Download Dissecting plant meiosis using Arabidopsis thaliana mutants

Journal of Experimental Botany, Vol. 54, No. 380,
Plant Reproductive Biology Special Issue, pp. 25±38, January 2003
DOI: 10.1093/jxb/erg041
Dissecting plant meiosis using Arabidopsis thaliana
mutants
Anthony P. Caryl1, Gareth H. Jones and F. Christopher H. Franklin
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Received 12 June 2002; Accepted 18 September 2002
Abstract
Meiosis is a key stage in the life cycle of all sexually
reproducing eukaryotes. In plants, specialized reproductive cells differentiate from somatic tissue. These
cells then undergo a single round of DNA replication
followed by two rounds of chromosome division to
produce haploid cells that then undergo further
rounds of mitotic division to produce the pollen grain
and embryo sac. A detailed cytological description of
meiosis has been built up over many years, based on
studies in a wide range of plants. Until recently, comparable molecular studies have proved too challenging, however, a number of groups are beginning to
use Arabidopsis thaliana to overcome this problem.
A range of meiotic mutants affecting key stages in
meiosis have been identi®ed using a combination of
screening for plants exhibiting reduced fertility and,
more recently, using a reverse genetics approach.
These are now providing the means to identify and
characterize the activity of key meiotic genes in
¯owering plants.
Key words: Arabidopsis, meiosis, meiotic mutant, reduced
fertility, T-DNA, transposon.
Introduction
The life cycles of all sexually reproducing eukaryotes are
dependent on the process of meiosis. This combines a
single round of DNA replication with two rounds of
chromosome division to produce haploid gametes and to
allow the process of genetic recombination to occur
producing novel combinations of genetic information. A
co-ordinated series of events during prophase I lead to
homologous chromosome pairing, synapsis to form
1
bivalents and recombination between non-sister chromatids within the bivalent. Reductional division then occurs
separating the homologous chromosomes during anaphase
I. The second meiotic division is equational and separates
the sister chromatids of each chromosome to give rise to
the haploid gametes necessary for sexual reproduction.
Meiosis in ¯owering plants has long been a subject of
research and has led to the identi®cation of many meiotic
mutants (Baker et al., 1976; Golubovskaya, 1979; Koduru
and Rao, 1981; Kaul and Murthy, 1985). Until recently, it
has not been possible to identify and characterize the genes
affected in these mutants at a molecular level. However,
this has begun to change with the identi®cation of a
number of meiotic mutants from Arabidopsis thaliana,
which have provided the means to clone a range of genes.
Analysis of these genes together with the development of
improved cytogenetic and immunocytological techniques
for Arabidopsis (Ross et al., 1996; Armstrong and Jones,
2001, 2003) are permitting more complete analyses of
meiotic chromosome behaviour to be undertaken. In this
article, recent progress in the isolation of meiotic mutants
in Arabidopsis and the contribution this is making to the
study of meiosis in plants is reviewed.
Meiosis in wild-type Arabidopsis thaliana: a
brief outline
The development of the male (anther) and female (ovule)
reproductive structures from somatic tissue initiate the
basic process of reproductive development in Arabidopsis
thaliana.
In male development, a group of cells within the
developing anther differentiate into archesporial cells.
These then give rise to the primary sporogenous cells
which differentiate into the pollen mother cells (PMCs) in
which meiosis occurs and primary parietal cells which give
To whom correspondence should be addressed. Fax: +44 (0)121 414 5925. E-mail: [email protected]
ã Society for Experimental Biology 2003
26 Caryl et al.
rise to the tapetum, endothecium and the middle layer of
the anther (Yang et al., 1999a). Each anther locule
contains around 30 PMCs, which enter and proceed
through meiosis with a high degree of synchrony
(Armstrong et al., 2001). Meiosis in the PMC produces a
tetrad of haploid microspores, which are then released
from a common wall and go on to develop into the male
gametophytes (pollen grains).
In the female reproductive tissues an archesporial cell at
the top of the ovule primordium differentiates from a
single hypodermal cell. This archesporial cell directly
differentiates into the megaspore mother cell (MMC).
Meiosis in the MMC results in the formation of four
haploid megaspores. The three spores closest to the
micropyle of the ovule undergo programmed cell death
leaving the chalazal megaspore to develop into the female
gametophyte (the embryo sac) (Yang et al., 1999a). It is of
interest to note that in common with many other
angiosperms there is a degree of asynchrony between
male and female meiosis, such that by the time male
meiosis is complete, female meiosis is still at prophase I
(Armstrong and Jones, 2001).
The development of improved cytogenetic techniques
using modi®ed chromosome spreading and DAPI (4¢,6diamidino-2-phenylindole) staining procedures have enabled the behaviour of the chromosomes during meiosis to
be examined in considerable detail (Ross et al., 1996,
1997; Armstrong and Jones, 2001). Following replication
and meiotic interphase the diffuse chromosomes begin to
condense, establishing short stretches of chromosome axes
that become discernible during the leptotene stage of
prophase I. Telomere pairing is also initiated during
leptotene (Armstrong et al., 2001), progressing into
zygotene where the pairing of homologous chromosomes
is ®rst clearly observed. At pachytene chromosome
synapsis is complete (Fig. 1a). During diplotene/diakinesis
the homologous chromosome pairs condense further and
desynapse leading to the visualization of discrete bivalents
linked by one to three chiasmata (Ross et al., 1996), the
sites of physical cross-over that arise from the events of
homologous recombination during prophase I (Fig. 1c). As
meiosis proceeds from prophase I into metaphase I the
highly condensed bivalents become attached to the meiotic
spindle and align at the equator of the cell with their
centromeres directed to opposite poles. At anaphase I
homologous chromosomes migrate to the opposite poles of
the cell. At telophase I (the dyad stage) the two chromosome groups partially decondense before the second
meiotic division begins. The chromosomes recondense at
prophase II and at metaphase II become aligned prior to the
second division (Fig. 1h). At this point sister chromatid
cohesion has broken down in the chromosome arms thus
permitting pairs of chromatids to be observed. The
chromatids are separated at anaphase II to produce four
tetrad nuclei each containing a haploid chromosome
content (Fig. 1j). Cytokinesis then occurs producing four
haploid cells (Ross et al., 1996; Armstrong and Jones,
2001).
Identi®cation of meiotic mutants in Arabidopsis
An extensive range of meiotic genes have been identi®ed
in a variety of organisms, notably budding and ®ssion yeast
(reviewed in: Roeder, 1997; Zickler and Kleckner, 1999;
Dresser, 2000; Davis and Smith, 2001). Hence, one might
anticipate that it should easily be possible to identify
homologues of these genes in Arabidopsis. To some extent
this is indeed the case, particularly for genes that encode
proteins catalysing homologous recombination. For example, DMC1 (Sato et al., 1995; Klimyuk and Jones, 1997;
Doutriaux et al., 1998), RAD51 (Doutriaux et al., 1998),
and SPO11 (Hartung and Puchta, 2000; Grelon et al.,
2001). However, it is clear that this is only possible for a
subset of meiotic genes, as in many cases homologues
cannot readily be identi®ed in silico. This seems to apply
particularly to genes encoding proteins involved in
chromosome morphogenesis such as synapsis of homologous chromosomes. For instance, the yeast ZIP1 and rat
SCP1 genes both encode proteins that comprise the
transverse ®laments of the synaptonemal complex. Yet,
whilst they appear functionally and structurally similar
they exhibit no primary sequence homology beyond that
expected between two coiled-coil proteins (Heyting,
1996). Inspection of the Arabidopsis genome for genes
encoding homologues to either or both proteins reveals a
range of candidates with little more than 20% sequence
identity (AP Caryl, NP Jackson, FCH Franklin, unpublished data). An added complication in assigning gene
function on the basis of homology arises from the ®nding
that the Arabidopsis genome has undergone an ancient
duplication (The Arabidopsis Genome Initiative, 2000).
Around 60% of the Arabidopsis genome is duplicated and
in many cases the genes within these duplicated regions
have undergone functional divergence. Thus, even in cases
where putative meiotic genes can be identi®ed on the basis
of homology there can be several potential candidates
whose functions have to be established.
Both these problems may be addressed using mutation.
To date, most emphasis and progress has been made as the
result of screens aimed at the de novo identi®cation of
meiotic mutants and subsequent gene isolation, which will
be the main focus of the rest of this article.
