|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 27 February 2008
doi: 10.1242/dev.017947
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan.
Author for correspondence (e-mail:
twada{at}psc.riken.jp)
Accepted 15 January 2008
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: Arabidopsis, Epidermis, MYB, Endoreduplication, Flowering
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). 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), 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 |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). 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 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).
Gene constructs
Primers
Primer sequences are listed in Table
1.
|
) 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 pBS-CPL3:2xGFP. 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.
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 pBS-CPL3p. 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 pBS-ETC1, -ETC2 and -TRY. The
SalI and BamHI-digested fragment of pBS-ETC1 was
ligated into the SalI and BamHI sites of binary vector
pBI101 (Clontech Laboratories) to create ETC1p::GUS. The
SalI and XbaI-digested 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 0.5xMS 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
2-week-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-4-chloro-3-indolyl-β-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 4-week-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). On-column
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 µg total RNA in a 20 µl 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, 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).
|
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-week-old
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 µg/ml
propidium iodide (PI) for 30 seconds and mounted in water. Confocal images
were obtained with a 40x 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).
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 β-galactosidase
assay was performed on each transformant using O-nitrophenyl
β-D-galactopyranoside (Sigma) as substrate.
|
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
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.
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).
|
). 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).
|
|
). 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. 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).
|
;
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 supplementary material), and there were no trichomes
produced (Fig. 2W,X,
Table 3, and see Fig. S5B in
the supplementary material).
|
), and that CPC, TRY, ETC1 and ETC2 bind to the
bHLH-domain-containing 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).
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 real-time 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 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' (non-stomate) 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.
|
|
|
;
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-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.
|
|
|
), 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 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.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Although
the typical plant MYB gene encodes a R2-R3 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).
|
;
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 CPC-like MYB proteins. CPL3p::GUS was mainly expressed around stomata (Fig. 4F-H), 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-wall-associated 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 cpl3-1 mutant itself has a reduced number of root hairs,
whereas etc1-1 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. 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).
|
). 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.
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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1335/DC1
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bagnall, D. J. (1993). Light quality and
vernalization interact in controlling late flowering in Arabidopsis ecotypes
and mutants. Ann. Bot.
71, 75.
Berger, F., Linstead, P., Dolan, L. and Haseloff, J. (1998). Stomata patterning on the hypocotyl of Arabidopsis thaliana is controlled by genes involved in the control of root epidermis patterning. Dev. Biol. 194,226 -234.[CrossRef][Medline]
Bernhardt, C., Lee, M. M., Gonzalez, A., Zhang, F., Lloyd, A.
and Schiefelbein, J. (2003). The bHLH genes GLABRA3 (GL3) and
ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis
root. Development 130,6431
-6439.
Bernhardt, C., Zhao, M., Gonzalez, A., Lloyd, A. and
Schiefelbein, J. (2005). The bHLH genes GL3 and EGL3
participate in an intercellular regulatory circuit that controls cell
patterning in the Arabidopsis root epidermis.
Development 132,291
-298.
Burssens, S., de Almeida Engler, J., Beeckman, T., Richard, C., Shaul, O., Ferreira, P., Van Montagu, M. and Inze, D. (2000). Developmental expression of the Arabidopsis thaliana CycA2;1 gene. Planta 211,623 -631.[CrossRef][Medline]
Churchman, M. L., Brown, M. L., Kato, N., Kirik, V., Hulskamp,
M., Inze, D., De Veylder, L., Walker, J. D., Zheng, Z., Oppenheimer, D. G. et
al. (2006). SIAMESE, a plant-specific cell cycle regulator,
controls endoreplication onset in Arabidopsis thaliana. Plant
Cell 18,3145
-3157.
