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Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

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., Phosphorylation of the HORMA domain protein ASY1 by the central cell cycle kinase promotes its recruitment onto meiotic chromosomes both via protein interactions and by countering disassembly ATPase PCH2.

International audience; During the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components.

To understand how the identity of an organ can be switched, we studied the transformation of lateral root primordia (LRP) into shoot meristems in Arabidopsis root segments. In this system, the cytokinin-induced conversion does not involve the formation of callus-like structures. Detailed analysis showed that the conversion sequence starts with a mitotic pause and is concomitant with the differential expression of regulators of root and shoot development. The conversion requires the presence of apical stem cells and only LRP at stages VI or VII can be switched. It is engaged as soon as cell divisions resume because their position and orientation differ in the converting organ compared to the undisturbed emerging LRP. By alternating auxin and cytokinin treatments, we showed that the root and shoot organogenetic programs are remarkably plastic because the status of the same plant stem cell niche can be reversed repeatedly within a set developmental window. Thus, the networks at play in the meristem of a root can morph in the span of a couple cell division cycles into those of a shoot, and back, through transdifferentiation.

A partner protein for meiotic snip Eukaryotes generate germ cells through meiotic recombination. This process initiates through breaks in genomic DNA catalyzed by the SPO11 protein. Vrielynck et al. and Robert et al. discover that SPO11, like topoisomerase VI enzymes, interacts with a partner protein (see the Perspective by Bouuaert and Keeney). This partner is required for proper meiotic recombination and is found in a wide range of eukaryotes, suggesting that it is a universal feature of the essential recombination step. Science , this issue p. 939 , 943 ; see also p. 916

Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review, we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an up-to-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.

The sixth author's name is spelled incorrectly. The correct name is: Afef Lemhemdi. The correct citation is: Girard C, Chelysheva L, Choinard S, Froger N, Macaisne N, Lemhemdi A, et al. (2015) AAA-ATPase FIDGETIN-LIKE 1 and Helicase FANCM Antagonize Meiotic Crossovers by Distinct Mechanisms. PLoS Genet 11(7): e1005369. doi:10.1371/journal.pgen.1005369

Meiotic crossovers (COs) generate genetic diversity and are critical for the correct completion of meiosis in most species. Their occurrence is tightly constrained but the mechanisms underlying this limitation remain poorly understood. Here we identified the conserved AAA-ATPase FIDGETIN-LIKE-1 (FIGL1) as a negative regulator of meiotic CO formation. We show that Arabidopsis FIGL1 limits CO formation genome-wide, that FIGL1 controls dynamics of the two conserved recombinases DMC1 and RAD51 and that FIGL1 hinders the interaction between homologous chromosomes, suggesting that FIGL1 counteracts DMC1/RAD51-mediated inter-homologue strand invasion to limit CO formation. Further, depleting both FIGL1 and the previously identified anti-CO helicase FANCM synergistically increases crossover frequency. Additionally, we showed that the effect of mutating FANCM on recombination is much lower in F1 hybrids contrasting from the phenotype of inbred lines, while figl1 mutation equally increases crossovers in both contexts. This shows that the modes of action of FIGL1 and FANCM are differently affected by genomic contexts. We propose that FIGL1 and FANCM represent two successive barriers to CO formation, one limiting strand invasion, the other disassembling D-loops to promote SDSA, which when both lifted, leads to a large increase of crossovers, without impairing meiotic progression., Author Summary Sexually reproducing species produce offspring that are genetically unique from one another, despite having the same parents. This uniqueness is created by meiosis, which is a specialized cell division. After meiosis each parent transmits half of their DNA, but each time this occurs, the 'half portion' of DNA transmitted to offspring is different from the previous. The differences are due to resorting the parental chromosomes, but also recombining them. Here we describe a gene—FIDGETIN-LIKE 1—which limits the amount of recombination that occurs during meiosis. Previously we identified a gene with a similar function, FANCM. FIGL1 and FANCM operate through distinct mechanisms. This discovery will be useful to understand more, from an evolutionary perspective, why recombination is naturally limited. Also this has potentially significant applications for plant breeding which is largely about sampling many 'recombinants' to find individuals that have heritable advantages compared to their parents.

Summary Background: Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe . In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans . The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae . Results: To unravel the crossover status in the plant Arabidopsis thaliana , we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals. Conclusions: Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.

