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.

In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.

These authors contributed equally to this work. SUMMARYThe movement of chromosomes during meiosis involves location of their telomeres at the inner surface of the nuclear envelope. Sad1/UNC-84 (SUN) domain proteins are inner nuclear envelope proteins that are part of complexes linking cytoskeletal elements with the nucleoskeleton, connecting telomeres to the force-generating mechanism in the cytoplasm. These proteins play a conserved role in chromosome dynamics in eukaryotes. Homologues of SUN domain proteins have been identified in several plant species. In Arabidopsis thaliana, two proteins that interact with each other, named AtSUN1 and AtSUN2, have been identified. Immunolocalization using antibodies against AtSUN1 and AtSUN2 proteins revealed that they were associated with the nuclear envelope during meiotic prophase I. Analysis of the double mutant Atsun1-1 Atsun2-2 has revealed severe meiotic defects, namely a delay in the progression of meiosis, absence of full synapsis, the presence of unresolved interlock-like structures, and a reduction in the mean cell chiasma frequency. We propose that in Arabidopsis thaliana, overlapping functions of SUN1 and SUN2 ensure normal meiotic recombination and synapsis.

. * For correspondence (fax +34 91 394 4844; e-mail pradillo@bio.ucm.es). † E. Sá nchez-Morá n and J.L. Santos acted as joint senior authors. SUMMARYThe eukaryotic recombinases RAD51 and DMC1 are essential for DNA strand-exchange between homologous chromosomes during meiosis. RAD51 is also expressed during mitosis, and mediates homologous recombination (HR) between sister chromatids. It has been suggested that DMC1 might be involved in the switch from intersister chromatid recombination in somatic cells to interhomolog meiotic recombination. At meiosis, the Arabidopsis Atrad51 null mutant fails to synapse and has extensive chromosome fragmentation. The Atdmc1 null mutant is also asynaptic, but in this case chromosome fragmentation is absent. Thus in plants, AtDMC1 appears to be indispensable for interhomolog homologous recombination, whereas AtRAD51 seems to be more involved in intersister recombination. In this work, we have studied a new AtRAD51 knock-down mutant, Atrad51-2, which expresses only a small quantity of RAD51 protein. Atrad51-2 mutant plants are sterile and hypersensitive to DNA double-strand break induction, but their vegetative development is apparently normal. The meiotic phenotype of the mutant consists of partial synapsis, an elevated frequency of univalents, a low incidence of chromosome fragmentation and multivalent chromosome associations. Surprisingly, nonhomologous chromosomes are involved in 51% of bivalents. The depletion of AtDMC1 in the Atrad51-2 background results in the loss of bivalents and in an increase of chromosome fragmentation. Our results suggest that a critical level of AtRAD51 is required to ensure the fidelity of HR during interchromosomal exchanges. Assuming the existence of asymmetrical DNA strand invasion during the initial steps of recombination, we have developed a working model in which the initial step of strand invasion is mediated by AtDMC1, with AtRAD51 required to check the fidelity of this process.

In plants, small non-coding RNAs (≈20–30 nt) play a major role in a gene regulation mechanism that controls development, maintains heterochromatin and defends against viruses. However, their possible role in cell division (mitosis and meiosis) still remains to be ascertained. ARGONAUTE (AGO) proteins are key players in the different small RNA (sRNA) pathways. Arabidopsis contains 10 AGO proteins belonging to three distinct phylogenetic clades based on amino acid sequence, namely: AGO1/AGO5/AGO10, AGO2/AGO3/AGO7, and AGO4/AGO6/AGO8/AGO9. To gain new insights into the role of AGO proteins, we have focused our attention on AGO2, AGO5, and AGO9 by means of the analysis of plants carrying mutations in the corresponding genes. AGO2 plays a role in the natural cis-antisense (nat-siRNA) pathway and is required for an efficient DNA repair. On the other hand, AGO5, involved in miRNA (microRNA)-directed target cleavage, and AGO9, involved in RNA-directed DNA methylation (RdDM), are highly enriched in germline. On these grounds, we have analyzed the effects of these proteins on the meiotic process and also on DNA repair. It was confirmed that AGO2 is involved in DNA repair. In ago2-1 the mean cell chiasma frequency in pollen mother cells (PMCs) was increased relative to the wild-type (WT). ago5-4 showed a delay in germination time and a slight decrease in fertility, however the meiotic process and chiasma levels were normal. Meiosis in PMCs of ago9-1 was characterized by a high frequency of chromosome interlocks from pachytene to metaphase I, but chiasma frequency and fertility were normal. Genotoxicity assays have confirmed that AGO9 is also involved in somatic DNA repair.

