Figure 7.

ICE1 is a transcriptional activator and its overexpression enhances the CBF regulon in the cold and improves freezing tolerance. (A) Schematic representation of the reporter and effector plasmids used in the transient expression assay. A GAL4-responsive reporter gene was used in this experiment. Nos, the terminator signal of the nopaline synthase gene; Ω, the translational enhancer of tobacco mosaic virus; GAL4 DB, the DNA-binding domain of the yeast transcription factor GAL4. (B) Relative luciferase activities after transfection with GAL4–LUC and 35S-GAL4–ICE1 or 35S-GAL4–ice1. To normalize values obtained after each transfection, a gene for luciferase from Renilla was used as an internal control. Luciferase activity is expressed in arbitrary units relative to the activity of Renilla luciferase [as described in Ohta et al. (2001)]. The values are averages of three bombardments, and error bars indicate standard deviations. (C) RNA blot analysis of ICE1 and cold-responsive gene expression in wild-type and ICE1 overexpressing transgenic (Super-ICE1) plants. Seedlings were either not treated (0 h) or treated with low temperature (0°C) for 3 or 6 h. Ethidium bromide stained rRNA bands are shown as loading control. (D) CBF3–LUC expression (indicated as luminescence intensity) in wild-type and ICE1 overexpressing transgenic (Super-ICE1) plants. (E) Improved survival of ICE1 overexpressing transgenic (Super-ICE1) plants after a freezing treatment.