The Arabidopsis Transcription Factor AtTCP15 Regulates Endoreduplication by Modulating Expression of Key Cell-cycle Genes  Li Zi-Yu , Li Bin , Dong Ai-Wu   Molecular Plant  Volume 5, Issue 1, Pages 270-280 (January 2012) DOI: 10.1093/mp/ssr086 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 The Expression of AtTCP15 Gene in Arabidopsis. (A) The expression level of AtTCP15 in major organs by real-time RT–PCR analysis. Quantification was normalized to Ubiquitin10 for each organ, and the value of cotyledon was arbitrarily fixed at 1. (B–I) AtTCP15 expression pattern was histochemically analyzed using a GUS reporter gene that was introduced into the wild-type (WT) plants: (B) early lateral root, (C) late lateral root, (D) mature lateral root, (E) vascular tissue in rosette leaves, (F) developing trichomes, (G) 1-day-old leaf after initiation, (H) 3-day-old leaf after initiation, (I) 8-day-old leaf after initiation. Scale bar: 5 mm (B–E); 1 mm (F); 0.5 mm (G); 2 mm (H); 1 cm (I). Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Phenotypes of AtTCP15:AtTCP15SRDX Transgenic Plants. (A) Schematic representation of the AtTCP15:AtTCP15SRDX construct. Black lines represent the 5′ and 3′ regulatory fragments of AtTCP15, and the open boxes represent the 5′ and 3′ untranslated regions. The blue box represents the open reading frame of AtTCP15 and the red box represents the SRDX repression domain consisting of 12 amino acids. (B–F) WT Col-0 (left panel) and AtTCP15:AtTCP15SRDX transgenic plant (right panel): (B) 7-day-old seedlings, (C) 28-day-old plants, (D) inflorescences, (E) siliques, (F) dissected siliques. Scale bar: 5 mm (B); 2 cm (C); 1 cm (D); 5 mm (E); 0.5 mm (F). Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Branch Number and Ploidy Level of Trichome Cells in AtTCP15:AtTCP15SRDX Transgenic Plants. (A, B) SEM images of mature trichome with three branches of WT Col-0 (A) and trichome with five branches of AtTCP15:AtTCP15SRDX transgenic plants (B). (C) Distribution of trichomes with different branches in WT Col-0 and AtTCP15:AtTCP15SRDX plants, respectively. (D–G) Representatives of DAPI stained trichome nuclei of WT Col-0 (D) and AtTCP15:AtTCP15SRDX (E–G). The branch number for each trichome is shown in the upper right of the picture. (H, I) In situ fluorescence was measured using DAPI-stained trichome nuclei of WT Col-0 (H) and AtTCP15:AtTCP15SRDX plants (I). Scale bar: 100 μm (A, B); 5 μm (D–G). Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Ploidy Level and Cell Size of Cotyledon Cells in AtTCP15:AtTCP15SRDX Transgenic Plants. (A) Ploidy level was shown by flow cytometric profiles using PI-stained nuclei. (B–E) Epidermal (B, C) and mesophyll (D, E) cells of WT Col-0 (B, D) and AtTCP15:AtTCP15SRDX (C, E). Scale bar: 100 μm (B, C); 50 μm (D, E). Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Phenotype of AtTCP15 Overexpression Plants. (A-D) 5-day-old seedlings of the WT Col-0 and pTA:AtTCP15–EYFP after germination on MS medium without (A, C) or with (B, D) 30 μM DEX. (E, F) Close-up of representative plants from (C) and (D). (G) Flow cytometric profiles of cotyledon and hypocotyl cells from pTA:AtTCP15–EYFP plants without or with DEX treatment, respectively. Scale bar: 5 mm (E); 2.5 mm (F). Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Phenotypes of Trichomes and Rosette Epidermal Cells in Plants Overexpressing AtTCP15. (A) SEM image of the representative trichome of pTA:AtTCP15–EYFP transgenic plants with DEX treatment. (B) DAPI staining of trichome nucleus of (A). (C) Distribution of trichomes with different branch numbers in pTA:AtTCP15–EYFP plants without or with DEX treatment. (D) In situ fluorescence measurements of DAPI-stained trichome nuclei from pTA:AtTCP15–EYFP plants induced by DEX. (E, F) SEM images of epidermal cells from rosette leaves of pTA:AtTCP15–EYFP plants without (E) or with (F) DEX treatment. (G) Flow cytometric analysis of rosette leaf cells from pTA:AtTCP15–EYFP plants induced by DEX. Scale bar: 100 μm (A, E, F); 5 μm (B). Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Cell-Cycle Control Genes Were Regulated by AtTCP15. (A) The relative expression levels of cell-cycle-related genes were measured by real-time RT–PCR, using 12-day-old seedlings of WT Col-0, AtTCP15:AtTCP15SRDX, and pTA:AtTCP15–EYFP plants induced by DEX for 24 h (AtTCP15 overexpression), respectively. Quantification was normalized to Ubiquitin10 and then to the value of each gene from the WT Col-0 (control), which was arbitrarily fixed at 1. Each cDNA sample was made in triplicate, and the consistent results from three separately prepared RNA samples were used. Bars show standard errors. (B, C) AtTCP15 protein binds to the CYCA2;3 (B) and RBR (C) promoters by ChIP combined with real-time PCR analysis, using 12-day-old seedlings of pTA:EYFP and pTA:AtTCP15–EYFP transgenic plants induced by DEX for 24 h, respectively. Diagrams of the CYCA2;3 and RBR upstream structures are shown above the ChIP analysis panels, in which the long and thick lines show promoter regions, and the short and thin lines marked by A, B, and C indicate the PCR fragments that contain the predicted TCP binding sites, marked by stars. Results were normalized to ACTIN2 and then to the value of each fragment from the pTA:EYFP transgenic plants, which was arbitrarily fixed at 1. Molecular Plant 2012 5, 270-280DOI: (10.1093/mp/ssr086) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions