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First published online October 12, 2007
doi: 10.1242/10.1242/dev.009597
1 Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4,
Canada.
2 Department of Molecular, Cell, and Developmental Biology, University of
Michigan, Ann Arbor, MI 48109, USA.
3 Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T
1Z4, Canada.
4 National Key Laboratory of Plant Molecular Genetics, Institute of Plant
Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai 200032, China.
* Authors for correspondence (e-mails: schiefel{at}umich.edu; jingui{at}interchange.ubc.ca)
Accepted 14 August 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Lateral inhibition, MYB, Pattern formation, Trichome, TRICHOMELESS1
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Pesch and Hülskamp, 2004;
Serna, 2005;
Schellmann et al., 2007).
Trichome patterning in Arabidopsis is controlled by several
transcription factors. According to their effect on trichome initiation, these
transcription factors can be divided into two groups: positive regulators and
negative regulators. Positive regulators include the WD-repeat protein
TRANSPARENT TESTA GLABRA1 (TTG1) (Galway et
al., 1994; Walker et al.,
1999), the R2R3 MYB-type transcription factor GLABRA1 (GL1)
(Oppenheimer et al., 1991),
the basic helix-loop-helix (bHLH) transcription factors GLABRA3 (GL3) and
ENHANCER OF GLABRA3 (EGL3) (Payne et al.,
2000; Zhang et al.,
2003) and the homeodomain protein GLABRA2 (GL2)
(Rerie et al., 1994;
Masucci et al., 1996). The
negative regulators include several small single-repeat R3 MYB transcription
factors, such as TRIPTYCHON (TRY)
(Schnittger et al., 1999;
Schellmann et al., 2002),
CAPRICE (CPC) (Wada et al.,
1997; Wada et al.,
2002) and ENHANCER OF TRY AND CPC 1 and 2 (ETC1 and ETC2)
(Esch et al., 2004;
Kirik et al., 2004a;
Kirik et al., 2004b).
These positive and negative regulators work together to control trichome
initiation and patterning in Arabidopsis. The R2R3 MYB-type
transcription factor GL1, a bHLH transcription factor (GL3 or EGL3), and TTG1
form a complex to induce the expression of GL2, which in turn induces
trichome formation in shoots (reviewed by
Schiefelbein, 2003;
Pesch and Hülskamp,
2004). The small MYB transcription factors TRY, CPC, ETC1 and
ETC2, inhibit the trichome cell type in the shoot, presumably by competing
with GL1 for binding GL3, thereby limiting the transcriptional activity of the
trichome initiation and patterning activator complex
(Hülskamp et al., 1994;
Wada et al., 1997;
Schellmann et al., 2002;
Esch et al., 2003).
Root hair patterning is largely controlled by the same components, except
that GL1 is replaced by another R2R3 MYB-type transcription factor, WEREWOLF
(WER), to form a complex with TTG1 and GL3/EGL3 to induce GL2
expression (Bernhardt et al.,
2003; Bernhardt et al.,
2005; Lee and Schiefelbein,
1999). However, the positive regulators for trichome initiation
function to inhibit root hair initiation, whereas negative regulators for
trichome initiation function to promote root hair initiation
(Schiefelbein, 2003;
Pesch and Hülskamp,
2004).
We report here the identification and functional analysis of a new negative regulator for trichome initiation and patterning, TRICHOMELESS1 (TCL1). TCL1 represents a previously unknown member of the single-repeat R3 MYB transcription factor family. We demonstrate that overexpression of TCL1 completely abolishes trichome formation on all organs examined, whereas a loss-of-function mutation in TCL1 confers unique, ectopic trichome formation along inflorescence stems and pedicels. These trichome phenotypes have not been reported in mutants of any other members of the single-repeat R3 MYB transcription factor family. Furthermore, we provide genetic evidence that TCL1 and CPC work synergistically to regulate trichome formation on inflorescence stems and pedicels. In an unexpected finding, we show that TCL1 is likely to act by negatively regulating GL1 expression. This provides new insight into the organ-specific control of epidermal patterning and suggests the existence of a novel regulatory loop in trichome patterning.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The cpc mutant is in the WS ecotypic background
(Wada et al., 1997). Double
mutants were generated by crossing single mutants, examining the F2 progeny
for putative mutant phenotype, and confirming their double-mutant status by
genotyping in F2 and subsequent generations. Similarly, tcl1-1 cpc
try triple mutants were generated by crossing tcl1-1 cpc with
tcl1-1 try double mutants. Plants were grown at 23°C with 14/10
hour photoperiod at approximately 120 µmol m-2
s-1. For seedlings used for phenotypic and RT-PCR analyses, seeds were surface-sterilized and grown on Murashige & Skoog (MS) basal medium with vitamins (Plantmedia, Dublin, OH) and 1% (w/v) sucrose, solidified with 0.6% (w/v) phytoagar (Plantmedia).
