Abstract

This article reviews the historical development of cytology and cytogenetics in Arabidopsis, and summarizes recent developments in molecular cytogenetics, with special emphasis on meiotic studies. Despite the small genome and small chromosomes of Arabidopsis, considerable progress has been made in developing appropriate cytogenetical techniques for chromosome analysis. Fluorescence in situ hybridization (FISH) applied to extended meiotic pachytene chromosomes has resulted in a standardized karyotype (idiogram) for the species that has also been aligned with the genetical map. A better understanding of floral and meiotic development has been achieved by combining cytological studies, based on both sectioning and spreading techniques, with morphometric data and developmental landmarks. The meiotic interphase, preceding prophase I, has been investigated by marking the nuclei undergoing DNA replication with BrdU. This allowed the subclasses of meiotic interphase to be distinguished and also provided a means to time the duration of meiosis and its constituent phases. The FISH technique has been used to analyse in detail the meiotic organization of telomeres and centromeric regions. The results indicate that centromere regions do not play an active role in chromosome pairing and synapsis; however, telomeres pair homologously in advance of general chromosome synapsis. The FISH technique is currently being applied to analysing the pairing and synapsis of interstitial chromosome regions through interphase and prophase I. FISH probes also allow the five bivalents of Arabidopsis to be identified at metaphase I and this has permitted an analysis of chiasma frequencies in individual bivalents, both in wild‐type Arabidopsis and in two meiotic mutants.

Received 12 June 2002; Accepted 12 September 2002

Introduction

Meiosis is a highly conserved process in eukaryotes, occupying a central role in the life cycles of all sexually reproducing organisms. Two successive rounds of chromosome segregation follow a single round of DNA replication, producing four haploid products. The segregation of homologous pairs of chromosomes at the first division is dependent on their prior pairing, synapsis and recombination at earlier stages. The second meiotic division serves to separate the two sister chromatids of each chromosome. Subsequent fertilization of male and female gametes restores the diploid state. An understanding of the meiotic process is pivotal to furthering research on reproduction, fertility, genetics and breeding, and in plants, has implications for crop production.

Analysis of meiosis in the flowering plant, Arabidopsis thaliana L. (2n=10), is a growing area of research. Arabidopsis has a number of positive attributes for meiotic studies, including a large pool of tagged meiotic mutants and the molecular tools available to characterize them (Caryl et al., 2003). As cytology and cytogenetics are central to meiotic studies, this review aims to bring together existing information on these important aspects of meiosis in wild‐type Arabidopsis.

The nuclear genomes of most angiosperms are typically several‐fold to 100 times larger than that of Arabidopsis (Bennett and Smith, 1991). The current estimate of the genome size of Arabidopsis is 125 MB, comprising 25 498 genes. One of the reasons for the small genome is that, unlike most angiosperms, less than 10% of its genome consists of repeated sequences, principally comprising the telomeres, the pericentromeric heterochromatin and the rDNA genes associated with chromosomes 2 and 4. Publication of the complete genome sequence of Arabidopsis has provided the foundation for the comparison of conserved processes in eukaryotes, identifying a wide range of plant‐specific gene functions and establishing rapid ways to identify genes, including those associated with meiosis (Arabidopsis Genome Initiative, 2000).

Development of cytogenetics in Arabidopsis

The relatively small nuclear genome of Arabidopsis is at odds with the general requirement of the cytogeneticist for large chromosomes and good chromosome morphology. Many of the cytogenetic advances of the twentieth century, including chromosome behaviour in meiosis, were based on species that met these requirements, such as Liliaceous plants, Amphibia and Orthopteran insects. Because of its small chromosomes and consequent poor chromosome morphology, the earliest Arabidopsis cytogenetic studies only confirmed the number of chromosomes in the complement (Jones and Heslop‐Harrison, 2000, and references therein). Early attempts at karyotype analysis were only moderately successful. Giemsa C‐banding led to improvements in chromosome identification together with recognition that the haploid genome contained two nucleolus organizing regions (NORs) and, ultimately, an alignment of the cytological chromosome map with the genetic linkage map (Steinitz‐Sears and Lee Chen, 1970; Ambros and Schweizer, 1976; Schweizer et al., 1987). These early cytogenetic studies of Arabidopsis relied on squashing techniques and conventional cytological stains, applied to root‐tip meristem preparations or pollen mother cells. Much clearer images of mitotic chromosomes of Arabidopsis were eventually obtained by staining with the DNA fluorochrome DAPI, and the locations of NORs (45S rDNA), 5S rDNA and pericentromeric heterochromatin blocks have been confirmed by fluorescence in situ hybridization (FISH) (Maluszynska and Heslop‐Harrison, 1991; Murata et al., 1997; Brandes et al., 1997).

