Assessment of DNA methylation changes in tissue culture of Brassica napus

Plant tissue culture, as a fundamental technique for genetic engineering, has great potential of epigenetic variation, of which DNA methylation is well known of importance to genome activity. We assessed DNA methylation level of explants during tissue culture of Brassica napus (cv. Yangyou 9), using high-performance liquid chromatography (HPLC) assisted quantification. By detecting methylation levels in hypocotyls cultured in mediums with different concentrations of hormones, we found dissected tissue cultured with 0.1 mg/L 2,4-D and 1.0 mg/L 6-BA, presented the lowest methylation level and highest induction rate of callus (91.0%). Different time point of cultured explants also showed obvious methylation variations, explants cultured after 6 and 21 days exhibited methylation ratios of 4.33 and 8.07%, respectively. Whereas, the methylation ratio raised to 38.7% after 30 days cultivation, indicating that methylation level of hypocotyls ranged during tissue culture. Moreover, we observed that the methylation level in callus is the highest during regeneration of rapeseed, following the regenerated plantlets and hypocotyls. This paper indicated the function of hormones and differentiation of callus is relevant to the methylation levels during tissue culture.     

Variations in leaf morphology and DNA methylation following in vitro culture of Malus xiaojinensis

Juvenile trees of Malus xiaojinensis produce leaves with three to five lobes; however, adult trees do not. Thus, M. xiaojinensis serves as a model plant for studies on rejuvenation of woody perennials. To better understand effects of in vitro culture, number of passages, and plant growth regulators on observed somaclonal variations in leaf morphology, total levels of DNA methylation and expression of associated genes, including DRM2 and MET1, of apical shoot meristems of M. xiaojinensis, were evaluated. Following 17 passages of nodal stem segment culture and four passages of callus culture, levels of DNA methylation and expression of DRM2 and MET1 were determined, and found to decrease from the 11th passage of nodal stem segment culture or adventitious bud culture and from the first passage of callus culture. Levels of DNA methylation increased with increasing 2,4-dichlorophenoxyacetic acid concentrations, but decreased significantly with increasing 6-benzyladenine (BA) concentrations. Expression of DRM2 and MET1 in regenerated plantlets were negatively correlated with BA concentrations in the culture medium. Overall, less than 11 passages of nodal stem segment or adventitious bud culture did not contribute to incidence of somaclonal variation, even in the presence of low concentrations of BA (<0.5?mg/L) in the culture medium. Moreover, when nodal stem segment cultures were maintained for up to 17 passages, complete rejuvenation was observed.?However, epigenetic changes were detected following the first passage of callus cultures.     

Different methylation pattern of melon satellite DNA sequences in hypocotyl and callus tissues

Satellite DNA sequences from Cucumis melo have been examined with respect to modification at CCGG sequences in hypocotyls and in callus tissues. For this purpose, restriction fragments given by HpaII and MspI were compared (both enzymes recognize CCGG sequences but have different sensitivity to methylation at this site). Whereas the methylation level of satellite DNA sequences is on average higher in hypocotyls than in callus tissues, the comparison of partially methylated repeat units of satellite DNA reveals that in callus tissues, all methylated restriction sites are doubly methylated.     

Epigenetic variation in the callus of <Emphasis Type="Italic">Brassica napus</Emphasis> under different inducement conditions

Tissue culture, a traditional technique broadly used for the genetic transformation and functional verification of target genes, induces epigenetic variations in transgenic acceptors of plants. This study compared the DNA methylation patterns during the callus formation of Brassica napus induced by different concentrations of 6-BA and 2,4-D through methylation-sensitive amplification polymorphism. The highest induction rate (85%) was observed in the hypocotyls cultured with 0.1 mg/L 2,4-D and the lowest methylation rate (25.09%) was detected in the hypocotyls cultured with 1.0 mg/L 6-BA. The methylation rates of the callus cultured with 0.2 and 0.05 mg/L 2,4-D were 29.99 and 28.31%, respectively. The callus induction rates were reduced to 79 and 80%. The methylation rates of the callus induced by 2.0 and 0.5 mg/L 6-BA were 28.17 and 33.98%, respectively. The callus induction rates were reduced to 76 and 74%. The expression analysis of methyltransferase under different induction conditions agreed with methylation modifications; therefore, the effects of hormones on callus induction may be partially indicated by methylation changes in B. napus genome.     

