Abstract
The phytohormone auxin and its directional transport through tissues are intensively studied. However, a mechanistic understanding of auxin-mediated feedback on endocytosis and polar distribution of PIN auxin transporters remains limited due to contradictory observations and interpretations. Here, we used state-of-the-art methods to reexamine the auxin effects on PIN endocytic trafficking. We used high auxin concentrations or longer treatments versus lower concentrations and shorter treatments of natural indole-3-acetic acid (IAA) and synthetic naphthalene acetic acid (NAA) auxins to distinguish between specific and nonspecific effects. Longer treatments of both auxins interfere with Brefeldin A-mediated intracellular PIN2 accumulation and also with general aggregation of endomembrane compartments. NAA treatment decreased the internalization of the endocytic tracer dye, FM4-64; however, NAA treatment also affected the number, distribution, and compartment identity of the early endosome/trans-Golgi network, rendering the FM4-64 endocytic assays at high NAA concentrations unreliable. To circumvent these nonspecific effects of NAA and IAA affecting the endomembrane system, we opted for alternative approaches visualizing the endocytic events directly at the plasma membrane (PM). Using total internal reflection fluorescence microscopy, we saw no significant effects of IAA or NAA treatments on the incidence and dynamics of clathrin foci, implying that these treatments do not affect the overall endocytosis rate. However, both NAA and IAA at low concentrations rapidly and specifically promoted endocytosis of photo-converted PIN2 from the PM. These analyses identify a specific effect of NAA and IAA on PIN2 endocytosis, thus, contributing to its polarity maintenance and furthermore illustrate that high auxin levels have nonspecific effects on trafficking and endomembrane compartments.
Highlights
A multitude of developmental processes throughout the lifecycle of a plant, such as organ formation, embryonic axis establishment, and tropic responses, are regulated by the local accumulation and asymmetric distribution of the growth regulating hormone auxin (Bargmann et al, 2013; Lavy and Estelle, 2016; Gallei et al, 2020)
We further provide: (1) a better characterization of the effects of naphthalene acetic acid (NAA) and IAA on different cellular processes including clathrin-mediated endocytosis (CME) and intracellular trafficking; (2) comparisons of the effects between NAA and IAA on different cargoes; and (3) the identification of a rapid, specific endocytic auxin effect, during which NAA and IAA, even at very low concentrations, promote internalization of PIN2. Both NAA and IAA interfere with BFA-induced intracellular cargo accumulation and endosomal aggregation It has been shown that co-treatment of different auxins and auxin-analogues, such as IAA, NAA, 2,4-dichlorophenoxyacetic acid (2,4-D), a-(phenyl ethyl-2-one)-indole-3-acetic acid (PEO-IAA), 2-NAA, and pin static acid with BFA leads to reduced intracellular cargo in BFA bodies (Paciorek et al, 2005; Robert et al, 2010; Jasik and Elmon, 2016; Oochi et al, 2019; Paponov et al, 2019b)
NAA but not IAA interferes with the structure and identity of the endomembrane system We further investigated the observed disparity between the effects of NAA and IAA on FM4-64 internalization (Figure 2, A–C and Supplemental Figure S3A)
Summary
A multitude of developmental processes throughout the lifecycle of a plant, such as organ formation, embryonic axis establishment, and tropic responses, are regulated by the local accumulation and asymmetric distribution of the growth regulating hormone auxin (Bargmann et al, 2013; Lavy and Estelle, 2016; Gallei et al, 2020). Auxin itself has the ability to dynamically change the subcellular localization of PINs at the PM by forming a feedback loop between auxin signaling and transport (Ravichandran et al, 2020). This was proposed to facilitate the rise of new polarized routes of auxin transport (Mazur et al, 2016; Prat et al, 2018; Mazur et al, 2020a). Auxin-mediated changes in PIN polarity were observed during apical-basal axis formation during embryogenesis (Robert et al, 2018, 2013; Wabnik et al, 2013) or during termination of shoot bending responses (Rakusovaet al., 2016, 2019)
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