3456 RESEARCH ARTICLE
Development 140, 3456-3467 (2013) doi:10.1242/dev.094698
© 2013. Published by The Company of Biologists Ltd
Mutual control of intracellular localisation of the patterning
proteins AtMYC1, GL1 and TRY/CPC in Arabidopsis
Martina Pesch*, Ilka Schultheiß*, Simona Digiuni, Joachim F. Uhrig and Martin Hülskamp‡
SUMMARY
Trichome and root hair patterning is governed by a gene regulatory network involving TTG1 and several homologous MYB and bHLH
proteins. The bHLH proteins GL3 and EGL3 are core components that serve as a regulatory platform for the activation of downstream
genes. In this study we show that a homologue of GL3 and EGL3, AtMYC1, can regulate the intracellular localisation of GL1 and TRY.
AtMYC1 protein is predominantly localised in the cytoplasm and can relocate GL1 from the nucleus into the cytoplasm. Conversely,
AtMYC1 can be recruited into the nucleus by TRY and CPC, concomitant with a strong accumulation of TRY and CPC in the nucleus.
When AtMYC1 is targeted to the nucleus or cytoplasm by nuclear localisation or export signals (NLS or NES), respectively, the
intracellular localisation of GL1 and TRY also changes accordingly. The biological significance of this intracellular localisation is
suggested by the finding that the efficiency of rescue of trichome number is significantly altered in NES and NLS fusions as compared
with wild-type AtMYC1. Genetic analysis of mutants and overexpression lines supports the hypothesis that AtMYC1 represses the
activity of TRY and CPC.
INTRODUCTION
The initiation of Arabidopsis thaliana trichomes serves as an
excellent model system with which to study how cells are specified
in a temporal and spatially defined manner by a gene regulatory
network (Balkunde et al., 2010; Tominaga-Wada et al., 2011; Grebe,
2012). In wild type, trichomes are formed at the leaf basis of young
leaves without a recognisable positional reference to other leaf
structures (Hülskamp et al., 1994), and the analysis of genetic
mosaics ruled out the possibility that the selection is based on cell
lineage (Larkin et al., 1996; Schnittger et al., 1999). It is therefore
postulated that trichome patterning is governed by cell-cell
interactions between initially equivalent cells (Ishida et al., 2008;
Pesch and Hülskamp, 2009; Balkunde et al., 2010).
In the current models of trichome patterning, a regulatory network
of positively and negatively acting genes explains how trichomes are
selected in a field of competent protodermal cells. The positive
regulators are represented by a group of three proteins: the WD40
protein TRANSPARENT TESTA GLABRA 1 (TTG1) (Koornneef,
1981; Galway et al., 1994; Walker et al., 1999); the R2R3 MYBrelated transcription factor GLABRA 1 (GL1) (Oppenheimer et al.,
1991); and the basic helix-loop-helix (bHLH)-like transcription factor
GLABRA 3 (GL3) (Koornneef et al., 1982; Hülskamp et al., 1994;
Payne et al., 2000). GL1 and GL3 act partially redundantly with their
homologues MYB23 and EGL3, respectively (Kirik et al., 2001;
Zhang et al., 2003; Kirik et al., 2005). The negative regulators are a
class of seven homologous R3 single-repeat MYB genes that act in a
partially redundant manner (Schellmann et al., 2002; Kirik et al.,
2004a; Kirik et al., 2004b; Wang et al., 2007; Tominaga et al., 2008;
Wang et al., 2008; Wester et al., 2009; Wang et al., 2010; Gan et al.,
2011). Among these, TRIPTYCHON (TRY) and CAPRICE (CPC)
Biocenter, Cologne University, Botanical Institute, Zülpicher Straße 47b, 50674
Cologne, Germany.
*These authors contributed equally to this work
‡
Author for correspondence ([email protected])
Accepted 17 June 2013
show the strongest single-mutant phenotypes (Hulskamp et al., 1994;
Wada et al., 1997; Schellmann et al., 2002). TTG1, an R2R3MYB
and a bHLH protein form a trimeric activator complex, in which
TTG1 and the MYB both bind to the bHLH protein (Payne et al.,
2000; Zhang et al., 2003; Zimmermann et al., 2004; Kirik et al., 2005;
Digiuni et al., 2008; Gao et al., 2008; Wang and Chen, 2008; Zhao et
al., 2008). The inhibition of the activator complex is governed by the
binding of an R3MYB to the bHLH protein, thereby replacing the
R2R3MYB (Payne et al., 2000; Bernhardt et al., 2003; Esch et al.,
2003). The immediate downstream gene of this machinery promoting
the differentiation of trichome cells is GLABRA 2 (GL2) (Rerie et al.,
1994).
The activators and inhibitors of trichome patterning are engaged
in a gene regulatory network (Benítez et al., 2007; Benítez et al.,
2008), from which two principles acting in parallel can be extracted
(Pesch and Hülskamp, 2009). First, the activator-inhibitor model
explains pattern formation by a feedback loop between the
activators and the inhibitors, in which the activators activate the
inhibitors, which in turn repress the activators. The inhibitors
mediate the cell-cell communication by intercellular movement.
Second, the activator depletion model explains patterning by the
removal of the mobile activator TTG1 in neighbouring cells through
trapping by GL3 in trichome initials (Bouyer et al., 2008; Balkunde
et al., 2011).
In addition to GL3 and EGL3, the bHLH gene AtMYC1 has been
shown to participate in the regulation of trichome patterning
(Symonds et al., 2011; Zhao et al., 2012). Formally, AtMYC1 acts as
a positive regulator of trichome initiation because trichome number
is reduced in mutants (Symonds et al., 2011; Zhao et al., 2012).
Promoter swap experiments showed that GL3/EGL3 can replace
AtMYC1, whereas AtMYC1 cannot rescue gl3 egl3 mutants,
suggesting a redundant but also a divergent function (Zhao et al.,
2012). The AtMYC1 gene was initially isolated in a screen for
Antirrhinum DELILA homologues in Arabidopsis (Urao et al.,
1996). Expression analyses suggest that AtMYC1 functions
downstream of the patterning genes but upstream of GL2.
Chromatin immunoprecipitation experiments have shown that
DEVELOPMENT
KEY WORDS: Arabidopsis thaliana, GL1, MYC1, TRY, Patterning, Trichome
Localisation of TRY and GL1 by AtMYC1
MATERIALS AND METHODS
Plant lines, growth conditions, transformation of plant material
and phenotypic analysis
Plants were grown on soil at 24°C with 16 hours light/day. Plant
transformations were performed by the floral dip method (Clough and Bent,
1998). We used the Col-0 wild type, gl3-3 egl3-3 (GK 545D05;
SALK_077439) and atmyc1-1 (Salk 057388) to transform 35S:GL3,
35S:EGL3 and 35S:AtMYC1 in the respective backgrounds. 5⬘AtMYC1:AtMYC1, 35S:YFP-AtMYC1, 35S:YFP-NLS-AtMYC1 and 35S:YFPNES-AtMYC1 were transformed in atmyc1-1 plants. 5⬘-TRY:GUS,
5⬘-CPC:GUS and 5⬘-GL2:GUS were transformed in Col-0 plants. The
respective T2 lines of 5⬘-TRY:GUS, 5⬘-CPC:GUS and 5⬘-GL2:GUS were
crossed with atmyc1-1. The 35S:GL1, gl1 (SalK_039478), try-JC and cpc-1
alleles (Wada et al., 1997; Larkin et al., 1999) were crossed with one selected
T2 line of 35S:AtMYC1 Col-0 and with atmyc1-1. The insertion of all atmyc1
alleles was confirmed: Salk_006354, Salk_057388, Salk_056899, Gabi_621F09 and Sail_277_H01. The different combinations of the atmyc1 mutants
were created by crossing of atmyc1-1, gl3-3 (GK 545D05) (Rosso et al., 2003;
Jakoby et al., 2008) and egl3 (Salk_019114) (Wester et al., 2009). The rescue
efficiency is presented by box-whisker plots (Tukey, 1977).
