ZYP1 is required for obligate cross-over formation and cross-over interference in Arabidopsis

Abstract

The synaptonemal complex is a tripartite proteinaceous ultrastructure that forms between homologous chromosomes during prophase I of meiosis in the majority of eukaryotes. It is characterized by the coordinated installation of transverse filament proteins between two lateral elements and is required for wild-type levels of crossing over and meiotic progression. We have generated null mutants of the duplicated Arabidopsis transverse filament genes zyp1a and zyp1b using a combination of T-DNA insertional mutants and targeted CRISPR/Cas mutagenesis. Cytological and genetic analysis of the zyp1 null mutants reveals loss of the obligate chiasma, an increase in recombination map length by 1.3- to 1.7-fold and a virtual absence of cross-over (CO) interference, determined by a significant increase in the number of double COs. At diplotene, the numbers of HEI10 foci, a marker for Class I interference-sensitive COs, are twofold greater in the zyp1 mutant compared to wild type. The increase in recombination in zyp1 does not appear to be due to the Class II interference-insensitive COs as chiasmata were reduced by ∼52% in msh5/zyp1 compared to msh5 These data suggest that ZYP1 limits the formation of closely spaced Class I COs in Arabidopsis Our data indicate that installation of ZYP1 occurs at ASY1-labeled axial bridges and that loss of the protein disrupts progressive coalignment of the chromosome axes.

Keywords: cross-over interference; meiosis; recombination; synaptonemal complex.

Fig. 1.

zyp1 null mutants are asynaptic. (A) The ZYP1 locus showing positions of T-DNA insertions and CRISPR/Cas mutations for the three zyp1 null mutants. Exons are represented by dark red boxes, and noncoding regions of the transcripts are shown in light red. (B) Coimmunofluorescence of ASY1 and ZYP1-C terminus on wild-type and zyp1a-2/zyp1b-1 meiotic prophase I chromosome spread preparations. (Scale bars, 10 µm for main panels and 1 µm for zoom.)

Fig. 2.

Chromosome axes align at a greater distance in zyp1. (A and B) Coimmunofluorescence of ASY1 and ZYP1-N terminus on wild-type and zyp1a-2/zyp1b-1 meiotic prophase I chromosome spread preparations (zoom panel is shown in white box in channel merge). (C and D) Enlarged region (white box from A and B, SMC3 channel) showing representative lines bisecting the homologous axes used to measure homologous distance. (E and F) Plot profiles of bisecting lines (from C and D). (G) Comparison of lateral distances between wild type and zyp1a-2/zyp1b-1. ***P < 0.001 (Wilcoxon rank sum test). (H) False-colored Z-stack projection of a zyp1 pachytene analog nucleus. (Scale bar, 10 µm.) (I) One pair of aligned chromosomes is traced and shown in orange and pink. (J) Enlarged region (white box in A) shows three features of zyp1 “pachytene” nuclei (1: axial entanglement, 2: axial bridge, and 3: twisted axes). (K) False color encoding for slice depth (z). (Scale bars, A [MERGE], B [MERGE], H: 10 µm; A [ZOOM], B [ZOOM], C, D, J: 1 µm.)

Fig. 3.

Coimmunofluorescence of ASY1 and ZYP1-C terminus on a late-zygotene wild-type nucleus by structured illumination microscopy. Images show dynamics of axis protein ASY1 unloading and transverse filament protein ZYP1 loading at synaptic forks. Insets and white arrows highlight localization at the axial bridges. (Scale bar, 10 µm.)

Fig. 4.

ZYP1 ensures formation of the obligate chiasma. (A) Meiotic atlas of DAPI-stained chromosome spreads from wild-type (A–H) and zyp1a-2/zyp1b-1 (I–P) pollen mother cells. (A and I) Leptotene. (B and J) Zygotene (and analog). (C and K) Pachytene (and analog). (D and L) Diplotene. (E and M) Diakinesis. (F and N) Metaphase I. (G and O) Metaphase II. (H and P) Telophase II. (Q–T) Examples of aberrant recombination and possible interlocks in zyp1a-2/zyp1b-1 at metaphase I. (Scale bars, 10 μm.)

Fig. 5.

Chromosome configurations at meiotic metaphase I. Chromosome configurations for all genotypes analyzed are presented relative to the five chromosome pairs of A. thaliana. The number of cells sampled for each line is shown in brackets.

Fig. 6.

Higher numbers of HEI10 foci persist in the absence of ZYP1. Coimmunofluorescence of HEI10 and SMC3 on meiotic prophase I chromosome spreads. (A and D) Zygotene/early pachytene (and analog). (B and E) Late pachytene and “pachytene”’/diplotene transition. (C and F) Diplotene. (Scale bars, 10 µm.) (G) Counts of HEI10 foci per cell for the wild type and zyp1a-2/zyp1b-1. *P < 0.05 and ***P < 0.001 for a two-sample Wilcoxon ranked sum test.

Fig. 7.

Recombination and double COs increase in zyp1. (A) Genetic map distance (centi-Morgan) determined by recombination in pollen FTLs in three intervals. (B) Interference ratio calculated in the I3bc double interval. (C) Illustration and examples of double COs observed in zyp1 fluorescent pollen tetrads and an NCO in wild type. (D) Genetic map distance (cM) determined by fluorescent seed-based assay of the 420 interval. (E) An example of a field of view from the fluorescent seed recombination assay.