RESEARCH ARTICLE 1335
Development 135, 1335-1345 (2008) doi:10.1242/dev.017947
Arabidopsis CAPRICE-LIKE MYB 3 (CPL3) controls
endoreduplication and flowering development in addition to
trichome and root hair formation
Rumi Tominaga, Mineko Iwata, Ryosuke Sano, Kayoko Inoue, Kiyotaka Okada* and Takuji Wada†
CAPRICE (CPC) encodes a small protein with an R3 MYB motif and promotes root hair cell differentiation in Arabidopsis thaliana.
Three additional CPC-like MYB genes, TRY (TRIPTYCHON), ETC1 (ENHANCER OF TRY AND CPC 1) and ETC2 (ENHANCER OF TRY AND
CPC 2) act in a redundant manner with CPC in trichome and root hair patterning. In this study, we identified an additional homolog,
CPC-LIKE MYB 3 (CPL3), which has high sequence similarity to CPC, TRY, ETC1 and ETC2. Overexpression of CPL3 results in the
suppression of trichomes and overproduction of root hairs, as has been observed for CPC, TRY, ETC1 and ETC2. Morphological
studies with double, triple and quadruple homolog mutants indicate that the CPL3 gene cooperatively regulates epidermal cell
differentiation with other CPC homologs. Promoter-GUS analyses indicate that CPL3 is specifically expressed in leaf epidermal cells,
including stomate guard cells. Notably, the CPL3 gene has pleiotropic effects on flowering development, epidermal cell size and
trichome branching through the regulation of endoreduplication.
KEY WORDS: Arabidopsis, Epidermis, MYB, Endoreduplication, Flowering
Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa
230-0045, Japan.
*Present address: National Institute for Basic Biology, 38 Nishigonaka, Myodaiji,
Okazaki, Aichi 444-8585, Japan
†
Author for correspondence (e-mail: [email protected])
Accepted 15 January 2008
ENHANCER OF TRY AND CPC 1, 2 and 3 (ETC1, ETC2 and
ETC3) (Schellmann et al., 2002; Kirik et al., 2004a; Kirik et al.,
2004b; Esch et al., 2004; Simon et al., 2007). The clustered trichome
phenotype of the try mutant indicates that TRY protein has a
regulatory role in trichome formation (Hulskamp et al., 1994;
Schellmann et al., 2002). ETC1 and ETC2 have been characterized
and their relationship with CPC and TRY genetically examined
(Kirik et al., 2004a; Kirik et al., 2004b; Esch et al., 2004). We have
recently identified a fourth CPC-like MYB, At4g01060,
independently of Simon et al. (Simon et al., 2007), and have named
it CPC-LIKE MYB 3 (CPL3).
Here, we examine the functions of the CPL3 gene in Arabidopsis.
CPL3 redundantly regulates root hair and trichome development
along with other CPC homologs. Notably, among the homologs,
only CPL3 has pleiotropic effects on flowering development and
epidermal cell size through the regulation of endoreduplication.
MATERIALS AND METHODS
Plant materials and growth conditions
The Arabidopsis thaliana Col-0 ecotype was used as wild type. The cpc2 mutant used in this study was described previously (Kurata et al., 2005).
The cpl3-1 mutant was isolated from a Wisconsin T-DNA population. We
backcrossed cpl3-1 to wild type and confirmed that all of the phenotypes
associated with cpl3-1 co-segregate with the T-DNA insertion. We
isolated the try-29760, etc1-1 and etc2-2 mutants from a SALK T-DNA
population. All mutants were in the Col-0 background. Double, triple and
quadruple mutants of cpc, try, etc1, etc2 and cpl3 were screened from F2
progeny using PCR to identify homozygous cpc-2, try-29760, etc1-1,
etc2-2 and cpl3-1 plants. Selected double, triple and quadruple mutants
were checked and documented in the F3 generation. The 35S::CPC and
CPCp::GUS transgenic lines were described previously (Wada et al.,
1997; Wada et al., 2002). Seeds were surface-sterilized and sown on 1.5%
agar plates as described previously (Okada and Shimura, 1990) and
grown out for observation of seedling phenotypes. Seeded plates were
kept at 4°C for 2 days and then incubated at 22°C under constant white
light. For each mutant line, at least ten individual 5-day-old seedlings
were assayed for root epidermis changes, and at least five 2-week-old
third leaves were assayed for trichomes. Plants were grown in soil at 22°C
under continuous light for determining flowering time, leaf size and
DEVELOPMENT
INTRODUCTION
The specification and patterning of cell types is a crucial feature of
development in multicellular organisms. In Arabidopsis thaliana,
the differentiation of epidermal cells has been used extensively as a
relatively simple model for analyzing cell fate specification. Several
types of epidermal cells are differentiated in Arabidopsis. For
example, root epidermal cells differentiate into either root hair cells
or hairless cells (Dolan et al., 1993). Leaf epidermal cells of
Arabidopsis can differentiate into trichomes, stomate guard cells and
pavement cells. Several regulatory factors are known to be involved
in this epidermal cell differentiation. For example, the glabra 2 (gl2)
and werewolf (wer) mutant phenotypes show conversion of hairless
cells to root hair cells (Masucci et al., 1996; Lee and Schiefelbein,
1999). GL2 encodes a homeodomain leucine-zipper protein, and
WER encodes an R2R3-type MYB protein that activates GL2
expression (Rerie et al., 1994; Di Cristina et al., 1996; Masucci et
al., 1996; Lee and Schiefelbein, 1999). GLABRA 3 (GL3) and
ENHANCER OF GLABRA 3 (EGL3) encode basic helix-loop-helix
(bHLH) proteins that affect hairless cell differentiation in a
redundant manner (Bernhardt et al., 2003). There are two other
bHLH genes, AtMYC1 (Urao et al., 1996) and TRANSPARENT
TESTA 8 (TT8) (Nesi et al., 2000), that are in the same subgroup as
GL3 and EGL3 (Heim, 2003).
CAPRICE (CPC) encodes a small protein with an R3 MYB motif,
and promotes root hair cell differentiation in Arabidopsis (Wada et
al., 1997). CPC protein moves from hairless cells to neighboring
hair-forming cells and represses expression of GL2 (Wada et al.,
2002). Arabidopsis has several additional CPC-like MYB sequences
in the Arabidopsis genome, including TRIPTYCHON (TRY),
1336 RESEARCH ARTICLE
Development 135 (7)
Gene constructs
Primers
Primer sequences are listed in Table 1.
35S::CPL3 construct
A 0.8 kb PCR-amplified linear CPL3 genomic fragment (primers
TW1167/TW1168) was subcloned into pBluescript SK+ (pBS; Stratagene)
using Pyrobest DNA polymerase (Takara, Tokyo, Japan) to make pBSCPL3. Next, the Acc65I to SalI fragment of pBS-CPL3 was ligated into the
Acc65I and SalI sites of the pCHF3 binary vector (Jarvis et al., 1998) to
create 35S::CPL3. PCR-generated constructs were completely sequenced
following isolation of the clones to check for amplification-induced errors.
Finally, the amplified and ligated constructs were cloned into
transformation vector pJHA212K (Yoo et al., 2005).
