1 Department of Biology, Yonsei University, Sinchon 134, Seoul 120-749,
Korea
2 Division of Molecular and Life Sciences, Pohang University of Science and
Technology, Pohang 790-784, Korea
3 Department of Molecular, Cellular, and Developmental Biology, University of
Michigan, Ann Arbor, MI 48109, USA
* Author for correspondence (e-mail: mmlee{at}yonsei.ac.kr)
Accepted 23 August 2005
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SUMMARY |
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Key words: WEREWOLF, CAPRICE, EMSA, Direct target, Root epidermis, Cell fate specification, Arabidopsis
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Introduction |
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In plants, the epidermal cells in the Arabidopsis root provide a
model system to study cell fate specification. The root epidermis is composed
of two kinds of cells, hair-bearing cells and non-hair cells, and their fates
are determined in a position-dependent manner
(Dolan et al., 1994;
Galway et al., 1994
). The
cells located on the periclinal cell wall of the underlying cortex
(N-position) differentiate into non-hair cells, and the cells located on the
anticlinal cell wall of the underlying cortex (H-position) differentiate into
hair-bearing cells. Molecular genetic studies have revealed that many putative
transcription factors are involved in this position-dependent cell fate
specification. For example, WEREWOLF (WER), GLABRA2
(GL2) and TRANSPARENT TESTA GLABRA1 (TTG1) are
known to promote the non-hair cell fate, so that in their cognate mutants,
most of the root epidermal cells differentiate into hair cells, regardless of
their position (Galway et al.,
1994
; Lee and Schiefelbein,
1999
; Masucci et al.,
1996
). WER encodes a putative transcription factor of the
MYB family, and it is preferentially expressed in the N-position cells
(Lee and Schiefelbein, 1999
).
The MYB transcription factor family is one of the largest families of
transcription factors in the Arabidopsis genome
(Reichmann et al., 2000
).
There are more than 180 MYB genes in Arabidopsis encoding one to
three repeats (R1, R2 and R3) of the MYB domain, which is
50 amino acids
long and has regularly spaced tryptophan residues
(Rosinski and Atchley, 1998
).
GL2, which encodes a homeodomain-leucine zipper (HD-Zip) protein, is
expressed in the N-position cells (Masucci
et al., 1996
; Rerie et al.,
1994
) in a WER-dependent manner
(Lee and Schiefelbein, 1999
).
TTG1 encodes a WD40-repeat containing protein
(Walker et al., 1999
) and its
expression pattern has not been reported yet. GLABRA3 (GL3)
and ENHANCER OF GLABRA3 (EGL3), which encode bHLH putative
transcription factors, are also reported to promote the non-hair cell fate in
a redundant manner (Bernhardt et al.,
2003
), although they are expressed in the H-position cells
(Bernhardt et al., 2005
). The
CAPRICE (CPC) gene encodes a single MYB repeat protein that
lacks any discernible transcriptional activation domain; mutation of this gene
causes a reduced number of hair cells, implying that it induces the hair cell
fate (Wada et al., 1997
). The
CPC protein probably acts in a partially redundant manner with the related
TRIPTYCHON (TRY) and ENHANCER OF TRY AND CPC1 (ETC1) proteins (Schellman et
al., 2001; Kirik et al.,
2004
).
To explain cell fate specification in the root epidermis, a competition
mechanism between the WER and CPC MYB proteins was proposed
(Lee and Schiefelbein, 1999).
