCCAACGAAGTCAG-3′). DYAD cDNA was amplified with the gene specific primers
3RR1 (5′-CATGGAAGAGACCTTACCAGTTCACATCA-3′) and
g2.2r7 (5′-AGCTAGTGATTATTGGAGAAACCTTGCG-3′).
In situ hybridization was carried out as described previously
(Siddiqi et al., 2000). A 1.26 kb coding region of DUET cDNA
lacking the PHD-finger domain was amplified using the primers
STMF1 (5′-GGTCATTTGGTATGTGTCAATGGTATGG-3′) and
STMR3 (5′-TCAATCTCAGTCACTACAAAATTTGACAAG-3′) and
subcloned into pGEM-T (Promega) for synthesis of RNA probes.
Molecular analysis of the duet mutant locus
Southern hybridization experiments using a Ds transposon-specific
probe derived from pWS31 (Sundaresan et al., 1995) was used to
establish copy number of the insertion. TAIL-PCR was used to
amplify sequences flanking the transposon insertion (Parinov et al.,
1999). The amplified product was sequenced. The site of insertion was
confirmed by Southern analysis using as the probe a genomic
fragment amplified using the primers Set3′ (5′-TCTCGGAGCAAGGTAATGGAG-3′) and R16 (5′-AAAGTTTGATCCAAAAACTGACTTTACAAA-3′) that were specific to At1g66170. To obtain
genomic sequences flanking the Ds element, Ds-specific primers from
both the 5′ and 3′ ends, Ds5-2 (5′-CGTTCCGTTTTCGTTTTTTACC3′) and Ds3-2 (5′-CCGGTATATCCCGTTTTCG-3′) were used in
combination with gene-specific primers set5′ (5′-GTAACTCACGTTCACGCGTTA-3′) and Set3′ respectively.
Double mutant analysis
duet (Ler) as the female parent was crossed to dyad (Col) as the male
parent. F1 and F2 were selected on MS plates containing kanamycin
at 50 µg/ml. 96 F2 plants were transferred to soil; 49 of these were
sterile. Plants homozygous for dyad were identified by examination
of ovules as described previously (Agashe et al., 2002). The presence
of the insertion and wild-type alleles at the duet locus was examined
by PCR using a gene-specific primer R12 (5′-ATTCTCTGAACTTGGAAACTCATACTTTGG-3′) in combination with Ds5-2 for the
insertion allele, and two gene-specific primers Dhf4 (5′-GTAGTAGATGGCCTGTGAGGAGACTAAT-3′) and STR5 (5′-TCTGC-
Arabidopsis meiotic chromosome organization 5977
AAATTCTTCACAGCAATTCG-3′) on either side of the insertion
site for the wild-type allele. DNA was isolated from one or two rosette
leaves using the Nucleon Phytopure kit (Amersham). Pollen viability
was measured using fluorescein diacetate according to the method of
Heslop-Harrison and Heslop-Harrison (Heslop-Harrison and HeslopHarrison, 1970). To determine the effect of duet/duet on female
fertility 10 plants that were homozygous for the duet allele and had
normal ovules (dyad/+ or +/+) were crossed as the female parent to
dyad as the male parent. All the siliques elongated and showed greater
than 50% seed set after crossing, indicating no significant defect in
female fertility.
Fluorescence microscopy of meiotic chromosomes
Analysis of meiotic chromosome spreads of male meiocytes was
carried out according to the method of Ross et al. (Ross et al., 1996)
with minor modifications (Agashe et al., 2002). Chromosomes were
observed on a Zeiss Axioplan2 imaging microscope using a 365 nm
excitation, 420 nm long-pass emission filter and a 100× oil objective.
The photographs were captured on an Axiocam CCD camera (Carl
Zeiss) using the Axiovision program (version 3.1) and were edited
with Adobe Photoshop 5.0.
Results
was caused by a transposon insertion, the mutant was crossed
as the female parent to a line that was homozygous for an
insertion carrying the Ac transposase expressed under control
of the 35S promoter (Sundaresan et al., 1995). The F1 plants
were fertile and the segregation of the mutant phenotype in the
F2 was consistent with it being a single gene recessive trait
(Table 2). Out of a total of 51 sterile F2 plants, 17 were found
to contain a revertant sector bearing one or more elongated
siliques (Fig. 1C). The reversion phenotype was confirmed for
nine independent sectors in the F3 generation by the presence
of fertile plants among the progeny. Multiple plant progeny
from seven of the nine sectors were tested by PCR for both the
presence and absence of the Ds at the original site of insertion
in At1g66170. Three out of the seven sectors lacked Ds
indicating that excision had occurred from both chromosomes
and was associated with the reversion to fertility. Genomic
DNA flanking the site of excision was amplified by PCR and
sequenced for multiple plants from each of the three sectors
lacking Ds (Fig. 1F). Two out of the three sectors contained
one wild-type and one mutant allele carrying the same 7 bp
footprint at the excision site. The third sector contained one
The duet mutant is male sterile because of a Ds
transposon insertion
The duet mutant was identified as a sterile line, SET8286,
carrying a Ds enhancer trap insertion (Parinov et al., 1999). An
examination of flowers showed that the mutant anthers lacked
pollen. To determine the reason for sterility, reciprocal crosses
were carried out to wild type. The results indicated that the
mutant was male sterile and that female fertility was normal
(Table 1). The anther filament did not fully elongate and remained
below the level of the stigma (Fig. 1E), a feature that has also
been observed in other male sterile lines (Sanders et al., 1999).
