RESUMO
Sugar beet (Beta vulgaris altissima) is a biennial root crop with an absolute requirement for cold exposure to bolt and flower, a process called vernalization. Global DNA methylation variations have been reported during vernalization in several plants. However, few genes targeted by DNA methylation during vernalization have been described. The objectives of this study were to identify differentially methylated regions and to study their involvement in bolting induction and tolerance. Restriction landmark genome scanning was applied to DNA from shoot apical meristems of sugar beet genotypes, providing a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence. Several differentially methylated regions exhibiting variations of gene-body DNA methylation and expression during cold exposure and/or between genotypes were identified, including an AROGENATE DEHYDRATASE and two RNA METHYLCYTOSINE TRANSFERASE sequences. One RNA METHYLCYTOSINE TRANSFERASE sequence displayed gene-body hypermethylation and activation of expression, while the other was hypomethylated and inhibited by cold exposure. Global RNA methylation and phenolic compound levels changed during cold exposure in a genotype-dependent way. The use of methyl RNA immunoprecipitation of total RNA and reverse transcription-PCR analysis revealed mRNA methylation in a vernalized bolting-resistant genotype for the FLOWERING LOCUS 1 gene, a repressor of flowering. Finally, Arabidopsis mutants for RNA METHYLCYTOSINE TRANSFERASE and AROGENATE DEHYDRATASE were shown to exhibit, under different environmental conditions, early or late bolting phenotypes, respectively. Overall, the data identified functional targets of DNA methylation during vernalization in sugar beet, and it is proposed that RNA methylation and phenolic compounds play a role in the floral transition.
Assuntos
Arabidopsis/enzimologia , Beta vulgaris/enzimologia , Flores/crescimento & desenvolvimento , Metiltransferases/metabolismo , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Beta vulgaris/genética , Beta vulgaris/crescimento & desenvolvimento , Beta vulgaris/metabolismo , Flores/enzimologia , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Metilação , Metiltransferases/genética , Proteínas de Plantas/genética , RNA de Plantas/genéticaRESUMO
During differentiation, in vitro organogenesis calls for the adjustment of the gene expression program toward a new fate. The role of epigenetic mechanisms including DNA methylation is suggested but little is known about the loci affected by DNA methylation changes, particularly in agronomic plants for witch in vitro technologies are useful such as sugar beet. Here, three pairs of organogenic and non-organogenic in vitro cell lines originating from different sugar beet (Beta vulgaris altissima) cultivars were used to assess the dynamics of DNA methylation at the global or genic levels during shoot or root regeneration. The restriction landmark genome scanning for methylation approach was applied to provide a direct quantitative epigenetic assessment of several CG methylated genes without prior knowledge of gene sequence that is particularly adapted for studies on crop plants without a fully sequenced genome. The cloned sequences had putative roles in cell proliferation, differentiation or unknown functions and displayed organ-specific DNA polymorphism for methylation and changes in expression during in vitro organogenesis. Among them, a potential ubiquitin extension protein 6 (UBI6) was shown, in different cultivars, to exhibit repeatable variations of DNA methylation and gene expression during shoot regeneration. In addition, abnormal development and callogenesis were observed in a T-DNA insertion mutant (ubi6) for a homologous sequence in Arabidopsis. Our data showed that DNA methylation is changed in an organ-specific way for genes exhibiting variations of expression and playing potential role during organogenesis. These epialleles could be conserved between parental lines opening perspectives for molecular markers.
Assuntos
Beta vulgaris/genética , Metilação de DNA/genética , Epigênese Genética/genética , Regulação da Expressão Gênica de Plantas/genética , Alelos , Beta vulgaris/fisiologia , Técnicas de Cultura de Células , Diferenciação Celular , Ilhas de CpG/genética , DNA de Plantas/química , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Especificidade de Órgãos , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/genética , Brotos de Planta/fisiologia , Plantas Geneticamente Modificadas , Regeneração , Análise de Sequência de DNARESUMO
An epigenetic control of vernalization has been demonstrated in annual plants such as Arabidopsis and cereals, but the situation remains unclear in biennial plants such as sugar beet that has an absolute requirement for vernalization. The role of DNA methylation in flowering induction and the identification of corresponding target loci also need to be clarified. In this context, sugar beet (Beta vulgaris altissima) genotypes differing in bolting tolerance were submitted to various bolting conditions such as different temperatures and/or methylating drugs. DNA hypomethylating treatment was not sufficient to induce bolting while DNA hypermethylation treatment inhibits and delays bolting. Vernalizing and devernalizing temperatures were shown to affect bolting as well as DNA methylation levels in the shoot apical meristem. In addition, a negative correlation was established between bolting and DNA methylation. Genotypes considered as resistant or sensitive to bolting could also be distinguished by their DNA methylation levels. Finally, sugar beet homologues of the Arabidopsis vernalization genes FLC and VIN3 exhibited distinct DNA methylation marks during vernalization independently to the variations of global DNA methylation. These vernalization genes also displayed differences in mRNA accumulation and methylation profiles between genotypes resistant or sensitive to bolting. Taken together, the data suggest that the time course and amplitude of DNA methylation variations are critical points for the induction of sugar beet bolting and represent an epigenetic component of the genotypic bolting tolerance, opening up new perspectives for sugar beet breeding.
Assuntos
Adaptação Fisiológica/genética , Beta vulgaris/genética , Beta vulgaris/fisiologia , Metilação de DNA/genética , Flores/fisiologia , Meristema/genética , Sequência de Bases , Beta vulgaris/enzimologia , Temperatura Baixa , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação da Expressão Gênica de Plantas , Genótipo , Meristema/enzimologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
In order to evaluate the permanent chromatin remodeling in plant allowing their high developmental plasticity, three sugarbeet cell lines (Beta vulgaris L. altissima) originating from the same mother plant and exhibiting graduate states of differentiation were analyzed. Cell differentiation has been estimated by the cell redox state characterized by 36 biochemical parameters as reactive oxygen species steady-state levels, peroxidation product contents and enzymatic or non-enzymatic protective systems. Chromatin remodeling has been estimated by the measurement of levels of DNA methylation, histone acetylation and corresponding enzyme activities that were shown to differ between cell lines. Furthermore, distinct loci related to proteins involved in cell cycle, gene expression regulation and cell redox state were shown by restriction landmark genome scanning or bisulfite sequencing to display differential methylation states in relation to the morphogenic capacity of the lines. DNA methylating, demethylating and/or histone acetylating treatments allowed to generate a collection of sugarbeet cell lines differing by their phenotypes (from organogenic to dedifferentiated), methylcytosine percentages (from 15.0 to 43.5%) and acetylated histone ratios (from 0.37 to 0.52). Correlations between methylcytosine or acetylated histone contents and levels of various parameters (23 or 7, respectively, out of 36) of the cell redox state could be established. These data lead to the identification of biomarkers of sugarbeet morphogenesis in vitro under epigenetic regulation and provide evidence for a connection between plant morphogenesis in vitro, cell redox state and epigenetic mechanisms.
