1 Department of Molecular, Cellular, and Developmental Biology, University of
Michigan, 830 North University Avenue, Ann Arbor, MI 48109, USA
2 Section of Molecular, Cell and Developmental Biology and the Institute for
Cellular and Molecular Biology, University of Texas, Austin, TX 78712,
USA
* Author for correspondence (e-mail: schiefel{at}umich.edu)
Accepted 5 November 2004
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SUMMARY |
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Key words: Epidermis, Pattern formation, Root hairs, Gene regulation, Cell differentiation, Embryogenesis, Cell communication, Hypocotyl, Arabidopsis thaliana
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Introduction |
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In Arabidopsis, root hair cells are specified in a
position-dependent manner, such that all cells located in a cleft between two
underlying cortical cells (designated the H position) develop as hair cells
and cells located outside a single cortical cell (designated the N position)
adopt the non-hair fate (Dolan et al.,
1994; Galway et al.,
1994
). Molecular genetic studies have shown that a suite of
putative transcription factors regulates the patterning of root hair cells in
Arabidopsis. These factors include a homeodomain protein, GLABRA2
(GL2) (Masucci et al., 1996
;
Rerie et al., 1994
); a
WD-repeat protein, TRANSPARENT TESTA GLABRA (TTG)
(Galway et al., 1994
;
Walker et al., 1999
); an R2R3
MYB-type transcription factor, WEREWOLF (WER)
(Lee and Schiefelbein, 1999
);
two closely related basic helix-loop-helix proteins, GLABRA3 and ENHANCER OF
GLABRA3 (Bernhardt et al.,
2003
); and three small MYB proteins, CAPRICE (CPC), TRIPTYCHON
(TRY) and ENHANCER OF TRIPTYCHON AND CAPRICE (ETC1)
(Kirik et al., 2004
;
Schellmann et al., 2002
;
Wada et al., 2002
;
Wada et al., 1997
). The GL2,
TTG, WER, GL3 and EGL3 appear to have a primary role in promoting the non-hair
fate, whereas the CPC, TRY and ETC1 are most important in specifying the hair
cell fate. The cell pattern is proposed to result from a lateral inhibition
mechanism that is mediated by CPC, TRY and ETC1
(Larkin et al., 2003
;
Schiefelbein, 2003
). The
transcription of CPC (and presumably of TRY and
ETC1) is promoted by a putative complex of TTG, WER, GL3 and EGL3 in
the N cell position, and these small MYB proteins inhibit the neighboring H
cells from adopting the non-hair fate, possibly by directly moving from
cell-to-cell and interfering with the WER function
(Schellmann et al., 2002
;
Schiefelbein, 2003
;
Wada et al., 2002
;
Wada et al., 1997
).
Although the importance of the putative complex containing GL3, EGL3, WER
and TTG is clear, the mechanisms that regulate the accumulation of these
components is poorly understood. In this study, we sought to analyze the
expression and regulation of GL3 and EGL3 during root
epidermis development. In prior work, we have analyzed mutants and
overexpression lines to show that GL3 and EGL3 are likely to act redundantly
to help specify both the hair and non-hair cell fates
(Bernhardt et al., 2003). The
gl3 egl3 double mutant was shown to have excessive root-hair cells,
while the overexpression of these genes caused an increased frequency of
non-hair cells. Furthermore, these bHLH proteins are required for the positive
transcriptional control of GL2 (which specifies the non-hair fate)
and CPC (which helps specify the hair cell fate), and they interact
with the WER and the CPC MYB proteins in yeast
(Bernhardt et al., 2003
). The
prediction from this work was that these bHLH genes might be expressed (and
their gene products accumulate) in both the H and N cells. Here, we use RNA
hybridization, promoter reporter fusions and genetic analyses to show that the
GL3/EGL3 genes are preferentially expressed in the H
position, and furthermore, that this expression pattern is controlled by
several of the known cell fate regulators, including the GL3/EGL3 proteins
themselves. Using a YFP translational fusion, we find that GL3 accumulates in
the nuclei of the N cells. These results suggest a new feedback loop in the
epidermal regulatory network that helps establish and reinforce the cell fate
pattern.
