The Arabidopsis Cdk1/Cdk2 homolog CDKA;1 controls chromosome axis assembly during plant meiosis.

Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.

ZmASY1 interacts with ZmPRD3 and is crucial for meiotic double-strand break formation in maize.

During meiosis, recombination-mediated pairing and synapsis of homologous chromosomes begin with programmed DNA double-strand breaks (DSBs). In yeast and mice, DSBs form in a tethered loop-axis complex, in which DSB sites are located within chromatin loops and tethered to the proteinaceous axial element (AE) by DSB-forming factors. In plants, the molecular connection between DSB sites and chromosome axes is poorly understood. By integrating genetic analysis, immunostaining technology, and protein-protein interaction studies, the putative factors linking DSB formation to chromosome axis were explored in maize meiosis. Here, we report that the AE protein ZmASY1 directly interacts with the DSB-forming protein ZmPRD3 in maize (Zea mays) and mediates DSB formation, synaptonemal complex assembly, and homologous recombination. ZmPRD3 also interacts with ZmPRD1, which plays a central role in organizing the DSB-forming complex. These results suggest that ZmASY1 and ZmPRD3 may work as a key module linking DSB sites to chromosome axes during DSB formation in maize. This mechanism is similar to that described in yeast and recently Arabidopsis involving the homologs Mer2/ZmPRD3 and HOP1/ZmASY1, thus indicating that the process of tethering DSBs in chromatin loops to the chromosome axes may be evolutionarily conserved in diverse taxa.

ASY1 acts as a dosage-dependent antagonist of telomere-led recombination and mediates crossover interference in Arabidopsis.

During meiosis, interhomolog recombination produces crossovers and noncrossovers to create genetic diversity. Meiotic recombination frequency varies at multiple scales, with high subtelomeric recombination and suppressed centromeric recombination typical in many eukaryotes. During recombination, sister chromatids are tethered as loops to a polymerized chromosome axis, which, in plants, includes the ASY1 HORMA domain protein and REC8-cohesin complexes. Using chromatin immunoprecipitation, we show an ascending telomere-to-centromere gradient of ASY1 enrichment, which correlates strongly with REC8-cohesin ChIP-seq data. We mapped crossovers genome-wide in the absence of ASY1 and observe that telomere-led recombination becomes dominant. Surprisingly, asy1/+ heterozygotes also remodel crossovers toward subtelomeric regions at the expense of the pericentromeres. Telomeric recombination increases in asy1/+ occur in distal regions where ASY1 and REC8 ChIP enrichment are lowest in wild type. In wild type, the majority of crossovers show interference, meaning that they are more widely spaced along the chromosomes than expected by chance. To measure interference, we analyzed double crossover distances, MLH1 foci, and fluorescent pollen tetrads. Interestingly, while crossover interference is normal in asy1/+, it is undetectable in asy1 mutants, indicating that ASY1 is required to mediate crossover interference. Together, this is consistent with ASY1 antagonizing telomere-led recombination and promoting spaced crossover formation along the chromosomes via interference. These findings provide insight into the role of the meiotic axis in patterning recombination frequency within plant genomes.

The Control of the Crossover Localization in Allium.

Meiotic crossovers/chiasmata are not randomly distributed and strictly controlled. The mechanisms behind crossover (CO) patterning remain largely unknown. In Allium cepa, as in the vast majority of plants and animals, COs predominantly occur in the distal 2/3 of the chromosome arm, while in Allium fistulosum they are strictly localized in the proximal region. We investigated the factors that may contribute to the pattern of COs in A. cepa, A. fistulosum and their F1 diploid (2n = 2x = 8C + 8F) and F1 triploid (2n = 3x = 16F + 8C) hybrids. The genome structure of F1 hybrids was confirmed using genomic in situ hybridization (GISH). The analysis of bivalents in the pollen mother cells (PMCs) of the F1 triploid hybrid showed a significant shift in the localization of COs to the distal and interstitial regions. In F1 diploid hybrid, the COs localization was predominantly the same as that of the A. cepa parent. We found no differences in the assembly and disassembly of ASY1 and ZYP1 in PMCs between A. cepa and A. fistulosum, while F1 diploid hybrid showed a delay in chromosome pairing and a partial absence of synapsis in paired chromosomes. Immunolabeling of MLH1 (class I COs) and MUS81 (class II COs) proteins showed a significant difference in the class I/II CO ratio between A. fistulosum (50%:50%) and A. cepa (73%:27%). The MLH1:MUS81 ratio at the homeologous synapsis of F1 diploid hybrid (70%:30%) was the most similar to that of the A. cepa parent. F1 triploid hybrid at the A. fistulosum homologous synapsis showed a significant increase in MLH1:MUS81 ratio (60%:40%) compared to the A. fistulosum parent. The results suggest possible genetic control of CO localization. Other factors affecting the distribution of COs are discussed.

