Fig. 5.

Myb10‐D binds to the secondary wall MYB‐responsive element (SMRE) in the NCED promoter and induces its expression in wheat (Triticeae aestivum). (a) The expression profiles of Myb10‐D (left) and NCED (right) 5‐30 DPA as detected by RNA‐seq and qPCR. (b) Schematic diagram of the 9‐cis‐epoxycarotenoid dioxygenase (NCED) promoter structure. The SMRE element is −192 to −199 bp away from the ATG of NCED (TraesCS6B02G298800). (c) Electrophoretic mobility shift assay (EMSA) of Mb10‐D with probes containing the binding motifs in the promoters of NCED. The presence (+) or absence (–) of specific probes is indicated. (d) Schematic diagram of various effector and reporter constructs of NCED. (e) Dual‐luciferase assay of Myb10‐D in the promoters of NCED in Nicotiana benthamiana leaves. (f) Significant differences (Student's t‐test: *, P < 0.05) detected in N. benthamiana leaves and Arabidopsis protoplasts. Firefly luciferase (LUC) activity was normalized to the Renilla luciferase (REN) activity (as an internal control). Values are means ± SD from three independent replicates for each construct. (g) Yeast one‐hybrid analysis of the interaction of Myb10‐D and the NCED promoter. The promoter was used for autoactivation tests in the presence of different concentrations of aureobasidin A (AbA) on SD/‐Ura medium, and physical interaction was determined on SD/‐Leu medium in the presence of corresponding AbA concentrations.