Chromosome Research 11: 205^215, 2003. # 2003 Kluwer Academic Publishers. Printed in the Netherlands
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Meiotic chromosome synapsis and recombination in Arabidopsis thaliana; an integration of cytological and molecular approaches
G. H. Jones, S. J. Armstrong, A. P. Caryl & F. C. H. Franklin School of Biosciences, The University of Birmingham, Birmingham, B15 2TT, UK; Tel: (0)121 414 5918; Fax: (0)121 414 5925; E-mail:
[email protected]
Key words: Arabidopsis, chromosomes, meiosis, recombination, synapsis
Abstract Arabidopsis has emerged as an important model for the analysis of meiosis in Angiosperm plants, creating an interesting and useful parallel to other model organisms. This development has been underpinned by advances in the molecular biology and genetics of Arabidopsis, especially the determination of its entire genome sequence. However, these advances alone would have been insuf¢cient without the development of improved methods for cytological analysis and cytogenetic investigation of meiotic nuclei and chromosomes. A basic descriptive framework of meiosis in Arabidopsis has been established based on these procedures. In addition, molecular cytogenetic and immunocytological techniques have provided supplementary detailed information on some aspects of meiosis. Gene identi¢cation and characterization have proceeded in parallel with these developments based on both forward and reverse genetic procedures utilising the considerable range of Arabidopsis genetic and molecular resources, such as T-DNA and transposon tagged lines as well as the genomic DNA database, in combination with cytological analysis. A diverse range of meiotic genes have been identi¢ed and analysed by these procedures and in selected cases they have been subjected to detailed functional analysis. This review focuses on genes that are involved in the key meiotic events of chromosome synapsis and recombination.
Introduction The last 15 years have witnessed a dramatic acceleration of meiosis research, stimulated largely by progress in the genetic and molecular dissection of meiosis in the budding yeast, Saccharomyces cerevisiae (Roeder 1997, Zickler & Kleckner 1998, 1999). The low amenability of yeast to conventional cytological analysis has been partially overcome by the application of molecular cytogenetic procedures such as immunolocalization and £uorescence in-situ hybridization
(FISH) and the development of ultrastructural methods for investigating synaptonemal complexes. However, it has been pointed out that yeast is a highly specialized unicellular micro-organism, possessing a tiny genome, and its meiotic processes may not therefore be entirely appropriate models for ‘higher’ eukaryotes with much larger and more complex genomes (Loidl 2000). Parallel studies of meiosis in animal model organisms such as Caenorhabditis elegans, Drosophila melanogaster and mouse are aimed, in part at least, at this question. Although meiosis has been extensively
206 investigated in higher plants for over a century, including the isolation and description of many meiotic mutants, until recently, the resources have not been available for an integration of cytological, genetic and molecular approaches to the analysis of plant meiosis. The adoption of Arabidopsis as a model plant system for molecular biology and genetics, the sequencing of its genome and improved cytogenetic and immunocytological methods, have created a situation where this is now a realistic possibility and there is widespread interest in exploiting Arabidopsis as a model species for meiosis research (Bhatt et al. 2001, Mercier et al. 2001a, Caryl et al. 2003). Apart from the inherent interest of understanding meiosis and its regulation in a plant system, and the comparative aspects referred to earlier, there are considerable potential practical applications of this knowledge in terms of improvements to plant production and quality. This review will focus on recent work that is contributing to a better understanding of the key meiotic events involved in homologous chromosome synapsis and recombination in Arabidopsis.
