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

Position dependency in developmental processes is a critical aspect for building the multicellular body of plants and requires intercellular communication. Intercellular communication is mediated by secreted signal molecules, such as small peptides(Matsubayashi et al., 2001). A mode of cellular communication in plants is the direct movement of signaling molecules through plasmodesmata, the cell wall pores that provide cytoplasmic continuity in plant cells. Plasmodesmata enable the transport of nutrients, as well as of macromolecules such as RNA and proteins(Crawford and Zambryski, 1999; Haywood et al., 2002), and have specific size exclusion limits (SELs) that depend on the developmental phase of the plant (Oparka et al.,1999). The cell-to-cell movement of macromolecules is generally considered to be crucial in plant development(Zambryski, 2004), and recent studies have underscored the significance of the cell-to-cell movement of plant transcription factors (Ruiz-Medrano et al., 2004). The intercellular movement of transcription factors in animal cells has also been described. Several animal homeodomain proteins,such as Engrailed (En), Hoxa5, Hoxb4, Hoxc8, Emx1, Emx2, Otx2 and Pax6, move intercellularly in cultured cells (ex vivo)(Prochiantz and Joliot, 2003). Intercellular movement of En protein is driven by secretion and internalization, and this secretion process is regulated by the phosphorylation of En itself (Joliot et al., 1998; Maizel et al.,1999; Maizel et al.,2002). The intercellular movement of animal transcription factors may provide a model for analysis of the movement of transcription factors in plants.

SHORT-ROOT (SHR), a member of the GRAS family of putative transcription factors, moves from stele cells to the endodermis, where it activates endodermal cell differentiation and cell division in Arabidopsis(Helariutta et al., 2000; Nakajima et al., 2001). When the SHR:GFP fusion protein was expressed in several tissues under the control of tissue-specific promoters, it was unable to move from phloem companion cells and epidermal cells, suggesting a requirement for tissue-specific SHR movement factors (Sena et al.,2004). Further investigation demonstrated that SHR must be localized in the cytoplasm to move intercellularly. However, the mere presence of SHR in the cytoplasm is not sufficient for movement(Gallagher et al., 2004).

The maize homeobox protein KNOTTED1 (KN1) controls leaf formation and has been shown to move from inner cells to epidermal cells, possibly through plasmodesmata (Lucas et al.,1995). Leaf injection experiments demonstrated that KN1 increases the SEL of plasmodesmata and induces the movement of the kn1 RNA/KN1 protein complex (Kragler et al.,2000; Lucas et al.,1995). Cell-to-cell movement of KN1:GFP fusion proteins was investigated in Arabidopsis using heterologous promoters. Like the natural protein, the fusion product was able to move from inner tissue layers to the leaf epidermis in Arabidopsis. By contrast, KN1:GFP moved from the epidermal L1 layer towards the inner cell layers in the shoot apical meristem. Thus, the movement of KN1 is regulated in a tissue-specific manner(Kim et al., 2002; Kim et al., 2003).

The mode of movement was investigated for the transcription factor LEAFY(LFY), one of the floral identity genes in Arabidopsis. By using GFP-fusion proteins controlled by the L1-specific promoter ATML1, the movement of LFY was shown to be non-targeted and driven by diffusion. This interpretation was supported by the correlation between cytoplasmic localization and the ability of LFY to move(Wu et al., 2003). These studies suggest that there are at least two modes of protein cell-to-cell movement, including non-targeted translocation by diffusion, and targeted,regulated transport. Targeted movement is thought to be mediated by specific interactions between the transported protein and plasmodesmata components,leading to an increase in the SEL. Plant viral movement proteins (MPs) provide the best-characterized case of targeted protein movement. Cytoskeletal elements, including actin filaments and microtubules, are involved in the targeted movement of MPs (Kawakami et al.,2004; Kragler et al.,2003). However, the movement mechanisms of endogenous proteins are obscure.

To elucidate the mechanisms of cell-to-cell movement of regulatory proteins, we are studying the small Myb-like protein CAPRICE (CPC), a positive regulator of root hair formation in Arabidopsis. CPC is a small protein of 94 amino acids, with a single Myb-R3 domain. Loss-of-function mutants develop a reduced number of normal-shaped root hairs(Wada et al., 1997). Although this morphological defect in cpc mutants is obviously related to hair-cell differentiation in trichoblasts (hair cells), CPC mRNA expression is detected only in atrichoblasts (hairless cells). Using CPC:GFP fusion products, we established that CPC was localized to the nuclei of all epidermal cells, implying that the CPC:GFP fusion protein moved from atrichoblasts to trichoblasts in the Arabidopsis root epidermis(Wada et al., 2002).

In the present study, we confirmed by immunohistochemistry that the native CPC protein is localized in hair cells. By employing truncated CPC proteins fused to GFP, we further demonstrated that two motifs, one in the N-terminal region and the other in the Myb domain, are responsible for the cell-to-cell movement of CPC. Amino acid substitution experiments on CPC:GFP indicated that both W76 and M78 in the Myb domain are critical for cell-to-cell movement. Moreover, the W76A mutation reduced the nuclear accumulation of CPC:GFP. We also expressed CPC:GFP in stele cells and in root hair cells by linking the gene construct to the SHR or EGL3 promoters, respectively. CPC:GFP moved from root hair cells to hairless cells, but not from the stele to the epidermis, indicating a tissue-specific regulation of CPC movement. Analyses with a secretion inhibitor, Brefeldin A, and an rhd3 mutant suggested that plasmodesmata are the route of CPC movement. Fusion of CPC to tandem-GFPs showed the ability of CPC to increase the SEL of plasmodesmata. Finally, we discuss the structural properties required for CPC movement in the Arabidopsis root epidermis.