Particle bombardment (Pesch and Hülskamp, 2011), protoplast isolation
(Col ecotype) and transfection (Spitzer et al., 2006) were performed as
described previously. Tobacco leaves were transiently transformed by
infiltration with Agrobacterium tumefaciens (Agrobacterium strain GV3101
pMP90RK) and co-infiltrated with the antisilencing strain RK19 (Voinnet
et al., 1999).
Constructs
Coding DNA sequence (CDS) entry clones of the different
patterning genes
CDSs were cloned in pENTR1A/pENTR4 (Invitrogen) or introduced
through BP recombination in pDONR201 (Invitrogen). Mutations in TRY,
CPC and GL1 were created by site-directed mutagenesis (supplementary
material Table S3). The vector pENTR1A-w/o-ccdB was created by EcoRI
digestion and religation to remove almost the entire Gateway recombination
cassette between the attB sequences of pENTR1A. This construct was used
in LR recombinations to produce expression vectors without CDS.
AtMYC1-w/o-Stop-pDONR201 was created via PCR and BP recombination
in pDONR201 using AtMYC1-pDONR201 as template (supplementary
material Table S3).
NLS-AtMYC1-pDONR201 and NES-AtMYC1-pDONR201 are
derivatives of AtMYC1-pDONR201. To generate these constructs, tandem
copies of BamHI and XhoI restriction sites were inserted directly after the
start codon of AtMYC1 in AtMYC1-pDONR201 to obtain BamHI-XhoIAtMYC1-pDONR201 (supplementary material Table S3). In the next step,
the SV40 NLS was generated by annealing the oligonucleotides BamHINLS-XhoI-for and BamHI-NLS-XhoI-rev encoding amino acids
LQPKKKRKVGG. The NES from protein kinase inhibitor (PKI) was
produced by annealing the BamHI-NES-XhoI-for and BamHI-NES-XhoIrev
oligonucleotides
encoding
the
amino
acids
LQNELALKLAGLDINKTGG. The annealed linkers were inserted into
BamHI-XhoI-AtMYC1-pDONR201 using the BamHI and XhoI restriction
sites (supplementary material Table S3).
Plant vectors
The 5⬘ AtMYC1 region (−1 to −1963) was cloned in pAM-PAT-GWPro35S
(GenBank AY436765) (supplementary material Table S3). The
complementation construct 5⬘-AtMYC1:AtMYC1 was created by
recombination of 5⬘-AtMYC1-pAMPAT with AtMYC1-pDONR201.
Reporter constructs were created by recombining pENTR-GUS-201
(Invitrogen) with 5⬘-TRY-pAM-PAT, 5⬘-CPC-pAM-PAT and 5⬘-GL2-pAMPAT (Weinl et al., 2005; Wester et al., 2009). 35S:CDS constructs were
generated by LR recombination of the respective CDS entry clones (GL3,
EGL3, AtMYC1) and pAM-PAT-GWPro35S (GenBank AY436765).
35S:AtMYC1-YFP was created by LR recombination of AtMYC1-w/o-StoppDONR201 and pEX-G-YFP (Feys et al., 2005). The N-terminal YFP, RFP
and CFP fusion constructs were created through recombination of the
respective entry clones or pENTR1A-w/o-ccdB with pENS-G-YFP, pENSG-CFP and pNmR (Feys et al., 2005) (gift of A. Schrader, Botanical
Institute, Cologne, Germany), respectively. The 35S:GFP-YFP construct
was described previously (Digiuni et al., 2008). For BiFC analysis, the
pBATL vectors pCL112 YFP N-term and pCL113 YFP C-term (kindly
provided by Sean Chapman, SCRI, Dundee, UK) were used after LR
recombination of the entry clones containing the different CDSs.
Yeast vectors
For the nuclear trapping system, the Gateway recombination cassette was
removed by HindIII and XbaI digestion from pBluescript-GW-RekA (gift
of S. Bieri, MPI for Plant Breeding, Cologne, Germany) and introduced
into pVT-U (Vernet et al., 1987) to create pVT-U-attR. Patterning genes
were introduced by LR recombination with the respective entry clones. The
plasmid vector pNS and pNS-GL3 were described previously (Ueki et al.,
1998; Balkunde et al., 2011). AtMYC1 CDS was cloned as a SalI-HindIII
fragment from SalI-AtMYC1-HindIII-pGEM-T-easy (supplementary
material Table S3) into the SalI site of pNS to create pNS-AtMYC1
(supplementary material Table S3). GL1 CDS was cloned as a SalI-XhoI
fragment into the respective sites of pNS to produce pNS-GL1. pNS-TRY
was created by cloning the TRY CDS as a PvuII-NcoI fragment into SalI
(blunted) and NcoI cut pNS.
To produce control vectors without CDS fusions, the destination vectors
(pC-ACT2-attR, pAS-attR and pVT-U-attR) were recombined with
pENTR1A-w/o-ccdB.
LUMIER vectors
To fuse luciferase or protein A to patterning proteins the two destination
vectors pcDNA3-Rluc-GW and pTREX-dest30-ntPrA (Blasche et al., 2013)
were recombined by LR reaction with the entry clones of interest or with
pENTR1A-w/o-ccdB.
Yeast assays
Yeast two-hybrid assays were performed as described previously (Gietz et
al., 1995). The nuclear trapping assays used pNS and pVT-U vectors that
were co-transformed in the yeast strain EGY48 (Clontech). The nuclear
import of the NESLexAD fusion proteins was assayed on medium lacking
leucine and histidine or lacking leucine, histidine and uracil.
LUMIER assays
For LUMIER assays, proteins were transiently co-expressed in
HEK293TN cells (BioCat/SBI LV900A-1) as hybrid proteins with
Staphylococcus aureus protein A or with the Renilla reniformis luciferase
DEVELOPMENT
GL3/GL1 bind to the AtMYC1 promoter chromatin (Morohashi and
Grotewold, 2009), suggesting transcriptional regulation of AtMYC1
by GL1 and GL3. In the root hair system, AtMYC1 is controlled by
WER and CPC in a similar manner to that described for GL3/EGL3
(Bernhardt et al., 2005; Bruex et al., 2012). Its function upstream of
GL2 was inferred from the finding that GL2 expression is reduced
in atmyc1 mutants (Zhao et al., 2012). The AtMYC1 protein has
been grouped in a phylogenetic tree together with GL3, EGL3 and
TT8 in subgroup IIIf of Arabidopsis bHLH proteins (Heim et al.,
2003). Yeast two-hybrid assays revealed that, in addition to GL3,
AtMYC1 protein also interacts with most of the other patterning
proteins including the above-mentioned members CPC, TRY,
TTG1, GL1 and MYB23 (Zimmermann et al., 2004; Tominaga et
al., 2008; Zhao et al., 2012). However, in contrast to GL3 and
EGL3, the AtMYC1 protein appears to be unable to form homo- or
heterodimers with GL3/EGL3 (Zhao et al., 2012).
Here, we explore the molecular basis of AtMYC1 function. We
show that GL2, TRY and CPC expression patterns are unchanged
in atmyc1 mutants and overexpression lines. In contrast to GL3 and
EGL3, AtMYC1 protein is located in the cytoplasm. When
AtMYC1 is co-expressed with GL1, GL1 is relocated into the
cytoplasm. Co-expression of AtMYC1 with TRY or CPC leads to
the recruitment of AtMYC1 into the nucleus, as well as to transport
of TRY/CPC from the cytoplasm into the nucleus. Our genetic
analysis indicates that AtMYC1 inhibits the function of TRY/CPC.
RESEARCH ARTICLE 3457
Development 140 (16)
3458 RESEARCH ARTICLE
tag fused to their N-terminus. The LUMIER assay is based on the
technique used by Barrios-Rodiles et al. (Barrios-Rodiles et al., 2005) and
was optimised for our proteins as follows. Constructs (1.5 µg plasmid
DNA) were co-transfected into 1×106 HEK293TN cells in 6-well plates
using 10 μl Lipofectamine 2000 (Invitrogen). After 48 hours, the medium
was removed and cells were washed with PBS. The cells were collected
by centrifugation (600 g, 15 minutes) and then lysed on ice in 50 µl icecold lysis buffer [20 mM Tris pH 7.5, 250 mM NaCl, 1.1% Triton X-100,
10 mM EDTA, 10 mM DTT, Protease Inhibitor Cocktail (Roche, 1 836
170) and benzonase (Novagen, 70746; 0.0125 units per µl final
concentration)] for 1 hour. Subsequently, lysates were centrifuged at
15,000 rpm (21,380 g) for 15 minutes at 4°C, and the supernatant was
mixed with 10 μl PBS-prewashed sheep anti-rabbit IgG-coated magnetic
beads (Invitrogen, Dynabeads M280; 2 mg/ml final concentration) and
incubated for 1 hour on ice. The beads were collected using a magnetic
holder (Neodym-Magnets, 1.3 Teslar) and luminescence measured in a
microtitre plate reader (BMG Fluostar Optima). Luciferase activity was
determined for the input and the pulldown. Lysis buffer, PBS and
untransformed cells served as a negative control. Pulldown efficiency was
calculated by: [RLU pulldown/(RLU input×5)]×100.
RNA extraction and real-time PCR
Real-time PCR reactions were performed using the SYBR Green Real-Time
PCR Master Mix (Applied Biosystems). To quantify RNA expression levels,
published primers pairs were used: CPC, TRY primers (Morohashi et al.,
2007), TRY primers (Zhao et al., 2008). Actin primers were used as an
endogenous control as previously described (Zhao et al., 2008). Three
biological replicates, each including two technical replicas, were measured.
Relative RNA levels were calculated according to the comparative Ct
(2–ΔΔCt) method.
Histochemistry and microscopy
GUS activity was assayed as described (Sessions et al., 1999). Light and
fluorescence microscopy were performed using a Leica DMRE microscope
or a Leica MZ16F binocular microscope. Images were processed using
Adobe Photoshop Elements 7.0. Co-bombardments in Arabidopsis cells and
BiFC analyses in leek cells were studied with a Leica TCS SP2 confocal
laser-scanning microscope using a UV HCX APO L 40.0×/0.80 W or
UV/HCX APO L U-V-I 63.0×/0.90 W water-immersion objective or a PL
FLUOTAR L 20.0×/0.40 BD dry objective. YFP was excited at 514 nm and
emitted light was detected at 520-560 nm. RFP was excited at 561 nm and
detected in the 570-650 nm range. Colocalisation studies with YFP and RFP
fusion proteins in leek cells were imaged with a Leica TCS SPE confocal
laser-scanning microscope using an ACS APO 10.0×/0.30 dry objective.
YFP was excited at 488 nm and emitted light captured at 511-550 nm. RFP
was excited at 561 nm and detected in the 570-602 nm range. Simultaneous
detection of RFP and YFP was performed using the sequential scanning
facility of the microscopes.
Quantification of fluorescence signal intensities was performed using
Leica LAS AF and Leica LCS software. Images were created by overlying
the single stack images of the cell. Each pixel in the confocal image was
assigned a greyscale intensity value that ranges from black (0) to white
(255). The pixel sum (sum of the measured greyscale intensity values) of the
overlying image was measured for two regions of interest (whole cell and
nucleus). The pixel sum for the whole cell was defined as 100% and the
nucleus pixel sum expressed as a percentage of this. Images of Arabidopsis
leaves were created using the Leica MZ16F binocular microscope and the
z-stack function of Leica Application Suite Version 3.7.0. Scanning electron
microscopy (SEM) images were taken with an FEI Quanta 250 FEG
scanning electron microscope.
Protein extraction and western blot
Transformed leaf material was homogenised in liquid nitrogen using a
mortar and pestle. Leaf powder was incubated with lysis buffer [150 mM
NaCl, 1% Triton X-100, 50 mM Tris HCl pH 8, 0.1% SDS, 10 mM DTT,
Roche Complete Protease Inhibitor Cocktail (1 tablet/40 ml)] for
30 minutes on ice, centrifuged at 10,000 g? for 15 minutes at 4°C, the
supernatant denatured by addition of 6× concentrated protein sample
buffer (0.375 M Tris HCl pH 6.8, 12% SDS, 60% glycerol, 0.6 M DTT,
0.06% Bromophenol Blue) and heated for 15 minutes at 95°C. Samples
were analysed by western blot using mouse anti-GFP IgG (Roche, 1:1000)
and anti-mouse IgG (Sigma, 1:50,000), both in 5% milk powder in PBST
(PBS with 0.1% Tween 20). For detection of protein bands the
SuperSignal West Femto Maximum Sensitivity Substrate (Thermo
Scientific) and the ImageQuant LAS 4000 (GE Healthcare) detection
system were used.
RESULTS
Genetic analysis of AtMYC1
As recently described for three alleles (Symonds et al., 2011; Zhao
et al., 2012), we found a strong reduction in trichome number in all
five atmyc1 alleles tested in the Col wild-type background. When
comparing the trichome number for each on the first two leaf pairs,
we found a similar degree of reduction in all alleles independent of
the T-DNA insertion position (supplementary material Table S1,
Fig. S1A). We focused our analysis on the atmyc1-1 allele (SALK057388), which does not produce a full-length mRNA and is
therefore considered a null allele (supplementary material Fig.
S1D).