Arabidopsis resources for meiosis research An obvious, but important requirement for meiosis research is ready non-seasonal availability of meiotic cells. Arabidopsis has a short life-cycle and £owering plants can be produced on demand at any time of the year, in 6^8 weeks from sowing. Floral development in Arabidopsis has been analysed in detail, based on the morphometric characteristics of £ower buds and their component parts, and has been divided into 12 stages (Smyth et al. 1990). In order to facilitate the rapid and e⁄cient preparation of meiocytes for cytogenetic or molecular analysis, £oral stage, as de¢ned by Smyth et al. (1990) has been linked with morphometric data, developmental landmarks and meiotic stage (Armstrong & Jones 2001, Armstrong et al. 2001). Mitotic metaphase chromosomes of Arabidopsis are notoriously small, although they are not unique or exceptional in this respect, and consequently cytogenetic analysis has been regarded as challenging. The development of improved tech-
G. H. Jones et al. niques, based on spreading and DAPI staining (Figure 1a^f), has meant that the cytogenetic analysis of Arabidopsis meiosis, both wild-type and mutant, is now routine (Ross et al. 1996, 1997, Mercier et al. 2001b). In addition, the application of molecular cytogenetic techniques, such as FISH, immunocytology and GFP-fusion, have greatly extended the scope for the analysis of normal and defective meiosis (Figure 1g^l). Electron microscopic techniques are also available or are being developed for analysing ultrastructural aspects of chromosome synapsis and recombination in Arabidopsis and also for immunogold localization of meiotic proteins (Albini 1994, Armstrong et al. 2002). Brassica oleracea, an important horticultural crop plant species with a⁄nities to agricultural crops such as B. napus (oil seed rape) is a close relative of Arabidopsis within the family Cruciferae. In addition to providing a potential outlet for transfer of knowledge from Arabidopsis to crop plants, the close a⁄nity and high level of genome homology and synteny between Arabidopsis and Brassica permits the functional analysis of Arabidopsis genes in the cytologically more amenable Brassica system (Armstrong et al. 2002). Both forward and reverse genetic approaches have been employed successfully to isolate and characterize meiotic genes from Arabidopsis. In the forward approach, populations of T-DNA transformed lines, such as the Feldmann (Feldmann & Marks 1987) and INRA-Versailles (Bechtold et al. 1993) collections, were screened for meiotic mutants, initially on the basis of reduced fertility. Con¢rmation of meiotic defects was established by cytogenetic analysis. Subsequently the mutated genes were cloned and characterized using one of several procedures that exploit the tagging T-DNA inserts. This procedure has been very productive and has resulted in the characterization of several meiotic genes. The reverse genetic approach (gene sequence to phenotype) depends on identifying homologues of known meiotic genes from other organisms (principally yeast) by screening the Arabidopsis genomic DNA database using one of the currently available search packages. Arabidopsis homologues can then be cloned and further investigated by functional analysis and also checked for the
Meiosis in Arabidopsis existence of a meiotic mutant phenotype using a PCR procedure to identify potential gene knockouts from pools of T-DNA or transposon tagged lines. In general, the reverse genetic approach has proved to be relatively useful for identifying genes that encode proteins catalysing steps in homologous recombination. These are genes that are expected to be relatively well conserved since the basic biochemistry of recombination is likely to be similar across a wide phylogenetic range. However, this approach has to date been less successful in identifying other genes such as those involved in chromosome morphogenesis. This appears to be because the proteins involved in these processes, while sharing functional and structural homology, are quite divergent at the level of primary sequence. This point is discussed in more detail later. In addition, a further complication in assigning genes on the basis of homology arises from the ¢nding that the Arabidopsis genome has undergone an ancient duplication (The Arabidopsis Genome Initiative 2000). This has resulted in duplication of about 60% of the Arabidopsis genome and, in many cases, the duplicated genes have undergone functional divergence. The context Meiosis in Arabidopsis, as in Angiosperms generally, is organized rather di¡erently in male and female organs and the fates of the meiotic products are also divergent. In anthers, sporogenous cells proliferate by asynchronous mitotic divisions until the appropriate number of presumptive meiocytes is achieved where they arrest in a G0 state. In response to an appropriate signal, the meiocytes (pollen mother cells) enter meiosis in synchrony and proceed through meiosis more or less synchronously. In Arabidopsis, the number of pollen mother cells per anther is approximately 50, though this may vary depending on the accession. Upon completion of meiosis all the haploid cell products develop into pollen grains, the male gametophytes. In ovules, archesporial cells give rise to single embryo sac mother cells which undergo meiosis to give four haploid cells, three of which degenerate leaving one to develop into the embryo sac, the female gametophyte.
207 Initiation of meiosis in yeast is a complex process requiring the convergence of a starvation signal (low N2, low glucose) and a genetic signal involving several regulatory genes (Kassir & Simchen 1989). In contrast, relatively little is known regarding the initiation of meiosis in plants. The existence of a mechanism to switch cells from a mitotic to a meiotic developmental pathway is con¢rmed by the observation that explantation of lily anthers into culture medium before late S/G2 results in reversion of the pollen mother cells to mitotic division (Ito & Takegami 1982). The involvement of a genetic component in this switch is suggested by the phenotype of the Zea mays mutant ameiotic that fails to enter meiosis and instead undergoes a mitosis-like division (Golubovskaya et al. 1997). A similar defect has been described in the Arabidopsis mutants swi1-1 and swi1-2, but con¢ned to female meiosis (Motamayor et al. 2000).
Chromosome synapsis and recombination According to classical descriptions of meiosis, the synapsis of homologous chromosomes and their subsequent desynapsis occur within prophase I, commencing in zygotene and ending in diplotene. This may be preceded by presynaptic homologue alignment (pairing), the extent and timing of which varies among di¡erent species (Loidl 1990, Schwarzacher 1997, Scherthan et al. 1998). During leptotene of prophase I, each chromosome develops a dense proteinaceous axis or core, referred to as the axial element or axial core. Typically, in most organisms, including Arabidopsis, the axial elements are preformed along the entire length of each chromosome before synapsis commences. However, the precise onset of leptotene is di⁄cult to determine since the earliest emergence of axial elements is very gradual and appears to coincide with considerable chromatin reorganization into linear structures. Labelling of the Arabidopsis meiotic S-phase with BrdU, and detection with anti-BrdU antibody, has permitted tracing the progress of meiocytes from interphase into and through prophase I. The time taken for cells to progress from the end of S-phase to leptotene, with fully formed axial elements, is between 10 and 14 h at 20 C (Armstrong and Jones
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Meiosis in Arabidopsis 2003 and unpublished observations). Fully formed pachytene synaptonemal complexes (SCs) have been observed by EM in Arabidopsis (Albini 1994; Figure 2), but the initiation and progression of synapsis during zygotene have not yet been characterized for this species. In other species, there is a distinct tendency for chromosome synapsis to commence at or near chromosome ends and to proceed to more internal chromosome regions, often involving several interstitial initiations (Loidl 1990). Detailed ultrastructural investigations of these early synaptic events, in several di¡erent plant species, have revealed that multiple axis convergences occur and that distinctive structures, so-called early nodules (ENs), are present at each convergence (Albini & Jones 1987, Anderson & Stack 1988, Anderson et al. 2001). ENs persist in the newly assembled SCs for the duration of zygotene and sometimes into very early pachytene. It is currently postulated that these ENs contain packages of proteins required to carry out early steps in recombination, resulting in the establishment of recombination intermediates, which also have a direct role in the initiation of homologue synapsis (Zickler & Kleckner 1999). The initiating event in this process is the introduction of a double-strand break (DSB) into the DNA of one homologue, followed by resection of one strand to give a single-stranded tail capable of invading the DNA duplex of the other homologue. It was initially thought that all these intermediates developed into complex joint molecules, termed double Holliday junctions (DHJs), that could resolve into reciprocal crossovers or non-crossovers. According to this view, ENs resolved as non-crossovers disappear at the zygotene-pachytene transition while a subset of intermediates
209 destined to become crossovers are marked by persistent late nodules (LNs). However, the latest evidence from yeast suggests that there may be two di¡erent pathways, with only a few recombination intermediates developing via DHJs into crossovers, while the majority of interactions develop by a di¡erent pathway into non-crossovers (Allers & Lichten 2001). The pattern of early synapsis, involving terminal or subterminal chromosome regions, is thought to be promoted by the presynaptic clustering of telomeres, and the pairing of homologous telomeres, on a small region of the inner nuclear membrane, forming the so-called bouquet con¢guration. FISH analysis of Arabidopsis telomeres during meiosis has revealed an interesting variation of this behaviour (Figure 1g^i; Armstrong et al. 2001). During early meiotic interphase, including S-phase, the unpaired telomeres are closely associated with the nucleolus. During G2/early leptotene the telomeres, still associated with the nucleolus, progressively associate in pairs and FISH evidence using subtelomeric markers clearly indicates that pairing involves homologous chromosomes. As leptotene progresses the paired telomeres dissociate from the nucleolus and become widely dispersed around the nuclear periphery. During zygotene, the telomeres show a loose clustering within one hemisphere of the nucleus, which may represent a degenerate or relic bouquet. It is proposed that, in Arabidopsis, the classical leptotene/zygotene bouquet is replaced functionally by nucleolus-associated telomere clustering. There is no evidence of preleptotene centromere pairing in Arabidopsis, although some association of homologous centromere regions has been noted in somatic nuclei (Fransz et al. 2002).
