DNA methylation alterations of upland cotton (Gossypium hirsutum) in response to cold stress

DNA methylation plays an important role in regulating gene expression in plants. In the experiment, we studied effects of cold on DNA methylation variation in upland cotton. Using the methylation-sensitive amplified polymorphism procedure, we chose 66 pairs of selective amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, cold triggered the demethylation of hemimethylated or internally full methylated cytosine. With the prolongation of cold treatment, the demethylation loci increased and the methylation loci decreased. Nevertheless, this change could be reverted when cotton was subsequently recovered under normal temperature. In addition, 29 polymorphic bands that appeared in the electrophoretogram were sequenced. By homologous alignment analysis, most of these 29 fragments were identified as genes or DNA clones involved in abiotic stress response. The variation in methylation loci existed at both coding and non-coding regions. Furthermore, the expression of the abiotic stress-related genes, GhCLSD (Seq21), GhARK (Seq22), GhARM (Seq15, Seq18, Seq19 and Seq21) and GhTPS (Seq8), were tested. The results revealed that cold treatment induced down-regulation of GhCLSD, GhARK and GhARM, but up-regulated the expression of GhTPS. These changes were in accordance with the alteration of DNA methylation. Thus, cold may affect the gene expression via changing the methylation status in the cytosine nucleotide.     

The influence of Al<Superscript>3+</Superscript> on DNA methylation and sequence changes in the triticale (× <Emphasis Type="Italic">Triticosecale</Emphasis> Wittmack) genome

Abiotic stressors such as drought, salinity, and exposure to heavy metals can induce epigenetic changes in plants. In this study, liquid chromatography (RP-HPLC), methylation amplified fragment length polymorphisms (metAFLP), and methylation-sensitive amplification polymorphisms (MSAP) analysis was used to investigate the effects of aluminum (Al) stress on DNA methylation levels in the crop species triticale. RP-HPLC, but not metAFLP or MSAP, revealed significant differences in methylation between Al-tolerant (T) and non-tolerant (NT) triticale lines. The direction of methylation change was dependent on phenotype and organ. Al treatment increased the level of global DNA methylation in roots of T lines by approximately 0.6%, whereas demethylation of approximately 1.0% was observed in NT lines. DNA methylation in leaves was not affected by Al stress. The metAFLP and MSAP approaches identified DNA alterations induced by Al³⁺ treatment. The metAFLP technique revealed sequence changes in roots of all analyzed triticale lines and few mutations in leaves. MSAP showed that demethylation of CCGG sites reached approximately 3.97% and 3.75% for T and NT lines, respectively, and was more abundant than de novo methylation, which was observed only in two tolerant lines affected by Al stress. Three of the MSAP fragments showed similarity to genes involved in abiotic stress.     

Periodic DNA methylation in maize nucleosomes and demethylation by environmental stress

When maize seedlings were exposed to cold stress, a genome-wide demethylation occurred in root tissues. Screening of genomic DNA identified one particular fragment that was demethylated during chilling. This 1.8-kb fragment, designated ZmMI1, contained part of the coding region of a putative protein and part of a retrotransposon-like sequence. ZmMI1 was transcribed only under cold stress. Direct methylation mapping revealed that hypomethylated regions spanning 150 bases alternated with hypermethylated regions spanning 50 bases. Analysis of nuclear DNA digested with micrococcal nuclease indicated that these regions corresponded to nucleosome cores and linkers, respectively. Cold stress induced severe demethylation in core regions but left linker regions relatively intact. Thus, methylation and demethylation were periodic in nucleosomes. The following biological significance is conceivable. First, because DNA methylation in nucleosomes induces alteration of gene expression by changing chromatin structures, vast demethylation may serve as a common switch for many genes that are simultaneously controlled upon environmental cues. Second, because artificial demethylation induces heritable changes in plant phenotype (Sano, H., Kamada, I., Youssefian, S., Katsumi, M., and Wabilko, H. (1990) Mol. Gen. Genet. 220, 441-447), altered DNA methylation may result in epigenetic inheritance, in which gene expression is modified without changing the nucleotide sequence.     