This explanation posits that epidermal cells with a relatively high level of
WER adopt the non-hair cell fate, whereas cells accumulating a high level of
CPC adopt the hair cell fate. However, the CPC gene was found to be
expressed in the N-position cells instead of the H-position cells, and this
expression is WER dependent (Lee and
Schiefelbein, 2002
; Wada et
al., 2002
). These results led to a lateral inhibition model for
epidermal cell fate specification (Lee and
Schiefelbein, 2002
). In this model, WER expression is
regulated by positional information, which causes slightly greater WER
accumulation in the N-position cells than in the H-position cells. This is
proposed to lead to greater transcription of GL2 and CPC in
the N-position cells. The GL2 induces the non-hair cell fate in these
N-position cells, whereas the CPC is proposed to move to the neighboring
H-position cells where it inhibits GL2 (and CPC) expression
and thereby promotes the hair cell fate
(Lee and Schiefelbein, 2002
;
Wada et al., 2002
). Therefore,
the WER MYB protein is proposed to be a master regulator, which controls both
the non-hair cell fate (by promoting GL2 transcription) and the hair
cell fate (by promoting CPC transcription).
Although this model fits the experimental results so far, it is necessary
to further test and refine the model with additional experiments. Most
importantly, we do not know yet whether the WER protein acts as a
transcription factor in this system. This assumption was based solely on its
amino acid sequence similarity with previously known MYB transcription factors
(Lee and Schiefelbein, 1999)
and its ability to transactivate transcription using a yeast one-hybrid assay
(Lee and Schiefelbein, 2001
).
Furthermore, we do not know whether WER influences CPC and
GL2 transcription directly or indirectly. To address these issues, we
have examined the localization and the transcriptional properties of the WER
protein. Taken together, our findings strongly suggest that WER is indeed a
transcriptional activator and directly induces CPC expression to
specify cell fates during root epidermis development.
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Materials and methods |
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Seeds were germinated and grown vertically on agarose-solidified medium
containing mineral nutrients at 22°C under continuous light
(Schiefelbein and Somerville,
1990).
Histochemical GUS staining
GUS activity was histochemically defined by staining 4-day-old seedlings as
described previously (Lee and
Schiefelbein, 2002).
Confocal microscopy
GFP expression in seedlings was examined using a BioRad Radiance 2100
scanning system with excitation at 488 nm and detection at 500-530 nm.
RNA extraction and northern blot analysis
Total RNA was extracted from the root tips of seedlings using the TRIZOL
reagent according to the manufacturer's protocol. Northern blot analysis with
a CPC gene fragment as a probe was described previously
(Lee and Schiefelbein, 2002).
The resulting signal was visualized with a Bioimage analyzer, BAS-2500 (Fuji
film).
Electrophoretic mobility shift assay
Labeling of DNA fragments for the electrophoretic mobility shift assay was
carried out using T4 polynucleotide kinase (Promega) and
[-32P]ATP (>3000 Ci/mmol, 10 mCi/ml) as described
previously (Sainz et al.,
1997
) and this end-labeled probe was purified on a gel.
Electrophoretic mobility shift assays were carried out as described
(Ausubel et al., 1988), except
that the binding reaction included 20 mM NaCl, 10 mM Tris Cl (pH 8.0), 1 mM
EDTA, 1 mM DTT, 5% glycerol, 30 µg/ml poly(dI-dC), 20,000 to 40,000 cpm of
probe and the WER protein. This reaction was incubated at room temperature for
30 minutes and resolved on 6% polyacrylamide gel in 0.5xTBE buffer. The
gel was dried and the signal was visualized using BAS-2500 (Fuji film).
Protein expression and purification
E. coli BL21 (DE3) transformed with pET-WER was treated with 1 mM
ß-D-thiogalactopyranoside. These bacterial cells were harvested after
3-hour incubation at 37°C, disrupted using a sonicator and were
centrifuged at 14,000 g for 20 minutes to remove debris. From
this extract, the expressed WER protein was purified using His·Bind
Quick 900 Cartridges according to the manufacturer's manual (Novagen), and was
dialyzed against storage buffer (25 mM Tris-Cl, pH 7.5, 25 mM NaCl, 10 mM
EDTA, 0.1% NP-40, 50% glycerol).