The requirement for clearer chromosome morphology in Arabidopsis meiosis led to an adaptation of a spreading method originally developed for preparing tomato meiotic pachytene chromosomes (Zhong et al., 1996). This improved methodology employs enzyme digestion of pollen mother cells (PMCs) or embryo‐sac mother cells (EMCs) followed by acid disassociation and air‐drying of cells directly onto slides, combined with DAPI staining (Ross et al., 1996; Armstrong et al., 1998; Armstrong and Jones, 2001). The development of molecular cytogenetic techniques together with improved cytology has led to Arabidopsis becoming a useful model for the investigation of chromatin organization both in meiosis and in the interphase cell (Lysak et al., 2001; Armstrong et al., 2001), and for the characterization of meiotic mutants (Ross et al., 1997; Caryl et al., 2003).

The Arabidopsis karyotype

Despite the improvement in methods for the analysis of mitotic chromosomes (see previous section), the mitotic metaphase chromosomes of Arabidopsis are very condensed and their morphology is not well suited for detailed karyotyping and physical mapping of chromosome landmarks. By contrast, extended pachytene bivalents of Arabidopsis, prepared by the spreading technique, are 20– 25 times longer than the condensed mitotic chromosomes and allow much greater precision for molecular cytogenetic analysis (Fransz et al., 1998). Each meiotic bivalent is made up of two fully synapsed homologous chromosome with DAPI bright pericentromeric regions (Ross et al., 1996). Two of the bivalents (chromosomes 2 and 4) have a second DAPI bright region associated with the nucleolar organizing regions (NORs). Measurements based on bivalent length, arm ratios and features of the DAPI bright regions, combined with FISH using 5S and 45S rDNA probes have produced a definitive pachytene karyotype (Fransz et al., 1998; Fig. 1). In agreement with earlier karyotyping exercises the chromosomes are numbered according to their corresponding linkage groups (Schweizer et al., 1987), despite the non‐concurrence with their physical rankings.

Figure 1 illustrates the karyotype of Arabidopsis (Ws). Chromosomes 1, 3 and 5 constitute a metacentric/ sub‐metacentric group of chromosomes while chromosomes 2 and 4 are both short acrocentric chromosomes with distinctly unequal arms. Chromosome 1 is the largest metacentric/sub‐metacentric chromosome and lacks any rDNA sites. Chromosome 2 is a small acrocentric chromosome having a short‐arm NOR corresponding to a 45S rDNA site. Chromosome 3 is the shortest metacentric/sub‐metacentric chromosome; this chromosome is variable between accessions with respect to possession, location and size of a 5S rDNA site. Chromosome 4 is another NOR‐bearing acrocentric chromosome, similar in overall size and organization to chromosome 2, but distinguished by possession of an invariant 5S rDNA site located proximally on the short arm. Chromosome 5 is the second longest metacentric/sub‐metacentric chromosome, additionally distinguished from chromosome 1 by possession of an invariant large 5S rDNA site located proximally on the shorter arm (Fig. 1).

In addition to its value for karyotyping purposes, Fransz et al. (2000) demonstrated the powerful nature of the FISH technique applied to spread pachytene bivalents for high resolution physical mapping at the cytological level. In this work an integrated cytogenetic map was produced for the short arm of chromosome 4, showing detailed positions of various multicopy and unique sequences relative to euchromatin and heterochromatic segments. Moreover, they demonstrated that a recombination cold spot was associated with a condensed heterochromatin knob and, conversely, high recombination regions were correlated with more exended euchromatin.

