Figure 5.

TaWRKY19 directly binds the TaNOX10 promoter. A, Putative TaWRKY19-binding motif GTCGCGGTCAA, located in the TaNOX10 promoter. The biotin-labeled probe and the mProbe contain the binding motif sequence and mutated sequence are shown. B, Binding of TaWRKY19 to the TaNOX10 promoter, as determined by ChIP-qPCR. Data are shown as means ± SD from three biological replicates. Statistical significance was determined by Student’s t test. **P < 0.01. C, EMSA titration, performed as described in Figure 2B, assessing TaWRKY19 binding to the TaNOX10 promoter, which is dependent on the presence of the W box. Probe-3′-biotin (lanes 1 to 7) or mProbe-3′-biotin (lane 8) = 0.0025 µM of biotinylated DNA probe added to the reaction. D, Schematic diagrams of the effector, reporter, and control constructs used in dual-LUC reporter assays. TaNOX10pro consists of 2,000-bp upstream of the start codon of TaNOX10 as in (A); TaNOX10proΔ-box includes a mutation in the W-box element and was used as a negative control. E, TaNOX10 promoter activity. The relative fLUC/rLUC activity was evaluated in wheat protoplasts transformed with TaNOX10pro and pGL3, respectively. TaNOX10pro indicates the intact TaNOX10 promoter. The empty vector pGL3 was used as a negative control. Statistical significance was determined by Student’s t test. Data are shown as means ± SD are from five replicates. *P < 0.05. F, TaWRKY19 compromises TaNOX10 promoter activity. +TaWRKY19-GFP indicates co-expression of TaWRKY19 with TaNOX10pro, TaNOX10proΔ-box, or the pGL3 control. GFP was used as a negative control. In (E) and (F), relative activity (fLUC/rLUC) is the ratio of bioluminescence intensity of fLUC (TaNOX10pro or TaNOX10proΔ-box) to rLUC (35S promoter). LUC activity (fLUC/rLUC) was detected by a Modulus Single Tube Luminometer (Promega, Madison, USA). Statistical significance was determined by Student’s t test. Data are shown as means ± SD are from five replicates. *P < 0.05.