Meiotic mutants in Arabidopsis have been identi®ed by
screening EMS (ethyl methanesulphonate), T-DNA and
transposon mutagenized populations for plants exhibiting
reduced fertility or sterility (Ross et al., 1997; Bhatt et al.,
1999; Sanders et al., 1999; Siddiqi et al., 2000; Mercier
et al., 2001a). Several studies based on this approach,
summarized in Table 1, have resulted in the isolation of a
range of meiotic mutants. In one case, as an additional
Meiotic mutants of Arabidopsis
27
Fig. 1. Meiosis in pollen mother cells of Arabidopsis thaliana. (a) Wild-type pachytene (taken from the dsy1 mutant) illustrating full chromosome
synapsis, (b) early prophase in asy1 showing unsynapsed chromosomes, (c) wild-type diplotene illustrating ®ve bivalents joined by chiasmata, (d)
diplotene in dsy1, no bivalents are present but homologous chromosomes are associated, (e) diplotene in asy1, no bivalents are present and
homologous chromosomes are not associated. The four nucleolus organizing chromosomes converge at the site of their association with the
nucleolus (arrowed). (f) Metaphase I in mei1/mcd1 illustrating the severe chromosome fragmentation which occurs in this mutant, (g) dyad stage
in the mei1/mcd1 mutant showing the acentric chromosome fragments which are not incorporated in either nucleus, (h) metaphase II in wild type
showing ®ve chromosomes at each pole, (i) metaphase II as seen in asy1 and dsy1 (this image taken from dsy1) showing the uneven division of
chromosomes that results from synaptic defects in the mutants, (j) wild-type tetrad (from tdm1), (k) chromosome extension and stretching
following metaphase III in the tdm1 mutant, (l) multiple nuclei resulting from the attempted third meiotic division in tdm1 (these will go on to
form polyads). Each panel shows a single DAPI stained spread PMC. Bar=20 mM.
screening step the inheritance of the `reduced-fertility' trait
was checked over several generations. The object was to
ensure that the mutant phenotype did not segregate as
fertile/reduced fertility, as this would be indicative of
reduced fertility caused by heterozygosity for chromosomal rearrangement rather than by meiotic gene mutation
(Ross et al., 1997). Before any putative meiotic mutant
may be con®rmed it is essential that it is subjected to a
thorough cytogenetical examination. This requirement
arises from the fact that although a screen based on a
reduced fertility phenotype will indeed identify meiotic
mutants, a wide range of other mutants defective in various
aspects of reproductive development may be expected to
possess a very similar phenotype. This has proved to be the
case with mutants affecting a range of processes from
sporocyte initiation (Yang et al., 1999a) to anther
dehiscence (Sanders et al., 1999) being identi®ed.
Identi®cation of meiotic genes using reverse
genetics
An alternative approach to screening for meiotic mutants
representing unknown genes is to generate or identify
mutations in known genes which either show homology to
28 Caryl et al.
Table 1. Identi®cation of (potential) meiotic mutants by screening for reduced fertility in mutagenized Arabidopsis thaliana
populations
Number of reduced
fertility mutants
identi®ed
Number of potential
meiotic mutants
Mutagen used in
the screen
References
6
40
20
140
8
44
855
46
17
791
2
na
na
16a
6
26
359
na
7b
32
T-DNA
EMS
T-DNA
T-DNA
T-DNA
T-DNA
EMS
EMS
EMS
T-DNA
Dawson et al., 1993
Chaudhury et al., 1994
Glover et al., 1996
Peirson et al., 1996
Ross et al., 1997
Sanders et al., 1999
Sanders et al., 1999
Siddiqi et al., 2000
Mercier et al., 2001a
Mercier et al., 2001b
a
b
From 50 lines which were studied in detail.
Number of complementation groups identi®ed in the screen.
meiotic genes from other species or have an expression
pattern suggestive of a meiotic role. As discussed above,
some Arabidopsis meiotic genes will have homology to
known meiotic genes from other species. In many
instances this will be re¯ected in an essentially identical
biological function. However, since there may be exceptions it will be important to identify and characterize
mutant alleles of these genes.
This is exempli®ed by the Arabidopsis ASY1 gene
which, despite exhibiting some homology to the yeast
HOP1 gene, appears to exhibit differences when examined
at a functional level (Hollingsworth et al., 1990; Smith and
Roeder, 1997; Kironmai et al., 1998; Caryl et al., 2000;
Armstrong et al., 2002).
Although expression of many Arabidopsis meiotic
genes can be detected in somatic tissues, some such as
DMC1, are up-regulated during meiosis (Klimyuk and
Jones, 1997) or possess meiosis-speci®c splice variants
such as SYN1/DIF1 (Bai et al., 1999). This suggests that it
may be possible to identify genes with meiotic roles by
expression pro®ling using microarray technology.
Identi®cation of knockouts in genes identi®ed on the
basis of sequence homology or expression pattern can be
done by screening the sites of T-DNA and transposon
insertions in individual lines from tagged populations.
These include the Feldmann (Feldmann and Marks, 1987),
INRA-Versailles (Bechtold et al., 1993), SALK (Salk
Institute Genomic Analysis Laboratory, http://signal.salk.
edu) and SAIL (Syngenta Arabidopsis Insertion Library,
http://www.nadii.com/pages/collaborations/garlic_®les/
GarlicDescription.html) T-DNA insertion lines and the
IMA (Parinov et al., 1999) and SLAT (Tissier et al., 1999)
transposon insertion lines. Several approaches can be used
to screen these tagged populations. PCR using gene- and
tag-speci®c primers can be performed on DNA pools made
from the tagged population. Alternatively, inverse PCR
(IPCR) can be used to amplify plant DNA adjacent to the
tag which can then be blotted and screened by hybridization to gene-speci®c probes. More recently, IPCR generated tags have been systematically sequenced to provide a
sequence database that can be queried for knockouts in a
gene of interest. An approach has also been developed to
allow high throughput screening of chemically mutagenized DNA. In denaturing high power liquid chromatography (DHPLC) plants are treated with a chemical
mutagen such as EMS. The gene of interest is then
ampli®ed by PCR, and the resulting products resolved by
HPLC where point mutations can easily be identi®ed
(McCallum et al., 2000). The identi®cation of gene
knockouts in Arabidopsis is reviewed in greater detail in
Bouche and Bouchez (2001).
If a particular gene knockout cannot be identi®ed it is
possible to generate one. Directed gene disruption by
homologous recombination, which is widely used in yeast
and mouse would, in theory, be the best approach, but,
unfortunately, this is not yet a realistic option in plants
(Mengiste and Paszkowski, 1999). However, several
alternative methods do exist, collectively known as posttranscriptional gene silencing; these include antisense
RNA, cosuppression and RNAi. These phenomena depend
on the generation or introduction of double-stranded RNA
based on the target gene sequence. This results in the
degradation of the corresponding mRNA, and in some
cases the methylation of the nuclear DNA with the result
that gene expression is reduced or eliminated (reviewed in
Bourque, 1995; Wang and Waterhouse, 2001; Hannon,
2002).
Meiotic mutants: from phenotype to gene
A variety of Arabidopsis meiotic mutants have now been
isolated. In a number of cases the corresponding gene has
been cloned and further characterization carried out. In the
following section current progress is reviewed. In general,
Meiotic mutants of Arabidopsis
an attempt was made to categorize the mutants on the basis
of their meiotic phenotype. In some cases, this has been
made dif®cult through a lack of data or a complex meiotic
phenotype, hence in these cases the categorization may
need to be re-evaluated when relevant new data emerges.
Chromosome cohesion
Newly replicated sister chromatids are associated from
their formation at meiotic S phase through to their
disjunction at anaphase II. Sister chromatid cohesion is
maintained by a multi-protein cohesin complex (reviewed
in van Heemst and Heyting, 2000; Lee and Orr-Weaver,
2001). At least two Arabidopsis meiotic mutants have been
described that are proposed to play a role in establishing or
maintaining sister chromatid cohesion.
Three mutant alleles of the SWITCH1(SWI1)/DYAD
gene have been isolated. All three alleles show similar
defects in female meiosis. Ten univalents are observed at
metaphase I which segregate evenly into two daughter
cells (a mitosis-like division). Agashe et al. (2002) report
that in the dyad mutant GUS (b-glucuronidase) expression
is driven from a DMC1 promoter during female meiosis
suggesting that this division may be meiotic rather than
mitotic in nature. Subsequently, these cells can undergo
either a second mitosis like division, or an aberrant second
division with uneven chromosome segregation giving rise
to a variable number (1±5) of daughter cells (Motamayor
et al., 2000; Siddiqi et al., 2000; Mercier et al., 2001a).
These cells then degenerate with no embryo sac produced
in the mutant.
Male meiosis is unaffected in swi1-1 and dyad
(Motamayor et al., 2000; Siddiqi et al., 2000), but severely
affected in swi1-2. No true leptotene, zygotene or
pachytene stages are observed in swi1-2, and no evidence
of synapsis is seen. Ten univalents are initially observed
during meiosis I followed by a premature loss of sister
chromatid cohesion. This begins along the chromosome
arms and later at the centromeres resulting in the
production of 20 chromatids before the end of meiosis I.
Chromosome condensation is also seen to be incomplete/
reduced. Uneven segregation of the chromatids during the
®rst and second meiotic divisions results in the production
of 1±10 unbalanced nuclei which subsequently degenerate
(Mercier et al., 2001a).
The swi1-1 allele is not completely penetrant as 3% of
the wild-type number of embryo sacs are observed
(Motamayor et al., 2000; Mercier et al., 2001a).
Heterozygotes between swi1-1 and swi1-2 show an
intermediate male meiosis phenotype (Mercier et al.,
2001a). A male-sterile, female-fertile mutant, ms4 identi®ed by Chaudhury et al. (1994) also results in the
production of dyads, which persist until late stages of
anther development then degenerate.
29
The SWI1 gene produces two alternatively spliced
transcripts encoding proteins of 578 and 635 amino
acids, respectively, differing only at the N termini. The
protein shows no clear homology to any other sequence in
the database (Mercier et al., 2001a). The swi1-1 allele,
which affects only female meiosis, was caused by a TDNA insertion just after the transcriptional start site of the
long transcript. This presumably results in no protein being
produced, or occasional secondary translational initiation
may produce small quantities of wild-type Swi1 protein
(Mercier et al., 2001a). The dyad allele results from a
frameshift mutation after amino acid 505 (and terminates
after a further 9) (Agashe et al., 2002). The swi1-2 allele,
which affects both male and female meiosis, was generated
by EMS and has a stop codon after amino acid 389 (of the
long transcript).