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Materials and methods |
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The following transgenic lines have been described previously:
GL3::GUS and EGL3::GUS
(Zhang et al., 2003),
GL2::GUS (Masucci et al.,
1996
), CPC::GUS (Wada
et al., 2002
), 35S::GL3
(Payne et al., 2000
),
35S::EGL3 (Zhang et al.,
2003
), 35S::CPC (Wada
et al., 1997
) and 35S::TRY
(Schellmann et al., 2002
).
Lines homozygous for multiple mutations and/or transgenes were constructed by crossing single mutant or transgenic plants, examining the F2 progeny for putative mutant phenotypes, and confirming the desired genotype in subsequent generations by backcrossing to single mutants and/or PCR-based tests.
For seedling analysis, Arabidopsis seeds were surface sterilized
and grown on agarose-solidified nutrient medium in vertically oriented petri
plates as previously described
(Schiefelbein and Somerville,
1990).
Microscopy
The histochemical analysis of plants containing the GUS reporter
constructs was performed on at least 20 four-day-old root tips for each strain
essentially as described (Masucci et al.,
1996). Root epidermal cells were deemed to be in the N position if
they were located outside a periclinal cortical cell wall, whereas cells in
the H position were located outside a radial wall between adjacent cortical
cells.
The distribution of epidermal cell types in the hypocotyl was analyzed in 25 nine-day-old seedlings for each strain by determining the number of stomata formed along the hypocotyl in two adjacent epidermal cell files, one located over anticlinal cortical cell walls and one located over periclinal cortical cell walls.
In situ RNA hybridization
The whole-mount in situ RNA hybridization procedure has been described
(de Almeida Engler et al.,
1994). The RNA probe was designed to hybridize to both
GL3 and EGL3 transcripts, so it included bp 550-1120 and bp
1400-1850 downstream from the start site of the EGL3-coding sequence
corresponding to the most similar region of the GL3 and EGL3 proteins but
excluding the bHLH signature region to eliminate the possibility of
cross-hybridization to other bHLH proteins.
Molecular biology methods
To construct the GL3::GL3-YFP translational fusion, pD2L-2
(Payne et al., 2000) was used
to provide a GL3 genomic DNA fragment containing the entire
GL3 gene, including
1 kb upstream of the start codon and 1 kb
downstream of the stop codon. The existing SacI and SalI
sites of pD2L-2 were destroyed and new SacI and SalI sites
were generated by inverse PCR at the 3' end of the GL3-coding
region. The EYFP-coding region was amplified from pEYFP (Clontech)
and fused in-frame to the GL3 3' end. A BamHI fragment
from this vector, containing the entire GL3::EYFP fusion, was
subcloned into the BglII site of the T-DNA vector pAL47
(Lloyd and Davis, 1994
). The
GL3-YFP fusion protein is predicted to be 96 kDa, whereas the predicted size
of the GL3 and EGL3 proteins is 70 kDa and 66 kDa, respectively. Plant
transformation was performed by the floral dip method
(Clough and Bent, 1998
).
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Results |
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GL3 and EGL3 function in the embryonic root and during hypocotyl epidermis development
The position-dependent patterning of root epidermal gene expression is
known to be established during early stages of embryogenesis
(Costa and Dolan, 2003;
Lin and Schiefelbein, 2001
).
To explore further the unexpected expression pattern of GL3 and
EGL3, we examined the GL3::GUS and EGL3::GUS
activity during embryogenesis. We found that GUS activity accumulated in the
same H cell pattern in the epidermis of the mature embryonic root as in the
postembryonic root (Fig.
3A).
|
The hypocotyl is known to be patterned by a similar network of
transcription factors as the root to define two position-dependent cell types:
cells capable of producing stomata and non-stomata cells
(Berger et al., 1998b;
Hung et al., 1998
). In gl3
egl3 embryos bearing either the GL2::GUS or the
CPC::GUS reporters, we detected a significantly reduced level of GUS
activity in the embryonic hypocotyl relative to the wild type
(Fig. 3B). This dependence of
GL2 and CPC expression on the presence of GL3/EGL3 in the
embryonic hypocotyl suggested a possible role for both proteins during
stomatal patterning in the hypocotyl. To explore this further, we analyzed
stomata distribution in the hypocotyls of nine-day-old seedlings of wild type
and gl3 egl3 double mutants. In contrast to the wild type, which has
stomata predominantly form over a radial cortical cell wall (analogous to the
H position of the root epidermis), the gl3 egl3 hypocotyls have an
increased number of stomata and they are present in both positions
(Table 1). This shows that GL3
and EGL3 are required for proper stomata patterning, and it strengthens the
close relationship between the mechanism for patterning the hypocotyl and root
epidermis in Arabidopsis.
