Epigenetic changes accompanying plant cell dedifferentiation and differentiation are reported in 35S ribosomal DNA (rDNA) of tobacco (Nicotiana tabacum). There was a reduction of CG and CNG methylation in both intergenic and genic regions of the rDNA cistron in fully dedifferentiated callus and root compared to leaf. The rDNA hypomethylation was not random, but targeted to particular rDNA gene families at units that are clustered within the tandem array. The process of hypomethylation was initiated as early as 2 weeks after the callus induction and established epigenetic patterns were stably maintained throughout prolonged culture. However, regenerated plants and their progeny showed partial and complete remethylation of units, respectively. Nuclear run-on assays revealed a 2-fold increase of primary (unprocessed) ribosomal RNA transcripts in callus compared to leaf tissue. However, the abundance of mature transcripts in callus was elevated by only about 25%. Fluorescence in situ hybridization analysis of interphase nuclei showed high levels of rDNA chromatin condensation in both callus and leaf, with substantially less decondensed rDNA than is observed in meristematic root-tip cells. It is likely that the regions of the rDNA locus showing decondensation correspond to the clusters of hypomethylated units that occur in the tandem array at each locus. The data together indicate that the establishment of pluripotency and cell proliferation occurring with callus induction is associated with enhanced ribosomal RNA gene expression and overall rDNA hypomethylation, but is not associated with material-enhanced relaxation of chromatin structure (decondensation) at rDNA loci.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno

Zobrazit více v PubMed

Amado L, Abranches R, Neves N, Viegas W (1997) Development-dependent inheritance of 5-azacytidine-induced epimutations in triticale: analysis of rDNA expression patterns. Chromosome Res 5: 445–450 PubMed

Anderson S, Lewis-Smith AC, Smith SM (1990) Methylation of ribosomal RNA genes in Petunia hybrida plants, callus cultures and regenerated shoots. Plant Cell Rep 8: 554–557 PubMed

Arnholdt-Schmitt B (2004) Stress-induced reprogramming. A role for global genome regulation? Plant Physiol 136: 2579–2586 PubMed PMC

Arnholdt-Schmitt B, Hesterich S, Neumann K-H (1995) Physiological aspects of genome variability in tissue culture, I. Growth phase-dependent differential DNA methylation of the carrot genome (Daucus carrota L.) during primary culture. Theor Appl Genet 91: 809–815 PubMed

Avivi Y, Morad V, Ben-Meir H, Zhao J, Kashkush K, Tzfira T, Citovsky V, Grafi G (2004) Reorganization of specific chromosomal domains and activation of silent genes in plant cells acquiring pluripotentiality. Dev Dyn 230: 12–22 PubMed

Brock GJR, Bird A (1997) Mosaic methylation of the repeat units of the human ribosomal RNA genes. Hum Mol Genet 6: 451–456 PubMed

Castilho A, Neves N, Rufini-Castiglione M, Viegas W, Heslop-Harrison JS (1999) 5-Methylcytosine distribution and genome organization in triticale before and after treatment with 5-azacytidine. J Cell Sci 112: 4397–4404 PubMed

Cerdido A, Medina FJ (1995) Subnucleolar location of fibrillarin and variation in its levels during the cell cycle and during differentiation of plant cells. Chromosoma 103: 625–634 PubMed

Chen ZJ, Pikaard CS (1997) Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance. Genes Dev 11: 2124–2136 PubMed PMC

Fan H, Yakura K, Miyanishi M, Sugita M, Sugiura M (1995) In vitro transcription of plant RNA polymerase I-dependent rRNA genes is species-specific. Plant J 8: 295–298 PubMed

Finnegan EJ, Kovac KA (2000) Plant DNA methyltransferases. Plant Mol Biol 43: 189–201 PubMed

Flavell RB, O'Dell M, Thompson WF (1988) Regulation of cytosine methylation in ribosomal DNA and nucleolus organizer expression in wheat. J Mol Biol 204: 523–534 PubMed

Fojtova M, Van Houdt H, Depicker A, Kovarik A (2003) Epigenetic switch from posttranscriptional to transcriptional silencing is correlated with promoter hypermethylation. Plant Physiol 133: 1240–1250 PubMed PMC

Fulnecek J, Matyasek R, Kovarik A, Bezdek M (1998) Mapping of 5-methylcytosine residues in Nicotiana tabacum 5S rRNA genes by genomic sequencing. Mol Gen Genet 259: 133–141 PubMed

Gaudino RJ, Pikaard CS (1997) Cytokinin induction of RNA polymerase I transcription in Arabidopsis thaliana. J Biol Chem 272: 6799–6804 PubMed

Glyn MC, Egertova M, Gazdova B, Kovarik A, Bezdek M, Leitch AR (1995) The influence of 5-azacytidine on the condensation of the short arm of rye chromosome 1R in Triticum aestivum L. root tip meristematic nuclei. Chromosoma 106: 485–492 PubMed

Goodspeed TH (1954) The Genus Nicotiana, Vol 16. Chronica Botanica, Waltham, MA

Grafi G (2004) How cells differentiate: lessons from plants. Dev Biol 268: 1–6 PubMed

Hasterok R, Maluszynska J (2000) Different rRNA gene expression in primary and adventitious roots of Allium cepa L. Folia Histochem Cytobiol 38: 181–184 PubMed

Hemleben V, Zentgraf U (1994) Structural organisation and regulation of transcription by RNA polymerase I of plant nuclear ribosomal genes. In L Nover, ed, Results and Problems in Cell Differentiation 20: Plant Promoters and Transcription Factors. Springer-Verlag, Berlin/Heidelberg, pp 3–24 PubMed

Hodurkova J, Vyskot B (2003) Histone H4 acetylation patterns during seed germination and early plant development. Biol Plant (Prague) 46: 23–28

Jaligot E, Rival A, Beule T, Dussert S, Verdeil JL (2000) Somaclonal variation in oil palm (Elaesis guineensis Jacg.): the DNA methylation hypothesis. Plant Cell Rep 19: 684–690 PubMed

Jupe ER, Zimmer EA (1990) Unmethylated regions in the intergenic spacer of maize and teosinte ribosomal RNA genes. Plant Mol Biol 14: 333–347 PubMed

Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43: 179–188 PubMed

Kenton A, Parokonny AS, Gleba YY, Bennett MD (1993) Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics. Mol Gen Genet 240: 159–169 PubMed

Kiss T, Szkukalek A, Solymosy F (1989) Nucleotide sequence of a 17S (18S) rRNA gene from tomato. Nucleic Acids Res 17: 2127. PubMed PMC

Komarova NY, Grabe T, Huigen DJ, Hemleben V, Volkov RA (2004) Organization, differential expression and methylation of rDNA in artificial Solanum allopolyploids. Plant Mol Biol 56: 439–463 PubMed

Kovarik A, Koukalova B, Bezdek M, Opatrny Z (1997) Hypermethylation of tobacco heterochromatic loci in response to osmotic stress. Theor Appl Genet 95: 301–306

Kovarik A, Koukalova B, Lim KY, Matyasek R, Lichtenstein CP, Leitch AR, Bezdek M (2000) Comparative analysis of DNA methylation in tobacco heterochromatic sequences. Chromosome Res 8: 527–541 PubMed

Kovarik A, Matyasek R, Lim KY, Skalická K, Koukalova B, Knapp S, Chase M, Leitch AR (2004) Concerted evolution of 18-5.8-26S rDNA repeats in Nicotiana allotetraploids. Biol J Linn Soc 82: 615–625

Kovarik A, Pires JC, Leitch AR, Lim KY, Sherwood A, Matyasek R, Rocca J, Soltis DE, Soltis PS (2005) Rapid concerted evolution of nuclear ribosomal DNA in two allopolyploids of recent and recurrent origin. Genetics 169: 931–944 PubMed PMC

Labra M, Ghiani A, Citterio S, Sgorbati S, Sala F, Vannini C, Ruffini-Castiglione M, Bracale M (2002) Analysis of cytosine methylation pattern in response to water deficit in pea root tips. Plant Biol 4: 694–699

Lawrence RJ, Earley K, Pontes O, Silva M, Chen ZJ, Neves N, Viegas W, Pikaard CS (2004) A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Mol Cell 13: 599–609 PubMed

Leitch AR, Schwarzacher T, Wang ML, Leitch IJ, Surlan-Momirovich G, Moore G, Heslop-Harrison JS (1993) Molecular cytogenetic analysis of repeated sequences in a long term wheat suspension culture. Plant Cell Tissue Organ Cult 33: 287–296

Lim KY, Kovarik A, Matyasek R, Bezdek M, Lichtenstein CP, Leitch AR (2000) Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units. Chromosoma 109: 161–172 PubMed

Lim KY, Skalická K, Koukalova B, Volkov RA, Matyasek R, Hemleben V, Leitch AR, Kovarik A (2004) Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana. Genetics 166: 1935–1946 PubMed PMC

Mathieu O, Jasencakova Z, Vaillant I, Gendrel A-V, Colot V, Schubert I, Tourmente S (2003) Changes in 5S rDNA chromatin organization and transcription during heterochromatin establishment in Arabidopsis. Plant Cell 15: 2929–2939 PubMed PMC

Mathieu O, Picard G, Tourmente S (2002) Methylation of an euchromatin-heterochromatin transition region in Arabidopsis thaliana chromosome 5 left arm. Chromosome Res 10: 455–466 PubMed

