Fig. 5: BIN2 phosphorylates SPL9 and the phosphorylation site in SPL9 is required for its normal function to promote vegetative phase change.

a A schematic diagram of the SPL9 protein. The TPKVT motif is the typical conserved phosphorylation recognition motif of the GSK3 kinase. The threonine in red indicates the phosphorylated site. b SPL9 is phosphorylated by BIN2 in vitro. SPL9-His was incubated with GST or BIN2-GST at 30 °C for 1 h. Protein was separated in a SDS-PAGE gel containing phos-tag regent and detected by anti-GST and anti-His antibodies. SPL9 (P), phosphorylated SPL9 with slower gel mobility shift. c In vitro kinase assay. Kinase assays were performed with purified GST-BIN2 and His-SPL9 (T) or mutant His-SPL9 (A). d Pie charts show phenotypic distribution of the first leaf with abaxial trichomes in the primary rSPL9-APKVA (T-A), rSPL9-TPKVT (T-T), and rSPL9-DPKVD (T-D) transgenic plants. About 70 primary independent transformants were characterized. e Phenotypic characterization of representative transgenic plants transformed with T-A, T-T, and T-D with comparable levels of the SPL9 transcript. 21-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# lines grown in short days were used for phenotypic analysis. Numbers indicate the first leaf with abaxial trichomes (n = 20 plants, ±SD). Different letters indicate significant difference between genotypes using one-way ANOVA at P < 0.001. Scale bar = 1 cm. f, g SPL9 transcript level (f) and protein level (g) in 14-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# transgenic lines in short days. Data are means ± SD from a representative experiment with three technical replicates for each sample. Different letters indicate significant difference using one-way ANOVA at P < 0.01. All experiments were repeated 3 times biologically. Numbers between two blots indicate the relative normalized value for each sample. The intensity of each sample was first normalized to its corresponding ACTIN, then the resultant value was normalized again to the value of T-T 1#. The band intensity was determined using image J. h Leaf length/width ratio of the first leaf from 40-day-old T-T 1#, 10#, T-A 2#, 6#, T-D 4#, 8#, and 50# plants. Different letters indicate significant difference between genotypes using one-way ANOVA at P < 0.001 (n = 10 plants, ±SD).