Figure 1.

The 130-bp key region of the promoter of TsVP1 is a direct binding target of TsNAC1. A, Multiple amino acid sequence alignment of TsNAC1 and its ortholog in Arabidopsis (RD26). Identical residues are shaded in black, and five subdomains (A–E) are indicated on the sequences. B, Transcriptional activity assay of TsNAC1 in yeast strain YM4271. TsNAC1 was cloned and fused with the GAL4 AD. The promoter of TsVP1 was cloned into pLacZi vector. Streaked transformants are shown on Yeast extract Peptone Dextrose medium with 0.003% Adenine hemisulfate (YPDA) and SD/-uracil medium. The activities of β-galactosidase were examined by X-gal staining. C, Electrophoretic mobility shifts assay (EMSA). Probes, TsNAC1-GST, competitors (130-bp unlabeled fragments), and mutated competitors at 50× and 200× molar excess were present (+) or absent (−) in each reaction. The protein-DNA complexes are marked by black arrowheads.