Figure 7.

PGAZAT is regulated by AtDOF4.7. A, Abscission-related gene expression in AtDOF4.7 overexpression lines. Real-time RT-PCR analysis was performed to quantify the transcript abundance of abscission-related genes in wild-type (WT) and AtDOF4.7 overexpression (S107 and S109) plants. Relative expression levels were calculated and normalized with respect to the corresponding gene transcript levels in the wild type. ACTIN was used as the internal control gene. The relative transcript levels were averaged over three biological replicates and are shown with the sd (error bars). B, Regulation of AtDOF4.7 on the PGAZAT promoter. The promoter activities are shown as relative GUS activity in transient cotransfection assays with the 35S::GUS control as a reference. C, The AtDOF4.7 protein binds to cis-DNA elements in the promoter region of PGAZAT. The underlined sequences indicate the core sequence of the DOF-binding elements. The reporter pHIS2 vector carrying the corresponding fragment and the effector pAD-AtDOF4.7 vector were cotransfected into yeast Y187 cells. Growth of the transfected yeast cells on 3-AT medium indicates that the AtDOF4.7 protein can bind to the PGAZAT promoter in a sequence-specific manner. D, Gel-shift analysis of the AtDOF4.7 protein. Electrophoretic mobility shift assays of the recombinant AtDOF4.7 protein fused to GST and the PGAZAT promoter fragment including the AAAG motif are shown. E1, A copy of the PGAZAT promoter fragment; E2, single-base mutation of the AAAG motif in E1; E3, four-base mutation of the AAAG motif in E1.