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First published online 31 March 2009
doi: 10.1242/dev.021733
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1 University of Cologne, Botanical Institute III, Gyrhofstr. 15, 50931 Cologne,
Germany.
2 University of Freiburg, Department of Biology, Schaenzlestr. 1, 79104
Freiburg, Germany.
3 University of Freiburg, Department of Mathematics and Physics,
Hermann-Herder-Str. 3a, 79104 Freiburg, Germany.
* Author for correspondence (e-mail: martin.huelskamp{at}uni-koeln.de)
Accepted 3 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Trichomes, Patterning, Arabidopsis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Serna, 2005) and one that
controls the spatial distribution of root hairs and trichomes on leaves
(Ishida et al., 2008;
Larkin et al., 2003;
Pesch and Hulskamp, 2004;
Schellmann et al., 2007). The
latter patterning system is based on an evolutionarily conserved gene cassette
consisting of three types of positive regulators comprising a WD40 protein,
R2R3 MYB-type transcription factors and bHLH transcription factors, and
negative regulators of the single-repeat R3 MYB transcription factor family.
The WD40 protein is encoded by the single-copy gene TRANSPARENT TESTA
GLABRA1 (TTG1) (Galway et
al., 1994; Walker et al.,
1999). The R2R3 MYB-type transcription factors are represented by
a small gene family, with GLABRA1 and MYB23 functioning in
trichome patterning and WEREWOLF in root hair patterning
(Kirik et al., 2005;
Kirik et al., 2001;
Lee and Schiefelbein, 1999;
Oppenheimer et al., 1991). The
bHLH factors are GLABRA3 and ENHANCER OF GLABRA3
(EGL3), which act redundantly in trichome and root hair patterning
(Bernhardt et al., 2005;
Payne et al., 2000;
Zhang et al., 2003). The
single-repeat R3 MYB proteins act as negative regulators and have been grouped
into a family of six genes (Wang et al.,
2007). The CAPRICE (CPC) gene was initially
identified as a major regulator of root hair development, whereas
TRIPTYCHON (TRY) is predominantly required for trichome
patterning (Kirik et al.,
2004a; Kirik et al.,
2004b; Schellmann et al.,
2007; Wada et al.,
1997). Subsequent analysis showed that CPC and
TRY act redundantly in root hair and trichome development
(Schellmann et al., 2007).
Also, two other members of the family, ENHANCER OF TRY AND CPC 1
(ETC1) and ETC2, act redundantly with TRY and
CPC (Esch et al.,
2004; Kirik et al.,
2004a; Kirik et al.,
2004b). In addition, TRICHOMELESS1 (TCL1) has
been shown to play an important role in trichome formation on the stem and the
pedicels (Wang et al., 2007).
In an initial report, the analysis of etc3 mutants focused on root
hair development (Simon et al.,
2007). More recently, further phenotypes caused by mutations in
ETC3 were studied and CPL3 (CPC-LIKE-MYB3) was
introduced as a second name for this gene
(Tominaga et al., 2008).
ETC3 seems to represent an unusual member of the single-repeat R3 MYB
class, as various unusual phenotypes were reported for the mutant including an
early flowering time phenotype, reduced trichome size and branching, longer
hypocotyls and general plant growth changes
(Tominaga et al., 2008). A
role for ETC3 in trichome formation is controversial. Tominaga and co-workers
reported a higher trichome density in the corresponding mutant and, in
contrast to the other members of the single-repeat R3 MYBs, a conspicuous
absence of trichome-specific expression
(Tominaga et al., 2008). Wang
and co-workers could not detect any difference in trichome number between
etc3 single mutants and the corresponding wild type
(Wang et al., 2008).
The positive regulators are thought to form a complex in which the R2R3 MYB
factor and TTG1 bind to GL3 or EGL3, and the negative regulation by the R3
single-repeat MYBs is postulated to be governed by their competition with MYB
factors for binding to GL3 and EGL3, thereby rendering the activator complex
inactive (Bernhardt et al.,
2003; Esch et al.,
2003; Payne et al.,
2000). Inhibition by direct binding to the GL1 promoter
was suggested by ChIP experiments for TCL1
(Wang et al., 2007).
Although the same machinery is central to the spatial regulation of root
hair and trichome patterning, the context is different. Whereas trichome
formation on rosette leaves takes place without a recognisable reference to
other leaf structures except for other trichomes
(Hulskamp and Schnittger,
1998; Larkin et al.,
1996), root hair patterning is strongly biased such that root
hairs are normally found only in epidermal cells overlying a cleft between two
underlying cortex cells (Berger et al.,
1998; Dolan et al.,
1994). For both systems, models that try to explain pattern
formation are based on a feedback loop in which the positive regulators
activate the negative regulators and the negative regulators inhibit the
activators, with the negative regulators being able to move between cells
(Digiuni et al., 2008;
Larkin et al., 1996;
Marks and Esch, 2003;
Pesch and Hulskamp, 2004;
Scheres, 2002). Recently,
evidence for a parallel second patterning system has been reported in which
the depletion of TTG1 around incipient trichomes contributes to pattern
formation (Bouyer et al.,
2008).
In this study we focus on the function of the single-repeat R3 MYB factors
in trichome patterning with emphasis on ETC3. Initially, we reassess the
published molecular and genetic data
(Tominaga et al., 2008;
Wang et al., 2008). We show
that ETC3 is expressed in trichomes, that trichome density is
increased in etc3 mutants and that this phenotype can be rescued by a
pETC3:ETC3 construct. Our data do not support most of the additional
phenotypes, such as the flowering time, the hypocotyl length and the trichome
size and branching phenotypes previously reported. Next, we compared the
molecular function of TRY, CPC, ETC1, ETC2 and ETC3. These factors differ in
their binding strength to GL3 and, using the yeast three-hybrid system, we
show that their capacity to compete with GL1 for binding to GL3 also differs.
Marked differences were found, with CPC being the most potent inhibitor
followed by ETC1, TRY, ETC3 and ETC2. We further show that ETC3 protein can
travel between cells. Promoter-swap experiments revealed that transcriptional
regulation, in particular, is important for the functional diversity.
Mathematical analysis suggests that the mobility of the inhibitors depends on
their affinity for GL3. This prediction is supported by a comparison of the
mobility of CPC and ETC3 in egl3 gl3 double mutants and 35S:GL3
lines.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
try-82 in Ler ecotype
(Hulskamp et al., 1994),
try-JC in Col ecotype (Larkin et
al., 1999), etc1 in Col ecotype
(Kirik et al., 2004a),
etc2 in WS ecotype (Kirik et al.,
2004b), etc3 in Col ecotype
(Simon et al., 2007) and
cpc try double mutant (Schellmann
et al., 2002). Plants homozygous for multiple mutations were
constructed by crossing single, double or triple mutants. The single and
multiple mutants were confirmed by PCR. All MS plates contained 1% sucrose.
The gl3 egl3 double mutant was created by crossing the single mutants
gl3 (Salk_118201) and egl3 (Salk_019114). The
GL3-overexpressing line was established by transformation of wild-type (Col)
plants with the pAM-PAT p35S:GL3 construct.
Agrobacterium-mediated transformation of Arabidopsis
plants was performed as described (Clough
and Bent, 1998).
Cytological methods
GUS staining using X-glucuronide was performed as described
(Vroemen et al., 1996).
Fourteen-day-old plants from soil were used for GUS staining of leaves. The
root expression analysis was performed with 5-day-old plants grown on MS
plates. Pictures were taken with a light microscope equipped with DISKUS
software (Carl H. Hilgers-Technisches Büro, Königswinter, Germany;
version 4.30.19).
For staining cell walls, a propidium iodide solution (0.3 mg/ml) was used. Plant material was incubated for 5 minutes at room temperature and washed twice in water.
Evaluation of root-hair numbers and stomata density
For quantification of root hairs, plants were grown on MS plates in a
vertical orientation for 5 days. Stomata numbers were determined on 3-week-old
leaves stained with propidium iodide. Using a confocal laser-scanning
microscope, a 1 mm2 area of the fourth leaf was scanned for the
statistical analysis.
Confocal laser-scanning microscopy (CLSM)
For CLSM, a Leica SP2 was used. Pictures were taken using the LCS software
and images processed using Adobe Photoshop 6.0.
The quantitative comparison of YFP signals in neighbouring cells was based on full stacks that were subsequently merged to one plane. The resulting raw image was analysed using Leica Confocal Histogram Quantification software. In order to determine how much signal of the initially targeted cell arrives in the neighbouring cell, the signal intensity of the nuclei was compared.
Nucleic acid analysis
Plants carrying the T-DNA insertion in the ETC3 gene were
identified by PCR on genomic DNA using primers ETC3-for
(5'-ATGGATAACCATCGCAGGAC-3') and LBa1
(5'-TGGTTCACGTAGTGGGCCATCG-3').
ETC3 cDNA was amplified from Col wild-type cDNA by PCR using primers ETC3-XbaI-for (XbaI site added, 5'-TCTAGAATGGATAACCATCGCAGGAC-3'), ETC3-SacI-rev (SacI site added, 5'-GAGCTCTCAATTTTTCATGACCCAA-3'). The PCR product was subcloned into pBluescript (pBS) with 5' XbaI and 3' SacI restriction sites downstream of GFP cDNA.
The ETC3 cDNA in pBS was amplified by PCR using primers ETC3-attB1 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGATAACCATCGCAGGACTAAGCA-3') and ETC3-attB2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAATTTTTCATGACCCAAAACCTC-3'). The PCR product was recombined in the DONR201 vector by the BP reaction (Invitrogen) and constitutes the ETC3 pEntry clone.
The GFP-ETC3 fusion fragment was amplified and recombined through BP in pDONR201 using primers GFP-attB1 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGGTGAGCAAGGGCGAGGAGCTGT-3') and ETC3-attB2. The YFP-ETC3 pEntry was created by exchanging the GFP for YFP in the entry clone.
A 2023 bp fragment upstream of the ETC3 start codon was used as
the putative 5' ETC3 promoter. The promoter fragment was
amplified by PCR from genomic Col DNA using primers 5'-pETC3-for
(5'-ATTCTGGATTCCCTATACATAAC-3') and 5'-pETC3-rev
(5'-GTCAAACGGCACCGTATTAC-3'). The PCR product was subcloned into
SmaI-cut pBS by blunt-end ligation. The promoter was cloned into the
pAM-PAT-GW p35S vector (GenBank AY436765) with 5'
Ecl136II/AscI (blunt) and 3' XhoI restriction
sites, resulting in an exchange of the 35S promoter for the ETC3
promoter. For the CPC promoter, a 1091 bp fragment upstream of the
start codon was used. For the TRY promoter, a 1.4 kb fragment
upstream of the TRY start codon was used
(Schellmann et al., 2002).
Both fragments were cloned into the pAM-PAT-GW vector, exchanging the
CPC or TRY for the 35S promoter.
The RUBISCO SMALL SUBUNIT 2B (RBCS-2B) promoter (pRbcS2b) was cloned from the pK1573 plasmid (gift from the David Jackson laboratory, Cold Spring Harbor, NY, USA) into the pAM-PAT-GW vector, exchanging for the 35S promoter.
