Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

The H3 histone chaperone NASPSIM3 escorts CenH3 in Arabidopsis.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co-expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N-terminal tail and the histone fold domain of non-nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co-immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein-protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis-associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.

MAP Kinase PrMPK9-1 Contributes to the Self-Incompatibility Response.

Mitogen-activated protein kinases (MAPKs) form important signaling modules for a variety of cellular responses in eukaryotic cells. In plants, MAPKs play key roles in growth and development as well as in immunity/stress responses. Pollen-pistil interactions are critical early events regulating pollination and fertilization and involve many signaling events. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants and also is known to utilize signaling to achieve incompatible pollen rejection. Although several pollen-expressed MAPKs exist, very little is known about their function. We previously identified a pollen-expressed MAPK (p56) from Papaver rhoeas that was rapidly activated during SI; several studies implicated its role in signaling to SI-induced programmed cell death involving a DEVDase. However, to date, the identity of the MAPK involved has been unknown. Here, we have identified and cloned a pollen-expressed P. rhoeas threonine-aspartate-tyrosine (TDY) MAPK, PrMPK9-1 Rather few data relating to the function of TDY MAPKs in plants currently exist. We provide evidence that PrMPK9-1 has a cell type-specific function, with a distinct role from AtMPK9 To our knowledge, this is the first study implicating a function for a TDY MAPK in pollen. We show that PrMPK9-1 corresponds to p56 and demonstrate that it is functionally involved in mediating SI in P. rhoeas pollen: PrMPK9-1 is a key regulator for SI in pollen and acts upstream of programmed cell death involving actin and activation of a DEVDase. Our study provides an important advance in elucidating functional roles for this class of MAPKs.

Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca2+ and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

The Papaver rhoeas S determinants confer self-incompatibility to Arabidopsis thaliana in planta.

Self-incompatibility (SI) is a major genetically controlled system used to prevent inbreeding in higher plants. S determinants regulate allele-specific rejection of "self" pollen by the pistil. SI is an important model system for cell-to-cell recognition and signaling and could be potentially useful for first-generation (F1) hybrid breeding. To date, the transfer of S determinants has used the complementation of orthologs to "restore" SI in close relatives. We expressed the Papaver rhoeas S determinants PrsS and PrpS in Arabidopsis thaliana. This enabled pistils to reject pollen expressing cognate PrpS. Moreover, plants coexpressing cognate PrpS and PrsS exhibit robust SI. This demonstrates that PrsS and PrpS are sufficient for a functional synthetic S locus in vivo. This transfer of novel S determinants into a highly divergent species (>140 million years apart) with no orthologs suggests their potential utility in crop production.

The condensin complexes play distinct roles to ensure normal chromosome morphogenesis during meiotic division in Arabidopsis.

Meiosis is a specialized cell division essential for sexual reproduction. During meiosis the chromosomes are highly organized, and correct chromosome architecture is required for faithful segregation of chromosomes at anaphase I and II. Condensin is involved in chromosome organization during meiotic and mitotic cell divisions. Three condensin subunits, AtSMC4 and the condensin I and II specific subunits AtCAP-D2 and AtCAP-D3, respectively, have been studied for their role in meiosis. This has revealed that both the condensin I and condensin II complexes are required to maintain normal structural integrity of the meiotic chromosomes during the two nuclear divisions. Their roles appear functionally distinct in that condensin I is required to maintain normal compaction of the centromeric repeats and 45S rDNA, whereas loss of condensin II was associated with extensive interchromosome connections at metaphase I. Depletion of condensin is also associated with a slight reduction in crossover formation, suggesting a role during meiotic prophase I.

SHUGOSHINs and PATRONUS protect meiotic centromere cohesion in Arabidopsis thaliana.