Primer sequences are listed in Table S1 in the supplementary material.

To create a plasmid with a gene encoding HA-tagged CPC, a CPCgenomic DNA fragment pBS-gCPC including the 1.3 kb 5′ region,the 0.9 kb coding region and the 0.45 kb 3′ regions(Wada et al., 1997) was used. HA1-F/HA1-R oligonucleotides (for HA peptide) were annealed and inserted into the 5′ portion of the coding region of a PCR-amplified linear CPC genome fragment with TK383 and TK384 (pBS-CPCp-HA-CPC). The XbaI and SalI fragments of pBS-CPCp-HA-CPC were cloned into the pJHA212K binary vector(Yoo et al., 2005).

To create plasmids with truncated CPC sequences, the 2×rsGFP-NosT fragment was amplified with TK106/TK107 from pRTL2-2×rsGFP(Crawford and Zambryski, 2000)as a template, and cloned into the pT7 blue T-vector (Novagen). EcoRV and XbaI fragments of 2×rsGFP-NosT were cloned into pBluescriptII SK+ (pBS-2×rsGFP-NosT). The CPC promoter region was amplified with TK100/TK101 using Pyrobest DNA polymerase (Takara, Japan), and was cloned into pT7 (pT-CPCp). pT-CPCp was digested with SphI, blunt-end filled with T4 DNA polymerase (Takara, Japan) and re-digested with SmaI. After pBluescriptII had been digested with SacI and EcoRV, and treated with T4 DNA polymerase to abolish the SacI site, the CPCp fragment was cloned into this modified pBluescriptII(pBS-CPCp). All truncated CPC-coding regions were amplified by PCR with the following primers: TK102/TK103 for NMG, TK104/TK103 for MG,TK102/TK128 for NG and TK 104/TK201 for MCG. TK102 was used as an upstream primer for the PCR-amplification of constructs of the truncated NM region. Downstream primers were TK186 for 1-79G, TK180 for 1-75G, TK181 for 1-65G,TK182 for 1-55G and TK183 for 1-45G. For additional plasmids containing the truncated NM region, the following primers were used: TK184/TK103 for 10-83G,TK185/TK103 for 21-83G and TK184/TK182 for 10-55G. Amplified, truncated CPC-coding regions were cloned into pT7, and digested with SacI and EcoRV. These fragments were cloned into the SacI and EcoRV sites of pBS-CPCp(pBS-CPCp-trCPC). A CPCp-trCPC fragment was created by digestion with SalI and EcoRV, and cloned into pBS-2×rsGFP-NosT (pBS-CPCp-trCPC-GFP-NosT). Finally, the entire region was cloned into the SalI and XbaI sites of the pJHA212K binary vector.

For the substitution of specific amino acids in the NM region of CPC,PCR-mediated mutagenesis was carried out on pBS-CPCp::CPC:GFP-NosT(Wada et al., 2002) using the QuickChange Site-Directed Mutagenesis Kit (Stratagene), with the primers:TK112/TK113 for K6A:K9A, TK320/TK321 for K79A, TK340/TK341 for W76A,TK342/TK343 for L77V and TK379/TK380 for M78A. Each mutagenized region was cloned into the SalI and XbaI sites of pJHA212K.

EGL3p::CPC:GFP was created by ligating the Klenow fragment blunt-ended SalI and BamHI fragment of the EGL3promoter from pBS-EGL3p (R.S., K.O. and T.W., unpublished) into the 5′portion (ClaI digestion and fill-in by Klenow) of pBS-CPC-GFP-NosT (pBS-EGL3p-CPC-GFP-NosT). The SalI and XbaI fragment from pBS-EGL3p-CPC-GFP-NosT was then ligated into pJHA212K.

The SHORT-ROOT promoter can be divided into two regions: a 1.8 kb upstream region (SHR-A) and a 0.7 kb downstream region (SHR-B)(Helariutta et al., 2000). These regions were amplified with TK233/TK240 and TK237/TK234, respectively. Amplified fragments were ligated into pT7 (pT-SHR-A or pT-SHR-B). The EcoRV/SmaI fragment of pT-SHR-B was cloned into pT-SHR-A (pT-SHRp). The SalI/SmaI fragment of pT-SHRp was ligated into the SalI/EcoRV sites of pBS-CPC-GFP-NosT(pBS-SHRp-CPC-GFP-NosT). The SalI/EcoRV fragment of the SCR (SCARECROW) promoter from pJH-SCRp-GFP-NosT(T.K., K.O. and T.W., unpublished) was replaced by the SalI/SmaI fragment of pT-SHRp.