ETC3/GFP-ETC3/YFP-ETC3 entry clones were recombined in the p35S pAM-PAT, 5'pETC3 pAM-PAT, 5'pTRY pAM-PAT or 5'CPC pAM-PAT vector by the LR reaction (Invitrogen).
The p35S:YFP-CPC construct was cloned by the LR reaction. The CPC entry clone was recombined in the 5'YFPpENS vector.
The GFP-ER tag cDNA was amplified from a subclone by PCR using primers GFP-ER-attB1 (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTGATGAAGACTAATCTTTTTCTCTTTCTCAT-3') and GFP-ER-attB2 (5'-GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAAGCTCATCATGTTT-3'). The PCR product was cloned in pDONR201 by the BP reaction and recombined in the 5'pETC3 pAM-PAT vector.
For RT-PCR of full-length cDNA, the ETC3-specific primers ETC3-for and ETC3-rev (5'-CAATTTTTCATGACCCAAA-3') were used. For amplification of the first and second exons, ETC3.1-for (5'-ATGGATAACCATCGCAGGAC-3') and ETC3.2-rev (5'-ATGCATTCGAGAGACCAAAT-3') were used. Actin primers served as control for DNA content: Actin-for (5'-TGCGACAATGGAACTGGAATG-3') and Actin-rev (5'-GGATAGCATGTGGAAGTGCATAC-3').
BiFC method in protoplasts
The pBATL vectors pCL112 YFP N-term and pCL113 YFP C-term (gift of Sean
Chapman, SCRI, Dundee, UK) were used. GL3 and GL3-
1-96 [truncated GL3
(Payne et al., 2000)] were
fused with the YFP N-term, and ETC1, ETC2, ETC3, CPC and TRY with the YFP
C-term vector. Protoplast isolation and transfection were performed as
described (Spitzer et al.,
2006).
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).
For two-hybrid assays, the GAL4 DNA-BD plasmid pAS2-1 and GAL4 DNA-AD plasmid
pAct from Clontech were used. GL3 was fused to the DNA-AD in pACT. GL1
(lacking 27 amino acids at the C-terminus), ETC1, ETC2, ETC3, CPC and TRY were
fused to the DNA-BD in pAS. For analysing protein-protein interactions, yeasts
were grown on media lacking histidine and supplemented with 5 mM 3-amino-1, 2,
4-triazole.
For three-hybrid assays, GL3 was fused to the DNA-AD in pAct. GL1
was fused to the GAL4 DNA-BD in the pBridge vector (Clontech). ETC1, ETC2,
ETC3, CPC and TRY were fused downstream of the methionine-repressible promoter
in pBridge. For analyzing competition between GL1 and one of the other
inhibitor proteins for binding to GL3, yeasts were grown on plates lacking
histidine. The plates contained different concentrations of methionine (0,
100, 250 or 500 µM) and were supplemented with 50 mM 3-amino-1, 2,
4-triazole.
Transient expression
Rosette leaves were co-bombarded with DNA-coated gold particles using the
Biolistic PDS-1000/He system (Bio-Rad). Gold particles (1.0 µm) were coated
with 400 ng of each DNA. Particles were bombarded into epidermal cells of
rosette leaves with 900-psi rupture disks under a vacuum of 26 inches of Hg.
The p35S:YFP-peroxisome (PTS) construct was used as a marker to identify the
transformed cells (Mathur et al.,
2002). The fluorescence analysis was carried out 7-15 hours
later.
Mathematical modelling
The effect of depletion on the mobility of the inhibitors was studied by
mathematical modelling. The inhibitor U is produced from a point
source (the targeted cell) with a rate 
. It diffuses into the tissue
with a rate constant D and is degraded with a rate constant 
.
U is also depleted by an irreversible binding to GL3 (G)
with a rate constant β. These assumptions lead to the following
one-dimensional second-order differential equation for the steady state
profile of U(X):
 | (1a) |
 | (1b) |
 | (1c) |
The production of U is stated in the boundary condition 1b.
Boundary condition 1c ensures that the profile of U declines to zero
for X
, as the source of U at X=0 is
the only source in the considered domain. In the following we assume that GL3
is spatially uniform, i.e. G(X)=constant. The movement
ability of the inhibitor is measured by its characteristic decay length (CDL),
, which is the distance from the source at which the inhibitor level
drops to 1/e (37%) of its source level. We use a scaling approach in order to
identify the effective parameters of the problem. The following substitutions
for the concentrations U and G and the spatial coordinate
X are introduced:
 |
The factor 
is the CDL of U for β0. Using these substitutions gives the new
dimensionless system:
 | (2a) |
 | (2b) |
|
| (2c) |
The solution of equation 2 is
where
and
=(0βg) are the only effective
parameters of the problem. Parameter has a convenient interpretation
because it is the ratio of two time scales: the time it takes one unit of
u to diffuse a distance of 0 and the time it takes
one unit of u to bind to one unit of g. The parameter
can be interpreted as a measure for the relative binding affinity of
u for g. The relative CDL of the inhibitor as plotted in the
inset of Fig. 5 is given by
.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Tominaga et al., 2008). The
deduced 75-amino-acid ETC3 protein shows high homology to TRY and CPC (38% and
50% amino acid identity, respectively). Our comparison of the genomic and cDNA
sequences showed that the ETC3 gene consists of three exons and two
introns (Fig. 1A). As a first step towards a functional analysis of the ETC3 gene, we isolated a T-DNA knockout line of the corresponding At4G01060 locus. The SALK line 094027 has an insertion after 354 bp in the second intron (Fig. 1A). RT-PCR analysis with primers in exons 1 and 2 before the insertion yielded no amplification product, indicating that the isolated mutant is a null (Fig. 1B).
We noticed that etc3 mutant plants exhibit a higher trichome
density. On leaves one and two, as well as on leaves three and four, 
50%
more trichomes were found as compared with the respective Columbia wild-type
background (Table 1;
Fig. 2A,B). In order to ensure
that this phenotype is due to a mutation in the ETC3 gene, we
expressed the ETC3 cDNA under the control of a 2023 bp upstream
putative promoter fragment. This pETC3:ETC3 construct rescued the trichome
phenotype completely (Table
1).
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). For a
better spatial resolution we used the 2023 bp promoter fragment shown to
rescue the trichome phenotype to drive the expression of the GUS marker gene
(pETC3:GUS). Plant lines containing this construct showed GUS expression in
the basal parts of leaves in young trichomes
(Fig. 1D) and in stomata
(Fig. 1E). Expression ceased
during trichome differentiation and was absent in mature trichomes. Because
Tominaga and co-workers were unable to detect ETC3 expression in
trichomes (Tominaga et al.,
2008), we reasoned that this might be due to differences in the
promoter fragments. To test this possibility we created a pETC3:GUS construct
using the same 2.4 kb promoter fragment as described by Tominaga et al.
(Tominaga et al., 2008).
Arabidopsis lines containing this construct revealed the same
expression pattern as we found for the 2023 bp fragment, including expression
in trichomes (see Fig. S1 in the supplementary material).
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).
Redundant function of ETC3 in trichome patterning
To further investigate the role of ETC3 in trichome patterning we
expressed a p35S:ETC3 construct in a wild-type background. As observed
previously for all other members of the TRY/CPC group,
overexpression of ETC3 resulted in a glabrous phenotype
(Fig. 2C;
Table 1).
A potential redundant functional overlap with the other four members of the TRY/CPC group was tested by creating double, triple and quadruple mutants (Table 2). As a first step, we compared the four possible double mutants of etc3 with the try/cpc group members. The single mutants fell into three phenotypic classes: try, which exhibited trichome clusters; cpc and etc2, which exhibited increased trichome number; and etc1, which was without a detectable trichome phenotype. The etc3 try double mutant displayed a weak synergistic phenotype. Trichome density was similar to that of etc3 mutants and cluster frequency was significantly increased compared with try mutants (Student's t-test, P=0.006). The cpc etc3 double mutant showed a similar phenotype to the cpc single mutant. The etc3 etc1 double mutant showed a significant increase in trichomes (P=0.003) indicating that ETC1 acts redundantly with ETC3. By contrast, the etc2 etc3 double mutant exhibited a trichome density similar to that of the etc3 single mutant. However, a redundant function of ETC2 with ETC3 was uncovered in the etc1 etc2 etc3 triple mutant, in which a significantly higher trichome number was found as compared with the etc1 etc3 double mutant (Fig. 2E; Table 2). A much stronger effect was found in the etc3 try cpc triple mutant, in which only a few large clusters of trichomes were found that contained more than 100 trichomes (Fig. 2F). A phenotypic enhancement was also observed for ectopic trichome formation on the hypocotyl (Fig. 2G,H). The phenotypic strength of this triple mutant was increased in the etc1 etc3 try cpc (Fig. 2J) and etc1 etc2 try cpc (Fig. 2I) quadruple mutants.
|
), whereas Wang and co-workers did not
(Wang et al., 2008). We analysed the root hair pattern with respect to the underlying cortex cells (Table 3), focusing on the etc3 mutant and cpc etc3 double mutant. In etc3 mutants the number of root hairs in trichoblast positions was significantly decreased (P<0.01). The cpc etc3 double mutant showed no difference compared with cpc, and the overexpression line p35S:ETC3 exhibited significantly more root hairs in the atrichoblast position (P<0.01) (Table 3).
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), we found no deviation from wild type in any of these lines
(data not shown).
ETC3 phenotypes unrelated to canonical TRY/CPC functions
Tominaga and co-workers reported several etc3 mutant phenotypes
that are not observed for any of the other TRY/CPC group
mutants. These include an early flowering phenotype under long-day conditions,
a reduction of trichome size and branching, an increase in hypocotyl length,
and drastic overall growth changes. Our combination of a null mutant and a
genomic rescue of this mutant enabled us to reassess these unusual
phenotypes.
The flowering time was studied under long-day conditions (16 hours light/8 hours dark) (see Table S1 in the supplementary material). Under these conditions, wild-type plants flowered after 27±1 days (n=20), whereas etc3 mutants flowered after 28±1 days (n=20). By that time, wild-type and etc3 plants had produced 13±1 and 15±1 rosette leaves, respectively. The same behaviour was found for plants kept under continuous light conditions. An early flowering phenotype was not observed.
In our hands, trichome size was indistinguishable in wild type and
etc3 mutants (see Fig. S2 in the supplementary material). The marked
reduction reported by Tominaga and co-workers
(Tominaga et al., 2008) was
not observed. Also, trichome branching was not affected in the etc3
mutant (see Table S2 in the supplementary material).
Hypocotyls were reported to almost double in length in etc3
mutants and to be shorter in 35S:ETC3 plants, which was correlated with
changes in the epidermal cell shape
(Tominaga et al., 2008). We
could not detect any difference in hypocotyl length between wild type,
etc3 and 35S:ETC3 mutants (see Fig. S3 in the supplementary
material), nor any epidermal cell shape changes (data not shown).
When comparing overall plant growth we noted moderate differences between wild-type, etc3, 35S:ETC3 and pETC3:ETC3 plants with respect to rosette leaf size and height (see Fig. S4 in the supplementary material).
|
Expression of ETC3 protein under the TRY or CPC promoter can rescue etc3 mutants
The overlapping redundancy of the five TRY/CPC genes
raises the question of whether the relative differences in their importance
for trichome patterning are due to differences in the regulation of their
expression or in protein function. As a first step, we compared the ability of
ETC3 to rescue the etc3 mutant under its own promoter, the
TRY promoter and the CPC promoter. All rescue experiments
described here were analysed in the first generation progeny (T1) using 45
plants. The three constructs rescued the etc3 phenotype equally well
(Table 1). This indicates that
the TRY, CPC and ETC3 promoters are interchangeable with
respect to etc3 rescue.
This result prompted us to consider to what extent the expression of ETC3 under these three promoters can rescue the cpc try etc3 triple mutant. To our surprise, we found qualitatively different results for the three constructs. As expected, the pETC3:ETC3 construct rescued the cpc try etc3 mutant to the same extent as the try cpc mutant. When using the pTRY:ETC3 and pCPC:ETC3 constructs, we observed a wide range of rescue phenotypes and often plants were rescued back to the try cpc phenotype or even exhibited an over-rescued phenotype resembling that of try. The over-rescue phenotype was never observed in pETC3:ETC3 lines. We noted one striking difference between pTRY:ETC3 and pCPC:ETC3 with respect to the intermediate phenotypes. In pTRY:ETC3 we observed huge, separated trichome clusters that were typically devoid of trichomes in their middle region (Fig. 2K). The pCPC:ETC3 construct, by contrast, showed only a few clusters and these were smaller than in the try cpc mutants and trichomes were arranged irregularly at a high density (Fig. 2L). Although we do not understand the exact basis for these different patterns, these results strongly suggest that the qualitatively different phenotypes of try and cpc are due to differences in the regulation of their expression.
Localisation and cell-to-cell movement of ETC3
The localisation of the ETC3 protein was initially determined using a
p35S:YFP-ETC3 construct. As shown in Fig.
3A, the protein was localised in the nucleus as well as in the
cytoplasm. Expression under the control of its own promoter revealed a more
differentiated pattern. We compared the expression and localisation of two
constructs. The pETC3:GFP-ER construct was used as a control to visualise the
expression pattern with a non-mobile GFP protein. In pETC3:GFP-ER lines,
expression was only found in single cells
(Fig. 3C). The ubiquitous
expression detected with the GUS lines was not observed, most likely because
the sensitivity of the GFP signal was much lower than in the GUS lines. In
pETC3:YFP-ETC3 lines, by contrast, cells around an incipient trichome showed a
clear YFP signal. Strikingly, the YFP-ETC3 signal in surrounding cells was
restricted to the nucleus, whereas in trichome initials YFP-ETC3 was found
both in the cytoplasm and in the nucleus
(Fig. 3B). This indicates that
ETC3 protein moves from the trichome initial into the neighbouring epidermal
cells.
We also demonstrated movement of ETC3 in two independent experiments. First, by expressing ETC3 in the subepidermis under the control of the RUBISCO promoter, in the construct pRbc:YFP-ETC3, in a wild-type background. If ETC3 were able to move into the epidermis one would expect a trichome reduction similar to that found in p35S:ETC3 lines. In these lines, a clear reduction of trichomes was observed (Fig. 3F; Table 1). Direct microscopic inspection revealed fluorescence in the epidermis suggesting that YFP-ETC3 protein or RNA has moved into the epidermis (Fig. 3E,F).
In a second experiment, p35S:GFP-ETC3 was transiently expressed in single leaf epidermal cells using the particle bombardment method. As shown in Fig. 3D, fluorescence was also found in cells around the cell expressing the construct. This movement was observed in 76% of all successfully transformed cells (n=100).
Protein-protein interactions of ETC3
The most important recognised protein-protein interaction of TRY and CPC
during pattern formation is their binding to GL3. As shown in
Fig. 4A, all five TRY/CPC group
proteins showed strong binding to GL3 in yeast two-hybrid assays. This
interaction was confirmed for TRY, CPC, ETC1, ETC2 and ETC3 with the BiFC
system (Fig. 4B). As a control
we used the truncated GL3 protein lacking the N-terminal 96 amino acids that
was previously shown not to bind to GL1, TRY, CPC, ETC1, ETC2 or ETC3 in yeast
two-hybrid assays (Kirik et al.,
2005; Payne et al.,
2000) (data not shown). The interaction of the TRY/CPC proteins
with GL3 competes with the interaction of GL1 with GL3. We used this situation
to study whether the TRY/CPC group proteins compete with GL1 with different
efficiency using the yeast three-hybrid system. Growth at different
concentrations of methionine was compared
(Fig. 4C-F). At 500 µM
methionine the methionine promoter is inactive
(Fig. 4F). At 250 µM
methionine CPC (4*) prevented GL1-GL3 interaction and ETC1
(1*) caused a clear growth reduction
(Fig. 4E). At 100 µM
methionine TRY (5*) prevented the GL1-GL3 interaction
(Fig. 4D), and without
methionine ETC3 (3*) could repress growth. ETC2 (2*)
showed only a little growth reduction (Fig.
4C). Together, these data suggest that CPC is the most potent
competitor followed by ETC1, TRY, ETC3 and ETC2.
|
,
which is the distance over which the level of the inhibitor drops to 1/e (37%)
of its source level. As shown in Fig.
5, a clear reduction in the movement ability of the inhibitor is
predicted for increasing binding affinities. For example, if the relative
binding affinity, , of the inhibitor of GL3 is increased from 1 to 10,
its decay length is decreased from 71% to 30% relative to its CDL in the
absence of any binding to GL3. Therefore, a strong binding affinity for GL3 is
expected to result in a less mobile inhibitor, whereas a weak binding affinity
should make the inhibitor more mobile as long as all other parameters are
constant. To experimentally test the prediction that higher binding affinity for GL3 decreases mobility, we compared the movement behaviour between single cells expressing YFP-CPC and YFP-ETC3 and their immediate neighbouring cells. In a first series of experiments we co-bombarded YFP-CPC or YFP-ETC3 with the peroxisome marker PTS in order to recognise the transformed cells. To evaluate the influence of GL3 on the movement behaviour we compared movement on rosette leaves of gl3 egl3 double mutants and a p35S:GL3-overexpression line (Table 4). Movement into neighbouring cells was detected in 74% and 66% of the targeted cells for YFP-ETC3 and YFP-CPC, respectively. Elevated GL3 levels significantly reduced the number of cells from which movement was observed. YFP-CPC movement was reduced to 22%, which corresponds to a reduction to 33% of the levels found in gl3 egl3 plants. As theoretically expected, YFP-ETC3 movement was much less affected by higher GL3 levels: 39% of the cells still showed movement, which corresponds to 53% of the levels found in gl3 egl3 plants.
|
50% in p35S:GL3 plants as compared
with gl3 egl3 plants (Table
5) (Student's t-test, P=0.000). The different
movement behaviour of CPC and ETC3 in the two mutants was evident from the
fact that neither showed a significantly different target/source ratio in
gl3 egl3 mutants (Table
5) (Student's t-test, P=0.129), but highly
significant differences in p35S:GL3 plants
(Table 5) (Student's
t-test, P=0.000). The robustness of all Student's
t-test results was confirmed using a non-parametric test according to
Mann-Whitney (U-test). Together, the data indicate that the presence
of GL3 reduces CPC mobility more efficiently than it reduces ETC3
mobility.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Functional diversity of R3 MYB transcription factors
Phylogenetic analysis groups TRY and ETC2 in one tree and
CPC, TCL1, ETC1 and ETC3 in a second tree
(Wang et al., 2007); within
this tree, CPC is separated from TCL1 and a second branch
containing ETC1 and ETC3. How does this evolutionary tree
fit with their functional adaptations in root hair development and trichome
formation?
Five of the six R3 MYB genes are involved in the regulation of root hair
formation, although to different extents. CPC has the most prominent
role and the corresponding mutants show a strong reduction of root hairs
(Wada et al., 1997). A
function for TRY and ETC1 was recognised in the respective
double mutants with cpc; the try and etc1 single
mutants are indistinguishable from wild type
(Kirik et al., 2004a;
Schellmann et al., 2002). A
function of TCL1 in root hair formation was recognised in a cpc etc1 etc3
tcl1 mutant (Wang et al.,
2008). A role of ETC3 in root hair formation was
suggested by Tominaga et al. (Tominaga et
al., 2008) and by our data presented here. However, such a role
was not found in two previous studies
(Simon et al., 2007;
Wang et al., 2008), which
might be explained by different growth conditions.
By contrast, all six R3 MYB genes play a role in trichome formation. This,
however, does not simply constitute redundant action, as found for
ETC1, but also functional diversification. TRY seems to be
important for the local selection of trichome cells as suggested by a cluster
phenotype in try mutants
(Hulskamp et al., 1994).
CPC, ETC2 and ETC3 seem to regulate the distance between
trichomes as indicated by a higher trichome density in these mutants
(Kirik et al., 2004b;
Schellmann et al., 2002).
Finally, TCL1 is important for organ-specific trichome regulation
(Wang et al., 2007), although
a role in pattern formation on leaves is also suggested by the tc1 cpc
etc1 etc3 phenotype (Wang et al.,
2008). The double and triple mutant analyses revealed plenty of
redundancies between these three regulatory aspects. The etc3 try and
etc2 try double mutants, for example, revealed an additional function
of ETC3 and ETC2 in the local selection process
(Kirik et al., 2004b). The
triple mutant combination of etc1 and etc2 with the try
cpc double mutant revealed a region specificity for ETC1 and
ETC2 in the regulation of the trichome formation of petioles
(Kirik et al., 2004a;
Kirik et al., 2004b).
Conversely, the synergistic enhancement of trichome formation on pedicels and
stem in the tcl1 cpc double mutants indicates a redundancy between
TCL1 and CPC (Wang et
al., 2007). Taken together, there is no obvious correlation
between the functional diversification and evolutionary distances of these
proteins.
Expression or protein function: which is relevant for functional diversification?
Differences in transcriptional regulation are the most obvious reason for
functional diversification. This is particularly evident for ETC2 and
TCL1, neither of which is expressed in the root
(Kirik et al., 2004b;
Wang et al., 2007). In all
other cases, the expression pattern is almost indistinguishable in root and
shoot. The expression levels seem to vary in RT-PCR experiments; however, it
is not possible to judge the relevance of this as the expression levels in the
relevant cell types (trichoblast or atrichoblast/trichomes or epidermal cells)
at the time when the pattern is established remain elusive. Also, our
promoter-swap experiments suggest relevant differences at the promoter level,
as etc3 try cpc triple mutants can be over-rescued when ETC3
is expressed under control of the CPC or TRY promoter.
Promoter-swap experiments also revealed examples demonstrating differences
at the protein level. In the root system the rescue of the cpc mutant
phenotype with CPC-promoter driven cDNAs of the R3 MYB family
revealed clear differences, such that ETC1 rescued best, followed by
ETC3, TRY and ETC2
(Simon et al., 2007). In the
same experimental set-up, the cpc trichome phenotype was rescued
equally well by all proteins except for TRY, which over-rescued. TCL1 seems to
have diverged the most among the R3 MYB group at the protein level. Whereas
overexpression of all the other genes causes supernumary root hairs,
TCL1 overexpression has no effect on root hair formation
(Wang et al., 2007). A
difference is also seen in the trichome system, such that TCL1
expression under the CPC promoter can rescue the cpc mutant
phenotype but expression under the TRY promoter cannot rescue the
try mutant phenotype (Wang et
al., 2007).
Protein properties: which aspects matter for patterning?
Currently, two mechanisms are proposed to explain how the R3 MYB proteins
inhibit the activators. First, it is suggested that TCL1 can bind directly to
the regulatory DNA regions thereby repressing their expression
(Wang et al., 2007), although
no evidence supporting such a mechanism is available for any of the other
group members. Second, it is suggested that GL1-GL3 dimerisation is inhibited
by binding of the inhibitors to GL3 (Esch
et al., 2003). We demonstrated here that this property is shared
by CPC, TRY, ETC1, ETC2 and ETC3. It is likely that TCL1 also shares this
function as it can bind to GL3 (Wang et
al., 2008).
Our finding that the five analysed inhibitors differed in their ability to
interfere with the GL1-GL3 interaction raised the question of whether this
might be relevant for their role in patterning. Theoretical models provide the
key criteria to formulate the relevant parameters
(Koch and Meinhardt, 1994):
(1) the binding strength to GL3 or to DNA; (2) the diffusion/transport rates;
and (3) the degradation rates. Our theoretical calculations suggest that if
all other parameters are constant, movement of the inhibitors should be
affected by their different binding strengths to GL3, such that ETC2 moves
best and CPC least. Focusing on the movement of YFP-CPC and YFP-ETC3 between
two cells, we provide two lines of evidence supporting the theoretical
prediction. First, we show that the number of transiently transformed cells
from which movement is observed depends on the presence of GL3 and that this
affects CPC movement more than that of ETC3. In a second line of experiments
we measured the fraction of YFP-CPC or YFP-ETC3 that had moved into the
neighbouring cell, and again CPC mobility was increased in the absence of GL3,
whereas ETC3 movement was not. It is important to note that these predictions
cannot be simply extended to the whole patterning process and to the
phenotypic differences between the mutants because a quantitative
determination of the expression levels in trichomes, a quantitative comparison
of the protein degradation rates and a quantitative evaluation of the
transport rates between the cells are missing. Future steps towards a
mechanistic understanding of the patterning systems will require not only an
unravelling of the genetic and molecular network, but also a combination of
quantitative experimental and theoretical approaches.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/9/1487/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Berger, F., Haseloff, J., Schiefelbein, J. and Dolan, L.
(1998). Positional information in root epidermis is defined
during embryogenesis an acts in domains with strict boundaries.
Curr. Biol. 8,421
-430.[CrossRef][Medline]
Bergmann, D. C. and Sack, F. D. (2007).
Stomatal development. Annu. Rev. Plant Biol.
58,163
-181.[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.
Bernhardt, C., Zhao, M., Gonzalez, A., Lloyd, A. and
Schiefelbein, J. (2005). The bHLH genes GL3 and EGL3
participate in an intercellular regulatory circuit that controls cell
patterning in the Arabidopsis root epidermis.
Development 132,291
-298.
Bouyer, D., Geier, F., Kragler, F., Schnittger, A., Pesch, M.,
Wester, K., Balkunde, R., Timmer, J., Fleck, C. and Hulskamp, M.
(2008). Two-dimensional patterning by a trapping/depletion
mechanism: the role of TTG1 and GL3 in Arabidopsis trichome formation.
PLoS Biol. 6,e141
.[CrossRef][Medline]
Clough, S. and Bent, A. (1998). Floral dip: a
simplified method for Agrobacterium-mediated transformation of Arabidopsis
thaliana. Plant J. 16,735
-743.[CrossRef][Medline]
Digiuni, S., Schellmann, S., Geier, F., Greese, B., Pesch, M.,
Wester, K., Dartan, B., Mach, V., Srinivas, B. P., Timmer, J. et al.
(2008). A competitive complex formation mechanism underlies
trichome patterning on Arabidopsis leaves. Mol. Syst.
Biol. 4,217
.[Medline]
Dolan, L., Duckett, C. M., Grierson, C., Linstead, P.,
Schneider, K., Lawson, E., Dean, C., Poethig, S. and Roberts, K.
(1994). Clonal relationships and cell patterning in the root
epidermis of Arabidopsis. Development
120,2465
-2474.
Esch, J. J., Chen, M., Sanders, M., Hillestad, M., Ndkium, S.,
Idelkope, B., Neizer, J. and Marks, M. D. (2003). A
contradictory GLABRA3 allele helps define gene interactions controlling
trichome development in Arabidopsis. Development
130,5885
-5894.
Esch, J. J., Chen, M. A., Hillestad, M. and Marks, M. D.
(2004). Comparison of TRY and the closely related At1g01380 gene
in controlling Arabidopsis trichome patterning. Plant
J. 40,860
-869.[CrossRef][Medline]
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]
Gietz, R. D. and Schiestl, R. H. (1995).
Transforming yeast with DNA. Methods Mol. Cell Biol.
5, 255-269.
Hulskamp, M. and Schnittger, A. (1998). Spatial
regulation of trichome formation in Arabidopsis thaliana. Semin.
Cell Dev. Biol. 9,213
-220.[CrossRef][Medline]
Hulskamp, M., Misera, S. and Jürgens, G.
(1994). Genetic dissection of trichome cell development in
Arabidopsis. Cell
76,555
-566.[CrossRef][Medline]
Ishida, T., Kurata, T., Okada, K. and Wada, T.
(2008). A genetic regulatory network in the development of
trichomes and root hairs. Annu. Rev. Plant Biol.
59,364
-386.
Kirik, V., Schnittger, A., Radchuk, V., Adler, K., Hulskamp, M.
and Baumlein, H. (2001). Ectopic expression of the
Arabidopsis AtMYB23 gene induces differentiation of trichome cells.
Dev. Biol. 235,366
-377.[CrossRef][Medline]
Kirik, V., Simon, M., Hulskamp, M. and Schiefelbein, J.
(2004a). The ENHANCER of TRY and CPC1 (ETC1) gene acts
redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell
patterning in Arabidopsis. Dev. Biol.
268,506
-513.[CrossRef][Medline]
Kirik, V., Simon, M., Wester, K., Schiefelbein, J. and Hulskamp,
M. (2004b). ENHANCER of TRY and CPC 2 (ETC2) reveals
redundancy in the region-specific control of trichome development of
Arabidopsis. Plant Mol. Biol.
55,389
-398.[CrossRef][Medline]
Kirik, V., Lee, M. M., Wester, K., Herrmann, U., Zheng, Z.,
Oppenheimer, D., Schiefelbein, J. and Hulskamp, M. (2005).
Functional diversification of MYB23 and GL1 genes in trichome morphogenesis
and initiation. Development
132,1477
-1485.
Koch, A. J. and Meinhardt, H. (1994).
Biological pattern formation: from basic mechanisms to complex structures.
Rev. Mod. Phys. 66,1481
-1507.[CrossRef]
Larkin, J. C., Young, N., Prigge, M. and Marks, M. D.
(1996). The control of trichome spacing and number in
Arabidopsis. Development
122,997
-1005.[Abstract]
Larkin, J. C., Walker, J. D., Bolognesi-Winfield, A. C., Gray,
J. C. and Walker, A. R. (1999). Allele-specific interactions
between ttg and gl1 during trichome development in Arabidopsis thaliana.
Genetics 151,1591
-1604.
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
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.[CrossRef][Medline]
Marks, M. D. and Esch, J. J. (2003). Initiating
inhibition: control of epidermal cell patterning in plants. EMBO
Rep. 4,24
-25.[CrossRef][Medline]
Mathur, J., Mathur, N. and Hulskamp, M. (2002).
Simultaneous visualization of peroxisomes and cytoskeletal elements reveals
actin and not microtubule-based peroxisome motility in plants.
Plant Physiol. 128,1031
-1045.
Oppenheimer, D. G., Herman, P. L., Sivakumaran, S., Esch, J. and
Marks, M. D. (1991). A myb gene required for leaf
trichome differentiation in Arabidopsis is expressed in stipules.
Cell 67,483
-493.[CrossRef][Medline]
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.
Pesch, M. and Hulskamp, M. (2004). Creating a
two-dimensional pattern de novo during Arabidopsis trichome and root hair
initiation. Curr. Opin. Genet. Dev.
14,422
-427.[CrossRef][Medline]
Schellmann, S., Schnittger, A., Kirik, V., Wada, T., Okada, K.,
Beermann, A., Thumfahrt, J., Jurgens, G. and Hulskamp, M.
(2002). TRIPTYCHON and CAPRICE mediate lateral inhibition during
trichome and root hair patterning in Arabidopsis. EMBO
J. 21,5036
-5046.[CrossRef][Medline]
Schellmann, S., Hulskamp, M. and Uhrig, J.
(2007). Epidermal pattern formation in the root and shoot of
Arabidopsis. Biochem. Soc. Trans.
35,146
-148.[CrossRef][Medline]
Scheres, B. (2002). Plant patterning: TRY to
inhibit your neighbors. Curr. Biol.
12,R804
-R806.[CrossRef][Medline]
Serna, L. (2005). Epidermal cell patterning and
differentiation throughout the apical-basal axis of the seedling.
J. Exp. Bot. 56,1983
-1989.
Simon, M., Lee, M. M., Lin, Y., Gish, L. and Schiefelbein,
J. (2007). Distinct and overlapping roles of single-repeat
MYB genes in root epidermal patterning. Dev. Biol.
311,566
-578.[CrossRef][Medline]
Spitzer, C., Schellmann, S., Sabovljevic, A., Shahriari, M.,
Keshavaiah, C., Bechtold, N., Herzog, M., Muller, S., Hanisch, F. G. and
Hulskamp, M. (2006). The Arabidopsis elch mutant reveals
functions of an ESCRT component in cytokinesis.
Development 133,4679
-4689.
Tominaga, R., Iwata, M., Sano, R., Inoue, K., Okada, K. and
Wada, T. (2008). Arabidopsis CAPRICE-LIKE MYB 3 (CPL3)
controls endoreduplication and flowering development in addition to trichome
and root hair formation. Development
135,1335
-1345.
Vroemen, C. W., Langeveld, S., Mayer, U., Ripper, G., Jurgens,
G., Van Kammen, A. and De Vries, S. C. (1996). Pattern
formation in the arabidopsis embryo revealed by position-specific lipid
transfer protein gene expression. Plant Cell
8, 783-791.[Abstract]
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.
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
-1349.
Wang, S., Kwak, S. H., Zeng, Q., Ellis, B. E., Chen, X. Y.,
Schiefelbein, J. and Chen, J. G. (2007). TRICHOMELESS1
regulates trichome patterning by suppressing GLABRA1 in Arabidopsis.
Development 134,3873
-3882.
Wang, S., Hubbard, L., Chang, Y., Guo, J., Schiefelbein, J. and
Chen, J. G. (2008). Comprehensive analysis of single-repeat
R3 MYB proteins in epidermal cell patterning and their transcriptional
regulation in Arabidopsis. BMC Plant Biol.
8, 81.[CrossRef][Medline]
Zhang, F., Gonzalez, A., Zhao, M., Payne, C. T. and Lloyd,
A. (2003). A network of redundant bHLH proteins functions in
all TTG1-dependent pathways of Arabidopsis.
Development 130,4859
-4869.
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