Figure 6.

TaBZR1 physically interacts with histone acetyltransferase TaHAG1. A) Luciferase complementation imaging (LCI) assays. The construct encoding nLUC-tagged TaHAG1 was co-infiltrated into the leaves of N. benthamiana plants along with a construct encoding cLUC-targeted TaBZR1. B) MBP pull-down assay showing the interaction of recombinant TaHAG1-MBP with TaBZR1-GST. TaBZR1-GST was detected using anti-GST antibody. TaBZR1-GST was incubated with immobilized MBP or TaHAG1-MBP. Immunoprecipitated fractions were probed by immunoblotting with an anti-MBP and anti-GST antibody. Protein size (kD) is provided to the right of the gel. C) Co-immunoprecipitation (Co-IP) showing the physical interaction of TaBZR1 and TaHAG1. Protein extracts from the leaves of N. benthamiana plants (Input) and after IP with GFP-trap beads were probed by immunoblotting using an anti-MYC antibody. D) Bimolecular fluorescence complementation (BiFC) assay showing that TaBZR1 interacts with TaHAG1 in nuclei. Constructs encoding TaBZR1 and TaHAG1 fused to the N terminus or the C terminus of yellow fluorescent protein (YFP) were co-infiltrated into the leaves of N. benthamiana plants. Scale bars, 50 μm. E) Ten-day-old wild-type Fielder and TaHAG1-OE (ox-1 and ox-2) lines were exposed to −10°C for 1.5 h, followed by recovery for 24 h. Scale bars, 5 cm. F) Survival rates of the wild-type and TaHAG1-OE lines after freezing treatment. Data are shown as means ± SD derived from measurements of at least 15 plants per genotype in three independent experiments. G) Electrolyte leakage of the wild-type and TaHAG1-OE lines in control and freezing conditions. Data are shown as means ± SD (n = 6). H) Relative TaSAMT1 transcript levels in wild-type Fielder and TaHAG1-OE plants. Data were normalized to TaACTIN and are shown as means ± SD (n = 3). The data in (F), (G), and (H) were analyzed by one-way ANOVA followed by an LSD test. Different lowercase letters indicate a significant difference (P < 0.05).