Characterization of Arabidopsis lines overexpressing bZIP53 and its mutant forms. (A) Representative images of each transgenic line expressing coding sequences of bZIP53-GFP (bZIP53ox), bZIP53Ser15,19Ala-GFP (bZIP53Aox), or bZIP53Ser15,19Asp-GFP (bZIP53Dox). (B) Box-plot of leaf rosette diameter in the transgenic plants overexpressing bZIP53 and its mutant forms. Different letters indicate significantly different diameters (ANOVA, P<0.05); 12 plants from three independent lines for each construct were used. The seeds from the transgenic lines and corresponding wild-type belonged to the same seed batch. (C) Western blot analysis of the transgenic lines using anti-GFP antibody (upper panel). Ponceau staining was used to confirm the equal transfer of proteins onto the membrane (lower panel). The correct size of the bZIP53-GFP protein (48 kDa) was confirmed with peqGOLD Prestained Protein-Marker (PEQLAB, Lot No.1422). (D) RT-qPCR analysis of transgenic lines. For each gene, the expression level in the wild-type was set to 1, and different lines are presented as the mean of relative fold-changes compared to the control. Data are means (±SD), n=3. A, substitution of Ser15 and Ser19 to Ala; D, substitution of Ser15 and Ser19 to Asp.