Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.

• Klíčová slova
• 35S rDNA copy number variations, CCGs and CHHs, CWGs, Tragopogon porrifolius ssp. porrifolius, bidirectional nucleolar dominance, methylation dynamics of CGs, transcriptional silencing/activation,
• MeSH
• chromozomy rostlin genetika   MeSH
• cytosin * metabolismus   MeSH
• epigeneze genetická MeSH
• genetická transkripce MeSH
• metylace DNA * MeSH
• regulace genové exprese u rostlin MeSH
• ribozomální DNA * genetika   MeSH
• umlčování genů * MeSH
• variabilita počtu kopií segmentů DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytosin * MeSH
• ribozomální DNA * MeSH

The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic interactions of rDNA loci, and epigenetic silencing. In this article, we focus on the early rDNA research in plants, performed mainly at the dawn of molecular biology in the 60 to 80-ties of the last century which presented a prequel to the modern genomic era. We discuss - from a personal view - the topics such as synthesis of rRNA precursor (35S pre-rRNA in plants), processing, and the organization of 35S and 5S rDNA. Cloning and sequencing led to the observation that the transcribed and processed regions of the rRNA genes vary enormously, even between populations and species, in comparison with the more conserved regions coding for the mature rRNAs. Epigenetic phenomena and the impact of hybridization and allopolyploidy on rDNA expression and homogenization are discussed. This historical view of scientific progress and achievements sets the scene for the other articles highlighting the immense progress in rDNA research published in this special issue of Frontiers in Plant Science on "Molecular organization, evolution, and function of ribosomal DNA."

• Klíčová slova
• epigenetics, hybridization, molecular evolution, nucleolar dominance, polyploidy, rDNA research history, rRNA precursor, rRNA processing,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Nucleolar dominance (ND) is an epigenetic, developmentally regulated phenomenon that describes the selective inactivation of 35S rDNA loci derived from one progenitor of a hybrid or allopolyploid. The presence of ND was documented in an allotetraploid grass, Brachypodium hybridum (genome composition DDSS), which is a polyphyletic species that arose from crosses between two putative ancestors that resembled the modern B. distachyon (DD) and B. stacei (SS). In this work, we investigated the developmental stability of ND in B. hybridum genotype 3-7-2 and compared it with the reference genotype ABR113. We addressed the question of whether the ND is established in generative tissues such as pollen mother cells (PMC). We examined condensation of rDNA chromatin by fluorescence in situ hybridization employing state-of-art confocal microscopy. The transcription of rDNA homeologs was determined by reverse-transcription cleaved amplified polymorphic sequence analysis. In ABR113, the ND was stable in all tissues analyzed (primary and adventitious root, leaf, and spikes). In contrast, the 3-7-2 individuals showed a strong upregulation of the S-genome units in adventitious roots but not in other tissues. Microscopic analysis of the 3-7-2 PMCs revealed extensive decondensation of the D-genome loci and their association with the nucleolus in meiosis. As opposed, the S-genome loci were always highly condensed and localized outside the nucleolus. These results indicate that genotype-specific loss of ND in B. hybridum occurs probably after fertilization during developmental processes. This finding supports our view that B. hybridum is an attractive model to study ND in grasses.

• Klíčová slova
• 35S rDNA, 3D-FISH, Brachypodium, allopolyploidy, nucleolar dominance, rRNA gene expression, secondary constriction,
• Publikační typ
• časopisecké články MeSH

The intergenic spacer (IGS) of rDNA is frequently built of long blocks of tandem repeats. To estimate the intragenomic variability of such knotty regions, we employed PacBio sequencing of the Cucurbita moschata genome, in which thousands of rDNA copies are distributed across a number of loci. The rRNA coding regions are highly conserved, indicating intensive interlocus homogenization and/or high selection pressure. However, the IGS exhibits high intragenomic structural diversity. Two repeated blocks, R1 (300-1250 bp) and R2 (290-643 bp), account for most of the IGS variation. They exhibit minisatellite-like features built of multiple periodically spaced short GC-rich sequence motifs with the potential to adopt non-canonical DNA conformations, G-quadruplex-folded and left-handed Z-DNA. The mutual arrangement of these motifs can be used to classify IGS variants into five structural families. Subtle polymorphisms exist within each family due to a variable number of repeats, suggesting the coexistence of an enormous number of IGS variants. The substantial length and structural heterogeneity of IGS minisatellites suggests that the tempo of their divergence exceeds the tempo of the homogenization of rDNA arrays. As frequently occurring among plants, we hypothesize that their instability may influence transcription regulation and/or destabilize rDNA units, possibly spreading them across the genome.

• Klíčová slova
• Cucurbita moschata, DNA-minisatellite, intragenomic structural heterogeneity, non-canonical DNA conformations, ribosomal DNA intergenic spacer,
• MeSH
• Cucurbita genetika   MeSH
• genetická variace * MeSH
• konformace nukleové kyseliny * MeSH
• mezerníky ribozomální DNA chemie   genetika   metabolismus   MeSH
• minisatelitní repetice * MeSH
• sekvenční analýza DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• mezerníky ribozomální DNA MeSH