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.     

Biscoclaurine alkaloids in callus tissues of Stephania cepharantha

Although callus tissues derived from tubers of Stephania cepharantha cannot synthesize the main alkaloids of the original plant, cepharanthine and isotetrandrine, they are able to synthesize biscoclaurine alkaloids, berbamine and aromorine, the latter not being found in the original plant. These results suggest that enzymes controlling specific methylation and methylenedioxy group formation are absent from the callus. The maximum content of total alkaloid in the callus tissues subcultured for 9 months was more than 3 times that of original plant. Alkaloid content was affected by addition of various auxins, IAA being most effective.     

Physiological Changes in Cultured Sorghum Cells in Response to Induced Water Stress : II. Soluble Carbohydrates and Organic Acids

Eight cultivars Sorghum bicolor (L.) Moench were grown as callus cultures under induced, prolonged water stress (8 weeks), with polyethylene glycol in the medium. Concentrations of soluble carbohydrates and organic acids in callus were measured at the end of the growth period to determine differences in response to prolonged water stress. Sucrose, glucose, fructose, and malate were the predominant solutes detected in all callus at all water potentials. All cultivars had high levels of solutes in the absence of water stress and low levels in the presence of prolonged water stress. However, at low water potentials, low levels of solutes were observed in drought-tolerant cultivar callus and high solute levels were observed in drought-susceptible cultivar callus. Estimated sucrose concentrations were significantly higher in water-stressed, susceptible cultivar callus. Large solute concentrations in susceptible cultivar callus were attributed to osmotic adjustment and/or reduced growth during water stress.     

Gene promoter methylation in colorectal cancer and healthy adjacent mucosa specimens: Correlation with physiological and pathological characteristics,and with biomarkers of one-carbon metabolism

We evaluated the promoter methylation levels of the APC, MGMT, hMLH1, RASSF1A and CDKN2A genes in 107 colorectal cancer (CRC) samples and 80 healthy adjacent tissues. We searched for correlation with both physical and pathological features, polymorphisms of folate metabolism pathway genes (MTHFR, MTRR, MTR, RFC1, TYMS, and DNMT3B), and data on circulating folate, vitamin B12 and homocysteine, which were available in a subgroup of the CRC patients. An increased number of methylated samples were found in CRC respect to adjacent healthy tissues, with the exception of APC, which was also frequently methylated in healthy colonic mucosa. Statistically significant associations were found between RASSF1A promoter methylation and tumor stage, and between hMLH1 promoter methylation and tumor location. Increasing age positively correlated with both hMLH1 and MGMT methylation levels in CRC tissues, and with APC methylation levels in the adjacent healthy mucosa. Concerning gender, females showed higher hMLH1 promoter methylation levels with respect to males. In CRC samples, the MTR 2756AG genotype correlated with higher methylation levels of RASSF1A, and the TYMS 1494 6bp ins/del polymorphism correlated with the methylation levels of both APC and hMLH1. In adjacent healthy tissues, MTR 2756AG and TYMS 1494 6bp del/del genotypes correlated with APC and MGMT promoter methylation, respectively. Low folate levels were associated with hMLH1 hypermethylation. Present results support the hypothesis that DNA methylation in CRC depends from both physiological and environmental factors, with one-carbon metabolism largely involved in this process.     

