Role of hydrogen peroxide pretreatment in heat-induced alteration of DNA methylation in cucumber leaves

To investigate whether exogenous hydrogen peroxide (H2O2) affects the heat-induced alteration of DNA methylation in H2O2-pretreated heat-stressed plants, cucumber seedlings were exposed to heat (42/38 °C, day/night) for 3 d after pretreatment with 1.5 mM H2O2 for 12 h, and then DNA methylation patterns were detected through a method of methylation sensitive amplified polymorphism (MSAP). Exogenous H2O2 mitigated growth suppression caused by heat. Meanwhile, using 120 pairs of primers, small percentages (16.2–16.4%) of DNA sequences were methylated in leaves when cucumbers were subjected to heat or pretreated with H2O2, and 20 MSAP loci were selected for differentially amplified fragments. Among which sequences of three MSAP fragments had identity with coding sequences of gene Csa026131, Csa012834 and Csa015520 in cucumbers, respectively. Compared to control, heat treatment caused methylation in 6 MSAP loci and led to demethylation in 4 loci, while the combined effect of H2O2 pretreatment and heat aroused methylation in 8 loci and induced demethylation in 6 loci. In comparison to heat treatment alone, the combination of H2O2 pretreatment and heat aroused 4 MSAP loci to be demethylated and caused 4 MSAP loci to be methylated, and it as well enhanced the transcript levels of gene Csa026131 and Csa012834 and decreased the expression of gene Csa015520. Results of bisulfite sequencing PCR showed that the cytosine methylation levels in gene Csa026131 and Csa015520 were different between heat and H2O2 + heat treatments. We propose H2O2 changes the heat-induced alteration of DNA methylation when it is used to mitigate heat stress of cucumbers.

► Heat and exogenous H2O2 both trigger DNA methylation variations.
► Compared to heat treatment, H2O2 + heat changes DNA methylation in some MSAP loci.
► Expression of three genes is different between heat and H2O2 + heat treatments.
► Cytosine methylation in two genes is also different between the two treatments.
► So H2O2 pretreatment changes heat-induced alteration of DNA methylation in plants.