Fig. 1: iP-NBD interacts with cytokinin perception and transport.

a Chemical structure of iP-NBD with excitation and emission wavelengths b Superposition of docking simulation of iP-NBD (in green) and natural ligand iP (in grey) in AHK2, AHK3 (upper row) and CRE1/AHK4 (left panel of lower row) receptor cavity showing the embedded position of the ligands. CRE1/AHK4 (right panel of lower row) with visualized entrance channel and the fluorophore fitting into an antechamber through the ethylene linker. c Competitive binding assay with Escherichia coli expressing AHK3 and CRE1/AHK4. Binding of [2-³H]iP (18 and 6 nM in the case of AHK3 and CRE1/AHK4, respectively) was assayed together with increasing concentrations of unlabelled iP and iP-NBD. The bars represent mean ± s.d., ***p < 0.001, **p < 0.01, *p < 0.05; n = 3 (Student’s t test). d Expression of the early cytokinin response gene ARR5 in 5-day-old seedlings of Col-0 treated with 0.01 µM iP, 0.1 µM iP, 1 µM iP, 10 µM iP-NBD, 100 µM iP-NBD or co-treatments of 0.1 µM iP + 10 µM iP-NBD, 0.1 µM iP + 100 µM iP-NBD or DMSO for 15 min (mean ± s.d., p < 0.05 by two-way ANOVA. n = 9–18; three technical replicates from three biological replicates per condition). e Kinetics of 5 µM iP-NBD uptake in Arabidopsis LRC cells of wild type (Col-0) pre-treated with 5 µM iP. f Kinetics of 5 µM iP-NBD uptake in root meristem epidermal cells of wild type (Col-0) and pup14. iP-NBD fluorescence was measured in four time points (0, 2, 7 and 12 min). The bars represent mean ± s.d., ***p < 0.001, **p < 0.01; n ≥ 20 (Student’s t test) (e, f).