Interplay of ribosomal DNA loci in nucleolar dominance: dominant NORs are up-regulated by chromatin dynamics in the wheat-rye system

Abstract

Background: Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions.

Methodology/principal findings: Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs.

Conclusions/significance: We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to 'mutual amphiplasty' in the nucleolar dominance process.

Figure 1. Quantitative real-time PCR of wheat rRNA in wheat and wheat addition lines.

A The melt curves for two replicates of cDNA isolated from wheat (blue), rye (black), wheat+1R1R (green), and wheat+7R7R (red) amplified with primers specific for wheat rRNA are shown. A single melt peak with Tm = 88.5 in the three genomes containing wheat genetic material indicate a single amplification product. Due to the specificity of primers for wheat rRNA, there is no amplification of rye rRNA. B Quantitative real-time PCR products separated by gel electrophoresis. The results indicate greater expression of wheat-specific rRNA in the addition line wheat+1R1R (1R) in comparison to wheat (W) and addition line wheat+7R7R (7R). No amplification product was observed in rye (R). Actin controls are shown and M is the molecular weight marker (1 kb⁺, 300 basepair band is shown on the left). C Transcription levels of wheat rRNAs in wheat and wheat+1R1R in respect to wheat+7R7R. Quantitative real-time PCR threshold cycles (Ct) were equilibrated with actin for wheat, wheat+1R1R, and wheat+7R7R (delta Ct). Wheat+7R7R mean delta Ct was utilized to calculate mean delta delta Ct (mean ΔΔ Ct) of two replicates of two wheat and wheat+1R1R cDNA dilutions. The graph illustrates – mean ΔΔ Ct ± standard deviation (SD) for wheat and wheat+1R1R, and the associated fold changes in transcription (2 ^{−mean ΔΔ Ct}).

Figure 2. Evaluation of NOR activity in wheat and wheat-rye addition line (wheat+1R1R).

Silver-staining technique in root-tip metaphase cells. A shows the schematic representation of the NOR bearing chromosomes (NORs black square) and the diagnostic silver bands (black circles) allowing the identification of nucleolar chromosomes 1B, 6B, and 1R. B shows a root-tip metaphase cell of the wheat-rye addition line (wheat+1R1R), with six Ag-NORs. In this plate the 1B and 6B NORs are classified as large while 1R NORs are classified as medium. Bar = 10 µm. C represents the graphic for the frequency of different classes of Ag-NORs for each rDNA locus in root-tip metaphase cells of wheat and wheat+1R1R. Values result from the analysis of 50 c-metaphase cells in each genotype, and the differential distribution for 1B and 6B NORs between the two lines was confirmed by Chi-squared test (p<0.001).

Figure 3. Ribosomal chromatin organization and transcription sites in wheat.

A–C Nucleolar labelling of rDNA chromatin (A, green) and of transcription sites detected by BrUTP incorporation (B, red) in root sections of wheat. A and B are projections of single confocal sections spanning the nucleolus; C is the merging of rDNA and BrUTP images. No transcription foci are detected in perinucleolar knobs of ribosomal chromatin (C, arrows) whereas intranucleolar rDNA is associated with BrUTP revealing transcriptional activity (orange labelling resulting from overlapping of green and red channels). D–F Nuclear transcriptional labelling detected by BrUTP incorporation. D, E and F are individual consecutive confocal sections of the nucleus. Transcription sites are overall dispersed throughout the nucleus and in the nucleolar compartment. Note the absence of transcription label in the chromatin encircling the nucleolus (indicated by the lines in D). Bar = 5 µm.

Figure 4. Ribosomal chromatin organization in wheat and wheat-rye addition line (wheat+1R1R) nuclei.

In situ hybridization to rDNA in root sections of wheat and wheat-rye addition line (wheat+1R1R). A–C and E–G are wheat single consecutive confocal sections of interphase nuclei with one or four nucleoli respectively (perinucleolar knobs are indicated with arrows). D and H are models from the complete 3D data stack shown in A–C and E–G, respectively, where intranucleolar dots are represented in yellow and perinucleolar knobs in red. The single nucleolus of the nucleus shown in A–D has five perinucleolar knobs (arrows in A, B and C). The nucleus shown in E–H has four nucleoli, where one perinucleolar knob is visible in nucleoli marked as a, b and d (arrows in F), while the fourth nucleolus (c) shows two perinucleolar knobs (arrows in G). I–L shows a wheat-rye (wheat+1R1R) interphase nucleus probed for the rDNA (green) and for rye chromosomes 1R (red). I–K are single confocal consecutive sections and L corresponds to the projection of the 3D data stack, showing the presence of two nucleoli. In these plates it is possible to distinguish the wheat and rye rDNA perinucleolar knobs, since the later overlap with 1R chromosomes (white arrows in J). Bar = 10 µm.