Chromosome Dynamics Visualized with an Anti-Centromeric Histone H3 Antibody in Allium

Kiyotaka Nagaki,Minoru Murata,Yasuhiko Mukai,Maki Yamamoto,Naoki Yamaji,Daniela Cimini

doi:10.1371/journal.pone.0051315

Kiyotaka Nagaki, Minoru Murata + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0051315

Copy DOI

Abstract

Due to the ease with which chromosomes can be observed, the Allium species, and onion in particular, have been familiar materials employed in cytogenetic experiments in biology. In this study, centromeric histone H3 (CENH3)-coding cDNAs were identified in four Allium species (onion, welsh onion, garlic and garlic chives) and cloned. Anti-CENH3 antibody was then raised against a deduced amino acid sequence of CENH3 of welsh onion. The antibody recognized all CENH3 orthologs of the Allium species tested. Immunostaining with the antibody enabled clear visualization of chromosome behavior during mitosis in the species. Furthermore, three-dimensional (3D) observation of mitotic cell division was achieved by subjecting root sections to immunohistochemical techniques. The 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and α-tubulin) were clearly revealed by immunohistochemical staining with the antibodies. The immunohistochemical analysis made it possible to establish an overview of the location of dividing cells in the root tissues. This breakthrough in technique, in addition to the two centromeric DNA sequences isolated from welsh onion by chromatin immuno-precipitation using the antibody, should lead to a better understanding of plant cell division. A phylogenetic analysis of Allium CENH3s together with the previously reported plant CENH3s showed two separate clades for monocot species tested. One clade was made from CENH3s of the Allium species with those of Poaceae species, and the other from CENH3s of a holocentric species (Luzula nivea). These data may imply functional differences of CENH3s between holocentric and monocentric species. Centromeric localization of DNA sequences isolated from welsh onion by chromatin immuno-precipitation (ChIP) using the antibody was confirmed by fluorescence in situ hybridization and ChIP-quantitative PCR.

Highlights

Allium species including onion (A. cepa), welsh onion (A. fistulosum), garlic (A. sativum) and garlic chives (A. tuberosum) have been used as foods and/or spices around the world
We searched for onion centromeric histone H3 (CENH3) in an onion expressed sequence tag (EST) database of the gene index project using the tblastn program with the amino acid sequence of OsCENH3 [15] as a query
To obtain full-length cDNA of the gene, we conducted a rapid amplification of cDNA ends (RACE) experiment using onion root cDNA as a template, and successfully amplified AceCENH3 (GenBank accession number AB600275)

Summary

Introduction

Allium species including onion (A. cepa), welsh onion (A. fistulosum), garlic (A. sativum) and garlic chives (A. tuberosum) have been used as foods and/or spices around the world. Given the lack of molecular markers on the centromeres, information concerning cell division is limited in comparison with that of animal cells, for the species analyzed here, and for other plant species possessing large chromosomes [2,3,4]. Investigation of Allium chromosomes at the molecular level has revealed some special features; the species lack typical Arabidopsistype telomere sequences, and their chromosome ends are maintained with subtelomeric tandem repeats [5]. A number of different tandem repeats have been identified in subtelomeric heterochromatins which may relate to function [6,7,8,9,10,11]. Other chromosomal regions including centromeres have yet to be analyzed

Methods

Results

Conclusion