Meiosis is a cellular division, essential for sexual reproduction. During meiotic prophase, homologous chromosomes of maternal and paternal origin recognize each other and associate, a process that is facilitated by their rapid movements in the nucleoplasm. Meiotic prophase movements rely on the attachment of the chromosomes to the nuclear envelope and on cytoplasmic forces transmitted to the chromosomes through the nuclear envelope. In plants, the limited accessibility and fragility of male meiocytes inside the anthers make live imaging approaches and chromosome movement analysis technically challenging. Indeed, these movements are very rapid, and analyzing them requires finding the optimal acquisition conditions. Here, an efficient method to capture rapid chromosome movements during male meiotic prophase in Arabidopsis thaliana is described, in time-lapse movies, by following the movements of fluorescent-tagged centromeres. It is based on anther dissection and image acquisition using confocal laser scanning microscopy (CLSM), in 3D and over time. Acquiring meiotic z-stacks every 7 s allows sufficient resolution to reconstruct and analyze the trajectories of tagged centromeres. The extracted data is subsequently processed, enabling the quantitative analysis of these chromosomal movements. The development of models from this data is essential for understanding the mechanism of rapid chromosome movement, including the nature and intensity of the forces driving this process.

Homologous pairing and recombination during meiosis are facilitated by rapid prophase movements (RPMs), which depend on chromosome attachment to the nuclear envelope (NE) and on cytoplasmic forces transmitted to the chromosomes through the NE, mediated by Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes. In plants, only the NE-associated SUN-domain proteins SUN1 and SUN2 have been identified as components of the RPM process. Here we show that, during meiosis, SUN1 and SUN2 form a LINC complex with the KASH-domain protein SINE3, which recruits the meiosis-specific kinesin PSS1 to the NE. These proteins accumulate at telomere-binding sites in the NE, and their loss disrupts telomere attachment and bouquet formation and abolishes RPMs. These defects lead to defective synapsis and clustered crossovers, resulting in chromosome mis-segregation. Our results establish that the mechanism underlying RPMs is conserved in Arabidopsis thaliana, with RPMs primarily facilitating homologous recognition rather than preventing non-homologous interactions. The rapid chromosomal movements during meiotic prophase in Arabidopsis thaliana depend on telomere-to-cytoskeleton attachment through the nuclear envelope via a protein complex composed of the kinesin PSS1, the KASH protein SINE3 and the carboxy-terminal SUN-domain proteins.

Formation of programmed DNA double-strand breaks is essential for initiating meiotic recombination. Genetic studies on Arabidopsis thaliana and Mus musculus have revealed that assembly of a type IIB topoisomerase VI (Topo VI)-like complex, composed of SPO11 and MTOPVIB, is a prerequisite for generating DNA breaks. However, it remains enigmatic if MTOPVIB resembles its Topo VI subunit B (VIB) ortholog in possessing robust ATPase activity, ability to undergo ATP-dependent dimerization, and activation of SPO11-mediated DNA cleavage. Here, we successfully prepared highly pure A. thaliana MTOPVIB and MTOPVIB-SPO11 complex. Contrary to expectations, our findings highlight that MTOPVIB differs from orthologous Topo VIB by lacking ATP-binding activity and independently forming dimers without ATP. Most significantly, our study reveals that while MTOPVIB lacks the capability to stimulate SPO11-mediated DNA cleavage, it functions as a bona fide DNA-binding protein and plays a substantial role in facilitating the dsDNA binding capacity of the MOTOVIB-SPO11 complex. Thus, we illustrate mechanistic divergence between the MTOPVIB-SPO11 complex and classical type IIB topoisomerases.