CPL3p::CPL3:GFP constructs
A 3.0 kb PCR-amplified linear CPL3 genomic fragment (primers
RT71/RT72) was digested with SalI and EcoRV and ligated into the SalI
and EcoRV sites of pBS-2xGFP (Kurata et al., 2005) to create pBSCPL3:2xGFP. PCR-generated constructs were completely sequenced
following isolation of the clones to check for amplification-induced errors.
Finally, the SalI to SacI fragment of pBS-CPL3:2xGFP was ligated into the
SalI and SacI sites of the pJHA212K binary vector (Yoo et al., 2005) to
create CPL3p::CPL3:GFP.
Table 1. Primers used in this study
Primer
TW1167
TW1168
RT46
RT47
RT48
RT49
RT50
RT51
RT71
RT72
RT88
RT89
RT126
RT127
EF1␣-F
EF1␣-R
GUS+00+
GUS+09–
CycA2;1-F
CycA2;1-R
CycA2;2-F
CycA2;2-R
CycA2;3-F
CycA2;3-R
CycA2;4-F
CycA2;4-R
CycA1;1-F
CycA1;1-R
Act2-F
Act2-R
SIM-F
SIM-R
CO-F
CO-R
FT-F
FT-R
SOC1-F
SOC1-R
Sequence (5⬘ to 3⬘)
ATATGGTACCTTTAACATAGAAACCGAC
ATATGTCGACATCTACGACTTAGCTTC
GGCCAGTCGACAGAAAACTCACTCACTATTCACATC
CGAGGATCCACGCTGCGTATTCATCTCAA
GGCCAGTCGACGCTTGGCTAGCTCATAAACG
CGATCTAGAACGGTTGGTATTATCCATAACTACT
GGCCAGTCGACCAGCCCTGAAAACAGCTAAGAA
CGAGGATCCGCGATGGTTATCCATGTCAAAC
ATATGTCGACCAGCCCTGAAAACAGCTAAGAA
ATATGATATCATTTTTCATGACCCAAAACCTCT
ATATGGATCCACGGTCAGTGTTATCCATTACTATT
ATATGTCGACCTCAATATATCAAATTCAAACATTCA
GATAACCATCGCAGGACTAAGC
TACAACGGAATATAATCGAAACAATC
ATGCCCCAGGACATCGTGATTTCAT
TTGGCGGCACCCTTAGCTGGATCA
ATGTTACGTCCTGTAGAAACCCCAA
CGTGCACCATCAGCACGTTAT
CGCTTCAGCGGTTTTCTTAG
ATCCTCCATTGCAAGTACCG
TGTATGTGTTGGCCGTAATG
TGGTGTCTCTTGCATGCTTA
CTCTATGCCCCTGAAATCCA
ACCTCCACAAGCAATCAAC
CAAAGCCTCCGATCTCAAAG
CTTGTCCGGTAGCTCTCCAG
CGATGACGAAGAAACGAGCA
TGGCATTAACGCAAACACTTG
CTGGATCGGTGGTTCCATTC
CCTGGACCTGCCTCATCATAC
TTCCGACCACAAGATTCCTC
AGAAGAACCGCTCGATCTCA
TGGCTCCTCAGGGACTCACTACAA
TTGACTCCGGCACAACACCAGT
GATACGAGTAACGAACGGTGAT
CCCCCTCTCATTTTTATTACAC
ATGAATTCGCCAGCTCCAAT
GCTTCATATTTCAAATGCTGCA
CPL3p::GUS constructs
A 2.4 kb PCR-amplified promoter region of CPL3 (primers RT50/RT51)
was digested with NotI and AccI and subcloned into pBS to create pBSCPL3p. PCR-generated constructs were completely sequenced following
isolation of the clones to check for amplification-induced errors. The SalI
and BamHI-digested fragment of pBS-CPL3p was ligated into the SalI and
BamHI sites of binary vector pBI101 (Clontech Laboratories, CA) to create
the CPL3p::GUS construct.
Promoter::GUS constructs
A 1.9 kb PCR-amplified promoter region of ETC1 (primers RT46/RT47),
a 3.0 kb PCR-amplified promoter region of ETC2 (primers RT48/RT49)
and a 3.0 kb PCR-amplified promoter region of TRY (primers RT88/RT89),
were digested with NotI and AccI and subcloned into pBS to create pBSETC1, -ETC2 and -TRY. The SalI and BamHI-digested fragment of pBSETC1 was ligated into the SalI and BamHI sites of binary vector pBI101
(Clontech Laboratories) to create ETC1p::GUS. The SalI and XbaIdigested fragments of pBS-ETC2 and pBS-TRY were ligated into SalI and
XbaI of binary vector pBI101 to create ETC2p::GUS and TRYp::GUS
constructs. At least three T3 lines were isolated on the basis of their
segregation ratios for kanamycin resistance for each transgenic line.
Transgenic plants
Plant transformation was performed by a floral dip method (Clough and Bent,
1998), and transformants were selected on a 0.5⫻MS agar plates containing
50 mg/l kanamycin. Homozygous transgenic lines were selected by
kanamycin resistance. We isolated at least twelve T1 lines for each construct
and selected at least six T2 and three T3 lines on the basis of their segregation
ratios for kanamycin resistance. For each transgenic line, at least ten individual
5-day-old seedlings were assayed for root hair numbers, and at least five 2week-old third leaves were assayed for trichome numbers. Promoter::GUS
transgenic lines were analyzed by PCR (primers GUS+00+/GUS+09–). At
least three individual plants were assayed for GUS activity in each of the three
transgenic lines. The CPL3p::GUS construct was introduced into the cpc-2,
try-29760, etc1-1, etc2-1 and cpl3-1 mutants by conventional crosses and F2
seedlings were analyzed by PCR. At least five plants from each transgenic line
were assayed for GUS activity.
Histology
Promoter::GUS plants were excised from the growth medium and
immersed in X-Gluc solution containing 1.0 mM X-Gluc (5-bromo-4chloro-3-indolyl-␤-glucuronide), 1.0 mM K3Fe(CN)6, 1.0 mM K4Fe(CN)6,
100 mM NaPi (pH 7.0), 100 mM EDTA and 0.1% Triton X-100. Primary
roots of 5-day-old seedlings were incubated at 37°C overnight. Cotyledons
of 5-day-old seedlings, 2-week-old rosette leaves and hypocotyls, and 4week-old inflorescences and siliques were incubated at 37°C for 3.5 hours.
In situ hybridization
In situ hybridization was as described (Kurata et al., 2003). DIG-labeled
antisense RNA probes for CPC, ETC1, ETC2 and CPL3 were generated by
transcribing pBS-cDNA (pBS-CPC, -ETC1, -ETC2 and -CPL3) digested
with HindIII, SpeI, XhoI and SpeI, respectively. T3 polymerase was used
for CPC, ETC1 and CPL3 probes, T7 polymerase for the ETC2 probe.
Semi-quantitative RT-PCR
RNA extraction and semi-quantitative RT-PCR reactions were as described
(Kurata et al., 2003). The CPL3 fragment was amplified with the
RT73/RT92 primer pair. EF (At1g07930) was amplified with the EF1␣F/EF1␣-R primer pair as described (Kurata et al., 2005).