Southern analysis indicated the presence of a single Ds
insertion in the duet mutant (data not shown). DNA flanking
the insertion site was isolated using TAIL PCR (Liu et al.,
1995) and sequenced. The sequence indicated the insertion to
be within the putative gene At1g66170. Southern analysis
using a wild-type genomic clone as a probe confirmed the site
of insertion (data not shown). To test whether the phenotype
Table 1. The duet mutation causes male sterility
Female parent
Wild type
duet
Wild type
Male parent
Number of
seeds per silique
duet
Wild type
Wild type
0
25.8±2.8
27.2±1.8
Reciprocal crosses between duet and wild type, both in the Ler background
were conducted to measure the seed yield. The results represent the mean and
standard deviation from a minimum of eight crosses.
Table 2. Segregation of the duet phenotype
Wild type:mutant
Observed
Expected
χ2
194:51
183.75:61.25 (3:1)*
228.66:16.33 (15:1)†
2.18, P>0.1
70.7, P<<0.001
*Mutant phenotype results from a single gene recessive mutation.
†Two unlinked mutations are responsible for the mutant phenotype.
Fig. 1. Wild-type (Ler), duet and revertant plants. (A) Wild-type
plant showing normal elongating siliques. (B) duet mutant plant with
short siliques. (C) Mutant plant with revertant sector showing
elongating silique (arrow). (D) Wild-type flower with long anther
filaments and plentiful pollen. (E) duet flower with short filaments
and anthers lacking pollen. (F) Wild-type cDNA and derived amino
acid sequence of DUET near the site of the Ds insertion. The duet
mutant sequence shows a 8 bp duplication (bold underlined). The
two excision alleles have a 7 and 8 bp footprint respectively (bold).
5978 Development 130 (24)
Research article
Fig. 2. Anther and pollen development in wild type and duet. Plastic cross-sections of anthers. (A,D,G) Wild type; (B,C,E,F,H,I) duet.
(A,B) Anthers with pollen mother cells at stage 5, all the layers of the anther are present in both genotypes. (C) duet microspore mother cell at
meiosis; pollen mother cells (PMCs) surrounded by a layer of callose. (D) Stage 7 anther showing tetrads held together by a layer of callose.
(E) duet anther with dyads, triads, tetrads: products of a defective meiosis. (F) Products of aberrant meiosis separate out, enlarge and undergo
nuclear division (arrowheads). (G) Stage 12 anther containing mature pollen. (H) Enlarged microspore-like cells (arrowheads) with a single
nucleus and lacking exine. (I) Late stage anther showing empty locules. CL, callose layer; E, epidermis; En, endothecium; Ml, middle layer;
PMC, pollen mother cell; T, tapetum; Tds, tetrads. Scale bars: 25 µm (A,B,C,F,G,I) and 12.5 µm (D,H,E).
wild-type and two mutant alleles carrying the 7 bp and an 8 bp
footprint, respectively. We found one plant that was
heteroallelic for the two mutant alleles and was sterile. This is
consistent with the development of the pollen that is carrying
the mutant excision allele being associated with a multiple
event that gave rise to the wild-type allele together with the
mutant allele in the sporophyte. These data show that the duet
mutant phenotype is due to the Ds transposon insertion.
Microsporogenesis is defective in duet
To examine the basis of male sterility and lack of pollen in the
duet mutant we carried out a stagewise analysis of anther and
pollen development by examining cleared anthers as well as
plastic sections. Early stages of pollen development
corresponding to anther stage 5 (Sanders et al., 1999) were
normal (Fig. 2). The endothecium and internal layers surrounding
the microsporocyte were indistinguishable from wild type as was
the appearance of the microsporocyte prior to meiosis. We
compared the time course of pollen development in wild type and
duet by examination of cleared anthers and ovules from
successive buds of inflorescences. Meiocyte division was
prolonged (Table 3) and extended across three to four successive
bud positions in the mutant inflorescence whereas in wild type it
covered no more than two successive bud positions. The major
product of division of the meiocyte was a pair of cells instead of
a normal tetrad that is observed in wild type (Fig. 3). Tetrads were
seen only rarely (Fig. 2E). The pair of cells separated and formed
enlarged cells that were uninucleate (Fig. 2H, Fig. 3I).
