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

Abstract

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.

Clathrin in plant defense signaling and execution

“…Pathway of PEPR1 receptor exocytosis, but not its endocytotic internalization and further transport, requires BFA-sensitive ARF GTPase GDP/GTP exchange factor function, and exocytosis seems to be the main factor controlling the incidence of inactive PEPRs at the PM (8). This observation is congruent with recent reinterpretation of PIN-FORMED2 (PIN2)-containing BFA bodies as also mostly coming from de novo biosynthetic anterograde PIN2 membrane transport (17). It is now clear that at least some of the published data based on the use of BFA secretion block and "recycling" from BFA bodies after the BFA washout need to be reinterpreted.…”

Clathrin Heavy Chain Subunits Coordinate Endo- and Exocytic Traffic and Affect Stomatal Movement

“…This other side of the traffic cycle at the plasma membrane is equally critical for physiology and development. The polarized localization of PIN proteins, for example, is maintained by the coordinated transport between the plasma membrane and endomembrane compartments to provide directional gradients of auxin in plants (Feraru and Friml, 2008;Kitakura et al, 2011;Jásik et al, 2013Jásik et al, , 2016. When endocytosis is disrupted, this polarized expression is lost and auxin gradients dissipate (Kitakura et al, 2011).…”

Auxin‐induced actin cytoskeleton rearrangements require AUX1

“…However, AUX1 is not redistributed in response to NAA (Kleine‐Vehn et al , ), which stimulates at least partial actin reorganization in Col‐0, WS and both aux1 mutants. Use of a photoconvertible PIN2 shows that it is not maintained at root epidermal cell PMs after auxin treatments, and most PIN2 in brefeldin A compartments is newly synthesized, not recycled (Jásik et al , ). This complicates actin's role in auxin response.…”