Fig 11. The phosphorylation of OsMADS23 by SAPK9 is required for its stability and transcriptional activity in an ABA-dependent manner.
(A) Cell-free degradation assays of GST-OsMADS23 or its different mutated versions (GST-OsMADS23T20A S36A and GST-OsMADS23T20D S36D) in wild type (WT) or sapk9 mutant with or without ABA treatment. GST-OsMADS23 and its mutated versions were detected by western blotting using anti-GST antibody. The Coomassie blue–stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. GST-OsMADS23 and its mutated versions were expressed in BL21 (DE3) and purified; an equal amount of each was incubated for different times at 30°C with equal amount protein extracts from leaves of 10-day-old wild-type and sapk9 plants, with or without 50 μM ABA treatment for 16 h. (B) and (C) Quantification analysis of the results described in (A). The relative levels of GST-OsMADS23 and its mutated versions in different protein extracts at 0 h were defined as 1. Data represent the means of three independent experiments. (D) Cell free degradation of GST-OsMADS23 or its different mutated versions with 50 μM MG132. GST-OsMADS23 and its mutated versions were detected by western blotting using anti-GST antibody. The Coomassie blue-stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (E) Time course of OsMADS23-GFP degradation when the protein extracts from OsMADS23-GFP plants were incubated with His or His-SAPK9. Equal amounts of plant crude extracts were added to equal amounts of the recombinant proteins in the in vitro cell-free degradation assays. OsMADS23-GFP was detected by western blotting using anti-GFP antibody. The Coomassie blue-stained ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (Rbc L) was used as a loading control. (F) Quantification analysis of the results in (E). The relative levels of OsMADS23-GFP at 0 h were defined as 1. Data represent the means of three independent experiments. (G) Schematic diagram of the constructs used in the transient transactivation assay. (H) SAPK9 as well as ABA can increase the transactivation activity of OsMADS23 in Nicotiana leaves. One half of the infiltrated leaves were incubated with 50 μM ABA for 16 h. Data represent the means of three independent experiments. *p < 0.05, **p < 0.01 (Student’s t test). These cell-free degradation assays were performed in the presence of ATP.