Subsequently nuclear division took place and cells were seen to
contain one to three nuclei. Normal microspores were not
observed and the exine was not formed. The enlarged cells later
degenerated, leaving an empty locule (Fig. 2I). Mutant meiocytes
at the time of division had a prominent callose wall (Fig. 2C)
Table 3. Pollen development in wild type and duet relative
to ovule stages
Ovule stage
1-2
2-1
2-2
2-3
2-4
2-5
3-1
3-2
Wild type
Microsporocytes
Tetrads
Free microspores (expanding)
Microspores with exine
Vacuolated microspores
Mature pollen
duet
Microsporocytes
Separated meiocytes
Dyads
Dyads (separating)
Uninucleate spores
Uni, binucleate spores
Multinucleate spores
Spores degenerate
Three inflorescences each for wild type and duet were analyzed. Ovule
stages are according to Schneitz et al., (Schneitz et al., 1995).
Arabidopsis meiotic chromosome organization 5979
Fig. 3. Stages of male meiosis and pollen development in wild type and duet. Optical sections of cleared anthers viewed under DIC optics.
(A-D) Wild type; (E-K) duet. (A,E) Stage 5 anthers with pollen mother cells (arrowheads). (B) Stage 7 anther containing tetrads (arrowhead).
(C) Microspores released from the tetrad generate an exine wall and become vacuolated (arrowhead). (D) Mature pollen. (F,G) Dyads
(arrowhead in F) formed after a defective meiosis. (H,I) Cells of the dyad separate and enlarge (arrowhead in H). (J,K) The microspore-like
cells released from the dyad enlarge and undergo nuclear division to form 2-3 nucleate cells (arrowhead in J), which later degenerate. Scale
bars: 12.5 µm.
whereas the pair of cells formed by division of the meiocyte
lacked a thick callose wall and separated into single cells. This
probably corresponds to loss of the callose wall from tetrads at
the time of microspore release in wild type. The stage at which
the pair of cells separated in the duet mutant corresponded to
ovule stage 2-4 which is later than the time of microspore release
in wild type (ovule stage 2-2; Table 3). Taken together these data
indicate that duet is defective in male meiotic progression.
DUET potentially encodes a PHD finger protein
Based on the predicted cDNA sequence of At1g66170, PCR
primers were designed to span the coding region. RT-PCR was
carried out using cDNA prepared from inflorescence mRNA.
The sequence of the cDNA obtained (GenBank accession no.
AY305007) indicated the presence of 3 exons potentially
encoding a protein of 704 amino acids and having a mass of
80.8 kDa (Fig. 4). The Ds insertion is in the third exon at a
position corresponding to aa 550 and hence likely to create a
null allele. The putative protein was similar to that of the AGI
annotation but contained an additional exon that was not
present in the annotated sequence. A potential nuclear
localization signal was found at amino acid residues 10-15.
A homology search using BLASTP 2.2.6 revealed the
presence of a PHD finger domain towards the C-terminal
portion of the predicted DUET protein, from aa 609-656. The
PHD domain is a modified zinc finger thought to be involved
in transcriptional regulation and chromatin organization
(Aasland et al., 1995). The closest known protein to DUET was
the MALE STERILITY 1 (MS1) protein (expectation value,
E=4×10–83), which has been proposed to be a transcriptional
regulator of male gametogenesis and also contains a C-terminal
PHD finger domain (Wilson et al., 2001). In addition, two
predicted genes from Arabidopsis (At1g33420, and At2g01810)
and one from rice (GenBank ID AC090882) showed similarity
to DUET. All three predicted proteins have a PHD domain
towards the C-terminal end. All five genes show similarity
throughout their length and hence constitute a gene family.
Apart from the above genes, which showed strong similarity to
DUET, weak similarity was detected to other Arabidopsis genes
including SWI1/DYAD (E=1×10–4). The SWI1/DYAD gene has
been shown to be required for chromosome cohesion in meiosis
and for female meiotic progression (Mercier et al., 2001;
Agashe et al., 2002). The SWI1/DYAD homology resides
towards the middle portion of the gene from aa 309 to 395.
DUET is expressed in male meiocytes
Expression of the DUET gene was examined using RT-PCR.
The presence of the transcript could be detected in the
5980 Development 130 (24)
Research article
Fig. 4. (A) Schematic representation of the DUET
gene. The exons are indicated with black boxes.