Isolation of the tcl1-1D mutant and molecular cloning of TCL1
A dominant mutant with glabrous leaves was isolated from an
activation-tagged mutant population (
10,000 plants) produced in a
gpa1-2 mutant background (Ullah
et al., 2001), and designated as tcl1-1D.
A plasmid rescue technique was used to clone the TCL1 gene locus.
Genomic DNA (20 µg) isolated from the tcl1-1D mutant was digested
with PstI, which left the right-border of the T-DNA intact. The
digestion products were purified, ligated, and transformed into
Escherichia coli DH5
. The transformants were selected on LB
plates containing 100 µg/ml ampicillin. Two independent colonies were
selected and the plasmid DNA was sequenced using T-DNA left-border
(5'-TTGACAGTGACGACAAATCG-3') and right-border
(5'-ATGTGATATCTAGATCCGAAAC-3') primers. The tcl1-1D
phenotypes were subsequently confirmed by recapitulation experiments.
Isolation of the loss-of-function allele tcl1-1
A T-DNA insertion mutant allele of TCL1, SALK_055460, was
identified from the SALK T-DNA Express Database
(http://signal.salk.edu/cgi-bin/tdnaexpress).
In this allele, the T-DNA insertion site is in the second intron of the
TCL1 gene. The insertion was confirmed by PCR and sequencing using a
TCL1-specific primer (5'-ATGGATAACACAAACCGTC-3') and the
T-DNA-specific primer JMLB1
(5'-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3'), and the mutant allele was
designated tcl1-1. Loss of detectable full-length TCL1
transcript in the tcl1-1 mutant was verified by RT-PCR.
Plasmid construction
To generate the 35S:HA-TCL1 construct, the full-length open
reading frame (ORF) of TCL1 (gene locus At2g30432) was amplified by
RT-PCR using RNA from 10-day-old light-grown Arabidopsis seedlings.
The PCR fragment was then cloned in frame with an N-terminal HA tag into the
pUC19 vector under the control of the double 35S enhancer promoter of
CaMV (Tiwari et al., 2003;
Wang et al., 2005). To
generate the 35S:TCL1-VP16 construct, VP16 was amplified by
PCR using GD-VP16 (Tiwari et al.,
2003) as template and fused in frame with TCL1 under the
control of the CaMV 35S promoter. The TCL1-GFP construct was
cloned by fusing TCL1 in frame with GFP, then subcloning
this into the pUC19 vector under the control of the TCL1 promoter (a
fragment that covers the region -1535 to +1 of the start codon of
TCL1). The PTCL1:GUS construct was cloned by
replacing the PAtOPF1 promoter in
PAtOPF1:GUS (Wang et
al., 2007) with the TCL1 promoter. Corresponding
constructs in pUC19 were digested with EcoRI, then subcloned into the
binary vector pPZP211 or pPZP221 for plant transformation
(Hajdukiewicz et al.,
1994).
Phylogenetic analysis
The phylogenetic tree for TCL1, TRY, CPC, ETC1, ETC2 and At4g01060
(Fig. 2D) was generated using
AliBee - Multiple Alignment software, release 2.0
(http://www.genebee.msu.su/services/malign_reduced.html).
Plant transformation and selection of transgenic plants
Plants of about 5 weeks of age and with several mature flowers on the main
inflorescence were transformed with various constructs via Agrobacterium
tumefaciens GV3101 by the floral dip method
(Clough and Bent, 1998).
