AtSPO11-1 is necessary for efficient meiotic recombination in plants

The Saccharomyces cerevisiae Spo11 protein catalyses DNA double-strand breaks (DSBs) that initiate meiotic recombination. The model plant Arabidopsis thaliana possesses at least three SPO11 homologues. T-DNA and ethyl-methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11-1 gene is disrupted. Both male and female meiocytes formed very few bivalents. Furthermore, no fully synapsed chromosomes were observed during prophase I. Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non-functional gametes. These meiotic aberrations were associated with a drastic reduction in meiotic recombination. Thus, our data show that initiation of meiotic recombination by SPO11- induced DSBs is a mechanism conserved in plants. Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.     

The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation

Recent studies in Saccharomyces cerevisiae have unveiled that meiotic recombination crossovers are formed by two genetically distinct pathways: a major interference-sensitive pathway and a minor interference-insensitive pathway. Several proteins, including the MSH4/MSH5 heterodimer and the MER3 DNA helicase, are indispensable for the interference-sensitive pathway. MSH4 homologs have been identified in mice and Arabidopsis and shown to be required for normal levels of crossovers, suggesting that the function of MSH4 may be conserved among major eukaryotic kingdoms. However, it is not known whether an MER3-like function is also required for meiosis in animals and plants. We have identified an Arabidopsis gene that encodes a putative MER3 homolog and is preferentially expressed in meiocytes. T-DNA insertional mutants of this gene exhibit defects in fertility and meiosis. Detailed cytological studies indicate that the mutants are defective in homolog synapsis and crossover formation, resulting in a reduction of bivalents and in the formation of univalents at late prophase I. We have named this gene ROCK-N-ROLLERS (RCK) to reflect the mutant phenotype of chromosomes undergoing the meiotic 'dance' either in pairs or individually. Our results demonstrate that an MER3-like function is required for meiotic crossover in plants and provide further support for the idea that Arabidopsis, like the budding yeast, possesses both interference-sensitive and insensitive pathways for crossover formation.     

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.     

Chromosome synapsis defects and sexually dimorphic meiotic progression in mice lacking Spo11

Spo11, a protein first identified in yeast, is thought to generate the chromosome breaks that initiate meiotic recombination. We now report that disruption of mouse Spo11 leads to severe gonadal abnormalities from defective meiosis. Spermatocytes suffer apoptotic death during early prophase; oocytes reach the diplotene/dictyate stage in nearly normal numbers, but most die soon after birth. Consistent with a conserved function in initiating meiotic recombination, Dmc1/Rad51 focus formation is abolished. Spo11(-/-) meiocytes also display homologous chromosome synapsis defects, similar to fungi but distinct from flies and nematodes. We propose that recombination initiation precedes and is required for normal synapsis in mammals. Our results also support the view that mammalian checkpoint responses to meiotic recombination and/or synapsis defects are sexually dimorphic.     

The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein

Progression through the meiotic cell cycle is an essential part of the developmental program of sporogenesis in plants. The duet mutant of Arabidopsis was identified as a male sterile mutant that lacked pollen and underwent an aberrant male meiosis. Male meiocyte division resulted in the formation of two cells instead of a normal tetrad. In wild type, male meiosis extends across two successive bud positions in an inflorescence whereas in duet, meiotic stages covered three to five bud positions indicating defective progression. Normal microspores were absent in the mutant and the products of the aberrant meiosis were uni- to tri-nucleate cells that later degenerated, resulting in anthers containing largely empty locules. Defects in male meiotic chromosome organization were observed starting from diplotene and extending to subsequent stages of meiosis. There was an accumulation of meiotic structures at metaphase 1, suggesting an arrest in cell cycle progression. Double mutant analysis revealed interaction with dyad, a mutation causing chromosome cohesion during female meiosis. Cloning and molecular analysis of DUET indicated that it potentially encodes a PHD-finger protein and shows specific expression in male meiocytes. Taken together these data suggest that DUET is required for male meiotic chromosome organization and progression.     

