Fig. 4: AsDOF25 positively regulates AsHSFA2c via transcription activation.
a A circular gene network diagram was displayed, with AsHSFA2c as the core, surrounded by genes associated with AsDOF family members, and gene connections represented connectivity. b EMSA assay showed that AsDOF25 protein bound the promoter of AsHSFA2c. GST-AsDOF25 was incubated with 5′-biotin-labeled DNA probes with AsHSFA2c promoter sequence (Biotin-AsHSFA2c) or mutated AsHSFA2c promoter sequence (Biotin-mAsHSFA2c). c Luciferase assay showed that AsDOF25 activated the AsHSFA2c promoter. N.benthamiana plants were co-inoculated with p35s::AsDOF25-D312/p35s::AsDOF25 and pAsHSFA2c::LUC. The p35s::AsDOF25-D312 plasmid contains only the transcriptional binding domain of AsDOF25 and lacks its transcriptional activation domain. The sample was collected at 48 hpi. A cooled CCD imaging apparatus (Roper Scientific) was used to capture luciferase images. Scale bar, 0.5 cm. qRT-PCR detection of LUC expression level. N.benthamiana plants were co-inoculated with p35s::AsDOF25-D312 and pAsHSFA2c::LUC as a negative control. d qRT-PCR detection of AsHSFA2c expression level in AsDOF25 knock-down mutants. Two-leaves-stage plant samples were collected. e, f The malfunction of AsDOF25 promoted the growth of plants. Whole-plant images of 2-week-old plants (e). Scale bars, 5 cm. Statistical analyses of plant height are presented in panel (f), n = 10. g, h Drought tolerance assessment of AsDOF25 knockdown lines. Photographs were taken under well-watered conditions and after a 2-day period of recovery with full irrigation post-drought treatment. Whole-plant images of 5-week-old plants (g). Scale bars, 5 cm. Values in (h) were means ± SD from three independent experiments. i Water loss rate statistics of AsDOF25 knock-down strains. The error bar represents the SD of three biological replicates. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, p ≥ 0.05.
