1 NYU, Department of Biology, The Silver Center, room 1009; 100 Washington
Square East, New York, NY 10003, USA
2 Duke University, Department of Biology, Box 91000, Durham, NC 27708, USA
Author for correspondence (e-mail:
philip.benfey{at}duke.edu)
Accepted 24 February 2004
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SUMMARY |
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Key words: Arabidopsis, Root, Radial pattern, SHORT ROOT (SHR), Protein movement, Intercellular trafficking
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Introduction |
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Our previous work provided evidence that the SHORT ROOT (SHR) protein, a
member of the GRAS family of putative transcription factors, acts as a
positional signal essential to radial pattern formation. Genetic analyses in
Arabidopsis first revealed a role for SHR in root radial patterning
(Benfey et al., 1993). The
radial organization of the root encompasses concentric rings of epidermis,
cortex, endodermis and pericycle, surrounding a central vascular cylinder that
together with the pericycle comprise the stele
(Dolan et al., 1993
). The
radial pattern is generated through stereotyped asymmetric divisions of a set
of stem cells ('initials') in the meristem and subsequent acquisition of
different cell fates (Dolan et al.,
1993
). The endodermis and cortex cell layers (the ground tissue)
are extended by two sequential divisions of the cortex/endodermis (co/en)
initial cells. The first anticlinal division (i.e. with the plane of division
perpendicular to the surface of the root) regenerates the initial cell and
produces a daughter cell which then undergoes a periclinal division (i.e. with
the plane of division parallel to the surface of the root) to form the first
cells of the endodermis and cortex layers. In loss-of-function shr
mutants, the periclinal division fails to take place
(Benfey et al., 1993
). This
results in a single tissue that has only cortex characteristics
(Benfey et al., 1993
;
Helariutta et al., 2000
),
indicating that SHR is necessary for both periclinal division of the co/en
initial daughter and for endodermal specification. SHR transcripts
were detected only in the stele, indicating that it acts in a
noncell-autonomous fashion (Helariutta et
al., 2000
). Evidence that SHR protein is able to move from the
stele provided a mechanism by which it could act as a positional signal in
radial patterning (Helariutta et al.,
2000
; Nakajima et al.,
2001
). We have shown with GFP transcriptional and translational
fusions, as well as with in situ mRNA hybridization and immunolocalization,
that SHR moves from the stele of the root to the first adjacent cell layer
(Nakajima et al., 2001
).
Several other transcription factors have been shown to exert their
non-cell-autonomous actions by intercellular movement, presumably through PD
(for reviews, see Barton, 2001;
Hake, 2001
;
Roberts and Oparka, 2003
;
Wu et al., 2002
). These
include the maize transcription factor KNOTTED1 (KN1)
(Lucas et al., 1995
), the
Antirrhinum MADS domain proteins DEFICIENS (DEF) and GLOBOSA (GLO)
(Perbal et al., 1996
), the
Arabidopsis floral identity protein LEAFY (LFY)
(Sessions et al., 2000
), and
the MYB-related CAPRICE (CPC) protein
(Wada et al., 2002
).
Our previous results indicated that regulation of SHR signaling occurred
both in the extent of travel of the signal (SHR protein moves into the
endodermis but no further) as well as competence to respond to the signal
(although SHR is present in stele cells they do not acquire endodermal
characteristics) (Helariutta et al.,
2000; Nakajima et al.,
2001
). Determining the mechanism by which SHR intercellular
trafficking from the stele is limited to the next cell layer is essential to
address how spatial control of the signal is regulated. Studying the
distribution of competence to respond to SHR by either periclinal cell
divisions or endodermal specification is important for understanding the role
of cellular competence in generating radial positional information.
Previously, we attempted to address these issues by driving SHR
expression with two different promoters. Expression by both the constitutive
35S promoter and the SCR promoter, which is dependent on
SHR activity for full activation and normally confers expression in
the cell layer adjacent to the stele, resulted in supernumerary cell layers,
many of which had endodermal characteristics
(Helariutta et al., 2000;
Nakajima et al., 2001
).
However, with both promoters it was impossible to determine if intercellular
movement of SHR had occurred. Moreover, we could not determine the origins of
those cells that responded to SHR by periclinal division or endodermal
specification. For both movement and competence the problem was that one
promoter is ubiquitously active and the other expresses in cells that normally
contain SHR.