Tissue culture-induced DNA methylation polymorphisms in repetitive DNA of tomato calli and regenerated plants

The propagation of plants through tissue culture can induce a variety of genetic and epigenetic changes. Variation in DNA methylation has been proposed as a mechanism that may explain at least a part of these changes. In the present study, the methylation of tomato callus DNA was compared with that of leaf DNA, from control or regenerated plants, at MspI/HpaII sites around five middle-repetitive sequences. Although the methylation of the internal cytosine in the recognition sequence CCGG varied from zero to nearly full methylation, depending on the probe used, no differences were found between callus and leaf DNA. For the external cytosine, small differences were revealed between leaf and callus DNA with two probes, but no polymorphisms were detected among DNA samples of calli or DNA samples of leaves of regenerated plants. When callus DNA cut with HindIII was studied with one of the probes, H9D9, most of the signal was found in high-molecular-weight DNA, as opposed to control leaf DNA where almost all the signal was in a fragment of 530 bp. Also, an extra fragment of 630 bp was found in the callus DNA that was not present in control leaf DNA. Among leaves of plants regenerated from tissue culture, the 630-bp fragment was found in 10 of 68 regenerated plants. This 630-bp fragment was present among progeny of only 4 of these 10 plants after selfing, i.e. it was partly inherited. In these cases, the fragment was not found in all progeny plants, indicating heterozygosity of the regenerated plants. The data are interpreted as indicating that a HindIII site becomes methylated in callus tissue, and that some of this methylation persists in regenerated plants and is partly transmitted to their progeny.     

White blood cell global methylation and IL-6 promoter methylation in association with diet and lifestyle risk factors in a cancer-free population

Altered levels of global DNA methylation and gene silencing through methylation of promoter regions can impact cancer risk, but little is known about their environmental determinants. We examined the association between lifestyle factors and levels of global genomic methylation and IL-6 promoter methylation in white blood cell DNA of 165 cancer-free subjects, 18–78 years old, enrolled in the COMIR (Commuting Mode and Inflammatory Response) study, New York, 2009–2010. Besides self-administrated questionnaires on diet and physical activity, we measured weight and height, white blood cell (WBC) counts, plasma levels of high sensitivity C-reactive protein (hs-CRP), and genomic (LINE-1) and gene-specific methylation (IL-6) by pyrosequencing in peripheral blood WBC. Mean levels of LINE-1 and IL-6 promoter methylation were 78.2% and 57.1%, respectively. In multivariate linear regression models adjusting for age, gender, race/ethnicity, body mass index, diet, physical activity, WBC counts and CRP, only dietary folate intake from fortified foods was positively associated with LINE-1 methylation. Levels of IL-6 promoter methylation were not significantly correlated with age, gender, race/ethnicity, body mass index, physical activity or diet, including overall dietary patterns and individual food groups and nutrients. There were no apparent associations between levels of methylation and inflammation markers such as WBC counts and hs-CRP. Overall, among several lifestyle factors examined in association with DNA methylation, only dietary folate intake from fortification was associated with LINE-1 methylation. The long-term consequence of folate fortification on DNA methylation needs to be further evaluated in longitudinal settings.     

Evidence for Sulfhydryl Involvement in Regulation of Phytoalexin Accumulation in Trifolium repens Callus Tissue Cultures

