Hydrogen sulfide regulation of the transcriptional profile of rice plants under drought stress
Hydrogen sulfide regulation of the transcriptional profile of rice plants under drought stress
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. Rice (Oryza sativa L., japonica, Jsendra cultivar, Spain) (Reig-Valiente et al., 2016) was used in this study. The rice growth and treatments were performed as previously (Zhang et al., 2024a). Briefly, 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 with 50 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. Total RNA was extracted from the leaves of the four different plant samples described above using the Qiagen RNeasy Plant Mini Kit. Four independent biological replicates of each sample were used for RNA-seq performed by Novogene Co. Ltd Total RNA was extracted from the leaves of the four different plant samples described above using the Qiagen RNeasy Plant Mini Kit. Four independent biological replicates of each sample were used for RNA-seq performed by Novogene Co. Ltd. Briefly, after previous analyses of RNA quantification, integrity and purity using Agilent 5400 analyzer.  Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.