1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad
500007, India
2 Department of Plant Biology and Agronomy, Life Sciences Addition 1002,
University of California, Davis, CA95616, USA
Author for correspondence (e-mail:
imran{at}ccmb.res.in)
Accepted 27 August 2003
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SUMMARY |
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Key words: Chromatin, Male sterility, Checkpoint, Cohesion, Synapsis
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Introduction |
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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 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.
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Materials and methods |
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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 x40 and x100 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
1x PBS overnight at 4°C. The inflorescences were then washed with
1x PBS, treated with osmium tetroxide for 3 hours, followed by
dehydration in a graded ethanol series (30%, 50%, 70%, 90%, 100% x5) 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'-CGTTCCGTTTTCGTTTTTTACC-3') 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'-TCTGCAAATTCTTCACAGCAATTCG-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
Heslop-Harrison, 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
100x 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.
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Results |
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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 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.
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The duet mutation interacts with dyad
The duet mutant phenotype is similar to what is seen for female
meiosis in the dyad mutant
(Siddiqi et al., 2000). In
each case a pair of cells is formed after meiosis instead of a tetrad. The
pair of cells do not develop further into functional gametes. For both
duet and dyad, the two-cell phenotype correlates with
defective progression through meiosis and the underlying cause appears to be
altered chromosome organization in both cases. The DYAD/SWI1 gene is
required for female meiotic progression, and for chromosome cohesion in male
and female meiosis (Mercier et al.,
2001
; Agashe et al.,
2002
). However, the dyad mutant allele is female specific
and shows normal pollen development and male fertility.
To test if both mutants are affected in a related aspect of chromosome organization during meiosis, we intercrossed duet and dyad as female and male parents respectively to test for genetic interaction. The F1 were fully fertile and showed normal pollen and embryo sac development (data not shown). F2 plants were genotyped with respect to the alleles present at the duet locus (duet/duet, duet/+, and +/+) and at the dyad locus (dyad/dyad, and +/), and various dose combinations of duet and dyad were examined for evidence of genetic interaction.
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 pollen grains was also lower for the interaction genotype. A rough
indication of this could be obtained from the total amount of pollen on the
slide. For the duet/+ dyad/dyad genotype the mean count of total
pollen grains per flower was 131±50 whereas for sibling plants that
were duet/+ +/ the mean pollen count was 326±171. Each
individual genotype duet/+ and dyad/dyad produced numbers of
viable pollen that were comparable to wild type
(Siddiqi et al., 2000; data
not shown). Analysis of pollen development in cleared anthers showed that male
meiocytes in duet/+ dyad/dyad plants underwent an aberrant division
to produce mostly dyads and triads, as well as some tetrads
(Fig. 8). The majority of
spores produced were defective and formed enlarged cells containing 1-3 nuclei
and that subsequently degenerated. The phenotype therefore broadly resembled
that of homozygous duet plants though it was less severe.
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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
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.
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Discussion |
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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
female-specific 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 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 tension-dependent 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.
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ACKNOWLEDGMENTS |
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Footnotes |
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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).
* These authors contributed equally to this work
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