Fig. 7: Functional relevance of the subcellular localization of NPH3.
a Quantification of hypocotyl phototropism (mean ± SD) in etiolated Arabidopsis nph3-7 seedlings expressing GFP:NPH3 variants (35S promoter) and exposed to unilateral blue light (BL) (1 μmol m−2 s−1, 24 h) (n ≥ 30 seedlings per experiment, three replicates). One-way ANOVA with Tukey’s post hoc test is shown, different letters mark statistically significant differences (P < 0.05). Center line: median, bounds of box: minima and maxima (25th and the 75th percentiles), whiskers: 1.5 × IQR (IQR: the interquartile range between the 25th and the 75th percentile). Exact P-values for all experiments are provided in the source data file. b Representative confocal microscopy images of hypocotyl cells from seedlings shown in a and either maintained in darkness (D), treated with BL (~6 min GFP laser) or re-transferred to D (1 h) after 30 min BL (1 μmol m−2 s−1) (R-D). Scale bars, 25 μm. c Immunoblot analysis of total protein extracts (7.5% SDS-PAGE) from seedlings essentially treated as shown in b. BL treatment: 1 μmol m−2 s−1, 30 min. Dashed line: expected shift in molecular mass, closed/open arrowheads: positions of “generally” phosphorylated/dephosphorylated NPH3 proteins, respectively. d In vivo interaction of RFP:NPH3 variants and phot1:GFP in transiently transformed (35S promoter) and dark-adapted N. benthamiana leaves. Microsomal proteins were immunoprecipitated using GFP beads. Immunoblot analysis of flowthrough and immunoprecipitate (IP) (11% SDS-PAGE) is shown. All experiments were performed at least three times with similar results. e Model depicting the light regime-triggered changes in the phosphorylation status, subcellular localization, and phototropic responsiveness of NPH3. BL-induced and phosphorylation-dependent (S744, blue) 14-3-3 association releases NPH3 from the PM into the cytosol followed by condensate formation. Residues phosphorylated in darkness (yellow) and dephosphorylated upon BL cause an electrophoretic mobility shift (“general” (de)phosphorylation). Re-transfer to darkness reverts all BL-triggered processes, finally resulting in PM re-association (middle panel). NPH3 variants either constitutively attached to (red flash, left panel) or constitutively detached from (red arrowhead, right panel) the PM are non-functional. Cycling of NPH3 between the PM and the cytosol is suggested to be essential for proper function.
