Insight into chromatin compaction and spatial organization in rice interphase nuclei

Abstract

Chromatin organization and its interactions are essential for biological processes, such as DNA repair, transcription, and DNA replication. Detailed cytogenetics data on chromatin conformation, and the arrangement and mutual positioning of chromosome territories in interphase nuclei are still widely missing in plants. In this study, level of chromatin condensation in interphase nuclei of rice (Oryza sativa) and the distribution of chromosome territories (CTs) were analyzed. Super-resolution, stimulated emission depletion (STED) microscopy showed different levels of chromatin condensation in leaf and root interphase nuclei. 3D immuno-FISH experiments with painting probes specific to chromosomes 9 and 2 were conducted to investigate their spatial distribution in root and leaf nuclei. Six different configurations of chromosome territories, including their complete association, weak association, and complete separation, were observed in root meristematic nuclei, and four configurations were observed in leaf nuclei. The volume of CTs and frequency of their association varied between the tissue types. The frequency of association of CTs specific to chromosome 9, containing NOR region, is also affected by the activity of the 45S rDNA locus. Our data suggested that the arrangement of chromosomes in the nucleus is connected with the position and the size of the nucleolus.

Keywords: 3D immuno-FISH; chromosome painting; chromosome territory; microscopy; rice; spatial organization.

Figure 1

STED analysis of chromatin condensation in G1 nuclei of young leaves and root meristem. DNA was stained by spirochrome (white). (A) Differences in DNA structure are clearly visible in zoomed pictures and heat maps. Red arrows indicate region of chromatin width measurement. (B) Graph of discrete chromatin fiber measurements. Leaf fibers measurements displayed two different groups of chromatin size (grey and pink plot) Scale bar: 2 µm.

Figure 2

(A) Graph representing average nucleolus/nucleus ration of each individual cell. (B) Differences in shape of the analyzed nuclei. Maximal intensity projection (MIP) of nuclear DNA stained with DAPI (blue). Nucleolus was visualized using fibrillarin immunolabeling (red).

Figure 3

Maximal intensity projection of confocal scanning of chromosomes and G1 nuclei of rice after immuno-FISH localization on flow sorted G1 nuclei of root meristem. (A) Visualization of centromere (yellow), short arm of chromosome 2 (2S) (pink), and long arm (2L) (green) on metaphase chromosomes. (B) Visualization of chromosome 2 (pink) and chromosome 9 (green) by oligo-painting FISH on prometaphase chromosomes. (C) Visualization of two separate chromosome territories corresponding to two homologous chromosomes. Long arm of chromosome 2 in pink, short arm of chromosome 2 in yellow. (D, E) DNA was counterstained with DAPI (blue). Scale bar: 3 µm. Immuno-FISH localization of specific probes on G1 nuclei of root meristem (D, F, G) and leaf tissue (E). DNA was counterstained with DAPI (blue). Scale bar: 2 µm.

Figure 4

Comparison between root and leaf chromosome arrangements. (A) Graph summarizing the chromosome associations in root and leaf tissues displayed in (B). (B) Models of individual arrangements created with BioRender.com, based on raw data observation captured by confocal microscopy.

Figure 5

3D models of CTs positioning in root and leaf G1 nuclei. (A) Spatial positioning of CTs specific to chromosome 2 (yellow) and 9 (green) and nucleoli (red). (B) Model showing spatial arrangement of the CTs and nucleoli with respect to the center and periphery of the nucleus. Shells of equal area depict regions of the nuclei, where signals of DAPI (white), and chromosome 2 (yellow) and chromosome 9 (green) were localized.

Figure 6

Oligo-painting FISH on root ultra-thin sections prepared by cryomicrotome. Centromeric probe (red), telomeric probe (green) and specific probe for chromosome 2 (yellow) were applied. Images displayed the evidence of Rabl configuration in xylem (A) and cortex (B) cells.