The Swi1 protein has been localized to the nucleus
during premeiosis and very early prophase using a green
¯uorescent protein (GFP) fusion construct driven by the
SWI1 promoter. The GFP signal suggests that the Swi1
protein co-localizes with DNA (Mercier et al., 2001a). It is
proposed that the Swi1 protein promotes the establishment
of sister chromatid cohesion, but is not responsible for its
maintenance, since the protein does not persist through
meiosis I. That the sister chromatids remain associated at
the centromeres in the swi1-2 mutant rather than immediately disassociating suggests that an additional early
acting, transient cohesion is promoted by factors independent of Swi1 (Mercier et al., 2001a).
Two groups have described mutations in a second gene
that has been associated with chromosome cohesion (Bai
et al., 1999; Bhatt et al., 1999). Mutations in the SYN1/
DIF1 gene result in a complex meiotic phenotype that
affects both male and female fertility. A defect can initially
be detected as early as leptotene when chromosome
condensation appears irregular (Bai et al., 1999), however
chromosomes appear to be normally synapsed during
pachytene (Bhatt et al., 1999). Extensive chromosome
fragmentation is clearly observed by anaphase I.
Chromosome segregation fails, with some chromosomes
and acentric chromosome fragments failing to attach to the
meiotic spindle (Peirson et al., 1997; Bai et al., 1999).
Some univalent chromosomes are also observed at postpachytene stages in syn1/dif1 mutants, suggesting that
sister chromatid cohesion may be compromised (Bhatt
et al., 1999). Further acentric fragments together with
chromosome bridges are seen at anaphase II. Subsequent
non-disjunction leads to the production of up to 8 polyads
containing variable amounts of DNA. A detailed examination of female meiosis has not been made in the syn1/dif1
mutants, but the ovules do not contain an obvious embryo
sac and excess callose is seen deposited around the
gametocyte (Bhatt et al., 1999).
The gene responsible for the syn1/dif1 phenotype has
been cloned. It has homology to the RAD21 (largely
30 Caryl et al.
mitotic)/REC8 (meiotic) cohesin family involved in sister
chromatid cohesion. In yeast mitosis six genes are known
to be required to establish sister chromatid cohesion, the
protein products of four of these going on to make up a
cohesion complex with the DNA. In Xenopus oocytes the
cohesion complex contains ®ve proteins, three of which
show homology to proteins in the yeast complex (Lee and
Orr-Weaver, 2001; Dong et al., 2001). In
Schizosaccharomyces pombe, rec8 mutants result in
impaired meiotic chromosome pairing, linear element
disruption, reduced recombination and premature sister
chromatid separation during meiosis (Molnar et al., 1995;
Watanabe and Nurse, 1999).
Two alternately spliced SYN1/DIF1 transcripts have
been identi®ed encoding proteins of 627 amino acids
(expressed at low levels in all tissues) and 617 amino acids
(expressed in buds only). Multiple mutant alleles have
been identi®ed; these include a T-DNA (Bai et al., 1999)
and ®ve transposon insertion lines (Bhatt et al., 1999). The
Syn1/Dif1 protein contains two potential nuclear localization signals and several PEST sequences typically found in
proteins that are subject to rapid turnover (Bai et al., 1999;
Bhatt et al., 1999). Some evidence that Syn1/Dif1 is acting
as a cohesin is provided by the fact that the protein can
partially substitute for the Mcd1 mitotic cohesin in yeast
complementation tests (Dong et al., 2001).
Arabidopsis has two other genes with homology to the
RAD21/REC8 cohesins, called SYN2 (encoding an 809
amino acid protein) and SYN3 (encoding a 692 amino acids
protein) (Dong et al., 2001). As yet no mutant alleles of
these genes are known. Both are most highly expressed in
regions of actively dividing cells suggesting a possible role
in mitosis, however, it is interesting to note that neither
will complement yeast cells de®cient in the Mcd1 mitotic
cohesin (Dong et al., 2001).
Recombination and meiotic DNA repair
Meiotic recombination has been studied extensively,
notably in yeast (reviewed in Roeder 1997; Zickler and
Kleckner, 1999; Sung et al., 2000). Many of the genes
encoding the proteins that catalyse this process have been
cloned and characterized. In general, these genes appear to
be highly conserved across a wide range of eukaryotes. For
example, DMC1 homologues are known in yeast (Bishop
et al., 1992), mouse (Habu et al., 1996), human (Habu
et al., 1996), and Arabidopsis (Sato et al., 1995). There are
now several characterized examples of Arabidopsis
mutants in recombination genes.
Meiotic recombination in higher eukaryotes is initiated
following double-strand break formation (DSB) by the
Spo11 protein (Keeney et al., 1997). The protein is a
member of a novel family of type II-like topoisomerases
(Bergerat et al., 1997). It possesses a transesterase activity
that catalyses double-strand breaks through formation of a
5¢-phosphotyrosyl linkage (Bergerat et al., 1997; Keeney
et al., 1997). In addition to initiating double-strand breaks
the protein is also required for chromosome synapsis in
yeast and mouse, but not in Drosophila and
Caenorhabditis elegans (Giroux et al., 1989; Dernburg
et al., 1998; McKim and Hayashi-Hagihara, 1998;
Romanienko and Camerini-Otero, 2000).
Three potential homologues of SPO11 have been
identi®ed in Arabidopsis. Evidence suggests that of one
these, AtSPO11-1, is required in meiosis (Grelon et al.,
2001). The gene encodes a protein of 362 amino acids and
is most highly expressed in reproductive tissues (Hartung
and Puchta, 2000; Grelon et al., 2001). Two mutant alleles
of AtSPO11-1 have been identi®ed amongst reduced
fertility plants isolated from T-DNA transformed and
EMS mutagenized populations (spo11-1-1, spo11-1-2,
respectively) (Grelon et al., 2001).
Examination of spo11-1-1 revealed that the mutant
formed few bivalents during both male and female
meiosis, leading to meiotic non-disjunction and the
production of polyads (as opposed to tetrads) with variable
DNA content during male meiosis. During female meiosis
the embryo sac typically fails to differentiate. No fully
synapsed chromosomes are detected at prophase I. As a
result of these defects the mutant exhibits a dramatic
reduction in meiotic recombination frequency (Grelon
et al., 2001).
The reduced chiasma frequency (7% of wild type) is an
interesting feature of spo11-1-1, which, based on the site of
T-DNA insertion in the ®rst exon, is expected to be a null
mutant. The corresponding Drosophila and C. elegans
spo11 mutants show no recombination (McKim et al.,
1998; McKim and Hayashi-Hagihara, 1998; Dernburg
et al., 1998). This suggests that, in Arabidopsis, some
DSBs are formed by an alternative route, such as one of the
other SPO11 homologues, or another plant speci®c
protein(s) or environmental DNA damage (Grelon et al.,
2001).
There is no compelling evidence to support a meiotic
role for the two remaining Arabidopsis SPO11 homologues, AtSPO11-2 and AtSPO11-3. Recent yeast twohybrid studies have revealed that Spo11-2 and Spo11-3
proteins can interact with a homologue of the archaebacterial topoisomerase B subunit (AtTOP6b) also found in
Arabidopsis. This ®nding taken in conjunction with their
expression patterns suggest they primarily function as
topoisomerases (Hartung and Puchta, 2001).
The yeast homologues of the Escherichia coli RecA
protein, Rad51 and Dmc1 have been shown to be essential
for inter-homologue recombination and, therefore, bivalent formation (Bishop et al., 1992; Shinohara et al., 1992).
In meiosis, these proteins work in conjunction to catalyse
invasion of the homologous non-sister duplex by singlestranded resected 3¢ ends from double-strand breaks.
Whilst the role of Dmc1 is speci®c to meiosis, Rad51 is
Meiotic mutants of Arabidopsis
also active in mitotic recombinational repair (Bishop et al.,
1992; Shinohara et al., 1992). Both genes appear to be
highly conserved in eukaryotes, including Arabidopsis
(Bishop et al., 1992; Morita et al., 1993; Shinohara et al.,
1993; Sato et al., 1995; Habu et al., 1996). To date there
are no reports of an Arabidopsis RAD51 mutation, possibly
re¯ecting the mitotic requirement for the protein, however
a T-DNA insertion mutant in AtDMC1 has been identi®ed
(Couteau et al., 1999). Homozygous dmc1 plants set 1.5%
viable seed compared to wild type (random segregation of
®ve chromosome pairs is expected to result in the
production of about 3% viable seed) with both male and
female fertility affected. In PMCs the primary cytological
defect appears to be in bivalent formation with ten
univalents observed at metaphase I, although occasional
bivalents are seen. Subsequent non-disjunction during
meiosis I and meiosis II results in polyads with variable
DNA content. Megaspore mother cells in the mutant arrest
just after meiosis and fail to go on to form the 8-nucleus
embryo sac (Couteau et al., 1999). The meiotic behaviour
of dmc1 highlights a feature found in other Arabidopsis
mutants such as asy1 (Ross et al., 1997), syn1/dif1 (Peirson
et al., 1997; Bhatt et al., 1999) and ms5/tdm1 (Chaudhury
et al., 1994; Ross et al., 1997) in that, despite gross defects,
meiosis does not arrest. In contrast, in other species such as
yeast and Drosophila (reviewed in Zickler and Kleckner,
1999; Roeder and Bailis, 2000) such lesions often trigger
meiotic arrest or in the case of mice (and perhaps C.
elegans), apoptosis (Romanienko and Camerini-Otero,
2000; Gartner et al., 2000; Roeder and Bailis, 2000). This
suggests that Arabidopsis and possibly other plants lack
the meiotic checkpoints found in other organisms.