Comparative transcriptomic analysis of thermally stressed Arabidopsis thaliana meiotic recombination mutants.

BACKGROUND: Meiosis is a specialized cell division that underpins sexual reproduction in most eukaryotes. During meiosis, interhomolog meiotic recombination facilitates accurate chromosome segregation and generates genetic diversity by shuffling parental alleles in the gametes. The frequency of meiotic recombination in Arabidopsis has a U-shaped curve in response to environmental temperature, and is dependent on the Type I, crossover (CO) interference-sensitive pathway. The mechanisms that modulate recombination frequency in response to temperature are not yet known. RESULTS: In this study, we compare the transcriptomes of thermally-stressed meiotic-stage anthers from msh4 and mus81 mutants that mediate the Type I and Type II meiotic recombination pathways, respectively. We show that heat stress reduces the number of expressed genes regardless of genotype. In addition, msh4 mutants have a distinct gene expression pattern compared to mus81 and wild type controls. Interestingly, ASY1, which encodes a HORMA domain protein that is a component of meiotic chromosome axes, is up-regulated in wild type and mus81 but not in msh4. In addition, SDS the meiosis-specific cyclin-like gene, DMC1 the meiosis-specific recombinase, SYN1/REC8 the meiosis-specific cohesion complex component, and SWI1 which functions in meiotic sister chromatid cohesion are up-regulated in all three genotypes. We also characterize 51 novel, previously unannotated transcripts, and show that their promoter regions are associated with A-rich meiotic recombination hotspot motifs. CONCLUSIONS: Our transcriptomic analysis of msh4 and mus81 mutants enhances our understanding of how the Type I and Type II meiotic CO pathway respond to environmental temperature stress and might provide a strategy to manipulate recombination levels in plants.

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.

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat.

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synaptonemal complex (SC) construction in hexaploid wheat (2n = 42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarization (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganization of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarization were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues.

Analyses of phenotype and ARGOS and ASY1 expression in a ploidy Chinese cabbage series derived from one haploid.

The aim of this research was to improve our understanding of how ploidy level influences phenotype and gene expression in Chinese cabbage (Brassica rapa L. ssp. pekinensis). Haploid plants (2n = 10) was induced by 0.2% colchicine to produce diploid (2n = 20) and tetraploid plants (2n = 40). The aneuploid (2n = 24) was also obtained by hybridization between diploid plants as the female and tetraploid plants. The ploidy levels of all plants were identified through chromosome counts and flow cytometry. Leaves and petals became larger as the ploidy level increased from haploid to diploid, and from aneuploid to tetraploid. Similarly, expression of ARGOS was regulated by genome size, increasing in parallel with the level of ploidy. Among the four ploidy types, expression was stronger in the floral buds than in the leaves. Expression by ASY1 also differed according to ploidy level, being highest in diploid plants, followed in order by tetraploids. Expression was similar between haploids and aneuploids at two stages-prior to and after meiosis-but was higher in the haploids during meiosis. When buds were compared within the same ploidy type at different stages, ASY1 expression was obviously higher during meiosis than either before or after. Our study demonstrated the generation and phenotype of a ploidy Chinese cabbage series derived from one haploid. Expression of genes ARGOS and ASY1 were modulated by genome size in this ploidy series, and the regulated patterns of the two genes was different.

Chromoanagenesis in the asy1 meiotic mutant of Arabidopsis.

