An easy protocol for studying chromatin and recombination protein dynamics during Arabidopsis thaliana meiosis: immunodetection of cohesins, histones and MLH1.
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.
Chelysheva
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L
Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, Versailles, France. Liudmila.Chelysheva@versailles.inra.fr
Grandont
L
L
Vrielynck
N
N
le Guin
S
S
Mercier
R
R
Grelon
M
M
D000251:Adenosine Triphosphatases / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D017360:Arabidopsis / Q000166:cytology* / Q000235:genetics* / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000276:immunology / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D032461:Chromosomes, Plant / Q000235:genetics / Q000378:metabolism; D003434:Crossing Over, Genetic; D020732:Cytogenetic Analysis / Q000379:methods*; D006657:Histones / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D008540:Meiosis / Q000235:genetics* / Q000502:physiology; D000070957:MutL Protein Homolog 1; D051381:Rats; D011995:Recombination, Genetic; D000097722:Cohesins
D000251:Adenosine Triphosphatases; D000906:Antibodies; D029681:Arabidopsis Proteins; D018797:Cell Cycle Proteins; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D000070957:MutL Protein Homolog 1; D000097722:Cohesins; C524390:MLH1 protein, Arabidopsis