The AtDMC1 gene which has been cloned encodes a 344
amino acid protein with 51.8% identity, 71.1% similarity
to the yeast Dmc1 protein (Sato et al., 1995; Klimyuk and
Jones, 1997; Doutriaux et al., 1998). AtDMC1 expression
can be detected in non-meiotic tissues by RT-PCR
(Klimyuk and Jones, 1997) and Northern blots
(Doutriaux et al., 1998). However, RNA in situ hybridization and promoter GUS fusion experiments suggest high
level expression is con®ned to meiotic tissues (Klimyuk
and Jones, 1997).
The Rad50 protein is essential for DSB repair in yeast
and is, therefore, required both for repair of DNA damage
and for meiosis (Alani et al., 1989) and is also involved in
telomere maintenance (Bertuch and Lundblad, 1998). A
mutant in the Arabidopsis RAD50 gene has been identi®ed.
Homozygous plants are sterile, but no detailed analysis of
meiosis in the mutant has been presented (Gallego et al.,
2001). It is not clear whether the phenotype affects both
male and female meiosis, although this would be expected
to be the case. Suspension cell cultures derived from the
mutant plants show hypersensitivity to the DNA damaging
agent MMS (methyl methanesulphonate) suggesting the
RAD50 gene also has a role in DNA repair in Arabidopsis
31
(Gallego et al., 2001). Cultured mutant cells undergo
reduction in telomere length, although telomerase activity
levels are the same as observed in wild-type cultured cells
(Gallego and White, 2001). Mutant plants display somatic
hyper recombination (8±10-fold higher than that observed
in heterozygotes) reminiscent of that seen in yeast rad50
mutants (Gottlieb et al., 1989; Gherbi et al., 2001). Mutant
plants grown on agar also appear stunted compared to wild
type although this phenotype is not apparent in soil-grown
plants. Nevertheless, it suggests that RAD50 also has a role
in mitotic growth (Gallego et al., 2001).
The RAD50 gene encodes a 1316 amino acid protein
with 29.8% identity to the human and 27.3% to the yeast
Rad50 proteins, respectively, and is expressed in all tissues
tested (by Northern blotting), and at a higher levels in
tissues containing rapidly dividing cells. The T-DNA
insertion in the rad50 mutant line is predicted to cause
premature truncation of the protein resulting in the loss of
the last 266 amino acids and so may not represent a null
mutant (Gallego et al., 2001).
Other Arabidopsis mutants have been described that are
potentially mutated in genes affecting meiotic recombination. Masson and Paszkowski, (1997) reported recombination rates in three X-ray-sensitive mutants isolated from an
EMS mutagenized population, which were also hypersensitive to the DNA damaging agents MMS and mitomycin
C. The xrs11 mutant showed normal levels of extrachromosomal recombination, but was defective in X-raymediated stimulation of homologous recombination in
such a manner as to suggest a signalling failure may be
involved. Meiotic recombination in this mutant was not
tested (Masson and Paszkowski, 1997). The xrs9 mutant
showed reduced fertility (50% of wild type), together with
reduced extrachromosomal and meiotic recombination. In
this it resembles yeast mutants of the RAD52 epistasis
group (Masson and Paszkowski, 1997). The xrs4 mutant
showed reduced extrachromosomal recombination, but
was found to exhibit meiotic hyper-recombination. This
mutant is, however, only 10% as fertile as the wild type
(Masson and Paszkowski, 1997).
The mei1 (also known as mcd1) mutant was isolated as a
tagged mutant from a T-DNA transformed population.
This mutant displays a DNA fragmentation phenotype
when examined at a cytological level, indicating that it is
unable to repair double-strand breaks. The mutant exhibits
a male-sterile (He et al., 1996) or male-reduced-fertility
phenotype (Ross et al., 1997) (presumably dependent on
the plant growth conditions). Premeiosis and early
prophase I in the mutant are apparently normal, however,
by early diplotene extensive chromosome fragmentation
can be observed. By metaphase I (Fig. 1f) the degree of
fragmentation can be seen to vary from cell to cell, with
some univalent chromosomes also present. At telophase I
some of the acentric chromosome fragments are excluded
from the daughter nuclei which can be clearly seen at the
32 Caryl et al.
dyad stage (Fig. 1g). Further fragmentation may also occur
during the second meiotic division. The products of male
meiosis are up to eight microspores with variable DNA
content, some chromatin fragments remain in the cytoplasm and never reach the nuclei (He et al., 1996; Ross
et al., 1997).
It is unclear at this stage which gene has been affected in
the mei1 mutant. He and Mascarenhas (1998) report that
the MEI1 gene is composed of three exons encoding an 89
amino acids protein with weak homology to human
acrosin-trypsin inhibitor (20% identity, 42% similarity).
RT-PCR and RACE-PCR experiments support the existence of this gene transcript (He and Mascarenhas, 1998).
However, a subsequent study suggests that the gene
encodes a larger predicted protein (of between 783 and
842 amino acids) (Yang and McCormick, 2002). This
predicted protein contains three BRCT domains, which are
found in proteins involved in double-strand break repair in
yeast and mouse meiosis (Herrmann et al., 1998; GarcõÂaDõÂaz et al., 2000). However, the issue is complicated by the
fact that the two proteins are predicted to be encoded on
complementary DNA strands at the MEI1 locus.
In yeast and other organisms several genes with
homology to the E. coli mismatch repair genes have
been demonstrated to be essential for the repair of doublestrand breaks induced during meiotic recombination
(Roeder, 1997). Mouse knockouts of mismatch repair
genes also show a range of meiotic phenotypes (Baker
et al., 1995, 1996; Edelmann et al., 1996). The Mlh1
protein, a homologue of the E. coli MutL gene, has been
shown to promote crossing over during meiosis in yeast
(Hunter and Borts, 1997). Arabidopsis possesses an MLH1
homologue encoding a 737 amino acid protein which
shows 56% similarity to the human protein (Jean et al.,
1999). A T-DNA insertion in the promoter of MLH1 has
been identi®ed. However, transcription levels in this
mutant are not affected, as the T-DNA sequences appear
to supply an alternative promoter and the line has no
mutant phenotype (Jean et al., 1999). Arabidopsis has two
other MutL homologues, PMS1 and MLHX (Jean et al.,
1999) as well as homologues of the E. coli MutS gene
termed MSH2, MSH3, MSH6-1, and MSH6-2 (Ade et al.,
1999). No mutants in these gene have been reported,
however work on the Msh2 protein suggests that it is a
functional MutS homologue (Ade et al., 2001).
Mutants affected in chromosome synapsis or
bivalent stability
A central feature of prophase I of meiosis is the juxtaposition and pairing of homologous chromosomes leading to
their intimate synapsis at pachytene (Zickler and Kleckner,
1999). In the majority of eukaryotes synapsis is mediated
by an evolutionarily conserved tripartite proteinaceous
structure, the synaptonemal complex (von Wettstein et al.,
1984; Zickler and Kleckner, 1999). Although, the exact
function of the synaptonemal complex remains to be
established it plays a key role in meiosis.
Several Arabidopsis mutants that show defects in
chromosome synapsis have been described. By using
detailed cytological examination these can be subdivided
into two categories; asynaptic, where chromosome synapsis is never achieved and desynaptic, where chromosome
synapsis occurs but is lost prematurely.
Two synaptic mutants were found as part of a screen for
meiotic mutants amongst the Feldmann collection of TDNA transformed Arabidopsis lines (Feldmann and
Marks, 1987; Ross et al., 1997). Asynaptic1 (asy1) is an
asynaptic mutant that leads to a male and female reducedfertility phenotype. Cytological examination of spread
DAPI stained pollen mother cells (PMCs) and embryo sac
mother cells (EMCs) reveal that asy1 homozygotes exhibit
a failure of homologous chromosome synapsis during
prophase I (Ross et al., 1997; Armstrong and Jones, 2001).
The mutant proceeds normally through premeiotic interphase with unpaired centromeres distributed throughout
the cell and unpaired telomeres closely associated with the
nucleolus. Telomere pairing is initiated in both asy1 and
wild type during leptotene (Armstrong et al., 2001).
Meiosis in the asy1 mutant then diverges from wild type as
there is no compelling evidence of synapsis, hence normal
pachytene cells are not found (Fig. 1b). At late diplotene
most chromosomes are seen to be univalent (Fig. 1e) with a
residual low bivalent frequency of 1.27±1.57 per cell
(Sanchez-Moran et al., 2001; Ross et al., 1997) in PMCs.
The residual chiasmata are predominantly sub-telomeric
(Sanchez-Moran et al., 2001), rather than exhibiting the
normal wild-type distribution. The lack of synapsis in asy1
results in univalent formation and meiotic non-disjunction
during meiosis I (a typical example of the uneven
chromosome distribution seen after meiosis I is shown in
Fig. 1i) and meiosis II giving rise to polyads containing
uneven numbers of chromosomes.
