Detection of oxidation products of 5-methyl-2'-deoxycytidine in Arabidopsis DNA

PLoS One. 2013 Dec 31;8(12):e84620. doi: 10.1371/journal.pone.0084620. eCollection 2013.


Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2'-deoxycytidine (5-FodC), 5-carboxyl-2'-deoxycytidine (5-CadC), 5-hydroxymethyl-2'-deoxyuridine (5-HmdU), and the (5'S) diastereomer of 8,5'-cyclo-2'-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 10(6) nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Arabidopsis / chemistry*
  • Arabidopsis / physiology
  • Chromatography, High Pressure Liquid
  • DNA, Plant / chemistry*
  • Deoxycytidine / analogs & derivatives*
  • Deoxycytidine / chemistry
  • Deoxycytidine / genetics
  • Deoxycytidine / metabolism
  • Deoxycytidine Monophosphate / analogs & derivatives
  • Deoxycytidine Monophosphate / chemistry
  • Deoxycytidine Monophosphate / metabolism
  • Epigenesis, Genetic / genetics
  • Epigenesis, Genetic / physiology*
  • Fluoresceins / chemistry
  • Fluoresceins / metabolism
  • Isotope Labeling
  • Molecular Structure
  • Nucleosides / metabolism*
  • Oxidation-Reduction
  • Tandem Mass Spectrometry
  • Thymidine / analogs & derivatives
  • Thymidine / chemistry
  • Thymidine / metabolism


  • 5-carboxyl-2'-deoxycytidine
  • 5-formyl-2'-deoxycytidine
  • DNA, Plant
  • Fluoresceins
  • Nucleosides
  • Deoxycytidine
  • Deoxycytidine Monophosphate
  • 5-hydroxymethyldeoxycytidylic acid
  • 5-hydroxymethyl-2'-deoxyuridine
  • 5-carboxyfluorescein diacetate
  • 5-methyldeoxycytidine
  • Thymidine