Real-time PCR
Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen). Oncolumn DNase I digestion was performed during RNA purification following
the protocol described in the RNeasy Mini Kit handbook. First-strand cDNA
was synthesized from 1 ␮g total RNA in a 20 ␮l reaction mixture using the
Prime Script RT Regent Kit (Takara). Real-time PCR was performed in a
Chromo4 Real-Time PCR Detection System (Bio-Rad, Hercules, CA) using
SYBR Premix Ex Taq (Takara). PCR amplification employed a 30 second
denaturing step at 95°C, followed by 5 seconds at 95°C and 30 seconds at
60°C with 45 cycles for CPL3 and 40 cycles for CYCA2;1, CYCA2;2,
DEVELOPMENT
ploidy. For flowering-related gene expression analyses (CO, FT and
SOC1), plants were grown in soil under long-day conditions (16 hours
light/8 hours dark).
CPL3 gene controls ploidy and flowering
RESEARCH ARTICLE 1337
Fig. 1. Gene structure and amino acid
sequences of Arabidopsis CPC
homologs.
(A) Sequence alignment of CPChomologous MYB proteins (CPL3, CPC, TRY,
ETC1, ETC2, Os01g43180 and
Os01g43230). Red outlined letters indicate
identical residues. (B) Phylogenetic tree
based on the amino acid sequences.
Numbers above branches are genetic
distances based on 10,000 bootstrap
replicates. The tree was obtained by the
neighbor-joining method using Genetyx ver.
11.2.7 software (Genetyx, Tokyo, Japan).
(C) Structure of CPC-homologous genes and
positions of mutations. The locations of
start and stop codons are indicated. Three
exon (boxes) and two intron (lines) positions
were determined by comparing the
genomic sequences with the cDNA
sequences. Positions of T-DNA insertions
and the identity of mutations are indicated
(cpc-2, try-029760, etc1-1, etc2-2 and cpl31). (D) Semi-quantitative RT-PCR analysis of
CPL3. EF (At1g07930) was used as a
control.
Ploidy analysis
Nuclei were extracted and stained with CyStain UV Precise P (Partec,
Münster, Germany) following the manufacturer’s protocol. Flow
cytometric analysis was performed by a Ploidy Analyzer PA flow cytometer
(Partec), according to the manufacturer’s instructions.
Microscopy
Light microscopy
Root phenotypes were observed using an Olympus Provis AX70
microscope and an Olympus SZH binocular microscope. For each mutant
or transgenic line, at least ten individual 5-day-old seedlings were analyzed
for root hair number and root GUS activity. For the observation of
trichomes, images were recorded with a VC4500 3D digital fine
microscope (Omron, Kyoto, Japan) or digital microscope (VH-8000;
Keyence, Osaka, Japan). At least five 2-week-old third true leaves were
analyzed for trichome number and GUS activity for each mutant or
transgenic line. For measurement of epidermal cell numbers, five 2-weekold third leaves were cleared with chloral hydrate:glycerol:water (8:2:1,
w:v:v) (Yadegari et al., 1994), and visualized using a Zeiss Axio-plan2
microscope (Carl Zeiss, Germany).
Confocal laser scanning microscopy (CLSM)
CPL3p::CPL3:GFP transgenic lines were stained with 5 ␮g/ml propidium
iodide (PI) for 30 seconds and mounted in water. Confocal images were
obtained with a 40⫻ water-immersion objective on a Zeiss LSM-Pascal or
a Zeiss LSM-510 Meta confocal laser scanning microscope using 488 nm
laser lines for GFP excitation. Image processing was with Photoshop
version 7.0 (Adobe Systems, CA).
Scanning electron microscopy (SEM)
To observe the phenotype of trichomes, rosette leaves or inflorescences
were attached to the stage and cooled in liquid nitrogen. Observations were
made in low vacuum with a scanning electron microscope (model
JSM5610-LV; JEOL, Akishima, Japan).
DEVELOPMENT
CYCA2;3, CYCA2;4, CYCA1;1, SIM, CO, FT, SOC1 and ACT2. Relative
mRNA levels were calculated by iQ5 software (Bio-Rad), and normalized to
the concentration of ACT2 mRNA. The primers were: CYCA2;1-F and
CYCA2;1-R for CYCA2;1; CYCA2;2-F and CYCA2;2-R for CYCA2;2;
CYCA2;3-F and CYCA2;3-R for CYCA2;3; CYCA2;4-F and CYCA2;4-R for
CYCA2;4; CYCA1;1-F and CYCA1;1-R for CYCA1;1; SIM-F and SIM-R for
SIM; CO-F and CO-R for CO; FT-F and FT-R for FT; SOC1-F and SOC1-R
for SOC1; and ACT2-F and ACT2-R for ACT2 (Czechowski et al., 2004;
Huang et al., 2005; Yoshizumi et al., 2006).
ANOVA (analysis of variance) was performed to determine the significance
of differences between cpl3, 35S::CPL3 or CPL3p::CPL3, and wild type.
1338 RESEARCH ARTICLE
Development 135 (7)
Yeast two-hybrid assay
Vectors and yeast strains were obtained from Clontech (Mountain View,
CA; MATCHMAKER Two-Hybrid System). CPC, TRY, ETC1, ETC2 and
CPL3 full-length proteins were fused to the GAL4 DNA-binding domain
in pGBT9. GL3, EGL3, AtMYC1 and TT8 full-length proteins were fused
with the GAL4 activation domain in pGAD424. Yeast strain Y187 was
transformed with the appropriate plasmids using carrier DNA and the
lithium acetate method (Kallal and Kurjan, 1997). Following the Yeast
Protocols Handbook (Clontech), a ␤-galactosidase assay was performed on
each transformant using O-nitrophenyl ␤-D-galactopyranoside (Sigma) as
substrate.
Epidermis phenotypes of CPL3 loss-of-function
mutants
The root hair phenotype of the cpc-1 (WS background) (Wada et al.,
1997) and cpc-2 (Col-0 background) mutant lines is characterized
by the formation of approximately one-fourth as many root hairs as
the wild type (see Fig. S1A in the supplementary material),
respectively. In the cpl3-1 line, the relative number of root hairs was
about 80% that of wild type (Fig. 2B, Table 2). There is a distinct
possibility that functional redundancy provided by the presence of
similar genes obscures the phenotype of each individual knockout
mutant. Therefore, we made cpl3 double mutants with each of the
other CPC-like MYB mutants (Table 2). It had already been reported
that the cpc-1 try-82 and cpc-1 etc1-1 double mutants have very few
root hairs (Schellmann et al., 2002; Kirik et al., 2004b). We
confirmed these observations using the cpc-2, try-29760 and etc1-1
alleles (see Fig. S1A in the supplementary material). Root hair
production in the cpl3 cpc double mutant was about 50% of that in
the cpc single mutant (see Fig. S1A in the supplementary material).
However, root hair production in cpl3 try, cpl3 etc1 and cpl3 etc2
double mutants was not significantly different to that in wild type
(Table 2).