Gene constructs and plant transformation
To make the chimeric genes, a genomic DNA fragment of the WER-coding region
without the stop codon was PCR amplified, and fused to the N terminus of the
GFP (no ER version) gene in frame (WER-GFP), to the N-terminus of the
transactivating domain of the herpes viral VP16
(Triezenberg et al., 1988) in
frame (WER-VP16) or to the N-terminus of the hormone binding domain
of the glucocorticoid receptor (Picad et al., 1988) in frame
(WER-GR).
To examine the subcellular localization of the WER protein, the PWER:WER-GFP construct was generated by fusing a 2.4 kb 5' flanking region DNA fragment and a 1.1 kb 3' flanking region DNA fragment from the WER gene to the WER-GFP chimeric gene. As a control, the PWER:GFP construct was made with the same GFP (no ER version).
For P35S:WER-VP16 (or
P35S:WER-GR), the chimeric
WER-VP16 DNA (or WER-GR) was inserted between 35S dual
promoter and terminator in pRTL2-GUS
(Restrepo et al., 1990). For
BD-WER-VP16, the WER-VP16 DNA fragment was fused to the GAL4
DNA-binding domain in the vector pGBT9. In this construct, we used the coding
region of the WER cDNA instead of genomic DNA. Constructions of the
BD-WER and the AD-WER have been described previously
(Lee and Schiefelbein, 1999
;
Bernhardt et al., 2003
).
To express and purify WER protein from bacteria, we made pET-WER. The coding region of the WER cDNA excluding the 3' 72 bp long fragment was PCR amplified. This truncated DNA fragment was inserted downstream of the T7 tag sequence in the vector of pET28(a) using EcoRI and SacI.
All the DNA fragments that were PCR amplified using Phusion DNA polymerase (Finnzyme) were sequenced and confirmed to be error-free.
We used pCB302 vector as a binary vector for plant transformation
(Xiang et al., 1999). Plant
transformation was achieved by electroporating constructs into the
Agrobacterium strain GV3101 followed by introduction into Arabidopsis
using the floral dip method as previously described
(Clough and Bent, 1998
). T1
seeds were harvested and transgenic plants were selected by spraying
commercially available BASTA three times a week for 2 weeks.
Yeast one-hybrid assay
To compare the transcriptional activation activities of the native WER
protein and the WER-VP16 chimeric protein, the yeast one-hybrid assay was
employed as described by Sadowski et al.
(Sadowski et al., 1992).
BD-WER construct was transformed into yeast strain Y190 and
lacZ reporter gene expression was assessed by measuring
ß-galactosidase activity using chlorophenol red-b-D-galactopyranoside
(CPRG; BMS) as a substrate.
To test WBSI and WBSII in vivo, we used the Matchmaker yeast one-hybrid system (Clontech) (Kumar et al., 1966) according to the manufacturer's manual. We synthesized three tandem copies of WBSI, WBSII or point mutated WBSIs. These were inserted upstream of the HIS3 gene in the vector pHISi and upstream of lacZ gene in pLacZi, and these were integrated into yeast strain YM4271 genome. AD-WER described above was introduced into this reporter strains and lacZ reporter gene expression was assessed.
Arabidopsis protoplast transient expression assay
Arabidopsis mesophyll protoplasts were isolated and transfected as
described previously (Hwang and Sheen,
2001). Protoplasts (2x104) were transfected with
40 µg plasmid DNA with different combinations of reporter
(PCPC:LUC), effector (WER) and internal control
(UBQ10-GUS). Protoplasts were incubated in WI [0.5 M mannitol, 4 mM MES (pH
5.7), 20 mM KCl]for 9 hours under light conditions at room temperature. The
luciferase reporter activity was determined by the Luciferase Assay System
(Promega). The GUS assay was performed as described previously
(Jang and Sheen, 1994
).
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Results |
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We generated a P35S:WER-GR construct
in which the DNA sequence for the hormone-binding domain of the glucocorticoid
receptor (Picard et al., 1988)
was fused to the WER-coding region and its expression was driven by
the CaMV 35S promoter (Fig.