Arrows indicate the primers used for cDNA isolation
and expression analysis. Coordinates are with respect
to BAC F15E12. (B) The predicted sequence of DUET
protein. The putative nuclear localization signal is in
bold. The region showing homology to DYAD is
underlined and the PHD-finger domain is boxed. The
inverted triangle after amino acid 550 indicates the Ds
transposon insertion site. (C) Analysis of DUET
expression by RT-PCR. Expression of the DUET gene
was examined in wild type (Wt) leaves (Lea),
inflorescence (Inf) and duet mutant inflorescence by
amplifying the cDNA synthesized from poly(A)+
mRNA. The shift in the size of the amplicon can be
observed when genomic DNA (gen) was used as
template. The constitutive GAPC gene was used as the
normalization control. (D) Comparison of DUET and
DYAD cDNA in pistils.
inflorescence but not in leaves (Fig. 4C). The mutant did not
show expression under these conditions and hence is likely to
be a null allele. We have previously demonstrated the presence
of DYAD mRNA specifically in male and female meiocytes
(Agashe et al., 2001). To test whether DUET expression is male
specific, we compared the levels of DUET message with that
of DYAD in dissected pistils using RT-PCR. We did not observe
expression of DUET whereas DYAD expression could be
detected under the same conditions (Fig. 4D). To determine the
DUET expression pattern in the inflorescence at the cellular
level we carried out RNA in-situ hybridization using antisense
RNA complementary to DUET cDNA excluding the PHD
domain as a probe. Expression was first observed in
sporogenous cells at late anther stage 4 (Sanders et al., 1999)
(Fig. 5A), reached a maximum in male meiocytes at anther
stage 5, prior to meiosis (Fig. 5C). Lower expression was
observed at anther stage 6, during meiosis (Fig. 5D) and
subsequently declined. A weak signal could be seen in very
young pistils in the placenta, corresponding to the presumptive
site of ovule initiation (Fig. 5B,C). We did not see expression
in female meiocytes or in ovules (Fig. 5E,F). The lack of
female meiocyte expression as well as a phenotype in what
appears to be a null allele, would suggest that DUET does not
have a function in the female meiocyte. We also examined
GUS reporter gene expression in plants hemizygous and
homozygous for the insertion but did not observe expression
in anthers at stages 5 to 6.
Aberrant meiotic chromosome organization and
metaphase 1 arrest in the duet mutant
Meiotic chromosome stages in wild type and duet were
analyzed in spread chromosome preparations from anthers of
Fig. 5. Expression of DUET
in male meiocytes: RNA in
situ hybridization of DUET
antisense RNA to sections of
flower buds. (A) Expression
is first seen in sporogenous
cells at anther stage 4.
(B,C) Maximal expression is
seen in microsporocytes at
anther stage 5. (D) Anther
stage 6, meiotic cells.
(E,F) No expression is seen
in the female meiocyte at
stage 2-3 corresponding to
pre-meiosis. (E) Antisense;
(F) sense control. Scale bars:
50 µm.
Arabidopsis meiotic chromosome organization 5981
meiotic stage buds using the method of Ross et al. (Ross et al.,
1996). Chromosome development in duet appeared normal
through early stages of meiotic prophase up to pachytene.
Abnormalities first became noticeable at diplotene when duet
chromosomes started to appear somewhat diffuse in comparison
to wild type (Fig. 6D,N). During diplotene and diakinesis, duet
chromosomes were observed to desynapse along much of their
length including centromeres, to a greater extent than was
observed for wild type (Fig. 6N-P). At diakinesis, duet
chromosomes were irregular and less distinct than wild type
(Fig. 6P,Q). Metaphase 1 in duet varied from nearly normal
(Fig. 6R) to a diffuse mass where individual chromosomes
could not be clearly distinguished (Fig. 6T). In wild type, male
meiotic stages extended across no more than two successive bud
positions in an inflorescence whereas in duet, meiotic stages
covered three to five bud positions. The total number of meiotic
stages in an inflorescence was also several times greater for duet
than for wild type. This could be approximately gauged from
the fact that 364 meiotic stages were counted from six
inflorescences in the case of wild type and 602 from three
Fig. 6. Chromosome analysis in spreads of male
meiocytes in wild type and duet. (A-J) Wild type;
(K-W) duet. (A,K) Chromosomes first become visible
as elongated strands during leptotene. (B,L) Synapsis
takes place during zygotene. (C,M) Synapsis is
complete at pachytene and chromosomes have a shorter
and thicker appearance. (D) Diplotene stage, when
bivalents have undergone partial decondensation.
(E) Late diakinesis showing five pairs of chromosomes
with chiasmata at their ends. (F) Metaphase I stage showing five condensed bivalents arranged on a metaphase plate. (G) Telophase I: five
chromosomes at each end are separated by an organelle band. (H) Metaphase II. (I) Anaphase II. (G-I) Arrows indicate the densely compacted
organelle band. (J) Telophase II where four groups of five chromosomes each have separated. (N) First apparent visible defect in duet at
diplotene. Chromosomes start to look diffuse and two bivalents have undergone partial desynapsis (arrowheads). (O) A more severe form of
desynapsis can be observed in the majority of bivalents. (P,Q) Disorganized diakinesis in duet with diffuse chromosomes including the
centromeric region. (R,S,T) Metaphase I. (U) Anaphase I. (V) Defective anaphase I stage with fragmented chromosome and laggards.
