Identification of ASYNAPTIC4, a Component of the Meiotic Chromosome Axis.

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.

The kinesin AtPSS1 promotes synapsis and is required for proper crossover distribution in meiosis.

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.

Suppression of Dwarf and irregular xylem Phenotypes Generates Low-Acetylated Biomass Lines in Arabidopsis.

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.

FANCM-associated proteins MHF1 and MHF2, but not the other Fanconi anemia factors, limit meiotic crossovers.

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.

MCM8 is required for a pathway of meiotic double-strand break repair independent of DMC1 in Arabidopsis thaliana.

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.

OSD1 promotes meiotic progression via APC/C inhibition and forms a regulatory network with TDM and CYCA1;2/TAM.

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.

The cyclin-A CYCA1;2/TAM is required for the meiosis I to meiosis II transition and cooperates with OSD1 for the prophase to first meiotic division transition.

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.

Water Deficit-Responsive QTLs for Cell Wall Degradability and Composition in Maize at Silage Stage.

The use of lignocellulosic biomass for animal feed or biorefinery requires the optimization of its degradability. Moreover, biomass crops need to be better adapted to the changing climate and in particular to periods of drought. Although the negative impact of water deficit on biomass yield has often been mentioned, its impact on biomass quality has only been recently reported in a few species. In the present study, we combined the mapping power of a maize recombinant inbred line population with robust near infrared spectroscopy predictive equations to track the response to water deficit of traits associated with biomass quality. The population was cultivated under two contrasted water regimes over 3 consecutive years in the south of France and harvested at silage stage. We showed that cell wall degradability and ß-O-4-linked H lignin subunits were increased in response to water deficit, while lignin and p-coumaric acid contents were reduced. A mixed linear model was fitted to map quantitative trait loci (QTLs) for agronomical and cell wall-related traits. These QTLs were categorized as "constitutive" (QTL with an effect whatever the irrigation condition) or "responsive" (QTL involved in the response to water deficit) QTLs. Fifteen clusters of QTLs encompassed more than two third of the 213 constitutive QTLs and 13 clusters encompassed more than 60% of the 149 responsive QTLs. Interestingly, we showed that only half of the responsive QTLs co-localized with constitutive and yield QTLs, suggesting that specific genetic factors support biomass quality response to water deficit. Overall, our results demonstrate that water deficit favors cell wall degradability and that breeding of varieties that reconcile improved drought-tolerance and biomass degradability is possible.

Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not.

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.

Flanking sequence tags in Arabidopsis thaliana T-DNA insertion lines: a pilot study.

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.

[Molecular mechanisms of meiosis in plants]. / Les mécanismes moléculaires de la méiose chez les plantes.

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.

Centromeric cohesion is protected twice at meiosis, by SHUGOSHINs at anaphase I and by PATRONUS at interkinesis.

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.

FANCM limits meiotic crossovers.

The number of meiotic crossovers (COs) is tightly regulated within a narrow range, despite a large excess of molecular precursors. The factors that limit COs remain largely unknown. Here, using a genetic screen in Arabidopsis thaliana, we identified the highly conserved FANCM helicase, which is required for genome stability in humans and yeasts, as a major factor limiting meiotic CO formation. The fancm mutant has a threefold-increased CO frequency as compared to the wild type. These extra COs arise not from the pathway that accounts for most of the COs in wild type, but from an alternate, normally minor pathway. Thus, FANCM is a key factor imposing an upper limit on the number of meiotic COs, and its manipulation holds much promise for plant breeding.

The Arabidopsis TFIID factor AtTAF6 controls pollen tube growth.

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.

AtREC8 and AtSCC3 are essential to the monopolar orientation of the kinetochores during meiosis.

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.

The meiotic protein SWI1 is required for axial element formation and recombination initiation in Arabidopsis.

We report the detailed characterization of SWITCH1 (SWI1) an Arabidopsis thaliana protein that has been linked with the establishment of sister chromatid cohesion during meiosis. Using a combination of cytological methods including immunolocalization of meiotic chromosome-associated proteins we show that SWI1 is required for formation of axial elements. Our studies reveal that the swi1-2 mutation prevents the formation of RAD51 foci during meiotic prophase and suppresses the chromosome fragmentation phenotype of the recombination-defective dif1-1 mutant. Together, these data suggest that SWI1 may be required for meiotic recombination initiation. Finally we raised an antibody against SWI1 and showed, by immunolocalization coupled with bromodeoxyuridine incorporation experiments, that SWI1 is expressed exclusively in meiotic G(1) and S phase. Thus, SWI1 appears to be required for early meiotic events that are at the crossroad of sister chromatid cohesion, recombination and axial element formation. The possible inter-relationship between these processes and the function of SWI1 are discussed.