Download Non-Cell-Autonomous Regulation of Root Hair

Non-Cell-Autonomous Regulation of Root Hair Patterning
Genes by WRKY75 in Arabidopsis1[W]
Louai Rishmawi, Martina Pesch 2, Christian Juengst, Astrid C. Schauss,
Andrea Schrader 2, and Martin Hülskamp 2*
Biocenter, Cologne University, Botanical Institute, 50674 Cologne, Germany (L.R., M.P., A.S., M.H.); Cluster
of Excellence on Plant Sciences, University of Cologne, D-50674 Cologne, Germany (L.R., M.H.); and Cluster of
Excellence-Cellular Stress Responses in Aging-Associated Diseases, Forschungszentrum, 50931 Cologne,
Germany (C.J., A.C.S.)
In Arabidopsis (Arabidopsis thaliana), root hairs are formed in cell files over the cleft of underlying cortex cells. This pattern is
established by a well-known gene regulatory network of transcription factors. In this study, we show that WRKY75 suppresses
root hair development in nonroot hair files and that it represses the expression of TRIPTYCHON and CAPRICE. The WRKY75
protein binds to the CAPRICE promoter in a yeast one-hybrid assay. Binding to the promoter fragment requires an intact WRKY
protein-binding motif, the W box. A comparison of the spatial expression of WRKY75 and the localization of the WRKY75
protein revealed that WRKY75 is expressed in the pericycle and vascular tissue and that the WRKY75 RNA or protein moves into
the epidermis.
The establishment of a pattern of files forming root
hairs and nonroot hairs in the root epidermis is a wellstudied developmental model system (Schiefelbein
et al., 2009; Tominaga-Wada et al., 2011; Grebe, 2012).
Root hair cells develop from cell files over the cleft
of two underlying cortical cells, whereas all other cells
develop into nonroot hair cells (Dolan et al., 1993,
1994; Galway et al., 1994). The genetic dissection of the
underlying pathway has enabled the isolation of essential genes and, subsequently, their molecular characterization (Schiefelbein, 2000; Pesch and Hülskamp,
2004; Ishida et al., 2008). Mutations in four genes lead
to the formation of extra root hairs in nonroot hair cell
positions, indicating their importance in the repression
of root hair formation. These are TRANSPARENT TESTA
GLABRA1 (TTG1), GLABRA3 (GL3), ENHANCER OF
GLABRA3 (EGL3), and WEREWOLF (WER; Galway
et al., 1994; Masucci and Schiefelbein, 1996; Lee and
Schiefelbein, 1999; Bernhardt et al., 2003). In addition,
a number of positive regulators of root hair formation
have been identified, including CAPRICE (CPC),
TRIPTYCHON (TRY), ENHANCER OF TRIPTYCHON
AND CAPRICE1 (ETC1), ETC2, and ETC3 (Wada et al.,
1
This work was supported by the Cluster of Excellence on Plant
Sciences (grant no. EXC 1028 to M.H.) and International Max Planck
Research Schools (fellowship to L.R.).
2
These authors contributed equally to the article.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Martin Hülskamp ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.113.233775
186
1997; Schellmann et al., 2002; Kirik et al., 2004a, 2004b;
Simon et al., 2007; Wester et al., 2009). Current models
explain root hair patterning by a lateral inhibition scenario with feedback loops: the R2R3MYB protein WER
(Lee and Schiefelbein, 1999), the basic helix-loop-helix
proteins GL3 and EGL3 (Bernhardt et al., 2003), and the
WD40 domain protein TTG1 (Walker et al., 1999) are
considered to form a trimeric complex (MBW complex)
that activates GLABRA2 (GL2) expression in nonroot
hair cells (Schiefelbein, 2003; Pesch and Hülskamp,
2004), which in turn represses the root hair cell fate
(Masucci et al., 1996). In addition, the R3MYB genes
CPC, TRY, ETC1, ETC2, and ETC3 are activated and are
thought to suppress the activity of the MBW complex
by binding to GL3/EGL3 protein, thereby replacing
WER in a competitive manner (Esch et al., 2003; Kirik
et al., 2004a, 2004b; Kurata et al., 2005; Ryu et al., 2005;
Tominaga et al., 2007). Cross talk between the cells is
mediated by the R3MYB proteins. These are expressed
predominantly in the nonroot hair cell files and move to
root hair cell file cells. Recently, it was shown that CPC
can move freely in the root meristematic region and that
it is trapped by EGL3 in root hair cell file cells (Kang
et al., 2013). In addition, GL3 that is expressed in root
hair cells moves in the opposite direction into the nonroot hair cells (Bernhardt et al., 2005). The position with
respect to the underlying cortical cells is governed by the
Leu-rich repeat receptor-like kinase, SCRAMBLED (SCM),
and JACKDAW (JKD; Kwak et al., 2005; Hassan et al.,
2010). It is postulated that JKD expression in the cortex
differentially regulates SCM activity, because the sizes of
the contact areas are different between epidermal cells
overlying cortex cells and epidermal cells over the cleft of
two underlying cortex cells (Hassan et al., 2010). SCM in
turn represses WER in nonroot hair cells. Mathematical
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WRKY75, a Root Hair Patterning Factor
models have been developed to analyze the interaction
schemes and highlight the importance of the reciprocal
movement behavior of the R3MYBs and GL3 proteins
(Savage et al., 2008). In addition to this gene regulatory
network, the plant hormones ethylene and auxin are involved in root hair development. Genetic and expression
analyses suggest that both hormones promote root hair
formation downstream of TTG1 and GL2 (Masucci and
Schiefelbein, 1996).