Phenotypes of transgenic plants were examined in the T1 generation, and
confirmed in T2 up to T4 generations. For all transgenic plants, at least five
transgenic lines with similar phenotypes were obtained.
Histochemical staining for ß-glucuronidase (GUS) activity
Histochemical staining for GUS activity used the substrate
5-bromo-4-chloro-3-indolyl ß-D-glucuronide (X-Gluc; Rose
Scientific, Edmonton, Alberta, Canada) and the general procedure described by
Ulmasov et al. (Ulmasov et al.,
1997).
Protoplast isolation, transfection and GUS activity assay
Protoplast isolation, transfection and the GUS activity assay were
undertaken as described previously (Tiwari
et al., 2003; Wang et al.,
2005; Wang et al.,
2007). GUS activities were measured using a Fluoroskan
Finstruments Microplate Reader (MTX Lab Systems, Vienna, VA).
Microscopy
Trichomes and root hairs were analyzed and photographed under a Leica MZ6
microscope equipped with a digital camera. The pattern of epidermal cell types
was determined as described previously
(Lee and Schiefelbein, 2002;
Kirik et al., 2004a;
Kirik et al., 2004b). Root
hair analysis used 10-day-old seedlings grown vertically on Petri plates.
For leaf trichome analysis, the first two leaves of soil-grown plants were
used. For stem or pedicel trichome analysis, adult soil-grown plants were
used.
The expression and localization of TCL1-GFP in transgenic plants expressing TCL1-GFP under the control of the TCL1 promoter were examined in 4-day-old seedlings.
RNA isolation and RT-PCR
Total RNA was isolated from seedlings and from the various tissues/organs
of adult plants using the Trizol reagent (Invitrogen Canada, Burlington,
Ontario, Canada). cDNA was synthesized using 1 µg total RNA by
oligo(dT)-primed reverse transcription, using the Omniscript RT Kit (Qiagen).
TCL1-specific primers (5'-ATGGATAACACAAACCGTC-3' and
5'-TCATTTGTGGGAGAAATAGTC-3') were used to amplify the full-length
ORF of TCL1. GFP-specific primers
(5'-ATGGTGAGCAAGGGCGAGGAG-3' and
5'-TTACTTGTACAGCTCGTCCATGCC-3') were used to check the expression
of TCL1-GFP. HA-specific (5'-TACCCTTACGATGTTCCTGATTAC-3')
and TCL1-specific (5'-TCATTTGTGGGAGAAATAGTC-3') primers
were used to check the expression of HA-TCL1. TTG1-specific primers
(5'-ATGGATAATTCAGCTCCAG-3' and
5'-TCAAACTCTAAGGAGCTGC-3') were used to check the expression of
TTG1. GL1-specific primers (5'-ATGAGAATAAGGAGAAG-3' and
5'-CTAAAGGCAGTACTCAACATC-3') were used to check the expression of
GL1. GL2-specific primers (5'-ATGTCAATGGCCGTCGACATGTC-3'
and 5'-TCTCGCAGCTTCTCTAGTTCCC-3') were used to check the
expression of GL2. GL3-specific primers
(5'-ATGGCTACCGGACAAAACAG-3' and
5'-AAGGAACGGGAAGCAAACCACTGTG-3') were used to check the expression
of GL3. ACTIN2 (amplified using
5'-CCAGAAGGATGCATATGTTGGTGA-3' and
5'-GAGGAGCCTCGGTAAGAAGA-3') was used as a control in all PCR
reactions.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was conducted according to Lawrence et al.
(Lawrence et al., 2004) and
Wang et al. (Wang et al.,
2007). Briefly, about 1.5 g of 10-day-old 35S:HA-TCL1
seedlings were cross-linked using 1% formaldehyde solution, ground with liquid
nitrogen, and sonicated using a Branson sonifier for 4x10 seconds at 40%
duty cycle and 20% power. Soluble chromatin was subject to ChIP using anti-HA
antibodies (Abgent) or rabbit preimmune sera. Chromatin-antibody complexes
were collected on salmon sperm DNA/protein A-agarose (Upstate). DNA-protein
cross-links were reversed at 65°C overnight, and the DNA purified and used
in PCR reactions. Primer pairs used for PCR were: GL1intronFW
(5'-TGGACAGTTGAAGAAGACAACATC-3') and GL1intronRV
(5'-CACATCTCTTTAGCCCTATCAACG-3'), and GL1UTRFW
(5'-TACACATAGGGACATACATATGCG-3') and GL1UTRRV
(5'-TAGTTTTGGTGTCGAAATTCCCGG-3').
