Fig. 8

BrrICE1.1 bound to the BrrCBF3 promoter. a The potential binding site of BrrICE1.1 is the MYC element (CATTTG) in the BrrCBF3 promoter. b Yeast one-hybrid assays showed that the MYC element mediates BrrICE1.1 binding to the BrrCBF3 promoter and that the BrrCBF3 promoters were mutated (deleted MYC element) to abolish the MYC element alone. The experiments were repeated three times with the same results. c BrrICE1.1 activated the binding activity of BrrCBF3 in vivo; N. benthamiana leaves were transformed with the positive control (35S::LUC) and negative control (35S::BrrICE1.1, BrrCBF3pro::LUC), and the interaction was detected. Representative images of N. benthamiana leaves 72 h after infiltration are shown. d ChIP experiment using BrrICE1.1-6flag transgenic hair root. The structure of the BrrCBF3 gene promoter. The primer sequence regions used for ChIP assays are marked with a horizontal line to the left of the TSS. The control primer sequence (GD) is on the left side of the TSS. ChIP-qPCR showing binding of BrrICE1.1 to the BrrCBF3 promoters in vivo. WT and BrrCBF3-GD were used as negative controls. e The expression of BrrCBF3 in BrrICE1.1-OE and BrrICE1.1-RNAi transgenic hair roots. f BrrICE1.1 regulatory network under freezing stress in turnip. In d and e, the data are the mean of three replicates ± SD, and the asterisks indicate significant differences compared with the control (*P < 0.05, **P < 0.01, Student’s t test)