Analysis of the trichome number in atmyc1-1 egl3 (Salk-019114)
and atmyc-1 gl3-3 double mutants compared with wild type showed
subtle but statistically significant differences (Table 1). Since egl3
and gl3-3 single mutants also exhibit a reduced trichome number,
this phenotype is considered additive. When we examined trichome
branch number in the single and double mutants, we found no
significant genetic effect on each other (Table 1).
Overexpression of AtMYC1 under the control of the CaMV 35S
promoter resulted in an ~2-fold increase in trichome number in the
Col background (supplementary material Fig. S2; Tables 2, 3). This
raised the question of whether 35S:AtMYC1 can rescue the gl3 egl3
phenotype. We compared 35S:GL3, 35S:EGL3 and 35S:AtMYC1
Table 1. Trichome and branch number in atmyc1-1 single and double mutants
Genotype
Col
atmyc1-1
gl3-3
atmyc1-1 gl3-3
egl3-3
atmyc1-1 egl3-3
Four-branched trichomes per
leaf pair (%)
Two-branched trichomes per
leaf pair (%)
1/2
3/4
1/2
3/4
1/2
3/4
56.2±4.8
23.6±2.5
47.2±3.9
17.1±2.4
52.5±4.5
16.8±1.2
180.4±19.9
64.4±7.4
145.5±14.7
62.4±4.9
139.5±19.5
45.8±3.1
11.3±4.7
8.5±5.3
0
0
7.9±3.1
7.9±2.9
10.6±3.1
18.0±5.5
0
0
11.3±3.5
11.1±4.6
0
0
18.5±6.5
17.1±8.1
0
0
0
0
29.6±5.8
24.5±5.9
0
0
atmyc-1, gl3-3, egl3-3, atmyc1-1 gl3-3 and atmyc1-1 egl3-3 were tested for significant difference to Col on leaves 1/2 or 3 /4. atmyc1-1 egl3-3 was compared with atmyc1-1
egl3-3 and atmyc1-1 gl3-3 was compared with atmyc1-1 gl3-3. All values were significantly different in a Student’s t-test with P<10–7, or in the case of a comparison of egl33 and Col on leaves 1/2 with P= 0.0158. The mean±s.d. is shown in each case (n=20).
DEVELOPMENT
Trichomes per leaf pair
Localisation of TRY and GL1 by AtMYC1
RESEARCH ARTICLE 3459
Table 2. Trichome number and cluster frequency in try atmyc1 and try 35S:AtMYC1
Trichome number per leaf pair
Genotype
Col
try-JC
atmyc1-1
atmyc1-1 try-JC
35S:AtMYC1
35S:AtMYC1 try-JC
Cluster (%)
1/2
3/4
1/2
3/4
62.5±6.3
55.5±6.1
26.0±3.3
36.0±5.5
127.4±14.9
96.2±19.1
241.0±31.3
188.8±23.2
104.8±16.0
115.5±15.3
492.4±51.1
388.9±71.2
0.1±0.4
7.6±4.1
0.0±0.0
16.4±9.8
0.4±0.4
7.8±1.8
0.1±0.4
11.4±3.1
0.1±0.4
20.0±5.3
0.5±0.4
7.4±2.4
Trichome numbers of try-JC, atmyc1-1, atmyc1-1try-JC, 35S:AtMYC and 35S:AtMYC1 try-JC were tested for significant difference to Col on leaf 1/2 and leaf 3/4.
35S:AtMYC1 try-JC was compared with try-JC and 35S:AtMYC, and atmyc1-1try-JC was compared with try-JC. All values were significant (Student’s t-test, P<10–5). The
mean±s.d. is shown in each case (n=20).
with respect to their ability to rescue the gl3 egl3 trichome
phenotype (Fig. 1A). We found full rescue with 35S:GL3, partial
rescue with 35S:EGL3, and no rescue with 35S:AtMYC1. This
indicates that, despite its function as an activator of trichome
development, AtMYC1 cannot replace GL3/EGL3 function. This is
consistent with the finding by Zhao and co-workers that AtMYC1
expression under control of the GL3 and EGL3 promoters cannot
rescue the gl3 egl3 double mutant (Zhao et al., 2012). Conversely,
35S:GL3 or 35S:EGL3 caused substantial overproduction of
trichomes in the atmyc1-1 mutant background (Fig. 1B), whereas
35S:AtMYC1 showed a moderate rescue. This indicates that GL3
and EGL3 do not require AtMYC1 to induce extra trichome
formation.
pTRY:GUS, pCPC:GUS and pGL2:GUS expression is
not altered in atmyc1-1 mutants
The finding that AtMYC1 cannot replace GL3 and EGL3 function
prompted us to assess the ability of AtMYC1 to regulate
downstream genes. We analysed the temporal and spatial expression
of pTRY:GUS, pCPC:GUS and pGL2:GUS in atmyc1-1 mutants and
the corresponding Col background. At all leaf developmental stages
we found indistinguishable expression patterns for pTRY:GUS,
pCPC:GUS and pGL2:GUS in atmyc1-1 mutants and wild type
(Fig. 2A-F; supplementary material Fig. S3A-F). Also,
overexpression of AtMYC1 did not alter the expression pattern of
pTRY:GUS, pCPC:GUS or pGL2:GUS (Fig. 2G-I; supplementary
material Fig. S3G-I). To test whether the expression levels of TRY,
CPC or GL2 are changed in atmyc1-1 mutants or AtMYC1
overexpression lines, we employed qPCR (Table 4). Although
overexpression of AtMYC1 had no substantial influence on the
expression levels, a slight but significant reduction was seen for
CPC and GL2 in the atmyc1 mutant. The latter is consistent with the
findings of a previous study (Zhao et al., 2012).
Interactions of AtMYC1 with other patterning
proteins
Using the yeast two-hybrid method, AtMYC1 has been reported to
interact with various proteins involved in trichome patterning,
including ETC3 (Tominaga et al., 2008), GL1 and TTG1 (Symonds
et al., 2011), WER and TRY (Zhao et al., 2012). The interactions
with GL1, WER and TRY were confirmed by bimolecular
fluorescence complementation (BiFC) (Zhao et al., 2012). We
aimed to confirm and extend the characterisation of these
interactions. Our yeast two-hybrid screens confirmed these
interactions and also revealed, as expected, that AtMYC1 interacts
with CPC (supplementary material Fig. S4). We further show that
single amino acid exchanges (R/D) in the MYB-R/B-like bHLH
transcription factor interface of the MYB factors (Zimmermann et
al., 2004) strongly reduce the interaction of GL3 with GL1, TRY
and CPC. These amino acid exchanges in the respective MYB
factors also eliminate the interaction with AtMYC1. These data
suggest that binding of AtMYC1 with the MYB factors occurs by
a similar mechanism to that employed with GL3.
To provide independent support of the interaction between
AtMYC1 and the other patterning proteins we used the
luminescence-based mammalian interactome (LUMIER) assay
(Barrios-Rodiles et al., 2005). Protein fusions to protein A (ProtA)
or to luciferase were expressed in human HEC293TN cells. We
immunoprecipitated the ProtA-tagged protein with IgG beads and
quantified the co-immunoprecipitated proteins by luciferase assay.
This confirmed the interaction of AtMYC1 with GL1, TRY, CPC
and TTG1 (Table 5).
