Zip4/Spo22 is required for class I CO formation but not for synapsis completion in Arabidopsis thaliana

Abstract

In budding yeast meiosis, the formation of class I interference-sensitive crossovers requires the ZMM proteins. These ZMM proteins are essential in forming a mature synaptonemal complex, and a subset of these (Zip2, Zip3, and Zip4) has been proposed to compose the core of synapsis initiation complexes (SICs). Zip4/Spo22 functions with Zip2 to promote polymerization of Zip1 along chromosomes, making it a crucial SIC component. In higher eukaryotes, synapsis and recombination have often been correlated, but it is totally unknown how these two processes are linked. In this study, we present the characterization of a higher eukaryote SIC component homologue: Arabidopsis AtZIP4. We show that mutations in AtZIP4 belong to the same epistasis group as Atmsh4 and eliminate approximately 85% of crossovers (COs). Furthermore, genetic analyses on two adjacent intervals of Chromosome I established that the remaining COs in Atzip4 do not show interference. Lastly, immunolocalization studies showed that polymerization of the central element of the synaptonemal complex is not affected in Atzip4 background, even if it may proceed from fewer sites compared to wild type. These results reveal that Zip4 function in class I CO formation is conserved from budding yeast to Arabidopsis. On the other hand, and contrary to the situation in yeast, mutation in AtZIP4 does not prevent synapsis, showing that both aspects of the Zip4 function (i.e., class I CO maturation and synapsis) can be uncoupled.

Figure 1. The Zip4 Family and Atzip4 Mutations

(A) Schematic representation of the AtZIP4 coding sequence. Exons are represented as black boxes and T-DNA insertions in Atzip4-1 and Atzip4-2 alleles are indicated. (B) Alignment of A. thaliana, S. cerevisiae, Homo sapiens, Oryza sativa, and Dario reno Zip4 homologues. The numbers indicate aa positions; identical aas are boxed in black whereas similar aas are boxed in gray. The positions of the T-DNA insertions in the mutant alleles are indicated.

Figure 2. Atzip4 Mutant Phenotype

(A) Comparison of wild-type and homozygous Atzip4-1 mutant plants after 30 d in the greenhouse. Arrows show siliques that elongate in wild type but not in mutant. (B–E) Male sporogenesis in wild type (B–C) and Atzip4 mutant (D–E) shown for Atzip4-1. Male meiocytes (PMCs) are shown in (B and D) within the anthers, and the product of meiosis (tetrads or polyads) is shown in (C and E). Wt, wild type.

Figure 3. DAPI Staining of Wild-Type (Ws) PMCs during Meiosis

(A) Leptotene, (B) zygotene, (C) pachytene, (D) diakinesis, (E) metaphase I, (F) end of anaphase I, (G) telophase I, (H) end of anaphase II, (I) end of meiosis. Bar, 10 μm.

Figure 4. DAPI Staining of Atzip4-1 PMCs during Meiosis

(A) Leptotene, (B) zygotene: The synapsed portion of (B) has been magnified and is presented in the boxed area of the figure. (C) Pachytene, (D) diakinesis, (E–H) metaphase I, (I) anaphase I, (J) metaphase II, (K) anaphase II, (L) end of meiosis. Bar, 10 μm.

Figure 5. Coimmunolocalization of ASY1 and AtDMC1 in Wild-Type and Mutant Meiocytes

Coimmunolocalization of ASY1 (red) and DMC1 (green) in wild-type (Ws) and Atzip4–1 (Atzip4) PMCs. For each cell, each single staining is shown as well as the overlay of both signals (merge). (A and D) Leptotene, (B and E) zygotene, (C and F) pachytene. Bar, 10 μm.

Figure 6. Coimmunolocalization of ASY1 (Red) and ZYP1 (Green) in Wild-Type (Ws) and Mutant (Atzip4) PMCs

Prophase I cells showing increasing level of synapsis (according to anti-ZYP1 labeling) are shown for both genotypes: absence of synapsis (leptotene, A and M), partial synapsis (zygotene, B–J and N–V), and full synapsis (pachytene, K–L and W–X). For all cells, only the merge signal is shown, but Figures S2 and S3 provide each single staining. Bar, 10 μm (bar presented on A applies for all cells except O).