To construct CPCp::CPC:tandem GFPs, three plasmids containing 1×GFP were constructed. 1×GFP was amplified with GFPspacer/multiGFP-r from pBS-CPCp::CPC:GFP(Wada et al., 2002), and cloned into the EcoRV/BamHI sites of pBluescriptII SK+(pBS-1×GFP-A). Two other 1×GFPs were amplified from pBS-2xrsGFP-NosT, with multiGFP-f/multiGFP-r and multiGFP-r/M13R primer pairs, and cloned into the pT7 (pT7-1×GFP-B)and PstI/BamHI sites of pBluescriptII SK+(pBS-1×GFP-C). The SalI/BamHI fragment of pBS-1×GFP-A was cloned into the SalI/BglII sites of pBS-1×GFP-C(pBS-2×GFP-NosT). For 3×GFP, the SalI/BamHI fragment of pT7-1×GFP-Bwas cloned into the SalI/BglII sites of pBS-1×GFP-C(pBS-2×GFP-NosT-2), then the SalI/BamHI fragment of pBS-1×GFP-Awas inserted into the SalI/BglII sites of pBS-2×GFP-NosT-2(pBS-3×GFP-NosT). The SalI/BamHI fragment of pT7-1×GFP-B was also cloned into the SalI/BglII sites of pBS-2×GFP-NosT-2(pBS-3×GFP-NosT-2). The pBS-4×GFP-NosT and pBS-5×GFP-NosT plasmids were constructed by repeated insertions of the amplified fragment as above. Next, the SalI/NcoI fragment of pBS-CPCp::CPC:GFP was cloned into the SalI/NcoI sites of each pBS-tandem GFPs-NosT (pBS-CPCp::CPC:tandem GFPs-NosT). Finally, the SalI/XbaI fragments of pBS-CPCp::CPC:tandem GFPs-NosT were ligated into pJHA212K.

Col-0 or Ws Arabidopsis ecotypes were used as wild type. The 35S::CPC transgenic line and cpc-1 have been described previously (Wada et al.,1997). The cpc-2 mutant (KG12704) used in this study was isolated from Kazusa T-DNA lines. cpc-2 contains a T-DNA insert in the second intron of CPC. This mutant was backcrossed to Col-0 twice. Seeds of rhd3-1 were provided from the Arabidopsis Biological Resource Center at Ohio State University (OH, USA). Lines homozygous for mutations containing the transgene were constructed by crossing mutant and transgenic plants, the phenotype and GFP fluorescence was then examined. Transformation was performed as described previously(Kurata et al., 2003). For the observation of seedlings, plants were grown on agar plates as described by Okada and Shimura (Okada and Shimura,1990). For the inhibitor experiment, BFA (ICN, BFA stock solution was 50 mM) was added in agar medium at the indicated concentrations.

In situ hybridization was as described by Kurata et al.(Kurata et al., 2003). A DIG-labeled antisense RNA probe for CPC:GFP was generated by transcribing NcoI-digested pBS-1xrsGFP (R.S., K.O. and T.W.,unpublished) using a T3 polymerase.

Roots were stained with 5 μg/ml propidium iodide (PI) for 30 seconds and mounted in water. Confocal images were obtained with a 40×water-immersion objective on a Zeiss LSM-Pascal or a Zeiss LSM-510 Meta confocal laser-scanning microscope using 488 nm laser lines for GFP excitation. Image processing was done with Adobe Photoshop version 7.0 (Adobe Systems, CA, USA).

For the observation of root hairs and trichomes, images of seedlings were recorded with a VC4500 3D digital fine scope (Omron, Kyoto, Japan). The pattern of epidermal cell types was determined according to the protocol of Lee et al. (Lee et al., 2002), with minor modifications. A light microscope equipped with differential interference contrast (Nomarski) optics was used to determine cell type and relative locations of epidermal cells. The proportions of trichoblasts and atrichoblasts in root epidermis were determined by examining a minimum of ten 5-day-old seedlings from each line. An epidermal cell was counted as a trichoblast if any protrusion was visible, regardless of its length.

RNA extraction and semi-quantitative RT-PCR reaction were as described by Kurata et al. (Kurata et al.,2003). CPC, CPC:GFP and HA:CPC fragments were amplified with RT128/TK629, RT128/TK628 and HA-F/RT-129 primer pairs. EF1α was amplified as described by Kurata et al.(Kurata et al., 2003).

The GST-CPC protein was prepared as described previously(Wada et al., 2002). The His-CPC fusion was expressed using pET28a in Escherichia coli BL21(DE3). Soluble His-CPC was purified with Ni-NTA agarose (Quiagen). Purified GST-CPC protein was injected into rabbits, and the antisera were affinity purified with coupled His-CPC (BioGate Company Limited, Gifu, Japan). In parallel, we extracted total soluble protein from cpc-2 mutant seedlings and immobilized it in a HiTrap NHS-activated HP column (Amersham Biosciences). The affinity-purified fraction was further absorbed to the cpc-2 protein gel for 7 hours at 4°C. The final unbound fraction was used for western blotting.

Total plant protein was extracted from 7-day-old seedlings with a protein extraction buffer containing 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% (w/v)Triton X-100, 1 mM PMSF, 50 μM Leupeptin and 11 mM 2-mercaptoethanol. Crude protein was separated by SDS-PAGE with SuperSep gradient gel (Wako Pure Chemical Industries, Osaka, Japan) and Prestained Protein Marker (New England BioLabs). Samples were transferred to nitrocellulose membranes by wet-electroblotting, and checked by Ponceau S staining. The blotted membrane was incubated with rabbit anti-CPC primary antibody (1:2000). Incubation with secondary HRP-conjugated anti-rabbit antibody and detection were according to the manufacturer's protocol (ECL Plus, Amersham Biosciences).