One additional regulator of root hair initiation is the
WRKY75 gene. In WRKY75 RNAi, an RNA interference
line, the number of root hairs is markedly increased,
indicating that it is a negative regulator of root hair
formation (Devaiah et al., 2007). A root hair-specific
function of WRKY75 is unlikely, as it has been implicated also in phosphate starvation, pathogen responses,
and senescence (Dong et al., 2003; Guo and Gan, 2004;
Devaiah et al., 2007; Encinas-Villarejo et al., 2009). The
WRKY75 gene encodes a WRKY protein containing a
single WRKY domain and a C2H2 zinc finger motif at
the C terminus. WRKY proteins act as transcription
factors through direct interaction of the WRKY domain
to the W box cis-regulatory elements in target promoter sequences (Rushton et al., 1995; Eulgem et al.,
1999; Ciolkowski et al., 2008). Consistent with this, the
WRKY75 protein localizes to the nucleus and regulates the
expression of other phosphate-response genes (Devaiah
et al., 2007).
Whether WRKY75-dependent regulation of root hair
formation involves the root hair patterning genes or
acts at the level of hormonal regulation has not been
studied. In this work, we aimed to unravel in more
detail the molecular mechanism by which WRKY75
regulates the initiation of Arabidopsis (Arabidopsis
thaliana) root hairs.
nonhair files (Fig. 1, B and C; Table I). This suggested
to us that WRKY75 is a negative regulator of root hair
formation. To test this, we generated lines expressing
WRKY75 under the control of the ubiquitous 35S
promoter (p35S:WRKY75). In these lines, significantly
fewer root hairs were found in the hair files (Fig. 1D;
Table I). In p35S:WRKY75, WRKY75 expression was
14.9 6 3.9 times higher than the wild-type expression in
quantitative real-time PCR experiments (Supplemental
Fig. S3). Together, these phenotypes suggest that
WRKY75 acts on the root hair patterning network
by suppressing root hair fate in the nonhair files
and by promoting root hair production in the root
hair files.
Transcriptional Regulation of Root Hair Patterning Genes
by WRKY75
To determine whether WRKY75 regulates the expression of patterning genes, we studied the expression levels
of selected candidates by real-time PCR (Fig. 2; Table 1;
Supplemental Tables S1 and S2). We focused our expression
analyses on the patterning process by isolating the RNA
from the root hair initiation zone. We observed a significant increase in the expression levels of CPC and TRY
in both wrky75 mutants and reduced expression levels
in the p35S:WRKY75 line. Consistently, we found the
RESULTS
WRKY75 Represses Root Hair Development
The previous finding that a WRKY75 RNAi line produces extra root hairs (Devaiah et al., 2007) raises the
question of whether WRKY75 acts on the root hair
patterning network, directly on GL2, or even later in the
hormonal control of root hair formation. To address this
question, we first analyzed the root hair pattern in
wrky75 mutants and WRKY75 overexpression lines.
In our work, we used two mutant lines: the previously
described WRKY75 RNAi line (Devaiah et al., 2007) and
the transfer DNA (T-DNA) insertion line N121525, called
wrky75-25 (Encinas-Villarejo et al., 2009). We confirmed
that wrky75-25 is a null mutant by reverse transcription
PCR (Supplemental Fig. S1). Next, we cloned the
T-DNA flanking region to map the T-DNA insertion.
