Fig. 5: COP1-mediated K63 ubiquitination inhibits GH3.5 activity.

a Auxin inhibition of primary root growth in Col, cop1-4, gh3.5-1, and cop1-4 gh3.5-1. Seedlings were grown in the dark on MS medium with IAA or without IAA (0, 0.1, and 0.2 μM). IAA-mediated inhibition of root growth is expressed as the percentage decrease in the root length of IAA-treated seedlings compared to control seedlings. Different lowercase letters above the histogram represent statistically significant differences (P < 0.05) determined by ordinary two-way ANOVA with Tukey’s post hoc test. b Nalacin inhibition of primary root growth in cop1-4. Etiolated seedlings were treated with 6 μM nalacin. Root growth inhibition is expressed as the percentage decrease in length relative to seedlings grown without nalacin. Data are presented as the mean ± SD, n = 3 biological replicates. For each replicate, n = 22 and 15 seedlings in (a) and (b), respectively. Statistically significant differences were determined by two-sided Student’s t test. c Preparation of GST-GH3.5 proteins linked to K63 ubiquitin chains by COP1. GST-GH3.5-boiled, inactive GH3.5 caused by boiling; GST-GH3.5-K63, GH3.5 linked to K63 chains by COP1; GST-GH3.5, GH3.5 without ubiquitination. The IAA-Asp produced by GH3.5 was quantified by analyzing the peak area from the UPLC-MS assay. The extracted ion chromatograms of IAA-Asp products are shown in (d), and the quantification of the IAA-Asp yield is shown in (e). NL, normalization level. Standard, authentic IAA-Asp standard. Data in (e) are presented as the mean ± SD (n = 3 biological replicates), statistically significant differences (P < 0.001) were determined by ordinary one-way ANOVA with Tukey’s post hoc test. f Michaelis–Menten diagram and kinetic parameters of GH3.5 for IAA-Asp. Initial velocities are plotted against the IAA concentration. The average values ± SD (n = 3 biological replicates) are shown. g Content of endogenous IAA and its conjugates in the dark. Data are presented as the mean ± SD (n = number of biological replicates). Statistically significant differences (P < 0.05) were determined by ordinary one-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file.