Methylation-sensitive Amplification Polymorphism Method Research Articles

Corona discharge plasma stimulated production of atropine in callus of Datura inoxia by DNA hypomethylation and gene regulation: a novel technology for plant cell and tissue culture.

Few investigations have tested the practical use of cold plasma as a novel technology to meet the requirements in the plant cell and tissue culture field. To fill the knowledge gap, we intend to respond to the question of whether plasma priming influenced DNA ultrastructure and the production of atropine (a tropane alkaloid) in Datura inoxia. Calluses were treated with the corona discharge plasma at time durations ranging from 0 to 300s. Significant increases (about 60%) in biomass were observed in the plasma-primed calluses. The plasma priming of calluses enhanced the accumulation of atropine about 2-fold. The plasma treatments increased proline concentrations and soluble phenols. The drastic increases in the activity of the phenylalanine ammonia-lyase (PAL) enzyme resulted from the applied treatments. Likewise, the plasma treatment of 180s upregulated the expression of the PAL gene by 8-fold. Also, the expression of the ornithine decarboxylase (ODC) and tropinone reductase I (TR I) genes were stimulated by 4.3-fold and 3.2-fold, respectively, in response to the plasma treatment. The putrescine N-methyltransferase gene displayed a similar trend to that of TR I and ODC genes following the plasma priming. Methylation sensitive amplification polymorphism method was employed to explore the plasma-associated epigenetic changes in DNA ultrastructure. The molecular assessment referred to DNA hypomethylation, validating an epigenetic response. This biological assessment study validates the hypothesis that plasma priming of callus is an efficient, cost-effective, and eco-friendly tool to enhance callogenesis efficiency, elicit metabolism, affect gene regulation, and modify chromatin ultrastructure in D. inoxia.

Methylation-sensitive amplification polymorphism analysis reveals the DNA methylation dynamics during capitulum development in Chrysanthemum lavandulifolium

Chrysanthemum lavandulifolium (Fischer ex Trautv.) Makino is a diploid plant belonging to the Asteraceae family, with typical capitula composed of female ray florets and bisexual disc florets. The differentiation and development of these two types of florets have long been important research focuses; however, the potential epigenetic mechanisms governing these processes have not been elucidated. In the present study, methylation-sensitive amplification polymorphism method was used to trace the dynamic changes of DNA methylation during capitulum development in C. lavandulifolium. DNA methylation patterns and levels were detected in the whole capitula during seven developmental stages, and the obtained results revealed that DNA demethylation was dominant during this process. In addition, DNA methylation patterns and levels showed significant differences between ray and disc florets. Moreover, the expression patterns of candidate genes potentially involved in the development processes of two types of florets were analyzed by real-time quantitative reverse transcription polymerase chain reaction, and correlation analysis indicated that the expression levels of ClPI, ClAG2, ClSEP1, ClCYC2c, ClCYC2d, and ClCYC2e were highly correlated with DNA methylation levels. These results indicate that DNA methylation may be involved in the differentiation and development of ray and disc florets. This study provides epigenetic insights into the capitulum development in C. lavandulifolium.

Recurrent Water Deficit and Epigenetic Memory in Medicago sativa L. Varieties

Global DNA methylation changes in response to recurrent drought stress were investigated in two common Greek Medicago sativa L. varieties (Lamia and Chaironia-Institute of Ιndustrial and Forage Crops). The water deficit was implemented in two phases. At the end of the first phase, which lasted for 60 days, the plants were cut at the height of 5 cm and were watered regularly for two months before being subjected to the second drought stress, which lasted for two weeks. Finally, the following groups of plants were formed: CC (controls both in phase I and phase II), CD2 (Controls in phase I, experiencing drought in phase II), and D1D2 (were subjected to drought in both phase I and phase II). At the end of phase II, samples were taken for global DNA methylation analysis with the Methylation Sensitive Amplification Polymorphism (MSAP) method, and all plants were harvested in order to measure the fresh and dry weight of roots and shoots. The variety Lamia responded better, especially the D1D2 group, compared to Chaironia in terms of root and shoot dry weight. Additionally, the shoots of Lamia had a constant water status for CD2 and D1D2 group of plants. According to DNA methylation analysis by the MSAP method, Lamia had lower total DNA methylation percentage after the second drought episode (D1D2) as compared to the plants CD2 that had experienced only one drought episode. On the other hand, the total DNA methylation percentage of Chaironia was almost the same in plants grown under recurrent drought stress conditions compared to control plants. In conclusion, the decrease of DNA methylation of Lamia stressed plants probably indicates the existence of an epigenetic mechanism that may render drought tolerance.

Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm.

DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A) transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation changes induced by hDNMT3A expression were intergenerationally inherited by offspring without transmission of the transgene, which provided evidence for the transmission of active endogenous-factors-induced epigenetic variations.

No consistent daily variation in DNA methylation detected in Populus nigra leaves by methylation-sensitive amplification polymorphism analysis

DNA methylation, an epigenetic mechanism used by cells to control gene expression, has an important biological role in plant development and environmental fitness. Since plant DNA methylation is closely related to environmental conditions, variation during the day is expected. Here, in genetically identical plants of Populus nigra clone N46, DNA methylation changes in leaves over a 24 h period were detected using the methylation-sensitive amplification polymorphism method. The results showed different DNA methylation patterns in mature poplar leaves: not only in individuals at the same time, but also in samples at each of the six time during the day. In addition, night samples had a higher percentage of methylation than in morning samples. However, no statistically significant differences were found among the samples gathered at different times. Similar results were obtained for three other P. nigra clones with different genetic backgrounds. Real time qPCR showed that the DNA methyltransferase genes Pt-MET1 and Pt-SOM1 involved in CG DNA methylation in poplar were stable over a 24 h period in leaves of P. nigra N46 compared with circadian-controlled genes. That could be part of the reason that methylation of CCGG sites is stable in those leaves. That DNA methylation differed even in genetically identical plants indicates the specificity of DNA methylation changes in their genomes. No statistically significant differences in methylation changes were found between day and night, suggesting that DNA methylation is more stable than expected and is unlikely to be involved in circadian regulation in plants.

Epigenetic mechanisms of salt tolerance and heterosis in Upland cotton (Gossypium hirsutum L.) revealed by methylation-sensitive amplified polymorphism analysis

In order to explore the molecular mechanism of salt tolerance and heterosis in cotton, a methylation-sensitive amplification polymorphism method based on capillary electrophoresis was used to analyze DNA methylation level in a cotton hybrid CCRI 29 and its two parents. The major results and conclusions are as follows: Firstly, salt tolerance test showed that CCRI 29 had higher salt-tolerance level than its both parents. The global DNA methylation level in CCRI 29 under salt treatment significantly increased, whereas the two parents did not change significantly between salt treatment and control. All kinds of variation of DNA methylation happened in cotton under salt treatment, and hypermethylation happened at a significantly higher rate than that of hypomethylation in CCRI 29 but not in its two parents. The results suggested that the increase of global DNA methylation level in cotton genome and also different methylation types played an important role in tolerance to salt treatment in cotton. Secondly, both hypomethylation and hypermethylation happened to different genes and some genes maintained the same methylation level after salt stress, which meant that complex gene expression alterations occurred when responding to salt stress in cotton, indicating the complicated characteristics of roles that specific genes played in salt tolerance. Thirdly, although most cytosine methylation sites in hybrid CCRI 29 shared the same status as that of at least one of the parents, the site number of hypomethylation is significantly higher than that of hypermethylation in CCRI 29 compared to parents under both control and salt stress, indicating that demethylation could be the mechanism to explain heterosis in cotton hybrid.

Analysis of methylation-sensitive amplified polymorphism in different cotton accessions under salt stress based on capillary electrophoresis

A methylation-sensitive amplification polymorphism method based on capillary electrophoresis was used to analyze DNA methylation levels in three cotton accessions, two salt-tolerant accessions CCRI 35 and Zhong 07 and one salt-sensitive accession CCRI 12. Many categories of DNA methylation happened in the three cotton accessions under salt treatment, including hypermethylation, hypomethylation, and other patterns. Hypermethylation happened at a significantly higher rate than that of hypomethylation in salt-tolerant accessions CCRI 35 and Zhong 07. On the contrary, in salt-sensitive accession CCRI 12, hypomethylation happened at a significantly higher rate than that of hypermethylation. In general, the global DNA methylation level significantly increased under salt stress in both salt-tolerant accessions CCRI 35 and Zhong 07, whereas there was no significant difference in the salt-sensitive accessions CCRI 12. Our results suggested that salt-tolerant cotton might have a mechanism of increasing the methylation level when responding to salt stress; the increase of the global level of DNA methylation and also different methylation patterns might play important roles in tolerance to salt stress in cotton. Some interesting genes were found through cloning and analysis of differently methylated DNA sequences, which might contribute to salt tolerance in cotton.

Analysis of DNA cytosine methylation patterns in maize hybrids and their parents

In order to better understand the molecular basis of heterosis in maize, the methylation-sensitive amplification polymorphism method was used to estimate patterns of cytosine methylation in seedling roots and leaves and 15-d postfertilization embryo and endosperm tissues of hybrids and their parental lines Zheng58 and Chang7-2. In all tissues, total relative methylation levels in the hybrids were lower than corresponding mid-parent values, with a higher number of demethylation events inferred for the hybrids. The trend of reduced methylation and increased demethylation in the hybrids relative to their parents may allow de-repression and possibly the expression of various genes associated with a hybrid phenotypic variation. To further investigate observed methylation pattern changes, we sequenced 50 differentially displayed DNA fragments. The BLAST analysis revealed that 13 fragments shared similarity with known functional proteins in maize or other plant species including proteins related to metabolism, transposons/retrotransposons, development, stress response, and signal transduction. The genes associated with these proteins may thus contribute significantly to maize hybrid vigour.

Changes in DNA Methylation Pattern at Two Seedling Stages in Water Saving and Drought-Resistant Rice Variety after Drought Stress Domestication

Recent studies revealed that DNA methylation plays an important role in plant growth and development. In this study, a water-saving and drought-resistant rice variety Huhan 3 was subjected to drought stress from tillering to grain-filling stages in six successive growth cycles. The variations in DNA methylation pattern between the original generation (G0) and the sixth generation (G6) were analyzed by using methylation sensitive amplification polymorphism method. The results revealed that the methylated loci accounted for 34.3% to 34.8% of the total loci. Among these methylated loci, 83.1% to 84.8% were full- and hyper-methylated and 15.2% to 16.9% were hemi-methylated. The DNA methylation level decreased from the three-leaf to four-leaf stages in Huhan 3. Differentially methylated loci (DML) between generations or/and between different developmental stages accounted for 4.0% of the total loci, most of which were only related to plant development (57.9%). Compared to G0, the DNA methylation pattern of G6 changed after drought domestication, at the three-leaf stage, de-methylation accounting for 59.1%, while at the four-leaf stage, re-methylation for 47.9%. Genome-wide alternations of DNA methylation were observed between the two seedling stages, and DML mainly occurred on the gene's promoter and exon region. The genes related to DML involved in a wide range of functional biology and participated in many important biological processes.

Analysis of DNA methylation patterns and levels in maize hybrids and their parents.

Heterosis is the superior performance of heterozygous individuals and has been widely exploited in plant breeding, although the underlying regulatory mechanisms still remain largely elusive. To understand the molecular basis of heterosis in maize, in this study, roots and leaves at the seedling stage and embryos and endosperm tissues 15 days after fertilization of 2 elite hybrids and their parental lines were used to estimate the levels and patterns of cytosine methylation by the methylation-sensitive amplification polymorphism method. The relative total methylation levels were lower in all the tissues of all hybrids than their corresponding mid-parent values, and the number of demethylation events was higher in the hybrids. These results implied that the decreasing trend and demethylation in hybrids relative to their parents may enable the derepression and possibly expression of many genes that were associated with the phenotypic variation in hybrids. To further analyze the observed methylation pattern changes, a total of 63 differentially displayed DNA fragments were successfully sequenced. Basic Local Alignment Search Tool analysis showed that 11 fragments shared similarity with known functional proteins in maize or other plant species, including metabolism, transposon/retrotransposon, development, stress response, and signal transduction, which indicated that these genes might play a significant role in maize hybrid vigor.

Adaptational significance of variations in DNA methylation in clonal plant Hierochloe glabra (Poaceae) in heterogeneous habitats

As a prominent epigenetic modification, cytosine methylation may play a critical role in the adaptation of plants to different environments. The present study sought to investigate possible impacts of differential levels of nitrogen (N) supply on cytosine-methylation levels of a clonal plant, Hierochloe glabra Trin. (Poaceae). For this purpose, nitrate was applied at concentrations of 0, 0.15, 0.30 and 0.45 g N kg–1 soil, and ecologically important morphological traits were measured. The methylation-sensitive amplification polymorphism method was also conducted to analyse the variations in DNA cytosine methylation. Our results showed that N addition reduced CHG cytosine-methylation levels markedly compared with control plants growing in homogeneous pots (P = 0.026). No substantial differences were observed in morphological traits at the end of the growing stage, except for the highest ratio of leaf area to leaf dry mass in the medium-N patch (P = 0.008). However, significant linear regression relationships were found between cytosine-methylation levels and morphological traits, such as bud number and rhizome length and biomass. In conclusion, the higher cytosine-methylation level may activate asexual reproduction to produce more offspring and expand plant populations, possibly helping clonal plants to adapt to heterogeneous habitats.