Immunohistochemistry was performed according to Nakajima et al.(Nakajima et al., 2001), with minor modifications. Five-day-old seedlings were fixed in 4% (w/v)paraformaldehyde in PBS [10 mM sodium phosphate (pH 7.5), 130 mM NaCl]overnight at 4°C, washed with PBS and passed through an ethanol series. Samples were cleaned with Histo-clear II (National Diagnostics, GA, USA), and embedded in Histologie (MERCK, Darmstadt, Germany). Sections (9 μm) were cut with a microtome (Leica RM2165, Nubloh, Germany) and placed on MAS-coated slides (MATSUNAMI GLASS IND, Osaka, Japan). After deparaffinization and rehydration, the sections were treated with 2N HCl for 5 minutes and 300μg/ml pronase (Roche) for 10 minutes at 37°C. Treatment with pronase was stopped by incubation in glycine-TBS [TBS: 25 mM Tris-HCl (pH 7.4), 137 mM NaCl, 2.68 mM KCl] and TBS wash. After blocking with TBST [TBS plus 0.05%(w/v) Tween 20] containing 1% (w/v) bovine serum albumin and 2% (v/v) goat serum for 5 hours at room temperature, the sections were incubated with anti-HA antibodies (Clone 3F10, Roche) at a 1:200 dilution in the blocking solution overnight at room temperature. Slides were washed six times with TBST for 10 minutes and incubated with alkaline phosphatase-conjugated anti-rat IgG(1:500, Zymed, CA, USA) for 2 hours at room temperature. After washing four times in TBST and twice in TBS, the signal was developed with NBT/BCIP solution (Western Blue, Promega) with 1 mM levamisole for 4 hours at room temperature.

In wild-type roots, root hair cells and hairless cells are arranged in alternating files in the root epidermis(Wada et al., 2002). Transgenic plants in which the expression of 2×rsGFP was driven by the CPC promoter (CPCp::GFP) showed GFP fluorescence exclusively in hairless cell files, and had normal root hairs, as well as trichomes on leaf surfaces (Fig. 1A-C). The 2×rsGFP construct was chosen because 1×GFP could move freely from hairless cells to hair cells in a non-targeted manner (Y.K. and K.O.,unpublished) (Crawford and Zanbryski,2000). By contrast, the roots of transgenic plants which expressed the CPC:2×rsGFP (CPC:GFP) fusion protein under the control of the CPC promoter (CPCp::CPC:GFP) exhibited GFP fluorescence in all epidermal cell nuclei, and developed ectopic hair cells in positions where hairless cells would usually develop (Fig. 1D,E). Moreover, CPCp::CPC:GFP plants lacked trichomes on the surface of rosette leaves (Fig. 1F). These phenotypes, observed in CPCp::CPC:GFP plants,resembled 35S::CPC overexpression lines(Wada et al., 1997). To exclude the possibility of the movement of CPC:GFP mRNA from hairless cells to hair cells, we checked the expression pattern of CPC:GFPmRNA by in situ hybridization in CPCp::CPC:GFP transgenic plants. CPC:GFP mRNA was detected in hairless cells with an antisense GFP probe (inset in Fig. 1D); this observation was similar to the wild-type transcripts detected with an antisense CPC probe(Wada et al., 2002). We also examined CPCp::CPC:GFP transgenes in the cpc-1 mutant background and observed GFP fluorescence in all epidermal cell nuclei, which was similar to the fluorescence distribution in the wild-type background (see Fig. S1 in the supplementary material). The cpc-1 plants containing CPCp::CPC:GFP showed normal hair formation in the hair-cell files and ectopic root hairs in the hairless-cell files, suggesting that CPC:GFP is functional in the cpc-1 mutant background (see Fig. S1 in the supplementary material).

To confirm the localization of CPC using immunohistochemistry, we used haemagglutinin A (HA)-tagged CPC. HA-tagged CPC was expressed under the control of the CPC promoter in the cpc-1 mutant(CPCp::HA:CPC in cpc-1). These transgenic plants showed a complemented phenotype without ectopic root hair formation(Fig. 2B), suggesting that the HA:CPC fusion protein behaved like the endogenous CPC protein. Immunostaining with anti-HA antibody of root transverse sections from CPCp::HA:CPCtransgenic cpc-1 plants led to strong signals in the nuclei of root hair cells, but not in the nuclei of hairless cells(Fig. 2C). Detection of CPC protein in the nuclei of root hair cells confirms the results obtained in CPC:GFP fusion plants (Fig. 1D), and supports our model that CPC moves from atrichoblasts to trichoblasts in the root epidermis. However, Fig. 2C shows that the amount of CPC protein in the nuclei of hairless cells falls below detectable levels. This is not consistent with the previous result shown in Fig. 1D.

The apparent disparity in accumulation levels between CPC:GFP and HA:CPC fusion proteins in hairless cells was addressed by western blot with proteins extracted from wild type (Col-0), CPCp::CPC:GFP transgenic plants, CPCp::HA:CPC transgenic cpc-1 plants, 35S::CPC and cpc-2. In transgenic plants expressing CPCp::CPC:GFP, a predicted band (65 kDa) corresponding to CPC:GFP was detected but there was no corresponding signal for HA:CPC in CPCp::HA:CPC transgenic cpc-1 plants (Fig. 3A). Transgenic plants expressing 35S::CPC yielded an 11 kDa band, which correlates with the predicted size of CPC. However, we did not detect a band corresponding to CPC in either the wild type (Col-0) or the cpc-2 mutant (Fig. 3A). Quantitation by RT-PCR with gene-specific primer pairs suggested that there was no significant difference in the amounts of mRNA between CPCp::CPC:GFP and CPCp::HA:CPC plants, but these transgenic plants had a higher concentration of CPC:GFP and HA:CPC fusion transcripts than wild type (Col-0) did(Fig. 3B). 35S:CPC had the highest concentration of CPC transcripts, whereas no CPCtranscripts were detected in the cpc-2 mutant, suggesting that cpc-2 was a null mutant (Fig. 3B). These data indicate that the CPC:GFP fusion protein is more stable than the HA:CPC protein is and that it accumulated in hairless cells. Therefore, a small amount of HA:CPC, and presumably also endogenous CPC, could be effectively transported into root hair cells from hairless cells. However,we used the CPC:GFP fusion for qualitative analyses of the cell-to-cell movement of CPC, as GFP fusion proteins can be conveniently and non-invasively monitored, and our focus is on the ability of CPC to move into hair cells. Furthermore, both CPC:GFP and HA:CPC accumulated in the nuclei of hair cells,implying that the movement activity of CPC:GFP from hairless cells to hair cells is equivalent to that of HA:CPC (and presumably endogenous CPC).

