Fig. 5: Overexpression of AeHSFA2-2 enhances AeEGY3 expression, and H2O2-induced polymerization of AeHSFA2-2 promotes its transcription activity.
Shoot phenotypes (a) and leaf survival rates (b) of MH-Z11 and AeHSFA2-2-FLAG overexpressing lines (OE-2, -3, and -5) after an 8-h-long exposure to 40 °C and followed by a 2 d recovery period. c qRT-PCR analysis of AeHSFA2-2 and AeEGY3 expression levels in MH-Z11 and AeHSFA2-2-FLAG-OE lines exposed to 40 °C for 0 and 1 h. d Western blot analysis of total proteins extracted from AeHSFA2-2-FLAG-OE-1 plants treated with or without 50 μM H2O2 for 6 h using anti-FLAG and anti-Actin antibodies. “M” stands for protein ladder. e Luciferase complementation imaging assays show the interaction between nLUC-AeHSFA2−2 and cLUC-AeHSFA2-2 in tobacco leaves. Luciferase fluorescence was observed after the tobacco leaves were injected and treated with water (control) or 10 μM Methyl Viologen (MV), a herbicide inducing ROS in the chloroplast, or 10 mM N,N’-Dimethylthiourea (DMTU), a H2O2 scavenger, for 3 h. f Dual-luciferase reporter assays in tobacco leaves transiently co-expressing 35S::AeHSFA2-2-FLAG and proEGY3MH::LUC. Luciferase fluorescence was observed after the tobacco leaves were injected and treated with 10 mM DMTU or 10 mM DTT for 3 h. g DLRA in tobacco leaves transiently co-expressing proAeHSP70−2::LUC with empty vector (EV) or 35S::AeHSFA2-2-FLAG. The quantitative measurements of the LUC/REN ratios were performed. h EMSA of AeHSFA2-2 binding to proAeHSP70-2. i qRT-PCR analysis of AeHSF70−2 expression levels in MH-Z11 and AeHSFA2-2-FLAG-OE lines exposed to 40 °C for 0 and 1 h. In (b, c, f, g, h), data represent mean ± SD (n = 3 biological replicates). Asterisks indicate significant differences by two-tailed Student’s t-test (**p < 0.01, ***p < 0.001). Source data are provided as a Source Data file.