3 Figure 1. Illustrations of meiotic stages, FISH and antibody Immunolocalization in meiotic nuclei of Arabidopsis and Brassica. (a^f) DAPI-stained spreads of meiotic stages in pollen mother cells of wild-type and mutant Arabidopsis thaliana; (a^c) examples of wild-type pachytene (a), diakinesis (b) and metaphase II (c); (e^f) give examples of corresponding stages in the asy1 mutant showing absence of synapsis in prophase I (d), presence of univalents rather than bivalents at diakinesis (e) and irregular chromosome distribution to the metaphase II groups (f). (g^i) FISH of telomere (red) and pericentric heterochromatin (green) DNA sequences to meiotic chromosomes of Arabidopsis; the telomeres are clustered around the nucleolus and partially paired (12 telomere signals visible) in meiotic interphase (g), but the centromeres are unpaired and widely scattered at this stage; at pachytene (h) the centromeres and telomeres are fully synapsed and dispersed through the nucleus; at metaphase I (i) the centomeres and telomeres show characteristic orientations in the ¢ve condensed bivalents. (j^l) Immunolocalization of Asy1 protein in meiotic chromosomes of Brassica oleracea using an Arabidopsis Asy1 antibody; in meiotic interphase/early leptotene (j), the antibody shows a punctate distribution over chromatin which becomes continuous along chromosome axes of synaptonemal complexes by pachytene (k) and eventually becomes fragmented and discarded from the chromosomes in diplotene (l). Bars ¼ 10 mm.
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Figure 2. Electron micrograph of a spread and silver-stained Arabidopsis pollen mother cell at pachytene of meiosis. The ¢ve chromosome pairs are fully synapsed, with the appearance of typical tripartite synaptonemal complexes. Two of the synaptonemal complexes, corresponding to chromosome pairs 2 and 4, are associated with a prominent nucleolus (n). Bar ¼ 2 mm.
Mutants defective in synapsis and/or recombination In classical cytogenetics, mutants with defects in chromosome association at late prophase I/ metaphase I were classi¢ed as asynaptic or desynaptic. Asynaptics exhibit a primary failure of homologous chromosome synapsis whereas, in desynaptics, initial synapsis occurs normally but the chromosomes subsequently undergo precocious desynapsis either due to an absence of crossovers (chiasmata) or a defect in sister chromatid cohesion. This distinction originally made sense since it was commonly believed that synapsis preceded recombination. In the light of our current understanding of early meiotic events, the distinction is less logical since it is now realised that defects in the establishment of recombination intermediates may have direct e¡ects on both synapsis and recombination. Nevertheless, with this reservation, it remains a useful practical distinction. Many synaptic mutants have been described cytogenetically in a variety of plant species (Kaul & Murthy 1985) but without the possibility of characterizing the genes and their mode of action at the molecular level. A number of mutants with
an asynaptic phenotype have now been identi¢ed in Arabidopsis and their molecular characterization gives an insight into some aspects of chromosome synapsis. These mutants and their corresponding wild-type genes fall into two distinct groups. The ¢rst group of genes is involved in some aspects of chromosome morphogenesis with a role in synapsis, either in the establishment of axial elements or other SC components, or in the interaction of these structures and associated proteins with chromatin. The second group is involved in the establishment of recombination intermediates that constitute the initial homologous chromosome interactions required for synapsis.
