Ahp2 (Hop2) function in Arabidopsis thaliana (Ler) is required for stabilization of close alignment and synaptonemal complex formation except for the two short arms that contain nucleolus organizer regions

Abstract

A cytological comparative analysis of male meiocytes was performed for Arabidopsis wild type and the ahp2 (hop2) mutant with emphasis on ahp2's largely uncharacterized prophase I. Leptotene progression appeared normal in ahp2 meiocytes; chromosomes exhibited regular axis formation and assumed a typical polarized nuclear organization. In contrast, 4',6'-diamidino-2-phenylindole-stained ahp2 pachytene chromosome spreads demonstrated a severe reduction in stabilized pairing. However, transmission electron microscopy (TEM) analysis of sections from meiocytes revealed that ahp2 chromosome axes underwent significant amounts of close alignment (44% of total axis). This apparent paradox strongly suggests that the Ahp2 protein is involved in the stabilization of homologous chromosome close alignment. Fluorescent in situ hybridization in combination with Zyp1 immunostaining revealed that ahp2 mutants undergo homologous synapsis of the nucleolus-organizer-region-bearing short arms of chromosomes 2 and 4, despite the otherwise "nucleus-wide" lack of stabilized pairing. The duration of ahp2 zygotene was significantly prolonged and is most likely due to difficulties in chromosome alignment stabilization and subsequent synaptonemal complex formation. Ahp2 and Mnd1 proteins have previously been shown, "in vitro," to form a heterodimer. Here we show, "in situ," that the Ahp2 and Mnd1 proteins are synchronous in their appearance and disappearance from meiotic chromosomes. Both the Ahp2 and Mnd1 proteins localize along the chromosomal axis. However, localization of the Ahp2 protein was entirely foci-based whereas Mnd1 protein exhibited an immunostaining pattern with some foci along the axis and a diffuse staining for the rest of the chromosome.

Sufficient Amounts of Functional HOP2/MND1 Complex Promote Interhomolog DNA Repair but Are Dispensable for Intersister DNA Repair during Meiosis in Arabidopsis

“…First, normal chromosome pairing was not observed during prophase I of male meiosis in any of the studied alleles (Figures 1Ae and 1Ai to 1Aa) (Schommer et al, 2003;Stronghill et al, 2010). As described before (Schommer et al, 2003), severe chromosomal defects (chromosome bridges and DNA fragmentation) could be observed from anaphase I to the end of meiosis in hop2-1 (Figures 1Af to 1Ah) in a large majority of the meiotic cells ( Figure 1B, 82%, n = 39).…”

“…The HOP2 wild-type protein is structured into an N-terminal domain, a putative coiled coil region, and a C-terminal domain. hop2-1 plants do not express the protein (Stronghill et al, 2010). Plants carrying the hypomorphic hop2-2 allele produce very low levels of the mRNA and anticipated very low levels of HOP2 protein.…”

“…Sequencing of the HOP2 gene in these alleles revealed the nature of each mutation. The original hop2-1 null-mutant (in the Landsberg erecta [Ler] background) has a T-DNA insertion in exon 4, does not produce functional mRNA, and lacks HOP2 protein ( Figure 2A) (Schommer et al, 2003;Stronghill et al, 2010). The hop2-2 allele has a 147-bp deletion 101 bp upstream of the start codon of the HOP2 gene (Figure 2A).…”

“…Nevertheless, the Arabidopsis HOP2/MND1 complex has not been shown to be involved in the promotion of IH repair via a DMC1-directed pathway since both IH and IS DNA repair are dysfunctional in the absence of any of these two proteins (Schommer et al, 2003;Kerzendorfer et al, 2006;Stronghill et al, 2010). Recently, we identified two mutant alleles of Arabidopsis HOP2 (De Muyt et al, 2009) that differ from the already characterized hop2-1 null mutant allele (previously named ahp2-1; renamed hop2-1 for nomenclature consistency; Schommer et al, 2003).…”

Homologous pairing and the role of pairing centers in meiosis

“…However, the short arms of chromosomes 2 and 4, where the two NORs are located, exhibit normal pairing frequencies and normal SC formation. These findings indicate that the NORs act as cis-acting pairing and synapsis initiation sites in ahp2 mutants (Stronghill et al, 2010), in a manner reminiscent of C. elegans PCs. The extent to which NORs contribute to pairing of chromosomes 2 and 4 in wild-type plants, and whether NORs exhibit similar behavior in other organisms, still remains to be determined.…”