3A). This construct was stably introduced into wer-1
mutant plants. In these P35S:WER-GR wer-1 plants,
WER activity is expected to depend on glucocorticoid [e.g. dexamethasone
(DEX)] because the translocation of the WER-GR recombinant protein into the
nucleus should require glucocorticoid. Furthermore, the addition of
cycloheximide, an inhibitor of translation, is expected to prevent indirect
transcriptional induction of genes following DEX treatment. We tested these
expectations by examining the expression of reporter gene
PGL2:GUS, which was introduced by crossing into
the P35S:WER-GR wer-1 line as a means to monitor
epidermal cell specification in response to various treatments. In the absence
of DEX, GUS activity was almost undetectable in the wer-1 mutant
harboring P35S:WER-GR
(Fig. 3C), just as in the
wer-1 mutant itself (Fig.
3C) (Lee and Schiefelbein,
1999
). However, when we applied 1 µM DEX to 4-day-old
transgenic seedlings for 3 hours, PGL2:GUS was
expressed in epidermal cells in a pattern indistinguishable from the
wer-1 mutant harboring P35S:WER
(Fig. 3C). Furthermore, when we
applied 10 µM cycloheximide (CHX) with 1 µM DEX, we could not detect any
GUS activity (Fig. 3C). In
addition to the PGL2:GUS expression, the
epidermal cell fate was also affected by the application of DEX. While the
4-day-old P35S:WER-GR wer-1 seedlings
grown without DEX showed the typical wer-1 mutant phenotype, the
seedlings grown on agarose media containing 1 µM DEX showed a random
distribution of epidermal cell types (Table
1), similar to the phenotype of the
P35S:WER wer-1 seedlings previously reported
(Lee and Schiefelbein, 2002
),
implying that the WER-GR can act like the native WER protein and postembryonic
functioning of WER is sufficient to change the cell fate. These results show
that 1 µM dexamethasone is sufficient for the WER-GR fusion to induce
PGL2:GUS expression and that 10 µM CHX is
effective to suppress translation of the transcribed GUS mRNA.
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Next, we tested whether these two binding sites had different affinities for the WER protein. When we used radiolabeled WBSI as a probe and the cold WBSII as a competitor, we were able to detect a slight reduction in WBSI-WER binding with increasing amounts of the cold competitor (Fig. 4E). By contrast, when we used WBSII as a probe and WBSI as a cold competitor, the WBSII-WER binding was decreased sharply with increasing amounts of the cold competitor (WBSI) (Fig. 4E). These reductions were not caused by competition with mutated sequences (Fig. 4E, lanes 6 and 7). This competitive gel shift assay suggests that the affinity of WBSI for the WER protein is much stronger than that of WBSII in vitro.
In vivo tests of the WER-binding sites in the CPC promoter
To validate these WER-binding sites in vivo, a yeast one-hybrid assay was
employed with these two binding sites and the WER protein. For the reporter
constructs, we made three tandem repeats of WBSI and WBSII, respectively,
inserted them upstream of the lacZ reporter gene promoter, and
introduced these constructs into yeast
(Fig. 5A). We also made yeast
reporter strains that have three tandem copies of two mutated versions of WBSI
in the lacZ promoter (Fig.
5A). For the effector construct, a DNA fragment including the
WER-coding region was fused to the GAL4 activation domain sequence
(AD-WER) and expressed in these reporter strains. We found
that the AD-WER was able to increase the ß-galactosidase activity by
threefold in the yeast harboring WBSI on its reporter gene promoter
(Fig. 5B). By contrast, there
was no increase in the ß-galactosidase activity when
AD-WER was expressed in the yeast harboring the two mutated
versions of WBSI (WBSI-M1 and WBSI-M2). These data clearly show that WBSI,
which we identified in vitro using EMSA
(Fig. 4), functions as a
WER-binding site in vivo. We were not able to detect a significant increase in
reporter expression when the AD-WER was expressed in yeast harboring WBSII on
its reporter gene promoter, perhaps because the ß-galactosidase activity
in this yeast without the WER protein was already very high (data not shown).