In meiosis, chromosome cohesion is maintained by the cohesin complex, which is released in a two-step manner. At meiosis I, the meiosis-specific cohesin subunit Rec8 is cleaved by the protease Separase along chromosome arms, allowing homologous chromosome segregation. Next, in meiosis II, cleavage of the remaining centromere cohesin results in separation of the sister chromatids. In eukaryotes, protection of centromeric cohesion in meiosis I is mediated by SHUGOSHINs (SGOs). The Arabidopsis genome contains two SGO homologs. Here we demonstrate that Atsgo1 mutants show a premature loss of cohesion of sister chromatid centromeres at anaphase I and that AtSGO2 partially rescues this loss of cohesion. In addition to SGOs, we characterize PATRONUS which is specifically required for the maintenance of cohesion of sister chromatid centromeres in meiosis II. In contrast to the Atsgo1 Atsgo2 double mutant, patronus T-DNA insertion mutants only display loss of sister chromatid cohesion after meiosis I, and additionally show disorganized spindles, resulting in defects in chromosome segregation in meiosis. This leads to reduced fertility and aneuploid offspring. Furthermore, we detect aneuploidy in sporophytic tissue, indicating a role for PATRONUS in chromosome segregation in somatic cells. Thus, ploidy stability is preserved in Arabidopsis by PATRONUS during both meiosis and mitosis.

The Fanconi anemia ortholog FANCM ensures ordered homologous recombination in both somatic and meiotic cells in Arabidopsis.

The human hereditary disease Fanconi anemia leads to severe symptoms, including developmental defects and breakdown of the hematopoietic system. It is caused by single mutations in the FANC genes, one of which encodes the DNA translocase FANCM (for Fanconi anemia complementation group M), which is required for the repair of DNA interstrand cross-links to ensure replication progression. We identified a homolog of FANCM in Arabidopsis thaliana that is not directly involved in the repair of DNA lesions but suppresses spontaneous somatic homologous recombination via a RecQ helicase (At-RECQ4A)-independent pathway. In addition, it is required for double-strand break-induced homologous recombination. The fertility of At-fancm mutant plants is compromised. Evidence suggests that during meiosis At-FANCM acts as antirecombinase to suppress ectopic recombination-dependent chromosome interactions, but this activity is antagonized by the ZMM pathway to enable the formation of interference-sensitive crossovers and chromosome synapsis. Surprisingly, mutation of At-FANCM overcomes the sterility phenotype of an At-MutS homolog4 mutant by apparently rescuing a proportion of crossover-designated recombination intermediates via a route that is likely At-MMS and UV sensitive81 dependent. However, this is insufficient to ensure the formation of an obligate crossover. Thus, At-FANCM is not only a safeguard for genome stability in somatic cells but is an important factor in the control of meiotic crossover formation.

The Papaver self-incompatibility pollen S-determinant, PrpS, functions in Arabidopsis thaliana.

Many angiosperms use specific interactions between pollen and pistil proteins as "self" recognition and/or rejection mechanisms to prevent self-fertilization. Self-incompatibility (SI) is encoded by a multiallelic S locus, comprising pollen and pistil S-determinants. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein, interact to trigger a Ca(2+)-dependent signaling network, resulting in inhibition of pollen tube growth, cytoskeletal alterations, and programmed cell death (PCD) in incompatible pollen. We introduced the PrpS gene into Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen: S-specific inhibition and hallmark features of Papaver SI. Our findings demonstrate that Papaver PrpS is functional in a species with no SI system that diverged ~140 million years ago. This suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. This will be of interest to biologists interested in the evolution of signaling networks in higher plants.

The RecQ helicase AtRECQ4A is required to remove inter-chromosomal telomeric connections that arise during meiotic recombination in Arabidopsis.