Genes encoding SC proteins or proteins involved in synapsis-associated chromosome morphogenesis Obvious candidates in this category are genes encoding protein components of the synaptonemal complex. Several such proteins have been identi¢ed from mammals and also from yeast, including components of the axial/lateral elements and the central element (Heyting 1996, Zickler & Kleckner
Meiosis in Arabidopsis 1999). As expected, knockout mutants of some of these genes in mouse and in yeast have asynaptic phenotypes (Yuan et al. 2000, Sym et al. 1993). Searching for Arabidopsis homologues to the major SC protein genes that have been identi¢ed and characterized in mammals and yeast has proved relatively unproductive. This is as expected given the low degree of evolutionary conservation that typi¢es this group of genes and proteins. Nevertheless, putative homologues of genes encoding the central element protein Zip1, showing low levels of identity/similarity to the yeast ZIP1 gene, have been tentatively identi¢ed (Mercier et al. 2001a; N. Jackson et al. unpublished data). Because of this low degree of conservation, insertional mutagenesis may well prove to be the best approach to identifying Arabidopsis genes encoding SC components or other proteins having synapsis-related chromosome morphogenesis roles. Two Arabidopsis genes (SWI1, ASY1) that have partially or completely asynaptic mutant phenotypes, but are not directly involved in the recombination pathway, have been identi¢ed by this route. The Arabidopsis ASY1 gene was identi¢ed by screening the Feldmann collection of T-DNA transformed lines for reduced fertility phenotypes, followed by cytogenetic analysis and cloning and characterization of the gene (Ross et al. 1997, Caryl et al. 2000). The asy1 mutant shows greatly reduced fertility and extensive disruption of meiosis due to a complete failure of chromosome synapsis (Figure 1d^f), despite having apparently normal axial element formation and exhibiting the wildtype pattern of telomere clustering and pairing (Armstrong et al. 2001). The amino-terminus region of the Asy1 protein shows 28% identity and 51% similarity to the Hop1 protein that is required for chromosome synapsis in yeast. This 250-aminoacid region comprises a so-called HORMA domain that is present in several chromatin-associated proteins. However, there is no detectable homology to the remainder of the Hop1 protein, which is known to contain sequences essential for the function of the yeast protein. Immunolocalization of the Asy1 protein to Arabidopsis and Brassica meiocytes (Figure 1j^l) indicates that it is associated with chromatin that is in close proximity to the axial elements and to the lateral elements of SCs, but is not a structural
211 component of axial/lateral elements (Armstrong et al. 2002). Nevertheless, it is clearly required for synapsis and its role might be to recruit the bases of chromatin loops to the developing axial elements, or some other chromatin/axial element interaction. Mutants of the SWI1 gene of Arabidopsis have interesting di¡erential e¡ects on male and female meiosis, the details of which di¡er in an allelespeci¢c manner. The swi1-1 allele has no apparent e¡ect on male meiosis but exhibits a defect in female meiosis that has been interpreted as replacement of meiosis by mitosis-like divisions (Motamayor et al. 2000). Female meiosis is similarly a¡ected in the independently isolated swi1-2 mutant but it also exhibits unique defects in male meiosis, not seen in the female meiocytes. Chromosome synapsis is totally suppressed in male meiosis of the swi1-2 mutant; hence its classi¢cation as an asynaptic mutant. Typical early-midprophase I stages are not seen; instead, the chromatin assumes a more di¡use appearance and there is a complete failure to develop typical meiotic axial elements. Eventually, the chromosomes condense to give discrete univalent structures, and eventually 20 separate chromatids, but their appearance is more mitotic than meiotic (Mercier et al. 2001b). It is proposed that the fundamental defect in this mutant is the failure to develop axial elements, and associated chromatin reorganization, which lead in turn to asynapsis and lack of chromatid arm and centromere cohesion. Evidently, however, although the SWI1 gene product is required for axial element formation, cohesion and synapsis, it is not a structural component of the axial element/SC. A SWI1-GFP fusion product indicates that the Swi1 protein is present in meiocytes only during meiotic interphase and very early prophase I, suggesting that it probably has a function upstream since these structures persist beyond this point. The predicted protein sequence shows no strong similarity to any known protein although there is limited similarity to a conserved region of a mammalian SMC gene family. Another Arabidopsis gene that may be included in this category is SYN1 (DIF1) since the corresponding syn1/dif1 mutants exhibit asynaptic phenotypes, albeit confounded with other meiotic defects, particularly chromosome fragmentation (Bhatt et al. 1999, Bai et al. 1999). The
212 SYN1/DIF1 gene encodes a putative homologue of the yeast REC8 cohesin gene. The yeast REC8 protein is a component of axial elements and is lost from chromosome arms and centromeres in the predicted order for a cohesin. The absence of REC8 protein in yeast causes chaotic chromosome segregation, which is consistent with its proposed cohesion function. RNAi depletion of the C. elegans homologue of REC8 induced a range of meiotic defects including asynapsis, univalent formation and separation of sister chromatids at diakinesis (consistent with a cohesion function) and also chromosome fragmentation at diakinesis (Pasierbek et al. 2001). The Arabidopsis SYN1/ DIF1 gene therefore appears to share some but not all the functions of the yeast and C. elegans homologues. The Arabidopsis gene certainly seems to be required for synapsis and for the repair of double-strand breaks but the mutant does not exhibit all the typical meiotic cohesion defects shown by C. elegans REC8-RNAi or Arabidopsis swi1-2.