Table 3. Trichome number and cluster frequency in cpc atmyc1 and cpc 35S:AtMYC1
Col
WS
cpc-1
cpc-1
atmyc1-1
atmyc1-1
atmyc1-1 cpc-1
35S:AtMYC1
35S:AtMYC1
35S:AtMYC1 cpc-1
Cluster (%)
Background
1/2
3/4
1/2
3/4
Col
WS
WS
WS/ Col
Col
WS/ Col
WS/ Col
Col
WS/ Col
WS/ Col
53.6±6.0
91.7±12.4
126.6±15.0
111.6±15.5
23.6±2.9
47.2±6.7
42.8±6.6
114.1±15.6
106.7±10.3
197.9±21.6
187.1±16.9
336.9±37.5
449.6±59.1
516.6±106.2
81.6±11.0
163.3±22.9
194.5±38.5
375.0±61.6
393.5±32.2
614.3 ±111.8
0.1±0.4
0.0±0.0
0.2±0.3
0.2±0.4
0.0±0.0
0.1±0.3
0.1±0.5
0.3±0.4
0.0±0.0
1.7±1.3
0.4±0.6
0.1±0.2
0.1±0.2
0.2±0.3
0.3±0.8
0.1±0.3
0.1±0.4
0.4±0.5
0.2±0.3
1.6±1.1
Trichome numbers of cpc-1 (WS/Col), atmyc1-1 (WS/Col), 35S:AtMYC (WS/Col) and 35S:AtMYC1 cpc-1 (WS/Col) were tested for significant difference to Col and WS on leaves
1/2 and 3/4. 35S:AtMYC1 cpc-1 (WS/Col) was compared with 35S:AtMYC (WS/Col) and cpc-1 (WS/Col), and atmyc1-1 cpc-1 was compared with cpc-1 and atmyc1-1. All values
were significant (Student’s t-test, P≤0.007). The mean±s.d. is shown in each case (n=20).
DEVELOPMENT
Trichome number per leaf pair
Genotype
3460 RESEARCH ARTICLE
Development 140 (16)
Fig. 1. Trichome number rescue efficiency of complementation
constructs. Box-whisker plots of the trichome number per leaf pair
(leaves three and four) of 35S:GL3, 35S:EGL3 and 35S:AtMYC1
complementation constructs (n=50 T1 plants) in mutant backgrounds
(A) gl3-3 egl3-3 and (B) atmyc1-1 as compared with the mutant itself and
the wild type (Col) (n=30). The coding DNA sequences (CDSs) of the
different bHLHs were expressed under the CaMV 35S promoter. Boxes
represent the range of the middle 50% of the data. The median value is
indicated by the line in the box. The vertical lines (whiskers) indicate the
variability outside the boxes (upper line indicating the 75th percentile
and the lower line the 25th percentile). The results of all rescue
experiments are significantly different (P<0.0001, Wilcoxon signed-rank
test) except for 35S:GL3 gl3-3 egl3-3, which is not significantly different
compared with the Col background (P=0.3527).
AtMYC1 is localised predominantly in the
cytoplasm
We used transient expression assays to determine the intracellular
localisation of AtMYC1. YFP-AtMYC1 localisation was studied in
Agrobacterium-transfected tobacco cells. In contrast to YFP-GL3
(Fig. 3C), we found YFP-AtMYC1 fluorescence predominantly in
the cytoplasm (Fig. 3B,E). This was unexpected for a bona fide
transcription factor. We therefore studied the localisation behaviour
in more detail. First, we confirmed the integrity of the YFPAtMYC1 fusion protein in a western blot using a GFP antibody
(Fig. 3L). We then analysed the localisation of AtMYC1 in four
additional cell types: Arabidopsis protoplasts (Fig. 3H,I),
Arabidopsis cotyledons (Fig. 3F), onion cells (Fig. 3K) and leek
Nucleocytoplasmic shuttling is cross-regulated by
AtMYC1, GL1, TRY and CPC
Our finding that AtMYC1 localisation differs from that of its MYB
interaction partners raised the question of whether AtMYC1 can
recruit them from the nucleus into the cytoplasm or vice versa. We
tested this by co-expressing RFP-AtMYC1 and YFP-GL1 or YFPTRY in Arabidopsis cotyledon cells (Fig. 4). YFP-GL1 is localised
mainly in the nucleus. In cells co-expressing RFP-AtMYC1 and
YFP-GL1 we found YFP-GL1 in the cytoplasm (Fig. 4;
supplementary material Fig. S5). Surprisingly, co-expression of
RFP-AtMYC1 and YFP-TRY led to the localisation of RFPAtMYC1 in the nucleus (Fig. 4). This was confirmed by a
quantitative analysis using the YFP and RFP tags reciprocally to
exclude biases caused by the tags (Tables 6, 7). The quantification
revealed that the co-expression of TRY and AtMYC1 also caused an
accumulation of TRY in the nucleus. When expressed alone,
between 40% and 67.5% of the fluorescent protein fused to TRY
was found in the cytoplasm. When co-expressed with AtMYC1,
TRY protein was almost exclusively found in the nucleus (95.1%,
92.9%, see Tables 6 and 7). When combining RFP-AtMYC1 and
YFP-CPC, we found similar, although slightly less drastic,
localisation behaviours as with TRY (Table 7). The relocalisation
was also seen in leek cells, demonstrating that this recruitment is
not dependent on cell type (supplementary material Fig. S5).
Upon co-expression of AtMYC1, GL1 and TRY, we found all
three in the nucleus (supplementary material Fig. S5), suggesting
that TRY can counteract the AtMYC1-dependent recruitment of
GL1 into the cytoplasm.
To further corroborate these findings, we studied the localisation
of the GL1-AtMYC1, TRY-AtMYC1 and CPC-AtMYC1
interactions using BiFC assays. The TRY-AtMYC1 and CPCAtMYC1 interactions occurred predominantly in the nucleus,
whereas the GL1-AtMYC1 interaction took place in the cytoplasm
(supplementary material Fig. S6).
We used the nuclear transportation trap (NTT) assay (Ueki et al.,
1998) to independently test the nuclear transport behaviour of
AtMYC1 in a more stringent manner. In the NTT assay the protein
under consideration is expressed as a translational fusion to the
artificial transactivator LexAD (LexA DNA-binding domain
combined with GAL4AD transactivation domain) fused to a nuclear
export signal (NES) from the HIV Rev protein. The NES can trigger
the nuclear export of proteins lacking a nuclear localisation signal
(NLS). A functional NLS is sufficient to overcome the NESmediated nuclear export resulting in activation of the LEUCINE2
(LEU2) reporter gene. Using the NTT assay we found that
AtMYC1, in contrast to GL3 (Balkunde et al., 2011), could not
activate the LEU2 reporter, indicating that it does not contain a
functional NLS. Significantly, additional expression of TRY or CPC
led to the activation of LEU2, whereas co-expression of GL1 did
DEVELOPMENT
epidermal cells (supplementary material Fig. S5). In all tested cell
types, AtMYC1 was localised predominantly in the cytoplasm. By
contrast, GL3 was localised, as expected, in the nucleus (Fig. 3G,J).
This visual impression was confirmed by quantifying the percentage
of nuclear and cytoplasmic fluorescence (Tables 6, 7). To test
whether the localisation of AtMYC1 depends on the construction of
the fusion proteins, we also studied the C-terminal fusion of YFP to
AtMYC1 (AtMYC1-YFP). Again, we found a predominantly
cytoplasmic localisation (Fig. 3I). Finally, we excluded the
possibility that fusion to the YFP protein affects localisation by
showing that an N-terminal fusion with RFP (RFP-AtMYC1) is also
found predominantly in the cytoplasm (Fig. 4, Table 6).
Localisation of TRY and GL1 by AtMYC1
RESEARCH ARTICLE 3461
Fig. 2. Expression analysis of TRY, CPC and GL2 in
atmyc1-1 and 35S:AtMYC1 mutants. (A-I) Expression of
pGL2:GUS (A,D,G), pTRY:GUS (B,E,H) and pCPC:GUS (C,F,I) was
analysed in Col (A-C), atmyc1-1 (D-F) and 35S:AtMYC1 (G-I)
in 10-day-old plants. GUS staining was performed for
16 hours. Scale bars: 2 mm.
Genetic interactions of AtMYC1 and MYB factors
The reduction in trichome number in atmyc1-1 mutants and the
supernumerary trichomes in 35S:AtMYC1 lines indicate that
AtMYC1 is a positive regulator of trichome development. At the
molecular level, AtMYC1 modulates the behaviour of GL1 by
recruiting it from the nucleus to the cytoplasm, and AtMYC1 is
recruited by TRY or CPC to the nucleus. This raises the question of
whether the interaction with GL1 and/or TRY and CPC is
biologically relevant. To test this, we analysed the genetic
interactions between AtMYC1, GL1, CPC and TRY using
combinations of overexpression lines and mutants. The try atmyc1
double mutant showed an increased cluster frequency, indicating
that the atmyc1 mutant enhances the try cluster phenotype (Table 2).
Overexpression of AtMYC1 in try mutants caused an increase in
trichome number, but no change in cluster frequency as compared
with try mutants (Table 2). Thus, extra trichome initiation by
overexpressed AtMYC1 is independent of the presence of TRY. The
atmyc1 cpc double mutants showed essentially additive phenotypes.
Table 4. Relative mRNA expression levels of GL2, TRY and CPC
in Col, atmyc1-1 and 35S:AtMYC1 backgrounds quantified by
real-time PCR
This experiment was performed with cpc1-1 in the Ws background
(Table 3) and repeated with the cpc1-2 allele in the Col background
in order to exclude the possibility that a quantitative trait locus
(QTL) modifies the trichome patterning phenotype in the double
mutant (supplementary material Table S2). Overexpression of
AtMYC1 in cpc mutants caused a slight increase in cluster
frequency. Lines overexpressing both GL1 and AtMYC1 showed a
substantial increase in trichome number (Fig. 6C). Frequently, we
found large clusters in which the outermost trichomes appeared
smaller than at the centre, suggesting that they had been initiated
later in leaf development (Fig. 6E). Overexpression of AtMYC1
has a similar genetic effect as the absence of TRY in cpc try and
35S:GL1 try mutant lines (Schnittger et al., 1998; Schellmann et al.,
2002), suggesting that AtMYC1 is negative regulator of TRY.
Targeting AtMYC1 into and out of the nucleus by
NLS and NES fusions
In order to test whether the intracellular localisation of AtMYC1 is
biologically relevant, we created N-terminal fusions with an NLS
(Kalderon et al., 1984) and with an NES (Wen et al., 1995;
Matsushita et al., 2003). We first tested the intracellular localisation
of the respective YFP-tagged fusion proteins by particle
bombardment in leek cells. As expected, YFP-NES-AtMYC1 was
Table 5. LUMIER assays to analyse the interaction of GL3 and
AtMYC1 with different patterning proteins
Luciferase activity: pulldown/input ratio (%)
Relative mRNA levels
Genotype
Col
atmyc1-1
35S:AtMYC1
GL2
TRY
CPC
1.00±0.00
0.44±0.06*
1.01±0.34
1.00±0.00
1.00±0.53
0.98±0.13
1.00±0.00
0.71±0.22‡
1.05±0.35
Expression levels in atmyc1-1 and 35S:AtMYC1 were normalised to wild-type (Col)
expression. ACTIN expression was used as an endogenous control. The standard
deviation of the three biological replicates is shown (n=3).
*GL2 expression was significantly reduced in atmyc1-1 mutants (Student’s t-test,
P<0.0004).
‡
CPC expression was significantly reduced in atmyc1-1 mutants (Student’s t-test,
P<0.04).
ProtA-GL1
ProtA-TRY
ProtA-CPC
ProtA-TTG1
ProtA*
Luc-GL3
Luc-AtMYC1
Luc*
49.7±13.1
52.2±5.2
47.2±4.0
10.6±1.5
0.4±0.1
15.4±0.8
64.4±13.2
33.4±15.8
19.9±4.3
0.4±0.0
0.0±0.0
0.1±0.0
0.1±0.0
0.1±0.0
0.1±0.0
Interaction was assessed using GL3 and AtMYC1 Renilla reniformis luciferase (Luc)
fusions and protein A (ProtA) fusions of the patterning proteins. The proteins were
co-expressed in human cells (HEK293TN) and co-immunoprecipitated with IgG
Dynabeads.
Data are mean±s.d. (n=3).
*Empty vector without CDS fusion.
DEVELOPMENT
not activate the reporter. These findings confirm the results of our
transient assays and support the conclusion that TRY and CPC can
trigger nuclear transport whereas GL1 cannot direct AtMYC1 into
the nucleus (Fig. 5).
Development 140 (16)
3462 RESEARCH ARTICLE
Fig. 3. Intracellular localisation of YFP-tagged AtMYC1.
(A-D) Confocal images of tobacco (Nicotiana benthamiana)
infiltrated leaves. Each combination was repeated five times with
more than 100 cells in each experiment and all showed the
intracellular localisation patterns represented here. (A) 35S:YFP, (B)
35S:YFP-AtMYC1, (C) 35S:YFP-EGL3, (D) 35S:YFP-GL3. (E) Higher
magnification of the nuclear region of B. (F,G) Transiently
transformed Arabidopsis thaliana cotyledon epidermis cells. More
than 25 cells in four independent experiments exhibited the
intracellular localisation patterns shown here. (F) 35S:YFP-AtMYC1,
(G) 35S:YFP-GL3. (H-J) Fluorescence microscopy images of transiently
transformed Arabidopsis protoplasts. More than 100 protoplasts
were analysed in three independent experiments with invariable
intracellular localisation patterns in all cases. (H) 35S:YFP-AtMYC1, (I)
35S:AtMYC1-YFP, (J) 35S:YFP-GL3. (K) 35S:YFP-AtMYC1 in an onion cell.
(L) Immunoblot with anti-GFP antibody detection of crude protein
extracts from the tobacco leaf samples, with YFP alone and
untransformed (WT) tobacco leaves as a negative control.
(M) Immunoblot with anti-GFP antibody detection of crude protein
extracts from tobacco leaf samples, with YFP alone and
untransformed tobacco leaves as a control.