The ASY1 gene has been cloned and encodes a 596
amino acid protein with signi®cant homology to the yeast
Hop1 protein over the ®rst 250 amino acids. This region
comprises a HORMA domain found in a number of
chromatin interacting proteins (Aravind and Koonin,
1998). The remainder of the protein is unique with no
homology to anything else in the database (Caryl et al.,
2000). The ASY1 gene was found to be expressed in all
tissues tested by RT-PCR. Yeast hop1 mutants exhibit
reduced levels of meiotic recombination and extremely
low levels of spore viability (Hollingsworth and Byers,
1989). Hop1 protein accumulates at multiple discrete foci
during prophase I of meiosis as a component of the axial
elements (Hollingsworth et al., 1990; Smith and Roeder,
1997), but is lost by the time full synapsis has been
achieved (Smith and Roeder, 1997) at pachytene. The
Hop1 protein has DNA binding activity, and contains a
Meiotic mutants of Arabidopsis
non-classical zinc ®nger (not seen in Asy1) around amino
acid 371 of the protein (Hollingsworth et al., 1990).
However, DNA binding activity, while enhanced in the
presence of zinc, is still observed even in the presence of
EDTA, suggesting the HORMA domain may also be
involved in DNA binding (Kironmai et al., 1998).
Immunolocalization studies using an antibody raised
against recombinant Asy1 protein have revealed the spatial
and temporal distribution of the protein. Asy1 is ®rst
observed as punctate chromatin associated foci during
meiotic interphase in PMCs. This signal becomes more
continuous during leptotene and zygotene, and is seen to be
associated with axial elements, but not the associated
chromatin loops. The protein begins to disappear from the
chromosomes as they desynapse. By late diplotene some
remaining protein may be observed as aggregates no
longer associated with chromatin (Armstrong et al., 2002).
A more detailed examination of Asy1 localization using
immunogold labelling has revealed that the protein is not
continuous along the axial/lateral elements, but instead is
found at discrete points along the chromatin associated
with these structures (Armstrong et al., 2002). Taken
together the asy1 mutant phenotype and protein localization suggests a potential role for Asy1 in homologous
chromosome juxtaposition during early prophase I with a
failure to complete homologous chromosome pairing
leading to a failure in SC assembly. Alternatively, if
transient or unstable pairing does occur in asy1 the role of
Asy1 may be to recruit the bases of chromatin loops to the
developing axial/lateral elements (Armstrong et al., 2002).
Desynaptic1 (dsy1) is a desynaptic mutant that leads to a
reduction in both male and female fertility. The dsy1
mutant proceeds normally through the early stages of
meiosis achieving apparently normal synapsis at pachytene
(Fig. 1a), however, by early diplotene homologous
chromosomes are seen to be loosely associated rather
than associated into bivalents by chiasmata (Fig. 1d), thus
suggesting a defect in chiasma formation or retention.
Occasional bivalents are, however, observed (frequency
0.75 per cell). A mixture of reductional and equational
division at anaphase I lead to the production of a variable
number of cells containing variable amounts of DNA. The
mutation affects both male and female fertility. However,
the line can be maintained as homozygotes, although a
high level of aneuploidy is seen in the progeny (SJ
Armstrong, personal communication). The gene involved
has not yet been identi®ed, but the mutation maps to
chromosome 3 between GL1 and HY2 (Ross et al., 1997).
Another desynaptic mutant dsy10 (Feldmann line 7219)
is both male and female sterile. The line also exhibits
asynchronous meiotic development both within and
between anthers (Peirson et al., 1996). Meiosis in the
mutant proceeds normally, with full synapsis achieved at
pachytene. However, at diplotene homologous chromosomes appear to separate precociously, giving rise to 10
33
univalents by diakinesis (Cai and Makaroff, 2001).
Occasional bivalents can be observed, usually joined at
the centromeres. Chromosome condensation in the mutant
is also less extensive than in wild type. Subsequent defects
in this mutant vary between cells; some chromosomes fail
to attach to the metaphase I spindle, whilst others attach
and segregation occurs. Chromosome bridges are often
associated with these segregating univalents. Some cells
arrest after meiosis I while others enter meiosis II (Cai and
Makaroff, 2001). At the tetrad stage a variety of cell types
can be observed, tetrads containing a variable number of
uninuclear and polynuclear microspores are observed,
along with cells which arrested after the ®rst meiotic
division. In 10% of cells precocious loss of sister
chromatid cohesion was observed giving rise to more
than 10 univalents at diakinesis, while other chromatids
remained attached only at the centromeres. Although the
primary defect in this mutant appears to be desynapsis, the
variable nature of the later stages may suggest that some
aspect of cell cycle control has been affected (Cai and
Makaroff, 2001)
Chromosome segregation mutants
In another group of meiotic mutants, the primary defect
appears to be in the function or regulation of the meiotic
spindle leading to defects in chromosome segregation.
A mutant with a Ds transposon insertion in the KATA/
ATK1 gene exhibits reduced male fertility (Chen et al.,
2002). Meiosis in PMCs appears normal up to metaphase I
where chromosomes fail to align correctly at the equator of
the cell. At anaphase I individual chromosome pairs may
be precocious or laggard with respect to segregation, with
some failing to separate at all, although some balanced
dyads are generated. Organelle distribution is also more
diffuse in the mutant. Chromatid segregation is also
affected during the second meiotic division, giving rise to
polyads with variable DNA content (Chen et al., 2002).
The meiotic spindle in the mutant appears abnormal
with less tubulin staining than wild type. Also the spindle
is diffuse and unfocused, especially at the poles and
appears less organized during meiosis I. During meiosis II,
minispindles form between the groups of chromosomes
produced in metaphase I (Chen et al., 2002).
The KATA/ATK1 gene has been cloned and appears to
be preferentially expressed in dividing tissues (Mitsui
et al., 1993; Chen et al., 2002). The gene encodes a protein
of 793 amino acids that has homology to kinesin. Kinesin
is a microtubule motor protein and is one of a group of
proteins that have been implicated in a complex range of
processes including spindle formation, position and
chromosome movement (reviewed in Sharp et al., 2000;
Goldstein, 2001). KatA/Atk1 protein was found to localize
to the mid-zone of the mitotic spindle and phragmoplast
(Liu et al., 1996). Recently, KATA was renamed ATK1 to
34 Caryl et al.
avoid confusion with the KAT1 potassium channel gene
(Chen et al., 2002; Schachtman et al., 1994) (although, it
should be noted that this nomenclature has already been
used for a homeodomain protein by Dockx et al., 1995). A
second closely related homologue of KATA/ATK1 is
present in the Arabidopsis genome. This gene, ATK5, is
91% similar to KATA/ATK1 at the protein level and may be
the reason why no mitotic or female meiotic phenotype is
seen in the katA/atk1 mutant (Chen et al., 2002). Two other
kinesin-like genes found in Arabidopsis, KATB and KATC
(Mitsui et al., 1994) also show homology to KATA/ATK1
(57% and 56% amino acid sequence identity, respectively)
(Chen et al., 2002).
Another mutant affecting chromosome segregation is
ask1. The ask1 (SKIP1-LIKE1) mutant isolated from a Ds
transposon insertion line is male sterile, but female fertile
(Yang et al., 1999b). Meiosis in pollen mother cells
appears to proceed normally with chromosome pairing
occurring, however, there is a failure of chromosome
segregation at anaphase I. Some bivalents fail to separate
entirely, with the chromosomes becoming stretched
instead. Meiosis II is also affected with some sister
chromatids failing to separate normally. This leads to the
production of polyads of variable size and chromosome
content (Yang et al., 1999b). Some ask1 mutant cells have
spindles that are longer than wild type, and it is these cells
which exhibit the highest degree of chromosome movement during meiosis I (Yang and Ma, 2001). The ASK1
gene also appears to have a non-meiotic role as homozygous plants display reduced rosette size and internode
length due to a reduction in cell numbers, together with
alterations in ¯oral organ development (Zhao et al., 1999).
The ASK1 gene encodes a protein of 160 amino acids
(Porat et al., 1998) and was found to be a homologue of the
yeast SKP1 gene which is involved in regulating the
mitotic cell cycle by targeting proteins for degradation via
the ubiquitin-mediated proteolysis pathway (Yang et al.,
1999b). The ask1 mutant contains a Ds transposon
insertion towards the middle of the gene and is thought
to be a null mutant (Yang et al., 1999b). The ASK1 gene is
expressed in all tissues tested by Northern blotting, but
maximum expression occurs in meristematic tissues (Porat
et al., 1998). The ASK1 gene is part of a large gene family
of at least nine members in Arabidopsis (Yang et al.,
1999b).
Cell cycle regulation mutants
The phenotypes of another group of meiotic mutants
suggest they may affect cell cycle regulation in some way.
In wild-type Arabidopsis PMC meiosis is synchronized
within the anther locules. The temperature-sensitive tam1
(tardy asynchronous meiosis) mutant (Magnard et al.,
2001) results in PMC meiosis becoming asynchronous
within the anther and was isolated from an EMS
mutagenized Arabidopsis population. Furthermore, dyad
meiotic products or pollen (containing two gametophytes
within one exine wall) are typically produced, apparently
as a result of a slower meiotic division set against wall
formation at the normal rate (i.e. the wall forms before
metaphase II has a chance to occur) (Magnard et al., 2001).
In addition, a range (2±7) of other microspore numbers are
also observed, some of which contain multiple nuclei. In
the quartet1-tam1 double mutant (where the products of
meiosis remain physically linked so they can be directly
followed) meiosis II was found to proceed, but at a slower
pace. The delay appears to be at the G2/M transition in
both meiosis I and meiosis II. Defects may also be
observed in chromosome condensation and segregation.
