Fig. 3: RIN1 interacts with STF1 and STF2 and reduces STF abundance in vitro and in vivo.

a Spatial expression of RIN1 in Wm82 stem tips at different periods as assayed by in situ hybridization. Arrows point to shoot apical meristems (SAM) and axillary meristems (AM). L: leaf primordium. Scale bars = 100 μm. Three independent biological replicates were performed. b Physical interactions between different genetypes of RIN1 and STF1 and STF2 in a yeast two-hybrid system. p53:T is a positive control and the empty pGAD vector is a negative control. c Pull-down assays verify the interaction between RIN1^Wm82 and STF1 and STF2 in vitro. Three independent biological replicates were performed. d BiFC analysis of physical interactions between RIN1^Wm82 and STF1 and STF2. YFP^C-tagged RIN1^Wm82 together with YFP^N-tagged STF1 and STF2, respectively, were transiently expressed in tobacco leaves. PRR5a-YFP^C and PRR5a-YFP^N were used for the negative controls. YFP^C: C-terminal YFP; YFP^N: N-terminal YFP. Scale bar = 20 μm. Three independent biological replicates were performed. e RIN1^HH43 promotes the degradation of STF1 and STF2 in vivo, and STF1 and STF2 are stabilized in protein extracts from NIL-rin1 plants compared to NIL-RIN1^HH43 plants in long-day photoperiods (16 h light/8 h dark). The membrane was probed with an antibody that recognizes both STF1 and STF2. Actin was used as a loading control. ZT: Zeitgeber time. Three independent biological replicates were performed. f In a cell-free degradation assay, RIN1^HH43 promotes the degradation of STF1 and STF2 in vitro, and both STF1–MBP and STF2–MBP are stabilized in protein extracts derived from NIL-rin1 plants over NIL-RIN1^HH43 plants grown under long-day photoperiods (16 h light/8 h dark). Actin was used as a loading control. Three independent biological replicates were performed. Black arrows in (c–e, f) represent the positions of protein ladder. Source data are provided as a Source Data file.