Fig. 7.

Binding of LcHB2 to LcCEL2 and LcCEL8 promoters and activation of their activities. LcHB2 activates the expression of LcCEL2 (A) and LcCEL8 (B) in vivo as indicated by transient dual-luciferase reporter assays in tobacco leaves. The ratio of LUC/REN expression of the empty vector (pEAQ) plus promoter was used as a calibrator (set to 1). Data are means (±SE) from six replicates. Significant differences between means were determined using Student’s t-test: ** P<0.01. (C) SDS-PAGE gel stained with Coomassie Brilliant Blue demonstrating affinity purification of the recombinant LcHB2 protein. (D, E) Electrophoretic mobility shift assays (EMSAs) showing the association of LcHB2 with the LcCEL2 and LcCEL8 promoters in vitro. Sequences of both the wild-type and mutant probes are shown at the top. The core HD-binding cis-elements are indicated in red and the mutant elements are indicated in green. Shifted bands, suggesting the formation of DNA–protein complexes, are indicated by arrows. ‘–’ and ‘+’ represent an absence or presence, respectively. ‘+++’ indicates increasing amounts of unlabeled or mutated probes introduced for competition. A non-biotin-labeled probe was added as an unlabeled competitor.