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First published online 16 November 2005
doi: 10.1242/dev.02139

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Cell-to-cell movement of the CAPRICE protein in Arabidopsis root epidermal cell differentiation

Tetsuya Kurata¹, Tetsuya Ishida¹, Chie Kawabata-Awai¹, Masahiro Noguchi¹, Sayoko Hattori¹, Ryosuke Sano^1,*, Ryoko Nagasaka¹, Rumi Tominaga¹, Yoshihiro Koshino-Kimura², Tomohiko Kato^3,, Shusei Sato³, Satoshi Tabata³, Kiyotaka Okada^1,2 and Takuji Wada^1,

¹ Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Kanagawa 230-0045, Japan
² Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
³ Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan

View larger version (100K): [in a new window]   Fig. 1. Phenotypes and distribution of GFP fluorescence in CPCp::GFP (A-C) and CPCp::CPC:GFP (D-F) transgenic plants. (A,D) Confocal laser scanning microscopy images of GFP fluorescence (green) and propidium iodide (PI) fluorescence (red) in the root epidermis of 5-day-old seedlings. (A) GFP is restricted to hairless cell files and is absent from the file of root hair cells (asterisk). (D) CPC:GFP has moved from hairless cell files to a hair cell file (asterisk; note CPC:GFP accumulation in the nuclei). The inset in D shows that CPC:GFP RNA was localized in hairless cells, as detected by in situ hybridization with an antisense GFP probe. (B,E) Distribution of root hairs in the root epidermis of 5-day-old seedlings. CPCp::CPC:GFP transgenic plants grow ectopic root hairs in positions normally occupied by hairless cells. (C,F) Phenotype of 8-day-old seedlings. No trichome formation was observed in CPCp::CPC:GFP transgenic plants (F). Scale bars: 50 µm in A; 200 µm in B; 1 mm in C.

View larger version (89K): [in a new window]   Fig. 2. Immunolocalization of HA-tagged CPC in roots of the cpc-1 mutant. (A) Schematic representation of the gene construct with the HA-tag at the N terminus of the CPC-coding region (CPCp::HA:CPC). (B) Root phenotype. (C) Immunostaining of HA:CPC in a root transverse section. Signals in nuclei of hair cells are stronger than those of non-hair cells. Root hair cells are marked by asterisks. Scale bars: 200 µm in B; 50 µm in C.

View larger version (33K): [in a new window]   Fig. 3. Western blot of whole-cell extracts and semi-quantitative RT-PCR analysis in Col-0 wild type (Wt), transgenic (CPCp::CPC:GFP, CPCp::HA:CPC cpc-1 and 35S::CPC) and cpc-2 seedlings. (A) CPC or CPC:GFP fusion proteins were detected with anti-CPC antibody, as indicated by the arrowheads. Molecular weights are shown on the right. Equal loading was confirmed by Ponceau staining of the blotted membrane. (B) Semi-quantitative RT-PCR analysis. One microgram of total RNA was used for each reverse transcription reaction. PCR: 26 cycles for CPC, CPC:GFP or HA:CPC; 22 cycles for the EF1 control.

View larger version (58K): [in a new window]   Fig. 4. Localization of truncated CPC in cells of the root epidermis. (A) Schematic representation of CPC, consisting of three non-overlapping domains (N, N-terminal domain; Myb, Myb domain; C, C-terminal domain), and the four truncated forms of the CPC:GFP fusion protein. At the bottom, the construct consisting of the CPC promoter, a truncated CPC and 2xrsGFP is shown. (B-E) CLSM images showing GFP fluorescence (green) and PI fluorescence (red) in the root epidermis of 5-day-old seedlings. (B,D) NMG moved from hairless cells to hair cells (hair cell file is highlighted by an asterisk), whereas NG did not. (C,E) Reduced movement of MG and MCG was indicated by the patchy occurrence of GFP fluorescence in hair cells. NMG, MG and MCG accumulated in the nuclei (B,C,E), while NG was dispersed throughout the hairless cells (D). Scale bar: 50 µm.

View larger version (41K): [in a new window]   Fig. 5. Identification of a signal domain that is necessary and sufficient for inter- and/or intracellular movement of the protein. (A) Diagram of the deletion mutants of CPC. The N-terminal domain is shown in red, the Myb domain in blue. (B-D) CLSM images showing GFP fluorescence (green) and PI fluorescence (red) in the root epidermis of 5-day-old seedlings. (B) Like full-length CPC fused to GFP, 1-79G moved from hairless cells to root hair cells (file of hair cells is indicated by an asterisk) and accumulated in the nuclei of both cell types. (C) In contrast to 1-79G, 1-75G remained distributed throughout the cytoplasm of the hairless cells. (D) A partial defect in CPC movement in 10-83G was indicated by the occurrence of the GFP signal only in a fraction of hair cells, with 1-79G and 10-83G accumulated in nuclei. (E) Diagrammatic representation of a signal domain (residues 1 to 79, as indicated by the yellow and green bar) that is necessary and sufficient for CPC cell-to-cell movement. The domain, amino acid residues 1 to 79, contains the N terminus and a part of the Myb domain. The regions whose deletions conferred defects in the intercellular movement of CPC:GFP were tentatively designated S1 and S2 (as indicated by the yellow bars). As the behavior of 1-75G shows (C), S2 also is required for the nuclear accumulation of CPC. Scale bar: 50 µm.

