Determination of
Oxidation Products of 5‑Methylcytosine
in Plants by Chemical Derivatization Coupled with Liquid Chromatography/Tandem
Mass Spectrometry Analysis
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Abstract
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