In this report, we investigate the regulation of SHR movement and the distribution of competence to respond to SHR using tissue-specific regulatory sequences from the SUC2, GL2 and WER genes, as well as the native SHR promoter. These promoters were used to drive expression of the translational fusion SHR::GFP in the phloem companion cells, maturing atrichoblasts, the epidermis with its initials and in the stele, respectively. We analyzed the ability of SHR::GFP to move from different cell types, the extent of movement when it occurred and the relationship of movement to subcellular localization. We found that in a wild-type background, movement occurs only from certain cell types but that the extent of movement does not depend on cell type. We also discovered that SCR may play a role in restricting movement. Competence to respond to SHR-mediated cell specification activity was broadly distributed in the outermost layer of the root, while competence to respond to the cell division activity of SHR appeared limited to the initials and involved induction of SCR. This broad competence to respond to SHR highlighted the importance of restricted SHR movement to generate the normal radial pattern in the root. Moreover, these results showed that SHR movement is not a pre-condition for activity.
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Materials and methods |
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Plants strains, transformation, and crosses
Arabidopsis seeds were surface sterilized and grown as described
previously (Benfey et al.,
1993).
The constructs in binary vectors were electroporated into
Agrobacterium, which were then used to transform Arabidopsis
(Columbia ecotype) following the floral dip method
(Clough and Bent, 1998).
Transgenic seedlings were selected on MS agar plates with 1% sucrose and 50
µg/ml kanamycin. For all the transgenes discussed, numerous individuals
from at least three independently transformed lines were analyzed.
The line pWER::SHR::GFP in a scr mutant background was
obtained by crossing a homozygous pWER::SHR::GFP line with a
homozygous scr-4 line (Fukaki et
al., 1998). The progeny was followed to the second generation and
genotyped for presence of the scr-4 allele and absence of wild-type
SCR.
The line pWER::SHR::GFP, pGL2::YFPER was obtained by crossing a homozygous pWER::SHR::GFP line with a homozygous pGL2::YFPER line and analyzing the F1 generation.
The line pWER::SHR::GFP, pSCR::YFPER was obtained by crossing a homozygous pWER::SHR::GFP line with a homozygous pSCR::YFPER line (a gift from Dr B. Scheres) and analyzing the F1 generation.
Confocal microscopy
Roots were counterstained in 10 µg/ml propidium iodide (PI) for 1
minute. Confocal images were obtained using a 63x water-immersion lens
on a Leica TCS SP2 spectral confocal laser-scanning microscope. In `GFP+YFP'
mode, we used the 488 nm Argon laser line to excite and collected in the range
493-536 nm (rendered in green) and 587-731 nm (rendered in red, collecting
emission from PI). In `YFP' mode, we used the 514+543nm Argon laser lines to
excite and collected in the ranges 555-587 nm (rendered in yellow) and 587-731
nm (rendered in red, collecting emission from PI).
Histochemical staining, immunolocalization and in situ hybridization
Roots from 4-7 day post-germination seedlings were fixed and embedded as
described previously (Fukaki et al.,
1998). For Casparian strip detection, 6 µm sections on slides
were incubated overnight in 0.1% berberine hemisulfate (Sigma) at room
temperature, rinsed with water, counterstained for 10 minutes in 0.5% Aniline
Blue WS (Polyscience) at room temperature, rinsed with water and transferred
for 5-10 minutes to 0.1% FeCl3 in 50% glycerol. The slides were
mounted with a drop of the same 0.1% FeCl3 solution
(Brundrett et al., 1988
;
Scheres et al., 1995
). Samples
were analyzed with a Leica DMRA2 epifluorescence microscope with FITC filters.
For immunolocalization, 6 µm sections were processed for staining with the
JIM13 antibody (Knox et al.,
1990
) as described previously
(Di Laurenzio et al., 1996
).
The samples were then analyzed by epifluorescence microscopy, as above. In
situ hybridization analysis was performed as described previously
(Di Laurenzio et al., 1996
).
The GFP-specific probe was first amplified by PCR to make the template
spanning nearly the entire coding region. The reverse primer was designed to
contain a T3 promoter site that was used to generate the antisense probes.
