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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.

Genetic recombination is important for generating diversity and to ensure faithful segregation of chromosomes at meiosis. However, few crossovers (COs) are formed per meiosis despite an excess of DNA double-strand break precursors. This reflects the existence of active mechanisms that limit CO formation. We previously showed that AtFANCM is a meiotic anti-CO factor. The same genetic screen now identified AtMHF2 as another player of the same anti-CO pathway. FANCM and MHF2 are both Fanconi Anemia (FA) associated proteins, prompting us to test the other FA genes conserved in Arabidopsis for a role in CO control at meiosis. This revealed that among the FA proteins tested, only FANCM and its two DNA-binding co-factors MHF1 and MHF2 limit CO formation at meiosis.

Meiosis is a special type of cell division present in all organisms that reproduce by sexual reproduction. It ensures the transition between the sporophytic and gametophytic state and allows gamete production through meiotic recombination and chromosome number reduction. In this paper, we describe a technique for the isolation of Arabidopsis thaliana male meiocytes. From this cellular material, it was then possible to develop large-scale transcriptome studies using CATMA microarrays and thus to obtain an overview of genes expressed during Arabidopsis meiosis. The expression profiles were studied with either stringent statistical criteria or by performing clustering. Both methods resulted in gene clusters enriched in meiosis-specific genes (from 14- to 55-fold). Analysis of these data provided a unique set of genes that will be pivotal to further analysis aimed at understanding the meiotic process.

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.

eskimo1-5 (esk1-5) is a dwarf Arabidopsis (Arabidopsis thaliana) mutant that has a constitutive drought syndrome and collapsed xylem vessels, along with low acetylation levels in xylan and mannan. ESK1 has xylan O-acetyltransferase activity in vitro. We used a suppressor strategy on esk1-5 to screen for variants with wild-type growth and low acetylation levels, a favorable combination for ethanol production. We found a recessive mutation in the KAKTUS (KAK) gene that suppressed dwarfism and the collapsed xylem character, the cause of decreased hydraulic conductivity in the esk1-5 mutant. Backcrosses between esk1-5 and two independent knockout kak mutants confirmed suppression of the esk1-5 effect. kak single mutants showed larger stem diameters than the wild type. The KAK promoter fused with a reporter gene showed activity in the vascular cambium, phloem, and primary xylem in the stem and hypocotyl. However, suppression of the collapsed xylem phenotype in esk1 kak double mutants was not associated with the recovery of cell wall O-acetylation or any major cell wall modifications. Therefore, our results indicate that, in addition to its described activity as a repressor of endoreduplication, KAK may play a role in vascular development. Furthermore, orthologous esk1 kak double mutants may hold promise for ethanol production in crop plants.

In yeast, the DMC1 gene is required for interhomolog recombination, which is an essential step for bivalent formation and the correct partition of chromosomes during meiosis I. By using a reverse genetics approach, we were able to identify a T-DNA insertion in AtDMC1, the Arabidopsis homolog of DMC1. Homozygotes for the AtDMC1 insertion failed to express AtDMC1, and their residual fertility was 1.5% that of the wild type. Complete fertility was restored in mutant plants when a wild-type copy of the AtDMC1 gene was reintroduced. Cytogenetical analysis points to a correlation of the sterility phenotype with severely disturbed chromosome behavior during both male and female meiosis. In this study, our data demonstrate that AtDMC1 function is crucial for meiosis in Arabidopsis. However, meiosis can be completed in the Arabidopsis dmc1 mutant, which is not the case for mouse or some yeast mutants.

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.