Recombination genes with roles in synapsis This group contains several potential members, the homologues of yeast genes that are known to be involved in early or intermediate steps of recombination and have a dual role in recombination and synapsis. However, the only clear-cut case that has been established for Arabidopsis, in which there is a fully characterized asynaptic mutant phenotype, is Atspo11 (Grelon et al. 2001). It has been established in yeast that Spo11 protein catalyses the formation of double-strand breaks, the initiating event in meiotic recombination, through formation of a 50 -phosphotyrosyl linkage. Homologues of SPO11 have been identi¢ed in a wide range of eukaryotes, including fungi, worms, £ies, mammals and plants, indicating that it is a highly conserved gene. Hartung & Puchta (2000) identi¢ed two Arabidopsis homologues of SPO11-1 and a third homologue has since been discovered. To date, only plants have been found to possess more than one homologue of this gene. The signi¢cance of this redundancy is unclear but it has recently been postulated that Spo11-2 and Spo11-3 function primarily as topoisomerases (Hartung & Puchta 2001). Disruption of the SPO11 gene
G. H. Jones et al. results in failure of synapsis in yeast and mouse, but not in Drosophila and C. elegans and these ¢ndings have stimulated much discussion regarding the interdependence of recombination and synapsis (Hunter et al. 2001). Grelon et al. (2001) report that disruption of the Arabidopsis SPO11-1 gene by T-DNA insertion or EMS mutagenesis causes perturbation of meiosis with very few bivalents formed and greatly reduced fertility. Examination of early prophase I showed that homologous chromosome synapsis does not occur in this mutant, therefore putting Arabidopsis in the same group as yeast and mouse regarding the interdependence of synapsis and recombination. Other potential members of this group are the Arabidopsis homologues of DMC1 and RAD51, well-conserved genes that are homologous to the yeast DMC1 and RAD51 genes that are in turn homologous to the E. coli RECA gene. The products of these genes work in conjunction to catalyse strand exchange between homologous DNA molecules; they di¡er mainly in that DMC1 is meiosis speci¢c whereas RAD51 has meiotic and mitotic activity. In addition, recent biochemical studies suggest that they may di¡er in the way in which they interact with their single-stranded DNA substrate (Masson & West 2001). RAD51 has been shown to localise to ENs in Lilium and Zea as well as in mouse and human (Zickler & Kleckner 1999), con¢rming its role in the establishment of recombination intermediates. Couteau et al. (1999) obtained an Arabidopsis mutant of DMC1 (Atdmc1) by conducting a PCR-based screen of the Feldmann collection of T-DNA lines, using the sequence of the Arabidopsis DMC1 gene. The mutant was almost sterile (1.5% residual fertility) and meiosis was severely disturbed. However, prophase I stages were not investigated and therefore the predicted asynaptic phenotype could not be con¢rmed. The absence, to date, of a RAD51 knockout could re£ect its mitotic activity and consequent requirement for normal vegetative growth and development. The Rad50 protein is essential for DSB repair in yeast and is thus required for repair of DNA damage and for meiotic recombination. Because of its role in the establishment of recombination intermediates in early prophase I, it is predicted to be required also for the establishment of chromosome synapsis. A T-DNA insertional mutation of
Meiosis in Arabidopsis the Arabidopsis RAD50 gene has been identi¢ed (Gallego et al. 2001). Homozygous mutant plants are sterile, suggesting a possible meiotic defect, but no cytological analysis of meiosis has been presented. In yeast, the Rad50 protein acts in a complex with Mre11 and Xrs2 (Johzuka & Ogawa 1995) and studies in Arabidopsis also indicate an interaction between Rad50 and Mre11 (DaoudalCotterell et al. 2002). Recently, two mutant alleles of MRE11 have been described in Arabidopsis (Bundock & Hooykaas 2002). One of these (Atmre11-1) has a T-DNA insert at the 50 end of the gene and exhibits a strong mutant phenotype