Fig. 3
Binding of AtNAP to the SAG202 promoter truncations and mutants in yeast and in planta. (a) Binding of AtNAP to the SAG202 promoter truncations revealed by yeast one-hybrid assay. The LacZ reporter gene driven by various SAG202 promoter truncations was used to test the binding ability of the GAD-AtNAP fusion protein. Red dash lines indicate promoter sequence that is highly conserved to the 9-bp (red letters) AtNAP binding site of the SAG113 promoter (Zhang and Gan, 2012). The immediate upstream bp of the translation start site was numbered as − 1. The CBP60g promoter (1727 bp in length) was also tested. (b) Failure of AtNAP binding to the SAG202 promoter that contains either transversion mutation or deletion of the motif sequence in yeast cells. (c) GUS staining of senescent leaves in transgenic Arabidopsis plants (WT or annap mutant background) harboring PSAG202W-GUS (W for WT), PSAG202T-GUS (T for transversion; the promoter contains the transversion mutation shown in (b)) or PSAG202D-GUS (D for deletion; the promoter contains the deletion mutation shown in (b)). (d) GUS enzymatic activities (expressed as nmol methylumbelliferone produced min − 1 mg − 1 protein) of senescent leaves of transgenic plants shown in (c). Data are mean values ± SD of five samples. Significant (P > 0.05) differences between means are indicated by different letters. ANOVA analysis with LSD test was used