Meiosis

Floral development

Meiosis occurs in the immature developing flower bud of Arabidopsis. The flower has a simple structure, typical of the Brassicaceae, with a calyx of four free sepals and a corolla of four petals. There are four long medial staments and two shorter lateral stamens. The superior gynaecium has two carpels whose locules are separated by a false septum (Smyth et al., 1990).

Inflorescences in Arabidopsis require a methodical approach if the meiotic stages are to be located and correctly identified. Floral development in Arabidopsis has been analysed in detail, based on morphometric characters of flower buds and their component parts, with 12 stages of floral development recognized (Smyth et al., 1990). In order to facilitate the rapid and efficient preparation of meiocytes for cytogenetic analysis, floral stage, as defined by Smyth et al. (1990) has been linked with morphometric data, developmental landmarks and meiotic stage (Armstrong and Jones, 2001; Armstrong et al., 2001; Table 1). The gross anatomy of anthers and ovules was determined from observations on resin‐embedded semi‐thin sections and lightly squashed whole mounts of anthers and/or gynoecia at appropriate developmental stages. Figure 2 illustrates the progression of meiosis in semi‐thin sections. Male and female meiosis in Arabidopsis, as in other angiosperms, are asynchronous. Prophase I of meiosis in embryo‐sac mother cells commences as the pollen mother cells arrive at the tetrad stage.

Anther and PMC development

The smallest flower buds that could be practically dissected from inflorescences and processed for spreading were about 0.2–0.3 mm long and coincided with late stage 7/early stage 8. Before this stage the archesporial cells undergo periclinal divisions to give rise to the primary parietal and primary sporogenous layers (Misra, 1962). Following the cessation of asynchronous archesporial mitotic divisions in anthers, the microsporocytes or pollen mother cells (PMCs) develop more or less synchronously through meiosis. By floral stage 8, the five layers of the stamen have differentiated. PMCs are surrounded by four layers: the tapetum, the middle layer, the endothecium and the epidermis, each of which is one cell thick (Owen and Makaroff, 1995; Fig. 2). The PMCs are about three times larger than tapetal cells. They have very thin walls at this stage and contain a large centrally located nucleus with a prominent nucleolus (Fig. 2A). On the other hand, the densely staining tapetal cells are smaller, and the nucleus and nucleolus are correspondingly smaller. Light squashes of anthers at this stage result in the extrusion of PMCs which tend to remain attached to each other, forming columns of cells. This feature is presumed to be due to the presence of intercellular cytoplasmic channels connecting the meiocytes at early stages of development (Owen and Makaroff, 1995). This is a common feature of pollen mother cells at early stages of development, and has been described in several different plant species (Esau, 1977). During later stages of meiosis these channels are closed coincident with the deposition of callose on PMC walls and extruded PMCs are consequently unconnected and easily separated. During floral stage 9 the remaining meiotic stages take place. In sections of Arabidopsis anthers the leptotene stage is difficult to distinguish due to the extreme fineness of the chromosome threads, but this stage is associated with movement of the nucleolus to the periphery of the nucleus. More definite chromatin threads are visible by zygotene due to the progressive condensation of the chromosomes and their synapsis into bivalent structures. At this stage the chromosomes are characteristically polarized to one side of the PMC nucleus, and deposition of callose onto PMC walls is evident. The tapetal cells become binucleate at zygotene–pachytene stages (Fig. 2B) as a result of a single synchronized mitotic division without subsequent cytokinesis. By pachytene, the chromosomes in the PMCs are fully synapsed, which in sections can, on closer observation, be visualized as thick threads (Fig. 2B). From pachytene onwards the PMCs have thick callose walls and the subsequent stages of divisions I and II are easily distinguished, culminating in the appearance of tetrads (Fig. 2C).

Ovule and EMC development

The gynoecium of Arabidopsis consists of two fused carpels that develop as a single cylinder. A total of 40–60 ovules are produced in four rows (Mansfield and Bowman, 1994). In the Arabidopsis accession Ws there are about 40 ovules in each gynoecium (Armstrong and Jones, 2001).

