Arabidopsis growing conditions and submergence treatment
Briefly, cDNA was prepared from 4-week rosette leaves of Arabidopsis and was diluted to 50 times. The diluted cDNA was then used as a template to amplify the WRKY33, which was inserted into a vector tagged by FLAG tag, under the control of the 35S promoter. The construct was transformed into Agrobacterium strain GV3101 [28], which was used to transform Arabidopsis using the floral dip method and identified by hygromycin screening followed by qRT-PCR analysis of their expression levels. All materials were grown at 22°C in a 16-hour light/8-hour dark cycle. Seeds were germinated on 1/2 MS medium (pH = 5.85) for seven days and then transplanted into soil.
For submergence treatments, 4-week-old plants were submerged 10 cm below the surface of the water in darkness for 65 hours. All submergence treatments started at 9:00 a.m. Twelve Col and WRKY33OE plants were used for submergence treatment every time. The total experiments were repeated three times.
For ChIP-sequencing, 4-week-old 35S:FLAG-WRKY33 transgenic plants were submerged 10 cm below the surface of the water in darkness for 24 hours. Then rosette leaves were collected for ChIP experiments. All submergence treatments started at 9:00 a.m.
Malondialdehyde Measurements
The Malondialdehyde (MDA) was measured according to a previous study [29]. 4-week-old rosette leaves of 10 plants treated by dark submergence were weighed and pulverized in 5% trichloroacetic acid buffer, and then mix the supernatant with 6.7% thiobarbituric acid and 5% trichloroacetic acid buffer. The materials were further incubated at 100°C for 0.5 hr, and then cooled to the room temperature. The absorbance was measured at 532, 450, and 600 nm with a spectrophotometer plate reader.
ChIP and ChIP-sequencing
Samples of 14-day-old seedlings of WRKY33OE1 and WRKY33OE2 plants were fixed using 1% formaldehyde and prepared for chromatin immunoprecipitation assays, as previously described [30]. The DNA-protein complexes were extracted from rosette leaves (2 g pooled leaf materials) of 4-week-old 35S:FLAG-WRKY33 OE1 and OE2 transgenic plants, and pulled down using anti:FLAG antibody (Sigma-Aldrich F1084) and protein A Agarose beads following the ChIP protocol [31]. The immunoprecipitated DNA fragments were dissolved in 40 μl ddH2O and then sent to the BGI (Shenzhen, China) company for the following experiment. 10% of the total DNA-protein complexes before the immunoprecipitation were used as the input DNA.
ChIP-Seq service was performed by BGI company (Shenzhen, China). The DNA is combined with End Repair Mix and incubated at 20°C for 30 min. We further purified the end-repaired DNA with QIAquick PCR Purification Kit (Qiagen), and added A-Tailing Mix and incubated at 37°C for 30 min. We combined the purified Adenylate 3 'Ends DNA, Adapter and Ligation Mix and incubated the ligation reaction at 20°C for 15 min. We purified the Adapter-ligated DNA with the QIAquick PCR Purification Kit. We conducted several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix to enrich the Adapter-ligated DNA fragments. Then the PCR products are selected (about 100–300 bp, including adaptor sequence) by running a 2% agarose gel to recover the target fragments. We purified the gel with QIAquick Gel Extraction kit (QIAGEN). The final library was quantitated in two ways: determining the average molecule length and sample integrity and purity using the Agilent 2100 bioanalyzer instrument (Agilent DNA 1000 Reagents) and quantifying the library by real-time quantitative PCR (QPCR). The double stranded PCR products were heat-denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) was formatted as the final library. Library was qualified by Qubit ssDNA kit. The sequencing was performed with the BGISEQ-500 sequencing system, featured by combinatorial probe-anchor synthesis (cPAS) and DNA Nanoballs (DNB) technology for superior data quality (BGI-Shenzhen, China).
The raw sequencing image data were examined by the Illumina analysis pipeline. ChIP-Seq reads were aligned to the Arabidopsis reference genome (TAIR10) by Bowtie [32] with at most 2 mismatches. The input group was used as a control. The results were visualized with IGV software. Reads that appeared more than twice at the same position on the same strand were discarded to remove PCR duplication. MACS2 (Model-based Analysis of ChIP-Seq) [33] was used to identify peaks using a q-value cutoff of 0.05.
Motif analysis
To identify possible binding motif of the WRKY33 transcription factor, the ChIP peak sequences were subjected to MEME (Multiple EM for Motif Elicitation)-ChIP [19]. The MEME-ChIP program uses two ab initio motif discovery algorithms: MEME [20], and DREME (Discriminative Regular Expression Motif Elicitation) [34], which uses regular expressions to search for short eukaryotic TF motifs that are missed by MEME.
Gene function of WRKY33 TF target genes
In order to determine the putative functions of the target gene WRKY33, all identifed genes with ChIP-Seq peaks in the upstream promoter region or the potential regulatory region downstream were subjected to annotation of the categories of ontological genes (GO) [21]. The default Fisher’s Exact Test and Benjamini-Yekutieli multiple test correction methods [35] were used to generate p-values for statistical significance and corresponding False Discovery Rate (FDR) values.
RNA extraction and quantification
Total RNA was isolated using the Biospin Plant Total RNA Extraction kit according to the user manual (Bioer Technology; Hangzhou, China), from the pooled three-week old rosette leaves of Col and 35S:FLAG-WRKY33 plants, and 1–2 μg total RNA was used for reverse transcription, using the PrimeScript RT reagent kit (Takara Cat# RR047A). A QuantiNova SYBR Green PCR Kit was used for qPCR reactions with qPCR-specific primers. The expression levels of putative target genes were compared with ArabidopsisACTIN genes.