A Feedback Mechanism Controlling SCRAMBLED Receptor Accumulation and Cell- Type Pattern in Arabidopsis  Su-Hwan Kwak, John Schiefelbein  Current Biology  Volume 18, Issue 24, Pages 1949-1954 (December 2008) DOI: 10.1016/j.cub.2008.10.064 Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 1 Localization of the SCM-GFP Fusion Protein (A and B) Four-day-old scm-2 SCM::SCM-GFP roots were stained with propidium iodide (red) for visualizing the cell wall and SCM-GFP accumulation (green) in longitudinal median view (A) and epidermal view (B). The upper part of (B) shows the underlying cortex. White asterisks indicate H cell files. The scale bar represents 50 μm. (C and D) Four-day-old scm-2 SCM::SCM-GFP roots were treated with water (C) or 1 M mannitol for 60 min for inducing plasmolysis (D). The scale bar represents 25 μm. (E–I) Accumulation of SCM-GFP in scm-2 SCM::SCM-GFP embryos in median view of heart stage (E), protoderm of heart stage (F), median view of torpedo stage (G), protoderm of torpedo stage (H), and epidermis of mature embryo (I). The scale bar represents 50 μm. Current Biology 2008 18, 1949-1954DOI: (10.1016/j.cub.2008.10.064) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 2 Inhibition of SCM-GFP Expression by Tissue-Specific RNAi (A–F) Confocal images for SCM-GFP (green, left) and propidium iodide (red, right) fluorescence with two separate channels from the same root for each RNAi line, showing (A) scm-2 SCM::SCM-GFP GL2::GUS (no RNAi), (B) scm-2 SCM::SCM-GFP GL2::GUS pSHR::SCM-RNAi, (C) scm-2 SCM::SCM-GFP GL2::GUS pSCR::SCM-RNAi, (D) scm-2 SCM::SCM-GFP GL2::GUS pCo2::SCM-RNAi, (E) scm-2 SCM::SCM-GFP GL2::GUS pWER::SCM-RNAi, and (F) scm-2 SCM::SCM-GFP GL2::GUS pSCM::SCM-RNAi. Asterisks indicate the tissue layer showing major reduction in SCM-GFP level caused by RNAi. The scale bar represents 50 μm. (G) Quantification of the epidermal cell-type pattern, showing frequencies of ectopic GL2::GUS-expressing cells in the H cell position (gray bars) and ectopic non-GL2::GUS-expressing cells in the N cell position (black bars). The mean and standard deviation are indicated for each line. Current Biology 2008 18, 1949-1954DOI: (10.1016/j.cub.2008.10.064) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 3 Tissue-Specific Expression of SCM-GFP in scm-2 Mutant Roots (A–O) Four-day-old roots of scm-2 GL2::GUS plants with different complementation constructs were stained with propidium iodide (red) and analyzed for SCM-GFP accumulation (green). Note that three independent lines transformed with the WER::SCM-GFP are presented here (−10, −4, and −14). Shown are (A) WER::SCM-GFP-10 median view, (B) WER::SCM-GFP-10 epidermal view, (C) WER::SCM-GFP-4 median view, (D) WER::SCM-GFP-4 epidermal view, (E) WER::SCM-GFP-14 median view, (F) WER::SCM-GFP-14 epidermal view, (G) Co2::SCM-GFP median view, (H) SCR::SCM-GFP median view, (I) SHR::SCM-GFP median view, (J) WER::SCM-GFP-10 SCR::SCM-GFP median view, (K) WER::SCM-GFP-10 SHR::SCM-GFP median view, (L) WER::SCM-GFP-4 Co2::SCM-GFP median view (M) GL3::SCM-GFP-5 median view, (N) GL3::SCM-GFP-5 epidermal view, and (O) 35S::SCM-GFP-5 epidermal view. The scale bar represents 50 μm. (P) Quantification of the epidermal cell-type pattern, showing frequencies of ectopic GL2::GUS-expressing cells in the H cell position (gray bars) and ectopic non-GL2::GUS-expressing cells in the N cell position (black bars) for each line. The mean and standard deviation are indicated for each line. Current Biology 2008 18, 1949-1954DOI: (10.1016/j.cub.2008.10.064) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 4 Effect of Transcription Factor Mutants on SCM Expression and Accumulation (A–F) Four-day-old roots of scm-2 SCM::SCM-GFP plants containing the indicated homozygous mutation(s) were analyzed by confocal microscopy. Green and red indicate SCM-GFP and propidium iodide, respectively. The underlying cortex layer is shown in the upper panels. Asterisks indicate the H cell files. The scale bar represents 50 μm. (G–L) Four-day-old roots of the indicated wild-type and mutants bearing the SCM::GUS reporter gene were analyzed for GUS expression. Asterisks indicate the epidermis tissue layer. The scale bar represents 50 μm. Current Biology 2008 18, 1949-1954DOI: (10.1016/j.cub.2008.10.064) Copyright © 2008 Elsevier Ltd Terms and Conditions

Figure 5 A Model for Indirect Autoregulatory Feedback Control of SCM Expression SCM signaling represses WER transcription in H cells, which leads to differential transcription factor activities in the H and N cells. As a result, SCM expression and accumulation is increased in the H cells (by CPC and TRY) and inhibited in the N cells (by the WER-GL3/EGL3-TTG1 complex). Solid lines indicate positive (represented by arrows) and negative (represented by blunt lines) transcriptional regulation. Dotted lines indicate protein movement. Current Biology 2008 18, 1949-1954DOI: (10.1016/j.cub.2008.10.064) Copyright © 2008 Elsevier Ltd Terms and Conditions