Volume 10, Issue 2, Pages 244-259 (February 2017) An Arabidopsis ABC Transporter Mediates Phosphate Deficiency-Induced Remodeling of Root Architecture by Modulating Iron Homeostasis in Roots  Jinsong Dong, Miguel A. Piñeros, Xiaoxuan Li, Haibing Yang, Yu Liu, Angus S. Murphy, Leon V. Kochian, Dong Liu  Molecular Plant  Volume 10, Issue 2, Pages 244-259 (February 2017) DOI: 10.1016/j.molp.2016.11.001 Copyright © 2017 The Author Terms and Conditions

Figure 1 Effect of Pi Availability on the Root Phenotype of WT and als3-3 Seedlings. (A) Morphology of WT and als3-3 seedlings grown on a Pi-sufficient (P+) or Pi-deficient (P−) medium at 8 and 11 DAG. Inset: magnified view of the root tip of a P− als3-3 seedling. In the inset, the red arrow indicates the tip of the primary root. (B) Close-up view of the root tips of 11-day-old seedlings shown in (A). The arrows indicate the boundary between meristematic and elongation zones. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 2 als3-3 Overaccumulates Fe3+ in Roots Under Pi Deficiency. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B) staining in the roots of 8-day-old WT and als3-3 seedlings grown on P+ Fe+ (P+), P− Fe+ (P−), and P− Fe− media. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 3 Fe Contents in the Roots of 8-Day-Old WT and als3-3 Seedlings Grown on P+ and P− Media. The values are means ± SD. The asterisk indicates a significant difference from the WT (t-test, P < 0.05). Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 4 Molecular Characterization of ALS3 and AtSTAR1 Genes and Root Phenotypes of als3 and atstar1. (A and B) Diagrams showing the structure of the ALS3 and AtSTAR1 genes, the positions of the point mutation in als3-1, and the position of the T-DNA insertions (empty triangles) in als3-2, als3-3, als3-4, and atstar1. The black box, gray box, and horizontal line represent the coding regions, untranslated regions, and introns, respectively. (C) Root phenotypes of the WT, als3-1, als3-2, als3-3, and als3-4 on P− medium. (D) Root phenotypes of the WT, als3-3, and two complementation lines on P− medium. (E) Root phenotypes of WT, als3-3, and atstar1 seedlings on P+ and P− media. In (C) to (E), the photographed seedlings were 8 days old. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 5 atstar1 Overaccumulates Fe3+ in Roots Under Pi Deficiency. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B) staining in the roots of the 8-day-old WT and atstar1 seedlings grown on P+ and P− media. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 6 ALS3 and AtSTAR1 Interact in Tonoplasts. Yeast two-hybrid (A), LCI (B), and BiFC (C–E) assays indicated physical interaction between ALS3 and AtSTAR1. (A) Yeast cells co-transformed with various combinations of the plasmids were grown on synthetic dropout medium without Leu or Trp (left panel) or synthetic dropout medium without Leu, Trp, His, or Ade (right panel) for 3 days at 30°C. The activation of the reporter genes by the SV40 large T antigen-Cub (large T-Cub) and NubG, the N-terminally truncated tumor suppressor protein p53 (NubG-Δp53), or ALS3-Cub and NubI was used as a positive control. (B) The ALS3-nLUC and cLUC-AtSTAR1 fusion genes were co-expressed in the leaves of N. benthamiana, and bioluminescence was captured with a CCD camera. (C) The ALS3-nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in the leaves of N. benthamiana, and fluorescence signals in the pavement cells were analyzed by confocal microscopy. The arrows indicate the positions of nucleus. (D) The fluorescence signals in the vacuoles isolated from the mesophyll protoplasts of the transformed leaves of N. benthamiana as shown in (C). (E) The ALS3-nYFP and cYFP-AtSTAR1 fusion genes were co-expressed in Arabidopsis protoplasts. Plasma membranes are stained red by FM4-64. The signal from the interaction between ALS3 and AtSTAR1 is shown in green. Inset: close-up view of part of the cell periphery showing the non-overlapping of plasma membrane and tonoplast. (F) Detection of expression of ALS3-HA and AtSTAR1-FLAG proteins in tobacco leaves by western blot using HA and FLAG antibodies. 1 and 2: total proteins extracted from uninfiltrated and Agrobacterium-infiltrated leaves of N. benthamiana. Rubsico was used as the protein loading control. (G) Detection of ALS3-HA and AtSTAR1-FLAG proteins in different cellular fractions. T, total proteins; S, soluble proteins; M, microsomal membrane fractions. V-ATPase, a tonoplast-specific marker. Rubisco was used as a soluble protein marker. (H) Sucrose density gradient centrifugation. Microsomal membranes were fractionated over a 20%–50% (w/w) sucrose gradient, and samples of each fraction (20 μl each) were analyzed by western blot using specific antibodies. H+-ATPase, a plasma membrane-specific marker. The black bars indicate the position of the major fractions where a given protein is located. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 7 UDP-Glc and UDP-GlcA Rescue the Mutant Phenotype of als3-3 and atstar1. The growth characteristics of 7-day-old als3-3 and atstar1 seedlings grown on P+, or P− media supplemented with various compounds (500 μM). Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 8 AtSTAR1 and ALS3 Interact to Form a Functional Transporter. (A) BiFC assays in Xenopus oocytes showing fluorescence (left panel) and bright-field merged (right panel) images resulting from the protein–protein interaction between AtSTAR1 and ALS3 (top row). No BiFC fluorescence was observed in the controls in which oocytes were co-injected with ALS3::nYFP and the complementary cYFP (bottom row). (B) Resting membrane potentials (measured in a standard ND96 bath solution) of cells injected with water (control), ALS3, or AtSTAR1, or co-injected with both AtSTAR1 and ALS3 (n = 12 cells in each case). (C) Electrogenic transport in control cells, cells injected with either AtSTAR1 or ALS3, or cells co-injected with AtSTAR1 and ALS3. Example of currents elicited in response to holding potentials ranging from 0 to −130 mV (in 10-mV increments with an inter-episode holding potential of −20 mV for 10 s) in AtSTAR1 + ALS3 co-injected cells (right panel), or cells injected only with water, AtSTAR1, or ALS3 (left panel). Symbols of each injection correspond to those shown in (B). Cells were bathed in standard ND96 (pH 7.5) solution. (D) Mean current–voltage relationships from recordings like those shown in (C). The straight and dotted arrows above the x axis indicate the reversal potential (Erev) recorded in cells co-injected with AtSTAR1 and ALS3 (full circles) and all other control and single injections. Symbols correspond to those in (B) and (C) (n = 10 cells). Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 9 Root Phenotypes of the WT and Various Mutants Grown on Agarose-Containing P+ and P− Media. (A) Eight-day-old seedlings of the WT, als3-3, lpr1lpr2, and als3-3lpr1lpr2. (B) Eight-day-old seedlings of the WT, als3-3, lpr1, and a genetic suppressor of als3-3, als3-3/lpr1. (C) Diagram illustrating the position of the point mutation in the LPR1 gene in als3-3/lpr1. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions

Figure 10 Suppression of Fe Accumulation in als3-3 Treated with UDP-Glc or Carrying a Mutation in LPR1. Fe accumulation patterns as indicated by Perls (A) and Perls/DAB (B) staining in the maturation zones of the roots of 8-day-old seedlings of the WT, UDP-Glc-treated als3-3, and various mutants grown on P− medium. Molecular Plant 2017 10, 244-259DOI: (10.1016/j.molp.2016.11.001) Copyright © 2017 The Author Terms and Conditions