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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subunit, gpa1-2
(Ullah et al., 2001), was used
as the parental genotype. This glabrous mutant was named trichomeless 1-1
Dominant (tcl1-1D) (Fig.
1). Compared with wild type and its parent, the tcl1-1D
mutant does not have any trichomes on the first pair of rosette leaves
(Fig. 1A,
Table 1), nor on successive
leaves, inflorescence stems, cauline leaves or floral organs (data not shown).
However, all other aspects of plant growth and development studied were
unaffected in the tcl1-1D mutant.
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) was used to identify the T-DNA insertion site in the
tcl1-1D mutant. As shown in Fig.
2A, in the tcl1-1D mutants the T-DNA was inserted in
chromosome 2 at a position that is 2115 bp upstream of the start codon of the
gene locus At2g30432, and 1339 bp downstream of the stop codon of the gene
locus At2g30440, with the four outward-facing 35S enhancers oriented
toward the gene locus At2g30432. RT-PCR analysis revealed that the transcript
level of gene locus At2g30432 was elevated
(Fig. 2B), indicating that the
TCL1 gene locus is most likely to be at At2g30432.
BLAST search analysis indicated that TCL1 encodes a protein that
is closely related to proteins encoded by the previously characterized genes
TRY, CPC, ETC1, ETC2, and by an uncharacterized gene locus At4g01060
(Fig. 2C)
(Kirik et al., 2004b;
Serna and Martin, 2006). All
these proteins have a conserved MYB region that is most closely related to the
R3 MYB domains of the R2R3 MYB gene family members in plants
(Wada et al., 1997;
Schellmann et al., 2002;
Esch et al., 2004;
Kirik et al., 2004a;
Kirik et al., 2004b).
Therefore, TCL1 represents a previously unknown member of the single-repeat R3
MYB transcription factor family. Results from phylogenetic analysis suggested
that TCL1 is more closely related to CPC than to TRY
(Fig. 2D).
To confirm that the phenotypes observed in the tcl1-1D mutant were caused by elevated expression of TCL1, we transformed wild-type Columbia (Col) plants with a binary vector containing the N-terminal HA-tagged full-length ORF of TCL1 expressed from the strong 35S promoter of the cauliflower mosaic virus (35S:HA-TCL1). As expected, overexpression of TCL1 recapitulated the tcl1-1D phenotypes (Fig. 1B). The transcript level of TCL1 in the transgenic lines was confirmed by RT-PCR using HA-specific and TCL1-specific primers (Fig. 1B).
TCL1 does not appear to affect root hair formation and patterning.
The number and pattern of root hair cells in the tcl1-1D mutant or in
plants overexpressing TCL1 were indistinguishable from those of
wild-type plants (Fig. 1C, and
see Table S1 in the supplementary material). Because overexpression of the
other single-repeat R3 MYBs (including CPC, TRY, ETC1 and ETC2) using the
35S promoter induces ectopic root hair cells
(Wada et al., 1997;
Schellmann et al., 2002;
Kirik et al., 2004a;
Kirik et al., 2004b), this
result indicates that the TCL1 protein may differ functionally from other R3
MYBs.
Loss-of-function mutation in TCL1 promotes trichome formation on inflorescence stems and pedicels
To further analyze the function of TCL1 in trichome formation, we
took a reverse genetic approach to seek loss-of-function alleles of
TCL1. By searching the SALK T-DNA Express Database
(http://signal.salk.edu/cgi-bin/tdnaexpress)
(Alonso et al., 2003), we found
one mutant allele, SALK_055460, in which TCL1 is interrupted by a
T-DNA insertion within its second intron
(Fig. 3A). The presence of the
T-DNA at the expected location was further verified by sequencing, and plants
homozygous for the T-DNA insertion at this locus were isolated by PCR-based
screening (data not shown). This mutant allele was named tcl1-1. The
expression of TCL1 was undetectable in tcl1-1 mutants by
RT-PCR (Fig. 3B), indicating
that tcl1-1 is likely to be a loss-of-function mutant allele of
TCL1.
