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
Author for correspondence (e-mail:
twada{at}psc.riken.jp)
Accepted 29 September 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Arabidopsis, Epidermis, CAPRICE, Myb, Protein movement
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CPCp::HA:CPC
To create a plasmid with a gene encoding HA-tagged CPC, a CPC
genomic 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
).
CPCp::truncatedCPC:GFP
To create plasmids with truncated CPC sequences, the
2xrsGFP-NosT fragment was amplified with TK106/TK107
from pRTL2-2xrsGFP
(Crawford and Zambryski, 2000)
as a template, and cloned into the pT7 blue T-vector (Novagen). EcoRV
and XbaI fragments of 2xrsGFP-NosT were
cloned into pBluescriptII SK+ (pBS-2xrsGFP-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-2xrsGFP-NosT (pBS-CPCp-trCPC-GFP-NosT).
Finally, the entire region was cloned into the SalI and XbaI
sites of the pJHA212K binary vector.
CPCp::substitutedCPC:GFP
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.
Ectopic expression controlled by heterologous promoters
EGL3p::CPC:GFP was created by ligating the Klenow fragment
blunt-ended SalI and BamHI fragment of the EGL3
promoter 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.
CPCp::CPC:tandem GFPs
To construct CPCp::CPC:tandem GFPs, three plasmids containing
1xGFP were constructed. 1xGFP 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-1xGFP-A). Two other 1xGFPs were amplified
from pBS-2xrsGFP-NosT, with multiGFP-f/multiGFP-r and multiGFP-r/M13R
primer pairs, and cloned into the pT7 (pT7-1xGFP-B)
and PstI/BamHI sites of pBluescriptII SK+
(pBS-1xGFP-C). The SalI/BamHI
fragment of pBS-1xGFP-A was cloned into the
SalI/BglII sites of pBS-1xGFP-C
(pBS-2xGFP-NosT). For 3xGFP, the
SalI/BamHI fragment of pT7-1xGFP-B
was cloned into the SalI/BglII sites of
pBS-1xGFP-C
(pBS-2xGFP-NosT-2), then the
SalI/BamHI fragment of pBS-1xGFP-A
was inserted into the SalI/BglII sites of
pBS-2xGFP-NosT-2
(pBS-3xGFP-NosT). The SalI/BamHI
fragment of pT7-1xGFP-B was also cloned into the
SalI/BglII sites of pBS-2xGFP-NosT-2
(pBS-3xGFP-NosT-2). The
pBS-4xGFP-NosT and
pBS-5xGFP-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.
Plant materials, growth conditions
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
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.
Microscopy
Confocal laser scanning microscopy (CLSM)
Roots were stained with 5 µg/ml propidium iodide (PI) for 30 seconds and
mounted in water. Confocal images were obtained with a 40x
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).
Light microscopy
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.
RT-PCR
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.
EF1
was amplified as described by Kurata et al.
(Kurata et al., 2003
).
Antibody preparation
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.
Western blot analysis
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
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 300
µg/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.
|
|
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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:CPC transgenic 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.
|
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.
A region spanning the N-terminal and Myb domains is required and sufficient for the cell-to-cell movement of CPC
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 2xrsGFP 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).
|
|
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.
Identification of active sites for cell-to-cell movement of CPC:GFP
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).
|
|
|
|
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:GFP
transgenic 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 2xGFP were expressed under the SCR promoter as a control,
it moved from the endodermis to the epidermis via the cortex, suggesting that
2xGFP may be passively transported in these layers (data not shown).
|
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.
Increase of SEL due to 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, 2x, 3x, 4x
and 5xGFP, were compared. CPC:tandem-GFPs were expressed under the
control of the CPC promoter. As a control, we examined the movement ability of
free 1xGFP and 2xGFP under the expression control of the CPC
promoter; CPCp::1xGFP and
CPCp::2xGFP. Free 2xGFP could not move out of
hairless cells but 1xGFP 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 (Mr of 1xGFP) and 54
kDa (Mr of 2xGFP) 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:2xGFP had GFP fluorescence in all epidermal cell nuclei
(Fig. 9A). For CPC:3xGFP,
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 (Mr of
CPC:4xGFP) when CPC fused to GFPs. Furthermore, intercellular movement
of CPC:GFP is apparently coupled with intracellular movement, resulting in
nuclear localization.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
Tissue-specific regulation of cell-to-cell 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 SHR
promoter, 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 CPC
promoter 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.
Plasmodesmata-mediated movement of CPC
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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/24/5387/DC1
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: Forestry Research Institute, Oji Paper Company Limited,
Kameyama, Mie 519-0212, Japan
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aoki, K., Kragler, F., Xoconostle-Cazares, B. and Lucas, W.
J. (2002). A subclass of plant heat shock cognate 70
chaperones carries a motif that facilitates trafficking through plasmodesmata.
Proc. Natl. Acad. Sci. USA
99,16342
-16347.
Bernhardt, C., Lee, M. M., Gonzalez, A., Zhang, F., Lloyd, A.
and Schiefelbein, J. (2003). The bHLH genes GLABRA3 (GL3) and
ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis
root. Development 130,6431
-6439.
Crawford, K. M. and Zambryski, P. C. (1999). Plasmodesmata signaling: many roles, sophisticated statutes. Curr. Opin. Plant Biol. 2,382 -387.[CrossRef][Medline]
Crawford, K. M. and Zambryski, P. C. (2000). Subcellular localization determines the availability of non-targeted proteins to plasmodesmatal transport. Curr. Biol. 10,1032 -1040.[CrossRef][Medline]
Di Laurenzio, L., Wysocka-Diller, J., Malamy, J. E., Pysh, L., Helariutta, Y., Freshour, G., Hahn, M. G., Feldmann, K. A. and Benfey, P. N. (1996). The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86,423 -433.[CrossRef][Medline]
Gallagher, K. L., Paquette, A. J., Nakajima, K. and Benfey, P. N. (2004). Mechanisms regulating SHORT-ROOT intercellular movement. Curr. Biol. 14,1847 -1851.[CrossRef][Medline]
Geldner, N., Friml, J., Stierhof, Y. D., Jurgens, G. and Palme, K. (2001). Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature 413,425 -428.[CrossRef][Medline]
Geldner, N., Anders, N., Wolters, H., Keicher, J., Kornberger, W., Muller, P., Delbarre, A., Ueda, T., Nakano, A. and Jurgens, G. (2003). The Arabidopsis GNOM ARF-GEF mediates endosomal recycling, auxin transport, and auxin-dependent plant growth. Cell 112,219 -230.[CrossRef][Medline]
Haupt, S., Cowan, G. H., Ziegler, A., Roberts, A. G., Oparka, K.
J. and Torrance, L. (2005). Two plant-viral movement proteins
traffic in the endocytic recycling pathway. Plant Cell
17,164
-181.
Haywood, V., Kragler, F. and Lucas, W. J.
(2002). Plasmodesmata: pathways for protein and ribonucleoprotein
signaling. Plant Cell
14,S303
-S325.
Helariutta, Y., Fukaki, H., Wysocka-Diller, J., Nakajima, K., Jung, J., Sena, G., Hauser, M. T. and Benfey, P. N. (2000). The SHORT-ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell 101,555 -567.[CrossRef][Medline]
Ishikawa, M., Soyano, T., Nishihama, R. and Machida, Y. (2002). The NPK1 mitogen-activated protein kinase kinase kinase contains a functional nuclear localization signal at the binding site for the NACK1 kinesin-like protein. Plant J. 32,789 -798.[CrossRef][Medline]
Ishiwatari, Y., Fujiwara, T., McFarland, K. C., Nemoto, K., Hayashi, H., Chino, M. and Lucas, W. J. (1998). Rice phloem thioredoxin h has the capacity to mediate its own cell-to-cell transport through plasmodesmata. Planta 205, 12-22.[CrossRef][Medline]
Joliot, A., Maizel, A., Rosenberg, D., Trembleau, A., Dupas, S., Volovitch, M. and Prochiantz, A. (1998). Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein. Curr. Biol. 8, 856-863.[CrossRef][Medline]
Kaffman, A. and O'Shea, E. K. (1999). Regulation of nuclear localization: a key to a door. Annu. Rev. Cell Dev. Biol. 15,291 -339.[CrossRef][Medline]
Kawakami, S., Watanabe, Y. and Beachy, R. N.
(2004). Tobacco mosaic virus infection spreads cell to cell as
intact replication complexes. Proc. Natl. Acad. Sci.
USA 101,6291
-6296.
Kim, J. Y., Yuan, Z., Cilia, M., Khalfan-Jagani, Z. and Jackson,
D. (2002). Intercellular trafficking of a KNOTTED1 green
fluorescent protein fusion in the leaf and shoot meristem of Arabidopsis.
Proc. Natl. Acad. Sci. USA
99,4103
-4108.
Kim, J. Y., Yuan, Z. and Jackson, D. (2003).
Developmental regulation and significance of KNOX protein trafficking in
Arabidopsis. Development
130,4351
-4362.
Kim, J. Y., Rim, Y., Wang, J. and Jackson, D.
(2005). A novel cell-to-cell trafficking assay indicates that the
KNOX homeodomain is necessary and sufficient for intercellular protein and
mRNA trafficking. Genes Dev.
19,788
-793.
Kirik, V., Simon, M., Huelskamp, M. and Schiefelbein, J. (2004a). The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev. Biol. 268,506 -513.[CrossRef][Medline]
Kirik, V., Simon, M., Wester, K., Schiefelbein, J. and Hulskamp, M. (2004b). ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol. Biol. 55,389 -398.[CrossRef][Medline]
Kotlizky, G., Katz, A., van der Laak, J., Boyko, V., Lapidot, M., Beachy, R. N., Heinlein, M. and Epel, B. L. (2001). A dysfunctional movement protein of tobacco mosaic virus interferes with targeting of wild-type movement protein to microtubules. Mol. Plant Microbe Interact. 14,895 -904.[Medline]
Kragler, F., Monzer, J., Xoconostle-Cazares, B. and Lucas, W.
J. (2000). Peptide antagonists of the plasmodesmal
macromolecular trafficking pathway. EMBO J.
19,2856
-2868.
Kragler, F., Curin, M., Trutnyeva, K., Gansch, A. and Waigmann,
E. (2003). MPB2C, a microtubule-associated plant protein
binds to and interferes with cell-to-cell transport of tobacco mosaic virus
movement protein. Plant Physiol.
132,1870
-1883.
Kurata, T., Kawabata-Awai, C., Sakuradani, E., Shimizu, S., Okada, K. and Wada, T. (2003). The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis. Plant J. 36,55 -66.[CrossRef][Medline]
Lee, J. Y., Yoo, B. C. and Lucas, W. J. (2000). Parallels between nuclear-pore and plasmodesmal trafficking of information molecules. Planta 210,177 -187.[Medline]
Lee, M. M. and Schiefelbein, J. (2002). Cell
pattern in the Arabidopsis root epidermis determined by lateral inhibition
with feedback. Plant Cell
14,611
-618.
Lloyd, A. M., Walbot, V. and Davis, R. W. (1992). Arabidopsis and Nicotiana anthocyanin production activated by maize regulators R and C1. Science 258,1773 -1775.[Medline]
Lucas, W. J., Bouché-Pillon, S., Jackson, D. P., Nguyen, L., Baker, L., Ding, B. and Hake, S. (1995). Selective trafficking of KNOTTED1 homeodomain protein and its mRNA through plasmodesmata. Science 270,1980 -1983.[Abstract]
Maizel, A., Bensaude, O., Prochiantz, A. and Joliot, A.
(1999). A short region of its homeodomain is necessary for
engrailed nuclear export and secretion. Development
126,3183
-3190.
Maizel, A., Tassetto, M., Filhol, O., Cochet, C., Prochiantz, A.
and Joliot, A. (2002). Engrailed homeoprotein secretion is a
regulated process. Development
129,3545
-3553.
Matsubayashi, Y., Yang, H. and Sakagami, Y. (2001). Peptide signals and their receptors in higher plants. Trends Plant Sci. 6,573 -577.[CrossRef][Medline]
Nakajima, K., Sena, G., Nawy, T. and Benfey, P. N. (2001). Intercellular movement of the putative transcription factor SHR in root patterning. Nature 413,307 -311.[CrossRef][Medline]
Nickel, W. (2003). The mystery of nonclassical protein secretion. A current view on cargo proteins and potential export routes. Eur. J. Biochem. 270,2109 -2119.[CrossRef][Medline]
Okada, K. and Shimura, Y. (1990). Reversible root tip rotation in Arabidopsis seedlings induced by obstacle-touching stimulus. Science 250,274 -276.
Oparka, K. J., Roberts, A. G., Boevink, P., Santa Cruz, S., Roberts, I., Pradel, K. S., Imlau, A., Kotlizky, G., Sauer, N. and Epel, B. (1999). Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. Cell 97,743 -754.[CrossRef][Medline]
Prochiantz, A. and Joliot, A. (2003). Can transcription factors function as cell-cell signalling molecules? Nat. Rev. Mol. Cell. Biol. 4, 814-819.[Medline]
Ritzenthaler, C., Nebenfuhr, A., Movafeghi, A., Stussi-Garaud,
C., Behnia, L., Pimpl, P., Staehelin, L. A. and Robinson, D. G.
(2002). Reevaluation of the effects of brefeldin A on plant cells
using tobacco Bright Yellow 2 cells expressing Golgi-targeted green
fluorescent protein and COPI antisera. Plant Cell
14,237
-261.
Ruiz-Medrano, R., Xoconostle-Cazares, B. and Kragler, F. (2004). The plasmodesmatal transport pathway for homeotic proteins, silencing signals and viruses. Curr. Opin. Plant Biol. 7,641 -650.[CrossRef][Medline]
Schellmann, S., Schnittger, A., Kirik, V., Wada, T., Okada, K.,
Beermann, A., Thumfahrt, J., Jurgens, G. and Hulskamp, M.
(2002). TRIPTYCHON and CAPRICE mediate lateral inhibition during
trichome and root hair patterning in Arabidopsis. EMBO
J. 21,5036
-5046.
Schiefelbein, J. W. and Somerville, C. (1990).
Genetic control of root hair development in Arabidopsis thaliana.
Plant Cell 2,235
-243.
Sena, G., Jung, J. W. and Benfey, P. N. (2004).
A broad competence to respond to SHORT ROOT revealed by tissue-specific
ectopic expression. Development
131,2817
-2826.
Wada, T., Tachibana, T., Shimura, Y. and Okada, K.
(1997). Epidermal cell differentiation in Arabidopsis determined
by a Myb homolog, CPC. Science
277,1113
-1116.
Wada, T., Kurata, T., Tominaga, R., Koshino-Kimura, Y.,
Tachibana, T., Goto, K., Marks, M. D., Shimura, Y. and Okada, K.
(2002). Role of a positive regulator of root hair development,
CAPRICE, in Arabidopsis root epidermal cell differentiation.
Development 129,5409
-5419.
Waigmann, E., Lucas, W. J., Citovsky, V. and Zambryski, P.
(1994). Direct functional assay for tobacco mosaic virus
cell-to-cell movement protein and identification of a domain involved in
increasing plasmodesmal permeability. Proc. Natl. Acad. Sci.
USA 91,1433
-1437.
Wu, X., Dinneny, J. R., Crawford, K. M., Rhee, Y., Citovsky, V.,
Zambryski, P. C. and Weigel, D. (2003). Modes of
intercellular transcription factor movement in the Arabidopsis apex.
Development 130,3735
-3745.
Yoo, S. Y., Bomblies, K., Yoo, S. K., Yang, J. W., Choi, M. S., Lee, J. S., Weigel, D. and Ahn, J. H. (2005). The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221,523 -530.[CrossRef][Medline]
Zambryski, P. (2004). Cell-to-cell transport of
proteins and fluorescent tracers via plasmodesmata during plant development.
J. Cell Biol. 164,165
-168.
Zambryski, P. and Crawford, K. (2000). Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants. Annu. Rev. Cell Dev. Biol. 16,393 -421.[CrossRef][Medline]
Zhang, F., Gonzalez, A., Zhao, M., Payne, C. T. and Lloyd,
A. (2003). A network of redundant bHLH proteins functions in
all TTG1-dependent pathways of Arabidopsis.
Development 130,4859
-4869.
Zheng, H., Kunst, L., Hawes, C. and Moore, I. (2004). A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus. Plant J. 37,398 -414.[CrossRef][Medline]
|