(W) Telophase I. The organelle band is absent. Scale bars: 12.5 µm.
5982 Development 130 (24)
The duet dyad double mutant flowers lacked viable pollen.
Stagewise analysis of pollen development in cleared anthers
(Fig. 8) showed meiocytes followed by enlarged uninucleate
cells that underwent further enlargement to produce binucleate
cells. We did not observe clear cytological evidence of meiotic
divisions. At a later stage anthers contained irregular enlarged
cells containing multiple nuclei and surrounded by an irregular
cell wall that resembled the exine. The exine-like structure was
not observed in the duet single mutant. Buds from plants that
were duet/+ dyad/dyad showed reduced numbers of pollen
grains compared to the corresponding single mutants. Anthers
from freshly opened flowers were dissected and tested for
pollen viability. The mean pollen viability for duet/+
dyad/dyad was 34±12% whereas for plants that were duet/+
+/– the pollen viability was 75±13%. The total number of
The duet mutation interacts with dyad
pollen grains was also lower for the interaction genotype. A
The duet mutant phenotype is similar to what is seen for female
rough indication of this could be obtained from the total
meiosis in the dyad mutant (Siddiqi et al., 2000). In each case
amount of pollen on the slide. For the duet/+ dyad/dyad
a pair of cells is formed after meiosis instead of a tetrad. The
genotype the mean count of total pollen grains per flower was
pair of cells do not develop further into functional gametes. For
131±50 whereas for sibling plants that were duet/+ +/– the
both duet and dyad, the two-cell phenotype correlates with
mean pollen count was 326±171. Each individual genotype
defective progression through meiosis and the underlying
duet/+ and dyad/dyad produced numbers of viable pollen that
cause appears to be altered chromosome organization in both
were comparable to wild type (Siddiqi et al., 2000; data not
cases. The DYAD/SWI1 gene is required for female meiotic
shown). Analysis of pollen development in cleared anthers
progression, and for chromosome cohesion in male and female
showed that male meiocytes in duet/+ dyad/dyad plants
meiosis (Mercier et al., 2001; Agashe et al., 2002). However,
underwent an aberrant division to produce mostly dyads and
the dyad mutant allele is female specific and shows normal
triads, as well as some tetrads (Fig. 8). The majority of spores
pollen development and male fertility.
produced were defective and formed enlarged cells containing
To test if both mutants are affected in a related aspect of
1-3 nuclei and that subsequently degenerated. The phenotype
chromosome organization during meiosis, we intercrossed duet
therefore broadly resembled that of homozygous duet plants
and dyad as female and male parents respectively to test for
though it was less severe.
genetic interaction. The F1 were fully fertile and showed
Examination of meiotic chromosome behavior revealed
normal pollen and embryo sac development (data not shown).
differences in duet/+ dyad/dyad plants from that observed in
F2 plants were genotyped with respect to the alleles present at
homozygous duet plants. The early stages of meiotic prophase
the duet locus (duet/duet, duet/+, and +/+) and at the dyad
were detected though aberrant chromosome morphology was
locus (dyad/dyad, and +/–), and various dose combinations of
apparent at the pachytene stage (Fig. 9B). Diakinesis was
duet and dyad were examined for evidence of genetic
variable with some being nearly normal and having all or a
interaction.
majority of the chromosomes retaining their bivalent structure
(Fig. 9C). In others, many of the chromosomes
dissociated into univalents (Fig. 9D). A more
60
extreme phenotype observed at diakinesis was
the loss of sister chromatid cohesion resulting in
50
dissociation of chromosomes into isolated
chromatids (Fig. 9E). The ensuing meiosis 1
40
divisions spanned the range from a normal
reductional division (these were relatively rare
and not observed, but their existence could be
30
inferred from the production of tetrads and viable
pollen) to an equational mitotic-like division in
20
which univalents separated into sister chromatids
(Fig. 9G,H), to an unequal division arising from
10
random segregation of univalents and single
chromatids (Fig. 9I,J). We did not observe a high
0
proportion of metaphase 1 arrest which
L
Z
P
DP
DI
M -I
A-I
T-I
M-II
A-II
T-II
TD
correlated with a reduction in the number of cell
Meiotic Chromosome Stages
pairs that were seen compared to the duet single
mutant. The homozygous double mutant
Fig. 7. Quantitation of different stages of male meiosis in wild type and duet.
combination was also examined. Here too
(White bars) Wild type; (Black bars) duet; leptotene (L), zygotene (Z), pachytene
bivalents were observed to separate into
(P), diplotene (DP), diakinesis (DI), metaphase I (M-I), anaphase I (A-I), telophase
univalents at diakinesis (Fig. 9L) and in some
I (T-I), metaphase II (M II), anaphase II (A-II), telophase (T-II) and tetrads (TD).
