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

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

BACKGROUND: DNA methylation plays a key role in development, contributes to genome stability, and may also respond to external factors supporting adaptation and evolution. To connect different types of stimuli with particular biological processes, identifying genome regions with altered 5-methylcytosine distribution at a genome-wide scale is important. Many researchers are using the simple, reliable, and relatively inexpensive Methylation Sensitive Amplified Polymorphism (MSAP) method that is particularly useful in studies of epigenetic variation. However, electrophoretic patterns produced by the method are rather difficult to interpret, particularly when MspI and HpaII isoschizomers are used because these enzymes are methylation-sensitive, and any C within the CCGG recognition motif can be methylated in plant DNA. RESULTS: Here, we evaluate MSAP patterns with respect to current knowledge of the enzyme activities and the level and distribution of 5-methylcytosine in plant and vertebrate genomes. We discuss potential caveats related to complex MSAP patterns and provide clues regarding how to interpret them. We further show that addition of combined HpaII + MspI digestion would assist in the interpretation of the most controversial MSAP pattern represented by the signal in the HpaII but not in the MspI profile. CONCLUSIONS: We recommend modification of the MSAP protocol that definitely discerns between putative hemimethylated mCCGG and internal CmCGG sites. We believe that our view and the simple improvement will assist in correct MSAP data interpretation.

• MeSH
• 5-Methylcytosine chemistry   MeSH
• DNA, Plant genetics   MeSH
• Epigenesis, Genetic MeSH
• DNA Methylation * MeSH
• Vertebrates genetics   MeSH
• Polymorphism, Genetic * MeSH
• Restriction Mapping MeSH
• Nicotiana genetics   MeSH
• Nucleic Acid Amplification Techniques methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• 5-Methylcytosine MeSH
• DNA, Plant MeSH

Telomere homeostasis is regulated at multiple levels, including the local chromatin structure of telomeres and subtelomeres. Recent reports demonstrated that a decrease in repressive chromatin marks, such as levels of cytosine methylation in subtelomeric regions, results in telomere elongation in mouse cells. Here we show that a considerable fraction of cytosines is methylated not only in subtelomeric, but also in telomeric DNA of tobacco BY-2 cells. Drug-induced hypomethylation (demonstrated at subtelomeric, telomeric, and global DNA levels) results in activation of telomerase. However, in contrast to mouse cells, the decrease in 5-methylcytosine levels and upregulation of telomerase do not result in any changes of telomere lengths. These results demonstrate the involvement of epigenetic mechanisms in the multilevel process of regulation of telomerase activity in plant cells and, at the same time, they indicate that changes in telomerase activity can be overridden by other factors governing telomere length stability.

• MeSH
• Adenine analogs & derivatives   pharmacology   MeSH
• Enzyme Activation drug effects   MeSH
• Cytidine analogs & derivatives   pharmacology   MeSH
• DNA, Plant chemistry   drug effects   MeSH
• Epigenesis, Genetic MeSH
• Transcription, Genetic drug effects   MeSH
• Cells, Cultured MeSH
• DNA Methylation drug effects   MeSH
• Nucleosomes drug effects   physiology   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Nicotiana cytology   drug effects   genetics   metabolism   MeSH
• Telomerase metabolism   MeSH
• Telomere chemistry   drug effects   metabolism   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
• Cytidine MeSH
• DNA, Plant MeSH
• Nucleosomes MeSH
• pyrimidin-2-one beta-ribofuranoside MeSH Browser
• Plant Proteins 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

Cytosine methylation levels and susceptibility to drug-induced hypomethylation have been studied in several Nicotiana tabacum (tobacco) DNA repetitive sequences. It has been shown using HapII, MspI, BamHI and Sau3AI methylation-sensitive restriction enzymes that the degree of 5'-mCmCG-3' methylation varied significantly between different repeats. There were almost saturation levels of 5-methylcytosine at the inner (3') cytosine position and variable degrees of methylation at the outer (5') cytosine at the enzyme recognition sites. The non-transcribed high copy satellite sequences (HRS60, GRS) displayed significant heterogeneity in methylation of their basic units while middle repetitive sequences (R8.1, GRD5, 5S rDNA) were more uniformly modified at both cytosine residues. Dihydroxypropyladenine (DHPA) treatment, which is thought to reduce DNA methyltransferase activity by increasing S-adenosylhomocysteine levels, resulted in extensive demethylation of the outer cytosine in all repeats, and the partial hypomethylation of cytosines at the inner positions in less densely methylated repeats such as HRS60 and GRS. The results suggest that hypomethylation of 5'-mCmCG-3' sites with DHPA is a gradual non-random process proceeding in the direction mCmCG-->CmCG-->CCG. The 18S-5.8S-25S rDNA was remarkably hypomethylated relative to the 5S rDNA at all restriction sites studied. Fluorescence in-situ hybridization showed that DNA decondensation within and between the 18S-5.8S-25S and 5S rDNA loci was variable in different nuclei. All nuclei had condensed and decondensed sequence. The chromatin of 18S-5.8S-25S rDNA was more readily digested with micrococcal nuclease than the 5S rDNA suggesting that the overall levels of decondensation were higher for 18S-5.8S-25S rDNA. Variable decondensation patterns within and between loci were also observed for GRS and HRS60. Cytosine methylation of the tobacco repeats is discussed with respect to transcription, overall levels of condensation and overall structure.

• MeSH
• Adenine analogs & derivatives   pharmacology   MeSH
• Cytosine metabolism   MeSH
• DNA, Plant drug effects   isolation & purification   metabolism   MeSH
• Transcription, Genetic MeSH
• Genome, Plant MeSH
• Heterochromatin metabolism   MeSH
• Interphase MeSH
• Plants, Toxic * MeSH
• Cells, Cultured MeSH
• DNA Methylation * MeSH
• Nucleotides metabolism   MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• DNA Restriction Enzymes MeSH
• DNA, Satellite MeSH
• Blotting, Southern MeSH
• Nicotiana genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• 9-(2,3-dihydroxypropyl)adenine MeSH Browser
• Adenine MeSH
• Cytosine MeSH
• DNA, Plant MeSH
• Heterochromatin MeSH
• Nucleotides MeSH
• DNA Restriction Enzymes MeSH
• DNA, Satellite MeSH