Summary: Drought stress can cause huge crop production losses. Drought resistance consists of complex traits, and is regulated by arrays of unclear networks at the molecular level. A stress-responsive NAC transcription factor gene SNAC1 has been reported for its function in the positive regulation of drought resistance in rice, and several downstream SNAC1 targets have been identified. However, a complete regulatory network mediated by SNAC1 in drought response remains unknown. In this study, we performed Chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq of SNAC1-overexpression transgenic rice (SNAC1-OE) lines and wild-type under normal and moderate drought stress conditions, to identify all SNAC1 target genes at a genome-wide scale by RNA-Seq analyses. We detected 980 differentially expressed genes (DEGs) in the SNAC1-OE lines compared to the wild-type control under drought stress conditions. By ChIP-Seq analyses, we identified 4,339 SNAC1-binding genes under drought stress conditions (SNAC1BGDs). By combining the DEGs and SNAC1BGDs, we identified 93 SNAC1-targeted genes involved in drought responses (SNAC1TGDs). Most SNAC1TGDs are involved in transcriptional regulation, response to water loss, and other processes related to stress responses. Moreover, the major motifs in the SNAC1BGDs promoters include a NAC recognition sequence (NACRS) and an ABA responsive element (ABRE). SNAC1-OE lines are more sensitive to ABA than wild-type. SNAC1 can bind to the OsbZIP23 promoter, an important ABA signaling regulator, and positively regulate the expression of several ABA signaling genes.
Overall Design: 4-leaf-stage SNAC1-overexpressing (SNAC1-OE) and ZH11 wild type (ZH11-WT) seedlings were harvested for RNA-seq and ChIP-seq, in triplicate, using Illumina Hiseq X Ten.
The transgenic and ZH11-WT seeds were sprouted on half-strength MS medium in the dark for 2-3 days at 28°C, and then 4-5 days in the greenhouse. The seedlings were then moved to pots with sand/paddy (1:3) soil under natural conditions at Wuhan, China.
Treatment Protocol:
After germination, the 1-week-old seedlings that were of uniform growth status were moved to pots filled with sandy soil. The transgenic and WT plants were grown in a half-and-half manner (10 plants each) in the pots. At the 4-leaf stage, the seedlings were subjected to drought stress treatment by stopping water for 10-15 days until the leaves were curled and turned yellow.
Extract Protocol:
The total RNAs were extracted from the leaves of SNAC1-OE lines and ZH11-WT plants using TRIzol reagent (InvitrogenTM) according to the manufacturer’s instructions. The ChIP assay was performed according to Chris Bowler (Bowler et al., 2004) with some modifications. Briefly, 3g shoot tissues from 4-leaf-stage seedlings were cross-linked in 1% formaldehyde by vacuum for 30 minutes. The chromatin was extracted on ice as described by Bowler method and sheared to 200-500 bp fragments by sonication. Subsequently, the DNA fragments were immunoprecipitated by custom-made anti-SNAC1 polyclonal antibody generated using full-length SNAC1 protein. The combined DNAs were eluted, purified, and dissolved in ddH2O.
Library Construction Protocol:
RNA-seq and ChIP-seq libraries were prepared for sequencing using standard Illumina protocols.