Because trichome initiation in the tcl1-1D mutant was suppressed,
we expected to see an increase in trichome initiation or altered trichome
patterning in the tcl1-1 mutant. We first checked trichome production
on rosette leaves. However, both trichome initiation and patterning in the
tcl1-1 mutant were indistinguishable from those in wild-type plants
(Table 1). Interestingly, we
observed a dramatic increase in trichome formation on the inflorescence stems
in the tcl1-1 mutant (Fig.
3C, Fig. 4). In
wild-type plants, in addition to a decline in adaxial trichome production on
successive cauline leaves (Telfer et al.,
1997), a decline of trichome production on successive
inflorescence stem internodes was also observed
(Gan et al., 2006). We found
that no matter how many internodes (usually 3-4) Col wild-type plants produced
on the main inflorescence stem, trichome production was restricted to the
region below the first flower on the main inflorescence stem. Only very few,
or no trichomes were formed on the internode just adjacent to the first flower
(Fig. 3C,
Fig. 4). However, in the
tcl1-1 mutant, the number of trichomes on the main inflorescence stem
internodes was dramatically increased (Fig.
3C, Fig. 4).
Moreover, trichomes also formed beyond the site of the first flower branch
(Fig. 3C,
Fig. 4). Trichome distribution
on the internodes of lateral branches was similar to that of the main
inflorescence stem (data not shown). Such ectopic trichome formations on the
inflorescence stems have not been reported for mutants of any other members of
the single-repeat R3 MYB transcription factor family.
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). Based
on these results, we conclude that TCL1 has a unique role in regulating
epidermal cell patterning by controlling trichome cell specification on the
inflorescence stems and pedicels. To confirm that the phenotype we observed in tcl1-1 mutants was due to the loss-of-function of TCL1, we transformed tcl1-1 mutants with a binary vector containing the full-length ORF of TCL1 fused in frame with GFP, driven by TCL1's own promoter (PTCL1:TCL1-GFP). We used a genomic DNA fragment that covers the region -1535 to +1 of the start codon of TCL1 to provide putative regulatory sequences for TCL1. When expressed from this putative regulatory region, the TCL1 ORF was able to complement the tcl1 mutant (Fig. 3C), indicating that this putative regulatory sequence is sufficient for normal TCL1 expression, and that the TCL1-GFP fusion protein is most likely functional. The complete rescue of the tcl1-1 phenotype by PTCL1:TCL1-GFP shows that the trichome phenotype in the tcl1-1 mutant is indeed due to the loss-of-function of TCL1.
Microscopic examination of the PTCL1:TCL1-GFP transgenic plants revealed that TCL1 is localized in the nucleus of epidermal cells, but TCL1-GFP fluorescence could also be detected in regions near to, or at, the plasma membrane (Fig. 3D).
Synergistic effect between TCL1 and CPC on trichome formation
Four single-repeat R3 MYB proteins, CPC, TRY, ETC1 and ETC2, have been
shown to repress trichome initiation in a redundant manner
(Schellmann et al., 2002;
Esch et al., 2004;
Kirik et al., 2004a;
Kirik et al., 2004b).
Overexpression of TCL1 also repressed trichome initiation
(Fig. 1A), implying that TCL1
might function redundantly with other single-repeat MYB proteins in regulating
trichome formation. Because TRY and CPC are the best characterized of the
known single-repeat R3 MYB transcription factors, we focused on testing
functional redundancy between TCL1 and TRY or CPC.
We generated double mutants between tcl1-1 and try or cpc. As shown in Table 1, tcl1-1 try double mutants have no significant difference in the number of trichomes or trichome clusters on their rosette leaves, as compared with the try single mutant. Furthermore, the tcl1-1 try double mutant had no significant change in root epidermal cell pattern (see Table S1 in the supplementary material). Similarly, tcl1 cpc double mutants were indistinguishable from the cpc single mutant in leaf trichome formation or in H- and N-root epidermal cell specification (Table 1, and see Table S1 in the supplementary material).
