Hydrogen sulfide mediates DNA methylation to enhance osmotic stress tolerance in Setaria italica L.

DNA methylation is an important form of epigenetic modification. It has a vital role in regulating plant growth and development, and also participates in plant response to various stresses. In recent years, hydrogen sulfide (H2S) has been shown to have similar functions as DNA methylation, but crosstalk between DNA methylation and H2S in the acquisition of drought resistance is unclear. In this study, foxtail millet (Setaria italica L.), a drought-resistant model crop, was selected as the experimental material to explore the subtle relationship between H2S and DNA methylation. The quantitative real-time (qRT)-PCR, bisulfite sequencing PCR (BSP), DNA methyltransferase (DNMT) activity detection and other techniques were used to analyze the differences of millet seedlings under osmotic stress, before and after H2S treatment. Osmotic stress induced the transcriptional expression and activity of key enzymes in H2S biosynthesis which regulated the accumulation of endogenous H2S. Physiological concentration of H2S (50 μM) can effectively alleviate the decrease of total DNMT activity and transcription level caused by osmotic stress, while the effective inhibitor of H2S biosynthesis, hydroxylamine (HA), can aggravate this change. Furthermore, transcription factors (TFs) responsive to both ‘osmotic stress’ and ‘H2S signal’ were screened. Six of them were selected to conduct further BSP analysis on seven CpG islands in their promoter regions. The results showed that, with H2S treatment, two CpG islands were hypermethylated, two were not, and three exhibited insensitivity to H2S. H2S signals may improve osmotic stress tolerance of foxtail millet by mediating DNA methylation.