The T-DNA insertion maps 143 bp downstream of the
stop codon (Supplemental Fig. S2). This suggests
that the 39 region is essential for the expression of
WRKY75.
Our analysis of the root hair pattern showed that
both wrky75 mutants exhibited ectopic root hairs in
Figure 1. Root hair phenotypes in the wild type and mutants. A, Wildtype root. B, wrky75-25 root with ectopic root hairs in nonhair positions indicated by arrows. C, WRKY75 RNAi root displaying ectopic
root hairs. D, p35S:WRKY75 root lacking root hairs in hair positions
indicated by dashed arrows. Bars = 100 mm.
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Rishmawi et al.
Table I. Root hair numbers in the patterning zone of the primary roots
Ten 7-d-old Arabidopsis plants were analyzed for each treatment. Values represent three biological
replicates (percentage 6 SD). P , 0.05 (Mann-Whitney test). Col-0, Columbia-0.
Root Hair Position
Nonroot Hair Position
Genotype
Root Hair Cell
Nonroot Hair Cells
Wild type (Col-0)
wrky75-25
WRKY75 RNAi
p35S:WRKY75
cpc-2
cpc-2 wrky75-25
p35S:WRKY75 cpc-2
95 6 6.0
99 6 3.6
96 6 5.8
70 6 9.7
34 6 11.0
39 6 6.4
21 6 8.5
5.0 6 6.0
1.0 6 3.6
4.0 6 5.8
30 6 9.7
66 6 11.0
61 6 6.4
79 6 8.5
corresponding changes in the expression levels of the
nonroot hair marker GL2 (Szymanski et al., 1998) and the
root hair marker ROOT HAIR DEFECTIVE6-LIKE4
(RSL4; Yi et al., 2010). In wrky75 mutants, the expression
levels of RSL4 were increased, whereas GL2 expression
levels were reduced. In the WRKY75 overexpression
line, the RSL4 expression levels were reduced and
those of GL2 were increased. Together, these data indicate
that WRKY75 regulates the expression of the root hair
patterning genes.
Changes in the expression levels can be explained by
higher but spatially correct expression or by temporal/
spatial changes. To distinguish between these two
possibilities, we focused on the spatial expression of
CPC and GL2 in roots using established promoter:GUS
marker lines (Fig. 3). In the wild type, CPC was
expressed in the nonroot hair files that can be recognized as continuous GUS-labeled stretches of cells and
weakly expressed in the stele (Wada et al., 2002). In
wrky75 mutants, we noted three differences (Fig. 3).
First, we found up to four adjoining cell files with
CPC expression, indicating that CPC is expressed in
hair and nonroot hair files. Second, we noted that
Root Hair Cell
0.3
25
29
0.0
0.0
0.0
0.0
6
6
6
6
6
6
6
1.5
9.6
6.4
100
100
100
100
Nonroot Hair Cells
99.7
75
71
100
100
100
100
6
6
6
6
6
6
6
1.5
9.6
6.4
100
100
100
100
nonroot hair files showed discontinuous labeling,
indicating that not all cells in nonroot hair cell positions express CPC. Third, CPC expression in the stele
was increased in wrky75 mutants (Supplemental
Fig. S4). Together, these data suggest that CPC expression
is affected in both epidermal cell files and the stele. In
p35S:WRKY75 lines, we found frequently that the CPCexpressing cell files were not continuous. CPC expression in the stele was barely detectable (Supplemental
Fig. S4). The analysis of pGL2:GUS revealed discontinuous GUS-expressing cell files in wrky75 mutants (Fig.
3, F and G). In p35S:WRKY75, we also found pCPC:GUS
expression in up to four immediately neighboring cell
files (Fig. 3H).
WRKY75 Protein Binds to the CPC Promoter through
the W Box
We tested the ability of WRKY75 protein to bind to
the CPC promoter using a yeast one-hybrid assay. It is
well established that WRKY proteins can bind to sixnucleotide-long consensus sequences (C/TTGACT/C)
Figure 2. Transcriptional regulation of
root hair patterning genes by WRKY75.
The relative expression of patterning
genes was compared by real-time PCR
(three biological samples, each with
three technical repeats). The wild-type
(wt) level was set to 1, and the expression changes in the mutants and
overexpression line were plotted.
Changes significantly different from the
wild type are marked with asterisks
(Student’s t test, P , 0.05). Error bars
represent SD.
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WRKY75, a Root Hair Patterning Factor
the CPC(2459 to 2378) promoter fragment through
the W box motif.
Genetic Analysis of WRKY75
If extra root hair formation in wrky75 mutants is
caused by the derepression of CPC, one would expect
that the cpc mutant root hair phenotype would be
epistatic to wrky75. To test this hypothesis, we created
the cpc-2 wrky75-25 double mutant. This double mutant showed only a few root hairs, as observed in
the cpc-2 mutant (Fig. 5; Table I). Overexpression of
WRKY75 in cpc-2 caused a further reduction in root
hair numbers. This is essentially an additive phenotype, suggesting that WRKY75 overexpression can
further reduce root hair number through additional
targets. One possible target is the TRY gene, as we
observed a reduced expression of TRY in p35S:WRKY75
lines.
WRKY75 Is Expressed in the Inner Part of the Root
Figure 3. Histochemical GUS expression analysis of CPC, GL2, and
WRKY75 in roots. The expression of CPC and GL2 is monitored
in pCPC:GUS (A–D) and pGL2:GUS (E–H) transgenic plants. The genetic background is indicated below each image. The expression of
WRKY75 is monitored in the pWRKY75:GUS:39-WRKY75 (I and J) and
pWRKY75:YFP-GUS:39-WRKY75 (K and L) wild-type (wt) Col-0 background. Bars = 50 mm.
called the W box (Rushton et al., 1995; Eulgem et al.,
1999; Ciolkowski et al., 2008). It was shown that the
region between –425 and –406 of the CPC promoter
contains a binding site of WER (Ryu et al., 2005). Therefore, we searched for W boxes near that region and chose
an 80-bp fragment [pCPC(2459 to 2378)] containing a
W box and the WER-binding site (Fig. 4A).
In the presence of WRKY75, the HIS3 reporter gene
driven by three tandem repeats of pCPC(2459 to
2378) was transcribed as indicated by yeast growth
on selective medium lacking His (Fig. 4B). To test
whether binding occurs through the W box, we performed this experiment with a promoter fragment in
which the W box was mutated. This promoter fragment failed to mediate WRKY75 binding. Binding of
WER was not impaired, indicating that the promoter
fragment was generally accessible. Together, our
data show that WRKY75 protein can bind directly to
As WRKY75 represses CPC, we expected it to be
coexpressed with CPC in nonroot hair files. As the
T-DNA insertion in the wrky75-25 allele was found in
the 39 region of the WRKY75 gene, we created a promoter:GUS construct containing a 2.2-kb upstream
region and a 627-bp 39 region (called pWRKY75:
GUS:39-WRKY75). The characterization of five lines in a
Col-0 background consistently revealed expression in
the inner region of the root (Fig. 3, I and L). Typically, the highest expression was observed close to
the root tip and weaker expression in the stele in upper
root regions. GUS staining for 9, 12, and 24 h revealed
that WRKY75 is not expressed in the early epidermal
cells in the root cap (Fig. 3J; Supplemental Fig. S5). The
same pattern was observed in lateral roots (Fig. 3K).
Plants transformed with constructs containing only the
2.2-kb upstream region (pWRKY75:GUS) showed no
GUS expression in 20 T1 seedlings.
WRKY75 Protein/RNA Moves from the Inner Part of the
Root to the Epidermis
As WRKY75 expression does not coincide with its
biological function in the epidermis, we tested the
possibility that WRKY75 RNA or WRKY75 protein
moves from the center of the root to the epidermis. To
test whether WRKY75 is non cell autonomous, we
created two constructs: pWRKY75:YFP-GUS:39-WRKY75
and pWRKY75:YFP-WRKY75:39-WRKY75. pWRKY75:YFPGUS:39-WRKY75 was used to precisely locate the expression of WRKY75 by yellow fluorescent protein (YFP)
fluorescence and to avoid artificial GUS staining caused
by GUS diffusion. The pWRKY75:YFP-WRKY75:39WRKY75 construct was used to study WRKY75
protein localization (Fig. 6). This construct rescued
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Rishmawi et al.
