Abstract
Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure–activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants.
Highlights
Auxin regulates diverse developmental and growth processes as well as tropic responses [1]
Root growth in the light-grown seedlings was less sensitive, with no effect observed at 1 μM trafficking and endocytosis inhibitor 1/TENin1 (TE1); 75 % root growth inhibition was observed at 50 μM TE1 (Supplementary Figure S1A)
Germination was unaffected by TE1, but at concentrations above 10 μM necrotic seedlings were observed after 7 days (Supplementary Figures S1B and S8A, top row)
Summary
Auxin regulates diverse developmental and growth processes as well as tropic responses [1]. Intracellular PIN2 accumulation could be reversed in seedlings transplanted to DMSO for 180 min after BFA and TE1 treatments respectively (Figure 2I), indicating that the TE1 effect was reversible. PIN2–GFP seedlings were pre-incubated in TE1 (showing TE1 bodies) followed by TE1 and BFA treatment
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