There is a clear relationship between meiotic progression and gynoecium length and morphology (Armstrong and Jones, 2001; Table 1). The smallest gynoecia (length 0.3–0.4 mm) have open pistils with a distinctly slotted appearance at their ends and appear at floral stage 9/10. Gynoecia at this stage contain megasporocytes or embryo‐sac mother cells (EMCs) that are at meiotic interphase. Larger gynoecia (length 0.5–0.8 mm) are characterized by closure of the pistil end and appearance of stigmatic papillae (still within floral stage 10). Meiotic stages from prophase I onwards are found in gynoecia in this length range. There is considerable synchrony in meiotic development between the different ovules within an inflorescence.

In female meiosis, the single‐celled archesporangium functions directly as the EMC and forms a distinctive cell even in the meiotic interphase and early meiotic stages. In sections, the EMC is seen to be more than three times larger than the surrounding cells of the nucellum (Armstrong and Jones, 2001; Webb and Gunning, 1990; Bajon et al., 1999). In lightly squashed preparations, the EMC is again seen to be clearly larger than any of the surrounding cells of the ovule. It contains a large nucleus with a prominent nucleolus and a large volume of cytoplasm with organelles.

Observations on sectioned and lightly squashed anthers and gynoecia are useful for determining the timing and progression of meiosis in relation to floral development, but they are not appropriate for detailed observations on chromosome organization and behaviour during meiosis. Conventional squashing and staining procedures using classical stains such as orcein or carmine are also unsuitable for analysis of Arabidopsis meiosis. This is principally because they fail to produce a sufficiently intense staining of the very fine extended chromosomes that characterize the prophase I stages of meiosis in Arabidopsis. Enzyme digestion/acid disassociation and air‐drying of meiocytes onto slides, combined with DAPI staining gives superior resolution of Arabidopsis chromosomes, particularly in the critical prophase I stages, and forms the basis for the following description of meiosis.

Male and female meiosis in Arabidopsis are very similar in most respects, with the exception of the tetrad stage. The following description is therefore based on the progression of meiosis in PMC, with references to female meiosis where necessary.

This description provides reference observations for interpreting the meiotic stages in Arabidopsis. It is beyond the scope of this article to provide figures to illustrate all the key stages of meiosis mentioned in the text. However, a series of images detailing the progression of meiosis in Arabidopsis PMCs is presented in an earlier paper (Ross et al., 1996; see also Fig. 1 in Caryl et al., 2003).

The meiotic interphase

Descriptions of meiosis commonly commence at the onset of prophase I, with the first appearance of distinct chromosome threads at leptotene, but there is a widespread and increasing recognition that meiotic interphase (sometimes termed premeiotic interphase) encompasses many important events despite its uniform and relatively undifferentiated appearance (Dover and Riley, 1977; Loidl, 1990; Zickler and Kleckner, 1998, 1999). Following the demonstration by Taylor and Macmaster (1954) that meiotic DNA replication occurs during meiotic interphase, and not prophase I as was originally thought, the meiotic S‐phase has been extensively analysed in a wide range of eukaryotes. A common feature of this S‐phase is its extended duration compared to somatic/mitotic S‐phases (John, 1990). The meiotic S‐phase also serves as a useful mid‐interphase marker, separating the unreplicated G1 phase from the replicated G2 phase. Several other important events are likely to occur during this interphase, including chromatin modification and the expression of many meiotic genes whose products are required for the successful completion of meiosis. Despite the general realization of the importance of the meiotic interphase, the analysis of meiosis in Arabidopsis has, to date, barely considered this phase. Immunocytological approaches are now being used to investigate both the S‐phase and the appearance of key meiotic proteins in Arabidopsis PMCs.

The duration of meiotic G2, and ensuing meiotic stages, may be determined by marking the S‐phase and subsequently sampling the marked population in a time‐course experiment. Previous experimental investigations involving meiotic S‐phase labelling in plants relied on tritiated thymidine, a radioisotopically labelled precursor of DNA, combined with autoradiographic detection of incorporated label (Bennett et al., 1971; Holm, 1977). An alternative labelling technique that avoids the time‐consuming autoradiographic detection is to mark the cells in S‐phase with the thymidine analogue bromodeoxyuridine (BrdU) that can be detected immunocytologically using anti‐BrdU antibody carrying a fluorescent tag.

