D homogenized in 4 mL ice-cold sodium phosphate buffer (50 mM, pH 7.eight) containing 1 polyvinylpyrrolidone. The homogenate was centrifuged at 12 000 g for 15 min at four oC. The supernatants have been employed because the crude extract for measurement of superoxide dismutase (SOD) (EC 1.15.1.1) and peroxidase (POD) (EC1.11.1.7) activities as well as the malondialdehyde (MDA) content material assay. The SOD activity was assayed by its capability to inhibit the photochemical reduction of nitro blue tetrazolium (NBT) (Giannopolitis and Ries, 1977). The POD activity was measured based on guaiacol oxidation (Possibility and Maehly, 1955). The lipid peroxidation level was assessed by measuring the thiobarbituric acid (TBA)reactive substances having a lipid peroxidation MDA assay kit (S0131, Beyotime, China). In situ histochemical localization of H2O2 and O2- In situ accumulation of H2O2 and O2- had been detected by histochemical staining with diaminobenzidine (DAB) and nitro blue tetrazolium (NBT), respectively. For localization of H2O2, leaves had been sampled and instantly vacuum-infiltrated in DAB option using a DAB color improvement kit (P0202, Beyotime, China). For O2- detection, a further set of leaves were vacuum-infiltrated inside a 1 mg mL-1 NBT resolution in 10 mM phosphate buffer (pH 7.eight). For each DAB and NBT staining, the infiltrated leaves were incubated at space temperature for 8 h, and then transferred to 70 ethanol to deplete chlorophyll and visualize the brown and blue spots for H2O2 and O2-, respectively. Microarray evaluation Leaves from WT and 3 transgenic lines have been collected ahead of and following five d of drought stress. An equal volume of leaves from three independent transgenic lines that were harvested around the same day was pooled as OE lines for RNA isolation. 4 samples had been collected at 10.00 h, which integrated WT 0 d, OE 0 d, WT five d and OE 5 d, and each and every sample was represented by two replicates. Total RNA was extracted utilizing TRIzol reagent (Invitrogen, USA). Chip hybridization and microarray analysis have been performed applying Affymetrix Microarray Services (CapitalBio Co., Beijing, China) (Shi et al., 2014). For array hybridization, 200 ng of total RNA was employed for first-strand and second-strand cDNA synthesis. The cRNA was labelled with a biotinylated ribonucleotide analogue and was fragmented with fragmentation buffer making use of the MessageAmpTM Premier RNA Amplification Kit (Ambion, #1792, USA). Soon after purification, 12.five g of labelled and fragmented cRNA probes had been hybridized to the Arabidopsis arrays using the Hybridization, Wash and Stain Kit (Affymetrix, #900720, USA). The arrays were scanned utilizing a GeneChipR Scanner 3000 (Affymetrix, #3000, USA) (Shi et al., 2014). The identification of differentially expressed genes was according to the fold transform two or 0.5 with P-values 0.05. Pathway enrichment evaluation was performed applying the Classification SuperViewer Tool (http:bar. Trilinolein web utoronto.cantoolscgi-binntools_classification_superviewer.cgi) (Provart and Zhu, 2003). Microarray information have been submitted to the Gene Expression Omnibus (GEO) database (accession number: GSE72050). Yeast one-hybrid assay The NACRS motif (acacgcatgt) and the mutant motif (acacAcaCAC) had been Chlorpyrifos-oxon site synthesized in 4 repeats. Each sequences had been cloned into the bait vector pAbAi according to the process described in the MatchmakerTM Gold Yeast One-Hybrid Library Screening System user manual (Clontech, CA, USA). The total CDS of VaNAC26 was cloned into the prey vector pGADT7 AD. Then, the yeast strains that contained t.