Meiotic rapid prophase chromosome movements (RPMs) require connections between the chromosomes and the cytoskeleton, involving SUN (Sad1/UNC-84)-domain-containing proteins at the inner nuclear envelope (NE). RPMs remain significantly understudied in plants, with respect to their importance in the regulation of meiosis. Here, we demonstrate that Arabidopsis thaliana meiotic centromeres undergo rapid (up to 500 nm/s) and uncoordinated movements during the zygotene and pachytene stages. These centromere movements are not affected by altered chromosome organization and recombination but are abolished in the double mutant sun1 sun2. We also document the changes in chromosome dynamics and nucleus organization during the transition from leptotene to zygotene, including telomere attachment to SUN-enriched NE domains, bouquet formation, and nucleolus displacement, all of which were defective in sun1 sun2. These results establish A. thaliana as a model species for studying the functional implications of meiotic RPMs and demonstrate the mechanistic conservation of telomere-led RPMs in plants.

Meiosis is a specialized eukaryotic division that produces genetically diverse gametes for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal exchanges, called crossovers, which recombine genetic variation. Meiotic crossovers are stringently controlled with at least one obligate exchange forming per chromosome pair, while closely spaced crossovers are inhibited by interference. In Arabidopsis, crossover positions can be explained by a diffusion-mediated coarsening model, in which large, approximately evenly spaced foci of the pro-crossover E3 ligase HEI10 grow at the expense of smaller, closely spaced clusters. However, the mechanisms that control HEI10 dynamics during meiosis remain unclear. Here, through a forward genetic screen in Arabidopsis, we identified high crossover rate3 (hcr3), a dominant-negative mutant that reduces crossover interference and increases crossovers genome-wide. HCR3 encodes J3, a co-chaperone related to HSP40, which acts to target protein aggregates and biomolecular condensates to the disassembly chaperone HSP70, thereby promoting proteasomal degradation. Consistently, we show that a network of HCR3 and HSP70 chaperones facilitates proteolysis of HEI10, thereby regulating interference and the recombination landscape. These results reveal a new role for the HSP40/J3-HSP70 chaperones in regulating chromosome-wide dynamics of recombination via control of HEI10 proteolysis.

We report the discovery and characterization of two small coiled-coil proteins that belong to the central element of the plant synaptonemal complex.

The synaptonemal complex (SC) is a proteinaceous structure that forms between homologous chromosomes during meiosis prophase. The SC is widely conserved across species, but its structure and roles during meiotic recombination are still debated. While the SC central region is made up of transverse filaments and central element proteins in mammals and fungi, few central element proteins have been identified in other species. Here we report the identification of two coiled-coil proteins, SCEP1 and SCEP2, that form a complex and localize at the centre of the Arabidopsis thaliana SC. In scep1 and scep2 mutants, chromosomes are aligned but not synapsed (the ZYP1 transverse filament protein is not loaded), crossovers are increased compared with the wild type, interference is lost and heterochiasmy is strongly reduced. We thus report the identification of two plant SC central elements, and homologues of these are found in all major angiosperm clades.

Most meiotic crossovers (COs), called class I crossovers, are produced by a conserved pathway catalyzed by the ZMM proteins; COs are limited in number, typically to 1–3 per chromosome, and are prevented from occurring close to one other by crossover interference 1-3 . In many species, CO number is subject to dimorphism between males and females, and a lower CO number is associated with shorter chromosome axes and stronger interference 4 . How the patterning of COs is imposed, however, remains poorly understood. Here, we show that overexpression of the ZMM protein HEI10 increases COs and reduces crossover interference but maintains sexual dimorphism; shorter axes length in female meiosis is still associated with fewer COs and stronger interference than in male meiocytes. Disrupting the synaptonemal complex (SC) by mutating ZYP1 also leads to an increase in class I COs but, in contrast, abolishes interference and disrupts the link between chromosome axis length and COs, with female and male meiocytes having the same CO frequency despite different axis lengths. Combining HEI10 overexpression and zyp1 mutation leads to a massive increase in class I COs and absence of interference, while axes lengths are still unaffected. These observations support, and can be effectively predicted by, a recently proposed coarsening model 5,6 in which HEI10 diffusion is funneled by the central element of the SC before coarsening into large, well-spaced CO-promoting droplets. Given the conservation of the components, this model may account for CO patterning in many eukaryotes.