This approach has been applied to the labelling of Arabidopsis meiotic S‐phase. By immersing cut stems bearing inflorescences in BrdU solution uptake of BrdU could be detected in a subset of meiotic interphase nuclei within 1 h and a high level of labelling within 2 h. In a time‐course experiment, cut stems were immersed in BrdU for 2 h and then transferred to water. Inflorescences were sampled at intervals, fixed and processed for detection of BrdU in cytological preparations. By this means the duration of meiotic G2 and all subsequent stages as far as tetrads has been determined, as well as the duration of S‐phase itself (SJ Armstrong, GH Jones, unpublished data). In addition, these experiments allowed positive identification of S‐phase meiocytes, as those labelled after short periods of exposure to BrdU, and also G2 cells as those that became labelled as cells passed from S‐phase to G2 at progressively later time points. By a process of elimination it was possible to identify G1 interphase cells as a category of cells, with distinctive morphology, that were not labelled at short sampling times after the pulse‐label. G1 cells were expected to be found in the next smallest buds to those containing S labelled cells. They were smaller than those of S‐phase and G2 but nevertheless were still larger than the surrounding somatic cells. They typically had a relatively large nucleolus and condensed pericentromeric heterochromatin. By contrast the S‐phase cells were larger and the pericentromeric heterochromatin was decondensed as evidenced by lack of DAPI bright regions. G2 cells showed a very similar nuclear organization to S‐phase in terms of nuclear size, nucleolus size and location, and chromatin condensation. The overall duration of meiosis in Arabidopsis measured at 20 °C, from S‐phase to the formation of tetrads, is 36 h.

Prophase I

Early leptotene proper is distinctive and is characterized by extensive stretches of unsynapsed chromosome axes having bead‐like chromomere differentiation along their length and the reappearance of condensed pericentromeric heterochromatin. However, the transition from late G2 to early leptotene is gradual and indefinable, with the appearance of short stretches of chromosome axes, that probably correspond to axial elements at the electron microscope level, and progressive condensation of the heterochromatin. This gradual transition makes precise determination of the relative durations of G2 and leptotene difficult. In early leptotene the nucleolus is a large structure, taking up as much as one‐third of the nuclear volume. It occupies a central position in the nucleus, but towards the end of the leptotene it moves progressively towards the nuclear periphery, where it remains throughout the rest of prophase I. Zygotene, by definition, is the stage of chromosome synapsis, and early zygotene is therefore characterized by the first indications of synapsis. The zygotene stage in Arabidopsis characteristically fails to produce well spread preparations. This appears to be due to a characteristic polarization and clumping of the chromosomes towards one side of the nucleus and is accompanied by a tendency for aggregation of the pericentromeric heterochromatin into a variable number of clumps. At these early meiotic prophase I stages in PMCs, the organelles are characteristically distributed. Typically, they accumulate at the opposite pole to the chromatin during late leptotene/zygotene. This arrangement was not observed in the EMC, which could be due to organizational differences between the single cell (EMC) compared to the many PMCs in the central column of the anther.

At pachytene, the chromosomes are much shorter compared to the earlier prophase stages (Fransz et al., 1998). All chromosomes are seen to be fully synapsed double structures, with obvious chromomere differentiation. At the electron microscope level the synapsed homologues are seen to be associated by typical tripartite synaptonemal complexes (SCs) running the entire length of each of the five bivalents (Albini, 1994). By late pachytene, the bivalents are quite well separated from each other and can often be fully traced. The only exception to this is a tendency for the NORs, located subterminally on the short arms of chromosomes 2 and 4, to be associated and it appears that they stay together through the rest of prophase I, up to and including diakinesis.

The transition from pachytene to diplotene involves the gradual and progressive separation or desynapsis of the homologues along most of their length, characterized at the EM level by the disruption and eventual disappearance of the SC. Once the chromosomes are fully desynapsed, except at chiasmata, the early diplotene nuclei are filled with extended and rather fuzzy single chromosome threads, corresponding to the so‐called diffuse stage described in many organisms including angiosperms. From this point onwards the five bivalents gradually condense to give discrete bivalent structures, associated by chiasmata, the degree of condensation increasing progressively from late diplotene, through diakinesis to its maximum at metaphase I.

