Chromosome dynamics visualized with an anti-centromeric histone H3 antibody in Allium

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.

Figure 1. Alignment and phylogenetic tree of CENH3s.

(A) Amino acid sequence alignment of CENH3s of A. fistulosum (AfiCENH3), A. cepa (AceCENH3), A. sativum (AsaCENH3), A. tuberosum (AtuCENH3) and Oryza sativa (OsCENH3), and a canonical histone H3 of O. sativa (OsHistoneH3). Identical, similar and weakly similar amino acids are indicated by asterisks, colons and dots, respectively. A red box indicates the amino acid residues used for the generation of antibody against AfiCENH3. Blue boxes correspond to helices in a histone fold domain. (B) Phylogenetic tree based on CENH3 amino acid sequences of plant species. A canonical histone H3 from O. sativa was used as an outgroup. Bootstrap values in 1000 tests are indicated on the branches.

Figure 2. Immunostaining of chromosomes of Allium species using an anti-AfiCENH3 antibody.

(A), (E), (I) and (M): DAPI stained chromosomes. (B), (F), (J) and (N): Immunosignals of an anti-AfiCENH3 antibody. (C), (G), (K) and (O): Immunosignals of an anti-α-tubulin antibody. (D): Merged image of (A–C). (H): Merged image of (E–G). (L): Merged image of (I–K). (P): Merged image of (M–O). (A–D): A. fistulosum. (E–H): A. cepa. (I–L): A. sativum. (M–P): A. tuberosum. Scale bar, 10 µm.

Figure 3. Immunohistochemical staining of onion root sections.

(A) Vertical slice of an onion root. The magenta and green boxes indicate columella and cortex, respectively. Anti-AfiCENH3, anti-α-tubulin and DAPI signals are indicated in red, green and blue, respectively. (B) and (C) are close-up images of the magenta and green boxes in (A), respectively. (D) Horizontal slice of an onion root. Anti-AfiCENH3, anti-α-tubulin and DAPI signals are indicated in red, green and blue, respectively. The magenta and green boxes indicate columella and cortex, respectively. (E) and (F) are close-up images of the magenta and green boxes in (D), respectively. (G) A 3D image constructed from a set of Z-stack pictures. Anti-AfiCENH3, anti-α-tubulin and DAPI signals are indicated in red, green and grey, respectively. Scale bar, 100 µm.

Figure 4. Comparison of acetic orcein staining, immunostaining and immunohistochemical staining in mitotic cells of onion.

Chromosome pictures of acetic-orcein-staining (A–E), general immunostaining (F–J), and immunostaining of sections (K–P). Red, green and gray signals in F∼P are signals of anti-AfiCENH3 antibody, anti-α-tubulin antibody and DAPI, respectively. Scale bar, 10 µm. (G–V) A set of diagrams based on the results in K–P.

Figure 5. FISH using the ChIP clones and related sequences.

(A), (E), (I), (M), (Q) and (U): DAPI stained A. fistulosum chromosomes. (B), (F), (J), (N) and (R): FISH signals of rDNA. (C): FISH signals of Afi11. (G): FISH signals of Afi19. (K): FISH signals of Afi54. (O): FISH signals of Afi56. (S): FISH signals of Afi61. (V): FISH signals of Afi19variant3-sub1 (W): FISH signals of AfiCen332-1508. (D): merged image of (A–C). (H): merged image of (E–G). (L): merged image of (I–K). (P): merged image of (M–O). (T): merged image of (Q–S). (X): merged image of (U–W). Scale bar, 10 µm.

Figure 6. Sequence and qPCR analysis of centromeric DNA sequences of A. fistulosum.

(A) ChIP-qPCR using anti-AfiCENH3 and chromatins from A. fistulosum. The columns and error bars represent the average relative enrichments (RE) and standard error from four independent ChIP experiments, respectively. Amplified regions of each qPCR are indicated by magenta boxes under the column in (B). 25SrDNA and 5SrDNA were used as non-centromeric controls. Statistical significance of differences between 25SrDNA and other sequences were tested using the Tukey’s Honesty Significant Difference test (*P<0.01). (B) A contig made by the ChIP (D) and Tail (E) clones. The blue and green arrows indicate the positions of two inverted repeats. (C) Arrow heads indicate primer position for the Tail-PCR. (D) Position of the ChIP clones. Regions containing non-homologous minisatellite and unique sequences in Afi19 are indicated by orange and gray boxes, respectively. (E) Position of the Tail-PCR clones. (F) Position of confirmed PCR products. (G) Afi19 and its variants. Regions containing non-homologous minisatellite and unique sequences in Afi19 are indicated by orange and gray boxes, respectively.