Cell cycle control is modified at meiosis compared to mitosis, because two divisions follow a single DNA replication event. Cyclin-dependent kinases (CDKs) promote progression through both meiosis and mitosis, and a central regulator of their activity is the APC/C (Anaphase Promoting Complex/Cyclosome) that is especially required for exit from mitosis. We have shown previously that OSD1 is involved in entry into both meiosis I and meiosis II in Arabidopsis thaliana; however, the molecular mechanism by which OSD1 controls these transitions has remained unclear. Here we show that OSD1 promotes meiotic progression through APC/C inhibition. Next, we explored the functional relationships between OSD1 and the genes known to control meiotic cell cycle transitions in Arabidopsis. Like osd1, cyca1;2/tam mutation leads to a premature exit from meiosis after the first division, while tdm mutants perform an aberrant third meiotic division after normal meiosis I and II. Remarkably, while tdm is epistatic to tam, osd1 is epistatic to tdm. We further show that the expression of a non-destructible CYCA1;2/TAM provokes, like tdm, the entry into a third meiotic division. Finally, we show that CYCA1;2/TAM forms an active complex with CDKA;1 that can phosphorylate OSD1 in vitro. We thus propose that a functional network composed of OSD1, CYCA1;2/TAM, and TDM controls three key steps of meiotic progression, in which OSD1 is a meiotic APC/C inhibitor., Author Summary In the life cycle of sexual organisms, a specialized cell division—meiosis—reduces the number of chromosomes from two sets (2n, diploid) to one set (n, haploid), while fertilization restores the original chromosome number. Meiosis reduces ploidy because it consists of two cellular divisions following a single DNA replication. In this study, we analyze the function of a group of genes that collectively controls the entry into the first meiotic division, the entry into the second meiotic division, and the exit from meiosis in the model plant Arabidopsis thaliana. We revealed a complex regulation network that controls these three key transitions.

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.

Meiosis halves the chromosome number because its two divisions follow a single round of DNA replication. This process involves two cell transitions, the transition from prophase to the first meiotic division (meiosis I) and the unique meiosis I to meiosis II transition. We show here that the A-type cyclin CYCA1;2/TAM plays a major role in both transitions in Arabidopsis. A series of tam mutants failed to enter meiosis II and thus produced diploid spores and functional diploid gametes. These diploid gametes had a recombined genotype produced through the single meiosis I division. In addition, by combining the tam-2 mutation with AtSpo11-1 and Atrec8, we obtained plants producing diploid gametes through a mitotic-like division that were genetically identical to their parents. Thus tam alleles displayed phenotypes very similar to that of the previously described osd1 mutant. Combining tam and osd1 mutations leads to a failure in the prophase to meiosis I transition during male meiosis and to the production of tetraploid spores and gametes. This suggests that TAM and OSD1 are involved in the control of both meiotic transitions., Author Summary In the life cycle of sexual organisms, a specialized cell division—meiosis—reduces the number of chromosomes from two sets (2n, diploid) to one set (n, haploid), while fertilization restores the original chromosome number. Meiosis reduces ploidy because it consists of two divisions following a single DNA replication. In this study, we identified genes that control the entry into the first and the second meiotic division in the model plant Arabidopsis thaliana. Plants lacking the CYCA1;2 gene execute a single division during meiosis producing functional diploid gametes and polyploid plants in the next generation. By combining this mutation with two others that affect key meiotic processes, we generated plants that produce diploid gametes through a mitotic-like division that are genetically identical to their parents. Furthermore, plants lacking CYCA1;2 and another previously described gene (OSD1) undergo no divisions during male meiosis, producing tetraploid pollen grains.

Five cytoplasmic male sterile lines of Nicotiana tabacum homozygous for a dominant rootless mutation [Rac-] were used as female parents in crosses with three male fertile varieties. Androgenetic haploids were selected by their ability to form plantlets with a normal root system, whereas hybrid plantlets failed to grow. High frequencies of androgenetic haploid plants were obtained with N. debneyi cytoplasm, and with one paternal genotype. Chromosome doubling was performed using three different methods (pollination, in vitro culture, acenaphthen) and cytoplasmic male sterile doubled haploid plants have been obtained. The results demonstrate the efficiency of this method for a rapid cytoplasm transfer in Nicotiana tabacum; the integrity of the cytoplasm is conserved. For the first time, we possess a counter selectable marker universally usable for Nicotiana tabacum and closely related species.