Metaphase I to telophase II

At metaphase I five condensed bivalents are present in wild‐type meiosis. They show co‐orientation on the metaphase I spindle, and each bivalent contains from one to three chiasmata (see below). Anaphase I achieves the separation of homologous whole chromosomes, whose sister chromatids remain associated at centromere regions. This stage is of brief duration and is rarely observed in Arabidopsis. Telophase I leads to the dyad stage in which the PMC contains two polar groups of chromosomes that partially decondense without achieving the full interphase condition. Partitioning of the cytoplasm does not directly follow the first nuclear division and is deferred until after the second division. During metaphase I and anaphase I the cytoplasmic organelles are excluded from the spindle and subsequently form a distinct band or saddle between the daughter nuclei that persists into the second division. Metaphase II and anaphase II follow the normal course, with separation of chromatids into daughter nuclei. Late anaphase II cells contain the expected four groups of five chromatids within a common cytoplasm, which gradually re‐form into tetrads of haploid interphase nuclei.

An overall conclusion from this survey is that meiosis in Arabidopsis follows a conventional pattern. Apart from an interesting difference in the mode of telomere clustering (described below) meiosis is comparable to that in other plant species that have been extensively analysed cytogenetically, such as maize, rye and tomato. Meiosis also appears to be very similar in PMCs and EMCs of Arabidopsis. The major difference concerns the fate of the haploid meiotic products. In anthers, the haploid cells (microspores) all develop into pollen grains, the male gametophytes. In ovules, on the other hand, the haploid meiotic products form a linear tetrad of megaspores, three of which abort leaving a single megaspore which undergoes mitotic division to give rise to the embryo‐sac, the female gametophyte.

Molecular cytogenetics of meiosis

Superimposed on this basic descriptive framework of Arabidopsis meiosis, molecular cytogenetic approaches have been applied to extend understanding of key meiotic events and processes. An illustration of this, as described earlier, is the use of BrdU labelling of replicating DNA, and detection by anti‐BrdU antibody, to identify and mark cells in meiotic S‐phase.

Immunocytology is also being applied to determine the time of expression of meiotic genes and the intranuclear locations of the proteins they encode. Several Arabidopsis meiotic genes have now been cloned and subsequently expressed in a bacterial in vitro system to yield recombinant protein products. These can then be used to generate antibodies to meiotic proteins which can be employed in immunolocalization studies. For example, expression of the Asy1 protein in Arabidopsis and Brassica oleracea has been investigated by this means. This protein is required for normal meiotic chromosome synapsis since an insertional mutant (asy1) has been shown to be completely asynaptic (Caryl et al., 2000). The protein is initially detected during meiotic interphase as numerous punctate foci distributed over the chromatin. As prophase I proceeds, the signal becomes increasingly continuous and is closely associated with the axial elements of unsynapsed chromosomes and with the synaptonemal complexes of synapsed chromosomes, but is not present in the chromatin loops extending from these structures. Immunogold labelling in conjunction with electron microscopy established that the protein localizes to regions of chromatin that are closely associated with the axial/lateral elements, rather than being a component of these structures. These observations indicate that the protein is required for morphogenesis of the synaptonemal complex, possibly by defining regions of chromatin that associate with the axial elements (Armstrong et al., 2002). Using a similar approach the expression and localization of several other meiotic proteins are currently being investigated.