Meiotic recombination is the fundamental process that produces balanced gametes and generates diversity within species. For successful meiosis, crossovers must form between homologous chromosomes. This condition is more difficult to fulfill in allopolyploid species, which have more than two sets of related chromosomes (homoeologs). Here, we investigated the formation, progression, and completion of several key hallmarks of meiosis in Brassica napus (AACC), a young polyphyletic allotetraploid crop species with closely related homoeologous chromosomes. Altogether, our results demonstrate a precocious and efficient sorting of homologous versus homoeologous chromosomes during early prophase I in two representative B. napus accessions that otherwise show a genotypic difference in the progression of homologous recombination. More strikingly, our detailed comparison of meiosis in near isogenic allohaploid and euploid plants showed that the mechanism(s) promoting efficient chromosome sorting in euploids is adjusted to promote crossover formation between homoeologs in allohaploids. This suggests that, in contrast to other polyploid species, chromosome sorting is context dependent in B. napus.

The eukaryotic RecA homologue Rad51 is a key factor in homologous recombination and recombinational repair. Rad51-like proteins have been identified in yeast (Rad55, Rad57 and Dmc1), plants and vertebrates (RAD51B, RAD51C, RAD51D, XRCC2, XRCC3 and DMC1). RAD51 and DMC1 are the strand-exchange proteins forming a nucleofilament for strand invasion, however, the function of the paralogues in the process of homologous recombination is less clear. In yeast the two Rad51 paralogues, Rad55 and Rad57, have been shown to be involved in somatic and meiotic HR and they are essential to the formation of the Rad51/DNA nucleofilament counterbalancing the anti-recombinase activity of the SRS2 helicase. Here, we examined the role of RAD51B in the model bryophyte Physcomitrella patens. Mutant analysis shows that RAD51B is essential for the maintenance of genome integrity, for resistance to DNA damaging agents and for gene targeting. Furthermore, we set up methods to investigate meiosis in Physcomitrella and we demonstrate that the RAD51B protein is essential for meiotic homologous recombination. Finally, we show that all these functions are independent of the SRS2 anti-recombinase protein, which is in striking contrast to what is found in budding yeast where the RAD51 paralogues are fully dependent on the SRS2 anti-recombinase function.

Summary Background At meiosis, two successive rounds of chromosome segregation lead to ploidy halving. This is achieved through a stepwise release of sister chromatid cohesion, along chromosome arms to allow homolog segregation at anaphase I and at centromeres to allow sister chromatid segregation at anaphase II. Cohesins, the protein complex that ensures cohesion, must then be protected at centromeres throughout meiosis, until the onset of anaphase II. Members of the Shugoshin protein family have been shown to protect centromeric cohesins at anaphase I, but much less is known about the protection of cohesion during interkinesis, the stage between meiosis I and meiosis II. Results Here, we (1) show that both Arabidopsis SHUGOSHINs paralogs are required for complete protection of centromeric cohesins during meiosis I, without apparent somatic function, and (2) identified PATRONUS (PANS1), a novel protein required for protection of meiotic centromeric cohesion. Although AtSGO1 and AtSGO2 protect centromeric cohesion during anaphase I, PANS1 is required at a later stage, during interkinesis. Additionally, we identified PANS2, a paralog of PANS1, whose mutation is synthetically lethal with pans1 suggesting that PANS genes are also essential for mitosis. PANS1 interacts directly with the CDC27b and the CDC20.1 subunit of the Anaphase Promoting Complex (APC/C), in a manner suggesting that PANS1 could be both a regulator and a target of the APC/C. Conclusions This study reveals that centromeric cohesion is actively protected at two successive stages of meiosis, by SHUGOSHINs at anaphase I and by PATRONUS at interkinesis.