It is possible that an endogenous yeast transcriptional activator is able to
bind to this WBSII in yeast cells.
|
Next, we made stable transgenic lines with the wild-type and three mutated versions of the CPC promoter fused to the GUS reporter gene (Fig. 6C). Although the wild-type version (PCPCWT:GUS) showed high level of GUS activity in the root epidermis (preferentially in the N-position cells), the mutant versions (PCPCM1:GUS, PCPCM2:GUS or PCPCM3:GUS, which have mutations at WBSI, WBSII or at both, respectively) showed very little GUS activity (Fig. 6D). Specifically, after 4 hours of incubation, no detectable GUS staining could be detected in these mutant versions, whereas the wild-type version showed strong GUS staining. After 24 hours of incubation, some of the seedlings harboring the mutant reporter genes showed very low GUS staining. These results show that the WER-binding sites are important for WER-dependent CPC promoter activity in the Arabidopsis root epidermis.
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Discussion |
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In this paper, we report that the WER protein is localized to the nucleus
in the Arabidopsis root epidermis
(Fig. 1C), that it is required
to directly induce CPC RNA accumulation
(Fig. 3), that it binds to
specific sites in the CPC promoter in vitro
(Fig. 4) and that these
WER-binding sites are important for WER-dependent CPC promoter activation in
vivo (Figs 5,
6). Together with the previous
finding of a transcriptional activation domain in the C terminus of WER
(Lee and Schiefelbein, 2001),
the results provided in this paper strongly suggest that WER indeed
encodes a transcriptional activator.
CPC is a direct target gene of WER
In animals, MYB proteins generally contain three MYB repeats (R1, R2 and
R3), and they bind to the consensus DNA sequence, YAACKG (Bidenkapp et al.,
1988; Golay et al., 1994;
Howe and Watson, 1991
). The R2
and R3 domains are important in this sequence specific binding and the R1
domain seems to stabilize the MYB-DNA interaction
(Howe et al., 1990
;
Saikumar et al., 1990
;
Tanikawa et al., 1993
). In
plants, two-repeat MYB proteins (R2R3 MYBs) are predominant
(Reichmann et al., 2000
;
Romero et al., 1998
), and they
differ in their DNA-binding specificity from the R1R2R3 MYB proteins from
animals (Solano et al., 1995
;
Williams and Grotewold, 1997
).
Plant R2R3 MYB proteins also show different binding specificity between
themselves. For example, maize C1 and P which are involved in flavonoid
biosynthesis show specific binding to a DNA sequence, CC(T/A)ACC
(Sainz et al., 1997
), which is
not related to the animal MYB binding sequence, YAACKG (MBSI). AtMYB2, which
functions in abscisic acid signaling in Arabidopsis
(Abe et al., 2003
), binds to a
DNA sequence, TGGTTAG, which is somewhat related to a DNA sequence, CNGTTR,
complementary to the MBSI (Abe et al.,
1997
). MYB.Ph3 from petunia shows dual DNA-binding specificity to
the DNA sequences consensus I (aaaAaaC(G/C)GTTA), which is similar to the
sequence CNGTTR (MBSI), and consensus II (aaaAGTTAGTTA) (MBSII)
(Solano et al., 1995
). This
consensus II cannot be recognized by animal c-MYB. Although MYB proteins are
believed to exert their roles by activating specific target genes that contain
MYB-binding sequences in their promoter regions, there are relatively few
studies that provide direct evidence for this assumption. One of the best
known examples involves P and C1, maize MYB genes. Using EMSA and a transient
expression system, P has been shown to regulate the a1 gene, one of
the flavonoid biasynthetic genes, and C1 has been shown to regulate many
flavonoid biosynthetic genes, a1, bz1 and a2
(Grotewold et al., 1994
;
Lesnick and Chandler, 1998
;
Sainz et al., 1997
).