|
We first examined the possible role of the upstream regulators in this system, WER and TTG. The EGL3::GUS transgene was introduced into the wer-1 and ttg-1 mutant backgrounds, and we found that each of the homozygous mutants exhibit ectopic GUS expression in the N cell position (Fig. 4A). This suggests that WER and TTG negatively regulate EGL3 expression in the N cells.
|
We next tested the effect of gl2 and rhd6 mutants on
EGL3 expression. Results from mutant and overexpression analyses
indicate that GL3/EGL3 act at an early stage of root epidermis development
(Bernhardt et al., 2003), so we
expected no effect of mutations in the later acting genes GL2 and
RHD6. Consistent with this, the EGL3::GUS pattern in
gl2 and rhd6 was indistinguishable from the wild type
(Fig. 4A).
Last, we examined the possibility that the GL3/EGL3 proteins themselves might regulate EGL3 promoter activity. Although roots of the gl3 EGL3::GUS and egl3 EGL3::GUS lines exhibit only weak ectopic GUS activity, the gl3 egl3 EGL3::GUS double mutant line possessed strong GUS expression in the N position (Fig. 4C). Furthermore, overexpression of either bHLH gene (via the 35S::GL3 and 35S::EGL3 constructs) caused a weak or modest reduction in EGL3::GUS expression. These results suggest an autoregulation of EGL3 expression: the EGL3 protein (together with GL3) is able to inhibit its own gene's promoter activity.
GL3 transcription is similarly controlled as EGL3 transcription
To determine whether the GL3 promoter activity is regulated in a
similar manner as EGL3, we introduced the GL3::GUS reporter
into selected genetic backgrounds. We found that the wer mutant, the
gl3 egl3 double mutant, and the 35S::CPC overexpression
construct caused ectopic GL3::GUS expression, whereas the cpc
try double mutant exhibited a much lower level of GL3::GUS
expression (Fig. 5). In
addition, GL3::GUS activity was not effected by the cpc
single mutant or the gl2 mutation (data not shown). These results are
all consistent with the effects of these factors on EGL3::GUS, and it
suggests that both genes are regulated similarly.
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Discussion |
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Despite their important role in specifying both epidermal cell types, we
used promoter reporter fusions to show that GL3 and EGL3 are
preferentially expressed in the developing hair cells. How might GL3/EGL3
affect specification of the N cells while being expressed predominantly in the
H cells? One possibility is that the GL3 and EGL3 proteins may not directly
function in the N cells but rather act through another yet unidentified factor
involved in a lateral signaling from H to N. However, this possibility is not
supported by yeast two-hybrid data, which suggest a direct interaction between
the WER and GL3/EGL3 (Bernhardt et al.,
2003). In a second scenario, GL3 and EGL3 could act in a non-cell
autonomous manner in that either the proteins themselves or their RNA is
moving from cells in the H position to cells in the N position. Our results
support this second scenario. Although in situ hybridization analysis
indicated that GL3 and EGL3 RNAs accumulate preferentially in cells in the H
position (similar to results from the promoter reporter fusion analysis), a
GL3-YFP construct was detected predominantly in the N cell position.
Thus, the requirement for GL3/EGL3 activity in the developing N cells may be
fulfilled by the movement of these proteins from H cells to N cells.
It is likely that the H-cell specific expression pattern of the GL3 and EGL3 genes is due, at least in part, to negative autoregulation at the transcriptional level. We found that functional GL3 and EGL3 genes are required to inhibit GL3/EGL3 gene transcription and RNA accumulation in the N position. We also found that overexpression of GL3 or EGL3 causes modest reduction in EGL3 transcription in the H position. Furthermore, we show that the putative partners of GL3/EGL3 action, WER and TTG, are also required to inhibit GL3/EGL3 in the N cells. Taken together, these data indicate that the GL3 and EGL3 gene transcription is negatively regulated by the putative WER/GL3/EGL3/TTG complex, which is likely to be most abundant in the N cell position.
However, the GL3 and EGL3 genes were found to be
positively regulated by the CPC and TRY proteins, which act in the H cell.