The TAM1 gene maps to chromosome 1 between the SSLP
marker nF22K20 and CAPS marker agp64 (Magnard et al.,
2001).
Two other mutants exhibiting asynchronous meiotic
development have been identi®ed by Peirson et al. (1996).
One, dsy10 (Cai and Makaroff, 2001), has been described
earlier in this review. In the other line, 7593, a putative TDNA tagged mutant, some microspores arrest prior to
meiosis, while others give rise to abnormal tetrads that
follow several different paths prior to collapse. Abnormal
callose deposition is observed in mutant microspores. The
7593 mutation only affects male fertility (Peirson et al.,
1996).
Another class of cell cycle mutant is represented by ms5
(also known as tdm1 and pollenless3). This was ®rst
identi®ed as an EMS mutant in which microsporocytes
initially appeared normal, but soon degenerated
(Chaudhury et al., 1994). A more detailed examination
of the cytology revealed that the pollen mother cells appear
to undergo two rounds of normal meiotic division.
However, at the end of meiosis II the cell attempts a
third division without any further DNA replication. The
chromosomes recondense and appear to become attached
to a spindle (determined on the basis of organelle
exclusion). The chromosomes then become extended and
stretched (Fig. 1k). Eventually interphase nuclei reform
containing random groups of chromatids (Fig. 1l), giving
rise to approximately eight microspores with variable
DNA content (Ross et al., 1997). Both the EMS ms5-1 and
T-DNA ms5-2/tdm1 alleles are sterile as homozygotes, but
the latter is semi-dominant showing reduced fertility as a
heterozygote under certain environmental conditions
(Ross et al., 1997; Glover et al., 1998; Luong, 1999). A
third allele pollenless3-2 is a deletion isolated from a TDNA transformed population (Sanders et al., 1999).
The MS5 gene encodes a 434 amino acids protein that
has no clear homology with other known proteins.
However, stretches of the protein have limited similarity
to the synaptonemal complex protein SCP1 from rat, and
the regulatory subunit of a cyclin-dependent kinase from
Xenopus (Glover et al., 1998). The ms5-1 allele is reduced
Meiotic mutants of Arabidopsis
to 129 amino acids (similar in size to pollenless 3-2) and
the ms5-2 allele is reduced to 322 amino acids (Glover
et al., 1998; Sanders et al., 1999). MS5 expression is
detected both in meiotic and non meiotic tissues by RTPCR, but RNA in situ hybridization detected MS5
transcripts only in meiotically dividing cells and indicated
that this RNA is lost prior to tetrad formation (Sanders
et al., 1999). Two other genes with homology to MS5
(POLLENLESS3-LIKE1 and POLLENLESS3-LIKE2) are
also present in Arabidopsis (Sanders et al., 1999).
Conclusion
To date, relatively few Arabidopsis meiotic mutants have
been both described and the corresponding genes cloned,
compared to organisms such as Saccharomyces cerevisiae.
The genes that have been identi®ed are, however, involved
in a diverse range of meiotic processes (chromosome
cohesion, recombination, synapsis, chromosome segregation, and cell cycle regulation). The studies described in
this article therefore highlight the value of Arabidopsis as a
model system
The identi®cation of meiotic mutants based on a reverse
genetics approach to identify T-DNA and transposon
tagged derivatives of known meiotic genes from other
species provides an excellent route to move the study of
plant meiosis to a more detailed level. Further application
of this approach will undoubtedly enable the identi®cation
of other meiotic genes in the future, especially as
additional tagged Arabidopsis populations have recently
become available to the research community. Other
reverse genetics techniques such as antisense RNA and
RNAi may also be useful in investigating the function of
meiotic gene homologues from other species when tagged
mutants are not available. However, this is not to say that
screening for reduced fertility mutants has become redundant as some meiotic genes may not exhibit suf®cient
homology at the level of primary amino acid sequence to
be readily identi®ed by homology searches in silico, or
may be unique to meiosis in plants. Moreover, both the
meiotic mutants and corresponding genes can be used in
conjunction with approaches such as transcriptomics,
proteomics and interaction cloning to provide yet another
powerful route towards the isolation of additional plant
meiotic genes.
Using these cloned genes it is possible to investigate the
molecular mechanisms that underpin plant meiosis. This
information may be combined with the detailed cytogenetic data that has been obtained over many decades and
recent additional information such as a timeline for
Arabidopsis meiosis established using BrdU (bromodeoxyuridine) labelling of meiotic S-phase cells (SJ Armstrong,
personal communication). An example of how this
approach may be used to dissect the interrelationship
between different aspects of meiosis may be seen in
35
Armstrong et al. (2001) where telomere pairing in the
asynaptic mutant asy1 was examined. This revealed that
while early telomere pairing occurs normally in the asy1
mutant this does not, however, progress to complete
pairing and synapsis. This clearly implies that early pairing
events are mechanistically distinguishable from pairing
progression and subsequent synapsis.
The application of immunocytology has been used in a
variety of species to investigate the distribution of meiotic
proteins. As mentioned earlier this is beginning to be
applied to meiotic proteins from Arabidopsis. This is an
important tool as it may be used to investigate the
distribution of meiotic proteins on individual chromosomes. Potential co-localization of meiotic proteins may
be used to infer protein±protein interactions that can then
be investigated in more detail. Protein localization can also
be helpful in developing hypotheses about gene function.
GFP fusion proteins such as SWI1-GFP can also be useful
in determining protein localization. They also have the
potential to allow real time visualization of meiosis in
plants.
Acknowledgement
This work was supported by the Biotechnology and
Biological Research Council (BBSRC).
References
Ade J, Belzile F, Philippe H, Doutriaux MP. 1999. Four
mismatch repair paralogues coexist in Arabidopsis thaliana:
AtMSH2, AtMSH3, AtMSH6-1, and AtMSH6-2. Molecular and
General Genetics 262, 239±249.
Ade J, Haffani Y, Beizile FJ. 2001. Functional analysis of the
Arabidopsis thaliana mismatch repair gene MSH2. Genome 44,
651±657.
Agashe B, Prasad CK, Siddiqi I. 2002. Identi®cation and analysis
of DYAD: a gene required for meiotic chromosome organization
and female meiotic progression in Arabidopsis. Development 129,
3935±3943.
Alani E, Subbiah S, Kleckner N. 1989. The yeast RAD50 gene
encodes a predicted 153 kDa protein containing a purine
nucleotide-binding domain and two large heptad-repeat regions.
Genetics 122, 47±57.
Aravind L, Koonin EV. 1998. The HORMA domain: a common
structural denominator in mitotic checkpoints, chromosome
synapsis and DNA repair. Trends in Biochemical Science 23,
284±286.
Armstrong SJ, Caryl AP, Jones GH, Franklin FCH. 2002. Asy1,
a protein required for meiotic chromosome synapsis, localizes to
axis-associated chromatin in Arabidopsis and Brassica. Journal
of Cell Science 115, 3645±3655.
Armstrong SJ, Franklin FCH, Jones GH. 2001. Nucleolusassociated telomere clustering and pairing precede meiotic
chromosome synapsis in Arabidopsis thaliana. Journal of Cell
Science 114, 4207±4217.
Armstrong SJ, Jones GH. 2001. Female meiosis in wild-type
Arabidopsis thaliana and in two meiotic mutants. Sexual Plant
Reproduction 13, 177±183.
Armstrong SJ, Jones GH. 2003. Meiotic cytology and
36 Caryl et al.
chromosome behaviour in wild-type Arabidopsis thaliana.
Journal of Experimental Botany 54, 1±10.
Bai X, Peirson BN, Dong F, Xue C, Makaroff CA. 1999. Isolation
and characterization of SYN1, a RAD21-like gene essential for
meiosis in Arabidopsis. The Plant Cell 11, 417±430.
Baker BS, Carpenter ATC, Esposito MS, Esposito RE, Sandler
L. 1976. The genetic control of meiosis. Annual Review of
Genetics 10, 53±134.
Baker SM, Bronner CE, Zhang L, et al. 1995. Male mice
defective in the DNA mismatch repair gene PMS2 exhibit
abnormal chromosome synapsis in meiosis. Cell 82, 309±319.
Baker SM, Plug AW, Prolla TA, et al. 1996. Involvement of
mouse MLH1 in DNA mis-match repair and meiotic crossing
over. Nature Genetics 13, 336±342.
Bechtold N, Ellis J, Pelletier G. 1993. In planta Agrobacteriummediated gene transfer by in®ltration of adult Arabidopsis
thaliana plants. Compte Rendu hebdomadaire des seÂances de
l'Academie des Sciences 316, 1194±1199.
Bergerat A, de Massy B, Gadelle D, Varoutas P-C, Nicolas A,
Forterre P. 1997. An atypical topoisomerase II from Archaea
with implications for meiotic recombination. Nature 386, 414±
417.
Bertuch A, Lundblad V. 1998. Telomeres and double-strand
breaks: trying to make ends meet. Trends in Cell Biology 8, 339±
342.
Bhatt AM, Lister C, Page T, Fransz P, Findlay K, Jones GH,
Dickinson HG, Dean C. 1999. The DIF1 gene of Arabidopsis is
required for meiotic chromosome segregation and belongs to the
REC8/RAD21 cohesin gene family. The Plant Journal 19, 463±
472.
Bishop DK, Park D, Xu L, Kleckner N. 1992. DMC1: a meiosisspeci®c yeast homolog of E. coli RecA required for
recombination, synaptonemal complex formation, and cell cycle
progression. Cell 69, 439±456.