Clough, S. J. and Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16,735 -743.[CrossRef][Medline]
Cooke, J., Nowak, M. A., Boerlijst, M. and Maynard-Smith, J. (1997). Evolutionary origins and maintenance of redundant gene expression during metazoan development. Trends Genet. 13,360 -364.[CrossRef][Medline]
Czechowski, T., Bari, R. P., Stitt, M., Scheible, W. R. and Udvardi, M. K. (2004). Real-time RT-PCR profiling of over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J. 38,366 -379.[CrossRef][Medline]
Di Cristina, M., Sessa, G., Dolan, L., Linstead, P., Baima, S., Ruberti, I. and Morelli, G. (1996). The Arabidopsis Athb-10 (GLABRA2) is an HD-Zip protein required for regulation of root hair development. Plant J. 10,393 -402.[CrossRef][Medline]
Dolan, L., Janmaat, K., Willemsen, V., Linstead, P., Poethig, S., Roberts, K. and Scheres, B. (1993). Cellular organisation of the Arabidopsis thaliana root. Development 119, 71-84.[Abstract]
Esch, J. J., Chen, M., Sanders, M., Hillestad, M., Ndkium, S.,
Idelkope, B., Neizer, J. and Marks, M. D. (2003). A
contradictory GLABRA3 allele helps define gene interactions controlling
trichome development in Arabidopsis. Development
130,5885
-5894.
Esch, J. J., Chen, M. A., Hillestad, M. and Marks, M. D. (2004). Comparison of TRY and the closely related At1g01380 gene in controlling Arabidopsis trichome patterning. Plant J. 40,860 -869.[CrossRef][Medline]
Folkers, U., Berger, J. and Hulskamp, M. (1997). Cell morphogenesis of trichomes in Arabidopsis: differential control of primary and secondary branching by branch initiation regulators and cell growth. Development 124,3779 -3786.[Abstract]
Gendreau, E., Hofte, H., Grandjean, O., Brown, S. and Traas, J. (1998). Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl. Plant J. 13,221 -230.[CrossRef][Medline]
Heim, M. A., Jakoby, M., Werber, M., Martin, C., Weisshaar, B.
and Bailey, P. C. (2003). The basic helix-loop-helix
transcription factor family in plants: a genome-wide study of protein
structure and functional diversity. Mol. Biol. Evol.
20,735
-747.
Huang, T., Bohlenius, H., Eriksson, S., Parcy, F. and Nilsson,
O. (2005). The mRNA of the Arabidopsis gene FT moves from
leaf to shoot apex and induces flowering. Science
309,1694
-1696.
Hulskamp, M., Misra, S. and Jurgens, G. (1994). Genetic dissection of trichome cell development in Arabidopsis. Cell 76,555 -566.[CrossRef][Medline]
Hung, C. Y., Lin, Y., Zhang, M., Pollock, S., Marks, M. D. and
Schiefelbein, J. (1998). A common position-dependent
mechanism controls cell-type patterning and GLABRA2 regulation in the root and
hypocotyl epidermis of Arabidopsis. Plant Physiol.
117, 73-84.
Imai, K. K., Ohashi, Y., Tsuge, T., Yoshizumi, T., Matsui, M.,
Oka, A. and Aoyama, T. (2006). The A-type cyclin CYCA2;3 is a
key regulator of ploidy levels in Arabidopsis endoreduplication.
Plant Cell 18,382
-396.
Jarvis, P., Chen, L. J., Li, H., Peto, C. A., Fankhauser, C. and
Chory, J. (1998). An Arabidopsis mutant defective in the
plastid general protein import apparatus. Science
282,100
-103.
Kallal, L. and Kurjan, J. (1997). Analysis of the receptor binding domain of Gpa1p, the G(alpha) subunit involved in the yeast pheromone response pathway. Mol. Cell. Biol. 17,2897 -2907.[Abstract]
Kirik, V., Simon, M., Wester, K., Schiefelbein, J. and Hulskamp, M. (2004a). The ENHANCER OF TRY AND CPC1 gene acts redundantly with TRIPTYCHON and CAPRICE in trichome and root hair cell patterning in Arabidopsis. Dev. Biol. 268,506 -513.[CrossRef][Medline]
Kirik, V., Simon, M., Huelskamp, M. and Schiefelbein, J. (2004b). ENHANCER of TRY and CPC 2 (ETC2) reveals redundancy in the region-specific control of trichome development of Arabidopsis. Plant Mol. Biol. 55,389 -398.[CrossRef][Medline]
Koornneef, M., Hanhart, C. J. and van der Veen, J. H. (1991). A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana. Mol. Gen. Genet. 229, 57-66.[Medline]
Kurata, T., Kawabata-Awai, C., Sakuradani, E., Shimizu, S., Okada, K. and Wada, T. (2003). The YORE-YORE gene regulates multiple aspects of epidermal cell differentiation in Arabidopsis. Plant J. 36,55 -66.[CrossRef][Medline]
Kurata, T., Ishida, T., Kawabata-Awai, C., Noguchi, M., Hattori,
S., Sano, R., Nagasaka, R., Tominaga, R., Koshino-Kimura, Y., Kato, T. et
al. (2005). Cell-to-cell movement of the CAPRICE protein in
Arabidopsis root epidermal cell differentiation.
