Figure 4. Analysis of the AtACBP6 5′-flanking region.

(A) Schematic diagram of AtACBP6pro::GUS constructs in which the AtACBP6 5′-flanking sequence was transcriptionally fused to the GUS reporter gene. Putative cis-elements on the AtACBP6 5′-flanking region (pAT452) and its deletion derivatives (pAT590, pAT591 and pAT592) are represented by boxes. SEF1, SOYBEAN EMBRYO FACTOR1; SEF4, SOYBEAN EMBRYO FACTOR4; Dof, DNA-binding with one finger; MBS, MYB-binding site; CRT, C-repeat; GUS, β-glucuronidase. The putative Dof-boxes are numbered 1, 2, and 3, corresponding to −490/−486, −421/−417 and −353/−349, respectively. (B) GUS staining of 7-day-old seedlings and cotyledonary-staged embryos from T₃ generation of Arabidopsis transformants containing AtACBP6pro::GUS construct pAT452 and its deletion derivatives (pAT590, pAT591 and pAT592). Bar in the upper panel represents 5 mm. Bar in the bottom panel represents 100 μm. (C) EMSA analysis showing the binding of nuclear extracts to the Dof-boxes in the AtACBP6 5′-flanking region. Probe 1 (ML2044/2045), Probe 2 (ML2046/2047) and Probe 3 (ML2048/2049), containing the three Dof-boxes at −490/−486, −421/−417 and −353/−349, respectively, are numbered 1, 2 and 3. Competitor contains 20×non-biotin-labelled probe; −, no competitor in the reaction. Nuclear proteins were extracted from 7-day-old Arabidopsis seedlings; −, no nuclear proteins in the reaction; +, nuclear proteins added in the reaction. Arrowhead indicates the DNA–protein binding complex formed in the presence of nuclear proteins extracted from 7-day-old Arabidopsis seedlings. Arrow indicates free unbound biotin-labelled probe.