The Arabidopsis MutS homolog AtMSH4 functions at an early step in recombination: evidence for two classes of recombination in Arabidopsis

Abstract

MSH4, a meiosis-specific member of the MutS-homolog family of genes, is required for normal levels of recombination and fertility in budding yeast, mouse, and Caenorhabditis elegans. In this paper, we report the identification and characterization of the Arabidopsis homolog of MSH4 (AtMSH4). We demonstrate that AtMSH4 expression can only be detected in floral tissues, consistent with a role in reproduction. Immunofluorescence studies indicate that its expression is limited to early meiotic prophase I, preceding the synapsis of homologous chromosomes. A T-DNA insertional mutant (Atmsh4) exhibited normal vegetative growth but a severe reduction in fertility, consistent with a meiotic defect; this was confirmed by cytological analysis of meiosis. RNAi-induced down-regulation of the MSH4 gene resulted in a similar fertility and meiotic phenotype. We demonstrate that prophase I chromosome synapsis is delayed and may be incomplete in Atmsh4, and metaphase I chiasma frequency is greatly reduced to approximately 15% of wild type, leading to univalence and nondisjunction. We show that these residual chiasmata are randomly distributed among cells and chromosomes. These features of chiasma frequency and distribution in Atmsh4 show close parallels to MSH4-independent crossovers in budding yeast that have been proposed to originate by a separate pathway. Furthermore, the characteristics of the MSH4-independent chiasmata in the Atmsh4 mutant closely parallel those of second-pathway crossovers that have been postulated from Arabidopsis crossover analysis and mathematical modeling. Taken together, this evidence strongly indicates that Arabidopsis possesses two crossover pathways.

Figure 1.

Nucleotide sequence of the Arabidopsis thaliana AtMSH4 cDNA including 5′- and 3′-untranslated regions. The deduced amino acid sequence of the AtMSH4 protein is presented below the cDNA.

Figure 2.

Map of the 5.6-kb At4g17380 locus showing the exon/intron organization of AtMSH4. The exons are represented by black boxes, the triangle shows where the SALK-136296 T-DNA has inserted, and the arrows indicate the positions of the primers used for the RT–PCR expression analysis (Fig. 3).

Figure 3.

Expression analysis shows tissue-specific expression of AtMSH4. (A) RT–PCR analysis of expression in wild-type leaf (L), stem (S), flower bud (B), and open flower (F), and Atmsh4 mutant flower bud (lane 5). (B) Control for A using housekeeping gene GAPD.

Figure 4.

(A–C) Immunolocalization of AtMSH4 protein (red) to early (A), mid (B), and late (C) wild-type prophase I nuclei, showing that AtMSH4 protein is present from mid-leptotene to late zygotene. Dual immunolocalization of AtMSH4 protein (red) with AtASY1 (E–G; green), AtRAD51 (I–K; green), and AtZYP1 (M–O; green), to early (E,I,M), mid (F,J,N), and late (G,K,O) wild-type prophase I nuclei. The patterns of colocalization with these three proteins allow a more precise description of the expression pattern of AtMSH4 (see text for details). (D,H,L,P) The corresponding immunolocalization patterns of these proteins in Atmsh4 mutant prophase I cells, showing expression of AtASY1, AtRAD51, and ATZYP1 but nonexpression of AtMSH4. Bar, 10 μm.

Figure 5.

(A–C) Representative metaphase I nuclei of wild-type (A) and Atmsh4 (B,C) cells after FISH to mark the locations of 5S rDNA (red) and 45S rDNA (green). (D–F) FISH of DNA probes to pachytene-stage nuclei of wild-type Arabidopsis. (D) The localization of the T-DNA insert (green) to chromosome 4 (arrowed); red signals show the locations of 5S rRNA loci on chromosomes 4 and 5. E and F illustrate single (E) and twin (F) BAC FISH signals (green) of the types recorded in the BAC synapsis assay. Bar, 5 μm.

Figure 6.

Representative meiotic stages from wild-type (A–F) and Atmsh4 (G–L) pollen mother cells. (A,G) Pachytene. (B,H) Early diplotene. (C,I) Diakinesis. (D,J) Metaphase I. (E,K) Metaphase II. (F,L) Tetrads. Pachytene and early diplotene are generally similar in appearance in wild type and Atmsh4 (but see text). At diakinesis (I) and metaphase I (J), some univalents are present in Atmsh4 that can lead to uneven chromosome distribution to the second division (K) and tetrad (L) nuclei. Bar, 10 μm.

Figure 7.

DAPI-stained metaphase I cells from RNAi plant 25 showing severely reduced chiasma frequencies. Cell in A has a single bivalent and eight univalents (1 chiasma); cell in B has three bivalents and four univalents (3 chiasmata). Bar, 10 μm.

Figure 8.

Electron micrographs of spread and silver-stained pachytene nuclei of wild-type (A) and Atmsh4 (B) cells. Synapsis is complete in wild type (A), but desynapsed regions are present in the Atmsh4 (B) example (arrowed). Bar, 2 μm.

Figure 9.

Observed (open triangles) and Poisson-predicted (solid circles) distributions of chiasma numbers per cell for Atmsh4 mutant (top) and wild type (bottom).

Figure 10.

Observed (open triangles) and Poisson-predicted (solid circles) distributions of chiasma numbers per chromosome 3 for Atmsh4 mutant (top) and wild type (bottom).