In a series of experiments, we used the wild-type background for the construction of transgenic plants because intact endogenous CPC is required for monitoring the intercellular movement of CPC fusions. If the mutated CPCs fused to GFP, which do not move intercellularly, were to be transformed into the cpc mutant background, GFP fluorescence might be observed in all epidermal cell files. Because CPC itself is indispensable for the transcriptional self-repression of CPC in hair cells(Wada et al., 2002), it would be unlikely that the defects of intercellular movement of mutated CPC could be detected.

To define which regions are required or sufficient for the cell-to-cell movement of CPC, we generated a series of truncated CPC proteins fused to the N terminus of 2×rsGFP and expressed them in transgenic plants. The CPC protein was divided into three non-overlapping domains: the N-terminal(residues 1 to 32), Myb (residues 33 to 83) and C-terminal (residues 84 to 94)domains (Fig. 4A). We made four constructs (Fig. 4A) and analyzed the ability of their expression products to move between cells. Transformant plant lines with NMG, containing the N-terminal and Myb (residues 1 to 83) domains, had GFP fluorescence in all epidermal cell nuclei, similar to the full-length CPC:GFP (Fig. 1D versus Fig. 4B). Lines containing MG, consisting of the Myb region only, had GFP signal mostly restricted to the nuclei of hairless cells(Fig. 4C). Transformants with the third product, NG, with only the N-terminal domain, showed neither the capability for cell-to-cell movement nor nuclear accumulation(Fig. 4D). MCG, containing the Myb and C-terminal (residues 33 to 94) domains, resembled MG: most of the GFP signal was observed in the nuclei of hairless cells(Fig. 4E). These results suggested that the region spanning from the N terminus to the Myb domain functions as a trafficking signal domain of CPC. In addition, the Myb domain appeared likely to contain the nuclear localization signal. To further delimit the CPC regions required for cell-to-cell movement, we constructed a series of NMG plasmids with deletions extending from both the N-terminal and C-terminal ends (Fig. 5A). 1-79G, with only four amino acids deleted from the C terminus of the Myb domain, gave results similar to those of NMG (Fig. 5B). By contrast, 1-75G transgenic plants had GFP fluorescence only in hairless cell files and lacked nuclear fluorescence(Fig. 5C). Thus, the four amino acids (WLMK) deleted in 1-75G are crucial for cell-to-cell movement and may also be required for a nuclear localization of CPC. Transgenic lines harboring CPC constructs with more extensive C-terminal deletions than 1-75G(i.e. 1-65G, 1-55G and 1-45G; Fig. 5A) had GFP signals similar to the 1-75G transgenic plants (data not shown).

Plasmid 10-83G, from which nine amino acids had been deleted at the N terminus of CPC (Fig. 5A), gave GFP fluorescence in most nuclei of hairless cell files(Fig. 5D), suggesting that the truncated fusion protein had lost intercellular movement ability. Similar results were obtained with plasmid 21-83G, in which the N terminus had been further shortened (data not shown).

From these deletion analyses, we concluded that CPC carries a signal domain(residues 1 to 79), which is necessary and sufficient for cell-to-cell movement, and which is made up of the whole N terminus and a part of the Myb domain (Fig. 5E). We tentatively named the two regions required for CPC movement S1 (sequence 1)and S2.

To identify crucial amino acid(s) in the S1 and S2 regions, we conducted site-directed mutagenesis. Alanine substitutions in both K6 and K9 (K6A:K9A)showed no difference in the distribution of GFP fluorescence when compared to the CPCp::CPC:GFP transgenic line(Fig. 6A). The K6A:K9A mutant transgenic line developed ectopic root hairs but no trichomes on leaves(Fig. 6B,C), suggesting that K6 and K9 are not required for cell-to-cell movement of CPC. We also structured mutants with alanine substitutions beyond the C-terminal border of S1 in the N-terminal domain of CPC (K12A:R13A, R14A:R15A, R15A:R16A and K19A:K21A), but no effects on CPC:GFP movement were observed (data not shown).

Three alanine substitutions (W76A, M78A, and K79A) and one valine substitution (L77V) were introduced into the S2 region. K79A and L77V mutants showed distributions of GFP fluorescence and morphological phenotypes like those of the CPCp::CPC:GFP line(Fig. 6D-I). Conversely, the GFP signal was mostly restricted to files of hairless cells in the mutants W76A and M78A (Fig. 6J,M; for a quantitative assessment see Table 1). In the W76A mutant, GFP fluorescence appeared to be dispersed in the cytoplasm (Fig. 6M). These results suggested that W76 was crucial for both cell-to-cell movement and nuclear accumulation. The fact that W76A had an almost normal root hair formation and a partial inhibition of trichome development(Fig. 6N,O; Table 2) is consistent with this interpretation. Furthermore, W76A in the cpc-2 mutant background had a reduced number of hair cells (see Fig. S2 in the supplementary material), suggesting that the W76A mutant form was not functional. The GFP signal of W76A in the cpc-2 background was observed to be dispersed in all epidermal cells, suggesting that the movement-defective W76A mutant form could not repress its own expression in hair cells in a cpc-2background, as there was no endogenous CPC present, and repression of CPC in hair cells did not occur. In M78A mutants, the cell-to-cell movement of CPC was defective but nuclear accumulation remained unaffected(Fig. 6J,L). The proportion of GFP-expressing cells in root hair-cell positions was higher in M78A than in W76A (Table 1). For M78A, we examined the effects on phenotype in the cpc-2 mutant background. M78A in the cpc-2 background produced hair formation in the hair-cell and hairless-cell files, and ectopic root hairs, as observed with wild-type CPC (see the Fig. S2 in the supplementary material). M78A in the cpc-2 background also produced GFP fluorescence in all epidermal cell nuclei for the same reason as W76A; that is, the movement-defective M78A form could not repress CPC expression in hair cells.

