Figure 4.

Interaction of TRAB1 with ABREs and activation of the ABRC of Osem promoter consisting of ABRE and CE3. (A) Electrophoretic mobility-shift assays of TRAB1 synthesized in vitro. Nucleotide sequences of ABREs (underlined) and its mutant used for probes of electrophoretic mobility-shift assays are listed on the top. The synthetic double-strand DNA fragments carried 4-bp 5′ overhangs at each end for fill-in labeling. Each different probe without specific competitors (Left) or motif A probe with an unlabeled competitor as indicated (Right) was incubated with TRAB1 protein (+TRAB1) synthesized in a rabbit reticulocyte cell-free system. “−TRAB1” indicates the control binding reactions with the reticulocyte lysate after protein synthesis reaction without added RNA templates. “×” indicates fold-molar excess. DNA–protein complexes were separated from free probes by PAGE. Arrows indicate the position of the bands of DNA–protein complexes specific to TRAB1 protein. Other bands derive from the protein present in the reticulocyte lysate. (B) Activation of the 55-bp ABRC of Osem by TRAB1. Ten micrograms of the ABRC-TATA-GUS reporter plasmid was cotransfected with the same amount of TRAB1 expression (p35S-ShΔ-TRAB1) or control (p35S-ShΔ-stop) plasmids, as indicated, into rice cell protoplasts by electroporation, and protoplasts were cultured in the absence (−) or presence (+) of 5 × 10⁻⁵ M ABA. The effects of TRAB1 and ABA are indicated by fold activation of normalized GUS activities relative to that obtained with the control plasmid in the absence of ABA. Other details are as described for Fig. 3. (C) The effect of VP1 overexpression on the activation of the 55-bp ABRC by TRAB1. Cotransfection experiments were conducted as in B except for the following: the amount of p35S-ShΔ-TRAB1 or its control plasmid was reduced to 5 μg; 30 μg of VP1 expression plasmid (p35S-Sh-OSVP1) or its corresponding control plasmid was included; and protoplasts were cultured only in the absence of ABA.