Volume 9, Issue 8, Pages 1197-1209 (August 2016) Conservation and Diversification of the SHR-SCR-SCL23 Regulatory Network in the Development of the Functional Endodermis in Arabidopsis Shoots  Eun Kyung Yoon, Souvik Dhar, Mi-Hyun Lee, Jae Hyo Song, Shin Ae Lee, Gyuree Kim, Sejeong Jang, Ji Won Choi, Jeong-Eun Choe, Jeong Hoe Kim, Myeong Min Lee, Jun Lim  Molecular Plant  Volume 9, Issue 8, Pages 1197-1209 (August 2016) DOI: 10.1016/j.molp.2016.06.007 Copyright © 2016 The Author Terms and Conditions

Figure 1 Protein Complexes of SHR and SCL23 Regulate Transcription of SCL23. (A and B) Expression of the ProSCL23:GUS transcriptional fusion in 10-day-old WT (A) and shr-2 (B) shoots. Scale bars represent 1 cm. (C) qRT–PCR of SCL23 mRNA abundance in WT and shr-2 shoots. The value for WT is set to 1 and the value relative to WT is shown. (D) Arabidopsis protoplast transient expression assays. Schematic representation of the effector and reporter plasmids is illustrated. The reporter consists of the SCL23 promoter (ProSCL23) and a firefly luciferase coding sequence (LUC). The effector plasmids have the coding regions of either SHR or SCL23 under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The relative LUC activity was measured, when SHR alone or SHR + SCL23 were transfected. The background value with the GUS effector is set to 1 as the control, and the values relative to the control are shown. (E) ChIP–PCR assays for SHR binding to the SCL23 promoter with ProSHR:SHR-GFP in shr-2. The representative SCL23 promoter regions from the translational start are indicated, and the known SHR-binding region in the SCR promoter (ProSCR) is included as a positive control. Regions exhibiting prominent enrichment of SHR binding to the SCL23 promoter regions, compared with the positive control (ProSCR), are highlighted in red. (F) ChIP–PCR for SHR binding to the SCL23 promoter with ProSHR:SHR-GFP in WT and scl23-1 shoots. The enrichment of SHR binding to the three prominent fragments in (E) was considerably reduced in the scl23-1 background. Error bars indicate mean ± SE from three biological replicates. Statistically significant differences were determined by Student's t-test (*p < 0.05). Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 2 Relationships of SHR, SCR, and SCL23 in the SHR-SCR-SCL23 Network. (A–F) Phenotypic analysis of 20-day-old shr-2 (A), shr-2 scl23-1 (B), scl23-1 (C), scr-3 (D), scr-3 scl23-1 (E), and wild-type (WT) (F) plants. Scale bar represents 1 cm. (G) Transient expression assays for the interactions of SHR, SCR, and SCL23 with the SCL23 promoter. The effector and reporter plasmids are schematically shown. The relative LUC activity was determined, when SCR alone, SCL23 alone, both SCR and SCL23, or the three GRAS TFs were transfected. The background value with the GUS effector is set to 1 as the control, and the values relative to the control are indicated. Error bars indicate mean ± SE from three biological replicates. Statistically significant differences were determined by Student's t-test (*p < 0.05; ** p < 0.01). Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 3 Phenotypic Analysis of SCL23-OX and SCL23-SRDX Shoots. (A–C) 20-day-old WT (A), SCL23-OX (B), and SCL23-SRDX (C) plants. Scale bar represents 1 cm. (D) Plant height of WT, SCL23-OX, and SCL23-SRDX plants at different days post germination (dpg). (E) Length of WT, SCL23-OX, and SCL23-SRDX leaves at 20 dpg. (F) Length of WT, SCL23-OX, and SCL23-SRDX petioles at 20 dpg. Error bars indicate mean ± SE (n = 6). Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 4 SCL23 Negatively Regulates Transcription of SHR in the Shoot. (A) qRT–PCR of SHR mRNA abundance in WT, SCL23-OX, and SCL23-SRDX shoots. The expression level of SHR in WT is set to one and the values relative to WT are shown. (B and C) Expression of the ProSHR:GUS transcriptional fusion in 10-day-old WT (B) and shr-2 (C) leaves. Scale bars represent 100 μm. (D) Transient expression assay for the interaction of SCL23 and the SHR promoter. Schematic illustration for the effector and reporter plasmids is shown. Addition of SCL23 alone reduced transcription of SHR, whereas SHR alone or SCR alone had nearly no influence on SHR expression. The background value is set to 1 as the control, and the values relative to the control are indicated. (E) ChIP–PCR assay for SCL23 binding to the SHR promoter. The representative SHR promoter regions from the translational start are indicated, and the SHR gene body region (SHR) is included as a negative control. Prominent enrichment of SCL23 binding to the SHR promoter fragment is shown in red. Error bars indicate mean ± SE from three biological replicates. Statistically significant differences were determined by Student's t-test (*p < 0.05; **p < 0.01). Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 5 SCL23 Can Restrict Intercellular Movement of SHR in the Hypocotyl. (A) Localization of the SHR-GFP recombinant protein in the endodermis of the WT hypocotyl. The arrowhead indicates the nuclear localization of SHR-GFP in the hypocotyl endodermis. (B) Localization of SHR-GFP in the SCL23-OX hypocotyl. No signal is detected in the endodermis. The epidermis (Ep), cortex (Co), and endodermis (En) layers are indicated. Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 6 Characterization of Hypocotyl Gravitropic Responses and Amyloplast Sedimentation. (A–E) Gravitropic responses of WT (A), shr-2 (B), scr-3 (C), SCL23-OX (D), and SCL23-SRDX (E) hypocotyls. Dark-grown seedlings at 3 dpg were rotated by an angle of 90° and incubated for 2 days. Scale bar represents 0.5 cm. (F–J) Amyloplast staining of WT (F), shr-2 (G), scr-3 (H), SCL23-OX (I), and SCL23-SRDX (J) hypocotyls. In response to the change of the gravity vector, amyloplasts precipitated in the bottom of the endodermis in the WT hypocotyl. By contrast, defects in amyloplast sedimentation were found in shr-2, scr-3, SCL23-OX, and SCL23-SRDX hypocotyls. Scale bar represents 100 μm. The arrow indicates the direction of gravity (g). The epidermis (Ep), cortex (Co), endodermis (En), and mutant (M) layers are indicated. Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 7 Characterization of the Hypocotyl Radial Pattern. (A–G) Transverse sections of the hypocotyls of WT (A), shr-2 (B), scr-3 (C), SCL23-OX (D), SCL23-SRDX (E), scr-3 scl23-1 (F), and triple-mutant (shr-2 scr-3 scl23-1) (G) seedlings. Both SCL23-OX and SCL23-SRDX hypocotyls contain three layers of the ground tissue (one endodermis and double cortex). Scale bar represents 40 μm. (H–N) Suberin staining of the hypocotyls of WT (H), shr-2 (I), scr-3 (J), SCL23-OX (K), SCL23-SRDX (L), scr-3 scl23-1 (M), and triple-mutant (shr-2 scr-3 scl23-1) (N) seedlings. The red arrowheads indicate positive cells of the innermost ground tissue by suberin staining. Unlike those in the shr-2 hypocotyl, both SCL23-OX and SCL23-SRDX hypocotyls have the endodermis with positive cells to suberin staining. Scale bar represents 25 μm. The epidermis (Ep), cortex (Co), endodermis (En), and mutant (M) layers are indicated. Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions

Figure 8 A Schematic Model for the Molecular Interactions of SHR, SCR, and SCL23 in the Development of the Shoot Endodermis. The master regulator SHR directly activates expression of both SCL23 and SCR in the endodermis and its equivalent tissues. When ectopically overexpressed, SCL23 can negatively regulate transcription of SHR. Both SCL23 and SCR interact with SHR, resulting in the restriction of SHR movement. SCR also can activate SCL23 expression. Based on what we know to date, SCR and SCL23 interact with each other at protein levels. Activation of SCR and SCL23 depend on their own presence in protein complexes, indicating autoregulation. Transcriptional controls (TC) are indicated in blue, and protein–protein interactions (PPI) are depicted in red. Arrows represent positive regulation, while bars denote negative regulation. Molecular Plant 2016 9, 1197-1209DOI: (10.1016/j.molp.2016.06.007) Copyright © 2016 The Author Terms and Conditions