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

Investigating the control of inter-homologue recombination and synapsis during meiosis in Arabidopsis thaliana

During meiosis, axis formation and synapsis of homologous chromosomes are tightly co-ordinated with meiotic recombination. This study investigates the influence of chromosome axis and synaptonemal complex proteins on meiotic crossover formation. The study involved the characterization of a novel protein, AtASY3, required for normal meiosis in Arabidopsis. Analysis of Atasy3 mutants revealed that loss AtASY3 compromises chromosome axis formation, synapsis and normal levels of crossover formation. Further analysis revealed that loss of AtASY3 disrupts the axial organization of AtASY1. In separate studies, colleagues found that AtASY3 and AtASY1 are able to interact. Together, these results suggest that AtASY3 is a functional homologue of the budding yeast axis protein, Red1. Since studies in budding yeast indicate that Red1 and Hop1 (homologue of AtASY1) play a key role in establishing a bias to favour inter-homologue recombination, this study suggests that AtASY3 and AtASY1 may have a similar role in Arabidopsis. The study also involved the analysis of the putative phosphorylation site, residue T295, on AtASY1. This revealed that T295 is essential for AtASY1-mediated crossover formation during meiosis. Additionally, a potential meiotic role for the RECQ DNA helicase, AtRECQ4B was investigated, however, the protein does not appear to be essential for Arabidopsis meiosis

Dual immunolocalisation of AtASY1 and AtZYP1 with recombination pathway proteins in wild-type and <i>Atasy3-1</i> meiotic prophase I nuclei.

<p>(A) AtASY1 (green) and γH2AX (red) on wild-type and (B) <i>Atasy3-1</i> at leptotene, (C) AtASY1 (green) and AtDMC1 (red) on wild-type and (D) on <i>Atasy3-1</i> at leptotene. (E) AtZYP1 (green) and AtMSH4 (red) on wild-type and (F) on <i>Atasy3-1</i> (AtMSH4 arrowed). (G) AtZYP1 (green) and AtMLH1 (red) on wild-type and (H) on <i>Atasy3-1</i> (AtMLH1 arrowed). DAPI (blue) for all. Bar, 10 µm.</p

Yeast two-hybrid analysis of AtASY3 and AtASY1 with schematic illustration of predicted domains and relative positions of amino acid residues.

<p>Plasmid constructs were co-transformed into Y2HGold yeast cells before plating on SD -Leu/-Trp (-LT), SD -Leu/-Trp/-His (-LTH) and SD -Leu/-Trp/-His/-Ade (-LTHA). Growth on –LTH and –LTHA confirms a protein-protein interaction between AtASY1 and AtASY3 that is dependent on AtASY3 residues 623–793.</p

Immunolocalization of AtASY3 and AtASY1.

<p>Immunolocalization of AtASY3 (red) on DAPI stained (blue) wild-type meiocytes during meiotic prophase I (A–F). (A) Late G2, (B) Leptotene, (C) Zygotene, (D) Pachytene, dual localization with AtZYP1 (green) (E) and merged image (F). Immunolocalization of AtASY1 (red) on DAPI stained wild-type meiocytes during prophase I (G–I). (G) Late G2, (H) Leptotene (deconvoluted image), (I) Pachytene (deconvoluted image). Bar, 10 µm. (J) Immunogold labelling of AtASY1 on <i>Crepis capillaris</i> at meiotic prophase I. Bar 100 nm.</p

Meiotic stages.

<p>From wild-type (A–F) and <i>Atasy3-1</i> (G–L) pollen mother cells. (A,G) Leptotene. (B,H) Pachytene. (C,I) Diakinesis. (D,J) Metaphase I. (E,K) Dyad. (F,L) Tetrad. Early prophase I stages in <i>Atasy3-1</i> are similar to that of wild-type, however, at pachytene (H) homologous chromosomes fail to undergo full synapsis in <i>Atasy3-1</i>. Some univalents are present at diakinesis (I) and metaphase I (J) in <i>Atasy3-1</i> which can lead to chromosome mis-segregation at meiotic divisions and give rise to unbalanced dyad (K) and tetrad (L) nuclei. Bar, 10 µm.</p

Immunolocalisation of chromosome axis proteins on <i>Arabidopsis</i> wild-type and chromosome axis mutants.

<p>At prophase I (A) AtASY3 (red) on <i>Atasy3-1</i>, (B) AtSMC3 (red) on wild-type and (C) <i>Atasy3-1</i>, (D) AtSYN1 (red) on wild-type and (E) <i>Atasy3-1</i>. (F) AtASY3 (red) on an <i>Atsyn1</i> mutant, G) AtASY1(red) on <i>Atasy3-1</i> at late G2 and (H) at late prophase I, (I) Immunolocalization of AtASY3 (red) on an <i>Atasy1</i> mutant at leptotene, (J) AtASY1(red) on an <i>Atspo11-1-4/Atasy3-1</i> mutant at late prophase I, (K) Immunolocalization of AtZYP1 (red) on wild-type at pachytene and (L) on <i>Atasy3-1</i> at late prophase I. DNA is stained with DAPI (blue) for all. Bar, 10 µm.</p