Functional CPC/TRY genes are necessary for GL3/EGL3
expression in the H position, and overexpression of the CPC/TRY genes
cause ectopic GL3/EGL3 promoter activity and RNA accumulation in the
N position. This may be due, in part, to the ability of CPC (and possibly TRY)
to inhibit WER gene expression in the H position
(Lee and Schiefelbein, 2002),
which would reduce the abundance of the WER complex and thereby indirectly
increase GL3 and EGL3 transcription.
Together, these results lead us to make the following proposal
(Fig. 8). In the N cell, the
abundant WER protein accumulation (presumably owing to the action of
positional cues) leads to the formation of sufficient WER/GL3/EGL3/TTG complex
to induce expression of the N-cell fate-promoting factor GL2 and the
lateral inhibitor CPC (Bernhardt
et al., 2003; Lee and
Schiefelbein, 2002
). The CPC protein (and possibly TRY) moves to
neighboring cells in the H position leading to the formation of an inactive
CPC/GL3/EGL3/TTG complex that prevents activation of GL2, thus
allowing for the specification of the hair cell fate
(Bernhardt et al., 2003
;
Lee and Schiefelbein, 2002
;
Wada et al., 2002
). At the
same time, while the accumulation of CPC (and possibly TRY) in H cells leads
to a reduction in WER (and CPC) expression, it also leads to
an increase in GL3 and EGL3 expression (this study). The GL3
(and likely EGL3) protein then acts in a lateral feedback loop by moving to
the neighboring N cells (possibly through plasmodesmata). This process is
likely to be efficient (perhaps driven by the constant removal of free bHLH
protein out of the equilibrium by binding to WER), as the GL3-YFP fusion
protein is found predominantly in N cells rather than being distributed in
equal intensity in all cell files. The additional GL3/EGL3 protein would
generate more of the WER/GL3/EGL3/TTG complex in the N cells, inducing
additional GL2 and CPC expression and also repression of
GL3 and EGL3 expression. In the end, this would mean that
essentially all of the GL3/EGL3 protein used in the N-cell complex formation
would come from the H cells.
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The explanation above suggests that bi-directional signaling, from the N to
the H cell (via CPC and possibly also TRY/ETC1) and from the H to the N cells
(via GL3 and possibly EGL3), is required for appropriate accumulation of
GL3/EGL3 in the N cell position during root epidermis development. The `back
and forth' signaling between cells proposed here is conceptually similar to
the kinds of bi-directional signaling identified in other cell specification
models, including the forespore/mother cell fate decision in Bacillus,
embryonic midgut and larval wing patterning in Drosophila, and vulval
cell specification in C. elegans
(Bondos and Tan, 2001;
Losick and Dworkin, 1999
;
Yoo et al., 2004
). However,
the signaling mechanism used in the Arabidopsis root epidermis
differs from these others because it involves the intercellular movement of
transcription factors, rather than receptor-mediated signaling, thereby
directly influencing gene expression and cell fates.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Berger, F., Hung, C. Y., Dolan, L. and Schiefelbein, J. (1998a). Control of cell division in the root epidermis of Arabidopsis thaliana. Dev. Biol. 194,235 -245.[CrossRef][Medline]
Berger, F., Linstead, P., Dolan, L. and Haseloff, J. (1998b). Stomata patterning on the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning. Dev. Biol. 194,226 -234.[CrossRef][Medline]
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.
Bondos, S. E. and Tan, X. X. (2001). Combinatorial transcriptional regulation: the interaction of transcription factors and cell signaling molecules with homeodomain proteins in Drosophila development. Crit. Rev. Eukaryot. Gene Expr. 11,145 -171.[Medline]
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
Costa, S. and Dolan, L. (2003). Epidermal
patterning genes are active during embryogenesis in Arabidopsis.
Development 130,2893
-2901.
de Almeida Engler, J., van Montagu, M. and Engler, G. (1994). Hybridization in situ of whole-mount messenger RNA in plants. Plant Mol. Biol. Reporter 12,321 -331.
Dolan, L. and Costa, S. (2001). Evolution and
genetics of root hair stripes in the root epidermis. J. Exp.
Bot. 52,413
-417.