A link between meiotic prophase progression and crossover control

During meiosis, most organisms ensure that homologous chromosomes undergo at least one exchange of DNA, or crossover, to link chromosomes together and accomplish proper segregation. How each chromosome receives a minimum of one crossover is unknown. During early meiosis in Caenorhabditis elegans and many other species, chromosomes adopt a polarized organization within the nucleus, which normally disappears upon completion of homolog synapsis. Mutations that impair synapsis even between a single pair of chromosomes in C. elegans delay this nuclear reorganization. We quantified this delay by developing a classification scheme for discrete stages of meiosis. Immunofluorescence localization of RAD-51 protein revealed that delayed meiotic cells also contained persistent recombination intermediates. Through genetic analysis, we found that this cytological delay in meiotic progression requires double-strand breaks and the function of the crossover-promoting heteroduplex HIM-14 (Msh4) and MSH-5. Failure of X chromosome synapsis also resulted in impaired crossover control on autosomes, which may result from greater numbers and persistence of recombination intermediates in the delayed nuclei. We conclude that maturation of recombination events on chromosomes promotes meiotic progression, and is coupled to the regulation of crossover number and placement. Our results have broad implications for the interpretation of meiotic mutants, as we have shown that asynapsis of a single chromosome pair can exert global effects on meiotic progression and recombination frequency.     

The Sgs1 helicase regulates chromosome synapsis and meiotic crossing over

BACKGROUND: In budding yeast, Sgs1 is the sole member of the RecQ family of DNA helicases. Like the human Bloom syndrome helicase (BLM), Sgs1 functions during both vegetative growth and meiosis. The sgs1 null mutant sporulates poorly and displays reduced spore viability. RESULTS: We have identified novel functions for Sgs1 in meiosis. Loss of Sgs1 increases the number of axial associations, which are connections between homologous chromosomes that serve as initiation sites for synaptonemal complex formation. In addition, mutation of SGS1 increases the number of synapsis initiation complexes and increases the rate of chromosome synapsis. Loss of Sgs1 also increases the number of meiotic crossovers without changing the frequency of gene conversion. The sgs1 defect in sporulation is due to checkpoint-induced arrest/delay at the pachytene stage of meiotic prophase. A non-null allele of SGS1 that specifically deletes the helicase domain is defective in the newly described meiotic functions of Sgs1, but wild-type for most vegetative functions and for spore formation. CONCLUSIONS: We have shown that the helicase domain of Sgs1 serves as a negative regulator of meiotic interchromosomal interactions. The activity of the wild-type Sgs1 protein reduces the numbers of axial associations, synapsis initiation complexes, and crossovers, and decreases the rate of chromosome synapsis. Our data argue strongly that axial associations marked by synapsis initiation complexes correspond to sites of reciprocal exchange. We propose that the Sgs1 helicase prevents a subset of recombination intermediates from becoming crossovers, and this distinction is made at an early stage in meiotic prophase.     

<Emphasis Type="Italic">Coprinus cinereus</Emphasis> Mer3 is required for synaptonemal complex formation during meiosis

Mer3 is an evolutionarily conserved DNA helicase that has crucial roles in meiotic recombination and crossover formation. We have identified the MER3 homolog in Coprinus cinereus (Ccmer3) and show that it is expressed in zygotene and pachytene meiocytes. Immunostaining analysis indicated that CcMer3 was localized on chromosomes at zygotene and pachytene and CcMer3 foci were more frequent on paired than unpaired chromosomes. We generated a C. cinereus mer3 mutant (#1) and found that it showed abnormal meiosis progression and underwent apoptosis after prophase I. Basidiospore production in #1 was reduced to 0.8% of the wild-type level; the spores showed slower germination at 25°C but were similar to the wild type at 37°C. Electron microscopic analysis of chromosome spreads revealed that axial elements were formed in the mutant but that synapsis was defective, resulting in a reduction in spore production. Our results demonstrate that CcMer3 is required for synaptonemal complex formation after axial elements align and is thus essential for homologous synapsis.     

The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.)

The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene transition but cluster slowly if at all as meiosis proceeds. Intermediate stages in telomere clustering including miniclusters are observed in pam1 but not in wild-type meiocytes. The tight bouquet normally seen at zygotene is a rare event. In contrast, the polarization of centromeres vs. telomeres in the nucleus at the leptotene-zygotene transition is the same in mutant and wild-type cells. Defects in homologous chromosome synapsis include incomplete synapsis, nonhomologous synapsis, and unresolved interlocks. However, the number of RAD51 foci on chromosomes in pam1 is similar to that of wild type. We suggest that the defects in homologous synapsis and the retardation of prophase I arise from the irregularity of telomere clustering and propose that pam1 is involved in the control of bouquet formation and downstream meiotic prophase I events.     