To examine the possibility of cell-to-cell movement of CPC in tissues other than the epidermis, we expressed CPC:GFP using the promoter of the SHORT-ROOT (SHR) gene. Control plants (SHRp::GFP)expressed GFP fluorescence preferentially in the stele (inset in Fig. 7A). This result is consistent with a previous study(Helariutta et al., 2000). Practically the same results were obtained from plants expressing SHRp::CPC:GFP (Fig. 7A,B), suggesting that the CPC:GFP fusion protein did not selectively move out of the stele. In contrast to epidermal cells, CPC:GFP did not specifically accumulate in the nuclei of stele cells(Fig. 7A,B). We confirmed the expression of the CPC:GFP fusion protein by western blot analysis in SHRp::CPC:GFP lines (data not shown). CPC:GFP expressed in stele cells had no effect on root hair formation. These results suggest that nuclear accumulation of CPC may be required for its action on root hair development,or that CPC functions with unidentified auxiliary factors that are not present in the stele because CPC has to be in the epidermis during root hair formation.

ENHANCER OF GL3 (EGL3), a bHLH-type transcriptional factor gene, has been characterized in several laboratories, including our own(Zhang et al., 2003; Bernhardt et al., 2003) (R.S.,K.O. and T.W., unpublished). The EGL3 promoter is active in the root epidermis, preferentially in hair cells (R.S. et al., unpublished). In plants carrying an EGL3p::CPC:GFP transgene, the fusion protein was localized in the nuclei of all epidermal cells(Fig. 7D,E). Control plants expressing EGL3p:GFP showed GFP fluorescence predominantly in hair cells (insets in Fig. 7D),suggesting that CPC:GFP expressed under the control of EGL3 promoter moved from the root hair cells to hairless cells. The protein is also functional in controlling root hair development, as EGL3p::CPC:GFPtransgenic plants grew ectopic root hairs(Fig. 7F). The differences in the behavior of CPC:GFP expressed either in the stele or the epidermis suggested that epidermis-specific components were required for cell-to-cell movement of CPC and also for its nuclear accumulation.

We also expressed the CPC:GFP in another ground tissue, the endodermis,with the endodermis-specific SCARECROW (SCR) promoter(Di Laurenzio et al., 1996). When 2×GFP were expressed under the SCR promoter as a control,it moved from the endodermis to the epidermis via the cortex, suggesting that 2×GFP may be passively transported in these layers (data not shown).

To examine whether the classical secretion process is involved in the movement of CPC, we performed two types of experiment. First, brefeldin A(BFA) blocks the secretory pathway from the endoplasmic reticulum (ER) to the plasma membrane by inhibiting vesicle formation at the Golgi apparatus(Ritzenthaler et al., 2002; Geldner et al., 2003). Seedlings treated with BFA had phenotypes similar to plants treated with an auxin-transport inhibitor, such as a reduction in root and hypocotyl gravitropism and elongation, as well as lateral root initiation (data not shown) (Geldner et al., 2001). CPC:GFP localization was not affected in CPCp::CPC:GFP transgenic plants treated with 10 μM BFA (Fig. 8A,B), whereas the aberrant phenotypes described above were observed (data not shown). This result strongly suggests that the BFA-sensitive secretion pathway is not involved in the movement of CPC:GFP.

Second, the rhd3-1 mutant allele was originally recognized and isolated by its phenotype of short wavy root hairs(Schiefelbein and Somerville,1990), and Zheng et al. (Zheng et al., 2004) reported that this mutant has a defect in the secretion of proteins to the extracellular space. We tested the movement of CPC:GFP in the rhd3-1 mutant. As shown in Fig. 8E, CPC:GFP was found in the nuclei of both hairless cells and root hair cells, indicating that the mutation does not affect the movement of CPC. Root hairs of the CPCp::CPC:GFP transgenic plant had short, wavy root-hairs on both root hair cell and hairless cell files(Fig. 8D), whereas the rhd3-1 mutant had short, wavy root hairs only on root hair cell files(Fig. 8C). This result is consistent with the model proposing that CPC is moved from hairless cells to neighboring hair cells in the rhd3-1 mutant. These two lines of evidence strongly suggest that Golgi-mediated secretion is not a mechanism of controlling the intercellular movement of CPC.