Chromoanagenesis is a catastrophic event that involves localized chromosomal shattering and reorganization. In this study, we report a case of chromoanagenesis resulting from defective meiosis in the MEIOTIC ASYNAPTIC MUTANT 1 (asy1) background in Arabidopsis thaliana. We provide a detailed characterization of the genomic structure of this individual with a severely shattered segment of chromosome 1. We identified 260 novel DNA junctions in the affected region, most of which affect gene sequence on 1 or both sides of the junction. Our results confirm that asy1-related defective meiosis is a potential trigger for chromoanagenesis. This is the first example of chromoanagenesis associated with female meiosis and indicates the potential for genome evolution during oogenesis. PLAIN LANGUAGE SUMMARY: Chromoanagenesis is a complex and catastrophic event that results in severely restructured chromosomes. It has been identified in cancer cells and in some plant samples, after specific triggering events. Here, we identified this kind of genome restructuring in a mutant that exhibits defective meiosis in the model plant system Arabidopsis thaliana.

COMET Functions as a PCH2 Cofactor in Regulating the HORMA Domain Protein ASY1.

The formation of the chromosome axis is key to meiotic recombination and hence the correct distribution of chromosomes to meiotic products. A key component of the axis in Arabidopsis is the HORMA domain protein (HORMAD) ASY1, the homolog of Hop1 in yeast and HORMAD1/2 in mammals. The chromosomal association of ASY1 is dynamic, i.e., ASY1 is recruited to the axis at early prophase and later largely removed when homologous chromosomes synapse. PCH2/TRIP13 proteins are well-known regulators of meiotic HORMADs and required for their depletion from synapsed chromosomes. However, no direct interaction has been found between PCH2/TRIP13 and the presumptive HORMAD substrates in any organism other than in budding yeast. Thus, it remains largely elusive how the dynamics of ASY1 and other meiotic HORMADs are controlled. Here, we have identified COMET, the Arabidopsis homolog of human p31comet, which is known for its function in the spindle assembly checkpoint (SAC), as a central regulator of ASY1 dynamics in meiosis. We provide evidence that COMET controls ASY1 localization by serving as an adaptor for PCH2. Because ASY1 accumulates in the cytoplasm in early prophase and is persistently present on chromosomes in comet, we conclude that COMET is required for both the recruitment of ASY1 to the nucleus and the subsequent removal from the axis. The here-revealed function of COMET as an adaptor for PCH2 remarkably resembles the regulation of another HORMAD, Mad2, in the SAC in yeast and animals, revealing a conserved regulatory module of HORMA-domain-containing protein complexes.

Observation of Extensive Chromosome Axis Remodeling during the "Diffuse-Phase" of Meiosis in Large Genome Cereals.

The production of balanced fertile haploid gametes requires the faithful separation of paired (synapsed) chromosomes toward the end of meiotic prophase I (desynapsis). This involves the timely dissolution of the synaptonemal complex during the pachytene-diplotene transition, a stage traditionally referred to as the "diffuse stage." In species with large genomes such as, barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.) we know most about the early stages of meiotic prophase I. There, synapsis initiates at the telomeric ends of chromosomes and progresses toward the centromeric regions through the ordered assembly of the synaptonemal complex (SC). Synapsis is impacted by recombination (crossing over, CO) which locally modifies the extent of chromatin compaction and extension. CO is uneven along the chromosomes, occurring mainly toward the telomeric regions resulting in a highly skewed distribution of recombination events. However, we know very little about the process of desynapsis which occurs during the "diffuse stage," where the synapsed and recombined chromosomes faithfully desynapse and separate into daughter cells. Here, using 3D-SIM super-resolution immuno-cytology combined with the use of antibodies directed against two crucial SC proteins, ASY1 and ZYP1, we followed the whole of meiosis I (i.e., both synapsis and desynapsis) in both barley and wheat. We showed that synapsis forms a characteristic tri-partite SC structure in zygotene (more clearly seen in barley). Toward the end of meiosis I, as the SC starts to disassemble, we show that extensive chromosome axis remodeling results in the formation of characteristic "tinsel-like" structures in both wheat and barley. By using a mutant (des10) that is severely compromised in polymerization of ZYP1during synapsis, we show that tinsel structure formation during SC dissolution is not dependant on full synapsis and may relate instead to changes in expansion stress. Our observations highlight a potentially new role for ASYNAPSIS1 (ASY1) in desynapsis, in addition to chromosome synapsis and cohesion.