Mouse pachytene checkpoint 2 (trip13) is required for completing meiotic recombination but not synapsis

In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice.     

Evidence That Masking of Synapsis Imperfections Counterbalances Quality Control to Promote Efficient Meiosis

Reduction in ploidy to generate haploid gametes during sexual reproduction is accomplished by the specialized cell division program of meiosis. Pairing between homologous chromosomes and assembly of the synaptonemal complex at their interface (synapsis) represent intermediate steps in the meiotic program that are essential to form crossover recombination-based linkages between homologs, which in turn enable segregation of the homologs to opposite poles at the meiosis I division. Here, we challenge the mechanisms of pairing and synapsis during C. elegans meiosis by disrupting the normal 1∶1 correspondence between homologs through karyotype manipulation. Using a combination of cytological tools, including S-phase labeling to specifically identify X chromosome territories in highly synchronous cohorts of nuclei and 3D rendering to visualize meiotic chromosome structures and organization, our analysis of trisomic (triplo-X) and polyploid meiosis provides insight into the principles governing pairing and synapsis and how the meiotic program is “wired” to maximize successful sexual reproduction. We show that chromosomes sort into homologous groups regardless of chromosome number, then preferentially achieve pairwise synapsis during a period of active chromosome mobilization. Further, comparisons of synapsis configurations in triplo-X germ cells that are proficient or defective for initiating recombination suggest a role for recombination in restricting chromosomal interactions to a pairwise state. Increased numbers of homologs prolong markers of the chromosome mobilization phase and/or boost germline apoptosis, consistent with triggering quality control mechanisms that promote resolution of synapsis problems and/or cull meiocytes containing synapsis defects. However, we also uncover evidence for the existence of mechanisms that “mask” defects, thus allowing resumption of prophase progression and survival of germ cells despite some asynapsis. We propose that coupling of saturable masking mechanisms with stringent quality controls maximizes meiotic success by making progression and survival dependent on achieving a level of synapsis sufficient for crossover formation without requiring perfect synapsis.     

Retinoblastoma protein is essential for early meiotic events in Arabidopsis

We have analysed the role of RBR (retinoblastoma related), the Arabidopsis homologue of the tumour suppressor Retinoblastoma protein (pRb), during meiosis. We characterise the rbr-2 mutation, which causes a loss of RBR in male meiocytes. The rbr-2 plants exhibit strongly reduced fertility, while vegetative growth is generally unaffected. The reduced fertility is due to a meiotic defect that results in reduced chiasma formation and subsequent errors in chromosome disjunction. Immunolocalisation studies in wild-type meiocytes reveal that RBR is recruited as foci to the chromosomes during early prophase I in a DNA double-strand-break-dependent manner. In the absence of RBR, expression of several meiotic genes is reduced. The localisation of the recombinases AtRAD51 and AtDMC1 is normal. However, localisation of the MutS homologue AtMSH4 is compromised. Additionally, polymerisation of the synaptonemal complex protein AtZYP1 is abnormal. Together, these data indicate that loss of RBR during meiosis results in a reduction of crossover formation and an associated failure in chromosome synapsis. Our results indicate that RBR has an important role in meiosis affecting different aspects of this complex process.     

The AtRAD51C gene is required for normal meiotic chromosome synapsis and double-stranded break repair in Arabidopsis

Meiotic prophase I is a complex process involving homologous chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be important for recombination and DNA repair in the mitotic cell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important for normal meiotic homolog pairing, synapsis, and repair of double-stranded breaks. In vertebrate cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous recombination and maintenance of genome integrity. However, the function of RAD51C in meiosis is not well understood. Here we describe the identification and analysis of a mutation in the Arabidopsis RAD51C ortholog, AtRAD51C. Although the atrad51c-1 mutant has normal vegetative and flower development and has no detectable abnormality in mitosis, it is completely male and female sterile. During early meiosis, homologous chromosomes in atrad51c-1 fail to undergo synapsis and become severely fragmented. In addition, analysis of the atrad51c-1 atspo11-1 double mutant showed that fragmentation was nearly completely suppressed by the atspo11-1 mutation, indicating that the fragmentation largely represents a defect in processing double-stranded breaks generated by AtSPO11-1. Fluorescence in situ hybridization experiments suggest that homolog juxtaposition might also be abnormal in atrad51c-1 meiocytes. These results demonstrate that AtRAD51C is essential for normal meiosis and is probably required for homologous synapsis.     