Trichome formation in the double mutants is more complicated
than root hair formation because of the three developmental aspects
of trichome formation: number, clustering and branching. There
Fig. 2. Phenotypes associated with CPC-homolog gain- and lossof-function mutants. (A-L) Root hair formation of a 5-day-old
Arabidopsis seedling showing no root hair phenotype (G,H), reduced
number of root hairs (B,C) and increased number of root hairs (I-L).
(M-X) Trichome formation on the 2-week-old third leaves showing
increased number of trichomes (N-T). No trichome formation was
observed in gain-of-function plants (U-X). Scale bars: 100 ␮m in A for
A-L; 1 mm in M-X.
Table 2. Phenotypes of root epidermal cells
Length of epidermal
cell (␮m)
Number of root
hairs per mm
Relative
hair number*
Col-0
180±10
41.3±4.3
74.2
cpl3
cpl3 cpc
cpl3 try
cpl3 etc1
cpl3 etc2
192±5
182±7
183±9
189±7
193±18
31.1±1.1
6.0±0.8
43.6±0.8
41.5±0.8
38.4±1.5
59.7
10.9
79.8
78.4
74.1
35S::CPL3#1
35S::CPL3#2
CPL3p::CPL3#1
CPL3p::CPL3#2
156±6
151±12
171±14
166±13
57.2±3.9
65.4±4.1
46.8±1.1
50.9±1.4
89.2
98.8
80.0
84.5
Genotype
Data, including s.d., were obtained from at least ten 5-day-old roots from each line.
*Relative hair number indicates the number of root hairs formed on a segment of
root with average length epidermal cells calculated from [length of epidermal cell
(␮m) ⫻ number of root hairs per mm]/100.
DEVELOPMENT
RESULTS
Identification of the CPL3 gene
A search of the Arabidopsis genome sequence revealed four MYB
gene sequences with high homology to CPC: TRY, ETC1, ETC2
and CPL3. The CPL3-encoded protein is closely related to the
proteins encoded by CPC-like MYB family members CPC, TRY,
ETC1 and ETC2, and two rice (Oryza sativa) homologs,
Os01g43180 and Os01g43230 (Fig. 1A). A phylogenetic tree based
on the alignment of amino acid sequences with the R2R3-type
MYB family members WER, GL1 and MYB23, indicated that the
two rice homologs are more closely related to the CPC-like MYB
family than to the R2R3-type MYB family, and that CPL3 is more
closely related to ETC1 than to the other CPC-homologous proteins
(Fig. 1B).
To assess the function of the CPL3 gene, we identified a T-DNA
mutant allele, cpl3-1, in the Wisconsin T-DNA collection. cpl3
plants have a T-DNA insertion in the first exon (Fig. 1C). Using
semi-quantitative RT-PCR, no CPL3 mRNA could be detected in the
cpl3 mutant, indicating that transcription of the CPL3 gene is
disrupted by the T-DNA insertion (Fig. 1D). We also screened
several T-DNA-tagged pools for knockouts of CPC-like MYB
genes, and identified the mutant lines etc1-1 (Kirik et al., 2004a) and
etc2-2. Additionally, ecotype Col-0 alleles of cpc and try mutants
were found and named cpc-2 (Kurata et al., 2005) and try-029760.
Thus, all of the mutants used in this study are in a Col-0 background.
CPL3 gene controls ploidy and flowering
RESEARCH ARTICLE 1339
Table 3. Leaf trichome number and cluster formation
Genotype
Col-0
cpl3
try
cpl3 try
cpl3 cpc
cpl3 etc1
cpl3 etc2
cpl3 try etc1
Trichomes per leaf
% Trichomes in clusters
39.4±3.6
72.5±4.6
42.4±2.8
53.3±1.8
106.3±9.3
67.4±4.2
94.6±3.4
68.2±3.5
0
0
23.7±3.6
29.9±3.2
0
0
0
55.4±6.2
0
0
0
0
0
0
0
0
35S::CPL3#1
35S::CPL3#2
CPL3p::CPL3#1
CPL3p::CPL3#2
Data, including s.d., were obtained from at least ten 2-week-old third leaves from
each line.
Table 4. Trichome branch numbers
Branches (br) / Trichome (%)
1 br
2 br
3 br
4 br
5 br
6 br
Col-0
cpl3
try
2±1
10±1
0
13±2
55±2
2±1
82±3
35±1
55±3
3±1
0
33±2
0
0
10±2
0
0
1±0.4
cpl3 try
cpl3 cpc
cpl3 etc1
cpl3 etc2
1±0.3
9±1
3±1
10±1
4±1
37±2
51±8
49±2
53±3
54±2
46±6
41±2
36±3
0
0
0
7±1
0
0
0
0
0
0
0
Data, including s.d., were obtained from at least five 2-week-old third leaves from
each line.
were more trichomes on cpc-2 than on wild type (see Fig. S1B in the
supplementary material), as had been reported using the WS allele,
cpc-1 (Schellmann et al., 2002). As was the case with cpc mutant
alleles, cpl3 plants also had more trichomes: roughly 80% more than
wild type (Table 3, Fig. 2N). The cpl3 cpc and cpl3 etc2 double
mutants had more trichomes than the parental single-mutant lines
(Table 3, Fig. 2O,Q). The combination of try-EM1 (Folkers et al.,
1997) with cpc-1 resulted in an increase in trichome clustering
(Schellmann et al., 2002). The cpl3 try double mutant had a slightly
increased percentage of clustered trichomes compared with the try
single mutant (Table 3). By contrast, there were no clusters on the
cpl3 single mutant, cpl3 cpc, cpl3 etc1, or cpl3 etc2 double mutants
(Fig. 2N,O,Q, Table 3).
The etc1-1 try-82 cpc-1 triple mutant produced a large number of
trichomes (Kirik et al., 2004b). In our experiment, the cpl3 try etc1
triple mutant had more clusters, though the trichome number was
similar to that of cpl3 (Fig. 2R, Table 3). The cpl3 cpc try triple
mutant had a more extreme phenotype, with heavy marginal clusters
(Fig. 2S). Each cluster in cpl3 cpc try was larger than any on cpc try,
but it was difficult to distinguish individual trichomes so they could
not be accurately counted (see Fig. S2 in the supplementary
material). The leaves of the cpl3 cpc try etc1 quadruple mutant were
entirely covered by trichomes (Fig. 2T), which is similar to that seen
in the transformant line 35S:GL1 35S:R (Larkin et al., 1994). Unlike
the etc1-1 try-82 cpc-1 triple mutant (Kirik et al., 2004b), the cpl3
cpc try etc1 quadruple mutant developed a large trichome cluster
covering even the midvein. Almost all of the adaxial epidermal cells
appeared to have differentiated into trichomes (Fig. 3A). Differential
interference contrast (DIC) images of the quadruple mutant showed
that the epidermal layer was completely made up of trichome cells,
to the exclusion of pavement cells, socket cells and guard cells (Fig.
Fig. 3. cpl3 cpc try etc1 quadruple mutant phenotype. (A) The
adaxial surface of rosette leaf of a cpl3 cpc try etc1 quadruple mutant
Arabidopsis was entirely covered by trichomes. (B) Pistil and stamen of
a cpl3 cpc try etc1 quadruple mutant were surrounded by trichomes.
(C) Adaxial epidermis of a rosette leaf of the cpl3 cpc try etc1
quadruple mutant. (D) Trichome phenotypes of a cpl3 cpc try etc1
quadruple mutant. Trichome phenotype of Col-0 (E) and cpl3 mutant
(F). Scale bars: 200 ␮m in A; 100 ␮m in B-F.
3C). Hypocotyls and inflorescences were also covered with
trichomes (Fig. 3B and see Fig. S3A in the supplementary material),
and almost every hypocotyl epidermal cell had differentiated into
trichomes (see Fig. S3B in the supplementary material). SEM
images of the adaxial surface of a true leaf showed that there was a
large variation in the size and branch number of individual trichomes
on the quadruple mutant (Fig. 3D).
try mutant trichomes have increased DNA content and more
branches than wild type (Hulskamp et al., 1994). By contrast, cpl3
mutant trichomes had consistently fewer branches than wild type,
with 55% of cpl3 trichomes having two branches (Table 4, Fig.
3E,F). These trichome phenotypes were also found on plants grown
in soil (see Fig. S4 in the supplementary material). Double mutants
with cpl3 and the other CPC homologs did not significantly further
reduce the branching observed in cpl3 (Table 4).
CPL3 has a similar function to CPC, TRY, ETC1 and
ETC2
CPL3 was expressed under the control of the 35S promoter to
produce an overexpressing line for comparison with the 35S::CPC,
35S::TRY, 35S::ETC1 and 35S::ETC2 lines (Wada et al., 1997;
Schellmann et al., 2002; Kirik et al., 2004a; Kirik et al., 2004b). As
with the overexpression lines of its homologs, 35S::CPL3 had more
than the normal number of root hairs, and no trichomes (Fig.
2I,J,U,V, Tables 2, 3, and see Fig. S5 in the supplementary material).
These results indicate that each of the CPC-like MYB homologs has
a similar function for root hair and trichome formation when
overexpressed under the control of the 35S promoter. When the
CPL3 gene’s own promoter was used to increase CPL3 expression
(CPL3p::CPL3), a relatively small amount of ectopic root hair
formation was observed (Fig. 2K,L, Table 2, and see Fig. S5A in the
DEVELOPMENT
Genotype
1340 RESEARCH ARTICLE
Development 135 (7)
Fig. 4. CPL3 gene expression. (A) Real-time PCR
analysis of CPL3 gene expression in Arabidopsis
organs. Total RNA was isolated from the indicated
tissues. (B-E) In situ hybridization patterns of CPChomologous genes at the shoot apex. Arrowheads in
B and C indicate CPC and ETC1 signals evident in
young trichomes. No signal for either ETC2 or CPL3
was detected in D and E. (F-M) Activity of CPL3p::GUS
reporter in 2-week-old leaves (F), in 5-day-old
cotyledons (G), hypocotyls (H) and roots (I), in 4-weekold inflorescence (J,K) and silique (L,M).
(N) Localization of CPL3-GFP fusion protein.
Fluorescence from the GFP fusion protein (green) and
propidium iodide (red) was observed with confocal
laser scanning microscopy. CPL3p::CPL3:GFP signal
localization in 2-week-old leaves. Scale bars: 50 ␮m in
B for B-E and in G,H,N; 100 ␮m in I; 200 ␮m in K,M; 1
mm in F,J,L.
Expression pattern of the CPL3 gene
CPL3 expression was examined in plant tissues using real-time
PCR. CPL3 was more strongly expressed in shoots (including a few
small true leaves) than in roots of seedlings (Fig. 4A). The relatively
strong expression of CPL3 in mature plants was specifically
observed in siliques and buds (Fig. 4A). Shellman et al. reported that
in trichomes, TRY expression was strongest, followed by CPC
(Schellmann et al., 2002). In our hands, in situ hybridization of shoot
meristem tissue indicated that the expression of CPC was stronger
than that of ETC1 in trichomes (Fig. 4B,C). We could not detect
ETC2 or CPL3 expression in young trichomes (Fig. 4D,E).
To analyze expression at the cellular level, we made CPL3
promoter-GUS fusions. CPL3p::GUS was expressed in young
leaves and mostly restricted to stomate guard cells in leaves,
cotyledons and hypocotyls (Fig. 4F-H). We could not detect
CPL3p::GUS in trichomes. CPL3 was detectable in roots by realtime PCR (Fig. 4A), but no CPL3p::GUS was detected in roots (Fig.
4I). Consistent with real-time PCR, strong CPL3p::GUS expression
was observed in inflorescences and developing seeds in siliques
(Fig. 4J-M).
Protein localization was determined using protein-2XGFP fusion
constructs driven by the CPL3 promoter (Fig. 4N). In
CPL3p::CPL3:GFP transgenic plants, a strong GFP signal was
observed in the guard cells (Fig. 4N), which was also consistent with
the expression pattern of the CPL3p::GUS construct (Fig. 4F). We
could not check for GFP localization in trichomes of these transgenic
plants because increased CPL3 gene dosage prevents the formation
of trichomes (Fig. 2W,X, Table 3, and see Fig. S5B in the
supplementary material). There was also no GFP signal in root
epidermis of CPL3p::CPL3:GFP plants, which is consistent with
the CPL3p::GUS data.
Intriguing features of the CPL3 gene
The guard cell-specific protein localization of CPL3 (Fig. 4N)
indicates some specific involvement with stomate initiation or
development, but there was no difference in the distribution or
cluster formation of guard cells among cpl3, 35S::CPL3,
CPL3p::CPL3 or wild-type plants (Table 5, and see Tables S1 and
S2 in the supplementary material). Also, the double mutant cpl3 etc2
did not show any aberrant stomata phenotype (see Table S3 in the
supplementary material). Similar to root hairs, stomates of
hypocotyls in wild-type Arabidopsis develop from epidermal cells
DEVELOPMENT
supplementary material), and there were no trichomes produced
(Fig. 2W,X, Table 3, and see Fig. S5B in the supplementary
material).
Previous analyses in yeast have shown that CPC protein binds to
the bHLH domain of the maize R protein (Wada et al., 2002), and
that CPC, TRY, ETC1 and ETC2 bind to the bHLH-domaincontaining GL3 protein (Kirik et al., 2004a; Kirik et al., 2004b; Esch
et al., 2003). To test whether CPL3 can also interact with GL3, we
performed a yeast two-hybrid analysis. All of the CPC homologous
proteins (CPC-BD, TRY-BD, ETC1-BD, ETC2-BD and CPL3-BD)
bound strongly to GL3-AD, and there also was significant binding
to EGL3-AD and AtMYC1-AD (see Fig. S6 in the supplementary
material).
CPL3 gene controls ploidy and flowering
RESEARCH ARTICLE 1341
Table 5. Stomatal density on cotyledons of the cpl3 mutant
and CPL3 overexpressing lines
Genotype
Col
cpl3
35S::CPL3#1
CPL3p::CPL3#1
2
A
FT
B
SOC1
2
Stomata per mm
Stomate index (%)
Clusters per mm
213±14
218±11
256±15
256±17
26.0±1.7
25.4±0.9
24.1±1.4
27.5±1.5
0
0
0
0
Data represent the mean±s.d. of at least five leaves per experiment.