RecQ helicases are a conserved group of proteins with a role in the maintenance of genome integrity. In Saccharomyces cerevisiae (budding yeast), meiotic recombination is increased in the absence of the RecQ helicase Sgs1. Here we investigated the potential meiotic role of the Sgs1 homologue AtRECQ4A and the closely related AtRECQ4B. Both proteins have been shown to function during recombination in somatic cells, but so far their meiotic role has not been investigated. Both AtRECQ4A and AtRECQ4B were expressed in reproductive tissues. Although immunolocalization studies showed that AtRECQ4A associates with recombination intermediates, we found no evidence that its loss or that of AtRECQ4B had a significant effect on meiotic cross-overs, suggesting functional redundancy with other RECQ family members. Nevertheless, pollen viability decreased in Atrecq4A, resulting in a reduction in fertility, although this was not the case in Atrecq4B. Cytological analysis revealed chromatin bridges between the telomeres of non-homologous chromosomes in Atrecq4A at metaphase I, in some instances accompanied by chromosome fragmentation at anaphase I. The bridges required telomeric repeats and were dependent on meiotic recombination. Immunolocalization confirmed the association of AtRECQ4A with the telomeres during prophase I, which we propose enables dissolution of recombination-dependent telomeric associations. Thus, this study has identified a hitherto unknown role for a member of the RECQ helicase family during meiosis that contributes to the maintenance of chromosome integrity. As telomere structure is generally conserved, it seems likely that these associations may arise during meiosis in other species, where they must also be removed.

Histone hyperacetylation affects meiotic recombination and chromosome segregation in Arabidopsis.

In this study, the meiotic role of MEIOTIC CONTROL OF CROSSOVERS1 (MCC1), a GCN5-related histone N-acetyltransferase, is described in Arabidopsis. Analysis of the over-expression mutant obtained by enhancer activation tagging revealed that acetylation of histone H3 increased in male prophase I. MCC1 appeared to be required in meiosis for normal chiasma number and distribution and for chromosome segregation. Overall, elevated MCC1 did not affect crossover number per cell, but has a differential effect on individual chromosomes elevating COs for chromosome 4, in which there is also a shift in chiasma distribution, and reducing COs for chromosome 1 and 2. For the latter there is a loss of the obligate CO/chiasma in 8% of the male meiocytes. The meiotic defects led to abortion in about half of the male and female gametes in the mutant. In wild type, the treatment with trichostatin A, an inhibitor of histone deacetylases, phenocopies MCC1 over-expression in meiosis. Our results provide evidence that histone hyperacetylation has a significant impact on the plant meiosis.

Identification of the pollen self-incompatibility determinant in Papaver rhoeas.

Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. 'Self' (incompatible) pollen is discriminated from 'non-self' (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca(2+)-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel approximately 20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

ASY1 mediates AtDMC1-dependent interhomolog recombination during meiosis in Arabidopsis.

ASY1 is an Arabidopsis protein required for synapsis and crossover formation during meiosis. The chronology of meiotic recombination has been investigated in wild type and an asy1 mutant. We observe a delay between the appearance of chromatin-associated AtSPO11-1 foci and DNA double-strand break (DSB) formation, which occurs contemporaneously with chromosome axis formation and transition of ASY1 from chromatin-associated foci to a linear axis-associated signal. DSBs are formed independently of ASY1 in an AtSPO11-1-dependent manner. They are partially restored in Atspo11-1-3 using cisplatin, but their control appears abnormal. Axis morphogenesis is independent of ASY1, but axis structure may be compromised in asy1. Localization of the strand exchange proteins AtRAD51 and AtDMC1 to the chromatin occurs asynchronously shortly after DSB formation, with AtDMC1 localizing in advance of AtRAD51. In wild-type nuclei, both recombinases form numerous foci that persist for approximately 12 h before gradually decreasing in number. In asy1, initial localization of AtDMC1 is normal, but declines abruptly such that interhomolog recombination is severely compromised. Limited ASY1-independent, DMC1-dependent interhomolog recombination remains, but appears restricted to subtelomeric sequences where the homologs are fortuitously in proximity. Thus, ASY1 plays a key role in coordinating the activity of the RecA homologs to create a bias in favor of interhomolog recombination.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen.

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.

A puromycin-sensitive aminopeptidase is essential for meiosis in Arabidopsis thaliana.