Figure 4. Yeast one-hybrid studies showing WRKY75 binding to a CPC promoter fragment. A, The 80-bp promoter sequence of
CPC used for the yeast one-hybrid assay. The W box is highlighted in gray, and the WER-binding site is underlined. B, Yeast onehybrid assay. Yeast strain YM4271 carrying a prey construct with three tandem repeats of 80 bp (2459 to 2378) of the CPC
promoter was cotransformed with WER (positive control) and WRKY75 fusions to the GAL4 activation domain or with an empty
vector (EV; negative control). Binding to the wild-type sequence of the promoter fragment (WBOX) was compared with a
fragment in which the W box was mutated (mWBOX). –LU, Medium lacking Leu and uracil; –HLU, medium lacking His, Leu,
and uracil.
the wrky75 mutant root hair phenotype, indicating
that the promoter sequences are sufficient for rescue
and that the YFP-WRKY75 fusion protein is functional (Supplemental Table S3). The WRKY75 transcript levels of plants carrying this construct in the
wrky75-25 background are lower than in Col-0
(Supplemental Fig. S6). This excludes the possibility
that artificially high expression levels lead to the
rescue. The expression of this construct in the wildtype Col-0 background did not cause a root hair
phenotype (Supplemental Table S4). In pWRKY75:
YFP-GUS:39-WRKY75 plants, YFP fluorescence (and
GUS staining; Fig. 3L) was found in the pericycle and
the vascular tissue of the root (Fig. 6B; Supplemental
Fig. S7), as observed with the pWRKY75:GUS:39-WRKY75
construct. In contrast, the YFP signal was also
found in the neighboring cell layers, including the
epidermis in pWRKY75:YFP-WRKY75:39-WRKY75
plants (Fig. 6A). This indicates that YFP-WRKY75
moves from the center to the epidermis of the primary root.
To independently confirm that WRKY75 can move
between different tissue layers, we expressed the
WRKY75 coding DNA sequence under the control of
the SCARECROW (SCR) promoter in the wrky75-25
mutant background (Helariutta et al., 2000). As expected, pSCR:GFPer (Fig. 7B; Supplemental Fig. S8C)
showed expression in the cortical/endodermis initial
cells and in the endodermis (Di Laurenzio et al.,
1996). In pSCR:YFP-WRKY75, the YFP signal was
detected in all cell layers (Fig. 7A; Supplemental Fig.
S8, A and B). Moreover, pSCR:YFP-WRKY75 was able
to rescue the wrky75-25 phenotype (Supplemental
Table S5). These results confirm that WRKY75 can
move between different tissue layers.
DISCUSSION
The previous finding that suppression of WRKY75
leads to an increased number of root hairs (Devaiah
et al., 2007) raised the question of how WRKY75
is tied in the root hair patterning or differentiation
machinery.
WRKY75 Regulates the Root Hair Patterning System
through Transcriptional Regulation of CPC
Our analysis of wrky75 mutants and WRKY75
overexpression lines revealed that WRKY75 acts as a
repressor of root hair formation. These genetic results are supported by changes in the expression of
the nonroot hair marker GL2 (Szymanski et al., 1998)
and the root hair marker RSL4 (Yi et al., 2010), such
that GL2 expression is decreased in wrky75 mutants
and increased in p35S:WRKY75 lines whereas RSL4
expression shows the opposite behavior. Our data
further indicate that these two markers are regulated by WRKY75 through the regulation of known
root hair patterning genes. Our expression analysis
of positive and negative regulators of root hair
patterning pointed to a regulation of CPC and TRY.
A temporal-spatial analysis of TRY was not considered, as the corresponding pTRY:GUS lines did
not show expression in wild-type roots (Schellmann
et al., 2002). The spatial analysis of CPC supported
these findings. As compared with the wild type, the
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WRKY75, a Root Hair Patterning Factor
specificity to the promoter is suggested by the finding
that mutations of the W box abolish the binding.
Nonautonomous Regulation of Epidermal Fate
by WRKY75
Figure 5. Genetic analysis of WRKY75. Light micrographs of roots in
the wild type (A), cpc-2 wrky75-25 (B), and cpc-2 p35S:WRKY75 (C)
are shown. Bars = 50 mm.
typical expression of pCPC:GUS in nonroot hair cells
(Wada et al., 2002) was reduced in p35S:WRKY75
lines, and ectopic expression in root hair cell files
was found in wrky75 mutants. The regulation of CPC
is likely to be driven through direct binding of
WRKY75 to the CPC promoter. This view is supported by our yeast one-hybrid data indicating that
WRKY75 can bind to the CPC promoter. Binding
Our finding that WRKY75 is transcriptionally expressed
in the inner part of the root and that YFP-WRKY75 is
found in all tissue layers suggests that WRKY75 regulates
root hair development in a non-cell-autonomous manner.
The promoter regions chosen for these experiments are
likely to reveal the correct expression pattern, as they are
sufficient for rescue and as they contain the region in
which a T-DNA insertion causes a wrky75 mutant phenotype. Thus, it is conceivable that WRKY75 RNA or
protein travels from the inner part of the root to the epidermis. We confirmed this movement behavior by also
showing that YFP-WRKY75 expressed under the control
of an endodermis-specific promoter can move to the epidermis and that it can rescue the wrky75 root hair phenotype. As two transcriptomic approaches studying
systematically the expression of genes in different root cell
types found WRKY75 in the epidermis (Birnbaum et al.,
2003; Lan et al., 2013), it is conceivable that WRKY RNA
can move between cells.
Figure 6. Intercellular motility of WRKY75. A,
Root of a wrky75-25 plant carrying pWRKY75:
YFP-WRKY75:39-WRKY75. B, Root of a Col-0
plant carrying pWRKY75:YFP-GUS:39-WRKY75.
Squares mark the region shown at higher magnification. Black stars mark lateral root cap cells,
white stars label epidermal cells, and red stars
mark cortical cells. Note that high laser intensity
and high-brightness/contrast modifications were
applied on the images to allow the visualization
of the signal, resulting in overexposure to the outer
layers (lateral root cap cells). Bars = 40 mm.
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Rishmawi et al.
Figure 7. Movement of WRKY75 when expressed
under the control of the SCR promoter. A, Root of
a wrky75-25 plant carrying pSCR:YFP-WRKY75.
B, Root of a Col-0 plant carrying pSCR:GFPer.
Squares mark the region shown at a higher magnification. Black stars mark lateral root cap cells,
white stars label epidermal cells, and red stars
mark cortical cells. Bars = 40 mm.
How Does WRKY75 Fit in the Root Hair
Patterning Models?
The current models explain the positional regulation
of root hair formation by regulatory feedback loops
of MBW proteins and their negative regulators, the
R3MYBs (Schiefelbein et al., 2009; Tominaga-Wada
et al., 2011; Grebe, 2012). The relative positions of root
hair and nonroot hair cells with respect to the cortical
cells are controlled by a repression of WER in root hair
cell positions by SCM. In addition, the SCHIZORIZA
gene had been implicated in epidermal differentiation
(Mylona et al., 2002; ten Hove et al., 2010). How does
WRKY75 fit into this network? Is WRKY75 involved in
the positioning of root hair and nonroot hair cell files,
possibly through JKD and SCM? We consider this to be
unlikely, as the corresponding mutants show distortions
of the overall radial pattern in the root. Our finding that
WRKY75 expression is rather unspecific in the inner part
of the root and the movement of WRKY75 RNA/
WRKY75 protein to the epidermal layer provides no
hint toward a biased regulation of root hair or nonroot
hair cell files. The recent report by Kang et al. (2013)
showing that expression of CPC in the stele can rescue
the cpc mutant phenotype suggests that epidermal cell
differentiation can be controlled by the inner part of the
root through the known patterning genes. It is possible
that WRKY75 operates in an analogous manner by
regulating gene expression in the epidermis in a noncell-autonomous manner. In this scenario, WRKY75
regulates CPC expression at two levels. First, in the
stele, where both genes are expressed, and second, in the
epidermis, through moving RNA/protein. WRKY75
seems to regulate root hair patterning by regulating only
a subset of the patterning genes. CPC and TRY are
clearly repressed, whereas other tested patterning genes
remain unaffected. Moreover, the binding of WRKY75 to
the CPC promoter in yeast one-hybrid assays suggests a
direct regulation rather than an indirect control through
JKD/SCM. This idea is supported by our finding that the
levels of WER expression are not changed in wrky75
mutants or WRKY75 overexpression lines. We speculate
that WRKY75 functions as a modulator of root hair formation through the direct regulation of CPC and possibly
GL2. In the wild type, WRKY75 contributes to the balance
of the expression levels of the two genes and, thereby, the
number of root hairs. In wrky75 mutants, the balance is
shifted and the number of root hairs increases in nonroot
hair cell files. Also, the reduction of root hairs in root hair
cell files in p35S:WRKY75 plants can be easily explained by
a reduction of CPC (and TRY) expression. Given that
WRKY75 mediates various environmental responses,
including pathogen attack and phosphate starvation,
this mechanism provides a simple and effective way to
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WRKY75, a Root Hair Patterning Factor
modulate root hair number in response to environmental stimuli.
25, WRKY75 RNAi, and p35S:WRKY75. The primers used are listed in
Supplemental Table S1.