If CPC protein moves via plasmodesmata, its movement, like other proteins or complex structures, is likely to have a size exclusion limit (SEL)(Zambryski and Crawford,2000). To investigate the effect of the SEL on CPC movement, the movement abilities of CPC fused to tandem GFPs, 2×, 3×, 4×and 5×GFP, were compared. CPC:tandem-GFPs were expressed under the control of the CPC promoter. As a control, we examined the movement ability of free 1×GFP and 2×GFP under the expression control of the CPC promoter; CPCp::1×GFP and CPCp::2×GFP. Free 2×GFP could not move out of hairless cells but 1×GFP moved freely between epidermal cells (see, for example Fig. 1A; data not shown). This result suggests that the SEL of plasmodesmata under these conditions is between 27 kDa (M_r of 1×GFP) and 54 kDa (M_r of 2×GFP) in the meristematic zone of root epidermis. In contrast to free GFP multimers, CPC increased the SEL of plasmodesmata for CPC:GFP multimer fusions. Transgenic plants expressing CPC:2×GFP had GFP fluorescence in all epidermal cell nuclei(Fig. 9A). For CPC:3×GFP,the nuclei of all epidermal cells showed GFP fluorescence and partially reduced nuclear localization, as some GFP fluorescence was dispersed in the cell (Fig. 9B). Larger multimers of GFP fused to CPC reduced the GFP signal in hair cells and nuclear-specific localization (Fig. 9C,D). These data demonstrated that the SEL of plasmodesmata specifically increases to around 119 kDa (M_r of CPC:4×GFP) when CPC fused to GFPs. Furthermore, intercellular movement of CPC:GFP is apparently coupled with intracellular movement, resulting in nuclear localization.

Although several transcription factors such as CPC have the ability to move from cell-to-cell in plants (Ruiz-Medrano et al., 2004), little is known about the mechanisms controlling these movements. To identify sequences required or sufficient for the cell-to-cell movement of CPC, we created a series of truncated proteins. Similar experiments have been conducted to elucidate the systemic movement of plant viruses. The movement protein (MP) of the tobacco mosaic virus (TMV)provides the best-characterized mechanism by which plant viruses move between cells. Using MP and fluorescent dextrans, the viral RNA-binding domain of TMV-MP was identified as a necessary component for cell-to-cell movement(Waigmann et al., 1994). Using a different approach, a series of deletions in the MP gene was tested for the ability to mediate the intercellular movement of TMV. Three N-terminal amino acids (residues 3 to 5) were found to be essential for transport in tobacco cells (Kotlizky et al., 2001). However, it remained unclear whether these three amino acids were the only ones necessary for the cell-to-cell movement of TMV. In contrast to the intracellular transport of proteins in and out of organelles, which is usually mediated by simple, short signal sequences, large sequence regions of MP may be necessary for the movement of TMV between cells. Another class of plant protein, including heat-shock cognate 70 chaperones and thioredoxin h, also moves from cell-to-cell, and motifs required for this movement were investigated, but no common conserved/similar amino-acid sequences have been identified, indicating that non-cell autonomous proteins have no consensus sequence among them for moving intercellularly(Aoki et al., 2002; Ishiwatari et al., 1998).

In the present study, we identified a domain that is necessary and sufficient for CPC cell-to-cell movement. The S1 region in the N-terminal domain whose truncation resulted in the inability of CPC to move consists of nine residues and is unique in the family of R3-type small Myb-like proteins. The other region, S2, in the Myb domain, which is also required for CPC movement, consists of four amino acids (residues 76 to 79). Both the S1 and S2 region required for CPC movement possess no significant homology to the amino acid sequences of known non-cell-autonomous proteins (NCAPs). Deletion of the S2 region abolished the nuclear accumulation of CPC. This is an interesting point, because the same region is thus apparently required in inter- and intracellular movement. This S2 region is neither rich in basic amino acids nor does it resemble a typical bipartite nuclear localization signal sequence(NLS) (Kaffman and O'Shea,1999), suggesting that it functions in an unknown type of nuclear import system. S2 overlaps with a region that is required for protein-protein interactions between CPC and R (residues 33 to 83)(Wada et al., 2002), a bHLH-type transcription factor from maize that affects root epidermal cell differentiation in Arabidopsis(Lloyd et al., 1992). Thus,the S2 region in the Myb domain functions as a part of multifunctional motif with roles in intercellular movement, nuclear localization (i.e. intracellular movement) and protein-protein interactions. A similar situation in which functional motifs overlap has been reported in the tobacco mitogen-activated protein kinase kinase kinase, NPK1, in which regions that function as a NLS and in protein-protein interactions are partly identical(Ishikawa et al., 2002). The crucial amino acids in the S2 region, W76 and M78, were identified by site-directed mutagenesis. Both residues are conserved in R3-type small Myb-like proteins (Fig. 6P). Thus, these conserved amino acids may be important for CPC movement. To clarify whether the two truncated sequences, S1 and S2, in the N-terminal and Myb domains are sufficient for cell-to-cell movement of CPC, we created a chimeric construct with a CPC promoter-driven gene in which 2×rsGFP was sandwiched between S1 and S2(CPCp::S1:2×rsGFP:S2). However, in the CPCp::S1:2×rsGFP:S2 transgenic plants, GFP fluorescence did not move from the hairless cells to hair cells (data not shown). These results indicate that the specific conformation of the native CPC protein is important for its cell-to-cell transport. An analysis of the three-dimensional structure of CPC will help to understand its intercellular movement structural requirements. Kim et al.(Kim et al., 2005) recently reported that the relatively large KNOX homeodomain (residues 256 to 326) is required and sufficient for KN1 movement. There is no detectable sequence homology/similarity between the domains of the two proteins (CPC versus KN1). Therefore, comparative analyses of the three dimensional structures of CPC and KN1 will help to understand the role of KNOX or other domains in cell-to-cell movement.