White clover (Trifolium repens L.) callus tissue cultures accumulated the phytoalexin medicarpin after treatment with sulfhydryl reagents. After 24-hour exposures to sulfhydryl reagents, maximum obtainable levels of medicarpin, determined by high performance liquid chromatography analysis, were found with 50 millimolar N-ethyl maleimide, 25 millimolar HgCl₂, 2 millimolar p-chloromercuribenzoic acid, and 0.5 millimolar iodoacetamide. Increased medicarpin levels were also observed in callus treated with p-chloromercuribenzene sulfonic acid, but the highest concentration tested (11.8 millimolar) did not produce the maximum response. After sulfhydryl treatment, medicarpin levels were unchanged for 4 to 6 hours, but steadily increased thereafter with maximum accumulation occurring by 48 to 50 hours for p-chloromercuribenzoic acid, p-chloromercuribenzene sulfonic acid, and HgCl₂ treated callus. Medicarpin levels did not increase in iodoacetamide-treated callus until 8 hours after sulfhydryl exposure, and medicarpin levels were still increasing linearly after 50 hours. Three other metabolic inhibitors, KCN, NaF, and Na₃AsO₄, did not exhibit elicitor activity, indicating cell death was not a factor in the response. Pretreatment of callus with 20 millimolar dithiothreitol followed by 40 millimolar N-ethyl maleimide did not produce the phytoalexin response. Preincubation with dithiothreitol also prevented elicitor activity of HgCl₂ and p-chloromercuribenzene sulfonic acid. These results suggested that dithiothreitol pretreatment somehow prevented sulfhydryl groups within the cell from reacting with the test compounds. These experiments established that the integrity of sulfhydryl groups is important in regulating phytoalexin accumulation in callus cells.     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

Variations in genomic DNA methylation during the long-term in vitro proliferation of oil palm embryogenic suspension cultures

Key message

The long-term proliferation of embryogenic cell suspensions of oil palm is associated with changes in both genomic methylation rates and embryogenic capacities.

Abstract

In the aim of exploring the relationship between epigenetic stability and the long-term in vitro proliferation of plant tissues, we have studied changes in genomic DNA methylation levels in embryogenic suspensions of oil palm (Elaeis guineensis Jacq.). Five embryogenic callus lines were obtained from selected hybrid seeds and then proliferated as suspension cultures. Each clonal line obtained from a single genotype was subdivided into three independent subclonal lines. Once established, cultures proliferated for 12 months and genomic DNA was sampled at 4 months intervals for the estimation of global DNA methylation rates through high performance liquid chromatography (HPLC) quantitation of deoxynucleosides. Our results show that in vitro proliferation induces DNA hypermethylation in a time-dependent fashion. Moreover, this trend is statistically significant in several clonal lines and shared between subclonal lines originating from the same genotype. Interestingly, the only clonal line undergoing loss of genomic methylation in the course of proliferation has been found unable to generate somatic embryos. We discuss the possible implications of genome-wide DNA methylation changes in proliferating cells with a view to the maintenance of genomic and epigenomic stability.     

Genome-wide methylation changes are associated with muscle fiber density and drip loss in male three-yellow chickens

In eukaryotes, DNA methylation is an important epigenetic modification involved in gene expression regulation. Meat quality traits are complicated traits that are controlled by many genes. Changes in the methylation levels of certain genes controlling meat quality traits will inevitably affect their expression levels, thereby affecting meat quality. The objectives of this study were to investigate the differences in the DNA methylation level in pectoral muscle tissues using fluorescent-labeled methylation sensitive amplified polymorphism and their relationships with meat quality traits in three-yellow chickens. The results showed that the differences in the DNA methylation level had a highly significant effect on muscle fiber density and drip loss (P < 0.01). However, no significant correlations were found between the DNA methylation levels and the other investigated traits (P > 0.05).     

Methylation patterns of two repetitive DNA sequences in tobacco tissue cultures and their regenerants

DNA methylation of two repetitive sequences in tobacco nuclear genome was studied in the course ofin vitro dedifferentiation and differentiation. Using 5-mC sensitivè restriction enzymes and DNA/DNA hybridization with 25S-rDNA probe it has been shown that during the early phase of callus induction prominent changes in the methylation pattern occur which are stably maintained during subsequent callus growth. The following protoplast recovery and plant regeneration have again displayed some more modifications of the methylation status. Comparing the patterns of R₀ plants with the original plant material and the calli it can be assumed that both share in the resulting methylation status. The experiments analyzing the HRS60 family of non-transcribed highly repetitive sequences have displayed a quite monotonous methylation status thus indicating no random methylation perturbations in silent DNA sequences.     

