Volume 7, Issue 12, Pages 1727-1739 (December 2014) SPINDLY, ERECTA, and Its Ligand STOMAGEN Have a Role in Redox-Mediated Cortex Proliferation in the Arabidopsis Root  Hongchang Cui, Danyu Kong, Pengcheng Wei, Yueling Hao, Keiko U. Torii, Jin Suk Lee, Jie Li  Molecular Plant  Volume 7, Issue 12, Pages 1727-1739 (December 2014) DOI: 10.1093/mp/ssu106 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Quantitative RT–PCR Assay, Showing Altered Transcript Levels of Genes Involved in Redox Homeostasis in spy-3 Root. At3g49120 (PERX33), At3g49110 (PERX34), At5g64120, At1g34510, At4g33420, and At5g67400 (PERX73) are peroxidases; At1g31710 is a copper amine oxidase; At1g20620 (CAT3) is a catalase; At1g19570 and At1g19550 are dehydroascorbate reductases; At4g25090 is a reactive oxygen-burst homolog. These genes were selected because microarray data showed that their expression level was altered by the spy mutation. The asterisks indicate the significance of the change by t-test. * p < 0.05; ** p < 0.01; *** p < 0.001. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Hydrogen Peroxide Assays Showing Elevated ROS Level in the spy Mutants. (A) Quantitative assay of H2O2 in the roots of 1-week-old seedlings grown in MS medium. The error bars represent standard deviation from triplicate measurements. The differences are highly significant (p < 0.001 or 0.01 for the Col versus spy-3 and Ler versus spy-8 comparisons, respectively; t-test). (B, C) Detection of H2O2 with dichloroflurescin diacetate. The numbers below different genotypes are the fluorescence intensity in the meristem zone (mean ± standard deviation, N = 15), as marked by arrowhead (left) and also outlined by broken line (right). Bars = 50 μm. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Middle-Cortex Formation Is Regulated by Cellular Redox Status. Confocal-microscopy images of wild-type (Col) (A, B) and spy-3 roots (C, D), 24h after transfer into water (A, C), 1mM H2O2 (B), or 1mM glutathione (D). The framed areas are shown on the right at a higher magnification. mc, middle cortex; c, cortex; e, endodermis. Bars = 20 μm. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 The Receptor Kinase ERECTA Is Required for Redox-Mediated Cortex Proliferation. Confocal microscopy images of wild-type (Ler, (A, B)), er-105(C, D), and spy-8 roots (E, F), 24h after transfer into water (A, C, E), 1mM H2O2 (B, D), or 1mM glutathione (F). c, cortex; mc, middle cortex; e, endodermis. Bars = 20 μm. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 ERECTA and STOMAGEN Regulate Redox-Mediated Cortex Proliferation through Intercellular Signaling. (A, C, E) Expression pattern of ERECTA (A), EPFL4 ((C), left), EPFL6 ((C), right), and STOMAGEN ((E), bright field in left and fluorescence in the right), as shown by promoter–reporter transgenes. The reporter gene is GUS in (A) and (C), and VENUS in (E). (B) Root radial pattern in er-105 mutant expressing ERECTA (ER) in the epidermis ((B), left), the xylem ((B), middle), or the phloem ((B), right) using the promoter of ML1, IRX3, or SUC2, respectively, after 24h of treatment with H2O2. (D, F) Root radial pattern in the epfl4 epfl6 double mutant (D) or transgenic plants that contain an artificial miRNA targeting STOMAGEN (ST-miR) (F), after 24h of treatment with water (control) or H2O2. c, cortex; mc, middle cortex; e, endodermis. Bars = 20 μm. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Effects on ROS on SPY Transcription, Alternative Splicing, and Protein Oxidization. (A) Diagram of alternative splicing of the SPY transcript, and the position of PCR primers for detection of the two isoforms. (B) RT–PCR assay of SPY transcript isoforms in the absence (Ctl) or presence of 1mM H2O2. The 18S rDNA was used as an internal control. The change is significant for Col (p < 0.05, t-test) but not for Ler. (C) Relative SPY transcript level in Col and Ler roots before and after treatment with water or 1mM H2O2 for 24h, as determined by real-time RT–PCR. The error bars represent standard deviations from triplicate measurements. (D) SPY–GFP protein level in roots after 24h of treatment with water and 1mM H2O2. Equal amounts of total protein extracts were loaded. (E) Western blot assay of the SPY–GFP protein in the presence (+) or absence of the reducing reagent dithiothreitol (DTT). The numbers on the left are the sizes of protein ladders. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Schematic of the Interplay between SPY and the STOMAGEN–ERECTA Signaling Pathway in ROS Sensing and Redox-Mediated Cortex Proliferation in the Arabidopsis Root. Under stress, plants accumulate ROS, which oxidize and activate STOMAGEN. STOMAGEN in turn activates ERECTA, which exerts its effect on mitosis in the endodermis through intercellular signaling. ROS also oxidizes and inactivates SPY, which normally represses middle-cortex formation by maintaining the expression of peroxidases and thus cellular redox. SPY acts downstream of ERECTA, but how SPY is affected by the ERECTA signaling pathway remains unknown. Molecular Plant 2014 7, 1727-1739DOI: (10.1093/mp/ssu106) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions