Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are important for the maintenance of genomic stability. Telomeres were considered as typical heterochromatic regions, but in light of recent results, this view should be reconsidered. Asymmetrically located cytosines in plant telomeric DNA repeats may be substrates for a DNA methyltransferase enzyme and indeed, it was shown that these repeats are methylated. Here, we analyse the methylation of telomeric cytosines and the length of telomeres in Arabidopsis thaliana methylation mutants (met 1-3 and ddm 1-8), and in their wild-type siblings that were germinated in the presence of hypomethylation drugs. Our results show that cytosine methylation in telomeric repeats depends on the activity of MET1 and DDM1 enzymes. Significantly shortened telomeres occur in later generations of methylation mutants as well as in plants germinated in the presence of hypomethylation drugs, and this phenotype is stably transmitted to the next plant generation. A possible role of compromised in vivo telomerase action in the observed telomere shortening is hypothesized based on telomere analysis of hypomethylated telomerase knockout plants. Results are discussed in connection with previous data in this field obtained using different model systems.

• MeSH
• Arabidopsis enzymology   genetics   metabolism   MeSH
• Cytosine metabolism   MeSH
• Telomere Homeostasis MeSH
• DNA Methylation * MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Plants genetics   metabolism   MeSH
• Telomerase metabolism   MeSH
• Telomere chemistry   metabolism   MeSH
• Telomere Shortening * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cytosine MeSH
• Telomerase MeSH

Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.

• MeSH
• Adenine analogs & derivatives   toxicity   MeSH
• Adenosylhomocysteinase antagonists & inhibitors   metabolism   MeSH
• DNA Primers genetics   MeSH
• Epigenesis, Genetic drug effects   physiology   MeSH
• Germination drug effects   physiology   MeSH
• DNA, Complementary genetics   MeSH
• Flowers anatomy & histology   physiology   MeSH
• DNA Methylation MeSH
• Statistics, Nonparametric MeSH
• Pollen physiology   MeSH
• Gene Expression Regulation, Plant drug effects   genetics   physiology   MeSH
• Plant Proteins metabolism   MeSH
• Blotting, Southern MeSH
• Nicotiana enzymology   physiology   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 9-(2,3-dihydroxypropyl)adenine MeSH Browser
• Adenine MeSH
• Adenosylhomocysteinase MeSH
• DNA Primers MeSH
• GLO protein, Nicotiana tabacum MeSH Browser
• DNA, Complementary MeSH
• Plant Proteins MeSH

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome.

• MeSH
• Diploidy MeSH
• DNA, Plant genetics   MeSH
• DNA (Cytosine-5-)-Methyltransferases classification   genetics   metabolism   MeSH
• Epigenesis, Genetic MeSH
• Gene Expression MeSH
• Phylogeny MeSH
• Genome, Plant MeSH
• Cloning, Molecular MeSH
• DNA Methylation genetics   MeSH
• Molecular Sequence Data MeSH
• Multigene Family * MeSH
• Polyploidy MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Genes, Plant * MeSH
• Base Sequence MeSH
• Selection, Genetic MeSH
• Nicotiana enzymology   genetics   MeSH
• Tissue Distribution MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Plant MeSH
• DNA (Cytosine-5-)-Methyltransferases MeSH

We studied the in trans-silencing capacities of a transgene locus that carried the neomycin phosphotransferase II reporter gene linked to the 35S promoter in an inverted repeat (IR). This transgene locus was originally posttranscriptionally silenced but switched to a transcriptionally silenced epiallele after in vitro tissue culture. Here, we show that both epialleles were strongly methylated in the coding region and IR center. However, by genomic sequencing, we found that the 1.0 kb region around the transcription start site was heavily methylated in symmetrical and non-symmetrical contexts in transcriptionally but not in posttranscriptionally silenced epilallele. Also, the posttranscriptionally silenced epiallele could trans-silence and trans-methylate homologous transgene loci irrespective of their genomic organization. We demonstrate that this in trans-silencing was accompanied by the production of small RNA molecules. On the other hand, the transcriptionally silenced variant could neither trans-silence nor trans-methylate homologous sequences, even after being in the same genetic background for generations and meiotic cycles. Interestingly, 5-aza-2-deoxy-cytidine-induced hypomethylation could partially restore signaling from the transcriptionally silenced epiallele. These results are consistent with the hypothesis that non-transcribed highly methylated IRs are poor silencers of homologous loci at non-allelic positions even across two generations and that transcription of the inverted sequences is essential for their trans-silencing potential.

• MeSH
• Alleles MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified genetics   metabolism   MeSH
• Kanamycin Kinase genetics   metabolism   MeSH
• DNA Methylation MeSH
• RNA, Untranslated analysis   MeSH
• Repetitive Sequences, Nucleic Acid * MeSH
• Genes, Reporter MeSH
• Nicotiana genetics   MeSH
• Transgenes * MeSH
• Gene Silencing * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Kanamycin Kinase MeSH
• RNA, Untranslated MeSH

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.

• MeSH
• Cell Differentiation * MeSH
• Chromatin chemistry   metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• In Situ Hybridization, Fluorescence MeSH
• Interphase MeSH
• Plant Roots genetics   MeSH
• Cells, Cultured MeSH
• Plant Leaves cytology   genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• DNA Methylation * MeSH
• Regeneration MeSH
• Gene Expression Regulation, Plant MeSH
• RNA, Ribosomal genetics   MeSH
• Nicotiana cytology   genetics   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• RNA, Messenger MeSH
• RNA, Ribosomal MeSH

Melandrium album (syn. Silene latifolia) is a model dioecious species in which the Y chromosome, present only in heterogametic males, plays both a male-determining and a strict female-suppressing role. We showed that treatment with 5-azacytidine (5-azaC) induces a sex change to androhermaphroditism (an-dromonoecy) in about 21% of male plants, while no apparent phenotypic effect was observed in females. All of these bisexual androhermaphrodites (with the standard male 24, AA + XY karyotype) were mosaics possessing both male and hermaphrodite flowers and, moreover, the hermaphrodite flowers displayed various degrees of gynoecium development and seed setting. Southern hybridization analysis with a repetitive DNA probe showed that the 5-azacytidine-treated plants were significantly hypomethylated in CG doubles, but only to a minor degree in CNG triplets. The bisexual trait was transmitted to two successive generations, but only when androhermaphrodite plants were used as pollen donors. The sex reversal was inherited with incomplete penetrance and varying expressivity. Based on the uniparental inheritance pattern of androhermaphroditism we conclude that it originated either by 5-azaC induced inhibition of Y-linked female-suppressing genes or by a heritable activation of autosomal female-determining/promoting genes which can be reversed, on passage through female meiosis, by a genomic imprinting mechanism. The data presented indicate that female sex suppression in M. album XY males is dependent on methylation of specific DNA sequences and can be heritably modified by hypomethylating drugs.

• MeSH
• Azacitidine pharmacology   MeSH
• DNA, Plant metabolism   MeSH
• Phenotype MeSH
• Methylation MeSH
• Gene Expression Regulation, Plant physiology   MeSH
• Plants genetics   metabolism   MeSH
• Reproduction MeSH
• Plant Development MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Azacitidine MeSH
• DNA, Plant MeSH