FISH is also being used to label chromosomal structures, or just chromosomal sites, in order to analyse the progress of chromosome pairing and synapsis. This approach has been applied successfully to the analysis of these events in several other organisms (reviewed in Zickler and Kleckner, 1998, 1999). In Arabidopsis, FISH was used to mark centromeres and telomeres, and their intranuclear arrangements were analysed from meiotic interphase through to late prophase I (Armstrong et al., 2001; Fig. 3). Centromere regions of Arabidopsis are unpaired, widely dispersed and peripherally located in nuclei during meiotic interphase, and they remain unpaired and unassociated throughout leptotene. Eventually they associate pairwise during zygotene, as part of the nucleus‐wide synapsis of homologous chromosomes. Telomeres, on the other hand, show a persistent association with the nucleolus throughout meiotic interphase. Variation in telomere signal number indicates that telomeres undergo pairing during this interval, preceding the onset of general chromosome synapsis. During leptotene the paired telomeres lose their association with the nucleolus and become widely dispersed. As the chromosomes synapse during zygotene, the telomeres reveal a loose clustering within one hemisphere, which may represent a degenerate or relic bouquet configuration. It is proposed that in Arabidopsis the classical leptotene/zygotene bouquet is absent and is replaced functionally by nucleolus‐associated telomere clustering. As an extension to this approach, FISH probes are currently being used to mark selected chromosomal sites in order to analyse the pairing and synaptic behaviour of non‐centromeric and non‐telomeric regions.

Chiasma analysis in Arabidopsis

Although chiasmata can occasionally be clearly resolved in diplotene and diakinesis of Arabidopsis, chiasma scoring at these stages has associated problems due to the difficulty of distinguishing relational twists from genuine chiasmata. In addition, the persistent association of the NORs with the nucleolus at this stage makes it virtually impossible to identify chiasmata in the short arms of chromosomes 2 and 4. Metaphase I is therefore preferable for chiasma scoring in this species, despite the highly condensed state of the bivalents. FISH with the repeated 5S and 45S rDNA probes can identify each of the five bivalents at metaphase I. Using this approach the chiasma frequencies of marked individual bivalents of the Ws accession of Arabidopsis have been analysed, as well as two meiotic mutants of Arabidopsis, one asynaptic (asy1) and one desynaptic (dsy1) (Sanchez‐Moran et al., 2001). The mutants were isolated from T‐DNA transformed lines created in the Ws background genotype (Ross et al., 1997). A wild‐type mean chiasma frequency of 9.24 per cell was determined from a sample of 50 cells, a value that is consistent with estimated genetic recombination values. Individual bivalent chiasma frequencies varied according to chromosome size; chromosome 1 had the highest mean chiasma frequency (2.14) while the acrocentric chromosomes had the lowest frequencies, 1.54 and 1.56 for chromosomes 2 and 4, respectively. Chiasma analysis of female meiosis is more demanding since each ovule contains but a single EMC and, consequently, the estimate of female chiasma frequency is based on fewer cells. A mean EMC chiasma frequency of 8.5 was obtained from a sample of ten diakinesis and metaphase I cells (Armstrong and Jones, 2001). Chiasma frequency has been shown to differ significantly between the sexes in several different species (John, 1990), but there is no consistency in the direction of the difference. In some cases male chiasma frequency is higher, while in others the female has a higher frequency; in yet other cases no difference was found. The observation of a lower chiasma frequency in Arabidopsis EMCs compared to PMCs is consistent with genetic recombination differences reported by Vizir and Korol (1990).

Conclusion

Arabidopsis is proving to be a convenient and powerful model system for the analysis of meiosis. The availability of good cytology combined with developments in molecular cytogenetics and molecular biology put it on a par with other ‘higher’ eukaryotic models such as Drosophila, C. elegans and mouse and, in addition, extend the range of model organisms to the plant kingdom. One of the aims of current meiosis research is to understand whether and to what extent meiotic processes and controls are conserved across all eukaryotes and, in this context, the inclusion of a plant model is very important. Meiosis is a highly complex process involving the action and interaction of very many genes. As a result of recent research in a number of model systems there is now a better understanding of these genes, the processes they determine and their regulation. However, a full understanding of the meiotic process is still a distant prospect and this field will continue to be a fascinating and rewarding field of endeavour for many years to come.

Acknowledgement

We are grateful to the BBSRC for their financial support of much of the work reported in this review.

Fig. 1. Karyotype of pachytene chromosomes of A. thaliana. Pericentromeric heterochromatin (grey), NOR (stippled) and 5SrDNA (black).

Fig. 1. Karyotype of pachytene chromosomes of A. thaliana. Pericentromeric heterochromatin (grey), NOR (stippled) and 5SrDNA (black).

