Susan J. Armstrong

Asy1, a protein required for meiotic chromosome synapsis, localizes to axis-associated chromatin in Arabidopsis and Brassica

The Arabidopsis thaliana ASY1 gene is essential for homologous chromosome synapsis. Antibodies specific to Asy1 protein and its homologue BoAsy1 from the related crop species Brassica oleracea have been used to investigate the temporal expression and localization of the protein in both species. Asy1 is initially detected in pollen mother cells during meiotic interphase as numerous punctate foci distributed over the chromatin. As leptotene progresses the signal appears to be increasingly continuous and is closely associated with the axial elements but not to the extended chromatin loops associated with them. By the end of zygotene the signal extends almost the entire length of the synapsed homologues, although not to the telomeres. The protein begins to disappear as the homologues desynapse, until by late diplotene it is no longer associated with the chromosomes. Immunogold labelling in conjunction with electron microscopy established that Asy1 localizes to regions of chromatin that associate with the axial/lateral elements of meiotic chromosomes rather than being a component of the synaptonemal complex itself. These data together with the previously observed asynaptic phenotype of the asy1 mutant suggest that Asy1 is required for morphogenesis of the synaptonemal complex, possibly by defining regions of chromatin that associate with the developing synaptonemal complex structure.

A homologue of the yeast HOP1 gene is inactivated in the Arabidopsis meiotic mutant asy1

Abstract.Synapsis of homologous chromosomes is a key event in meiosis as it is essential for normal chromosome segregation and is implicated in the regulation of crossover frequency. We have previously reported the identification and cytological characterisation of a T-DNA-tagged asynaptic mutant of Arabidopsis thaliana. We have demonstrated that this mutant, asy1, is defective in meiosis in both males and females. Cloning and nucleotide sequencing of the ASY1 gene has revealed that it encodes a polypeptide of 596 amino acids that exhibits similarity to the HOP1 gene of Saccharomyces cerevisiae, which is known to encode a protein essential for synaptonemal complex assembly and normal synapsis. Expression studies indicate that, in common with a number of other Arabidopsis meiotic genes, ASY1 exhibits low-level expression in a range of plant tissues. Southern analysis coupled with database searching has resulted in the identification of an ASY1 homologue, ASY2. Although asy1 exhibits a strong asynaptic phenotype, a residual low level of synapsis indicates that ASY1 and ASY2 may exhibit a low degree of functional redundancy.

Cytological characterization of four meiotic mutants of Arabidopsis isolated from T-DNA-transformed lines

A secondary screen of the Feldmann collection of T-DNA transformed Arabidopsis lines identified several meiotic mutants. We used a spreading technique combined with DAPI staining in a detailed cytogenetic analysis of meiotic chromosome behaviour in four of these mutants, all of which are putatively T-DNA tagged and therefore candidates for molecular and functional analysis of the mutated genes. Two of them are defined as ‘synaptic’ mutants, showing greatly reduced association of homologous chromosomes at metaphase I: one is asynaptic, showing failure of synapsis during prophase I, whereas the other is desynaptic and is characterized by normal but non-maintained synapsis. Another mutant is defective in meiotic cell cycle control and undergoes a third meiotic division, resembling a second division but without an additional round of chromosome duplication. A further mutant shows meiosis-limited chromosome disruption, resulting in extensive chromosome fragmentation combined with other defects. All four mutants experience very irregular chromosome distribution during the meiotic divisions, resulting in abnormal numbers and/or sizes of microspores, with resulting reduced fertility.

Pathways to meiotic recombination in Arabidopsis thaliana

Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.

Reduced meiotic crossovers and delayed prophase I progression in AtMLH3-deficient Arabidopsis.

Characterization of AtMLH3, the Arabidopsis homologue of the prokaryotic MutL mismatch repair gene, reveals that it is expressed in reproductive tissue where it is required for normal levels of meiotic crossovers (COs). Immunocytological studies in an Atmlh3 mutant indicate that chromosome pairing and synapsis proceed with normal distribution of the early recombination pathway proteins. Localization of the MutS homologue AtMSH4 occurs, suggesting that double Holliday junctions (dHjs) are formed, but the MutL homologue AtMLH1, which forms a heterocomplex with AtMLH3, fails to localize normally. Loss of AtMLH3 results in an ∼60% reduction in COs and is accompanied by a substantial delay of ∼25 h in prophase I progression. Analysis of the chiasma distribution in Atmlh3 suggests that dHj resolution can occur, but in contrast to wild type where most or all dHjs are directed to form COs the outcome is biased in favour of a non‐CO outcome by a ratio of around 2 to 1. The data are compatible with a model whereby the MutL complex imposes a dHj conformation that ensures CO formation.