Development 132,5387
-5398.
Larkin, J. C., Oppenheimer, D. G., Lloyd, A. M., Paparozzi, E. T. and Marks, M. D. (1994). Roles of the GLABROUS1 and TRANSPARENT TESTA GLABRA Genes in Arabidopsis Trichome Development. Plant Cell 6,1065 -1076.[Abstract]
Lee, H., Suh, S. S., Park, E., Cho, E., Ahn, J. H., Kim, S. G.,
Lee, J. S., Kwon, Y. M. and Lee, I. (2000). The AGAMOUS-LIKE
20 MADS domain protein integrates floral inductive pathways in Arabidopsis.
Genes Dev. 14,2366
-2376.
Lee, M. M. and Schiefelbein, J. (1999). WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning. Cell 99,473 -483.[CrossRef][Medline]
Leiva-Neto, J. T., Grafi, G., Sabelli, P. A., Dante, R. A., Woo,
Y. M., Maddock, S., Gordon-Kamm, W. J. and Larkins, B. A.
(2004). A dominant negative mutant of cyclin-dependent kinase A
reduces endoreduplication but not cell size or gene expression in maize
endosperm. Plant Cell
16,1854
-1869.
Masucci, J. D., Rerie, W. G., Foreman, D. R., Zhang, M., Galway, M. E., Marks, M. D. and Schiefelbein, J. W. (1996). The homeobox gene GLABRA2 is required for position-dependent cell differentiation in the root epidermis of Arabidopsis thaliana. Development 122,1253 -1260.[Abstract]
Nesi, N., Debeaujon, I., Jond, C., Pelletier, G., Caboche, M.
and Lepiniec, L. (2000). The TT8 gene encodes a basic
helix-loop-helix domain protein required for expression of DFR and BAN genes
in Arabidopsis siliques. Plant Cell
12,1863
-1878.
Okada, K. and Shimura, Y. (1990). Reversible
root tip rotation in Arabidopsis seedlings induced by obstacle-touching
stimulus. Science 250,274
-276.
Rerie, W. G., Feldmann, K. A. and Marks, M. D.
(1994). The GLABRA2 gene encodes a homeo domain protein required
for normal trichome development in Arabidopsis. Genes
Dev. 8,1388
-1399.
Rosinski, J. A. and Atchley, W. R. (1998). Molecular evolution of the Myb family of transcription factors: evidence for polyphyletic origin. J. Mol. Evol. 46, 74-83.[CrossRef][Medline]
Schellmann, S., Schnittger, A., Kirik, V., Wada, T., Okada, K., Beermann, A., Thumfahrt, J., Jurgens, G. and Hulskamp, M. (2002). TRIPTYCHON and CAPRICE mediate lateral inhibition during trichome and root hair patterning in Arabidopsis. EMBO J. 21,5036 -5046.[CrossRef][Medline]
Schnittger, A., Weinl, C., Bouyer, D., Schobinger, U. and
Hulskamp, M. (2003). Misexpression of the cyclin-dependent
kinase inhibitor ICK1/KRP1 in single-celled Arabidopsis trichomes reduces
endoreduplication and cell size and induces cell death. Plant
Cell 15,303
-315.
Simon, M., Lee, M. M., Lin, Y., Gish, L. and Schiefelbein, J. (2007). Distinct and overlapping roles of single-repeat MYB genes in root epidermal patterning. Dev. Biol. 311,566 -578.[CrossRef][Medline]
Sugimoto-Shirasu, K. and Roberts, K. (2003). "Big it up": endoreduplication and cell-size control in plants. Curr. Opin. Plant Biol. 6, 544-553.[CrossRef][Medline]
Szymanski, D. B. and Marks, M. D. (1998).
