Determination of oxidation products of 5-methylcytosine in plants by chemical derivatization coupled with liquid chromatography/tandem mass spectrometry analysis.

Cytosine methylation (5-methylcytosine, 5-mC) in DNA is an important epigenetic mark that has regulatory roles in various biological processes. In plants, active DNA demethylation can be achieved through direct cleavage by DNA glycosylases, followed by replacement of 5-mC with cytosine by base excision repair (BER) machinery. Recent studies in mammals have demonstrated 5-mC can be sequentially oxidized to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC) by Ten-eleven translocation (TET) proteins. The consecutive oxidations of 5-mC constitute the active DNA demethylation pathway in mammals, which raised the possible presence of oxidation products of 5-mC (5-hmC, 5-foC, and 5-caC) in plant genomes. However, there is no definitive evidence supporting the presence of these modified bases in plant genomic DNA, especially for 5-foC and 5-caC. Here we developed a chemical derivatization strategy combined with liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method to determine 5-formyl-2'-deoxycytidine (5-fodC) and 5-carboxyl-2'-deoxycytidine (5-cadC). Derivatization of 5-fodC and 5-cadC by Girard's reagents (GirD, GirT, and GirP) significantly increased the detection sensitivities of 5-fodC and 5-cadC by 52-260-fold. Using this method, we demonstrated the widespread existence of 5-fodC and 5-cadC in genomic DNA of various plant tissues, indicating that active DNA demethylation in plants may go through an alternative pathway similar to mammals besides the pathway of direct DNA glycosylases cleavage combined with BER. Moreover, we found that environmental stresses of drought and salinity can change the contents of 5-fodC and 5-cadC in plant genomes, suggesting the functional roles of 5-fodC and 5-cadC in response to environmental stresses.