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stress

DNA methylation,one of the most important epigenetic phenomena,plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli.In the present study,a rnethylation-sensitive amplified polymorphism (MSAP) analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress.Consistent with visibly different phenotypes in response to salt stress,epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salttolerant FL478 and salt-sensitive IR29.In addition,most tissue-specific DNA methylation loci were conserved,while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes.Strikingly,salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29 than in the FL478.This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage,and helps to further elucidate the implications of DNA methylation in crop growth and development.     

DNA methylation increases throughout Arabidopsis development

We used amplified fragment length polymorphisms (AFLP) to analyze the stability of DNA methylation throughout Arabidopsis development. AFLP can detect genome-wide changes in cytosine methylation produced by DNA demethylation agents, such as 5-azacytidine, or specific mutations at the DDM1 locus. In both cases, cytosine demethylation is associated with a general increase in the presence of amplified fragments. Using this approach, we followed DNA methylation at methylation sensitive restriction sites throughout Arabidopsis development. The results show a progressive DNA methylation trend from cotyledons to vegetative organs to reproductive organs.     

Vernalization-induced changes of the DNA methylation pattern in winter wheat.

Vernalization is a cold treatment that induces or accelerates flowering and insures that temperate-zone plants will not flower until after winter. There is evidence that vernalization results in DNA demethylation that induces flowering. Differences in DNA methylation can be determined using methylation-sensitive amplified fragment length polymorphisms (AFLPs). Methylation-sensitive AFLPs utilize restriction enzyme isoschizomers that are differentially sensitive to methylation, producing polymorphisms related to methylation differences as opposed to sequence differences. Near-isogenic lines (NILs) have been developed for spring vs. winter habit in wheat (Triticum aestivum) and allow for the study of a single vernalization locus. In this study, differences in the methylation pattern were determined for spring and winter NILs, as well as for unvernalized and vernalized individuals. Winter wheat was more highly methylated than spring wheat and methylation-related AFLPs were produced between winter and spring wheat. Changes in the methylation pattern were observed at the end of vernalization, one week after the end of vernalization, and four weeks after the end of vernalization of winter wheat. However, the most methylation differences were observed one week after removal of winter wheat from cold treatment. Our data suggest that there is not only a vernalization-induced demethylation related to flower induction, but there is also a more general and non-specific demethylation of sequences unrelated to flowering. Two methylation-related AFLPs induced by vernalization were shared among all of the winter NILs.     

Drought-induced site-specific DNA methylation and its association with drought tolerance in rice (Oryza sativa L.)

An indica pyramiding line, DK151, and its recurrent parent, IR64, were evaluated under drought stress and non-stress conditions for three consecutive seasons. DK151 showed significantly improved tolerance to drought. The DNA methylation changes in DK151 and IR64 under drought stress and subsequent recovery were assessed using methylation-sensitive amplified polymorphism analysis. Our results indicate that drought-induced genome-wide DNA methylation changes accounted for ～12.1% of the total site-specific methylation differences in the rice genome. This drought-induced DNA methylation pattern showed three interesting properties. The most important one was its genotypic specificity reflected by large differences in the detected DNA methylation/demethylation sites between DK151 and IR64, which result from introgressed genomic fragments in DK151. Second, most drought-induced methylation/demethylation sites were of two major types distinguished by their reversibility, including 70% of the sites at which drought-induced epigenetic changes were reversed to their original status after recovery, and 29% of sites at which the drought-induced DNA demethylation/methylation changes remain even after recovery. Third, the drought-induced DNA methylation alteration showed a significant level of developmental and tissue specificity. Together, these properties are expected to have contributed greatly to rice response and adaptation to drought stress. Thus, induced epigenetic changes in rice genome can be considered as a very important regulatory mechanism for rice plants to adapt to drought and possibly other environmental stresses.     

Detection of changes in DNA methylation induced by low-energy ion implantation in Arabidopsis thaliana

