Transient 5-azacytidine treatment of leaf explants from potato plants with transcriptionally silenced transgenes allows de novo regeneration of plants with restored transgene expression at the whole plant level. Transgenes introduced into plant genomes frequently become silenced either at the transcriptional or the posttranscriptional level. Transcriptional silencing is usually associated with DNA methylation in the promoter region. Treatments with inhibitors of maintenance DNA methylation were previously shown to allow reactivation of transcriptionally silenced transgenes in single cells or tissues, but not at the whole plant level. Here we analyzed the effect of DNA methylation inhibitor 5-azacytidine (AzaC) on the expression of two silenced reporter genes encoding green fluorescent protein (GFP) and neomycin phosphotransferase (NPTII) in potato plants. Whereas no obvious reactivation was observed in AzaC-treated stem cuttings, transient treatment of leaf segments with 10 μM AzaC and subsequent de novo regeneration of shoots on the selective medium with kanamycin resulted in the production of whole plants with clearly reactivated expression of previously silenced transgenes. Reactivation of nptII expression was accompanied by a decrease in cytosine methylation in the promoter region of the gene. Using the plants with reactivated GFP expression, we found that re-silencing of this transgene can be accidentally triggered by de novo regeneration. Thus, testing the incidence of transgene silencing during de novo regeneration could be a suitable procedure for negative selection of transgenic lines (insertion events) which have an inclination to be silenced. Based on our analysis of non-specific inhibitory effects of AzaC on growth of potato shoots in vitro, we estimated that AzaC half-life in the culture media is approximately 2 days.

• Keywords
• 5-Azacytidine, De novo regeneration, Methylation, Reactivation, TGS, Transgene silencing,
• MeSH
• Azacitidine pharmacology   MeSH
• Plants, Genetically Modified drug effects   genetics   metabolism   MeSH
• DNA Methylation drug effects   genetics   MeSH
• Gene Expression Regulation, Plant drug effects   genetics   MeSH
• Solanum tuberosum drug effects   genetics   metabolism   MeSH
• Transgenes drug effects   genetics   MeSH
• Gene Silencing MeSH
• Green Fluorescent Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Azacitidine MeSH
• Green Fluorescent Proteins MeSH

In plants, genome duplication followed by genome diversification and selection is recognized as a major evolutionary process. Rapid epigenetic and genetic changes that affect the transcription of parental genes are frequently observed after polyploidization. The pattern of alternative splicing is also frequently altered, yet the related molecular processes remain largely unresolved. Here, we study the inheritance and expression of parental variants of three floral organ identity genes in allotetraploid tobacco. DEFICIENS and GLOBOSA are B-class genes, and AGAMOUS is a C-class gene. Parental variants of these genes were found to be maintained in the tobacco genome, and the respective mRNAs were present in flower buds in comparable amounts. However, among five tobacco cultivars, we identified two in which the majority of paternal GLOBOSA pre-mRNA transcripts undergo exon 3 skipping, producing an mRNA with a premature termination codon. At the DNA level, we identified a G-A transition at the very last position of exon 3 in both cultivars. Although alternative splicing resulted in a dramatic decrease in full-length paternal GLOBOSA mRNA, no phenotypic effect was observed. Our finding likely serves as an example of the initiation of homoeolog diversification in a relatively young polyploid genome.

• Keywords
• Alternative splicing, Floral genes, Flowering, Polyploidy, Tobacco,
• MeSH
• Alternative Splicing genetics   MeSH
• Point Mutation genetics   MeSH
• Exons genetics   MeSH
• Transcription, Genetic * MeSH
• Homeodomain Proteins biosynthesis   genetics   MeSH
• Nucleotides genetics   MeSH
• Polyploidy MeSH
• RNA Precursors genetics   MeSH
• Gene Expression Regulation, Plant MeSH
• Plant Proteins biosynthesis   genetics   MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• GLOBOSA protein, plant MeSH Browser
• Homeodomain Proteins MeSH
• Nucleotides MeSH
• RNA Precursors MeSH
• Plant Proteins MeSH

