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. 2016 Jan 23;14(7):1633–1645. doi: 10.1111/pbi.12526

Figure 2.

Figure 2

MdbHLH104 directly activates the expression of MdAHA8 gene. (a) qRT‐PCR assays for MdbHLH104 and MdAHA genes in transgenic apple lines. (b) Illustration of the MdAHA8 promoter region indicating the presence of E‐box DNA motifs. Transverse lines show the positions of primers used in the ChIP‐PCR experiment. ChIP assays were performed using the 35S::GFP and 35S::MdbHLH104‐GFP apple calli. A region containing E‐box in the actin promoter is negative control. (c) MdbHLH104 binds to the E‐box motifs present in the MdAHA8 promoter in vitro, as indicated by an EMSA method. The MdAHA8 promoter fragment containing the E‐box motifs was incubated with His‐MdbHLH104 protein. Competition for MdbHLH104 binding was performed with 50× and 100× unlabelled probes (wt) or G‐box‐mutated probes (mut). His was used as the control. Mut indicates mutated probes. ‘+’ indicates presence, and ‘−’ indicates absence. (d) and (e) GUS staining assay and activity analysis of MdAHA8 expression promoter using PM d AHA 8 ::GUS and 35S::MdbHLH104‐GFP+ PM d AHA 8 ::GUS transgenic apple calli. GUS activity was measured using a 4‐methylumbelliferyl‐d‐glucuronide assay. The data represent the means ± SD of three independent experiments.