Fig. 3
HSFA2 activates SGIP1 transgenerationally through binding to its promoter. a, b HSFA2 remained upregulated in the progeny of heat-stressed plants at both transcript (a) and protein (b) levels. c HSFA2 binds to the SGIP1 promoter HSEs in vitro and in vivo. Left: EMSA shows the direct binding of His-HSFA2 to the HSE-P4 of SGIP1. The arrow indicates the shifted bands. Excess unlabeled probe (500 ng and 1000 ng) outcompeted the labeled probe (right lanes). Sequence of the probe is shown and the motif is highlighted in red. A mutant HSE and a downstream sequence SGIP1-NC (negative control) were used as negative controls. Right: ChIP-qPCR validation of HSFA2 occupancy at the HSE regions of the SGIP1 promoter. Data are shown as relative fold enrichments over the background (Col). The SGIP1-NC locus was used as the negative-control locus. The locations of 4 HSEs are indicated by triangles above the gene model. The regions validated by ChIP-qPCR were marked by a bar below the gene model. d SGIP1 transcript levels in Col and hsfa2 plants grown at 22 °C and 30 °C. Gene expression was shown as mean ± SD (n = 3). ACTIN2 was analyzed as an internal control (a, d). Lowercase letters indicate significant differences, as determined by the post hoc Tukey’s HSD test (a, c, d). e The abundance of SGIP1 protein increased in three transgenic lines overexpressing HSFA2-MYC, as immunodetected with anti-SGIP1 and anti-MYC antibodies. ACTIN served as a loading control (b, e) and the signals were quantified (b)