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Results |
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Within the stele, symplastic connections have been documented between the
phloem companion cells (CC) and sieve elements (SE) (for reviews, see
Oparka and Turgeon, 1999;
Ruiz-Medrano et al., 2001
;
Van Bel, 2003
). The filament
protein Phloem Protein 1 (PP1, 96 kDa) has been shown to move from the phloem
CC to SE in Cucurbita maxima leaves
(Clark et al., 1997
;
Leineweber et al., 2000
). In
addition, GFP (27 kDa) is able to move from the phloem CC throughout the root
meristem when expressed under the promoter of the SUC2
sucrose-H+ symporter gene (pSUC2), which is known to be
active in the phloem CC (Imlau et al.,
1999
; Truernit and Sauer,
1995
) (Fig. 1A,
compare with Fig. 1B,D).
|
To determine if SHR::GFP is able to move from the phloem CC, we produced transgenic lines where the SHR::GFP fusion protein (87 kDa) is driven by the SUC2 promoter. In the root of pSUC2::SHR::GFP lines, fluorescence was localized to the nuclei of the phloem CC (Fig. 1C,E). We did not detect any signal in other tissues (Fig. 1C,E, compare with Fig. 1B,D), indicating that the fusion protein is not able to move. This lack of movement from cells that support movement of other proteins raises the possibility that factors present where SHR is normally expressed are required for SHR::GFP movement. An alternative explanation is that factors inhibiting SHR::GFP movement are present in the phloem CC.
SHR::GFP moves only one cell distance through supernumerary endodermal layers
We next addressed the role of cell type in limiting the extent of movement.
SHR moves from the stele to the endodermis, but does not move into the
adjacent cortex. This suggested that there might be some restriction in
symplastic connectivity between endodermis and cortex that limits the extent
of radial movement of SHR. A prediction from this hypothesis would be that SHR
should be able to move from one presumptive endodermal cell to another. We
tested this in plants with multiple ground tissue layers all of which acquire
endodermal characteristics. This occurs when SHR is driven by the
SCR promoter (pSCR)
(Nakajima et al., 2001). Into
these plants we introduced SHR::GFP driven by the native SHR
promoter.
We detected the fusion protein in the stele and in the first layer of
ground tissue in contact with it, but not in the tissues further away
(Fig. 2). At the subcellular
level, we detected SHR::GFP in both nuclei and cytoplasm within the stele but
only in nuclei of the first ground tissue layer (arrowheads in
Fig. 2). In a few instances, a
faint signal was detected in a second cell next to the first ground tissue
layer. This could be attributed to the fact that after a periclinal division,
SHR is known to persist for a brief time in both daughter cells
(Nakajima et al., 2001).
|
SHR::GFP is not able to move from the epidermis in a wild-type background
To further investigate the role of cell type in regulating SHR movement we
asked if SHR::GFP is able to move from a cell type that does not normally
contain SHR protein. We chose to focus these experiments on the epidermis
because there are well-characterized promoters that confer expression in
subsets of epidermal cells in different stages of development. The promoter of
the homeobox gene GLABRA2 (GL2) is active in the root
epidermis, with preferential expression in the atrichoblasts (non-hair cells).
Expression becomes detectable in the meristematic zone, but not in the
initials, and is maintained in the elongation zone and in at least part of the
differentiation zone (Lin and
Schiefelbein, 2001; Masucci et
al., 1996
) (inset in Fig.
3A).
|
Competence to respond to SHR-mediated cell division is limited to initial cells
From the observation of four independent pGL2::SHR::GFP transgenic
lines, we saw no evidence of perturbation in radial patterning, indicating
that cell division processes had not been altered in these plants
(Fig. 3A). We conclude that
cells of the atrichoblast lineage above the initials do not appear to be
competent to respond to the cell division promoting activity of SHR.
We hypothesized that competence to respond to SHR by generating periclinal
divisions may reside in the initial cells in which the GL2 promoter
does not confer detectable expression. To test this hypothesis, we drove
expression of the SHR::GFP translational fusion using the 5'
and 3' regulatory sequences from the WEREWOLF (WER)
gene, which is transcribed in the epidermis, a region of the lateral root cap
(LRC) and the epidermal/LRC (ep/LRC) initials
(Lee and Schiefelbein, 1999)
(inset in Fig. 3C).
The pWER::SHR::GFP transgenic lines showed a dramatic perturbation of the radial pattern with an excess of periclinal cell divisions (Fig. 3C-E). The resulting supernumerary tissues appeared organized in concentric layers (Fig. 3E), although the number of layers varied between independent transgenic lines. These results together suggest that, in the epidermal lineage, only the initials are competent to respond to SHR with periclinal divisions.
