Fig. 4. MdWRKY11 binds to the promoter and activates the expression of MdHMA5, which encodes a Cu-transporting P_1B-type ATPase.

aMdHMA5 expression in transgenic and control apple plants. b Binding of MdWRKY11 to the W-box of the MdHMA5 promoter indicated by chromatin immunoprecipitation (ChIP)-qPCR. The position of the W-box is indicated by the gray bar. The ChIP signal was quantified as the percentage of immunoprecipitated DNA out of the total input DNA, as determined by qPCR. A parallel experiment without antibodies and the promoter of MdDRB served as the negative control. c Electrophoretic mobility shift assay (EMSA) results confirming the in vitro binding of MdWRKY11 to the MdHMA5 promoter fragment. The arrow indicates the position of a protein–DNA complex after incubation with GST-MdWRKY11 and the biotin-labeled DNA probe MdHMA5. Both the probe containing a W-box and the probe (m) containing a mutated W-box were synthesized according to the sequence of the MdHMA5 promoter. d Relative GUS activity normalized with respect to luciferase (LUC) activity in transiently transformed apple calli expressing 35S::MdWRKY11-GFP, proMdHMA5::GUS, and 35S::LUC; the relative GUS activity in transiently transformed apple calli expressing 35S::GFP, proMdHMA5::GUS, and 35S::LUC served as the control. The data are the means±SDs of triplicate experiments. The asterisks indicate values that are significantly different from those of the control (Student’s t-test): *P < 0.05; **P < 0.01