Bouche N, Bouchez D. 2001. Arabidopsis gene knockout:
phenotypes wanted. Current Opinion in Plant Biology 4, 111±
117.
Bourque JE. 1995. Antisense strategies for genetic manipulations
in plants. Plant Science 105, 125±149.
Cai X, Makaroff CA. 2001. The dsy10 mutation of Arabidopsis
results in desynapsis and a general breakdown in meiosis. Sexual
Plant Reproduction 14, 63±67.
Caryl AP, Armstrong SJ, Jones GH, Franklin FCH. 2000. A
homologue of the yeast HOP1 gene is inactivated in the
Arabidopsis meiotic mutant asy1. Chromosoma 109, 62±71.
Chaudhury AM, Lavithis M, Taylor PE, Craig S, Singh MB,
Signer ER, Knox RB, Dennis ES. 1994. Genetic control of male
fertility in Arabidopsis thaliana: structural analysis of premeiotic
developmental mutants. Sexual Plant Reproduction 7, 17±28.
Chen C, Marcus A, Li W, Hu Y, Calzada J-PV, Grossniklaus U,
Cyr RJ, Ma H. 2002. The Arabidopsis ATK1 gene is required for
spindle morphogenesis in male meiosis. Development 129, 2401±
2409.
Couteau F, Belzile F, Horlow C, Grandjean O, Vezon D,
Doutriaux MP. 1999. Random chromosome segregation without
meiotic arrest in both male and female meiocytes of a dmc1
mutant of Arabidopsis. The Plant Cell 11, 1623±1634.
Davis L, Smith GR. 2001. Meiotic recombination and chromosome
segregation in Schizosaccharomyces pombe. Proceedings of the
National Academy of Sciences, USA 98, 8395±8402.
Dawson J, Wilson ZA, Aarts MGM, Braithwaite AF, Briarty
LG, Mulligan BJ. 1993. Microspore and pollen development in
six male-sterile mutants of Arabidopsis thaliana. Canadian
Journal of Botany 71, 629±638.
Dernburg AF, McDonald K, Moulder G, Barstead R, Dresser
M, Villeneuve AM. 1998. Meiotic recombination in C. elegans
initiates by a conserved mechanism and is dispensable for
homologous chromosome synapsis. Cell 94, 387±398.
Dockx J, Quaedvlieg N, Keultjes G, Kock P, Weisbeek P,
Smeekens S. 1995. The homeobox gene ATK1 of Arabidopsis
thaliana is expressed in the shoot apex of the seedling and in
¯owers and in¯orescence stems of mature plants. Plant
Molecular Biology 28, 723±737.
Dong F, Cai X, Makaroff CA. 2001. Cloning and characterization
of two Arabidopsis genes that belong to the RAD21/REC8 family
of chromosome cohesin proteins. Gene 271, 99±108.
Doutriaux MP, Couteau F, Bergounioux C, White C. 1998.
Isolation and characterization of the RAD51 and DMC1 homologs
from Arabidopsis thaliana. Molecular and General Genetics 257,
283±291.
Dresser ME. 2000. Meiotic chromosome behavior in
Saccharomyces cerevisiae and (mostly) mammals. Mutation
Research 451, 107±127.
Edelmann W, Cohen PE, Kane M, et al. 1996. Meiotic pachytene
arrest in MLH1-de®cient mice. Cell 85, 1125±1134.
Feldmann KA, Marks MD. 1987. Agrobacterium-mediated
transformation of germinating seeds of Arabidopsis thaliana: a
non-tissue culture approach. Molecular and General Genetics
208, 1±9.
Gallego ME, White CI. 2001. RAD50 function is essential for
telomere maintenance in Arabidopsis. Proceedings of the
National Academy of Sciences, USA 98, 1711±1716.
Gallego ME, Jeanneau M, Granier F, Bouchez D, Bechtold N,
White CI. 2001. Disruption of the Arabidopsis RAD50 gene leads
to plant sterility and MMS sensitivity. The Plant Journal 25, 31±
41.
GarcõÂa-DõÂaz M, DomõÂnguez O, LoÂpez-FernaÂndez LA, et al. 2000.
DNA polymerase lambda (Pol lambda), a novel eukaryotic DNA
polymerase with a potential role in meiosis. Journal of Molecular
Biology 301, 851±867.
Gartner A, Milstein S, Ahmed S, Hodgkin J, Hengartner MO.
2000. A conserved checkpoint pathway mediates DNA damage±
induced apoptosis and cell cycle arrest in C. elegans. Molecular
Cell 5, 435±443.
Gherbi H, Gallego ME, Jalut N, Lucht JM, Hohn B, White CI.
2001. Homologous recombination in planta is stimulated in the
absence of Rad50. EMBO Reports 2, 287±291.
Giroux CN, Dresser ME, Tiano HF. 1989. Genetic control of
chromosome synapsis in yeast meiosis. Genome 31, 88±94.
Glover J, BloÈmer KC, Farrell LB, Chaudhury AM, Dennis ES.
1996. Searching for tagged male-sterile mutants of Arabidopsis.
Plant Molecular Biology Reporter 14, 330±342.
Glover J, Grelon M, Craig S, Chaudhury A, Dennis E. 1998.
Cloning and characterization of MS5 from Arabidopsis: a gene
critical in male meiosis. The Plant Journal 15, 345±356.
Goldstein LSB. 2001. Molecular motors: from one motor many
tails to one motor many tales. Trends in Cell Biology 11, 477±
482.
Golubovskaya IN. 1979. Genetic control of meiosis. International
Review of Cytology 58, 247±290.
Gottlieb S, Wagstaff J, Esposito RE. 1989. Evidence for two
pathways of meiotic intrachromosomal recombination in yeast.
Proceedings of the National Academy of Sciences, USA 86, 7072±
7076.
Grelon M, Vezon D, Gendrot G, Pelletier G. 2001. AtSPO11-1 is
necessary for ef®cient meiotic recombination in plants. EMBO
Journal 20, 589±600.
Habu T, Taki T, West A, Nishimune Y, Morita T. 1996. The
mouse and human homologs of DMC1, the yeast meiosis-speci®c
homologous recombination gene, have a common unique form of
exon-skipped transcript in meiosis. Nucleic Acids Research 24,
470±477.
Meiotic mutants of Arabidopsis
Hannon GJ. 2002. RNA interference. Nature 418, 244±251.
Hartung F, Puchta H. 2000. Molecular characterization of two
paralogous SPO11 homologues in Arabidopsis thaliana. Nucleic
Acids Research 28, 1548±1554.
Hartung F, Puchta H. 2001. Molecular characterization of
homologues of both subunits A (SPO11) and B of the
archaebacterial topoisomerase 6 in plants. Gene 271, 81±86.
He C, Tirlapur U, Cresti M, Peja M, Crone DE, Mascarenhas
JP. 1996. An Arabidopsis mutant showing aberrations in male
meiosis. Sexual Plant Reproduction 9, 54±57.
He C, Mascarenhas JP. 1998. MEI1, an Arabidopsis gene required
for male meiosis: isolation and characterization. Sexual Plant
Reproduction 11, 199±207.
Herrmann G, Lindahl T, SchaÈr P. 1998. Saccharomyces
cerevisiae LIF1: a function involved in DNA double-strand
break repair related to mammalian XRCC4. EMBO Journal 17,
4188±4198.
Heyting C. 1996. Synaptonemal complexes: structure and function.
Current Opinion in Cell Biology 8, 389±396.
Hollingsworth NM, Byers B. 1989. HOP1: a yeast meiotic pairing
gene. Genetics 121, 445±462.
Hollingsworth NM, Goetsch L, Byers B. 1990. The HOP1 gene
encodes a meiosis-speci®c component of yeast chromosomes.
Cell 61, 73±84.
Hunter N, Borts RH. 1997. Mlh1 is unique among mismatch repair
proteins in its ability to promote crossing-over during meiosis.
Genes and Development 11, 1573±1582.
Jean M, Pelletier J, Hilpert M, Belzile F, Kunze R. 1999.
Isolation and characterization of AtMLH1, a MutL homologue
from Arabidopsis thaliana. Molecular and General Genetics 262,
633±642.
Kaul MLH, Murthy TGK. 1985. Mutant genes affecting higher
plant meiosis. Theoretical and Applied Genetics 70, 449±466.
Keeney S, Giroux CN, Kleckner N. 1997. Meiosis-speci®c DNA
double-strand breaks are catalyzed by Spo11, a member of a
widely conserved protein family. Cell 88, 375±384.
Kironmai KM, Muniyappa K, Freidman DB, Hollingsworth
NM, Byers B. 1998. DNA-binding activities of Hop1 protein, a
synaptonemal complex component from Saccharomyces
cerevisiae. Molecular and Cell Biology 18, 1424±1435.
Klimyuk VI, Jones JDG. 1997. AtDMC1, the Arabidopsis
homologue of the yeast DMC1 gene: characterization,
transposon-induced allelic variation and meiosis-associated
expression. The Plant Journal 11, 1±14.
Koduru PRK, Rao MK. 1981. Cytogenetics of synaptic mutants in
higher plants. Theoretical and Applied Genetics 59, 197±214.
Lee JY, Orr-Weaver TL. 2001. The molecular basis of sisterchromatid cohesion. Annual Review of Cell and Developmental
Biology 17, 753±777.
Liu B, Cyr RJ, Palevitz BA. 1996. A kinesin-like protein, KatAp,
in the cells of Arabidopsis and other plants. The Plant Cell 8,
119±132.
