Mathilde Grelon

AtSPO11-1 is necessary for efficient meiotic recombination in plants.

The Saccharomyces cerevisiae Spo11 protein catalyses DNA double‐strand breaks (DSBs) that initiate meiotic recombination. The model plant Arabidopsis thaliana possesses at least three SPO11 homologues. T‐DNA and ethyl‐methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11‐1 gene is disrupted. Both male and female meiocytes formed very few bivalents. Furthermore, no fully synapsed chromosomes were observed during prophase I. Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non‐functional gametes. These meiotic aberrations were associated with a drastic reduction in meiotic recombination. Thus, our data show that initiation of meiotic recombination by SPO11‐ induced DSBs is a mechanism conserved in plants. Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.

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.

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.

Ogura cytoplasmic male-sterility (CMS)-associated orf138 is translated into a mitochondrial membrane polypeptide in male-sterile Brassica cybrids

Transcription of a putative mitochondrial gene (orf138) has previously been correlated with Ogura cytoplasmic male-sterility (CMS) in rapeseed cybrids. In this paper, studies performed on a Brassica cybrid with a different organization of the orf138 locus confirm this association. We also show that mitochondria isolated from male-sterile rapeseed plants synthesize a polypeptide of 19 kDa, which is absent in fertile revertants. Antibodies against a glutathione S-transferase-ORF138 fusion protein were raised to establish that this 19 kDa polypeptide is the product of orf138. The anti-ORF138 serum was used to demonstrate that the orf138 translation product occurs only in sterile cybrids and co-purifies with the mitochondrial membrane fraction.

The molecular biology of meiosis in plants.

Meiosis is the cell division that reshuffles genetic information between generations. Recently, much progress has been made in understanding this process; in particular, the identification and functional analysis of more than 80 plant genes involved in meiosis have dramatically deepened our knowledge of this peculiar cell division. In this review, we provide an overview of advancements in the understanding of all aspects of plant meiosis, including recombination, chromosome synapsis, cell cycle control, chromosome distribution, and the challenge of polyploidy.

Zip4/Spo22 Is Required for Class I CO Formation but Not for Synapsis Completion in Arabidopsis thaliana

In budding yeast meiosis, the formation of class I interference-sensitive crossovers requires the ZMM proteins. These ZMM proteins are essential in forming a mature synaptonemal complex, and a subset of these (Zip2, Zip3, and Zip4) has been proposed to compose the core of synapsis initiation complexes (SICs). Zip4/Spo22 functions with Zip2 to promote polymerization of Zip1 along chromosomes, making it a crucial SIC component. In higher eukaryotes, synapsis and recombination have often been correlated, but it is totally unknown how these two processes are linked. In this study, we present the characterization of a higher eukaryote SIC component homologue: Arabidopsis AtZIP4. We show that mutations in AtZIP4 belong to the same epistasis group as Atmsh4 and eliminate approximately 85% of crossovers (COs). Furthermore, genetic analyses on two adjacent intervals of Chromosome I established that the remaining COs in Atzip4 do not show interference. Lastly, immunolocalization studies showed that polymerization of the central element of the synaptonemal complex is not affected in Atzip4 background, even if it may proceed from fewer sites compared to wild type. These results reveal that Zip4 function in class I CO formation is conserved from budding yeast to Arabidopsis. On the other hand, and contrary to the situation in yeast, mutation in AtZIP4 does not prevent synapsis, showing that both aspects of the Zip4 function (i.e., class I CO maturation and synapsis) can be uncoupled.

Epigenetic Remodeling of Meiotic Crossover Frequency in Arabidopsis thaliana DNA Methyltransferase Mutants

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.

A High Throughput Genetic Screen Identifies New Early Meiotic Recombination Functions in Arabidopsis thaliana

Meiotic recombination is initiated by the formation of numerous DNA double-strand breaks (DSBs) catalysed by the widely conserved Spo11 protein. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation; however, unlike Spo11, few of these are conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we took advantage of a high-throughput meiotic mutant screen carried out in the model plant Arabidopsis thaliana. A collection of 55,000 mutant lines was screened, and spo11-like mutations, characterised by a drastic decrease in chiasma formation at metaphase I associated with an absence of synapsis at prophase, were selected. This screen led to the identification of two populations of mutants classified according to their recombination defects: mutants that repair meiotic DSBs using the sister chromatid such as Atdmc1 or mutants that are unable to make DSBs like Atspo11-1. We found that in Arabidopsis thaliana at least four proteins are necessary for driving meiotic DSB repair via the homologous chromosomes. These include the previously characterised DMC1 and the Hop1-related ASY1 proteins, but also the meiotic specific cyclin SDS as well as the Hop2 Arabidopsis homologue AHP2. Analysing the mutants defective in DSB formation, we identified the previously characterised AtSPO11-1, AtSPO11-2, and AtPRD1 as well as two new genes, AtPRD2 and AtPRD3. Our data thus increase the number of proteins necessary for DSB formation in Arabidopsis thaliana to five. Unlike SPO11 and (to a minor extent) PRD1, these two new proteins are poorly conserved among species, suggesting that the DSB formation mechanism, but not its regulation, is conserved among eukaryotes.

A CDC45 Homolog in Arabidopsis Is Essential for Meiosis, as Shown by RNA Interference–Induced Gene Silencing

CDC45 is required for the initiation of DNA replication in yeast and cell proliferation in mammals and functions as a DNA polymerase α loading factor in Xenopus. We have cloned a CDC45 homolog from Arabidopsis whose expression is upregulated at the G1/S transition and in young meiotic flower buds. One-third of Arabidopsis 35S:CDC45 T1 RNA interference lines are partially to completely sterile, and the proportion of sterile plants is increased by using a dmc1 promoter. T1 plants have decreased levels of the CDC45 transcript and contain 21- to 23-bp RNA fragments specific to the CDC45 gene. T2 transgenic lines, in which small RNA fragments are still present, were used to analyze S-phase entry by 5-bromodeoxyuridine incorporation, which was not altered compared with that in the wild type. However, microarray data show that other cell cycle genes are upregulated or downregulated. T2 plants also have highly reduced fertility. The severity of the phenotype is correlated with the levels of the CDC45 transcript and small RNA fragments. Severe chromosome fragmentation arising during meiosis, which is not the result of a defect in the repair of SPO11-induced double strand breaks, leads to abnormal chromosome segregation and defective pollen and ovule development.

The Arabidopsis HEI10 Is a New ZMM Protein Related to Zip3

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.