W76 is crucial not only for cell-to-cell movement but also for intracellular movement to nuclei. In addition, increasing the number of GFP in CPC:GFP fusions also affects both inter- and intracellular movement. Nuclear accumulation and intercellular movement of proteins depend on membrane pores,nuclear pores or plasmodesmata (Lee et al., 2000), and it is tempting to speculate that some structural elements of CPC may play similar roles in its transport through the nuclear membrane and plasmodesmata. Some other factor(s) may also have a functional similarity for transporting proteins through nuclear membranes and plasmodesmata. In the case of KN1 movement, a movement-defective mutation (M6)overlapped with a potential nuclear localization signal(Lucas et al., 1995). Coupling nuclear accumulation with the intercellular movement of protein has been also observed in the case of SHR. The shr-5 mutant allele, which converts threonine 289 into isoleucine (T289I), showed defects in both nuclear accumulation and intercellular movement(Gallagher et al., 2004).

Plasmodesmata-mediated cell-to-cell movement of proteins may proceed by either of two modes: non-targeted movement by simple diffusion, or targeted and regulated transport. In the first case, soluble proteins can cross plasmodesmata if the SEL is greater than the actual size of the protein molecule (Zambryski and Crawford,2000). Free GFP and LFY move by this mechanism(Crawford and Zambryski, 2000; Wu et al., 2003), but CPC:GFP does not. Rather, it has to be transported by a targeted movement that requires dilation of the plasmodesmata. This process is probably mediated by specific interactions between the transported protein and components of the plasmodesmata (Haywood et al.,2002). Thus, some specific factor must interact with CPC during targeted movement in the root epidermis. W76A transgenic plants in the cpc-2 background showed reduced numbers of root hairs; the same phenotype as cpc-2. The reason for the failure of complementation with W76A mutants may be the defective nuclear accumulation or reduced repressor activity of CPC in transcriptional regulation. By contrast, the substitution M78A only reduced the cell-to-cell movement of CPC, suggesting that M78 is specifically involved in the intercellular movement of CPC.

The SHR:GFP fusion protein expressed under control of the stele-specific SHR promoter moved from the stele to the adjacent endodermis(Nakajima et al., 2001). When the expression of the CPC:GFP fusion protein was driven by the SHRpromoter, CPC:GFP was not able to leave the stele. Apparently, factors required for CPC movement are absent from stele cells. As CPC:GFP expressed in the stele also did not accumulate in the nuclei, CPC apparently requires tissue-specific factors for nuclear import. Although the CPC:GFP fusion protein expressed under the control of the hairless-cell-specific CPCpromoter moved from hairless cells to root hair cells, the direction of movement was reversed when expression was limited to hair cells by the hair cell-specific EGL3 promoter. Thus, CPC can move through the epidermal layer, implying that the specific factors required for CPC movement are present in the epidermis as a whole. Similar tissue-specificity in the regulation of protein movement has been observed with SHR and KN1. When SHR:GFP is expressed in phloem companion cells by the tissue-specific SUC2 (sucrose-H⁺ symporter gene) promoter, it is unable to exit from the companion cells. SHR:GFP also remains trapped in root epidermal cells when expressed there through specific promoters(Sena et al., 2004). In the case of KN1, movement is directional depending on the developmental state of the tissue. In leaves, the GFP:KN1 fusion protein was able to move from mesophyll cells to epidermal cells, but not in the opposite direction. In the shoot apical meristem, however, GFP:KN1 can move from epidermal (L1) to inner layers (L2, L3) (Kim et al.,2003). Such developmentally regulated competency for protein movement probably depends on the quantity or quality of tissue-specific factors required to support movement.

Based on what is currently known about the transport of transcriptional regulatory proteins, there are two possible modes of intercellular movement of CPC; one is via plasmodesmata-mediated trafficking, the other is secretion-internalization-dependent apoplastic transport, such as the transport of homeodomain proteins in animal cell-cultured systems (ex vivo)(Prochiantz and Joliot,2003).

Localized accumulations of GFP:KN1 have been observed in the periphery of epidermal cells expressing the protein(Kim et al., 2003). These foci appeared to co-localize with plasmodesmata. We could not detect similar focal accumulations in cells expressing CPC:GFP. However, it still appears likely that CPC travels through plasmodesmata, as its movement was not inhibited by BFA nor by the rhd3 mutation. Furthermore, CPC increased the SEL between root epidermal cells, and does not have the putative signal sequences commonly observed in secretory proteins, supporting the conclusion that CPC moves from cell to cell through plasmodesmata or possibly by some as yet unknown mechanism. Recently, nonclassical protein secretion systems that are not inhibited by BFA have been reported(Nickel, 2003), and Haupt et al. (Haupt et al., 2005) have shown that the membrane trafficking (endocytic) pathway is employed in the intercellular movement of viral movement protein. Thus, further investigation is needed to clarify the involvement of unknown mechanism(s), including the endocytic pathway, on CPC movement.

In conclusion, this study has revealed the complex structural requirements for CPC cell-to-cell movement. A domain containing both the N-terminal domain and a part of Myb domain is required and sufficient to move the CPC:GFP fusion protein. Amino acid substitution experiments defined the critical residues,W76 and M78, for CPC movement, and suggested that CPC moves from atrichoblasts to trichoblasts in a regulated, targeted manner. Furthermore, substitution experiments indicated a coupling of intercellular and intracellular movement to nuclei, as W76 was required for both. We have also demonstrated that tissue-specific factors are involved in the cell-to-cell movement of CPC. These findings open the way for further investigations into the mechanisms and biological functions of CPC movement.

We thank Drs Yoshihiro Koshino-Kimura for providing unpublished results; Ji Hoon Ahn (Korea University), Katrina M. Crawford, Patricia C. Zambryski (UC,Berkeley) for providing plasmid materials; the Arabidopsis Biological Resource Center at Ohio State University, OH, USA for providing seeds; and Kenji Nakajima (Nara Institute of Science and Technology) for advice regarding immunohistochemistry.