Fig. 2.

Transactivation assays and EMSAs revealed that the NGA family proteins activated and directly bound to the NCED3 promoter in vitro. (A) Schematic diagram of the reporter and effector constructs. (B and C) Transactivation assays of the NGA family proteins using the various NCED3 promoters as reporters. The error bars indicate the SD from three replicate samples. Asterisks indicate statistically significant differences of reporter activities from the vector control. *P < 0.05 (Bonferroni-corrected Student’s t test). The 35S:ELUC plasmid was also cotransfected in each experiment as an internal control. (B) Reporters of 3, 2.4, and 1 kbp. (C) Reporters of 198-, 161-bp, and 3-kbp mNBE. (D and E) Schematic diagrams of the probes of the NCED3 promoters and EMSA using the recombinant NGA2 (D) and NGA1 (E) protein. The migration positions of the protein–DNA complexes are represented by arrows. Mutated nucleotides in each probe are underlined, and an orange box indicates the position of the NBE. Incubation with 100- or 1,000-fold competitors was performed in the presence of the recombinant NGA1 proteins to confirm specific binding to the probes.