The WUSCHEL Related Homeobox Protein WOX7 Regulates the Sugar Response of Lateral Root Development in Arabidopsis thaliana  Danyu Kong, Yueling Hao, Hongchang Cui  Molecular Plant  Volume 9, Issue 2, Pages 261-270 (February 2016) DOI: 10.1016/j.molp.2015.11.006 Copyright © 2016 The Author Terms and Conditions

Figure 1 WOX7 Has a Negative Role in Lateral Root Development. See also Supplemental Figure 1. (A–C) WOX7 expression pattern in the root, as shown by the WOX7pro:GUS reporter construct. Shown in A and B are primary roots, and in C lateral roots and lateral root primordia. Bars = 50 μm. (D) Diagram showing the location of the T-DNA in wox7-1 (Salk_065801) and wox7-2 (SAIL_409_H12). (E) Primary root length in 14-day-old seedlings of the wild type (WT), wox7-1, wox7-2, and two independent lines of the 35S:WOX7-GFP transgenic plants. (F) Time-course study of the number of lateral roots (LRs) per seedling in the wild type (WT), wox7-1 mutant, and the 35S:WOX7-GFP transgenic line. Visible lateral roots were counted 1–6 days (d) after 5-day-old seedlings were transferred to fresh growth medium. p < 0.05 for all measurements 3–6 days after transfer. t test. In E and F, the error bars represent standard deviations; N = 50. Molecular Plant 2016 9, 261-270DOI: (10.1016/j.molp.2015.11.006) Copyright © 2016 The Author Terms and Conditions

Figure 2 WOX7 Acts as a Repressor in Lateral Root Morphogenesis. (A and B) Root length in 12-day-old seedlings of WT, wox7-1, and independent lines of transgenic plants containing the WOX7pro:WOX7-VP16 construct in the wox7-1 background (V1-V3). (C) Number of lateral roots per seedling in WT, wox7-1, and WOX7pro:WOX7-VP16 transgenic plants. Seeds were planted on MS medium containing 1% sucrose, and lateral roots were counted starting 6 days after sowing. p < 0.05 for measurements 9–12 days after germination. t test, N = 50. The error bars represent standard deviations; N = 50. (D) Confocal microscopy showing the subcellular localization of the WOX7-GFP fusion protein expressed under the 35S promoter. Molecular Plant 2016 9, 261-270DOI: (10.1016/j.molp.2015.11.006) Copyright © 2016 The Author Terms and Conditions

Figure 3 WOX7 Represses Lateral Root Initiation and Primordia Growth through Direct Repression of CYCD6;1. See also Supplemental Figure 3. (A) Number of lateral root primordia (LRP) with GUS activity from the CYCB1;1:GUS reporter at different stages of lateral root development, in WT and wox7-1. (B) Number of cells with GUS activity from the CYCB1;1:GUS reporter, at different stages of lateral root development, in WT and wox7-1. In both A and B, Ten-day-old seedling were examined and pairwise comparisons were made between the wild type and the wox7 mutant. The asterisks mark those with significance difference (p < 0.05. t test). Error bars represent standard deviations; N = 30. (C) Number of lateral roots per seedling in WT and cycd6;1 mutant seedlings 6–12 days after sowing. The error bars represent standard deviations. * Indicate significance difference (p < 0.05. t test, N = 50). (D) Real-time ChIP-PCR assay showing WOX7 binding to the CYCD6;1 promoter. Molecular Plant 2016 9, 261-270DOI: (10.1016/j.molp.2015.11.006) Copyright © 2016 The Author Terms and Conditions

Figure 4 WOX7 Does Not Affect Auxin Transport or Sensing, but its Expression Is Affected by Auxin. (A and B) PIN1:PIN1-GUS expression in lateral root primordia in A, WT, or B, the wox7-1 mutant. (C and D) DR5:GFP expression in C, WT, or D, wox7-1. Bright field images of the roots are shown at right. (E–G) WOX7:GFP expression in lateral root primordia on E, MS medium; F, MS medium containing 0.2 μM (F); or G, MS medium containing 0.5 μM NAA. Bright field images of the roots are shown in the lower panel. Ten-day-old seedlings grown in MS medium were transferred to MS medium with or without NAA, and GFP was examined 24 hrs after treatment. Dotted lines in A–G and white lines in E–G are the outlines of lateral primordia and primary roots, respectively. Numbers are signal intensity for GUS staining or GFP fluorescence, as measured with Image J (mean and SD from at least 10 lateral root primordia at the same developmental stage). Bars = 20 μm. Molecular Plant 2016 9, 261-270DOI: (10.1016/j.molp.2015.11.006) Copyright © 2016 The Author Terms and Conditions

Figure 5 WOX7 Regulates Lateral Root Formation in a Sugar-Dependent Manner. (A) Expression of the WOX7pro:GFP transgene in lateral root primordia on MS medium with or without sucrose, or MS medium containing various concentrations of glucose. Bright field images of the roots are shown in the lower panel. Seeds were germinated in MS medium containing 1% sucrose; 5-day-old seedlings were transferred to MS containing no sugar, 1% sucrose, or 1%, 2%, 4.5% glucose. GFP signal was examined after 24 hrs of treatment. Numbers give signal intensity for GUS staining or GFP fluorescence, as measured with Image J (mean and SD from at least 10 lateral root primordia at the same developmental stage). Bars = 20 μm. (B–E) Number of lateral roots (LRs) per seedling in WT, wox7-1, and a 35S:WOX7-GFP transgenic line 1–6 days (d) after 5-day-old seedlings were transferred to MS medium with B, no sugar; C, 1% sucrose; D and E, 1% or 4.5% glucose as well as 1% sucrose. p < 0.05, t test for all data points 2d after transfer; N = 50. Error bars represent standard deviations. Molecular Plant 2016 9, 261-270DOI: (10.1016/j.molp.2015.11.006) Copyright © 2016 The Author Terms and Conditions

Figure 6 WOX7 Is Not Involved in Stress-Regulated Lateral Root Formation. (A–D) Expression of the WOX7:GFP transgene in lateral root primordia on A, MS medium; or B, MS medium containing 50 mM; or C, containing 100 mM NaCl; or D, containing 200 mM mannitol. White lines and dotted lines are outlines of primary roots and lateral root primordia, respectively. 5-day-old seedlings were transferred to MS containing NaCl or mannitol, and GFP signal was examined after 24 hrs of treatment. Numbers give signal intensity for GUS staining or GFP fluorescence, as measured with Image J (mean and SD from three technical replicates). Bars = 20 μm. (E–G) Number of lateral roots (LRs) per seedling in WT and wox7-1 various days (d) after 5-day-old seedlings were transferred to E, MS medium; F, MS medium containing 100 mM NaCl; or G, containing 200 mM mannitol. p > 0.05, t test; N = 50. Error bars represent standard deviations. Molecular Plant 2016 9, 261-270DOI: (10.1016/j.molp.2015.11.006) Copyright © 2016 The Author Terms and Conditions