FIGURE 8.

Effect of inserting the R6 motif in the GGP promoter. (A) Schematic of the engineered GGP promoter fused to the LUC reporter gene in pGreen0800LUC representing the position of two R6 insertions upstream of the 5′ UTR and uORF (open box and dash line respectively). (B) Dual luciferase assay of the GGP promoter harboring the R6 motif fused to the LUC reporter. Promoter constructs were co-infiltrated in N. benthamiana with MdMYB10 and MdbHLH3. Luminescence of LUC and REN was measured 3 days post-infiltration and expressed as a ratio of LUC to REN. Data represent means (± SE) of four replicate reactions. (C) The GGP cDNA, placed under its own promoter engineered with two copies of the R6 motif and harboring or not a single point mutation of the uORF start codon (GGPmut-R12_pro:GGP and GGPwt-R12_pro-GGP, respectively), or placed under the control a 35S promoter (35S_pro:GGP), or the corresponding empty vector (EV) were co-infiltrated with AcMYB110 (or the MYB8 negative control). Ascorbate content was measured at 5 dpi. Data represent means (± SE) of six biological replicates. One-way ANOVA test was applied and resulting grouping information was obtained using the Tukey method, columns sharing the same letters are not significantly different at P < 0.05.