Effects of Auxins on PIN-FORMED2 (PIN2) Dynamics Are Not Mediated by Inhibiting PIN2 Endocytosis

By using the photoconvertible fluorescence protein Dendra2 as a tag we demonstrated that neither the naturally occurring auxins indole-3-acetic acid and indole-3-butyric acid, nor the synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid nor compounds inhibiting polar auxin transport such as 2,3,5-triiodobenzoic acid and 1-N-naphthylphthalamic acid, were able to inhibit endocytosis of the putative auxin transporter PIN-FORMED2 (PIN2) in Arabidopsis (Arabidopsis thaliana) root epidermis cells. All compounds, except Indole-3-butyric acid, repressed the recovery of the PIN2-Dendra2 plasma membrane pool after photoconversion when they were used in high concentrations. The synthetic auxin analogs 1-naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid showed the strongest inhibition. Auxins and auxin transport inhibitors suppressed also the accumulation of both newly synthesized and endocytotic PIN2 pools in Brefeldin A compartments (BFACs). Furthermore, we demonstrated that all compounds are also interfering with BFAC formation. The synthetic auxin analogs caused the highest reduction in the number and size of BFACs. We concluded that auxins and inhibitors of auxin transport do affect PIN2 turnover in the cells, but it is through the synthetic rather than the endocytotic pathway. The study also confirmed inappropriateness of the BFA-based approach to study PIN2 endocytosis because the majority of PIN2 accumulating in BFACs is newly synthesized and not derived from the plasma membrane.