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doi: 10.1242/10.1242/dev.00111

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Role of a positive regulator of root hair development, CAPRICE, in Arabidopsis root epidermal cell differentiation

Takuji Wada1, Tetsuya Kurata1, Rumi Tominaga1, Yoshihiro Koshino-Kimura1,2, Tatsuhiko Tachibana2,*, Koji Goto3, M. David Marks4, Yoshiro Shimura5 and Kiyotaka Okada1,2,{dagger}

1 Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-Ku, Yokohama, Kanagawa 230-0045, Japan
2 Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
3 Research Institute for Biological Science, Kayo-cho, Jobo, Okayama 716-1241, Japan
4 Department of Genetics and Cell Biology, University of Minnesota, St. Paul, MN 55108-1095, USA
5 Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan
* Present address: Chugai Pharmaceutical Co. Ltd., 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan



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  		Fig. 1. Structure of Arabidopsis roots. Drawings of a transverse section showing the cellular organization (A), and root meristems of wild type (B), caprice (cpc) mutant (C), and gl2, ttg, wer and 35S::CPC mutants (D). (A) The wild-type root is made up of five tissues, outermost is the epidermis, then inside that is cortex, endodermis, pericycle and vascular tissue. Epidermal cells are of two types: hair cells and hairless cells. The stele includes pericycle and vascular tissue. A few hairs are formed randomly in the cpc mutant (C), whereas hairs are formed in all of the epidermal cells in the gl2, ttg and wer mutants, and in the 35S::CPC transgenic plant (D).

 


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  		Fig. 2. Patterns of root hair formation in the primary roots of 5-day-old seedlings. (A,H) Wild type, (B) cpc, (C) 35S::CPC, (D,I) gl2-1, (E) 35S::R, (F,J) 35S::RN and (G,K) GL2 promoter::CPC in cpc. Asterisk indicates cell files forming root hairs. (A to G and H to K are of the same magnification. Scale bars in A (for A-G) 200 µm; in H (for H-K) 50 µm.

 


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  		Fig. 5. Interaction between CPC and R in yeast cells. (A) Schematic representation of the CPC and R proteins. Regions required for the interaction are indicated. (B) Interaction between CPC and R. (C) Interaction between intact CPC and truncated R. GBD-bound CPC and the series of GAD-bound R were used as bait and prey, respectively. (D) Interaction between truncated CPC and the N-terminal region of R. GBD-bound R of residues 1-371 and a series of GAD-bound CPC were used as bait and prey, respectively. The activity of ß-galactosidase was assayed using three to six independently transformed yeast lines.

 


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  		Fig. 3. Spatial expression pattern of CPC promoter::GUS and GL2 promoter::GUS in different mutant backgrounds. Transverse (A-J) and longitudinal (K-P) sections of 5-day-old seedlings were stained with X-Gluc. (A-F) Expression of CPC::GUS in wild type (A), cpc (B), ttg-1 (C), 35S::R (D), rhd6-1 (E) and ctr-1 (F). (G-J) Expression of GL2::GUS in wild type (G), cpc (H), ttg-1 (I), and 35S::R (J). (K-M) Expression of CPC::GUS in wild type (K), 35S::CPC (L), 35S::R (M). (N-P) Expression of GL2::GUS in wild type (N), 35S::CPC (O), 35S::R (P). Photos and are of the same magnification. Scale bars in A (for A-J) and K (for K-P), 50 µm.

 


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  		Fig. 4. In situ hybridization pattern of CPC and GL2 in roots sections. (A-N) Hybridization of transverse sections using the following probes: antisense of CPC (A-F), antisense of GL2 (G-L), and sense of CPC (M,N). Photos were taken at the same magnification in (A-C,G-I,M) Bright-field, (D-F,J-L,N) dark-field illumination. (A,D,G,J,M,N) Wild type, (B,E) gl2-1, (C,F,I,L) ttg-1, (H,K) cpc. Scale bar: 25 µm.

 


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  		Fig. 6. In vitro binding of CPC to R. The intact and truncated R proteins were translated in vitro, labeled with [35S]methionine, incubated with or without the purified GST-CPC protein or with the GST protein, and adsorbed on glutathione-Sepharose. The Sepharose-bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Lane 1: intact R protein before incubation; lane 2: N-terminal region of R (residues 1-312) before incubation; lane 3: C-terminal region of R (residues 372-525) before incubation; lane 4: intact R incubated with GST-CPC; lane 5: N-terminal region of R incubated with GST-CPC; lane 6: C-terminal region of R incubated with GST-CPC; lane 7: intact R incubated with GST; lane 8: N-terminal region of R incubated with GST; lane 9: C-terminal region of R incubated with GST.

 


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  		Fig. 7. Analysis of CPC promoter::CPC:GFP transgenic roots. Confocal images of (A) CPC promoter:GFP, (B) CPC promoter::CPC::GFP and (C) CPC promoter::NLS:GFP. Asterisks in A and C indicate hairless cell file. Scale bar, 30 µm.

 




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