SHR::GFP does not move from the epidermis even in the presence of supernumerary layers
We then asked if SHR::GFP was now able to move within these ectopic layers.
In observations of three independent transgenic lines, we detected GFP signal
only in the outermost tissue, where SHR::GFP appeared localized to the nuclei
(Fig. 3C,D). In the very few
cases when SHR::GFP was detected in a cell not in the outermost layer, the
morphology always suggested that it resulted from a recent cell division of a
neighboring cell in the outermost tissue layer, which also contained SHR::GFP.
In conclusion, SHR::GFP does not move from the epidermis even in the presence
of supernumerary layers.
Competence to respond to SHR-mediated cell divisions is through SCR
SHR has been shown to act through SCR to induce the periclinal division
that generates endodermis and cortex
(Helariutta et al., 2000).
Moreover we have previously shown that the supernumerary divisions induced by
expression of SHR behind a SCR promoter are dependent on
active SCR (Helariutta et al.,
2000
; Nakajima et al.,
2001
). In plants expressing SHR::GFP driven by the
WER regulatory elements, we asked whether the resulting ectopic
periclinal divisions were dependent on SCR. To address this question, we
crossed a pWER::SHR::GFP line exhibiting a large number of
supernumerary layers into the scr-4 mutant background. We observed a
scr phenotype, lacking not only the supernumerary layers but also one
of the two ground tissues (Fig.
4A), demonstrating that SCR is required for the periclinal
divisions induced in the ep/LRC initials by ectopic SHR::GFP.
|
|
Competence to respond to SHR-mediated cell specification is widely distributed in the epidermis
To determine the competence to respond to SHR-mediated cell specification
activity, we looked for two independent endodermis-specific markers in roots
of pGL2::SHR::GFP and pWER::SHR::GFP transgenic lines. We
used a histochemical stain that reveals the suberin in the endodermis-specific
hydrophobic cell wall deposit known as Casparian strip (as well as the lignin
in differentiating xylem cells) (Brundrett
et al., 1988) (Fig.
6A) and the JIM13 monoclonal antibody specific for an
arabinogalactan epitope found in the endodermis (as well as in a subset of
stele cells) (Dolan and Roberts,
1995
) (Fig. 6B). In
both transgenic lines, evidence of Casparian strip deposition was found not
only in the normal location of the endodermis (arrowheads in
Fig. 6C,E) but also in some
cells of the outermost tissue layer (arrows in
Fig. 6C,E). JIM13
immunostaining was detected in even a greater number of cells in the outermost
tissue (Fig. 6D,F), confirming
the endodermal character of these cells. These results indicate that the outer
layer cells are competent to respond to the cell specification role of SHR.
Moreover, SHR::GFP is synthesized in these cells, rather than moving into
them, and yet is able to induce endodermal characteristics. This argues
against a model in which movement is a prerequisite for activity.
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Discussion |
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It has been proposed that symplastic movement of molecules can occur by two
mechanisms: simple diffusion is possible if the molecule is smaller than the
basal size exclusion limit (SEL) of the PD, while active movement requiring a
dilation of the PD can occur for bigger molecules
(Zambryski and Crawford,
2000). The process involved in triggering PD dilation is not
understood, but it appears that some form of specific interaction between the
trafficking protein and the PD apparatus is required
(Haywood et al., 2002
).
When the SHR::GFP fusion protein is expressed in the stele under the
control of the SHR promoter, it moves into the adjacent tissue layer,
but not further (Nakajima et al.,
2001) (Fig. 8A,C). One hypothesis for this limited trafficking is that SHR::GFP can move
passively from any cell in which it is expressed, and that movement is limited
by a symplastic barrier at the endodermal/cortex boundary. Neither part of
this hypothesis could be tested using the constitutive 35S promoter
or the SCR promoter. Here, we first used the SUC2 promoter
to express SHR in the phloem CC, which have been shown to be symplastically
connected to other cells (Oparka and
Turgeon, 1999
; Ruiz-Medrano et
al., 2001
; Van Bel,
2003
). The lack of any movement of the fusion protein from the
phloem CC, was our first indication that factors present in the source tissue
may play an important part in determining the ability of SHR::GFP to move.