Mitsuhara I, Shirasawa-Seo N, Iwai T, Nakamura S, Honkura R, Ohashi Y (2002) Release from post-transcriptional gene silencing by cell proliferation in transgenic tobacco plants: possible mechanism for noninheritance of the silencing. Genetics 160: 343–352 PubMed PMC

Moscone EA, Matzke MA, Matzke AJ (1996) The use of combined FISH/GISH in conjunction with DAPI counterstaining to identify chromosomes containing transgene inserts in amphidiploid tobacco. Chromosoma 105: 231–236 PubMed

Olhoft PM, Phillips RL (1999) Genetic and epigenetic instability in tissue culture and regenerated progenies. In HR Lerner, ed, Plant Responses to Environmental Stresses: From Phytohormones to Genome Reorganization. Marcel Dekker, New York, pp 111–148

Pikaard CS (2000) The epigenetics of nucleolar dominance. Trends Genet 16: 495–500 PubMed

Reichheld JP, Chaubet N, Shen WH, Renaudin JP, Gigot C (1996) Multiple A-type cyclins express sequentially during the cell cycle in Nicotiana tabacum BY2 cells. Proc Natl Acad Sci USA 93: 13819–13824 PubMed PMC

Santoro R, Grummt I (2001) Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription. Mol Cell 8: 719–725 PubMed

Sardana R, O'Dell M, Flavell R (1993) Correlation between the size of the intergenic regulatory region, the status of cytosine methylation of rRNA genes and nucleolar expression in wheat. Mol Gen Genet 236: 155–162 PubMed

Skalická K, Lim KY, Matyásek R, Koukalová B, Leitch AR, Kovarík A (2003) Rapid evolution of parental rDNA in a synthetic tobacco allotetraploid line. Am J Bot 90: 988–996 PubMed

Smulders MJM, Rus-Kortekaas W, Vosman B (1995) Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants. Theor Appl Genet 91: 1257–1264 PubMed

Soppe WJ, Jasencakova Z, Houben A, Kakutani T, Meister A, Huang MS, Jacobsen SE, Schubert I, Fransz PF (2002) DNA methylation controls histone H3 lysine 9 methylation and heterochromatin assembly in Arabidopsis. EMBO J 21: 6549–6559 PubMed PMC

Tariq M, Saze H, Probst AV, Lichota J, Habu Y, Paszkowski J (2003) Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin. Proc Natl Acad Sci USA 100: 8823–8827 PubMed PMC

Torres-Ruiz RA, Hemleben V (1994) Pattern and degree of methylation in ribosomal RNA genes of Cucurbita pepo L. Plant Mol Biol 26: 1167–1179 PubMed

van Blokland R, van der Geest N, Mol JN, Kooter JM (1994) Transgene-mediated suppression of chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J 6: 861–877

Van Houdt H, Kovarik A, Van Montagu M, Depicker A (2000) Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes. FEBS Lett 467: 41–46 PubMed

Viera R, Queiroz A, Morais L, Barao A, Mello-Sampavo T, Viegas WS (1990) 1R chromosome nucleolus organiser region activation by 5-azacytidine in wheat×rye hybrids. Genome 33: 707–712

Volkov RA, Borisjuk NV, Panchuk II, Schweizer D, Hemleben V (1999) Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum. Mol Biol Evol 16: 311–320 PubMed

Vyskot B, Gazdova B, Siroky J (1993) Methylation patterns of two repetitive DNA sequences in tobacco tissue cultures and their regenerants. Biol Plant (Prague) 35: 321–327

Vyskot B, Koukalova B, Kovarik A, Sachambula L, Reynolds D, Bezdek M (1995) Meiotic transmission of a hypomethylated repetitive DNA family in tobacco. Theor Appl Genet 91: 659–664 PubMed

Watson JC, Kaufman LS, Thompson WF (1987) Developmental regulation of cytosine methylation in the nuclear ribosomal RNA genes of Pisum sativum. J Mol Biol 193: 15–26 PubMed

Williams L, Zhao J, Morozova N, Li Y, Avivi Y, Grafi G (2003) Chromatin reorganization accompanying cellular dedifferentiation is associated with modifications of histone H3, redistribution of HP1, and activation of E2F-target genes. Dev Dyn 228: 113–120 PubMed

Personal Perspectives on Plant Ribosomal RNA Genes Research: From Precursor-rRNA to Molecular Evolution

The origin of exon 3 skipping of paternal GLOBOSA pre-mRNA in some Nicotiana tabacum lines correlates with a point mutation of the very last nucleotide of the exon

Dynamics and Reversibility of the DNA Methylation Landscape of Grapevine Plants (Vitis vinifera) Stressed by In Vitro Cultivation and Thermotherapy

Silenced rRNA genes are activated and substitute for partially eliminated active homeologs in the recently formed allotetraploid, Tragopogon mirus (Asteraceae)

Epigenetic switches of tobacco transgenes associate with transient redistribution of histone marks in callus culture

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles