Fig. 1: Nitrate deficiency negatively regulates MdBT2 expression.

a Anthocyanin pigmentation phenotype of four-week-old apple tissue culture GL-3 plantlets treated with 5 mM KCl (nitrate deficiency) or 5 mM KNO₃ (sufficient nitrate) under constant light for 10 d. b Anthocyanin content in apple tissue culture GL-3 plantlets treated with 5 mM KCl or 5 mM KNO₃. The error bars indicate the SDs of three biological replicates, with at least 6 plantlets per replicate. The asterisks indicate significant differences (***P < 0.001) based on Student’s t test. c Expression analysis of MdBT2 in response to nitrate deficiency. Four-week-old apple plantlets were treated with nitrogen-depleted Hoagland nutrient solution supplemented with 5 mM KCl or 5 mM KNO₃ for 3 days. The 18s gene was used as an internal reference. The error bars indicate the SDs of three independent biological replicates. The asterisks indicate significant differences (**P < 0.01) based on Student’s t test. d Cell-free degradation assays of MdBT2 in response to nitrate treatment. Four-week-old apple plantlets were treated with 5 mM KCl or 5 mM KNO3 for 3 days, and then the total protein extracts from these plantlets and recombinant GST-MdBT2 proteins were incubated together for the indicated times. For the proteasome inhibitor MG132 treatment, total protein samples extracted from 5 mM KCl-treated apple plantlets were pretreated with 50 μM MG132 for 0.5 h before sampling began. Anti-GST antibody was used for immunoblotting. ACTIN was used as loading control. The protein levels at 0 h were set to 1