Mini-chromosome maintenance (MCM) 2–9 proteins are related helicases. The first six, MCM2–7, are essential for DNA replication in all eukaryotes. In contrast, MCM8 is not always conserved in eukaryotes but is present in Arabidopsis thaliana. MCM8 is required for 95% of meiotic crossovers (COs) in Drosophila and is essential for meiosis completion in mouse, prompting us to study this gene in Arabidopsis meiosis. Three allelic Atmcm8 mutants showed a limited level of chromosome fragmentation at meiosis. This defect was dependent on programmed meiotic double-strand break (DSB) formation, revealing a role for AtMCM8 in meiotic DSB repair. In contrast, CO formation was not affected, as shown both genetically and cytologically. The Atmcm8 DSB repair defect was greatly amplified in the absence of the DMC1 recombinase or in mutants affected in DMC1 dynamics (sds, asy1). The Atmcm8 fragmentation defect was also amplified in plants heterozygous for a mutation in either recombinase, DMC1 or RAD51. Finally, in the context of absence of homologous chromosomes (i.e. haploid), mutation of AtMCM8 also provoked a low level of chromosome fragmentation. This fragmentation was amplified by the absence of DMC1 showing that both MCM8 and DMC1 can promote repair on the sister chromatid in Arabidopsis haploids. Altogether, this establishes a role for AtMCM8 in meiotic DSB repair, in parallel to DMC1. We propose that MCM8 is involved with RAD51 in a backup pathway that repairs meiotic DSB without giving CO when the major pathway, which relies on DMC1, fails., Author Summary Species that reproduce sexually have two copies of each chromosome, inherited from their father and mother. During a special cell division called meiosis, these two chromosomes are mixed by homologous recombination to give genetically unique chromosomes that will be transmitted to the next generation. This recombination process is initiated by DNA breaks that must be repaired efficiently to maintain fertility. Using the model plant Arabidopsis thaliana we revealed here that the gene AtMCM8 is required to repair a subset of these DNA breaks. However MCM8 appears to not be required for recombination with the homologous chromosome. Instead MCM8 appears to be involved in a safety system that operates to repair DNA breaks that have not been used for homologous recombination. Interestingly the equivalent gene also has an essential meiotic function in the fly and the mouse. However the three species require MCM8 for different aspects of meiosis.

Plant meiosis studies have enjoyed a fantastic boom in recent years with the use of Arabidopsis thaliana as an important model species for developmental studies because of its small genome, short life cycle, and large mutant collections. Unlike other eukaryotic models, plant meiosis does not display strict checkpoints and rarely commits to apoptotic processes, which makes it possible to investigate the whole meiotic process (spanning from premeiotic interphase to spore formation) in knockout mutants. In this chapter we describe a protocol for immunolabelling Arabidopsis and Brassica meiotic proteins on robustly spread chromosomes. This protocol allows the detection of a large range of proteins on well-preserved chromosomes and throughout the entire meiotic process.

Meiosis is a specialized eukaryotic cell division that generates haploid gametes required for sexual reproduction. During meiosis, homologous chromosomes pair and undergo reciprocal genetic exchange, termed crossover (CO). Meiotic CO frequency varies along the physical length of chromosomes and is determined by hierarchical mechanisms, including epigenetic organization, for example methylation of the DNA and histones. Here we investigate the role of DNA methylation in determining patterns of CO frequency along Arabidopsis thaliana chromosomes. In A. thaliana the pericentromeric regions are repetitive, densely DNA methylated, and suppressed for both RNA polymerase-II transcription and CO frequency. DNA hypomethylated methyltransferase1 (met1) mutants show transcriptional reactivation of repetitive sequences in the pericentromeres, which we demonstrate is coupled to extensive remodeling of CO frequency. We observe elevated centromere-proximal COs in met1, coincident with pericentromeric decreases and distal increases. Importantly, total numbers of CO events are similar between wild type and met1, suggesting a role for interference and homeostasis in CO remodeling. To understand recombination distributions at a finer scale we generated CO frequency maps close to the telomere of chromosome 3 in wild type and demonstrate an elevated recombination topology in met1. Using a pollen-typing strategy we have identified an intergenic nucleosome-free CO hotspot 3a, and we demonstrate that it undergoes increased recombination activity in met1. We hypothesize that modulation of 3a activity is caused by CO remodeling driven by elevated centromeric COs. These data demonstrate how regional epigenetic organization can pattern recombination frequency along eukaryotic chromosomes., Author Summary The majority of eukaryotes reproduce via a specialized cell division called meiosis, which generates gametes with half the number of chromosomes. During meiosis, homologous chromosomes pair and undergo a process of reciprocal exchange, called crossing-over (CO), which generates new combinations of genetic variation. The relative chance of a CO occurring is variable along the chromosome, for example COs are suppressed in the centromeric regions that attach to the spindle during chromosome segregation. These patterns correlate with domains of epigenetic organization along chromosomes, including methylation of the DNA and histones. DNA methylation occurs most densely in the centromeric regions of Arabidopsis thaliana chromosomes, where it is required for transcriptional suppression of repeated sequences. We demonstrate that mutants that lose DNA methylation (met1) show epigenetic remodeling of crossover frequencies, with increases in the centromeric regions and compensatory changes in the chromosome arms, though the total number of crossovers remains the same. As crossover numbers and distributions are subject to homeostatic mechanisms, we propose that these drive crossover remodeling in met1 in response to epigenetic change in the centromeric regions. Together these data demonstrate how domains of epigenetic organization are important for shaping patterns of crossover frequency along eukaryotic chromosomes.

In numerous species, the formation of meiotic crossovers is largely under the control of a group of proteins known as ZMM. Here, we identified a new ZMM protein, HEI10, a RING finger-containing protein that is well conserved among species. We show that HEI10 is structurally and functionally related to the yeast Zip3 ZMM and that it is absolutely required for class I crossover (CO) formation in Arabidopsis thaliana. Furthermore, we show that it is present as numerous foci on the chromosome axes and the synaptonemal complex central element until pachytene. Then, from pachytene to diakinesis, HEI10 is retained at a limited number of sites that correspond to class I COs, where it co-localises with MLH1. Assuming that HEI10 early staining represents an early selection of recombination intermediates to be channelled into the ZMM pathway, HEI10 would therefore draw a continuity between early chosen recombination intermediates and final class I COs., Author Summary During meiosis two successive chromosomal divisions follow a single S phase, resulting in the formation of four haploid cells, each with half of the parental genetic material. This ploidy reduction occurs during the first meiotic division, when homologous chromosomes (paternal and maternal) are separated from each other. For this to happen, homologous chromosomes associate in bivalents, where each chromosome is linked to its homologue by chiasmata. These chiasmata reflect the formation of crossovers (COs), one of the manifestations of the exchange of genetic material occurring during homologous recombination. In most species, the final number of COs represents only a small proportion of all meiotic recombination events (4% in Arabidopsis thaliana). The mechanisms that drive the choice of recombination intermediates that will mature into COs are still unknown. In this study, we identified the HEI10 protein that is present as numerous foci on chromosome axes during early meiotic prophase and is retained until the end of prophase at a limited number of sites corresponding to COs. We also showed that HEI10 is necessary for the formation of most of the COs. HEI10 is therefore a good candidate for a ZMM protein involved in generating continuity between chosen early recombination intermediates and final COs.

No Crossing Over To ensure the correct division of chromosome during the reduction division of meiosis, homologous chromosomes undergo double-strand breaks that—through crossing over and recombination—link the homologs together (and importantly introduce diversity into the genomes of gametes). But only a minority of these crossovers results in recombination—most are directed into non-crossover pathways. Lorenz et al. (p. 1585 ), working in the yeast Schizosaccharomyces pombe , and Crismani et al. (p. 1588 ), working in the higher plant Arabidopsis thaliana , looked for the factors that limit crossovers and promote non-crossover pathways. The homolog of the human Fanconi anemia complementation group M (FANCM) helicase protein was found to be a major meiotic anti-recombinase, which could drive meiotic recombination intermediates into the non-crossover pathway.

L'article original est publié par The American Society of Plant Biologists; Meiotic crossovers are necessary to generate balanced gametes and to increase genetic diversity. Even if crossover number is usually constrained, recent results suggest that manipulating karyotype composition could be a new way to increase rossover frequency in plants. In this study, we explored this hypothesis by analyzing the extent of crossover variation in a set of related diploid AA, allotriploid AAC, and allotetraploid AACC Brassica hybrids. We first used cytogenetic methods to describe the meiotic behavior of the different hybrids. We then combined a cytogenetic estimation of class I crossovers in the entire genome by immunolocalization of a key protein, MutL Homolog1, which forms distinct foci on meiotic chromosomes, with genetic analyses to specifically compare crossover rates between one pair of chromosomes in the different hybrids. Our results showed that the number of crossovers in the allotriploid AAC hybrid was higher than in the diploid AA hybrid. Accordingly, the allotetraploid AACC hybrid showed an intermediate behavior. We demonstrated that this increase was related to hybrid karyotype composition (diploid versus allotriploid versus allotetraploid) and that interference was maintained in the AAC hybrids. These results could provide another efficient way to manipulate recombination in traditional breeding and genetic studies.

SummaryCrossovers (COs) are essential for the completion of meiosis in most species and lead to new allelic combinations in gametes [1]. Two pathways of meiotic crossover formation have been distinguished. Class I COs, which are the major class of CO in budding yeast, mammals, Caenorhabditis elegans, and Arabidopsis, depend on a group of proteins called ZMM and rely on specific DNA structure intermediates that are processed to form COs [2–6]. We identified a novel gene, SHOC1, involved in meiosis in Arabidopsis. Shoc1 mutants showed a striking reduction in the number of COs produced, a similar phenotype to the previously described Arabidopsis zmm mutants. The early steps of recombination, revealed by DMC1 foci, and completion of synapsis are not affected in shoc1 mutants. Double mutant analysis showed that SHOC1 acts in the same pathway as AtMSH5, a conserved member of the ZMM group [7]. SHOC1 is thus a novel gene required for class I CO formation in Arabidopsis. Sequence similarity studies detected putative SHOC1 homologs in a large range of eukaryotes including human. SHOC1 appears to be related to the XPF endonuclease protein family, which suggests that it is directly involved in the maturation of DNA intermediates that lead to COs.

During meiosis, homologous chromosomes recognize each other, align, and exchange genetic information. This process requires the action of RecA-related proteins Rad51 and Dmc1 to catalyze DNA strand exchanges. The Mnd1–Hop2 complex has been shown to assist in Dmc1-dependent processes. Furthermore, higher eukaryotes possess additional RecA-related proteins, like XRCC3, which are involved in meiotic recombination. However, little is known about the functional interplay between these proteins during meiosis. We investigated the functional relationship between AtMND1, AtDMC1, AtRAD51, and AtXRCC3 during meiosis in Arabidopsis thaliana. We demonstrate the localization of AtMND1 to meiotic chromosomes, even in the absence of recombination, and show that AtMND1 loading depends exclusively on AHP2, the Arabidopsis Hop2 homolog. We provide evidence of genetic interaction between AtMND1, AtDMC1, AtRAD51, and AtXRCC3. In vitro assays suggest that this functional link is due to direct interaction of the AtMND1–AHP2 complex with AtRAD51 and AtDMC1. We show that AtDMC1 foci accumulate in the Atmnd1 mutant, but are reduced in number in Atrad51 and Atxrcc3 mutants. This study provides the first insights into the functional differences of AtRAD51 and AtXRCC3 during meiosis, demonstrating that AtXRCC3 is dispensable for AtDMC1 focus formation in an Atmnd1 mutant background, whereas AtRAD51 is not. These results clarify the functional interactions between key players in the strand exchange processes during meiotic recombination. Furthermore, they highlight a direct interaction between MND1 and RAD51 and show a functional divergence between RAD51 and XRCC3., Author Summary During meiosis, two rounds of chromosome segregation follow a single round of chromosome duplication, leading to the production of haploid gametes. It is during this specialised cell division that genetic traits are recombined, achieved by mutual exchange of DNA sequences of homologous chromosomes. Recombination events must be well controlled to ensure that: (i) they occur between homologous chromosomes rather than sister chromatids and (ii) they occur between true homologous sequences and not duplicated sequences present elsewhere in the genome. At one of the early steps of homologous recombination, a single-strand DNA molecule recognizes and invades the targeted homologous sequence. Many proteins, which are conserved throughout the kingdoms, are involved in this crucial step of DNA homology search. In this study, we analyze the role of a series of such proteins and their complex functional relationships in the model plant A. thaliana, shedding new light on this highly regulated step of meiotic recombination.

International audience; Interspecific crosses contribute significantly to plant evolution enabling gene exchanges between species.The efficiency of interspecific crosses depends on the similarity between the implicated genomes as high levels of genome similarity are required to ensure appropriate chromosome pairing and genetic recombination. Brassica napus (AACC) is an allopolyploid, resulting from natural hybridization between Brassica rapa (AA) and Brassica oleracea (CC), both being diploid species derived from a common ancestor.To study the relationships between genomes of these Brassica species, we have determined simultaneously the pairing and recombination pattern of A and C chromosomes during meiosis of AAC triploid hybrids, which result from the interspecific cross between natural B. napus and B. rapa. Different AAC triploid hybrids and their progenies have been analysed using cytogenetic, BAC-FISH, and molecular techniques. In 71% of the pollen mother cells, homologous A chromosomes paired regularly, and usually one chromosome of each pair was transmitted to the progeny. C chromosomes remained mainly univalent, but were involved in homoeologous pairing in 21.5% of the cells, and 13% of the transmitted C chromosomes were either recombined or broken. The rate of transmission of C chromosomes depended on the identity of the particular chromosome and on the way the hybrid was crossed, as the male or as the female parent, to B. napus or to B. rapa. Gene transfers in triploid hybrids are favoured between A genomes of B. rapa and B. napus, but also occur between A and C genomes though at lower rates.