Yeast One-Hybrid Analysis
MATERIALS AND METHODS
Plant Material, Growth, and Root Hair and
Cytological Analyses
The following Arabidopsis (Arabidopsis thaliana) lines were used in this
study: wrky75-25 (Encinas-Villarejo et al., 2009), WRKY75 RNAi (Devaiah et al.,
2007), and cpc-2 (Kurata et al., 2005). Double mutants were created by crossing. The homozygous lines were confirmed by PCR. The pGL2:GUS and pCPC:
GUS lines (Pesch et al., 2013) in p35S:WRKY75, WRKY75 RNAi, and wrky75-25
backgrounds were created by crossing with the respective Col-0 reporter lines.
All lines are in the Col-0 wild-type background. Plants were transformed by
the floral dip method (Clough and Bent, 1998).
For root hair analysis, seeds were sterilized by adding 100% (v/v) ethanol
followed by 4% (v/v) HCl and washed three times with water, vernalized at 4°
C for 3 d in the dark, and planted on Murashige and Skoog plates supplemented with 1% (w/v) Suc. Seedlings were grown under long-day conditions
(16 h of light/8 h of dark) for 7 d. Plates were positioned vertically to avoid
root penetration in the medium. Seedlings were fixed (50% [v/v] methanol,
10% [v/v] acetic acid) at 4°C for 24 h, transferred to 80% (v/v) ethanol for 2 h,
washed three times with water, and analyzed by transmission light microscopy. The files were defined by their relative positions to the cortical cells. For
each seedling, the number of root hairs was determined for 10 cells in the root
hair cell file and 10 cells in the nonroot hair cell position.
GUS staining was done as described previously (Vroemen et al., 1996).
Plasmid Construction
The coding DNA sequence (CDS) of WRKY75 was cloned into pDONR201
amplified from Col-0 CDS by Gateway technology (Invitrogen) using primers
with attB sites (Supplemental Table S1). The coding sequence of WER was
amplified from Landsberg erecta CDS and cloned with SalI and NotI restriction
sites in pENTR1A. GUS-pDONR201 was obtained from Invitrogen. The constructs p35S:WRKY75 (pAMPAT-GW), pCPC:GUS (pCPC-pAMPAT), pGL2:GUS
(pGL2-pAMPAT), WRKY75-pC-ACT2-attR, WER-pC-ACT2-attR, p35S:GUS
(pAMPAT-GW), YFP-WRKY75 (pENSG-YFP), and YFP-GUS (pENSG-YFP)
were created by Gateway LR recombination (Invitrogen) with the respective
entry clones (Weinl et al., 2005; Wester et al., 2009).
For pAMPAT-GW-pWRKY75:GUS:39-WRKY75, the WRKY75 promoter was
amplified using Asc1-pWRKY75 F and Xho1-pWRKY75 R primers (Supplemental
Table S1) and introduced in pJET1.2.b (Fermentas). pWRKY75 was recovered using
AscI/XhoI and introduced to GUS-pAMPAT-GW to obtain pWRKY75:GUS
(pAMPAT-GW). The downstream 627 bp of WRKY75 was isolated with
39WRKY75 F and 39WRKY75 R (Supplemental Table S1) and introduced to
pWRKY75:GUS (pAMPAT-GW) using Mss1.
For pWRKY75:YFP-WRKY75:39-WRKY75 (pENSG) and pWRKY75:YFPGUS:39-WRKY75 (pENSG), the same strategy was used to introduce pWRKY75
and 39-WRKY75 in pENSG-YFP. The WRKY75 coding sequence and GUS
coding sequence were introduced in a second step by LR reaction (Clontech).
For pSCR:GPFer (pAMPAT), first the SCR promoter was amplified using
Asc1-pWRKY75 F and Xho1-pWRKY75 R primers (Supplemental Table S1)
and introduced in pJET1.2.b (Fermentas). pSCR was recovered using AscI/
XhoI and introduced to GFPer-pAMPAT-GW to obtain pSCR:GFPer (pAMPAT-GW).
pSCR:YFP-GUS (pENSG) and pSCR:YFP-WRKY75 (pENSG) were created
in a similar way to pWRKY75:YFP-WRKY75:39-WRKY75.
Vectors containing the CPC promoter fragments wild-type and mutated W
box were created by sequentially cloning three equal promoter fragments
differing in the attached restriction sites (EcoRI and KpnI, KpnI and BamHI, and
BamHI and XbaI) in puc18. The fragments were either produced by PCR or
ordered as long oligonucleotides. The trimers were introduced in the EcoRI
and XbaI sites in pHISi (Invitrogen).