In plants, silencing is usually accompanied by DNA methylation and heterochromatic histone marks. We studied these epigenetic modifications in different epialleles of 35S promoter (P35S)-driven tobacco transgenes. In locus 1, the T-DNA was organized as an inverted repeat, and the residing neomycin phosphotransferase II reporter gene (P35S-nptII) was silenced at the posttranscriptional (PTGS) level. Transcriptionally silenced (TGS) epialleles were generated by trans-acting RNA signals in hybrids or in a callus culture. PTGS to TGS conversion in callus culture was accompanied by loss of the euchromatic H3K4me3 mark in the transcribed region of locus 1, but this change was not transmitted to the regenerated plants from these calli. In contrast, cytosine methylation that spread from the transcribed region into the promoter was maintained in regenerants. Also, the TGS epialleles generated by trans-acting siRNAs did not change their active histone modifications. Thus, both TGS and PTGS epialleles exhibit euchromatic (H3K4me3 and H3K9ac) histone modifications despite heavy DNA methylation in the promoter and transcribed region, respectively. However, in the TGS locus (271), abundant heterochromatic H3K9me2 marks and DNA methylation were present on P35S. Heterochromatic histone modifications are not automatically installed on transcriptionally silenced loci in tobacco, suggesting that repressive histone marks and cytosine methylation may be uncoupled. However, transient loss of euchromatic modifications may guide de novo DNA methylation leading to formation of stable repressed epialleles with recovered eukaryotic marks. Compilation of available data on epigenetic modification of inactivated P35S in different systems is provided.

• Keywords
• DNA methylation, callus, dedifferentiation, histone modification, tobacco, transgene silencing,
• MeSH
• Chromatin genetics   metabolism   MeSH
• Epigenesis, Genetic * MeSH
• Plants, Genetically Modified genetics   metabolism   MeSH
• Histones genetics   metabolism   MeSH
• Bony Callus metabolism   MeSH
• DNA Methylation MeSH
• Gene Expression Regulation, Plant MeSH
• Nicotiana genetics   metabolism   MeSH
• Transgenes MeSH
• Gene Silencing MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• Histones MeSH

It has been well established that trans-acting small RNAs guide promoter methylation leading to its inactivation and gene silencing at the transcriptional level (TGS). Here we addressed the question of the influence of the locus structure and epigenetic modifications of the target locus on its susceptibility for being paramutated by trans-acting small RNA molecules. Silencing was induced by crossing a 35S promoter silencer locus 271 with two different 35S-driven transgene loci, locus 2 containing a highly expressed single copy gene and locus 1 containing an inverted posttranscriptionally silenced (PTGS) repeat of this gene. Three generations of exposure to RNA signals from the 271 locus were required to complete silencing and methylation of the 35S promoter within locus 2. Segregating methylated locus 2 epialleles were obtained only from the third generation of hybrids, and this methylation was not correlated with silencing. Strikingly, only one generation was required for the PTGS locus 1 to acquire complete TGS and 35S promoter methylation. In this case, paramutated locus 1 epialleles bearing methylated and inactive 35S promoters segregated already from the first generation of hybrids. The results support the hypothesis that PTGS loci containing a palindrome structure and methylation in the coding region are more sensitive to paramutation by small RNAs and exhibit a strong tendency to formation of meiotically transmissible TGS epialleles. These features contrast with a non-methylated single copy transgenic locus that required several generations of contact with RNA silencing molecules to become imprinted in a stable epiallele.

• MeSH
• Alleles MeSH
• Epigenesis, Genetic MeSH
• Transcription, Genetic MeSH
• Plants, Genetically Modified genetics   MeSH
• Genomic Imprinting MeSH
• RNA, Small Interfering genetics   MeSH
• DNA Methylation * MeSH
• Promoter Regions, Genetic MeSH
• Gene Expression Regulation, Plant * MeSH
• RNA Interference MeSH
• Nicotiana genetics   MeSH
• Transgenes genetics   MeSH
• Silencer Elements, Transcriptional genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Small Interfering 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