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.

International audience; Initiation of transcription mediated by RNA polymerase II requires a number of transcription factors among which TFIID is the major core promoter recognition factor. TFIID is composed of highly conserved factors which include the TATA-binding protein (TBP) and about 14 TBP-associated factors (TAFs). Recently, the complete Arabidopsis TAF family has been identified. To obtain functional information about Arabidopsis TAFs, we analyzed a T-DNA insertion mutant for AtTAF6. Segregation analysis showed that plants homozygous for the mutant allele were never found, indicating that inhibition of the AtTAF6 function is lethal. Genetic experiments also revealed that the male gametophyte was affected by the attaf6 mutation since significant reduced transmission of the mutant allele through the male gametophyte was observed. Detailed histological and morphological analysis showed that the T-DNA insertion in AtTAF6 specifically affects pollen tube growth, indicating that the transcriptional regulation of only a specific subset of genes is controlled by this basal transcription factor.

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.

Meiosis is a key step in diploid sexual reproduction. Apart from its cytological description, the molecular mechanisms involved in this specialized cell division are being deciphered in plants thanks to the model plant Arabidopsis thaliana. While some meiotic mutants of Arabidopsis confirm the central role of functions that have been described either in yeast or in mice, others led to the identification of previously unknown genes. Numerous plants also exist as polyploids, which represent a special case with regard to meiosis.

Our understanding of plant meiosis is rapidly increasing thanks to the model Arabidopsis thaliana. Here we present the results of a screening for meiotic mutants carried out with a library containing 30 719 T-DNA insertion lines. An average of one mutant per 1000 lines was recovered. Several phenotypic classes could be distinguished and are presented. In parallel, 39 proteins known to be involved in meiosis in non-plant organisms were chosen and a search was performed for homologue sequences in the completed Arabidopsis thaliana genome. Approximately 30% of the meiotic related sequences showed similarities with one or several Arabidopsis putative genes. The relevance of forward versus reverse genetics in order to characterize meiotic functions is discussed.

Eight hundred and fifty Arabidopsis thaliana T-DNA insertion lines have been selected on a phenotypic basis. The T-DNA flanking sequences (FST) have been isolated using a PCR amplification procedure and sequenced. Seven hundred plant DNA sequences have been obtained revealing a T-DNA insertion in, or in the immediate vicinity of 482 annotated genes. Limited deletions of plant DNA have been observed at the site of insertion of T-DNA as well as in its left (LB) and right (RB) T-DNA signal sequences. The distribution of the T-DNA insertions along the chromosomes shows that they are essentially absent from the centrometric and pericentrometric regions.

We have characterized a new gene, SWI1, involved in sister chromatid cohesion during both male and female meiosis inArabidopsis thaliana. A first allele, swi1.1, was obtained as a T-DNA tagged mutant and was described previously as abnormal exclusively in female meiosis. We have isolated a new allele,swi1.2, which is defective for both male and female meiosis. Inswi1.2 male meiosis, the classical steps of prophase were not observed, especially because homologs do not synapse. Chromatid arms and centromeres lost their cohesion in a stepwise manner before metaphase I, and 20 chromatids instead of five bivalents were seen at the metaphase plate, which was followed by an aberrant segregation. In contrast, swi1.2 female meiocytes performed a mitotic-like division instead of meiosis, indicating a distinct role for SWI1 or a different effect of the loss of SWI1 function in both processes. TheSWI1 gene was cloned; the putative SWI1 protein did not show strong similarity to any known protein. Plants transformed with a SWI1–GFP fusion indicated that SWI1 protein is present in meiocyte nuclei, before meiosis and at a very early stage of prophase. Thus, SWI1 appears to be a novel protein involved in chromatid cohesion establishment and in chromosome structure during meiosis, but with clear differences between male and female meiosis.