including severe developmental defects, sterility and hypersensitivity to genotoxic agents. However, the basis of the reported sterility is unknown since this line has not been examined cytologically. Msh4 and Msh5 proteins are homologues of E. coli MutS, a component of the mismatch repair system. They were originally discovered in yeast, where they are implicated in intermediate or late steps in recombination. Zickler & Kleckner (1999) have argued that Msh4/5 may act at a relatively early (intermediate) step in recombination, compared to Mlh1/3 (see next section), and this is supported by the ¢nding that MSH5 mouse knockouts have an asynaptic phenotype (de Vries et al. 1999). Data-base searching of the Arabidopsis genome identi¢ed putative MSH4 and MSH5 homologues (Mercier et al. 2001a; unpublished) as well as homologues of other MUTS genes (MSH2, MSH3, MSH6-1 and MSH6-2) (Ade et al. 1999). However, mutations of these genes have not been isolated yet in Arabidopsis.
Genes required for late steps in recombination The preceding section has already covered genes required for early or intermediate steps in recombination, since these steps may also be required for synapsis as well as recombination. As mentioned earlier, most of these early events result in non-crossovers and possibly involve a di¡erent pathway from crossovers. It is known from yeast that the conversion of recombination intermediates, speci¢cally double Holliday junctions, into crossovers requires the products of several late-acting genes, in particular homologues of the E. coli MUTL mismatch repair genes. Arabidopsis
213 homologues of these genes have been identi¢ed by searching the genome database. MLH1 is a MUTL homologue that is known to promote crossing over during meiosis in yeast (Hunter & Borts 1997) and to be required for crossing over and chiasma formation in mouse (Baker et al. 1996). Arabidopsis possesses an MLH1 homologue encoding a 737 amino acid protein which shows 56% similarity to the human protein (excluding 3 hypervariable regions) (Jean et al. 1999). A T-DNA insertion in the promoter of Arabidopsis MLH1 has been identi¢ed but the line has no mutant phenotype because, by chance, the T-DNA sequence appears to provide an alternative promoter and transcription levels are not a¡ected. However, mouse knockouts of MLH1 show a range of meiotic phenotypes, including extensive univalence, but chromosome synapsis is una¡ected. Immunolocalization of Mlh1 protein shows that it is present as discrete foci on pachytene bivalents of mouse and human, whose numbers and locations match closely the numbers and distributions of chiasmata. Other MUTL homologues identi¢ed in Arabidopsis include PMS1 and MLHx (possibly MLH3) (Jean et al. 1999) but no mutations of these genes have been identi¢ed to date.
Conclusion Comparative studies of meiosis in plants as compared to other eukaryotes including fungal and animal models will extend our understanding of the basic mechanisms involved and their evolution. There are also strong practical reasons for achieving a better understanding of the mechanisms and controls of meiosis in plants. The integrated approach to plant meiosis described in this review has already achieved signi¢cant progress, both in terms of mechanistic aspects and the isolation and characterization of meiotic genes. However, there is still much to learn. While it appears that genes and proteins involved in catalysing steps in homologous recombination are well conserved between plants and other eukaryotes, other genes encoding proteins with more structural roles in synaptonemal complex and chromatin organization show relatively little conservation. This may, or may not,
214 indicate substantial structural/functional divergence in meiotic mechanisms between di¡erent eukaryotic groups. Further research will be necessary to identify and characterize further meiotic genes and to elucidate the functional details of protein^protein and DNA^protein interactions in meiotic nuclei.
Acknowledgements We gratefully acknowledge ¢nancial support from The Biotechnology and Biological Science Research Council.
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