Figure 7.

TaBZR1 and TaHAG1 cooperate to activate TaSAMT1-B transcription. A, B) ChIP-qPCR validation of TaHAG1-binding sites in the TaSAMT1-B promoter. Enrichment of the P1, P2, and P3 fragments of the TaSAMT1-B promoter was determined in chromatin precipitated with anti-GFP antibodies in TaHAG1-GFP lines. C, D) Levels of H3K9ac (C) and H3K14ac (D) modification along the TaSAMT1-B promoter region under control and freezing conditions. Data are shown as means ± SD (n = 3). E) Dual luciferase reporter assays in wheat protoplasts showing the ability of TaHAG1 and TaBZR1 to transactivate TaSAMT1-B promoter activity before and after freezing treatment (4°C in dark for 2 h). Left, diagrams of the effector and reporter constructs. Right, ratio of firefly luciferase (LUC) to Renilla luciferase (REN) indicating the transactivation ability of TaBZR1 and/or TaHAG1. Data are shown as means ± SD (n = 3). F, G) Enrichment of H3K9ac (F) and H3K14ac (G) in the TaSAMT1-B promoter region in wild-type KN199 and TaBZR1-OE lines under control and freezing conditions. Data are shown as means ± SD (n = 3). H) LCI assays. The construct encoding nLUC-tagged TaBZR1 was co-infiltrated into N. benthamiana leaves with a construct encoding cLUC-tagged TaHAG1 before and after cold treatment. Data are shown as means ± SD (n = 7). The relative luminescence values in control were set to 1. The data were analyzed by one-way ANOVA followed by an LSD test. Different lowercase letters indicate a significant difference (P < 0.05). I) A proposed working model for the TaHAG1–TaBZR1 cooperation in activating TaSAMT1 transcription for freezing tolerance. TaBZR1 alone establishes a basic transregulation system for activating the TaSAMT1 expression through its binding to the E-box; the interaction of TaHAG1 with TaBZR1 is necessary for enhanced transregulation via increasing histone acetylation. Higher TaSAMT1 levels in turn result in accumulation of MeSA and enhanced freezing tolerance.