The Interplay of RecA-related Proteins and the MND1–HOP2 Complex during Meiosis in Arabidopsis thaliana

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.

Spatiotemporal Asymmetry of the Meiotic Program Underlies the Predominantly Distal Distribution of Meiotic Crossovers in Barley

This work characterizes factors involved in the predominantly distal location of meiotic crossovers in barley. Recombination initiates first in the distal regions and later in the interstitial regions; manipulating meiotic progression with higher temperatures produced more interstitial crossovers that could improve mapping of agronomical traits and reduce linkage drag. Meiosis involves reciprocal exchange of genetic information between homologous chromosomes to generate new allelic combinations. In cereals, the distribution of genetic crossovers, cytologically visible as chiasmata, is skewed toward the distal regions of the chromosomes. However, many genes are known to lie within interstitial/proximal regions of low recombination, creating a limitation for breeders. We investigated the factors underlying the pattern of chiasma formation in barley (Hordeum vulgare) and show that chiasma distribution reflects polarization in the spatiotemporal initiation of recombination, chromosome pairing, and synapsis. Consequently, meiotic progression in distal chromosomal regions occurs in coordination with the chromatin cycles that are a conserved feature of the meiotic program. Recombination initiation in interstitial and proximal regions occurs later than distal events, is not coordinated with the cycles, and rarely progresses to form chiasmata. Early recombination initiation is spatially associated with early replicating, euchromatic DNA, which is predominately found in distal regions. We demonstrate that a modest temperature shift is sufficient to alter meiotic progression in relation to the chromosome cycles. The polarization of the meiotic processes is reduced and is accompanied by a shift in chiasma distribution with an increase in interstitial and proximal chiasmata, suggesting a potential route to modify recombination in cereals.

Inter-Homolog Crossing-Over and Synapsis in Arabidopsis Meiosis Are Dependent on the Chromosome Axis Protein AtASY3

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.

A and C Genome Distinction and Chromosome Identification in Brassica napus by Sequential Fluorescence in Situ Hybridization and Genomic in Situ Hybridization

The two genomes (A and C) of the allopolyploid Brassica napus have been clearly distinguished using genomic in situ hybridization (GISH) despite the fact that the two extant diploids, B. rapa (A, n = 10) and B. oleracea (C, n = 9), representing the progenitor genomes, are closely related. Using DNA from B. oleracea as the probe, with B. rapa DNA and the intergenic spacer of the B. oleracea 45S rDNA as the block, hybridization occurred on 9 of the 19 chromosome pairs along the majority of their length. The pattern of hybridization confirms that the two genomes have remained distinct in B. napus line DH12075, with no significant genome homogenization and no large-scale translocations between the genomes. Fluorescence in situ hybridization (FISH)—with 45S rDNA and a BAC that hybridizes to the pericentromeric heterochromatin of several chromosomes—followed by GISH allowed identification of six chromosomes and also three chromosome groups. Our procedure was used on the B. napus cultivar Westar, which has an interstitial reciprocal translocation. Two translocated segments were detected in pollen mother cells at the pachytene stage of meiosis. Using B. oleracea chromosome-specific BACs as FISH probes followed by GISH, the chromosomes involved were confirmed to be A7 and C6.

Physical mapping of DNA repetitive sequences to mitotic and meiotic chromosomes of Brassica oleracea var. alboglabra by fluorescence in situ hybridization

As part of a programme to integrate the genetic and physical chromosome maps of Brassica oleracea we have carried out fluorescence in situ hybridization (FISH) to mitotic and meiotic chromosomes of B. oleracea var. alboglabra, using repeat sequence DNA probes. We have confirmed that there are three 18S-5.8S-25S rDNA sites in the haploid genome and accurately determined their locations for the first time; two occur subtelomerically on the short arms of the two satellited acrocentric chromosomes (chromosomes 4 and 7), whereas the third site is adjacent to the centromere on the short arm of a large submetacentric chromosome (chromosome 2). 5S rDNA sequences are located on the long arm of this same chromosome, with closely adjacent major and minor loci. A highly repeated sequence (pBcKB4) colocalizes with the pericentromeric heterochromatin on all chromosomes, but six chromosomes exhibit very strong signals whereas the remaining three show only weak signals. The construction of a partial karyotype for B. oleracea var. alboglabra, based on several major cytogenetical landmarks provided by FISH localization of repetitive DNA sequences, provides a valuable framework for the future physical mapping of nonrepetitive sequences.