This was confirmed by expression of SHR::GFP in the epidermal lineage, which
did not support movement (Fig.
8D,E). The lack of SHR::GFP movement from both phloem CC and
epidermal cells cannot be attributed to impaired symplastic connections
between these cells and their neighbors, as non-targeted GFP driven by the
same promoters is able to move throughout all root tissues
(Imlau et al., 1999
) (M.
Cilia, personal communication). This suggests that factors required for
SHR::GFP movement are absent in the phloem CC, the epidermis and the
endodermis. Alternatively, factors inhibiting movement might be present in
these cells.
|
This result also provides new insight into the generation of the ectopic
cell layers in plants containing the pSCR::SHR construct
(Nakajima et al., 2001). The
lack of movement of SHR::GFP from the endodermis to the adjoining endodermal
layers would suggest that the ectopic layers are not produced by movement from
one layer to the next. Immunolocalization of SHR in wild-type roots indicated
that SHR was normally partitioned in roughly equal amounts after the
periclinal division of the co/en initial daughter
(Nakajima et al., 2001
). This
would suggest that the extra divisions in the pSCR::SHR lines are
likely to be the result of increased levels of SHR in the external daughter
(produced by a positive-feedback loop of SHR on the SCR promoter).
These increased levels would then trigger another round of division, and this
process could be repeated.
A role for SCR in limiting intercellular movement
A surprising result was that SHR::GFP moved from epidermal cells in the
scr mutant background and that movement correlated with a change in
subcellular localization (Fig.
8F). Because we showed that in those transgenic lines the
SCR promoter is not active in the epidermal tissue above the
initials, formally this suggests a non-cell autonomous effect of SCR
on SHR::GFP movement. This could be achieved through perdurance of SCR protein
produced in the initials. A more plausible explanation is that there is
normally an indirect effect either of SCR expressed in the initials or
expressed in the endodermis resulting in nuclear localization of SHR::GFP,
which inhibits movement. Alternatively, the change of radial pattern in the
scr mutant could have an effect on subcellular localization and
movement of SHR::GFP.
Subcellular localization has been suggested to be one relevant parameter in
the regulation of intercellular protein movement
(Crawford and Zambryski, 2000).
A recent analysis of a number of GFP-tagged proteins expressed in the shoot
apical meristem showed a positive correlation between cytoplasmic accumulation
and intercellular movement (Wu et al.,
2003
). In particular, a functional translational fusion between
GFP and the transcription factor LEAFY has been proposed to move by diffusion
in the shoot apical meristem (Sessions et
al., 2000
; Wu et al.,
2003
). The fact that SHR::GFP accumulation in the cytoplasm of the
source tissue correlates with its movement suggested that this might be a
necessary and sufficient condition for movement. However, analysis of a mutant
form of SHR suggests that although necessary for movement, cytoplasmic
localization is not a sufficient condition (K. L. Gallagher, A. J. Paquette,
K. Nakajima and P.N.B., unpublished). This raises the possibility that either
tissue-specific factors or post-translational modifications of SHR could be
involved in regulating movement. Interestingly, protein phosphorylation has
been associated with the regulation of the tobacco mosaic virus movement
protein TMV-MP intercellular trafficking
(Citovsky et al., 1993
;
Waigmann et al., 2000
) and a
similar mechanism has been recently proposed for KN1
(Kim et al., 2003
).
There is a broad competence to respond to SHR-mediated cell specification
Because cell specification has been shown to be dependent on position in
most plant tissues (Kidner et al.,
2000; van den Berg et al.,
1997
), it follows that cues are required to define a location
along the radial axis. SHR is expressed in the stele and it is necessary for
the correct differentiation of the endodermal tissue, into which it moves
(Helariutta et al., 2000
;
Nakajima et al., 2001
). In the
simplest scenario, SHR could then be the positional cue `instructing' the
ground tissue in contact with the stele to acquire endodermal fate. This
raises the question of whether a pre-pattern of competence exists to respond
to SHR or whether SHR alone is sufficient to induce endodermal fate in any
cell in which it is present. We know that SHR is not sufficient to induce
endodermal differentiation in the stele
(Helariutta et al., 2000
).