Table 6. Effect of CPL3 on hypocotyl stomate patterns
Genotype
Col-0
cpl3
35S::CPL3#1
CPL3p::CPL3#1
Number of
stomata
Stomata in
S position (%)
Stomata in
N position (%)
1.8±0.3
1.8±0.4
2.0±0.4
2.4±0.4
84±12
73±15
54±16
77±12
16±11
27±7
46±10
23±10
Data represent the mean±s.d. of ten hypocotyls per experiment.
C
CO
Table 7. Flowering time and leaf numbers at flowering time of
the cpc, try, etc1, etc2 and cpl3 knockout mutants and of the
35S::CPL3-overexpressing transformant line
Genotype
Col-0
cpl3
cpc
try
etc1
etc2
35S::CPL3#1
Flowering time (days)
Number of leaves
37.6±0.5
28.9±0.5
39.6±0.5
36.0±0.8
40.6±0.8
39.0±0.5
41.1±1.1
17.4±0.6
8.2±0.3
19.2±0.6
14.3±0.6
19.3±0.8
14.1±1.9
28.5±1.7
D
CPL3
Data represent the mean±s.d. of at least ten plants per experiment.
Fig. 5. Expression of flowering-related genes in the cpl3 mutant
and transgenic Arabidopsis plants expressing CPL3. Real-time PCR
analyses of FT (A), SOC1 (B), CO (C) and CPL3 (D) genes at three
developmental stages. Expression levels were normalized to ACT2
expression. Relative expression levels: expression levels of each gene in
cpl3, 35S::CPL3 and CPL3p::CPL3 relative to wild type at 7 days. RNA
was isolated from 7-, 14- and 21-day-old rosette leaves grown under
long-day conditions. The experiment was repeated four times. Error
bars indicate s.d.
fold higher, and that of SOC1 3.2-fold higher in cpl3 than in wild
type (Fig. 5A,B). Expression of CO in cpl3 was not significantly
different from that in wild type (Fig. 5C). These results suggest the
involvement of CPL3 in flowering regulation by repressing FT and
SOC1 expression.
In addition to the altered flowering phenotype, cpl3 mutant plants
were much larger and CPL3-overexpressing plants were much smaller
than wild type (Fig. 6A). We confirmed this dwarf phenotype of
CPL3-overexpressing plants with three 35S::CPL3 and five
CPL3p::CPL3 independent transgenic lines. The fresh weight of cpl3
mutants was about 50% greater than that of wild type, and
CPL3p::CPL3 plants were about 30% the fresh weight of wild type
(Fig. 6B). Leaf epidermal cells in the cpl3 mutant were remarkably
larger, and cells from CPL3p::CPL3 were smaller than those of wild
type (Fig. 6C-E). This translates into a significant difference in overall
plant size. For example, the cpl3 mutant third leaf was about 13%
larger, and the CPL3p::CPL3-overexpressing line was about half the
size, of wild type. However, there was no significant difference in leaf
cell numbers (Table 8). These observations demonstrate that enhanced
DEVELOPMENT
that overlie two cortical cell files (Berger et al., 1998; Hung et al.,
1998), a location known as the ‘S’ (stomate) position. Therefore, we
also checked the position of stomates on hypocotyls. About 84% of
stomates are located in the S position and 16% in the ‘N’ (nonstomate) position in Col-0 plants (Table 6). There was no significant
difference in the positions of hypocotyl stomata among cpl3,
CPL3p::CPL3 and wild-type plants (Table 6, and see Table S4 in the
supplementary material). 35S:CPL3 transgenic lines, however,
showed a significant difference in hypocotyl stomate distribution,
with 54% of its stomata at the S position and 46% at the N position
(Table 6, and see Table S5 in the supplementary material). These
observations suggest that CPL3 is involved in the distribution of
hypocotyl guard cells.
In addition to the effects on root hair and trichome formation, cpl3
mutant plants were affected in flowering time. As shown in Table 7,
cpl3 mutant plants flowered earlier than wild type (28.9±0.5 versus
37.6±0.5 days) and with fewer leaves (8.2±0.3 versus 17.4±0.6). By
contrast, cpc, try, etc1 and etc2 mutant plants were not significantly
different from wild type (Table 7). 35S::CPL3 transgenic plants
flowered slightly later than wild type (41.1±1.1 versus 37.6±0.5
days) and with more leaves (28.5±1.7 versus 17.4±0.6) (Table 7, and
see Table S6 in the supplementary material). To clarify the effect of
the CPL3 gene on flowering, the expression of some floweringrelated genes was examined in plant lines with altered CPL3
expression by real-time PCR. FLOWERING LOCUS T (FT),
SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and
CONSTANS (CO) play a central role in controlling floral transition
(flowering) (Bagnall, 1993; Lee et al., 2000; Koornneef et al., 1991).
Expression of FT and SOC1 were higher in the cpl3 mutant than in
wild type under long-day (LD) conditions during leaf development
(Fig. 5A,B). In 21-day-old leaves, expression of FT was about 9.5-
1342 RESEARCH ARTICLE
Development 135 (7)
Fig. 6. Phenotypes of cpl3 mutant and transgenic
plants expressing CPL3. (A) Soil-grown rosettes from
4-week-old Col-0, cpl3, etc2 cpl3, 35S::CPL3,
CPL3p::CPL3#1 and CPL3p::CPL3#2 Arabidopsis plants.
(B) Fresh weight of rosette leaves per plant was
calculated from the means (±s.d.) of a minimum of five
rosettes from each line. (C-E) Microscopic analysis of leaf
epidermis of Col-0, cpl3 and CPL3p::CPL3. The study
was carried out in the middle region of the leaf blade of
2-week-old plants. (F-H) Hypocotyls from 2-week-old
Col-0, cpl3 and 35S::CPL3. (I-K) Hypocotyl epidermis of
Col-0, cpl3 and 35S::CPL3. Scale bars: 5 mm in A; 100
␮m in C,I; 200 ␮m in F.
genes CYCA2;1, CYCA2;2, CYCA2;3, CYCA2;4 and CYCA1;1,
which have been reported to be involved in endoreduplication in
Arabidopsis (Yoshizumi et al., 2006; Imai et al., 2006), and of SIM,
which is a cell cycle regulator controlling the onset of
endoreduplication (Churchman et al., 2006). Expression of these
genes in Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3 was analyzed
by real-time PCR (Fig. 8). Although expression of CYCA2;1 and
CYCA2;2 in the cpl3 mutant seemed to be somewhat reduced, and
in overexpressers (35S::CPL3 and CPL3p::CPL3) seemed to be
increased, compared with wild type (Fig. 8A,B), no significant
changes were observed for any of the CYCA genes (Fig. 8A-E).
Compared with wild type, expression of SIM was higher in the cpl3
mutant and lower in 35S::CPL3 and CPL3p::CPL3 during leaf
development (Fig. 8F). Significant reduction in expression was
observed in 30-day-old 35S::CPL3 and 12-day-old CPL3p::CPL3
plants compared with wild type (Fig. 8F). These results suggest that
increased ploidy in the cpl3 mutant and decreased ploidy in CPL3
overexpressers are involved in the function of SIM.