GaMYB2/FIBER FACTOR 1 (FIF1), a MYB gene from
cotton, is expressed in developing cotton fibers and has been shown to
regulate RD22-like1 (RDL1) using the yeast one-hybrid assay
and heterologous transgenic plant analyses
(Wang et al., 2004
). In
addition, one of the MYB proteins in Arabidopsis, AtMYB2, has been
shown to regulate a dehydration-responsive gene, rd22, and an alcohol
dehydrogenase gene, AtADH1, using a transient expression experiment
(Abe et al., 1997
;
Hoeren et al., 1998
).
In this paper, we used the GR-inducible system to identify CPC as
a direct target of the WER protein, because CPC transcripts increased
significantly in response to WER-GR induction (in the wer-1 mutant
background) in the presence of cycloheximide. We then showed that the WER
protein has binding specificity to two sequences in the CPC promoter
(Fig. 4B). These two sequences,
WBSI (AgtaGTTa) and WBSII (CAACtg), are imperfectly complementary to each
other, but are move closely related to MBSII and MBSI, respectively. The
binding affinities of WER to these two sites are different at least in vitro.
Similarly, the maize A1 gene has two P binding sites in its promoter
(Grotewold et al., 1994), and
these two sites have different affinities for P protein in vitro
(Sainz et al., 1997
). Both of
these sites are functional in vivo and either site is sufficient for P
activation in vivo (Sainz et al.,
1997
). In the CPC promoter, we find that the two
WER-binding sites also function in vivo (Figs
5,
6); however, both of them are
required for the proper transcriptional activation of the gene, in contrast to
the activation of a1 gene expression by P. Apparently, WER binds to
these two sites separately as we could not detect any physical interaction
between WER proteins in yeast (data not shown).
WER influences the fate of H-position cells
Previously, we proposed a lateral inhibition model in the cell fate
specification mechanism in the Arabidopsis root epidermis
(Lee and Schiefelbein, 2002).
In that model, WER is responsible to specify the hair cell fate as well as the
non-hair cell fate. Here we obtain further support for this model. In the
wer-1 mutant plants harboring P35S:WER-VP16, the
PGL2:GUS-expressing cells and non-expressing cells could
be found in both positions (Fig.
2C). This is similar to the PGL2:GUS
expression pattern in the wer-1 mutant harboring
P35S:WER or P35S:WER-GR (+DEX)
(Fig. 3C) (Lee and Schiefelbein, 2002
).
In prior studies, it has been found that the inhibition of
PGL2:GUS expression is partly dependent on CPC
(Lee and Schiefelbein, 2002
).
Transcription of CPC takes place in the N-position cells and a
CPC-GFP fusion protein accumulates in both-position cells
(Lee and Schiefelbein, 2002
;
Wada et al., 2002
). Taken
together, these findings suggest that WER influences the fate of cells in the
H position through its effect on CPC. Specifically, it appears that WER
induces transcription of CPC directly in the N-position cells, CPC
protein moves to the neighboring H-position cells, and this CPC protein
induces the hair cell fate in these H-position cells.
Conclusion
In root epidermal cell fate specification, a cascade of several
transcription factors plays an important role. This report provides direct
evidence regarding the regulatory interactions between some of these genes.
Other than CPC, GL2 is another strong candidate for a direct target
of WER. The GL2 promoter contains several putative MYB-binding sites
and bHLH-binding sites, and some of these are located close, like the sites in
the CPC promoter. It will be interesting to see whether WER binds to
these sites and directly regulates GL2 expression. In addition, it
will be interesting to see how GL2 expression is induced by the WER
and suppressed by the CPC using biochemical and molecular approaches. Finally,
it will be important to understand how the CPC protein suppresses the
expression of some genes (GL2, CPC and WER), while it
induces the expression of GL3 and EGL3.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
---|
While this manuscript was under review, another article was published that
indicates WER is a regulator of CPC transcription
(Koshino-Kimura et al.,
2005).
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