An Easy Protocol for Studying Chromatin and Recombination Protein Dynamics during <i>Arabidopsis</i><i>thaliana</i> Meiosis: Immunodetection of Cohesins, Histones and MLH1

Abstract

Plant meiosis studies have enjoyed a fantastic boom in recent years with the use of Arabidopsis thaliana as a model not only for molecular genetics and genomics but also for cytogenetics. In this article we describe a new protocol for immunolabelling meiotic proteins that allows the detection of a large range of proteins on strongly spread chromosomes throughout the entire meiotic process. We used this method to immunodetect MLH1, a crucial component of the meiotic recombination machinery, and found that it can be visualised as foci from pachytene to diakinesis, where it co-localises with chiasmata. The mean MLH1 foci number per meiotic cell at diakinesis was 8.4 for WS-4 and 9.95 for Col-0, with the number of foci per bivalent ranging from 1 to 5. We also analysed MLH1 distribution within bivalents and found that they were not restricted to specific chromosomal regions. The analysis of MLH1 foci formation in the Atzip4 mutant, where class I crossover (CO) formation is prevented, revealed that residual chiasmata were not labelled by MLH1, strongly suggesting that MLH1 antibodies only label class I COs in Arabidopsis. It thus appears that the ‘obligatory CO’ is systematically labeled by MLH1 and is generated through the class I pathway.

The Fanconi Anemia Ortholog FANCM Ensures Ordered Homologous Recombination in Both Somatic and Meiotic Cells in Arabidopsis

“…We then analyzed the localization of At-MLH1 in fancm-1 and wild-type meiocytes at pachytene. The MutL homolog At-MLH1 is a component of late recombination nodules, and the number of MLH1 foci corresponds to the number of interference-sensitive COs (Marcon and Moens, 2003;Jackson et al, 2006;Chelysheva et al, 2010). In the wild type, the mean number of MLH1 foci per cell was 9.8 with a range of 8 to 12 ( Figure 4B).…”

Spatiotemporal Asymmetry of the Meiotic Program Underlies the Predominantly Distal Distribution of Meiotic Crossovers in Barley

“…Based on studies in budding yeast, it is proposed that Mlh1 acts with Mlh3 as a heterodimer to ensure that dHjs are resolved as COs rather than non-COs (Hunter and Borts, 1997;Wang et al, 1999). Immunolocalization studies in a variety of organisms indicate that both MLH1 and MLH3 form foci on pachytene chromosomes that correspond to the sites of COs (Plug et al, 1998;Marcon and Moens, 2003;Jackson et al, 2006;Chelysheva et al, 2010). Dual immunolocalization of MLH1 in conjunction with ZYP1 was conducted on prophase I chromosome spread preparations.…”

Crossovers Get a Boost inBrassicaAllotriploid and Allotetraploid Hybrids

“…therein). In Arabidopsis, immunolocalization of MLH1 antibodies in diakinesis PMCs reveals distinct foci on chromatin, which correspond to positions of interfering (class I) crossovers (Chelysheva et al, 2010). In order to compare the numbers of MLH1 foci in all hybrids, in which not only bivalents but also an additional number of multivalents were observed (Table 1), we chose to relate the number of MLH1 foci to the number of "recombining chromosomes"; this number was estimated in every PMC by dividing the total number of MLH1 foci by the number of chromosomes involved in recombination events (i.e., that showed at least one MLH1 focus).…”

“…The anti-Arabidopsis thaliana MLH1 polyclonal antibody was described by Chelysheva et al (2010), and the purified serum was used at a dilution of 1:15.…”

“…The slides were mounted in DAPI (2 mg/mL) in Vectashield antifade mounting medium. Fluorescence immunolocalization of MLH1 was performed on squashes according to the method described by Chelysheva et al (2010). All observations were made using a Leica (http://www.leica.com) DMRXA2 microscope; photographs were taken using a CoolSNAP HQ (Roper; http://www.…”