Table 8. Number of leaf epidermal cells in the cpl3 knockout
mutant and CPL3p::CPL3-overexpressing transformant line
Genotype
Col-0
cpl3
CPL3p::CPL3#1
Leaf size (mm2)
Pavement cells
(per mm2)
Pavement
cells/leaf
16.8±0.3
19.0±1.3
9.5±0.9
181±19
158±26
311±14
3035±311
3014±535
2960±306
Data represent the mean±s.d. of five leaves and at least 20 cells per experiment.
DEVELOPMENT
growth of cpl3 was caused by hypertrophy rather than hyperplasia of
leaf cells. In addition, hypocotyl cells and hypocotyls of cpl3 were
elongated, but those of 35S::CPL3 were rounded, resulting in slightly
shortened hypocotyls (Fig. 6F-K).
Because endoreduplication is generally thought to provide a
mechanism for increasing cell size, and the try mutation increases
trichome endoreduplication (Szymanski and Marks, 1998), we
analyzed the ploidy of leaf cells from wild-type, cpl3, 35S::CPL3
and CPL3p::CPL3 plants. In the cpl3 mutant, the number of 32C
and 16C cells was increased in 16-day-old first leaves (Fig. 7A;
the proportions of 32C and 16C were 0% and 26.9% for Col-0,
and 2.6% and 29.2% for cpl3, respectively). This result suggests
that the altered phenotypes of the cpl3 mutant, including
hypertrophic cell growth, are associated with an increase in
endoreduplication. By contrast, 35S:CPL3 and CPL3p::CPL3 had
reduced DNA levels in 16-day-old first leaves (Fig. 7A; the
proportions of 4C and 2C for Col-0 were 19.6% and 16.9%, for
35S::CPL3 24.3% and 27.4%, and for CPL3p::CPL3 33.1% and
26.4%, respectively). In 3-week-old third leaves, the ploidy
differences were observed more clearly (Fig. 7B; the proportions
of 16C for Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3 were 2.1%,
12.0%, 0.7% and 1.0%, respectively). This study demonstrates
that CPL3 is likely to have some role in ploidy-dependent
epidermal cell growth in Arabidopsis.
Endoreduplication is a type of cell cycle that skips the cell
division steps of mitosis and thus affects cell cycle-related gene
expression. We examined the expression of the cell cycle-related
RESEARCH ARTICLE 1343
A
(%)
100
32C
16C
8C
4C
2C
80
60
40
20
0
8d
16d
Col
8d
16d
cpl3
8d
16d
35S::CPL3
8d
16d
CPL3p::CPL3
B
(%)
120
100
32C
16C
8C
4C
2C
80
60
40
20
0
Col
cpl3
35S::CPL3
CPL3p::CPL3
Fig. 7. Loss of CPL3 function increases polyploidy levels.
(A) Relative ratios of each cell ploidy of 8- and 16-day-old first leaves of
Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3 Arabidopsis plants. (B) Relative
ratios of each cell ploidy of 3-week-old third leaves of Col-0, cpl3,
35S::CPL3 and CPL3p::CPL3. Approximately 5000 nuclei were counted
in Col-0, cpl3, 35S::CPL3 and CPL3p::CPL3 tissues.
DISCUSSION
Redundancy of CPL3 and CPC-like MYB genes in
epidermal differentiation
Historically, cpc was isolated as a mutant with few root hairs (Wada
et al., 1997). Although the typical plant MYB gene encodes a R2R3 type MYB region (Rosinski and Atchley, 1998), CPC encodes a
small R3-type MYB of only 94 amino acids. Not long after isolation
of CPC, TRY was isolated as a CPC-homologous gene from a
trichome-clustering mutant (Schellmann et al., 2002). More recently,
ETC1, ETC2 and ETC3 were isolated as enhancers of try and cpc
(Kirik et al., 2004a; Kirik et al., 2004b; Esch et al., 2004; Simon et
al., 2007). We also independently isolated these three genes by
homology with CPC. In this paper, we describe the isolation of
CPL3, and provide evidence that it has a redundant function with the
other homologs of CPC. In addition to its MYB function, CPL3 has
epistatic effects on a number of crucial plant development and
growth mechanisms. From the observation of double, triple and
quadruple mutant phenotypes, it is clear that CPC plays the
dominant role in the regulation of root hair formation, and TRY
plays the dominant role in trichome formation, but all five CPC-like
MYB genes, including CPL3, have a redundant function in root hair
and trichome formation (Figs 2, 3, Tables 2, 3, and see Fig. S1 in the
supplementary material). The reduction in root hair number
compared with wild type was significant in the cpl3-1 single mutant,
but not in cpl3 try, cpl3 etc1 or cpl3 etc2 double mutants (Table 2).
Because CPC represses its own expression (Wada et el., 2002), it is
possible that CPC-homologous genes also repress CPC expression.
Thus, disruption of the other CPC-homologous genes in the cpl3
mutant background may enhance CPC expression, which leads to
the formation of many root hairs. The conversion of almost all
adaxial surface leaf cells and hypocotyl cells into trichome cells in
the cpc try etc1 cpl3 quadruple mutant indicates that CPL3 either
directly or indirectly shares a similar function with its homologs,
because the cpc try etc1 triple mutant had a number of epidermal cell
types other than trichome cells (Fig. 3, and see Fig. S3A in the
supplementary material).
When CaMV 35S is used as a promoter for the expression of CPL3,
the number of root hairs is increased, and no trichomes are formed in
transformant lines, as observed in 35S::CPC, 35S::TRY, 35S::ETC1
and 35S::ETC2 transformants (Fig. 2, Tables 2, 3) (Wada et al., 1997;
Schellmann et al., 2002; Kirik et al., 2004a; Kirik et al., 2004b). Yeast
two-hybrid analyses showed that CPC, TRY, ETC1, ETC2 and CPL3
are capable of interacting with GL3, EGL3 and AtMYC1 (see Fig. S6
in the supplementary material). These findings demonstrate that CPL3
and the other CPC-like MYB proteins have a similar binding function.
Although EGL3 had higher promoter activity and RNA accumulation
than GL3, the mutant phenotype of egl3 was ‘weaker’ than that of gl3
(Bernhardt et al., 2003; Bernhardt et al., 2005). The strong trichome
deficiency and proliferation of root hair phenotypes of the gl3 mutant
are probably due to the strong binding activity of GL3 with the CPClike MYB proteins.
CPL3p::GUS was mainly expressed around stomata (Fig. 4FH), but the cpl3 mutant, cpl3 etc2 double mutant and CPL3
overexpressors did not have aberrant stomatal phenotypes (Table
5, and see Tables S1, S2, S3 in the supplementary material). We
counted stomates on cotyledons, which do not differ much in size
regardless of their genotype. Therefore, stomatal density is
apparently relatively constant (Table 5). The stomatal phenotypes
of true leaves were also observed several times. Thus, we
concluded that CPL3 did not affect stomate formation in leaves,
although the gene is expressed there. The expression patterns of
CPC homologs have been roughly classified into two groups.
CPCp::GUS, TRYp::GUS and ETC1p::GUS are expressed mainly
in roots and trichomes, and ETC2p::GUS and CPL3p::GUS are
expressed in young leaves and guard cells (see Fig. S7 in the
supplementary material). Thus, GUS expression by the CPC-like
MYB family is found in tissues throughout the entire plant body.