GLABROUS1 overexpression and TRIPTYCHON alter the cell cycle and trichome cell
fate in Arabidopsis. Plant Cell
10,2047
-2062.
Thomas, J. H. (1993). Thinking about genetic redundancy. Trends Genet. 9, 395-399.[CrossRef][Medline]
Urao, T., Yamaguchi-Shinozaki, K., Mitsukawa, N., Shibata, D. and Shinozaki, K. (1996). Molecular cloning and characterization of a gene that encodes a MYC-related protein in Arabidopsis. Plant Mol. Biol. 32,571 -576.[CrossRef][Medline]
Wada, T., Tachibana, T., Shimura, Y. and Okada, K.
(1997). Epidermal cell differentiation in Arabidopsis determined
by a Myb homolog, CPC. Science
277,1113
-1116.
Wada, T., Kurata, T., Tominaga, R., Koshino-Kimura, Y.,
Tachibana, T., Goto, K., Marks, M. D., Shimura, Y. and Okada, K.
(2002). Role of a positive regulator of root hair development,
CAPRICE, in Arabidopsis root epidermal cell differentiation.
Development 129,5409
-5419.
Yadegari, R., Paiva, G., Laux, T., Koltunow, A. M., Apuya, N.,
Zimmerman, J. L., Fischer, R. L., Harada, J. J. and Goldberg, R. B.
(1994). Cell differentiation and morphogenesis are uncoupled in
Arabidopsis raspberry embryos. Plant Cell
6,1713
-1729.
Yoo, S. Y., Bomblies, K., Yoo, S. K., Yang, J. W., Choi, M. S., Lee, J. S., Weigel, D. and Ahn, J. H. (2005). The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221,523 -530.[CrossRef][Medline]
Yoshizumi, T., Tsumoto, Y., Takiguchi, T., Nagata, N., Yamamoto,
Y. Y., Kawashima, M., Ichikawa, T., Nakazawa, M., Yamamoto, N. and Matsui,
M. (2006). Increased level of polyploidy1, a conserved
repressor of CYCLINA2 transcription, controls endoreduplication in
Arabidopsis. Plant Cell
18,2452
-2468.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
This article has been cited by other articles:
|
|
 |
|
  Compiled by, F. Tooke, T. Chiurugwi, and N. Battey Flowering Newsletter bibliography for 2008 J. Exp. Bot., June 23, 2009; (2009) erp154v1. [Full Text] [PDF]
|
|
|
|
 |
|
 H.-F. Zhu, K. Fitzsimmons, A. Khandelwal, and R. G. Kranz CPC, a Single-Repeat R3 MYB, Is a Negative Regulator of Anthocyanin Biosynthesis in Arabidopsis Mol Plant, June 2, 2009; (2009) ssp030v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Wester, S. Digiuni, F. Geier, J. Timmer, C. Fleck, and M. Hulskamp Functional diversity of R3 single-repeat genes in trichome development Development, May 1, 2009; 136(9): 1487 - 1496. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Wang and J.-G. Chen Arabidopsis Transient Expression Analysis Reveals that Activation of GLABRA2 May Require Concurrent Binding of GLABRA1 and GLABRA3 to the Promoter of GLABRA2 Plant Cell Physiol., December 1, 2008; 49(12): 1792 - 1804. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Maes, D. Inze, and A. Goossens Functional Specialization of the TRANSPARENT TESTA GLABRA1 Network Allows Differential Hormonal Control of Laminal and Marginal Trichome Initiation in Arabidopsis Rosette Leaves Plant Physiology, November 1, 2008; 148(3): 1453 - 1464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Jakoby, D. Falkenhan, M. T. Mader, G. Brininstool, E. Wischnitzki, N. Platz, A. Hudson, M. Hulskamp, J. Larkin, and A. Schnittger Transcriptional Profiling of Mature Arabidopsis Trichomes Reveals That NOECK Encodes the MIXTA-Like Transcriptional Regulator MYB106 Plant Physiology, November 1, 2008; 148(3): 1583 - 1602. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