In this study, megasporogenesis of the plant model Arabidopsis thaliana was investigated by electron microscopy for the first time. The data described here could constitute a reference for future investigations of Arabidopsis mutants. During the beginning of meiosis the megaspore mother cell shows a polarity created by unequal distribution of organelles in the cytoplasm. Plastids accumulate in the chalazal region and long parallel saccules of endoplasmic reticulum, small vacuoles and some dictyosomes are found in the micropylar region. Plasmodesmata are abundant in the chalazal cell wall. The nucleus is almost centrally localized and contains a prominent excentric nucleolus and numerous typical synaptonemal complexes. After the second division of meiosis the four megaspores are separated by thin cell walls crossed by numerous plasmodesmata and do not show significant cellular organization. The young functional megaspore is characterized by a large nucleus and a large granular nucleolus. The cytoplasm is very electron dense due to the abundance of free ribosomes and contains the following randomly distributed organelles: mitochondria, a few short saccules of endoplasmic reticulum, dictyosomes and undifferentiated plastids. However, there is no apparent polarity, except for the distribution of some small vacuoles which are more abundant in the micropylar region of the cell. The degenerating megaspores are extremely electron dense and do not show any substructure.

Many genes are thought to be expressed during the haploid phase in plants, however, very few haploid-specific genes have been isolated so far. T-DNA insertion mutagenesis is a powerful tool for generating mutations that affect gametophyte viability and function, as disruption of a gene essential for these processes should lead to a defect in the transmission of the gametes. Mutants can therefore be screened on the basis of segregation distortion for a reporter resistance gene contained in the T-DNA. We have screened the Versailles collection of Arabidopsis transformants for 1:1 KanR:KanS segregation after selfing, focussing on gametophyte mutations which show normal transmission through one gametophyte and cause lethality or dysfunction of the other. Only 1.3% (207) of the 16,000 lines screened were scored as good candidates. Thorough genetic analysis of 38 putative T-DNA transmission defect lines (Ttd) identified 8 defective gametophyte mutants, which all showed 0 to 1% T-DNA transmission through the pollen. During the screen, we observed a high background of low-penetrance mutations, often affecting the function of both gametophytes, and many lines which were likely to carry chromosomal rearrangements. The reasons for the small number of retained lines (all male gametophytic) are discussed, as well as the finding that, for most of them, residual T-DNA transmission is obtained through the affected gametophyte.

In this paper, we describe a novel plant mutant affected exclusively in the female mitosis-meiosis switch. The major effect of the swi1 mutation in Arabidopsis thaliana L. is to delay megasporogenesis events by inserting additional mitotic divisions of the mega- sporocyte. As a result of this delay, megagametogenesis is also affected. The absence of cellular polarity in the megasporocytes was also observed. Ovule ontogenesis is not affected by the mutation. The swi1 mutant is particularly interesting for studying sporophyt-gametophyte interactions. The swi1 mutation, obtained from a T-DNA tagging experiment, is monogenic recessive and mapped on chromosome five, at 16 cM from the yellow inflorescence marker.

National audience

La méiose est une étape-clé de la reproduction sexuée chez les organismes diploïdes. Au-delà de sa description cytologique, les mécanismes moléculaires qui entrent en jeu dans cette division spécialisée commencent à être décryptés chez les plantes grâce à la plante-modèle Arabidopsis thaliana. Si certains mutants méiotiques d’Arabidopsis confirment le rôle de fonctions centrales déjà décrites, par exemple, chez la levure ou chez la souris, d’autres ont permis d’identifier des gènes dont la fonction est inconnue à ce jour. De nombreuses plantes existent aussi à l’état de polyploïdes, ce qui pose un problème particulier du point de vue de la méiose., Meiosis is a key step in diploid sexual reproduction. Apart from its cytological description, the molecular mechanisms involved in this specialized cell division are being deciphered in plants thanks to the model plant Arabidopsis thaliana. While some meiotic mutants of Arabidopsis confirm the central role of functions that have been described either in yeast or in mice, others led to the identification of previously unknown genes. Numerous plants also exist as polyploids, which represent a special case with regard to meiosis.