Meiotic crossovers are limited in number and are prevented from occurring close to each other by crossover interference. In many species, crossover number is subject to sexual dimorphism, and a lower crossover number is associated with shorter chromosome axes lengths. How this patterning is imposed remains poorly understood. Here, we show that overexpression of the Arabidopsis pro-crossover protein HEI10 increases crossovers but maintains some interference and sexual dimorphism. Disrupting the synaptonemal complex by mutating ZYP1 also leads to an increase in crossovers but, in contrast, abolishes interference and disrupts the link between chromosome axis length and crossovers. Crucially, combining HEI10 overexpression and zyp1 mutation leads to a massive and unprecedented increase in crossovers. These observations support and can be predicted by, a recently proposed model in which HEI10 diffusion along the synaptonemal complex drives a coarsening process leading to well-spaced crossover-promoting foci, providing a mechanism for crossover patterning.

Meiotic crossovers (COs) have intriguing patterning properties, including CO interference, the tendency of COs to be well-spaced along chromosomes, and heterochiasmy, the marked difference in male and female CO rates. During meiosis, transverse filaments transiently associate the axes of homologous chromosomes, a process called synapsis that is essential for CO formation in many eukaryotes. Here, we describe the spatial organization of the transverse filaments in Arabidopsis (ZYP1) and show it to be evolutionary conserved. We show that in the absence of ZYP1 (zyp1azyp1b null mutants), chromosomes associate in pairs but do not synapse. Unexpectedly, in absence of ZYP1, CO formation is not prevented but increased. Furthermore, genome-wide analysis of recombination revealed that CO interference is abolished, with the frequent observation of close COs. In addition, heterochiasmy was erased, with identical CO rates in males and females. This shows that the tripartite synaptonemal complex is dispensable for CO formation and has a key role in regulating their number and distribution, imposing CO interference and heterochiasmy.

In the current meiotic recombination initiation model, the SPO11 catalytic subunits associate with MTOPVIB to form a Topoisomerase VI-like complex that generates DNA double strand breaks (DSBs). Four additional proteins, PRD1/AtMEI1, PRD2/AtMEI4, PRD3/AtMER2 and the plant specific DFO are required for meiotic DSB formation. Here we show that (i) MTOPVIB and PRD1 provide the link between the catalytic sub-complex and the other DSB proteins, (ii) PRD3/AtMER2, while localized to the axis, does not assemble a canonical pre-DSB complex but establishes a direct link between the DSB-forming and resection machineries, (iii) DFO controls MTOPVIB foci formation and is part of a divergent RMM-like complex including PHS1/AtREC114 and PRD2/AtMEI4 but not PRD3/AtMER2, (iv) PHS1/AtREC114 is absolutely unnecessary for DSB formation despite having a conserved position within the DSB protein network and (v) MTOPVIB and PRD2/AtMEI4 interact directly with chromosome axis proteins to anchor the meiotic DSB machinery to the axis.

Meiotic crossovers (COs) are important for reshuffling genetic information between homologous chromosomes and they are essential for their correct segregation. COs are unevenly distributed along chromosomes and the underlying mechanisms controlling CO localization are not well understood. We previously showed that meiotic COs are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis thaliana. Here, we report that in axr1-/-, male meiocytes show a strong defect in chromosome pairing whereas the formation of the telomere bouquet is not affected. COs are also redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of transposable elements (TEs) in the CHH context in axr1-/- meiocytes. Through examining somatic methylomes, we found axr1-/- affects DNA methylation in a plant, causing hypermethylation in all sequence contexts (CG, CHG and CHH) in TEs. Impairment of the main pathways involved in DNA methylation is epistatic over axr1-/- for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings reveal that the neddylation pathway not only regulates hormonal perception and CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells.

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.