CPL3p::GUS expression was reduced in the cpc and etc1
backgrounds (see Fig. S8 in the supplementary material). These
results suggest that the members of this regulatory protein
family play different roles. Given the complexity of regulatory
cascades and the contributions of phytohormones, cell-wallassociated proteins and cytoskeleton structures, it is nonetheless
likely that this gene family has fairly direct control over
epidermal differentiation, development and integration for the
entire plant.
Intriguing features of the CPL3 gene
Although the general effect of CPL3 on cell fate is similar to that
of CPC, TRY, ETC1 and ETC2, CPL3 has several characteristics
that make it distinct from the other CPC homologs. First, the cpl31 mutant itself has a reduced number of root hairs, whereas etc11 and etc2-2 have normal root hair numbers (Fig. 2B, and see Fig.
S1A in the supplementary material). cpl3-1 also produces an
increased number of trichomes, similar to cpc-2 (Fig. 2N, and see
Fig. S1B in the supplementary material). Secondly, most stomata
are distributed in the S position in wild-type hypocotyls, but
transformant line 35S::CPL3 trichomes are evenly distributed in
both the S and N positions (Table 6, and see Table S4, S5 in the
supplementary material). Thirdly, cpl3 plants have an early
flowering phenotype (Table 7, and see Table S6 in the
supplementary material). Expression of the FT and SOC1 genes
in the cpl3 mutant was increased compared with wild type (Fig.
DEVELOPMENT
CPL3 gene controls ploidy and flowering
1344 RESEARCH ARTICLE
12 days
16 days
20 days
30 days
A
2.5
D
1.5
12 days
16 days
20 days
30 days
CYCA2;4
CYCA2;1
1
1.5
1
0.5
0.5
Col-0
cpl3
35S::CPL3
B
12 days
16 days
20 days
30 days
3
CYCA2;2
2
1
0
Col-0
cpl3
35S::CPL3
C
CYCA2;3
1.5
Col-0
cpl3
35S::CPL3
E
12 days
16 days
20 days
30 days
CYCA1;1
1
0.5
0
Col-0
cpl3
35S::CPL3
F
12 days
16 days
20 days
30 days
SIM
2
*
*
1
0
CPL3p::CPL3
3
1
0.5
CPL3p::CPL3
1.5
CPL3p::CPL3
12 days
16 days
20 days
30 days
2
0
CPL3p::CPL3
Relative expression levels
Relative expression levels
0
Fig. 8. CYCA and SIM expression in cpl3 mutant
and transgenic Arabidopsis plants expressing
CPL3. Real-time PCR analysis of CYCA2;1 (A),
CYCA2;2 (B), CYCA2;3 (C), CYCA2;4 (D), CYCA1;1 (E)
and SIM (F) in wild type, cpl3, 35S::CPL3 and
CPL3p::CPL3 at four developmental stages. Expression
levels of each gene were normalized to ACT2
expression. Relative expression levels: expression levels
of each gene relative to wild type at 12 days. The
experiment was repeated four times. Error bars
indicate s.d. Student’s t-test, *P<0.020 versus wild
type.
0
Col-0
cpl3
35S::CPL3
CPL3p::CPL3
Col-0
cpl3
35S::CPL3
5A,B). A flowering regulatory cascade model would thus include
repression of FT and SOC1 by CPL3. CPL3 expression is
significantly higher in 35S::CPL3 and CPL3p::CPL3
transformant lines than in wild type during leaf development (Fig.
5D). If CPL3 directly represses FT and SOC1, their expression in
35S::CPL3 and CPL3p::CPL3 plants would be reduced
significantly (Fig. 5A,B). One possibility is that CO or some other
factor overcomes the function of CPL3 to repress FT and SOC1.
Fourthly, cpl3 plants have an abnormally large growth phenotype,
and CPL3 overexpressers have a dwarf phenotype (Fig. 6). The
hypertrophic cell phenotype of cpl3 is associated with an increase
in endoreduplication (Fig. 7). CPL3 is thus likely to play an
essential role in ploidy-dependent epidermal cell growth in
Arabidopsis. Endoreduplication is generally thought to provide a
mechanism for increasing cell size (Sugimoto-Shirasu and
Roberts, 2003), although the correlation between ploidy level and
cell size is not always high (Leiva-Neto et al., 2004; Gendreau et
al., 1998; Schnittger et al., 2003).
Previous studies have shown that a mutation in TRY leads to
increased endoreduplication in trichomes and reduced
endoreduplication in the epidermis (Szymanski and Marks, 1998).
This is the opposite of what happens in the cpl3 mutant. Because a
mutation in CPL3 leads to an increase in endoreduplication in the
epidermis (Fig. 7), a decrease in trichome branching might be the
result of reduced endoreduplication in trichomes (Fig. 3F, Table 4).
It has been postulated that TRY is expressed in trichomes, reducing
endoreduplication in those cells, followed by diffusion into
neighboring cells to mediate lateral inhibition (Schellmann et al.,
2002). Thus, we propose a model in which CPL3 is expressed in
young leaf epidermal cells and represses endoreduplication, after
which it affects neighboring trichome cells by slightly promoting
endoreduplication.
CPL3p::CPL3
A2-type cyclins play an important role in regulating
endoreduplication in Arabidopsis (Burssens et al., 2000; Imai et al.,
2006; Yoshizumi et al., 2006). CYCA2;1 is expressed in various
differentiated cells, such as guard cells (Burssens et al., 2000), and
loss of CYCA2;3 increases polyploidy in mature true leaves (Imai et
al., 2006). However, we could not detect any significant change in
the expression of CYCA genes in cpl3 mutant and transgenic
Arabidopsis plants expressing CPL3. SIM is a cell cycle regulator
that controls endoreduplication onset in Arabidopsis (Churchman et
al., 2006). SIM transcript levels are increased in GL3-overexpressing
lines and decreased in the gl3 egl3 double mutant (Churchman et al.,
2006). Because CPL3 can bind to GL3 and EGL3 (see Fig. S6 in the
supplementary material), it might inhibit GL3 and/or EGL3 function
to induce expression of SIM (Fig. 8F).
The CPC-like MYB gene families are thought to have evolved by
gene duplication (Fig. 1C). Gene family members that have not
completely diverged functionally and thus retain some functional
redundancy may represent intermediate stages of regulatory
specification (Thomas, 1993; Cooke et al., 1997). As such, they can
provide considerable potential for adaptive or evolutionary
responses to environmental changes or occupation of a new
ecological niche through selection for the most advantageous cell
size and flowering time.
We thank Dr Yoshizumi for technical advice, M. Sato, K. Toyooka, M.
Wakazaki, Y. Miyazaki, H. Oka and T. Gohara for technical assistance, and T.
Araki, S. Yamaguch, Y. Kamiya, T. Ishida and T. Kurata for useful suggestions.
This work was supported in part by Grants-in-Aid from the Ministry of
Education, Science, Sports and Culture of Japan (15770152).
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1335/DC1
DEVELOPMENT
2
Development 135 (7)
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DEVELOPMENT
CPL3 gene controls ploidy and flowering