Table 6. The percentage of nuclear localised RFP-AtMYC1, YFPGL1 and YFP-TRY depends on co-expression partners
Nuclear localised protein (%)
Co-bombarded constructs
RFP-AtMYC1
RFP-AtMYC1
RFP-AtMYC1
RFP*
RFP*
GFP-YFP
YFP-GL1Δ27
YFP-TRY
YFP-GL1Δ27
YFP-TRY
Data are mean±s.d. (n=3).
*Empty vector without CDS fusion.
RFP
YFP
n
4.6±2.7
8.4±7.3
90.3±4.8
7.8±3.5
4.6±2.7
10.6±5.1
9.6±4.4
95.1±2.8
93.0±5.4
67.5±25.6
18
7
15
8
11
atmyc1-1 lines we found an average of 151.3±92 trichomes on the
first four leaves (n=50 T1 lines). Compared with 35S:YFP-AtMYC1,
the 35S:YFP-NES-AtMYC1 construct exhibited significantly less
efficient rescue of atmyc1-1 (112.1±44.5; n=51 T1 lines; Wilcoxon
signed-rank test, P=0.02). Conversely, the 35S:YFP-NLS-AtMYC1
construct rescued the atmyc1-1 mutant significantly better
(176.5±81.5; n=50 T1 lines; Wilcoxon signed-rank test, P=0.005).
Together, these data show that changing the intracellular localisation
of AtMYC1 changes its biological activity.
DISCUSSION
AtMYC1 is one of the genes regulating trichome and root hair
patterning in Arabidopsis thaliana. It belongs to the large family of
Table 7. The percentage of nuclear localised YFP-AtMYC1, RFPGL1 and RFP-TRY depends on co-expression partners
Nuclear localised protein (%)
Co-bombarded constructs
YFP-AtMYC1
YFP-AtMYC1
YFP-AtMYC1
YFP-AtMYC1
YFP-AtMYC1
YFP*
YFP*
YFP*
YFP*
YFP*
RFP*
RFP-GL1
RFP-GL1Δ27
RFP-TRY
RFP-CPC
RFP*
RFP-GL1
RFP-GL1Δ27
RFP-TRY
RFP-CPC
Data are mean±s.d. (n=3).
*Empty vector fusion.
YFP
RFP
n
6.7±1.8
6.5±3.5
5.3±1.8
97.4±8.9
87.1±20.6
25.0±3.5
30.7±8.7
24.9±5.0
19.5±3.7
29.3±6.1
15.2±3.5
7.3±3.2
9.6±5.3
92.9±8.8
72.7±19.5
25.9±4.3
96.4±3.2
55.4±10.2
40.0±8.8
37.2±6.8
34
25
21
21
23
32
22
27
20
24
DEVELOPMENT
found in the cytoplasm and YFP-NLS-AtMYC1 in the nucleus
(Fig. 7). YFP-NLS-AtMYC1 and RFP-TRY co-expression resulted
in an accumulation of both fusion proteins in the nucleus (Fig. 7).
In cells co-expressing YFP-NES-AtMYC1 and RFP-TRY we found
the RFP signal in the nucleus and the cytoplasm, rather than
predominantly in the nucleus (Fig. 7). The co-expression of YFPNLS-AtMYC1 or YFP-NES-AtMYC1 with CFP-GL1 led to an
accumulation of CFP in the nucleus or cytoplasm, respectively
(Fig. 7). Together, these data indicate that the mislocalisation of
AtMYC1 triggers a change in the intracellular localisation of TRY
and GL1.
In a second set of experiments, we constitutively expressed YFPAtMYC1, YFP-NES-AtMYC1 and YFP-NLS-AtMYC1 in atmyc1
mutants and assessed the rescue of trichome number. As individual
transformants may show a wide range of phenotypes, we studied at
least 50 T1 plants (Pesch and Hülskamp, 2011) in order to catch the
full phenotypic spectrum for our comparisons. In 35S:YFP-AtMYC1
Localisation of TRY and GL1 by AtMYC1
RESEARCH ARTICLE 3463
Fig. 5. Yeast nuclear transportation trap (NTT) assay to study the
nuclear transport of AtMYC1. NESLexAD alone and N-terminal fusions
to AtMYC1 and GL3 were co-expressed with various untagged patterning
proteins as indicated above each column. (A) NESLexAD and the
untagged protein constructs were selected on medium lacking histidine
and uracil, respectively. (B) The entry of NESLexAD into the nucleus leads
to reporter gene expression (LEU2), which enables the growth on
medium lacking leucine. 1, empty control vector without CDS.
bHLH factors in Arabidopsis, which contains many key regulators
of plant development. A phylogenetic analysis of the bHLH domains
of 133 bHLH proteins placed AtMYC1 in subgroup IIIf (Heim et
al., 2003). In addition to AtMYC1, this subgroup contains three
further members, GL3, EGL3 and TT8, which are all involved in
TTG1-dependent pathways related to trichome, root hair and seed
coat development and anthocyanin production (Broun, 2005; Koes
et al., 2005; Ramsay and Glover, 2005; Lepiniec et al., 2006; Serna
and Martin, 2006; Balkunde et al., 2010; Feller et al., 2011;
Tominaga-Wada et al., 2011). In support of this phylogenetic
classification, several observations have suggested that AtMYC1
acts redundantly with GL3 and EGL3 in the root hair patterning
system (Bruex et al., 2012) and in the context of trichome patterning
(Zhao et al., 2012). In addition, AtMYC1 shows interactions with
other proteins, similar to those reported for GL3, EGL3 and TT8, in
the yeast two-hybrid assay (Zimmermann et al., 2004; Tominaga et
al., 2008; Zhao et al., 2012) and in pulldown experiments (this
study). However, two lines of genetic evidence indicate that the
molecular function of AtMYC1 differs from that of GL3, EGL3 and
TT8. First, in contrast to the gl3 egl3 double mutant the atmyc1 gl3
or atmyc1 egl3 double mutants show an additive phenotype but not
an enhancement of the single-mutant phenotypes of gl3 or egl3.
Second, the expression of AtMYC1 under the control of the GL3 or
EGL3 promoter (Zhao et al., 2012) or the 35S promoter (this study)
cannot rescue the gl3 egl3 mutant phenotype. A divergent function
of AtMYC1 is consistent with two phylogenetic studies that analysed
147 bHLH factors in Arabidopsis or bHLH factors in rice and
Genetic interpretation of the nucleocytoplasmic
shuttling of AtMYC1, GL1 and TRY/CPC
Our observations of the quantitative relocalisation of AtMYC1,
TRY/CPC and GL1 are based on the overexpression of the
respective proteins. Therefore, we do not know the relative
distribution of these proteins under native conditions. Our attempts
to study this in planta have thus far failed because AtMYC1, TRY
or GL1 fusions with YFP or RFP were below the detection limit
when expressed under the endogenous promoters. Nevertheless, it
is evident that the four proteins have the property to balance each
DEVELOPMENT
Fig. 4. Subcellular colocalisation of the AtMYC1 and MYB
transcription factors in Arabidopsis cotyledons. For each combination,
the RFP (left) and YFP (middle) channels and a merge (right) are shown.