This study evaluated changes in DNA methylation in Arabidopsis thaliana plants grown from seeds implanted with low-energy N(+) and Ar(+) ions. Methylation-sensitive amplified polymorphism (MSAP) testing revealed altered DNA methylation patterns after ion implantation at doses of 1 × 10(14) to 1 × 10(16) ions/cm(2). Comparison of the MSAP electrophoretic profiles revealed nine types of polymorphisms in ion-implanted seedlings relative to control seedlings, among which four represented methylation events, three represented demethylation events, and the methylation status of two was uncertain. The diversity of plant DNA methylation was increased by low-energy ion implantation. At the same time, total genomic DNA methylation levels at CCGG sites were unchanged by ion implantation. Moreover, a comparison of polymorphisms seen in N(+) ion-implanted, Ar(+) ion-implanted, and control DNA demonstrated that the species of incident ion influenced the resulting DNA methylation pattern. Sequencing of eight isolated fragments that showed different changing patterns in implanted plants allowed their mapping onto variable regions on one or more of the five Arabidopsis chromosomes; these segments included protein-coding genes, transposon and repeat DNA sequence. A further sodium bisulfite sequencing of three fragments also displayed alterations in methylation among either different types or doses of incident ions. Possible causes for the changes in methylation are discussed.     

大花蕙兰子房授粉前后基因组DNA 胞嘧啶甲基化状态的MSAP 分析

应用甲基化敏感扩增多态性(Methylation sensitive amplified polymorphism, MSAP) 技术分析了大花蕙兰( Cymbidium hybridium) 授粉前后子房DNA 甲基化状态的变化(甲基化水平和甲基化差异模式) 。采用72 对引物进行选择性扩增, 共得到5892 条带, 其中748 条带为甲基化多态性带。结果显示DNA 甲基化在大花蕙兰子房发育过程中发生频繁, 从授粉前后子房的总扩增位点甲基化水平(14%和11. 4%) 和全甲基化率(9.5%和7.8% ) 来看, 授粉后都略低于未授粉子房, 表明子房在授粉后的发育过程中在某些位点发生了去甲基化。除甲基化水平有变化外, 大花蕙兰子房授粉前后的DNA 甲基化模式也存在较大差异, 共检测到14 种带型, 分为两大类( Ⅰ 和Ⅱ 型)。其中, 授粉前后DNA 甲基化状态保持不变的位点少, 只占25.6% , 归为Ⅰ型; 大部分检测位点( 占74.4% , 归为Ⅱ型) 的DNA 甲基化模式在授粉前后存在显著差异。上述结果表明, 大花蕙兰子房发育过程中以DNA 甲基化为代表的表观遗传调控起重要作用。本研究的开展将促进对与大花蕙兰子房发育相关的甲基化差异片段及受DNA 甲基化调控的关键基因的克隆, 进而为从表观遗传学这一新角度揭示大花蕙兰子房发育的分子机制奠定基础。     

Evaluation of Genetic and Epigenetic Modification in Rapeseed （Brassica napus） Induced by Salt Stress

Salinity is an important limiting environmental factor for rapeseed production worldwide. In this study, we assessed the extent and pattern of DNA damages caused by salt stress in rapeseed plants. Amplified fragment length polymorphism （AFLP） analysis revealed dose-related increases in sequence alterations in plantlets exposed to 10-1000 mmol/L sodium chloride. In addition, individual plantlets exposed to the same salt concentration showed different AFLP and selected region amplified polymorphism banding patterns. These observations suggested that DNA mutation in response to salt stress was random in the genome and the effect was dose-dependant. DNA methylation changes in response to salt stress were also evaluated by methylation sensitive amplified polymorphism （MSAP）. Three types of MSAP bands were recovered. Type Ⅰ bands were observed with both isoschizomers Hpa Ⅱ and Msp Ⅰ, while type Ⅱ and type Ⅲ bands were observed only with Hpa Ⅱ and Msp Ⅰ, respectively. Extensive changes in types of MSAP bands after NaCI treatments were observed, including appearance and disappearance of type Ⅰ, Ⅱ and Ⅲ bands, as well as exchanges between either type Ⅰand type Ⅱ or type Ⅰ and type Ⅲ bands. An increase of 0.2-17.6% cytosine methylated CCGG sites were detected in plantlets exposed to 10- 200 mmol/L salt compared to the control, and these changes included both de novo methylation and demethylation events. Nine methylation related fragments were also recovered and sequenced, and one sharing a high sequence homology with the ethylene responsive element binding factor was identified. These results demonstrated clear DNA genetic and epigenetic alterations in planUets as a response to salt stress, and these changes may suggest a mechanism for plants adaptation under salt stress.     