Luong QT. 1999. Molecular characterization of a `three-division'
mutant of Arabidopsis thaliana. PhD thesis, University of
Birmingham, UK, 42±51.
Magnard J-L, Yang M, Chen Y-CS, Leary M, McCormick S.
2001. The Arabidopsis gene TARDY ASYNCHRONOUS
MEIOSIS is required for the normal pace and synchrony of cell
division during male meiosis. Plant Physiology 127, 1157±1166.
Masson JE, Paszkowski J. 1997. Arabidopsis thaliana mutants
altered in homologous recombination. Proceedings of the
National Academy of Sciences, USA 94, 11731±11735.
McCallum CM, Comai L, Greene EA, Henikoff S. 2000.
Targeted
screening
for
induced
mutations.
Nature
Biotechnology 18, 455±457.
McKim KS, Green-Marroquin BL, Sekelsky JJ, Chin G,
37
Steinberg C, Khodosh R, Hawley RS. 1998. Meiotic synapsis
in the absence of recombination. Science 279, 876±878.
McKim KS, Hayashi-Hagihara A. 1998. mei-W68 in Drosophila
melanogaster encodes a SPO11 homolog: evidence that the
mechanism for initiating meiotic recombination is conserved.
Genes and Development 12, 2932±2942.
Mengiste T, Paszkowski J. 1999. Prospects for the precise
engineering of plant genomes by homologous recombination.
Biological Chemistry 380, 749±758.
Mercier R, Grelon G, Vezon D, Horlow C, Pelletier G. 2001b.
How to characterize meiotic functions in plants? Biochimie 83,
1023±1028.
Mercier R, Vezon D, Bullier E, Motamayor JC, Sellier A,
LefeÁvre F, Pelletier G, Horlow C. 2001a. Switch1 (Swi1): a
novel protein required for the establishment of sister chromatid
cohesion and for bivalent formation at meiosis. Genes and
Development 15, 1859±1871.
Mitsui H, Yamaguchi-Shinozaki K, Shinozaki K, Nishikawa K,
Takahashi H. 1993. Identi®cation of a gene family (KAT)
encoding kinesin-like proteins in Arabidopsis thaliana and the
characterization of secondary structure of KatA. Molecular and
General Genetics 238, 362±268.
Mitsui H, Nakatani K, Yamaguchi-Shinozaki K, Shinozaki K,
Nishikawa K, Takahashi H. 1994. Sequencing and
characterization of the kinesin-related genes KATB and KATC
of Arabidopsis thaliana. Plant Molecular Biology 25, 865±876.
Molnar M, Bahler J, Sipiczki M, Kohli J. 1995. The REC8 gene
of Schizosaccharomyces pombe is involved in linear element
formation, chromosome pairing and sister-chromatid cohesion
during meiosis. Genetics 141, 61±73.
Morita T, Yoshimura Y, Yamamoto A, Murata K, Mori M,
Yamamoto H, Matsushiro A. 1993. A mouse homolog of the
Escherichia coli RecA and Saccharomyces cerevisiae RAD51
genes. Proceedings of the National Academy of Sciences, USA 90,
6577±6580.
Motamayor JC, Vezon D, Bajon C, Sauvanet A, Grandjean O,
Marchand M, Bechtold N, Pelletier G, Horlow C. 2000. Switch
(swi1), an Arabidopsis thaliana mutant affected in the female
meiotic switch. Sexual Plant Reproduction 12, 209±218.
Parinov S, Sevugan M, De Y, Yang W-C, Kumaran M,
Sundaresan V. 1999. Analysis of ¯anking sequences from
Dissociation insertion lines: a database for reverse genetics in
Arabidopsis. The Plant Cell 11, 2263±2270.
Peirson BN, Owen HA, Feldmann KA, Makaroff CA. 1996.
Characterization of three male-sterile mutants of Arabidopsis
thaliana exhibiting alterations in meiosis. Sexual Plant
Reproduction 9, 1±16.
Peirson BN, Bowling SE, Makaroff CA. 1997. A defect in
synapsis causes male sterility in a T-DNA-tagged Arabidopsis
thaliana mutant. The Plant Journal 11, 659±669.
Porat R, Lu P, O'Neill SD. 1998. Arabidopsis SKP1, a homologue
of a cell cycle regulator gene, is predominantly expressed in
meristematic cells. Planta 204, 345±351.
Roeder GS. 1997. Meiotic chromosomes: it takes two to tango.
Genes and Development 11, 2600±2621.
Roeder GS, Bailis JM. 2000. The pachytene checkpoint. Trends in
Genetics 16, 395±403.
Romanienko PJ, Camerini-Otero RD. 2000. The mouse SPO11
gene is required for meiotic chromosome synapsis. Molecular
Cell 6, 975±987.
Ross KJ, Fransz P, Jones GH. 1996. A light microscope atlas of
meiosis in Arabidopsis thaliana. Chromosome Research 4, 507±
516.
Ross KJ, Fransz P, Armstrong SJ, Vizir I, Mulligan B, Franklin
FCH, Jones GH. 1997. Cytological characterization of four
38 Caryl et al.
meiotic mutants of Arabidopsis isolated from T-DNAtransformed lines. Chromosome Research 5, 551±559.
Sanchez-Moran E, Armstrong SJ, Santos JL, Franklin FCH,
Jones GH. 2001. Chiasma formation in Arabidopsis thaliana
accession Wassileskija and in two meiotic mutants. Chromosome
Research 9, 121±128.
Sanders PM, Bui AQ, Weterings K, McIntire KN, Hsu Y-C, Lee
PY, Truong MT, Beals TP, Goldberg RB. 1999. Anther
developmental defects in Arabidopsis thaliana male-sterile
mutants. Sexual Plant Reproduction 11, 297±322.
Sato S, Hotta Y, Tabata S. 1995. Structural analysis of a RecA-like
gene in the genome of Arabidopsis thaliana. DNA Research 2,
89±93.
Schachtman DP, Schroeder JI, Lucas WJ, Anderson JA, Gaber
RF. 1994. Expression of an inward-rectifying potassium channel
by the Arabidopsis KAT1 cDNA. Science 258, 1654±1658.
Sharp DJ, Rogers GC, Scholey JM. 2000. Microtubule motors in
mitosis. Nature 407, 41±47.
Shinohara A, Ogawa H, Ogawa T. 1992. Rad51 protein involved
in repair and recombination in S. cerevisiae is a RecA-like
protein. Cell 69, 457±470. (Erratum in Cell 1992 71, following
page 180.)
Shinohara A, Ogawa H, Matsuda Y, Ushio N, Ikeo K, Ogawa T.
1993. Cloning of human, mouse and ®ssion yeast recombination
genes homologous to RAD51 and RecA. Nature Genetics 4, 239±
243. (Erratum in Nature Genetics 1993 5, 312.)
Siddiqi I, Ganesh G, Grossniklaus U, Subbiah V. 2000. The
DYAD gene is required for progression through female meiosis in
Arabidopsis. Development 127, 197±207.
Smith AV, Roeder GS. 1997. The yeast Red1 protein localises to
the cores of meiotic chromosomes. Journal of Cell Biology 136,
957±967.
Sung P, Trujillo KM, Van Komen S. 2000. Recombination factors
of Saccharomyces cerevisiae. Mutation Research 451, 257±275.
The Arabidopsis Genome Initiative. 2000. Analysis of the
genome sequence of the ¯owering plant Arabidopsis thaliana.
Nature 408, 796±815.
Tissier AF, Marillonnet S, Klimyuk V, Patel K, Torres MA,
Murphy G, Jones JGD. 1999. Multiple independent defective
suppressor-mutator transposon insertions in Arabidopsis: a tool
for functional genomics. The Plant Cell 11, 1841±1852.
van Heemst D, Heyting C. 2000. Sister chromatid cohesion and
recombination in meiosis. Chromosoma 109, 10±26.
von Wettstein D, Rasmussen SW, Holm PB. 1984. The
synaptonemal complex in genetic segregation. Annual Review
of Genetics 18, 331±413.
Wang M-B, Waterhouse PM. 2001. Application of gene silencing
in plants. Current Opinion in Plant Biology 5, 146±150.
Watanabe Y, Nurse P. 1999. Cohesin Rec8 is required for
reductional chromosome segregation at meiosis. Nature 400,
461±464.
Yang M, Hu Y, Lodhi M, McCombie WR, Ma H. 1999b. The
Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and
may control homologue separation. Proceedings of the National
Academy of Sciences, USA 96, 11416±11421.
Yang M, Ma H. 2001. Male meiotic spindle lengths in normal and
mutant Arabidopsis cells. Plant Physiology 126, 622±630.
Yang M, McCormick S. 2002. The Arabidopsis MEI1 gene likely
encodes a protein with BRCT domains. Sexual Plant
Reproduction 14, 355±357.
Yang W-C, Ye D, Xu J, Sundaresan V. 1999a. The
SPOROCYTELESS gene of Arabidopsis is required for initiation
of sporogenesis and encodes a novel nuclear protein. Genes and
Development 13, 2108±2117.
Zhao D, Yang M, Solva J, Ma H. 1999. The ASK1 gene regulates
development and interacts with the UFO gene to control ¯oral
organ identity in Arabidopsis. Developmental Genetics 25, 209±
223.
Zickler D, Kleckner N. 1999. Meiotic chromosomes: Integrating
structure and function. Annual Review of Genetics 33, 603±754.