Puromycin-sensitive aminopeptidases (PSAs) participate in a variety of proteolytic events essential for cell growth and viability, and in fertility in a broad range of organisms. We have identified and characterized an Arabidopsis thaliana mutant (mpa1) from a pool of T-DNA tagged lines that lacks PSA activity. This line exhibits reduced fertility, producing shorter siliques (fruits) bearing a lower number of seeds compared with wild-type plants. Cytogenetic characterization of meiosis in the mutant line reveals that both male and female meiosis are defective. In mpa1, early prophase I appears normal, but after pachytene most of the homologous chromosomes are desynaptic, thus, by metaphase I a high level of univalence is observed subsequently leading to abnormal chromosome segregation. Wild-type plants treated with specific inhibitors of PSA show a very similar desynaptic phenotype to that of the mutant line. A fluorescent PSA-specific bioprobe, DAMPAQ-22, reveals that the protein is maximally expressed in wild-type meiocytes during prophase I and is absent in mpa1. Immunolocalization of meiotic proteins showed that the meiotic recombination pathway is disrupted in mpa1. Chromosome pairing and early recombination appears normal, but progression to later stages of recombination and complete synapsis of homologous chromosomes are blocked.

The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis.

MSH4, a meiosis-specific member of the MutS-homolog family of genes, is required for normal levels of recombination and fertility in budding yeast, mouse, and Caenorhabditis elegans. In this paper, we report the identification and characterization of the Arabidopsis homolog of MSH4 (AtMSH4). We demonstrate that AtMSH4 expression can only be detected in floral tissues, consistent with a role in reproduction. Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes. A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent with a meiotic defect; this was confirmed by cytological analysis of meiosis. RNAi-induced down-regulation of the MSH4 gene resulted in a similar fertility and meiotic phenotype. We demonstrate that prophase I chromosome synapsis is delayed and may be incomplete in Atmsh4, and metaphase I chiasma frequency is greatly reduced to approximately 15% of wild type, leading to univalence and nondisjunction. We show that these residual chiasmata are randomly distributed among cells and chromosomes. These features of chiasma frequency and distribution in Atmsh4 show close parallels to MSH4-independent crossovers in budding yeast that have been proposed to originate by a separate pathway. Furthermore, the characteristics of the MSH4-independent chiasmata in the Atmsh4 mutant closely parallel those of second-pathway crossovers that have been postulated from Arabidopsis crossover analysis and mathematical modeling. Taken together, this evidence strongly indicates that Arabidopsis possesses two crossover pathways.

Activation of a putative MAP kinase in pollen is stimulated by the self-incompatibility (SI) response.

Mitogen-activated protein kinases (MAPKs) operate downstream of receptor-ligand interactions, playing a pivotal role in responses to extracellular signals. The self-incompatibility (SI) response in Papaver rhoeas L. triggers a Ca2+-dependent signalling cascade resulting in inhibition of incompatible pollen. We have investigated the possible involvement of MAPKs in SI. We report the enhanced activation of a 56 kDa protein kinase (p56) in SI-induced pollen and provide evidence that p56 has MAPK activity. This provides an important advance in our understanding of the SI response. We believe this is the first direct biochemical demonstration of activation of a MAPK during SI.

The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis.

We report the detailed characterization of SWITCH1 (SWI1) an Arabidopsis thaliana protein that has been linked with the establishment of sister chromatid cohesion during meiosis. Using a combination of cytological methods including immunolocalization of meiotic chromosome-associated proteins we show that SWI1 is required for formation of axial elements. Our studies reveal that the swi1-2 mutation prevents the formation of RAD51 foci during meiotic prophase and suppresses the chromosome fragmentation phenotype of the recombination-defective dif1-1 mutant. Together, these data suggest that SWI1 may be required for meiotic recombination initiation. Finally we raised an antibody against SWI1 and showed, by immunolocalization coupled with bromodeoxyuridine incorporation experiments, that SWI1 is expressed exclusively in meiotic G(1) and S phase. Thus, SWI1 appears to be required for early meiotic events that are at the crossroad of sister chromatid cohesion, recombination and axial element formation. The possible inter-relationship between these processes and the function of SWI1 are discussed.

Dissecting plant meiosis using Arabidopsis thaliana mutants.

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 identified 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 flowering plants.