Figure 6.

FvMAPK3 phosphorylated and degraded FvWRKY50 protein under low temperature to affect anthocyanin accumulation in strawberry fruit. (A) Luciferase complementary imaging assays showing the interaction between FvWRKY50 and FvMAPK3 in vivo. Various constructs were co-infiltrated into N. benthamiana leaves, and LUC activity was captured. (B) Y2H assays showing the interaction between FvWRKY50 and FvMAPK3. The interaction between FvMYB10 and FvMAPK3 was the positive control. (C) Anthocyanin accumulation phenotypes of WT and FvWRKY50 CR 2-9 fruits at 10°C for low temperature treatment. White stage fruits of WT and FvWRKY50 CR 2-9 were harvested and cultured in 88 mM sucrose solution at 10°C. Fruits were photographed at different days after treatment. (D) Anthocyanin accumulation-related gene expression of WT and FvWRKY50 CR 2-9 fruits at 4 days after low temperature treatment. (E) FvMAPK3 phosphorylated FvWRKY50 in vitro. His-FvMKK4^DD, GST-FvMAPK3, and His-FvWRKY50 recombinant proteins were incubated in kinase reaction buffer and then detected by SDS–PAGE. The autoradiograph (top) and CBB staining (bottom) of the proteins are indicated. (F) Cell-free degradation assay of recombinant His-FvWRKY50. Total proteins of WT and FvMAPK3-OE fruits were extracted and incubated with recombinant His-FvWRKY50 protein at 25 and 4°C with 50 mM MG132 or DMSO. His-FvWRKY50 protein was detected with anti-His antibody and tubulin was detected with anti-tubulin.