International audience; The success of the first meiotic division relies (among other factors) on the formation of bivalents between homologous chromosomes, the monopolar orientation of the sister kinetochores at metaphase I and the maintenance of centromeric cohesion until the onset of anaphase II. The meiotic cohesin subunit, Rec8 has been reported to be one of the key players in these processes, but its precise role in kinetochore orientation is still under debate. By contrast, much less is known about the other non-SMC cohesin subunit, Scc3. We report the identification and the characterisation of AtSCC3, the sole Arabidopsis homologue of Scc3. The detection of AtSCC3 in mitotic cells, the embryo lethality of a null allele Atscc3-2, and the mitotic defects of the weak allele Atscc3-1 suggest that AtSCC3 is required for mitosis. AtSCC3 was also detected in meiotic nuclei as early as interphase, and bound to the chromosome axis from early leptotene through to anaphase I. We show here that both AtREC8 and AtSCC3 are necessary not only to maintain centromere cohesion at anaphase I, but also for the monopolar orientation of the kinetochores during the first meiotic division. We also found that AtREC8 is involved in chromosome axis formation in an AtSPO11-1-independent manner. Finally, we provide evidence for a role of AtSPO11-1 in the stability of the cohesin complex.

Within Fabaceae, legume species have a variable genome size, chromosome number, and ploidy level. The genome distribution of ribosomal genes, easily detectable by fluorescent in situ hybridization (FISH), is a good tool for anchoring physical and genetic comparative maps. The organisation of 45S rDNA and 5S loci was analysed by FISH in the 4 closely related species: Pisum sativum, Medicago truncatula, Medicago sativa (2 diploid taxa), and Lathyrus sativus. The 2 types of rDNA arrays displayed interspecific variation in locus number and location, but little intraspecific variation was detected. In the model legume, M. truncatula, the presence of 2 adjacent 45S rDNA loci was demonstrated, and the location of the rDNA loci was independent of the general evolution of the genome DNA. The different parameters relative to clustering of the rDNA loci in specific chromosome regions and the possible basis of rDNA instability are discussed.Key words: ribosomal genes, FISH, Medicago species, Pisum sativum, Lathyrus sativus, rDNA mobility.

Crossovers are essential for the completion of meiosis. Recently, two pathways of crossover formation have been identified on the basis of distinct genetic controls. In one pathway, crossover inhibits the occurrence of another such event in a distance-dependent manner. This phenomenon is known as interference. The second kind of crossover is insensitive to interference. The two pathways function independently in budding yeast. Only interference-insensitive crossovers occur in Schizosaccharomyces pombe. In contrast, only interference-sensitive crossovers occur in Caenorabditis elegans. The situation in mammals and plants remains unclear. Mer3 is one of the genes shown to be required for the formation of interference-sensitive crossovers in Saccharomyces cerevisiae.To unravel the crossover status in the plant Arabidopsis thaliana, we investigated the role of the A. thaliana MER3 gene through the characterization of a series of allelic mutants. All mer3 mutants showed low levels of fertility and a significant decrease (about 75%) but not a total disappearance of meiotic crossovers, with the number of recombination events initiated in the mutants being similar to that in the wild-type. Genetic analyses showed that the residual crossovers in mer3 mutants did not display interference in one set of adjacent intervals.Mutation in MER3 in Arabidopsis appeared to be specific to recombination events resulting in interference-sensitive crossovers. Thus, MER3 function is conserved from yeast to plants and may exist in other metazoans. Arabidopsis therefore has at least two pathways for crossover formation, one giving rise to interference-sensitive crossover and the other to independently distributed crossovers.

Plant-parasitic nematodes Meloidogyne spp induce an elaborate permanent feeding site characterized by the redifferentiation of root cells into multinucleate and hypertrophied giant cells. We have isolated by a promoter trap strategy an Arabidopsis thaliana formin gene, AtFH6, which is upregulated during giant cell formation. Formins are actin-nucleating proteins that stimulate de novo polymerization of actin filaments. We show here that three type-I formins were upregulated in giant cells and that the AtFH6 protein was anchored to the plasma membrane and uniformly distributed. Suppression of the budding defect of the Saccharomyces cerevisiae bni1Delta bnr1Delta mutant showed that AtFH6 regulates polarized growth by controlling the assembly of actin cables. Our results suggest that AtFH6 might be involved in the isotropic growth of hypertrophied feeding cells via the reorganization of the actin cytoskeleton. The actin cables would serve as tracks for vesicle trafficking needed for extensive plasma membrane and cell wall biogenesis. Therefore, determining how plant parasitic nematodes modify root cells into giant cells represents an attractive system to identify genes that regulate cell growth and morphogenesis.