Yeast one-hybrid analyses were carried out using the MATCHMAKER OneHybrid System (Clontech). First, the yeast strain YM4271 was transformed with
pCPC(2459 to 2378)-pHISi or pCPC(2459 to 2378, mutated)-pHISi. Second,
the selected vectors expressing the GAL4 activation domain fusion proteins
were transformed into YM4271 carrying the promoter fragments and grown
on medium without uracil and Leu. Finally, the binding activities were determined by the ability of yeast to grow on selective medium without His, Leu,
and uracil. Three single colonies from each combination were tested.
pC-ACT2-ccdB (empty vector) served as a negative control and was described
before (Pesch et al., 2013).
Microscopy and Image Acquisition
Fluorescence microscopy and bright-field differential interference contrast
microscopy were performed with a Leica DMRB 5500 microscope equipped
with the Leica Application Software AF (Leica Microsystems). Images of unstained roots were generated by light microscopy as described previously
(Failmezger et al., 2013), and GUS-stained roots were acquired using a Leica
DMRA microscope (Leica Microsystems) equipped with DISKUS software
(Carl H. Hilgers-Technisches Büro).
Confocal laser-scanning microscopy was performed using a Leica TCS-SP8
confocal microscope equipped with the Leica LAS AF software or a Zeiss Meta
510 equipped with ZEN 2012 software. For imaging, a Leica 1.2 numerical
aperture, 633 water objective was used. The propidium iodide staining was
excited using a white light laser at 561 nm, and the GFP/YFP signal was
excited using an argon laser at 488/514 nm. Images were analyzed with Imaris
7.0 (Bitplane). For protein localization, seedlings were stained with 100 mg mL21
propidium iodide for 1 min followed by washing the samples with water. The
analyses were done by sequential scanning starting with the respective higher
wavelength.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: TTG1 (AT5G24520, The
Arabidopsis Information Resource [TAIR] locus 2153914), TRY (AT5G53200,
TAIR locus 2163766), GL2 (AT1G79840, TAIR locus 2017874), CPC (AT2G46410,
TAIR locus 2005503), WER (AT5G14750, TAIR locus 2185470), WRKY75
(AT5G13080, TAIR locus 2179862), SCR (AT3G54220, TAIR locus 2080345),
and RSL4 (AT1G27740, TAIR locus 2199221).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Agarose gel electrophoresis showing RT-PCR for
the wrky75-25 mutant.
Supplemental Figure S2. Nucleotide sequence showing the position of
T-DNA insertion in the wrky75-25 mutant.
Supplemental Figure S3. Quantitative real-time PCR of mRNA obtained
from root hair-patterning zone of 7-d-old seedlings grown on Murashige
and Skoog media.
Quantitative Real-Time PCR
Supplemental Figure S4. Expression analysis of CPC in the stele.
Using the stereomicroscope at 103 magnification, the root hair patterning
zone (the area where the first root hairs appear in the differentiation zone) was
determined. Seedlings were cut using a sharp blade to collect the root part
containing the patterning zone including the root cap. RNA extraction and realtime PCR were performed as described previously (Kwon et al., 2006). Relative
mRNA was normalized to the concentration of ELONGATION FACTOR a
A4 mRNA. The expression levels of genes of wild-type plants were set to
1 and subsequently used to calculate the relative changes of genes in wrky75-
Supplemental Figure S5. Time series for pWRKY75:GUS:3’WRKY75 GUS
staining.
Supplemental Figure S6. Quantitative real-time PCR of mRNA obtained
from root hair-patterning zone of 7-d-old seedlings grown on Murashige
and Skoog media.
Supplemental Figure S7. Three-dimensional reconstruction of pWRKY75:
YFP-GUS:39WRKY75.
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193
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Rishmawi et al.
Supplemental Figure S8. Movement of WRKY75 when expressed under
the SCR promoter.
Supplemental Table S1. Primer list.
Supplemental Table S2. Real-time PCR results.
Supplemental Table S3. Summary of root hair phenotype of wrky75-25 T1
plants transformed with pWRKY75:YFP-WRKY75 39-WRKY75.
Supplemental Table S4. Root hair numbers in the patterning zone of the
primary roots; 7-d-old Arabidopsis plants were analyzed for each
treatment.
Supplemental Table S5. Summary of root hair phenotype of wrky75-25 T1
plants transformed with pSCR:YFP-WRKY75.
ACKNOWLEDGMENTS
We thank Birgit Kernebeck for excellent technical assistance.
Received December 6, 2013; accepted March 17, 2014; published March 27,
2014.
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