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three‐dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre‐meiotic G₂ and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.     

Genetic collection of meiotic mutants of rye Secale cereale L

Genetic collection of meiotic mutants of winter rye Secale cereale L. (2n = 14) was created. Mutations were detected in inbred F2 generations after self-fertilization of the F1 hybrids, obtained by individual crossing of rye plants (cultivar Vyatka) or weedy rye with plants from autofertile lines. The mutations cause partial or complete plant sterility and are maintained in collection in a heterozygous state. Genetic analysis accompanied by cytogenetic study of meiosis has revealed six mutation types. (1) Nonallelic asynaptic mutations sy1 and sy9 caused the formation of only axial chromosome elements in prophase and anaphase. The synaptonemal complexes (SCs) were absent, the formation of the chromosome "bouquet" was impaired, and all chromosomes were univalent in meiotic metaphase I in 96% (sy1) and 67% (sy2) of cells. (2) Weak asynaptic mutation sy3, which hindered complete termination of synapsis in prophase II. Subterminal asynaptic segments were always observed in the SC, and at least one pair of univalents was present in metaphase I, but the number of cells with univalents did not exceed 2%. (3) Mutations sy2, sy6, sy7, sy8, sy10, and sy19, which caused partially nonhomologous synapsis: change in pairing partners and fold-back chromosome synapsis in prophase I. In metaphase I, the number of univalents varied and multivalents were observed. (4) Mutation mei6, which causes the formation of ultrastructural protrusions on the lateral SC elements, gaps and branching of these elements. (5) Allelic mutations mei8 and mei10, which caused irregular chromatin condensation along chromosomes in prophase I, sticking and fragmentation of chromosomes in metaphase I. (6) Allelic mutations mei5 and mei10, which caused chromosome hypercondensation, defects of the division spindle formation, and random arrest of cells at different meiotic stages. However, these mutations did not affect the formation of microspore envelopes even around the cells, whose development was blocked at prophase I. Analysis of cytological pictures of meiosis in double rye mutants reveled epistatic interaction in the mutation series sy9 > sy1 > sy3 > sy19, which reflects the order of switching these genes in the course of meiosis. The expression of genes sy2 and sy19 was shown to be controlled by modifier genes. Most meiotic mutations found in rye have analogs in other plant species.     

Semisterile meiotic mutant <Emphasis Type="Italic">sy11</Emphasis> with heterologous chromosome synapsis in rye <Emphasis Type="Italic">Secale cereale</Emphasis> L.

A study was made of the expression and inheritance of the sy11 mutation, which alters homologous chromosome synapsis in meiotic prophase I of rye. The abnormal phenotype proved to be determined by a recessive allele of a single sy11 gene. Univalents and multivalents were observed in homozygotes for the mutant allele. Analysis of the synaptonemal complex revealed a combination of homologous and nonhomologous synapsis in the mutant. The nonhomologous synapsis frequency significantly decreased in the course of meiotic prophase I in the mutant. The number of chiasmata per bivalent in metaphase I was 1.1 ± 0.01 versus 1.8 ± 0.01 in wild-type plants, and the number of univalents was 2.7 ± 0.06 versus 0.5 ± 0.05 in wild-type plants. As a result, a broad range of abnormalities was observed at subsequent stages of meiosis and led to the formation of defective microspores. Mutant plants were semisterile.     

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana . The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.     

Meiotic mutants of rye Secale cereale L.

Summary A mutant form of weedy rye characterized by male and female sterility and having a hereditary block in the chromosome synapsis has been found and described. Genetic analysis has shown the synapsis block to be determined by the recessive allele of a gene designated as sy-1. Electron microscopy of surface-spread microsporocyte nuclei revealed the complete absence of the synaptonemal complex over the whole meiotic prophase I, although the axial cores were perfectly formed by each chromosome. Only univalents were observed at metaphase I, their average number ranging from 13.1 to 14.0 per cell. A precocious distribution of univalents at the poles is observed at metaphase I. All of the later stages of meiosis were irregular and resulted in the formation of abnormal microspores. Thus, the mutant proves to be asynaptic because of the blocked initiation of synapses at prophase I.     

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.