Figure 5.

Ponalrestat is an inhibitor of monooxygenase YUCs. A, root lengths of 3-day-old eto1–2 etiolated seedlings grown on 1/2 MS medium containing ponalrestat (5 μm), Kyn (1.5 μm), or DMSO as the mock control with co-treatments of increasing concentrations of IPA. Bars represent mean ± S.D. of at least 10 seedlings. Statistical differences between the groups were calculated with ANOVA (with Tukey's HSD post hoc test). Data points with different letters are significantly different at p < 0.01. B, root lengths of Col-0 or yucQ with single or combinatorial treatments of 1 μm ACC, 5 μm ponalrestat, and 100 nm IAA. Bars represent mean ± S.D. of at least 10 seedlings. Statistical differences between the groups were calculated with ANOVA (with Tukey's HSD post hoc test). Data points with different letters are significantly different at p < 0.01. C, DARTS assay. Recombinant GST-YUC2 was pretreated with increasing concentration of ponalrestat (0.5 mm to 5 mm, along with a control without ponalrestat pretreatment) then digested by Pronase protease. Protein levels were detected using GST antibody. As a control, GST tag was subjected to the same digestion treatment. D, in vitro assay of GST-YUC2 enzymatic activity. Purified GST-YUC2 recombinant protein was used for the enzyme activity assay. FAD and NADPH were added to the reaction buffer as cofactors. IPA was fed to YUC2 in the presence of increasing concentration of ponalrestat (0.1 mm, 1 mm, 5 mm, and 10 mm). The IAA product was detected by LC/MS, and the relative enzyme activity was defined as the level of product. Bars represent mean ± S.D. of three replicates experiments. Statistical differences between the groups were calculated with ANOVA (with Tukey's HSD post hoc test). Bars with different letters are significantly different at p < 0.01. All the above experiments were repeated for at least three times with similar results.