Hydrogen sulfide regulation of the DNA methylation profile of rice plants under drought stress
Hydrogen sulfide regulation of the DNA methylation profile of rice plants under drought stress
Germinated seeds were sown in soil under a photoperiod of 12 h of white light (100 μmol m–2 s–1) at 28 C and 12 h of darkness at 28 C, and grown for 25 days under physiological water regime. Then, plants were divided into two batches, one water-irrigated and other treated with50 μM NaHS (as hydrogen sulfide donor compound) for an additional 10 d. After this period, each batch was subsequently divided into two new batches and subjected to water irrigation or drought for another 15 d. At the end of the whole treatment, four different plant samples were obtained as follows: control (C, water-irrigated plants during all period), sulfide-treated (S, sulfide-treated plants without any drought stress), drought (D, drought-treated plants with no-sulfide treatment), and sulfide-pretreated plus drought (S_D, sulfide-pretreated and after drought subjected plants). Leaves from different plant samples were collected, frozen in liquid nitrogen and stored at -80 C for further analysis. Germinated seeds were sown in soil under a photoperiod of 12 h of white light (100 μmol m–2 s–1) at 28 C and 12 h of darkness at 28 C, and grown for 25 days under physiological water regime. Then, plants were divided into two batches, one water-irrigated and other treated with50 μM NaHS (as hydrogen sulfide donor compound) for an additional 10 d. After this period, each batch was subsequently divided into two new batches and subjected to water irrigation or drought for another 15 d. Germinated seeds were sown in soil and grown for 25 days under physiological water regime. Then, plants were divided into two batches, one water-irrigated and other treated with50 μM NaHS (as hydrogen sulfide donor compound) for an additional 10 d. After this period, each batch was subsequently divided into two new batches and subjected to water irrigation or drought for another 15 d. At the end of the whole treatment, four different plant samples were obtained as follows: control (C, water-irrigated plants during all period), sulfide-treated (S, sulfide-treated plants without any drought stress), drought (D, drought-treated plants with no-sulfide treatment), and sulfide-pretreated plus drought (S_D, sulfide-pretreated and after drought subjected plants). Leaves from different plant samples were collected, frozen in liquid nitrogen and stored at -80 C for further analysis. DNA was extracted from leaves of two plant samples: control (C, water-irrigated plants during all period) and sulfide-treated (S, sulfide-treated plants without any drought stress) using the Qiagen DNeasy Plant Mini Kit. Four independent biological replicates of each sample were used  Fragmented DNA of 200-400 bp was bisulfite treated to generated single strand DNA in which unmethylated cytosine were converted into uracil and methylated cytosine unchanged. Double strand DNA was synthesized, subjected to PCR amplification, and the library was ready after size selection.