Transcriptional activation and DNA-binding activities of BdCBF1. A. Nuclear localization of BdCBF1. The BdCBF1-GFP fusion, in which the GFP-coding sequence was fused in-frame to the 3' end of BdCBF1 cDNA, was expressed transiently in Brachypodium protoplasts and visualized by fluorescence microscopy and differential interference contrast microscopy. Auto, autofluorescence. Scale bar = 10 μm. B and C. Transcriptional activation activity assays in Brachypodium protoplasts. The reporter and effector vectors used were diagrammed (B). UAS, upstream sequence; Ω, translational enhancer; DB, DNA binding. The GAL4 transient expression assays were carried out using Brachypodium protoplasts (C). Vector control, transformation with the effector vector without gene inserts. ARF5M and ARF1M, transformations with the effector vectors containing ARF5M gene (activator control) and ARF1M gene (repressor control), respectively . Five measurements were averaged and statistically treated using the Student t-test (*P < 0.01). Bars indicate standard error of the mean. D. EMSA assays. Recombinant BdCBF1-His fusion protein and radiolabeled DNA were used. Both wild-type and mutated CRT sequences (BS and mBS, respectively) were included in the assays. Excess amounts of unlabeled BS (x100) DNA fragments were also included in the assays to verify specific binding of BdCBF1 to the CRT sequences. Arrowhead indicates protein-DNA complexes. BS, CRT sequence; mBS, mutated BS.