Note the large increase in the proportion of metaphase I cells in duet.
meiocytes chromosome cohesion was lost
Percent total
inflorescences for duet. In most of the buds there was an excess
of metaphase 1 stages in the duet mutant (Fig. 7), whereas these
were relatively few in wild type. The fraction of anaphase 1
meiocytes was also greater for duet. A clear feature of duet
meiocytes was the absence of a band of organelles characteristic
of wild type and prominently visible starting at telophase 1 in
the central portion of the meiocyte (Fig. 6G). Instead organelles
were dispersed throughout the cytoplasmic region of the male
meiocyte in the case of duet (Fig. 6W).
These data confirm and extend the analysis of male meiocyte
division in the duet mutant. The results indicate that duet has
a defect in chromosome organization during male meiosis and
is also defective in male meiotic progression. Many of the
meiocytes arrest at metaphase 1.
Research article
Arabidopsis meiotic chromosome organization 5983
Fig. 8. Interactions between duet and dyad during male meiosis and pollen development. Optical sections of cleared anthers viewed under DIC
optics. (A-D) duet dyad double mutant; (E-L) duet/+ dyad/dyad. (A,E) Normal-looking meiocytes (arrowheads). (B) Defective microspores
lacking exine (arrowhead). (C) Binucleate spores. (D) Late stage defective binucleate and multinucleate spores (arrow and arrowhead,
respectively) surrounded by an uneven wall resembling the exine; not observed in the duet single mutant. (F) Dyad (arrowhead). (G) Undivided
triads (arrowhead). (H) Round spores containing 1-3 nuclei. (I) Normal-looking tetrads (arrowhead). (J) Normal-looking developing pollen.
(K) Abnormal multinucleate structures undergoing shrinkage (arrowhead). (L) Shrunken pollen. Scale bar: 12.5 µm.
resulting in isolated chromatids (Fig. 9M). At prometaphase
between eight and ten thick and diffuse chromosomes could
generally be counted, most probably they were a mixture of
univalents and bivalents (Fig. 9N). Male meiotic chromosomes
in the dyad mutant showed no apparent differences from wild
type. In the case of duet/+ also, the vast majority of meiotic
structures observed were normal, in agreement with the
essentially wild-type levels of viable pollen that were measured
in freshly opened flowers. However, in one inflorescence out
of four examined we observed a single bud in which a minority
(<10%) of meioses were aberrant. Chromosomes in this bud
showed defects in diakinesis that were similar to those
observed in homozygous duet, and some meiocytes were
arrested at metaphase 1 (data not shown). Separated
microspores were the major species present in this bud
indicating that the majority of meiocytes had completed
meiosis, and that the minority, showing defective meiotic
structures, were defective in progression.
The increased severity of defects in pollen development
resulting from defects in male meiosis that were observed in
duet/+ dyad/dyad plants, coupled with the loss of chromosome
cohesion provide evidence of a synergistic interaction between
the duet and dyad mutant alleles.
To test for possible effects of duet on female meiosis we
examined cleared ovules as well as female meiosis in duet dyad
double mutant plants (Fig. 9U-W). The ovule phenotype was
identical to that of the dyad mutant. In both cases the majority
of ovules showed a single division meiosis and the presence of
two enlarged cells in place of an embryo sac. Cytogenetic
analysis of female meiosis in duet dyad plants also indicated
that chromosome behavior was the same as that described
earlier for dyad, which was shown to undergo an equational
meiosis 1 division (Agashe et al., 2002). No additional effects
were observed from the presence of the duet mutant allele in
a dyad background, on the integrity or appearance of
chromosomes. We also examined female fertility of duet/duet
5984 Development 130 (24)
Research article
Fig. 9. Interactions between duet and dyad: meiotic
chromosome stages. (A-J) duet/+ dyad/dyad.
(K-N,W) duet dyad double mutant. (O-Q) duet/+
heterozygote. (R-T,V) dyad mutant. (A) Early
zygotene. (B) Pachytene stage showing thickened but
irregular chromosomes. (C) Diakinesis. Five bivalents
are visible. (D) Aberrant diakinesis in which
chromosomes have desynapsed to form ten univalents.
(E) Extreme diakinesis in which both synapsis and
sister chromatid cohesion have been lost to yield single chromatids. (F) Early anaphase 1 undergoing mixed segregation in which both
univalents and bivalents are involved. (G) Late anaphase 1 showing approximately equal separation of chromosomes. Eight to ten chromosomes
are present at each pole indicating an equal division. (H) Telophase 1. Equal division. Ten chromosomes are present at each pole. (I) Telophase
1. Unequal division. (J) Dyad formed after unequal division. (K) Zygotene. (L) Diakinesis involving 2 bivalents and 6 univalents. (M) Extreme
diakinesis containing mostly single chromatids. (N) Prometaphase 1 having eight to ten thick diffuse chromosomes. (O,R) Normal diakinesis.