Meiotic crossovers (COs) shape genetic diversity by mixing homologous chromosomes at each generation. CO distribution is a highly regulated process. CO assurance forces the occurrence of at least one obligatory CO per chromosome pair, CO homeostasis smoothes out the number of COs when faced with variation in precursor number and CO interference keeps multiple COs away from each other along a chromosome. In several organisms, it has been shown that cytoskeleton forces are transduced to the meiotic nucleus via KASH- and SUN-domain proteins, to promote chromosome synapsis and recombination. Here we show that the Arabidopsis kinesin AtPSS1 plays a major role in chromosome synapsis and regulation of CO distribution. In Atpss1 meiotic cells, chromosome axes and DNA double strand breaks (DSBs) appear to form normally but only a variable portion of the genome synapses and is competent for CO formation. Some chromosomes fail to form the obligatory CO, while there is an increased CO density in competent regions. However, the total number of COs per cell is unaffected. We further show that the kinesin motor domain of AtPSS1 is required for its meiotic function, and that AtPSS1 interacts directly with WIP1 and WIP2, two KASH-domain proteins. Finally, meiocytes missing AtPSS1 and/or SUN proteins show similar meiotic defects suggesting that AtPSS1 and SUNs act in the same pathway. This suggests that forces produced by the AtPSS1 kinesin and transduced by WIPs/SUNs, are required to authorize complete synapsis and regulate maturation of recombination intermediates into COs. We suggest that a form of homeostasis applies, which maintains the total number of COs per cell even if only a part of the genome is competent for CO formation., Author Summary In species that reproduce sexually, diploid individuals have two copies of each chromosome, inherited from their father and mother. During a special cell division called meiosis, these two sets of chromosomes are mixed by homologous recombination to give genetically unique chromosomes that will be transmitted to the next generation. Homologous recombination processes are highly controlled in terms of number and localization of events within and among chromosomes. Disruption of this control (a lack of or improper positioning of homologous recombination events) causes deleterious chromosome associations in the offspring. Using the model plant Arabidopsis thaliana we reveal here that the AtPSS1 gene is required for proper localization of these homologous recombination events along the genome. We also show that AtPSS1, which belongs to a family of proteins able to move along the cytoskeleton, is likely part of a module that allows cytoplasmic forces to be transmitted through the nucleus envelope to promote chromosome movements during homologous recombination progression.

A genetic study finds the neddylation pathway (known to-date for post-translational protein modification) is involved in regulating crossover localization but not crossover number during meiosis in Arabidopsis., Crossovers (COs) are at the origin of genetic variability, occurring across successive generations, and they are also essential for the correct segregation of chromosomes during meiosis. Their number and position are precisely controlled, however the mechanisms underlying these controls are poorly understood. Neddylation/rubylation is a regulatory pathway of posttranslational protein modification that is required for numerous cellular processes in eukaryotes, but has not yet been linked to homologous recombination. In a screen for meiotic recombination-defective mutants, we identified several axr1 alleles, disrupting the gene encoding the E1 enzyme of the neddylation complex in Arabidopsis. Using genetic and cytological approaches we found that axr1 mutants are characterised by a shortage in bivalent formation correlated with strong synapsis defects. We determined that the bivalent shortage in axr1 is not due to a general decrease in CO formation but rather due to a mislocalisation of class I COs. In axr1, as in wild type, COs are still under the control of the ZMM group of proteins. However, in contrast to wild type, they tend to cluster together and no longer follow the obligatory CO rule. Lastly, we showed that this deregulation of CO localisation is likely to be mediated by the activity of a cullin 4 RING ligase, known to be involved in DNA damage sensing during somatic DNA repair and mouse spermatogenesis. In conclusion, we provide evidence that the neddylation/rubylation pathway of protein modification is a key regulator of meiotic recombination. We propose that rather than regulating the number of recombination events, this pathway regulates their localisation, through the activation of cullin 4 RING ligase complexes. Possible targets for these ligases are discussed., Author Summary During meiosis, two successive chromosomal divisions follow a single S phase, resulting in the formation of four haploid cells, each with half of the parental genetic material. This reduction in chromosome number occurs during the first meiotic division, when homologous chromosomes (paternal and maternal) are separated from each other. For this to happen, homologous chromosomes associate in structures called bivalents, where each chromosome is linked to its homologue by a point of contact known as chiasmata. These chiasmata reflect the formation of crossovers (COs), one of the manifestations of the exchange of genetic material occurring during homologous recombination. CO number varies little at around two per chromosome pair, and they tend to be evenly spaced on chromosomes. Thus, CO number and distribution are very tightly controlled. However, the mechanisms underlying these controls are very poorly understood. In this study, we identified a regulatory pathway of meiotic recombination. We show that this pathway does not regulate the amount of recombination events per se, but instead controls their localisation, as when it is defective, CO events cluster together in a few regions of the genome, leading to bivalent shortage and progeny aneuploidy with incorrect numbers of chromosomes. This regulatory pathway is a posttranslational protein modification system called neddylation (or rubylation in plants), known to be required for numerous cellular processes in eukaryotes. We identify an enzyme of the neddylation complex as a major regulator of meiotic recombination in Arabidopsis and show that this process may be also conserved in mammals.