Epigenetic regulation of photoperiodic flowering

The cytidine analogue 5-azacytidine, which causes DNA demethylation, induced flowering in the non-vernalization-requiring plants Perilla frutescens var. crispa, Silene armeria and Pharbitis nil (synonym Ipomoea nil) under non-inductive photoperiodic conditions, suggesting that the expression of photoperiodic flowering-related genes is regulated epigenetically by DNA methylation. The flowering state induced by DNA demethylation was not heritable. Changes in the genome-wide methylation state were examined by methylation-sensitive amplified fragment length polymorphism analysis. This analysis indicated that the DNA methylation state was altered by the photoperiodic condition. DNA demethylation also induced dwarfism, and the induced dwarfism of P. frutescens was heritable.Key words: 5-azacytidine, DNA methylation, photoperiodic flowering, epigenetics, methylation-sensitive amplified fragment length polymorphism, CpG island, dwarfism     

Flowering and dwarfism induced by DNA demethylation in Pharbitis nil

Flowering and dwarfism induced by 5‐azacytidine and zebularine, which both cause DNA demethylation, were studied in a short‐day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non‐inductive long‐day (LD) conditions. The flower‐inducing effect of 5‐azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non‐inductive photoperiodic conditions. These results suggest that the flowering‐related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A₃ but not by t‐zeatin or kinetin. The change in the genome‐wide methylation state was examined by methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD‐specific fragments were extracted from the gel of the MS‐AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins.     

Transcript profiling in Arabidopsis reveals complex responses to global inhibition of DNA methylation and histone deacetylation

Blocking histone deacetylation with trichostatin A (TSA) or blocking cytosine methylation using 5-aza-2'-deoxycytosine (aza-dC) can derepress silenced genes in multicellular eukaryotes, including animals and plants. We questioned whether DNA methylation and histone deacetylation overlap in the regulation of endogenous plant genes by monitoring changes in expression of approximately 7800 Arabidopsis thaliana genes following treatment with azadC, TSA, or both chemicals together. RNA levels for approximately 4% of the genes were reproducibly changed 3-fold or more by at least one treatment. Distinct subsets of genes are up-regulated or down-regulated in response to aza-dC, TSA, or simultaneous treatment with both chemicals, with little overlap among subsets. Surprisingly, the microarray data indicate that TSA and aza-dC are often antagonistic rather than synergistic in their effects. Analysis of green fluorescent protein transgenic plants confirmed this finding, showing that TSA can block the up-regulation of silenced green fluorescent protein transgenes in response to aza-dC or a ddm1 (decrease in DNA methylation 1) mutation. Our results indicate that global inhibition of DNA methylation or histone deacetylation has complex, nonredundant effects for the majority of responsive genes and suggest that activation of some genes requires one or more TSA-sensitive deacetylation events in addition to cytosine demethylation.     

Epigenetic Changes during Hepatic Stellate Cell Activation

Background and Aims

Hepatic stellate cells (HSC), which can participate in liver regeneration and fibrogenesis, have recently been identified as liver-resident mesenchymal stem cells. During their activation HSC adopt a myofibroblast-like phenotype accompanied by profound changes in the gene expression profile. DNA methylation changes at single genes have been reported during HSC activation and may participate in the regulation of this process, but comprehensive DNA methylation analyses are still missing. The aim of the present study was to elucidate the role of DNA methylation during in vitro activation of HSC.

Methods and Results

The analysis of DNA methylation changes by antibody-based assays revealed a strong decrease in the global DNA methylation level during culture-induced activation of HSC. To identify genes which may be regulated by DNA methylation, we performed a genome-wide Methyl-MiniSeq EpiQuest sequencing comparing quiescent and early culture-activated HSC. Approximately 400 differentially methylated regions with a methylation change of at least 20% were identified, showing either hypo- or hypermethylation during activation. Further analysis of selected genes for DNA methylation and expression were performed revealing a good correlation between DNA methylation changes and gene expression. Furthermore, global DNA demethylation during HSC activation was investigated by 5-bromo-2-deoxyuridine assay and L-mimosine treatment showing that demethylation was independent of DNA synthesis and thereby excluding a passive DNA demethylation mechanism.

Conclusions

In summary, in vitro activation of HSC initiated strong DNA methylation changes, which were associated with gene regulation. These results indicate that epigenetic mechanisms are important for the control of early HSC activation. Furthermore, the data show that global DNA demethylation during activation is based on an active DNA demethylation mechanism.