(P,S) Metaphase 1. (Q,T) Telophase 1. (U,V) Cleared ovules of dyad (U) and duet dyad (V). (W) Metaphase 1. Scale bars: 12.5 µm (A-T,W)
and 25 µm (U,V).
dyad/+ plants and did not observe any reduction in fertility
(data not shown). We therefore did not find any evidence to
support a role for DUET in female meiosis.
Discussion
Progression through the meiotic cell cycle requires
orchestration of a set of events that includes the assembly of
chromosomes for reductional division, pairing of homologous
chromosomes, recombination and segregation of homologues
to opposite poles of the meiosis 1 spindle (reviewed by Dawe,
1998). Defects in meiotic chromosome organization can result
in delayed progression through the meiotic cell cycle. The role
of chromosomal checkpoints in monitoring and ordering the
phases of the mitotic and meiotic cell cycle is well documented
in yeast and animals (Roeder and Bailis, 2000; Handel et al.,
1999). In plants there is little information on the control of
meiotic progression and the role of chromosomal checkpoints
in meiosis (Garcia et al., 2003). Mutations in the yeast DMC1
gene cause arrest as a result of activation of the pachytene
Arabidopsis meiotic chromosome organization 5985
checkpoint (Bishop et al., 1992; Lydall et al., 1996). However,
a mutation in AtDMC1, the Arabidopsis homolog of DMC1
does not cause arrest. Instead it leads to random chromosome
segregation in meiosis and the production of defective spores
(Couteau et al., 1999). Hence the control of meiotic
progression in plants may differ from that in yeast.
The properties of the DUET gene described above indicate
that it is required for male meiotic chromosome organization
and suggest that in the absence of DUET function, male
meiocytes undergo defective progression through the meiotic
cell cycle. In the duet mutant, male meiocytes went through a
single division to produce a pair of cells instead of a normal
tetrad. The defective spores produced did not complete
gametogenesis and degenerated. The phenotype resembled that
observed in the dyad mutant of Arabidopsis in which the
majority of female meiocytes divide singly to give a dyad
followed by an arrest in further development of the female
gametophyte (Siddiqi et al., 2000). Single division meiosis has
also been described for the spo12, spo13 and slk19 mutants of
yeast (Klapholz and Esposito, 1980; Zeng and Saunders, 2000;
Kamieniecki et al., 2000).
The DUET gene was cloned by transposon tagging with a
Ds element and found to encode a putative PHD finger domain
protein. The gene is closely related to the MS1 gene, which has
been proposed to be a transcriptional regulator of male
gametogenesis in Arabidopsis (Wilson et al., 2001), to two
other putative Arabidopsis genes, At1g33420 and At2g01810,
and to a putative rice gene (GenBank ID AC090882). The PHD
finger is a modified zinc finger and is found in a number of
proteins that play a role in chromatin organization and
transcriptional regulation and include members of the
Trithorax and Polycomb groups (reviewed by Aasland et al.,
1995). In plants the PHD finger has been found in a
transcriptional regulator of genes involved in defense against
pathogens (Korfhage et al., 1994) and in genes that are required
for reproductive development and fertility: the PICKLE gene
is required to prevent re-expression of embryonic traits in
germinated seedlings and encodes a CHD3 domain protein that
has been proposed to act as a regulator to promote the transition
from embryonic to postembryonic development (Ogas et al.,
1999); overexpression of the SHL gene has been show to lead
to early flowering and defective reproductive development
whereas antisense inhibition caused dwarfism and delayed
growth (Mussig et al., 2000). Hence, in both plants and animals
PHD finger genes play a role in developmental transitions.
Recently the PHD finger domain has also been found in
proteins that act as E3 ubiquitin ligases (Cosoy and Ganem,
2003). This latter class of PHD finger proteins are localized to
the membrane or cytoplasm. The PHD finger in DUET is more
similar to that found in proteins that act as chromatin
remodeling factors or transcriptional regulators. The DUET
gene also showed limited similarity to SWI1/DYAD a gene that
has been demonstrated to be required for chromosome
cohesion during meiosis in Arabidopsis (Mercier et al., 2001;
Agashe et al., 2002).
Expression of the DUET gene in the inflorescence appeared
to be specific to the male meiocyte. Earliest expression was
detected in stage 4 anthers at a time that corresponds to the
presence of sporogenous cells. Maximal expression was seen
at anther stage 5 prior to meiosis, after which the signal
declined. Since the expression and phenotype for DUET and
the related MS1 gene appears to be sex specific (Wilson et al.,
2001; Ito and Shinozaki, 2002), it is possible that along with
the other two closely related putative genes At1g33420 and
At2g01810, they define a family of transcriptional regulators
that function during male meiosis and gametogenesis.