In human cells and in Saccharomyces cerevisiae, BLAP75/Rmi1 acts together with BLM/Sgs1 and TopoIIIalpha/Top3 to maintain genome stability by limiting crossover (CO) formation in favour of NCO events, probably through the dissolution of double Holliday junction intermediates (dHJ). So far, very limited data is available on the involvement of these complexes in meiotic DNA repair. In this paper, we present the first meiotic study of a member of the BLAP75 family through characterisation of the Arabidopsis thaliana homologue. In A. thaliana blap75 mutants, meiotic recombination is initiated, and recombination progresses until the formation of bivalent-like structures, even in the absence of ZMM proteins. However, chromosome fragmentation can be detected as soon as metaphase I and is drastic at anaphase I, while no second meiotic division is observed. Using genetic and imunolocalisation studies, we showed that these defects reflect a role of A. thaliana BLAP75 in meiotic double-strand break (DSB) repair -- that it acts after the invasion step mediated by RAD51 and associated proteins and that it is necessary to repair meiotic DSBs onto sister chromatids as well as onto the homologous chromosome. In conclusion, our results show for the first time that BLAP75/Rmi1 is a key protein of the meiotic homologous recombination machinery. In A. thaliana, we found that this protein is dispensable for homologous chromosome recognition and synapsis but necessary for the repair of meiotic DSBs. Furthermore, in the absence of BLAP75, bivalent formation can happen even in the absence of ZMM proteins, showing that in blap75 mutants, recombination intermediates exist that are stable enough to form bivalent structures, even when ZMM are absent.

In many species, sex-related differences in crossover (CO) rates have been described at chromosomal and regional levels. In this study, we determined the CO distribution along the entire Arabidopsis thaliana Chromosome 4 (18 Mb) in male and female meiosis, using high density genetic maps built on large backcross populations (44 markers, >1,300 plants). We observed dramatic differences between male and female map lengths that were calculated as 88 cM and 52 cM, respectively. This difference is remarkably parallel to that between the total synaptonemal complex lengths measured in male and female meiocytes by immunolabeling of ZYP1 (a component of the synaptonemal complex). Moreover, CO landscapes were clearly different: in particular, at both ends of the map, male CO rates were higher (up to 4-fold the mean value), whereas female CO rates were equal or even below the chromosomal average. This unique material gave us the opportunity to perform a detailed analysis of CO interference on Chromosome 4 in male and female meiosis. The number of COs per chromosome and the distances between them clearly departs from randomness. Strikingly, the interference level (measured by coincidence) varied significantly along the chromosome in male meiosis and was correlated to the physical distance between COs. The significance of this finding on the relevance of current CO interference models is discussed., Author Summary Meiotic crossovers between homologous chromosomes ensure their proper segregation to generate ultimately gametes. They also create new allelic combinations which contribute to the diversity of traits among individuals. In all eukaryotes, the number and the localization of crossovers along chromosomes are not random. In addition, crossovers are not independent of each other: the occurrence of a crossover lowers the probability that another crossover arises in its vicinity. The mechanism of this phenomenon, called “crossover interference,” is one of the most challenging puzzles that geneticists have been faced with in the last century. In this paper, we precisely described the distribution of crossovers along Chromosome 4 of the model plant species Arabidopsis thaliana, separately in male and female meiosis. Interestingly, we observed that crossovers are 1.7 more numerous in male than in female meiosis, and this increase is especially marked at the ends of the chromosome. Moreover, our results provide the first evidence that the level of interference along a chromosome is not a constant and is correlated with the physical distance between crossovers. These results shed new light on the determinism of crossover localization and could have important outcomes on the relevance of current models of crossover interference.