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We generated tcl1-1 cpc try triple mutants and found that they did not differ significantly from the cpc try double mutants in terms of the number of trichomes and trichome clusters on their rosette leaves (Table 1), or in root epidermal cell pattern (see Table S1 in the supplementary material). As expected, tcl1-1 cpc try triple mutants formed trichome clusters on the inflorescence stems beyond the site of the first flower branch, and on pedicels (Fig. 5). These results support a predominant role for TCL1, compared to that of other members of the single-repeat R3 MYB transcription factor family, in regulating trichome cell specification on inflorescence stem and pedicels.
TCL1 suppresses the expression of GL1
To gain insight into the mechanism by which TCL1 affects trichome
initiation and patterning, we examined the expression of the known trichome
initiation and patterning positive regulators TTG1
(Galway et al., 1994;
Walker et al., 1999),
GL1 (Oppenheimer et al.,
1991), GL3 (Payne et
al., 2000; Zhang et al.,
2003; Bernhardt et al.,
2003; Bernhardt et al.,
2005) and GL2 (Rerie
et al., 1994; Masucci et al.,
1996) in TCL1 overexpression plants and in
tcl1-1 mutants. As expected, the expression of GL2 was
reduced in TCL1 overexpression plants
(Fig. 6A), presumably because
TCL1, like TRY and CPC, can bind to GL3, thus blocking the interaction between
GL1 and GL3, an interaction that is required to form the GL1-GL3-TTG1
activator complex. According to one current model, inhibition of the formation
of this complex would result in the suppression of expression of the trichome
initiation positive regulator GL2
(Larkin et al., 2003;
Schiefelbein, 2003;
Schellmann et al., 2007).
Unexpectedly, however, the GL1 transcript level was also dramatically
reduced in plants overexpressing TCL1. The transcript level of
TTG1 and GL3, the other two members in the proposed
activator complex, was not affected (Fig.
6A), indicating that such a repression is GL1-specific.
Consistent with this, we found that the expression of a GL1::GUS
reporter was dramatically reduced in plants overexpressing TCL1
(Fig. 6B). These results raise
the possibility that TCL1 affects trichome formation by directly suppressing
GL1 expression. In this scenario, the reduced expression of
GL2 in plants overexpressing TCL1 might be a consequence of
reduced expression of GL1, because a reduced availability of GL1
would decrease the transcriptional activity of the overall activator complex.
To investigate this further, we examined the expression levels of GL1
and GL2 in the developing inflorescence of tcl1-1 mutants by
RT-PCR, because TCL1 was expressed at a relatively higher level in
inflorescence than in other tissues/organs
(Fig. 6C). GL1 and
GL2 transcripts were found to be increased in the tcl1-1
mutant, as compared with the wild type
(Fig. 6D), supporting the
possibility that TCL1 negatively regulates GL1 in the developing
inflorescence epidermis.
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; Wang et al.,
2005). As expected, the TCL1 protein alone could not activate or
repress reporter gene expression when it was recruited to the promoter region
(Gal4) of the GUS reporter gene
(Fig. 7A,B). However, when TCL1
was fused with a heterologous activator domain, VP16, the TCL1-VP16 fusion
protein was able to function efficiently as an activator
(Fig. 7A,B). We therefore
generated transgenic plants overexpressing this TCL1-VP16 fusion protein.
We reasoned that if GL1 is the target for TCL1, one would expect
that the transcription of GL1 would be elevated in
35S:TCL1-VP16 plants owing to the direct binding of TCL1 to the
cis-acting regulatory elements of GL1 and the concurrent activation
of GL1 transcription by VP16. In addition, we would predict that the
35S:TCL1-VP16 plants should phenocopy plants overexpressing
GL1. Indeed, multiple transgenic lines of 35S:TCL1-VP16
plants displayed dramatically reduced number of trichomes on their leaves and
inflorescence stems, some of which had glabrous stems
(Fig. 7C), thus phenocopying
plants overexpressing GL1 (Larkin
et al., 1994). Consistent with this, we detected elevated levels
of GL1 transcript (in contrast to largely unchanged levels of
TTG1 and GL3 transcripts) in these transgenic lines
(Fig. 7D). