Volume 8, Issue 7, Pages 1103-1114 (July 2015) Subcellular Redistribution of Root Aquaporins Induced by Hydrogen Peroxide  Michael M. Wudick, Xiaojuan Li, Valeria Valentini, Niko Geldner, Joanne Chory, Jinxing Lin, Christophe Maurel, Doan-Trung Luu  Molecular Plant  Volume 8, Issue 7, Pages 1103-1114 (July 2015) DOI: 10.1016/j.molp.2015.02.017 Copyright © 2015 The Author Terms and Conditions

Figure 1 Analysis of H2O2-Induced Redistribution of AtPIPs by Means of Subcellular Fractionation. Roots of plants cultivated in hydroponics were treated with cycloheximide alone (mock) or in combination with 0.5 mM H2O2 for 15 min, and used for extraction of microsomal membranes, which were subsequently separated in a continuous sucrose gradient. Sucrose concentration varied from 45% to 21% (w/w) throughout the gradient. Immunological detection in the indicated fractions was carried out using antibodies raised against AtPIP1s and AtPIP2s (see Materials and Methods). The bands detected correspond to ∼26 kDa. Two individual blots were aligned and joined to visualize all representative fractions of the same gradient. Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 2 Single-Particle Tracking Analysis of AtPIP2;1-GFP Dynamics in the Plasma Membrane. (A) Histograms showing the distribution of the diffusion coefficients (D) of AtPIP2;1-GFP fusions expressed in root epidermal cells and exposed to mock or 0.5 mM H2O2 treatments for 30 min. Gaussians were used to fit the data. N, number of cells analyzed; n, number of particles tracked. (B) Histograms showing the distribution of different diffusion modes (Brownian, directed, restricted, mixed) obtained from the same set of single-particle tracks as in (A). The asterisk indicates a significant difference between mock and H2O2 treatments (Student t-test, P = 0.005). Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 3 Density of AtPIP2;1-GFP Fusions in the Plasma Membrane Monitored by Fluorescence Correlation Spectroscopy. Root epidermal cells expressing AtPIP2;1-GFP fusions were exposed to mock treatments or treatments with H2O2, A23, NAA, H2O2 supplemented with A23 or NAA, BFA or A23 supplemented with BFA, as described in the text. N, number of cells analyzed. The asterisks indicate a significant difference between treatments (Student t-test, P < 0.001). Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 4 Analysis of the Redistribution of Fluorescently Tagged AtPIP2;1 in Response to H2O2. (A–D) Confocal microscopy images of Arabidopsis root epidermal cells expressing AtPIP2;1 tagged with either mCherry (A, C) or GFP (B, D). Roots were treated with mock (A, B) or 0.5 mM H2O2 solutions (C, D) for 30 min. Arrows in (C) and (D) indicate localization of the constructs in intracellular spherical bodies, whereas fuzzy intracellular fluorescent signals are marked with asterisks. Bar, 10 μm. (E) The percentage of root cells with an intracellular GFP/mCherry signal was determined for plants treated for 30 min with mock (open bars) or 0.5 mM H2O2 solutions (closed bars). n, number of cells. Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 5 Co-labeling Studies in Root Epidermal Cells of AtPIP2;1 Constructs with Different Subcellular Fluorescent Markers. The subcellular localization of AtPIP2;1 tagged with mCherry (magenta) or GFP (green) (left columns) was analyzed by confocal fluorescence microscopy for co-localization with distinct markers (middle columns; names indicated on the left side of the figure). The superposition of the images is shown in the right columns. The studies were performed in plants subjected for 30 min to mock treatment (right) or treatment with 0.5 mM H2O2 (left). Arrows indicate the localization of AtPIP2;1 constructs in intracellular compartments. Bars, 5 μm in (A) and 10 μm in (B). Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 6 Effects of Tyrphostin A23 (A23) on H2O2-Induced Relocalization of AtPIP2;1-mCherry. (A–E) The figure shows representative confocal fluorescence microscopic images of Arabidopsis root epidermal cells expressing AtPIP2;1-mCherry and subjected to the following treatments: 30 min with DMSO (A), 30 min with 50 μM BFA (B), 30 min with 30 μM A23 (C), pretreatment for 30 min with 30 μM A23, prior to a combined treatment for 30 min with A23 and BFA (D), or A23 and 0.5 mM H2O2 (E). The asterisk indicates accumulation of AtPIP2;1-mCherry in a BFA body (B), whereas arrows in (E) show its accumulation in intracellular spherical bodies. (F) Graph of the percentage of cells with an intracellular mCherry signal after 30-min treatments with DMSO (Mock), 30 μM A23, 0.5 mM H2O2, and after pretreatment for 30 min with A23 prior to a concomitant 30-min treatment with 0.5 mM H2O2 and A23. n, number of cells. Bar, 10 μm. Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 7 Effects of NAA on H2O2-Induced Internalization of AtPIP2;1-mCherry. Roots of plants expressing AtPIP2;1-mCherry were treated for 30 min with either ethanol (Mock), 5 μM NAA (NAA), or 0.5 mM H2O2 (H2O2), or pre-treated with NAA for 30 min prior to a 30-min treatment with 0.5 mM H2O2 and NAA (H2O2 + NAA). The figure shows the percentage of cells with an intracellular mCherry signal after treatment. n, number of cells. Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 8 Effects of H2O2 on the Abundance of AtPIP2 in Roots. (A) Roots were treated with 0.5 mM H2O2 supplemented with 50 μM cycloheximide for 0, 15, 30, 60, or 120 min. Total proteins from roots were extracted and immunodetection by ELISA was carried out using anti-PIP2 antibodies. ELISA tests were done in triplicate. Signal in arbitrary units (a.u.). (B) Time-dependent effects of H2O2 on the abundance of AtPIP2;1 as revealed through epifluorescence of roots stably expressing a photo-activatable version of GFP fused to AtPIP2;1. The fluorescence images of root sections in the absence (upper part) or in the presence (lower part) of 0.5 mM H2O2 were acquired immediately (0 min) or 120 min after photo-activation (left). The images were used to measure the overall mean gray values of given root sections (right). N, number of roots. Bar, 100 μm. Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions

Figure 9 Subcellular Trafficking Mechanisms Controlling AtPIP Localization in Response to H2O2. The figure shows a schematic overview of the routes and compartments of the plant endomembrane system. The representative markers that were analyzed (see text) are indicated in blue. Markers that did not colocalize are crossed out with a red bar. The inhibitory effects of NAA, but not of A23 on H2O2-induced PIP relocalization are shown. Molecular Plant 2015 8, 1103-1114DOI: (10.1016/j.molp.2015.02.017) Copyright © 2015 The Author Terms and Conditions