The appearance of chromosomes during early stages of
meiotic prophase in the duet mutant was normal up to
pachytene. Differences from wild type first became noticeable
at diplotene with chromosomes appearing more irregular and
diffuse in the mutant. This would suggest that either the timing
of DUET action is at the onset of diplotene or else that DUET
may act earlier, but its absence may lead to visible changes in
chromosome structure only at a later stage when the
synaptonemal complex is disassembled and most of the sister
chromatid cohesion is removed at diplotene. The difference
between duet and wild type was accentuated at diakinesis and
culminated in a high proportion of meiocytes showing arrest at
metaphase 1. The appearance of chromosomes at metaphase 1
was variable and distinctly different from wild type. The
metaphase 1 phenotype ranged from nearly normal looking
structures to ones in which the chromosomes appeared as an
irregular mass towards the center of the cell in which individual
chromosomes could not be clearly distinguished. A distinct
characteristic of the mutant meioses was the absence of the
organelle band which is a prominent feature found at the center
of the cell of wild-type meiocytes at telophase 1. Instead, the
organelles in the mutant meiocytes appeared more evenly
distributed throughout the cytoplasm. The reason for this could
be that the localization of mitochondria and plastids in the
dividing meiocyte requires expression of specific genes during
meiosis, and that the expression of these genes is adversely
affected in the mutant. Alternatively the cause could be a more
general disruption of cytoplasmic and cytoskeletal organization
that affects organelle transport and localization in the meiocyte.
The finding of defects in chromosome organization in meiosis
caused by disruption of the DUET gene extends the role of
PHD finger proteins to include functions that are specific to
meiosis.
Double mutant combinations of duet with dyad revealed
genetic interaction manifesting in defects during male meiosis.
The effect was most apparent in plants that were duet/+
dyad/dyad. Whereas the individual dyad/dyad and duet/+
plants showed no or very weak effects on pollen development,
the combination resulted in strong defects in male meiosis.
Microsporocytes showed defective division patterns and the
products were dyads, triads and tetrads. Most of the
microspores produced were defective and degenerated.
However a minority of spores did develop into viable pollen.
At the cellular level, the defective divisions of the male
meiocyte were similar to those in duet/duet, although less
severe. The duet dyad double mutant showed a progression
defect that was more severe than in the duet single mutant as
the male meiocytes failed to divide. At the chromosomal level
there were additional defects that were not observed in either
single mutant. In duet/+ dyad/dyad meiocytes, chromosomes
lost synapsis or cohesion prior to meiosis 1 and segregated
unequally in many cases. Loss of cohesion was not observed
in either the dyad or duet single mutants. dyad is a femalespecific allele and shows normal male meiosis and pollen
development (Siddiqi et al., 2000). The stronger allele swi1-2
is male sterile and shows loss of sister chromatid cohesion
5986 Development 130 (24)
during prophase of male meiosis (Mercier et al., 2001). Hence
the loss in sister chromatid cohesion as a result of the
interaction may be interpreted to mean that hemizygous duet/+
enhances the dyad mutant phenotype. The genetic interaction
between duet and dyad could be specific and the genes may act
at the same level of chromosome architecture. The presence of
the PHD finger in DUET implicates it as functioning in the
control of transcription at the level of chromatin organization
whereas DYAD/SWI1 has been shown to function in
chromosome organization and cohesion. If DUET does in fact
function as a transcriptional regulator, this would point to a
close connection between cohesion and the control of
transcription at the chromatin level during meiosis. The
formation of dyads and the fact that meiosis is more extended
in the duet mutant clearly suggests a defect in meiotic
progression. Analysis of single division meiosis in the spo13
mutant of yeast has shown that the basis for the progression
defect is the activation of the spindle checkpoint. In the absence
of the spindle checkpoint, spo13 mutants undergo normal
meiotic progression and form four spores (Shonn et al., 2002;
Lee et al., 2002). There is at present limited information on the
control of meiotic progression in plants. Immunolocalization
of a maize homologue of the yeast spindle checkpoint protein
MAD2 has shown that it is expressed in meiosis and localized
to the kinetochore where it functions through a tensiondependent mechanism (Yu et al., 1999). Hence the basic
apparatus for the spindle checkpoint is conserved in plants. A
recent study has identified a mutant sog1 that suppresses
gamma radiation-induced arrest and also affects pollen
development (Preuss and Britt, 2003). The further analysis of
mutants defective in meiotic progression should provide
information on the existence of chromosomal checkpoints in
plant meiosis.
This work was supported by the Council for Scientific and
Industrial Research, Government of India. T.V.R., B.A. and J.K. are
recipients of CSIR Research Fellowships. This work was also partly
supported by grants from NSTB Singapore to V.S. We thank Dr Shashi
Singh for help with sectioning of plant material and Mehar Sultana
for synthesis of oligonucleotides. We also acknowledge the ABRC for
DNA clones and seed material.
Note added in proof
While the manuscript was under review, Yang et al. reported
the cloning and expression analysis of the MMD gene, and
analysis of the mmd mutant. The MMD gene is identical to
DUET (Yang, X. et al., 2003).
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