Fig. 6: PavBPC6 binds to the promoter of PavAGL9, inhibiting its transcription and participating in the regulation of the blooming phenophase.

a Yeast one-hybrid (Y1H) analysis demonstrating the binding of PavBPC6 to the PavAGL9 promoter on selective medium lacking Ura and Trp, supplemented with 100 mg/mL X-Gal. b Electrophoretic mobility shift assay (EMSA) indicating the interaction of PavBPC6 with PavAGL9 promoter fragments containing the motif. Unlabeled probes were utilized for the competition assay; “−” and “+” denote the absence and presence of the binding, respectively. c Schematic overview of the LUC reporter vector containing the PavAGL9 promoter, alongside effector vectors containing PavBPC6. d Transient LUC imaging assays reveal that PavBPC6 represses the transcription of the proPavAGL9:LUC reporter. Representative images show LUC activity in N. benthamiana leaves 48 h post-infiltration. Three independent experiments with similar results were performed. e Dual-Luciferase (Dual-LUC) assay depicting promoter activity expressed as the ratio of LUC to 35S:Renilla (REN). Data are presented as means ± SD of fifteen independent measurements (n = 15 biological replicates), with significant differences evaluated using two-sided Student’s t-test (centerline, median; box limits, first and third quartiles; whiskers, minimum and maximum values). f The phenotypes resulting from the transient overexpression of PavBPC6 in flower buds of sweet cherry. Scale bar = 1 cm. g GFP signal in the transiently transformed flower buds was detected using a fluorescence microscope. Two independent experiments with similar results were performed. h Expression profiles of PavAGL9 in transient transgenic buds of sweet cherry. Data are presented as mean ± SD (n = 3 biological replicates). Statistical significance between groups was assessed using a two-sided Student’s t-test. i Yeast two-hybrid (Y2H) analysis demonstrating the interaction between PavAGL9 and PavSEP1, as well as PavPMADS2. Protein interactions were screened on a selective medium lacking Trp, His, Leu, and Ade, and supplemented with 20 mM X-α-Gal. AD-T and BD-lam were co-transferred as negative controls, while AD-T and BD-53 served as positive controls. j Bimolecular fluorescence complementation (BiFC) assay illustrating the interaction between PavAGL9 and PavSEP1 as well as PavPMADS2 in tobacco cells. MYC-pXY104 and 106J2-pXY106 pairs were used as a positive control. Yellow fluorescence represents the YFP fusion protein signal, while red fluorescence marks the nucleus using p2300-mCherry. Images show overlays of fluorescence and light views. Two independent experiments with similar results were performed. k Co-Immunoprecipitation (Co-IP) assay showing interaction of PavAGL9 with PavSEP1. l Co-IP assay demonstrating the interaction of PavAGL9 with PavPMADS2. PavSEP1-Flag and PavPMADS2-Flag were each co-expressed with PavAGL9-YFP in tobacco leaves. Source data are provided as a Source data file.