Volume 25, Issue 10, Pages 1381-1388 (May 2015) Root Cap-Derived Auxin Pre-patterns the Longitudinal Axis of the Arabidopsis Root  Wei Xuan, Dominique Audenaert, Boris Parizot, Barbara K. Möller, Maria F. Njo, Bert De Rybel, Gieljan De Rop, Gert Van Isterdael, Ari Pekka Mähönen, Steffen Vanneste, Tom Beeckman  Current Biology  Volume 25, Issue 10, Pages 1381-1388 (May 2015) DOI: 10.1016/j.cub.2015.03.046 Copyright © 2015 Elsevier Ltd Terms and Conditions

Current Biology 2015 25, 1381-1388DOI: (10.1016/j.cub.2015.03.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 1 Auxin Receptor-Dependent Auxin Signaling Is Required for Prebranch Site Formation (A) Schematic representation of the distribution of LRP and LRs along the primary root in 8-day-old Col-0 and tir1afb2 seedlings (n = 10). Red triangles indicate individual seedlings. The color code corresponds to different LRP stages and is explained in (B). (B) Schematic representation of a seedling with indication of the OZ and color-coded LRP stages and LRs. (C) Root phenotype and DR5:Luciferase expression in roots of Col-0 and tir1afb2 double mutant seedlings. Bright-field images were taken from 8-day-old seedlings (the scale bar represents 0.5 cm), and DR5:Luciferase was recorded in 3-day-old seedlings (the scale bar represents 0.1 cm). (D) Quantification of the number of LRs, prebranch sites, and LRP in 8-day-old Col-0 and tir1afb2 seedlings (n = 10). Error bars represent SD. (E) Quantification of DR5:Luciferase signal in 3-day-old Col-0 and tir1afb2 seedlings following prebranch site formation over 20 hr (n = 15). MZ, meristem zone; OZ, oscillation zone. Error bars represent SD. See also Figure S1. Current Biology 2015 25, 1381-1388DOI: (10.1016/j.cub.2015.03.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 2 IBA-to-IAA Conversion Regulates the Oscillation Amplitude and the Prebranch Site Number (A and B) Analysis of DR5:Luciferase expression and quantification of the number of prebranch sites, LRs, and LRP, the primary root (PR) length, and gravitropic index in 8-day-old Col-0, ibr1ibr3ibr10 triple mutant, and ech2ibr1ibr3ibr10 quadruple mutant seedlings. Images were overlayed with bright-field images. Red arrows indicate prebranch sites. OZ, oscillation zone. Error bars represent SD (n = 10). p < 0.05 by one-way ANOVA and Tukey’s test as post hoc analysis; letter code labels the significant differences between the genotypes. (C–E) Boxplots showing the quantification of the oscillation frequency and amplitude of DR5:Luciferase and the prebranch site number in 3-day-old seedlings. (F) Boxplots showing the number of LRs derived from the oscillations, determined after another 4 days. Error bars represent SD (p < 0.05 by one-way ANOVA and Tukey’s test as post hoc analysis; letter code labels the significant differences between the genotypes, n > 15 seedlings). (G) Kymograph of DR5:Luciferase intensity along the primary root of 3-day-old transgenic lines after treatment with or without IBA over 20 hr. DR5 luminescence intensity is color coded (see color code in the bottom left corner of the panels) and plotted following the primary root elongation (y axis) and time (x axis). The dashed lines indicate the position of the OZ over time. Current Biology 2015 25, 1381-1388DOI: (10.1016/j.cub.2015.03.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 3 An Auxin Source Derived from Root Cap-Specific IBA-to-IAA Conversion Regulates Root Patterning (A) DR5:GUS expression in root tips of 3-day-old Col-0 and ibr3 seedlings after 24 hr treatment with or without 3 μM IBA or 0.1 μM IAA. Percentages indicate the proportion of seedlings showing the identical DR5 expression pattern in the root meristem within a population. PX, protoxylem pole; Ep, epidermis; OZ, oscillation zone. The scale bar represents 200 μm. (B) Schematic representation of a longitudinal section of the primary Arabidopsis root indicating the expression domains of the different promoters and enhancer trap lines used. The enhancer trap lines J3411, J0951, and J1092 were expressed in different lateral root cap tissue domains. J0121 was used to drive expression in the pericycle associated with the xylem poles. The IBR3 and GLV5 promoters were lateral root cap and columella specific, respectively. (C and D) Root phenotypes and LR numbers of enhancer trap lines trans-activating IBR3 gene expression in the ibr3 mutant background. 3-day-old seedlings were treated with or without 1 μM IBA or 5 μM Naxillin for 5 days. The scale bar representes 1 cm. Error bars represent SD (n > 10). p < 0.05 by one-way ANOVA and Tukey’s test as post hoc analysis; letter code labels the significant differences between the genotypes. Current Biology 2015 25, 1381-1388DOI: (10.1016/j.cub.2015.03.046) Copyright © 2015 Elsevier Ltd Terms and Conditions

Figure 4 MAKR4 Acts Downstream of the IBA-to-IAA Conversion to Regulate LR Development (A) Schematic representation of a seedling with indication of the root zone (red box) used for transcript profiling of Col-0 and ibr1ibr3ibr10 triple mutant seedlings after IBA treatment. The scale bar represents 0.5 mm. (B) pMAKR4:CBG99 expression in 3-day-old seedlings. Luciferase image was overlayed with bright-field (BF) image in the right panel. (C) Kymograph based on quantification of pMAKR4:CBG99 intensity along the primary root during 24 hr. The scale bar represents 0.2 cm. (D) Comparison of the periodic time and the number of prebranch sites in 3-day-old pMAKR4:CBG99 and DR5:Luciferase lines during 24 hr. Error bars represent SD (n > 15 seedlings). (E and F) Localization of pMAKR4:GFP-MAKR4 protein in prebranch site of propidium iodide-stained 3-day-old seedlings (E) and during LR initiation in the pGATA23:nGFP seedlings background (F). Red arrows indicate localization of MAKR4 in the plasma membranes near the anticlinal cell walls of two adjacent pericycle founder cells before nuclear migration. White arrows indicate localization of MAKR4 in the plasma membranes adjacent to newly formed anticlinal cell walls of the small daughter cells after asymmetric cell division. The scale bar represents 20 μM. (G–I) LR phenotype, DR5:Luciferase expression, prebranch site number, LRP number, and LRP distribution in estradiol-inducible amiMAKR4-3 line. 3-day-old seedlings were transferred to ½ MS medium supplemented with or without 3 μM estradiol for 5 more days, after which prebranch sites and LRP were counted in the newly formed part of the primary root (n = 11). Red arrows and red dashed lines indicate the position of the root apex at the moment of transfer in (G). Red triangles indicate individual seedlings in (H). The scale bar represents 0.5 cm. Error bars represent SD. (J) Time line of the changes in activities of the signaling components used in this study during the root pre-patterning process. Current Biology 2015 25, 1381-1388DOI: (10.1016/j.cub.2015.03.046) Copyright © 2015 Elsevier Ltd Terms and Conditions