View larger version (64K): [in a new window]   Fig. 6. Localization of amino acid substitution mutants of CPC in roots, and phenotypes of the mutant plants. (A,D,G,J,M) CLSM images of GFP fluorescence (green) and PI fluorescence (red) in the root epidermis of 5-day-old seedlings. (B,E,H,K,N) Root hair phenotypes of 5-day-old seedlings. (C,F,I,L,O) Trichome phenotypes of 9-day-old seedlings. K6A:K9A, L77V and K79A moved from hairless cells to hair cells, and accumulated in the nuclei of both cell types (A,D,G). The plants developed ectopic root hairs (B,E,H) but no trichomes, thus resembling lines expressing CPC:GFP (C,F,I). The capability for cell-to-cell movement was reduced in M78A, but it accumulated in the nuclei (J). M78A formed ectopic root hairs but no trichomes (K,L). The W76A form of the protein did not move from hairless cells to root hair cells, and GFP-derived fluorescence was dispersed throughout the cells (M). Normal root hair formation and trichome formation were observed in this mutant (N,O). (P,Q) Alignment of the N termini, and of the C-terminal regions of the Myb domains, respectively, of CPC and homologous small Myb proteins. Small Myb protein sequences were compiled from Schellmann et al. (Schellmann et al., 2002) (TRY) and Kirik et al. (Kirik et al., 2004a; Kirik et al., 2004b) (ETC1 and ETC2). The S1 region is unique to CPC, whereas W76 and M78 in S2 are conserved in all small Myb proteins. Scale bars: 50 µm in A; 200 µm in B; 1 mm in C.

View larger version (80K): [in a new window]   Fig. 7. Tissue distribution of the CPC:GFP fusion protein expressed under the control of heterologous promoters. The figure shows longitudinal (A,D) and transverse (B,E) CLSM images with GFP (green) and PI fluorescence (red) in SHRp::CPC:GFP (A,B) and EGL3p::CPC:GFP (D,E) transgenic plants at 5 days post germination, and the corresponding root hair phenotypes (C,F). (A) Images are focused on the stele. When CPC:GFP is controlled by the stele-specific SHR promoter, the GFP signal is restricted to stele cells, where it is distributed throughout the cytoplasm (A). No signal was detected in the root cortex or the epidermis (B). No ectopic root hairs were observed in these plants (C). By contrast, GFP fluorescence was restricted to the epidermis when CPC:GFP expression was controlled by the hair cell-specific EGL3 promoter (D,E). The signal occurred in all epidermal cells and accumulated in nuclei. Root hairs were formed ectopically in these plants (F). Insets in A,D show the distribution of GFP fluorescence in SHRp::GFP and EGL3p::GFP control plants, respectively. The asterisk in D highlights a hair-cell file. ep, epidermis; co, cortex; en, endodermis. Scale bars: 50 µm in A,B; 200 µm in C,F.

View larger version (137K): [in a new window]   Fig. 8. Effects of BFA and the rhd3 mutation on CPC:GFP movement. (A,B,E) CLSM images of GFP (A,B,E) and PI (E) fluorescence in root epidermis of 5-day-old seedlings grown on media supplemented with (A) 0 µM BFA or (B)10 µM BFA. (C,D) Root hair phenotypes of (C) rhd3-1 and (D) rhd3-1 CPCp::CPC:GFP 5-day-old seedlings. (E) CLSM image of the root epidermis of D. Asterisks in A, B and E indicate hair-cell files. Scale bars: 50 µm in A; 200 µm in C; 50 µm in E.

View larger version (81K): [in a new window]   Fig. 9. Inter- and intracellular movement of CPC fused to tandem GFPmers. (A-D) CLSM images of GFP fluorescence (green) and propidium iodide (PI) fluorescence (red) in the root epidermis of 5-day-old seedlings. The CPC promoter-driven CPC was fused to 2xGFP (A), 3xGFP (B), 4xGFP (C) and 5xGFP (D). GFP fluorescence was observed in epidermal cell nuclei with 2xGFP or 3xGFP fusions (A,B), but patchy GFP fluorescence in hair cells was observed in CPC:4xGFP and CPC:5xGFP lines (C,D). CPC fused to greater than 3xGFP reduced the nuclear-specific localization (C,D).