Cardiovascular disease risk factors and DNA methylation at the LINE-1 repeat region in peripheral blood from Samoan Islanders

Lower levels of LINE-1 methylation in peripheral blood have been previously associated with risk of developing non-communicable conditions, the most well-explored of these being cancer, although recent research has begun to link altered LINE-1 methylation and cardiovascular disease. We examined the relationship between LINE-1 methylation and factors associated with metabolic and cardiovascular diseases through quantitative bisulfite pyrosequencing in DNA from peripheral blood samples from participants of the Samoan Family Study of Overweight and Diabetes (2002–03). The sample included 355 adult Samoans (88 men and 267 women) from both American Samoa and Samoa. In a model including all sample participants, men had significantly higher LINE-1 methylation levels than women (p = 0.04) and lower levels of LINE-1 methylation were associated with higher levels of fasting LDL (p = 0.02) and lower levels of fasting HDL (p = 0.009). The findings from this study confirm that DNA “global” hypomethylation (as measured by methylation at LINE-1 repeats) observed previously in cardiovascular disease is associated with altered levels of LDL and HDL in peripheral blood. Additionally, these findings strongly argue the need for further research, particularly including prospective studies, in order to understand the relationship between LINE-1 DNA methylation measured in blood and risk factors for cardiovascular disease.Key words: cardiovascular disease, HDL, LDL, LINE-1, DNA methylation, Samoa     

Cytokinin Differences in In Vitro Cultures and Inflorescences from Normal and Mantled Oil Palm (Elaeis guineensis Jacq.)

The mantled abnormality phenotype of the oil palm affects fruit development and thus jeopardizes oil yield. Cytokinins have been implicated in the development of the mantled phenotype. Endogenous cytokinin levels in the normal and mantled phenotypes were compared to determine whether levels of specific cytokinins are associated with mantling. Endogenous cytokinins were identified and quantified in in vitro cultures and inflorescences from normal and mantled oil palms. Twenty-two isoprenoid cytokinins, comprising the zeatin, dihydrozeatin, and isopentenyladenine types, were quantified. Total cytokinin levels, particularly of trans-zeatin and isopentenyladenine types, increased during the in vitro culture process, with the highest levels detected at the proliferating polyembryoid stages. The cytokinins were present mainly in their inactive 9-glucoside forms during in vitro culture. On the other hand, the predominant trans-zeatin cytokinins in inflorescences were present mainly in their ribotide forms, suggesting a metabolic pool of cytokinins for conversion to biologically active free bases or ribosides. Levels of specific cytokinins were significantly different in tissues at different stages. Mantled developed inflorescences contained higher levels of isopentenyladenine 9-glucoside compared with normal inflorescences. Mantled-derived callus tissues had higher isopentenyladenine levels but significantly lower levels of trans-zeatin 9-glucoside, dihydrozeatin riboside, and dihydrozeatin riboside 5′-monophosphate cytokinins compared with normal-derived callus. It would be of considerable interest to verify these specific cytokinin differences in more callus cultures and clones.     

Global DNA methylation levels in girls with and without a family history of breast cancer

Lower levels of global DNA methylation in white blood cell (WBC) DNA have been associated with adult cancers. It is unknown whether individuals with a family history of cancer also have lower levels of global DNA methylation early in life. We examined global DNA methylation in WBC (measured in three repetitive elements, LINE1, Sat2 and Alu, by MethyLight and in LINE1 by pyrosequencing) in 51 girls aged 6–17 years. Compared to girls without a family history of breast cancer, methylation levels were lower for all assays in girls with a family history of breast cancer and statistically significantly lower for Alu and LINE1 pyrosequencing. After adjusting for age, body mass index (BMI) and Tanner stage, only methylation in Alu was associated with family history of breast cancer. If these findings are replicated in larger studies, they suggest that lower levels of global WBC DNA methylation observed later in life in adults with cancer may also be present early in life in children with a family history of cancer.Key words: Alu, DNA global methylation, early life exposure, epigenetics, LINE1, methylight, pyrosequencing, Sat2