Dolan, L., Duckett, C., Grierson, C., Linstead, P., Schneider, K., Lawson, E., Dean, C., Poethig, R. S. and Roberts, K. (1994). Clonal relations and patterning in the root epidermis of Arabidopsis. Development 120,2465 -2474.[Abstract]
Galway, M. E., Masucci, J. D., Lloyd, A. M., Walbot, V., Davis, R. W. and Schiefelbein, J. W. (1994). The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root. Dev. Biol. 166,740 -754.[CrossRef][Medline]
Hulskamp, M., Misra, S. and Jurgens, G. (1994). Genetic dissection of trichome cell development in Arabidopsis. Cell 76,555 -566.[Medline]
Hung, C. Y., Lin, Y., Zhang, M., Pollock, S., Marks, M. D.
and Schiefelbein, J. (1998). A common
position-dependent mechanism controls cell-type patterning and GLABRA2
regulation in the root and hypocotyl epidermis of Arabidopsis.
Plant Physiol. 117,73
-84.
Kirik, V., Simon, M., Huelskamp, M. and Schiefelbein, J. (2004). 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]
Koornneef, M., Dellaert, S. W. M. and van der Veen, J. H. (1982). EMS- and radiation-induced mutation frequencies at individual loci in Arabidopsis thaliana (L.) Heynh. Mutat. Res. 93,109 -123.[Medline]
Larkin, J. C., Brown, M. L. and Schiefelbein, J. (2003). How do cells know what they want to be when they grow up? Lessons from epidermal patterning in Arabidopsis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 54,403 -430.[CrossRef][Medline]
Lee, M. M. and Schiefelbein, J. (1999). WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning. Cell 99,473 -483.[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.
Lin, Y. and Schiefelbein, J. (2001). Embryonic
control of epidermal cell patterning in the root and hypocotyl of Arabidopsis.
Development 128,3697
-3705.
Lloyd, A. and Davis, R. W. (1994). Functional expression of the yeast FLP/FRT site-specific recombination in Nicotiana tobacum. Mol. Gen. Genet. 242,653 -657.[Medline]
Losick, R. and Dworkin, J. (1999). Linking
asymmetric division to cell fate: teaching an old microbe new tricks.
Genes Dev. 13,377
-381.
Masucci, J. D. and Schiefelbein, J. W. (1994).
The rhd6 mutation of Arabidopsis thaliana alters root-hair initiation through
an auxin- and ethylene-associated process. Plant
Physiol. 106,1335
-1346.
Masucci, J. D., Rerie, W. G., Foreman, D. R., Zhang, M., Galway,
M. E., Marks, M. D. and Schiefelbein, J. W. (1996).
The homeobox gene GLABRA2 is required for position-dependent cell
differentiation in the root epidermis of Arabidopsis thaliana.
Development 122,1253
-1260.
Payne, C. T., Zhang, F. and Lloyd, A. M.
(2000). GL3 encodes a bHLH protein that regulates trichome
development in arabidopsis through interaction with GL1 and TTG1.
Genetics 156,1349
-1362.
Rerie, W. G., Feldmann, K. A. and Marks, M. D. (1994). The GLABRA2 gene encodes a homeo domain protein required for normal trichome development in Arabidopsis. Genes Dev. 8,1388 -1399.[Abstract]
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. (2003). Cell-fate specification in the epidermis: a common patterning mechanism in the root and shoot. Curr. Opin. Plant Biol. 6, 74-78.[CrossRef][Medline]
Schiefelbein, J. W. and Somerville, C. (1990).
Genetic control of root hair development in Arabidopsis thaliana.
Plant Cell 2,235
-243.
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.
Walker, A. R., Davison, P. A., Bolognesi-Winfield, A. C., James,
C. M., Srinivasan, N., Blundell, T. L., Esch, J. J., Marks, M. D. and
Gray, J. C. (1999). The TRANSPARENT TESTA GLABRA1 locus,
which regulates trichome differentiation and anthocyanin biosynthesis in
Arabidopsis, encodes a WD40 repeat protein. Plant Cell
11,1337
-1350.
Yoo, A. S., Bais, C. and Greenwald, I. (2004).
Crosstalk between the EGFR and LIN-12/Notch pathways in C. elegans vulval
development. Science
303,663
-666.
Zhang, F., Gonzalez, A., Zhao, M., Payne, C. T. and Lloyd, A.
M. (2003). A network of redundant bHLH proteins functions in
all TTG1-dependent pathways of Arabidopsis.
Development 130,4859
-4869.