High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis.

Along with its essential role in the maintenance of genome integrity, DNA methylation takes part in regulation of genes which are important for plant development and stress response. In plants, DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM) involving two plant-specific RNA polymerases - PolIV and PolV. The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation - DNA methyltransferase (MET1, CMT3, and DRM2), the largest subunits of PoIIV and PolV (NRPD1 and NRPE1 respectively) and the DNA demethylase ROS1. We also examined the high-temperature effect on two protein-coding genes - At3g50770 and At5g43260 whose promoters contain transposon insertions and are affected by DNA-methylation, as well as on the AtSN1, a SINE-like retrotransposon. To assess the involvement of PolIV and PolV in heat stress response, the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1 and NRPE1. The results demonstrate coordinated up-regulation of the DRM2, NRPD1 and NRPE1 in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress. The ROS1 expression was confirmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation. The increased expression of At3g50770 in response to elevated temperature correlated with reduced promoter DNA methylation, while the stress response of At5g43260 did not show inverse correlation between promoter methylation and gene expression. Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.