Fig 3. Glucose induces the phosphorylation of the MdbHLH3 protein at the Ser³⁶¹ site.

(A) Glucose induced the mobility shift of the MdbHLH3 protein, which was abolished by the phosphorylation inhibitor calf intestine alkaline phosphatase (CIP) in the 35S::MdbHLH3-Myc transgenic apple calli. Note: MdbHLH3-P represents phosphorylated MdbHLH3 protein unless noted otherwise in this study. (B) Collision-induced dissociation mass spectrum showing the phosphorylation of Ser-361, a glucose-induced phosphorylation site in MdbHLH3. Top panel: the structural diagram of MdbHLH3 protein and its phosphorylation site. Bottom panel: the phosphorylation sites were identified using LC-MS/MS. MdbHLH3-Myc protein from transgenic apple calli was affinity purified as in (A) before being subjected to in-gel digestion with AspN. (C) Glucose induced the phosphorylation of the MdbHLH3 protein, which was abolished by CIP in WT apple calli. Western blotting was conducted with an anti-MdbHLH3^S361 antibody specifically against the phosphorylation site. (D) and (E) The glucose-induced phosphorylation of MdbHLH3 protein depends on glucose concentration (D) and treatment time (E). The WT apple calli was treated with different concentrations of glucose (0, 1%, 3% or 6%) for 30 min (D), or treated with 6% glucose for different times (0, 10, 20, 30, or 60 min) (E). A Western blotting assay was performed with an anti-MdbHLH3^S361 antibody.