However, we have shown that it is sufficient to induce at least some aspects
of endodermal fate determination in the supernumerary layers between the stele
and the epidermis in plants expressing pSCR::SHR
(Nakajima et al., 2001
). To
extend this analysis to the outermost layer of the root, we asked if cells of
the epidermal lineage are able to acquire endodermal characteristics in
response to SHR. At least two independent endodermal markers could be found in
cells of the epidermal lineage when SHR::GFP is expressed there.
Thus, competence to develop endodermal characteristics in response to SHR
appears to exist in all root tissues along the radial axis external to the
stele. Moreover, expression of SHR::GFP by the GL2 promoter solely in
maturing epidermal cells is sufficient to confer endodermal fate. This broad
competence to respond to SHR indicates that development of a single endodermal
layer in contact with the stele depends on tight regulation of SHR movement.
Without this tight regulation, endodermal characteristics would be found
throughout the radial axis of the root external to the stele. The ability to
induce endodermal characteristics in the epidermal lineage in the absence of
SHR::GFP movement also provides evidence that movement is not a pre-condition
for activity.
It is interesting to note that expression of SHR::GFP in the epidermal
lineage did not result in a complete transformation of this tissue to
endodermis. Root hairs were still made by trichoblasts and the
atrichoblast-specific reporter pGL2::YFPER was active. We
previously reported that, in pSCR::SHR, pGL2::GUS transgenic roots, a
very small number of cells in the outermost position of the supernumerary
layers contained SHR protein in their nuclei and did not express GUS
from the GL2 promoter (Nakajima
et al., 2001). In these plants, there appeared to have been a more
complete transformation to endodermis. A likely explanation is that the cells
containing SHR originated in the internal supernumerary layers (expressing SHR
and not GL2), and were `pushed' into the outermost position due to
cell division events associated with the strongly perturbed radial
pattern.
There is a restricted competence to respond to SHR-mediated periclinal cell divisions
We have previously shown that either ubiquitous SHR expression
driven by the 35S promoter or more restricted expression with the
SCR promoter resulted in a perturbed radial pattern with
supernumerary cell layers (Helariutta et
al., 2000; Nakajima et al.,
2001
). In both cases, SCR expression was shown to be
induced in the supernumerary layers as well as in the co/en initials
(Helariutta et al., 2000
;
Nakajima et al., 2001
).
Moreover, induction of ectopic cell layers by the pSCR::SHR transgene
was dependent on active SCR (Nakajima et
al., 2001
).
We have extended our understanding of the potential for SHR to alter radial patterning by showing that the ep/LRC initials are competent to respond to SHR by expressing SCR and producing supernumerary cell layers. By contrast, expression of SHR::GFP restricted to more mature epidermal cells did not result in SCR expression, suggesting that only the initials are competent to express SCR upon SHR induction. In the case of pWER::SHR::GFP, production of the supernumerary tissues disappeared in a scr mutant background, indicating that the competence to respond to SHR by periclinal cell divisions is mediated by SCR in these initial cells that normally never see SHR or SCR.
We cannot formally exclude the possibility that instead of acting in the ep/LRC initials, small amounts of SHR::GFP moved into the co/en initials (where endogenous SHR is already present) and there induced SCR-dependent extra periclinal cell divisions. However, whenever the pattern allowed us to morphologically recognize the exact location of the co/en initials, we did not detect any GFP in these cells. Moreover, in pSHR::SHR::GFP transgenic roots in a wild-type background, where the fusion protein moved into the co/en initials, we never observed any significant alteration of the radial pattern (data not shown). The difference in competence to respond to SHR in different cell types suggests the presence of factors distributed in a tissue-specific manner. These factors could for example dimerize with SHR to either activate or inhibit the transcription of target genes, or modify SHR activity by producing post-translational modifications.
In conclusion, our analysis of tissue-specific ectopic expression of SHR::GFP has revealed a complex picture of the regulation of its intercellular trafficking. Movement by diffusion seems insufficient to explain the features we observed. Rather, tissue-specific factors seem likely to play a role in regulating movement of SHR::GFP. Restriction of SHR movement to the first layer external to the stele is crucial to radial patterning, as competence to respond to SHR-mediated cell specification appears widespread along the radial axis, while competence to respond to SHR-mediated periclinal cell divisions resides in initial cells (Fig. 9). This non-uniform distribution of competence to respond to SHR also suggests tissue-specific localization of factors essential for SHR activity.
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ACKNOWLEDGMENTS |
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Footnotes |
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