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. 2013 Mar 19;8(3):e58713. doi: 10.1371/journal.pone.0058713

Figure 6. Key residues of AP2 domains that underlie selectivity of cis-elements binding, and regulation of the TdCor410b promoter activity.

Figure 6

(A) Multiple sequence alignment of selected AP2 domains using PROMALS3D (44). Representative sequences are coloured according to predicted secondary structures (red: α-helix, blue: β-strand). The black box indicates the boundaries of the AP2 domains. The positions of highly conserved Pro residues in the ERF sequences and of variable non-proline residues in the DREB sequences are highlighted in yellow and green, respectively. The positions of two Pro residues conserved in selected cereal ERF sequences are highlighted in cyan, while the positions of the corresponding Arg residues are highlighted in grey. Consensus of secondary structure elements indicates the position of β-sheets (black arrows) and of an α-helix (purple). The degree of conservation of residues is shown above the sequences by black and brown numbers with a conservation index of 5 and higher. (B) Influence of conserved proline residue substitutions in the AP2 domain of TaERF4a on recognition of the GCC-box. TaDREB3 was used as a negative control and TaERF5a as a positive control of interaction with the GCC-box. Mutation of Pro26 to Arg26 (underlined) has no influence on interaction of the TaERF4a variant with the cis-element. Mutation of Pro42 to Arg42 (underlined and boxed in blue) lead to restoration of interaction and consequent growth of yeast on the selective (-Leu, -His, + 5 mM 3-AT) medium. (C) Regulation of the activity of the TdCor410b promoter and of the artificial promoter with substitution of the CRT element for a tandem of three GCC-boxes by representatives of each isolated ERF subfamily, and variants of TaERF4a with mutations in the AP2 domain. TFs were tested in a transient expression assay in a wheat cell culture. Either pTdCor410b-GUS or 3×GCCbox-GUS constructs were co-bombarded with pUbi-GFP (GFP; negative control), pUbi-TaERF4a (TaERF4a), pUbi-TaERF4a mutated at Pro26 (TaERF4a m1), pUbi-TaERF4a mutated at Pro42 (TaERF4a m2), pUbi-TaERF4a mutated at Pro26 and Pro42 (TaERF4a m1+2), pUbi-TaERF6 (TaERF6), or pUbi-TaERF5a (TaERF5a).