Fig. 3. The kinase activity of CTR1 is not necessary for ACC-induced CTR1 nuclear translocation.

a Seedlings were grown on MS medium with or without 10 μM ACC for 3 d and photographed. Scale bar, 5 mm. The graph represents the quantification of hypocotyl lengths of seedlings grown on MS without ACC. Significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test to compare the results to the WT (black) and ctr1-2 (blue) controls. Data represent the means and SE. b Seedlings expressing GFP-CTR1-1 from its native promoter were grown on MS medium with or without AgNO₃. The graph represents the ratio of nuclear-localized CTR1 in dark-grown seedlings treated with ACC or AgNO₃. Error bars, SE (n = 3 seedlings). c, Fractionation analysis of ctr1-1 mutant seedlings treated with or without ACC and silver nitrate. d Overexpression of CTR1-1 from 35S promoter leads to nuclear localization of CTR1 in the presence of silver nitrate and ACC. Error bars, SE (n = 3 seedlings). e, f Seedlings expressing GFP-∆NT-CTR1-1 from its native promoter (e) or the CaMV 35S promoter (f) were grown on MS medium with AgNO₃ or treated with ACC. The graph represents the ratio of nuclear-localized CTR1 in dark-grown seedlings treated with ACC or AgNO3. Error bars, SE (n = 3 seedlings). g In vitro phos-tag analysis of active and inactive ΔNT-CTR1 in Arabidopsis protoplasts. h The indicated bait and prey constructs were co-transformed into the AH109 yeast strain, and the transformed yeast were grown on selection medium. i Tobacco leaves were infiltrated with Agrobacterium transformed with ΔNT-CTR1 or ΔNT-CTR1^AAA plasmid construct, followed by a 3 d incubation and visualization of nuclear signals by confocal microscopy. All scale bars represent 50 μm except the scale bar in (a). The areas below the hook and above the elongation zone of hypocotyls of dark-grown seedlings were used for imaging. Two-tailed Student’s t test was performed to determine statistical significance for (b, d, e, f). n.s, statistically not significant.