Abstract
Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-2′-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.
Highlights
In plants, NOR locus is composed of multiple repeats of ribosomal genes that encode the 45S pre-ribosomal transcript, which is processed to generate mature 18S, 5.8S and 26–28S ribosomal RNA (rRNA) molecules
The inhibition of DNA methylation by 5-aza-29-dC relocated NOR-2 locus closer to the nucleolus/nucleoli, we found no cytological evidence of its transcriptional activity, suggesting that rRNA genes residing within this locus probably exist as pseudogenes
NOR-1 and the NOR-2 can be separated by their size Four fluorescence in situ hybridization (FISH) signals representing two 45S rDNA loci, referred to as the major (NOR-1) and the minor (NOR-2) locus, were identified in interphase nuclei of the common oak root tip cycling cells, consistent with our previous findings [35]
Summary
NOR (nucleolar organizer region) locus is composed of multiple repeats of ribosomal (rRNA) genes that encode the 45S pre-ribosomal transcript, which is processed to generate mature 18S, 5.8S and 26–28S rRNA molecules. Epigenetic mechanisms regulating the transcriptional state of rRNA genes associated with rDNA chromatin organization have been well studied in plant hybrid species exhibiting a phenomenon known as nucleolar dominance. In these hybrids, rDNA loci of only one parent are transcriptionally active while rDNA loci of the other parent stay silent [5,6]. Silent rRNA genes can be reactivated by the use of inhibitors of DNA methylases and histone deacetylases, further confirming the direct involvement of these epigenetic mechanisms in the transcriptional regulation of rRNA genes [7,9,10] Both CpG methylation by MET1 (Methyltransferase 1) and siRNA-guided
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