Fig. 2. Semi‐thin sections through young flower buds of A. thaliana, stained with toluidine blue, showing complete anther locules containing PMCs in meiotic interphase (A), pachytene (B) and tetrads (C) surrounded by four cell layers. (Bar=10 µm).

Fig. 2. Semi‐thin sections through young flower buds of A. thaliana, stained with toluidine blue, showing complete anther locules containing PMCs in meiotic interphase (A), pachytene (B) and tetrads (C) surrounded by four cell layers. (Bar=10 µm).

Fig. 3. FISH of telomere (red) and pericentromeric heterochromatin (CENs) (green) probes to wild‐type A. thaliana PMC nuclei. (A) S‐phase nucleus showing extended CENs and unpaired telomeres clustered around the nucleolus. (B) early leptotene nucleus showing dispersed CENs and paired telomeres. (C) Pachytene/very early diplotene nucleus showing paired CENs and telomeres; arrow indicates interstitial telomere locus adjacent to CEN on chromosome 1. (D) Diplotene/diakinesis nucleus showing five bivalents. (E) Metaphase I nucleus showing five fully condensed and aligned bivalents. (F) Tetrad stage showing four haploid nuclei; note telomeres have reassociated with the reformed nucleoli (Bar=10 µm).

Fig. 3. FISH of telomere (red) and pericentromeric heterochromatin (CENs) (green) probes to wild‐type A. thaliana PMC nuclei. (A) S‐phase nucleus showing extended CENs and unpaired telomeres clustered around the nucleolus. (B) early leptotene nucleus showing dispersed CENs and paired telomeres. (C) Pachytene/very early diplotene nucleus showing paired CENs and telomeres; arrow indicates interstitial telomere locus adjacent to CEN on chromosome 1. (D) Diplotene/diakinesis nucleus showing five bivalents. (E) Metaphase I nucleus showing five fully condensed and aligned bivalents. (F) Tetrad stage showing four haploid nuclei; note telomeres have reassociated with the reformed nucleoli (Bar=10 µm).

Table 1.

Summary of the relationship between floral development and cytological landmarks

Floral stagea Bud size (mm) Gynoecium length during meiosis Cytology Meiotic stage 
8. Anther and filament regions already separated (stage 7). Locules present in the long stamens. Primordia of petals visible <0.3  Anthers differentiated into five tissue layers. The innermost cells, the PMCs are surrounded by the tapetum, the middle cell layer, the endothecium, and the epidermis Meiotic interphase in the PMCs 
 
9. Petal primordia stalked at base 0.3–0.4  Tapetal cells become binucleate at zygotene/early pachytene stages Meiosis in PMCs 
 
10. Petals level with the short stamens. Stamens green <0.5 0.3–0.4 Gynoecium cylinder deeply slotted.  Microspores. Meiotic interphase in EMC. 
  0.5 Closure of cylinder, appearance of stigmatic papillae Meiosis in EMCs 
 
11. Anthers green, but changing to yellow as gynoecium enlarges <0.7 0.6–0.8 Stigmatic papillaedevelop Meiosis in EMCs 
Floral stagea Bud size (mm) Gynoecium length during meiosis Cytology Meiotic stage 
8. Anther and filament regions already separated (stage 7). Locules present in the long stamens. Primordia of petals visible <0.3  Anthers differentiated into five tissue layers. The innermost cells, the PMCs are surrounded by the tapetum, the middle cell layer, the endothecium, and the epidermis Meiotic interphase in the PMCs 
 
9. Petal primordia stalked at base 0.3–0.4  Tapetal cells become binucleate at zygotene/early pachytene stages Meiosis in PMCs 
 
10. Petals level with the short stamens. Stamens green <0.5 0.3–0.4 Gynoecium cylinder deeply slotted.  Microspores. Meiotic interphase in EMC. 
  0.5 Closure of cylinder, appearance of stigmatic papillae Meiosis in EMCs 
 
11. Anthers green, but changing to yellow as gynoecium enlarges <0.7 0.6–0.8 Stigmatic papillaedevelop Meiosis in EMCs 

a As described by Smyth et al. (1990).

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