Maintenance and precise regulation of sister chromatid cohesion is essential for faithful chromosome segregation during mitosis and meiosis. Cohesin cofactors contribute to cohesin dynamics and interact with cohesin complexes during cell cycle. One of these, PDS5, also known as SPO76, is essential during mitosis and meiosis in several organisms and also plays a role in DNA repair. In yeast, the complex Wapl-Pds5 controls cohesion maintenance and colocalizes with cohesin complexes into chromosomes. In Arabidopsis, AtWAPL proteins are essential during meiosis, however, the role of AtPDS5 remains to be ascertained. Here we have isolated mutants for each of the five AtPDS5 genes (A–E) and obtained, after different crosses between them, double, triple, and even quadruple mutants (Atpds5a Atpds5b Atpds5c Atpds5e). Depletion of AtPDS5 proteins has a weak impact on meiosis, but leads to severe effects on development, fertility, somatic homologous recombination (HR) and DNA repair. Furthermore, this cohesin cofactor could be important for the function of the AtSMC5/AtSMC6 complex. Contrarily to its function in other species, our results suggest that AtPDS5 is dispensable during the meiotic division of Arabidopsis, although it plays an important role in DNA repair by HR.

Little is known how patterns of cross-over (CO) numbers and distribution during meiosis are established. Here, we reveal that cyclin-dependent kinase A;1 (CDKA;1), the homolog of human Cdk1 and Cdk2, is a major regulator of meiotic recombination in Arabidopsis. Arabidopsis plants with reduced CDKA;1 activity experienced a decrease of class I COs, especially lowering recombination rates in centromere-proximal regions. Interestingly, this reduction of type I CO did not affect CO assurance, a mechanism by which each chromosome receives at least one CO, resulting in all chromosomes exhibiting similar genetic lengths in weak loss-of-function cdka;1 mutants. Conversely, an increase of CDKA;1 activity resulted in elevated recombination frequencies. Thus, modulation of CDKA;1 kinase activity affects the number and placement of COs along the chromosome axis in a dose-dependent manner.

Different histone modifications often modify DNA-histone interactions affecting both local and global structure of chromatin, thereby providing a vast potential for functional responses. Most studies have focused on the role of several modifications in gene transcription regulation, being scarce on other aspects of eukaryotic chromosome structure during cell division, mainly in meiosis. To solve this issue we have performed a cytological analysis to determine the chromosomal distribution of several histone H3 modifications throughout all phases of both mitosis and meiosis in different plant species. We have chosen Aegilops sp. and Secale cereale (monocots) and Arabidopsis thaliana (dicots) because they differ in their phylogenetic affiliation as well as in content and distribution of constitutive heterochromatin. In the species analyzed, the patterns of H3 acetylation and methylation were held constant through mitosis, including modifications associated with "open chromatin". Likewise, the immunolabeling patterns of H3 methylation remained invariable throughout meiosis in all cases. On the contrary, there was a total loss of acetylated H3 immunosignals on condensed chromosomes in both meiotic divisions, but only in monocot species. Regarding the phosphorylation of histone H3 at Ser10, present on condensed chromosomes, although we did not observe any difference in the dynamics, we found slight differences between the chromosomal distribution of this modification between Arabidopsis and cereals (Aegilops sp. and rye). Thus far, in plants chromosome condensation throughout cell division appears to be associated with a particular combination of H3 modifications. Moreover, the distribution and dynamics of these modifications seem to be species-specific and even differ between mitosis and meiosis in the same species.