Application of reversed-phase high-performance liquid chromatography with fluorimetric detection for simultaneous assessment of global DNA and total RNA methylation in Lepidium sativum: effect of plant exposure to Cd(II) and Se(IV)

Abstract

In the present work, application of the previously established reversed-phase liquid chromatography procedure based on fluorescent labeling of cytosine and methylcytosine moieties with 2-bromoacetophenone (HPLC-FLD) is presented for simultaneous evaluation of global DNA and total RNA methylation at cytosine carbon 5. The need for such analysis was comprehended from the recent advances in the field of epigenetics that highlight the importance of non-coding RNAs in DNA methylation and suggest that RNA methylation might play a similar role in the modulation of genetic information, as previously demonstrated for DNA. In order to adopt HPLC-FLD procedure for DNA and RNA methylation analysis in a single biomass extract, two extraction procedures with different selectivity toward nucleic acids were examined, and a simplified calibration was designed allowing for evaluation of methylation percentage based on the ratio of chromatographic peak areas: cytidine/5-methylcytidine for RNA and 2'-deoxycytidine/5-methyl-2'-deoxycytidine for DNA. As a proof of concept, global DNA and total RNA methylation were determined in Lepidium sativum hydroponically grown in the presence of different Cd(II) or Se(IV) concentrations, expecting that plant exposure to abiotic stress might affect not only global DNA but also total RNA methylation. The results obtained showed the increase of DNA methylation in the treated plants up to concentration levels 2 mg L(-1) Cd and 1 mg L(-1) Se in the growth medium. For higher stressors' concentration, global DNA methylation tended to decrease. Most importantly, an inverse correlation was found between DNA and RNA methylation levels (r = -0.6788, p = 0.031), calling for further studies of this particular modification of nucleic acids in epigenetic context.

Nitrogen acquisition in Agave tequilana from degradation of endophytic bacteria

“…: 15 N 3 -dC 1.13 × 10 2 ± 13.93; 15 N 5 -dA 4.50 × 10 1 ± 9.05) than plants treated with the heat-killed Bacillus (a.u. : 15 N 3 -dC 3.40 × 10 1 ± 26.51; 15 N 5 -dA 1.10 × 10 1 ± 6.83)2829303132.…”

“…3 and Supplementary Fig. S9 to 11)29303132. Mass spectrometry analyses were performed with the following parameters: collision excitation potential, 10 V; collision activated dissociation gas flow, medium; pause time, 5 ms; dwell time, 300 ms; curtain gas, 12 psi; ion source, 5500 V; temperature, 650°C; gas 1 and gas 2, 40 psi; declustering potential, 31 V and entrance potential, 10 V.…”

The new insights into cadmium sensing

“…In the latter case, increased methylation was correlated with elevated expression of methylotrasferase, indicating the de novo methylation process (Yang et al, 2007; Greco et al, 2012). Hypermethylation was also noted in garden cress in response to lower cadmium concentrations; however, more intense cadmium stress caused a decrease in the levels of DNA methylation (Yanez Barrientos et al, 2013). In turn, in Gracilaria dura exposure to cadmium resulted in a decrease in methylated DNA (Kumar et al, 2012).…”

Methylation on RNA: A Potential Mechanism Related to Immune Priming within But Not across Generations

“…DNA methylation is highly variable in eukaryotes and in some insect species, such as the beetle Tribolium castaneum , in which no detectable nuclear DNA methylation has been found (Zemach et al, 2010) whereas in another beetle, Nicrophorus vespilloides , there is strong evidence (Cunningham et al, 2015). Another recently discovered mechanism is a covalent modification that takes place in different types of RNA (5mC); but the biological significance of this mechanism remains unknown (Mattick et al, 2009; Motorin et al, 2010; Yanez-Barrientos et al, 2013; Yan et al, 2015; Chen et al, 2016). Despite the traditional view that the function of RNAs is to act as messengers between DNA and protein synthesis, recent evidence suggests that RNAs are also involved in the regulation of genome organization and gene expression (Morris and Mattick, 2014; Chen et al, 2016).…”

“…This species exhibit immune priming across generations (Moret, 2006; Zanchi et al, 2012). We used a novel procedure in analytical chemistry based on hydrolysis of DNA/RNA to nucleosides followed by selective fluorescent labeling of cytosine moieties with 2-bromoacetophenone and reversed-phase liquid chromatography separation (López-Torres et al, 2011; Yanez-Barrientos et al, 2013). This procedure allows for high precision and accuracy in the analysis of sub-microgram amounts of nucleic acids and it is validated by other methods to measure methylated DNA or RNA (López-Torres et al, 2011; Yanez-Barrientos et al, 2013).…”