Arabidopsis leaves were transformed biolistically and analysed by confocal
microscopy. Scale bars: 50 μm.
Arabidopsis (Toledo-Ortiz et al., 2003; Li et al., 2006). These studies
placed AtMYC1 in a clade distinct from GL3, EGL3 and TT8.
A possible molecular function can be deduced from the
intracellular localisation of AtMYC1 protein. In contrast to a
previous study (Zhao et al., 2012), we found AtMYC1
predominantly in the cytoplasm. As the AtMYC1 CDS was
successfully used for the rescue experiments and shows the
expected protein-protein interactions in yeast and in pulldown
assays, the AtMYC1 sequence used in this study is fully functional.
AtMYC1 was consistently found in the cytoplasm of cells from four
different plant species (tobacco, onion, leek and Arabidopsis) with
N- and C-terminal YFP fusions and with an RFP fusion. We
confirmed our results with the yeast NTT system, which is
specifically designed to test the nuclear transport of proteins. Thus,
our results consistently show that AtMYC1 predominantly localises
in the cytoplasm. Mislocalisation of AtMYC1 protein, either to the
cytoplasm or to the nucleus, showed that the correct localisation of
the protein is important for its regulatory function. It is interesting
to note that AtMYC1 contains a monopartite NLS sequence at its Nterminus that is predicted to target the protein predominantly to the
nucleus with a similar efficiency as that predicted for GL3
(supplementary material Table S4). This suggests that the AtMYC1
NLS sequence is masked under normal conditions either by protein
folding or binding to additional factors, which are likely to be
general factors as the cytoplasmic localisation was observed in
various species and cell types.
3464 RESEARCH ARTICLE
Development 140 (16)
Fig. 6. 35S:AtMYC1 enhances the 35S:GL1 phenotype.
(A-C) Rosette leaves of (A) 35S:GL1, (B) atmyc1-1 35S:GL1, (C)
35S:AtMYC1 35S:GL1. (D,E) SEM of trichomes on wild-type Col
(D) and 35S:AtMYC1 35S:GL1 (E) leaves. Scale bars: 1 mm in AC; 200 μm in D,E.
with an increased trichome number in 35S:AtMYC1 lines. A
repression of TRY activity by AtMYC1 could explain the
substantial increase in trichome number and the slightly increased
production of trichome clusters in 35S:AtMYC1 cpc and the large
trichome clusters in 35S:AtMYC1 35S:GL1 lines because this
phenotype is reminiscent of the 35S:GL1 try phenotype (Schnittger
et al., 1998; Szymanski and Marks, 1998). Also, the decrease in
trichome number by 50% compared with the wild type in the
atmyc1 mutant can be interpreted as being caused by enhanced
activity of CPC or TRY. The recruitment of GL1 by AtMYC1 from
the nucleus into the cytoplasm is likely to impair the activity of GL1.
Fig. 7. Subcellular colocalisation of GL1 or TRY
with NLS or NES fusions of AtMYC1. Single leek
cells expressing the respective fusion proteins
after transformation by particle bombardment. For
each combination, the YFP (left), CFP (middle) and
RFP (right) channels are shown. Scale bars: 50 μm.
DEVELOPMENT
other’s localisation. Given that GL1 and TRY/CPC are involved in
the transcriptional regulation of other genes, it is likely that their
relocalisation changes their activity: the recruitment of AtMYC1
by TRY/CPC and concomitant accumulation of TRY/CPC in the
nucleus reflects the fact that AtMYC1 binding to TRY/CPC can
modulate the balance between nuclear and cytoplasmic TRY/CPC
protein. The in vivo net effect is likely to be a suppression of both
TRY and CPC activity. This is suggested by several genetic
observations. If AtMYC1 represses TRY and/or CPC one would
expect lines overexpressing AtMYC1 to share phenotypic aspects
of try and cpc mutants. A repression of CPC activity is consistent
Localisation of TRY and GL1 by AtMYC1
RESEARCH ARTICLE 3465
Acknowledgements
We thank Birgit Kernebeck for excellent technical assistance; Irene
Klinkhammer for cell suspension culture work; Hans-Peter Bollhagen (AG
Büschges, Zoological Institute, University of Cologne) for help with SEM;
Sigfried Werth for photographic documentation; and L. Leson for the
installation of the magnetic holder. Vectors were kindly provided as follows:
pAM-PAT-GW by B. Ülker (MPIZ, Cologne); pET3A–Ubi-PrA by M. Koegl
(German Cancer Research Center, Heidelberg); pBluescript-GW-RekA by S.
Bierei (MPIZ, Cologne); TRY, CPC, GL3, TTG1 entry clones by U. Herrmann
(Department of Biosciences, Oslo, Norway); EGL3 and AtMYC1 entry clone by
I. Zimmermann; pENTR1A-w/o-ccdB by Arp Schnittger (Institut de Biologie
Moléculaire des Plantes du CNRS, Strasbourg, France); and TTG1Δ26 entry
clone by Rachappa Balkunde (Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY, USA).
Funding
This work was funded by the SFB572 from the Deutsche
Forschungsgemeinschaft (DFG).
However, such a repressive role of AtMYC1 on GL1 is not
consistent with our genetic finding that the reduced trichome
number phenotype of 35S:GL1 plants is rescued in a 35S:AtMYC1
background. This suggests that the impact of AtMYC1 on the
localisation of GL1 has little biological relevance or that the impact
on TRY/CPC overwrites this effect. The latter is consistent with the
observation that the relocalisation of GL1 by AtMYC1 is
counteracted in the presence of TRY protein.
Integrating AtMYC1 into the patterning gene
network
How is AtMYC1 integrated in the gene regulatory network? A putative
repressive function of AtMYC1 on TRY/CPC is difficult to
functionally integrate into existing models. One attractive possibility
for its function can be inferred from the finding that the AtMYC1
promoter is a direct target of the GL1-GL3/EGL-TTG1 activator
complex (Morohashi and Grotewold, 2009). This suggests that the
activator complex, and therefore also GL1, might activate the
expression of AtMYC1 as well as of TRY/CPC. On top of this
regulation at the transcriptional level we propose that the protein
activities of TRY/CPC and GL1 are coupled through AtMYC1. As
AtMYC1 can recruit GL1 from the nucleus into the cytoplasm and
TRY/CPC from the cytoplasm into the nucleus the regulatory effect
should be antagonistic (Fig. 8). In our model, the concentration of
GL1 in the nucleus is reduced in the presence of AtMYC1, whereas
the concentration of TRY in the nucleus is increased. It is possible
that, in addition to this cell-autonomous effect, a reduced level of
cytoplasmic TRY/CPC would lead to reduced lateral movement and
therefore to less repression in the neighbouring cells. Consistent with
this scenario, the distance between trichomes is markedly increased
in atmyc1 mutants as compared with wild type. This type of negativefeedback loop could serve not only to fine-tune the network but would
also provide an additional mechanism to modulate lateral repression.
Competing interests statement
The authors declare no competing financial interests.
Author contributions
M.P., I.S. and S.D. designed and performed experiments; J.F.U. designed
experiments; and M.H. designed experiments and wrote the manuscript.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.094698/-/DC1
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