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.     

叶锈菌胁迫下的小麦基因组MSAP分析

内源DNA甲基化是真核生物表观遗传调控的重要组成部分, 在真核生物的基因表达调控中具有重要的作用。生物胁迫为植物提供一种内在的表观遗传进化动力。研究生物胁迫下DNA甲基化的变异模式, 有助于全面理解DNA甲基化的表观调控生物学功能。小麦近等基因系TcLr19、TcLr41及其感病亲本Thatcher在苗期对叶锈菌生理小种THTT、TKTJ分别表现为小种特异性抗病反应和感病反应。文章利用甲基化敏感扩增多态性(Methylation-sensitive amplified polymorphism, MSAP)技术分析了小麦的甲基化水平, 同时比较了苗期在生物胁迫前后基因组DNA胞嘧啶甲基化模式。用60对MSAP引物对接种前后的小麦DNA进行全基因组筛选, 没有直接分离得到接菌前后的甲基化模式的差异, 结果初步表明, 叶锈菌并没有诱导稳定且特异的植物基因组DNA胞嘧啶位点的甲基化模式变化, 但发现TcLr41及其感病亲本Thatcher之间存在表观遗传学差异。     

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.     

甲基化敏感扩增多态性技术(MSAP)在生态学中的应用

DNA甲基化是生物体内最为重要的表观遗传修饰形式之一,在生态学上的应用越来越广泛。在收集、整理生态表观遗传学相关文献的基础上,介绍了甲基化敏感扩增多态性技术(MSAP)的原理、优势与局限性及其在生态学上的应用和展望。MSAP因其应用广泛、操作简便等优点成为研究DNA甲基化水平的有力工具,特别是在探究生物体如何快速适应生境变化以及外来入侵生物如何突破遗传瓶颈等问题上。MSAP技术能够很好地揭示生物种群内部或种群之间的表观遗传差异,是对遗传多样性、遗传变异研究的有力补充。     

DNA methylation changes detected by methylation-sensitive amplified polymorphism in the Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>) in response to salinity stress

Salinity is a considerable factor to the survival and distribution for a majority of marine organisms, the drawbacks of which are becoming a serious issue of aquaculture. DNA methylation, an extensively studied epigenetic modification in eukaryotes, plays a significant role in the regulation of gene expression in responding to environmental changes and triggering evolutionary consequences. The Pacific oyster Crassostrea gigas, as a eurythermal and euryhaline species, is considered to be tolerant to salinity fluctuation. In this study, fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis was used to investigate the frequency and variation of DNA methylation in C. gigas under different salinity and time. The results showed that total methylation level was generally on a downward trend. At lower salinity, the total methylation level decreased at the earlier process and then increased during experiment process, but continued to shrink at the rest salinity. Fully methylation tended to better reflect the dynamics of total methylation. Recovery treatment showed that the extent and pattern of DNA methylation were difficult to return to the normal level in this research. The sequencing and BLAST analysis indicated that in salt stress most of the selected bands were closely related to the metabolism of nucleic acids and proteins, tropomyosin, and cellular transport, effecting on different biological processes of C. gigas. This work provides useful data to further elucidate the molecular mechanisms of salt stress response and tolerance in invertebrates.     

Detection of DNA methylation pattern in thidiazuron-induced blueberry callus using methylation-sensitive amplification polymorphism

During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm^-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development.     

Assessment of changes in DNA methylation by methylation-sensitive amplification polymorphism in Jatropha curcas L. subjected to salinity stress

The present study assesses the changes in DNA methylation in leaf and root tissues of Jatropha curcas L., induced by salinity stress using methylation sensitive amplification polymorphism (MSAP) markers. Seedlings of 21 days (d) grown under controlled conditions were subjected to 0–100 mM salinity treatment for 24 h (1 d). Immediate changes in DNA methylation and polymorphism in methylated DNA in whole genome of both leaves and roots were assessed using 10 selective combinations of MSAP primers. In root and leaves 70.06% and 57.89% methylation was observed respectively. Similarly 67.22% and 71.21% polymorphism was observed in methylated DNA from root and leaf tissues respectively. Compared with control, the percentage of methylation and methylation polymorphism in roots of plants under different dosages of salinity was found in the order of 50 mM < 25 mM = 100 mM < 75 mM and 75 mM < 25 mM < 50 mM < 100 mM respectively. Similarly percentage of methylation and methylation polymorphism in leaves of plants treated with different levels of salinity was found in order of 75 mM < 25 mM < 50 mM < 100 mM and 50 mM < 25 mM < 100 mM < 75 mM respectively. The MSAP analysis showed that under salt stress homologous nucleotide sequences in genome from control and salt treated plants of J. curcas showed different patterns of methylation; which suggest that these fragments probably play an important role to induce immediate adaptive responses in Jatropha under salinity stress.     

Genetic analysis of involvement of ETR1 in plant response to salt and osmotic stress

The involvement of ethylene and ethylene receptor Ethylene Response 1 (ETR1) in plant stress responses has been highlighted. However, the physiological processes involved remain unclear. In this study, we have investigated the physiological response of two alleles etr1-1 and etr1-7 mutants during germination and post-germination seedling development in response to salt and osmotic stress. The etr1-1 mutants showed increased sensitivity to osmotic (200 mM or higher mannitol) and salt stress (50 mM NaCl or higher) during germination and seedling development, whereas the etr1-7 mutants displayed enhanced tolerance to the severe stresses (500 mM mannitol or 200 mM NaCl). These results provide physiological and genetic evidence that ethylene receptor ETR1 modulates plant response to abiotic stress. Furthermore, the etr1-1 and etr1-7 mutants showed different responses to exogenous abscisic acid (ABA) inhibition. The etr1-1 mutants were more sensitive to ABA than the wild type during germination, and young seedling development. In sharp contrast, the etr1-7 mutants showed enhanced insensitivity to ABA treatment (>1 μM ABA) in post-germination development including root elongation and greening of cotyledons of the treated seedlings, although the germination was not greatly altered at the tested doses of ABA. The results suggest that ETR1-modulated stress response may mediate ABA. Youning Wang and Tao Wang contributed equally to this report.     

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism （AFLP） to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP （methylation sensitive amplification polymorphism） ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5 ＇ -CCGG-3 ＇ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.     

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.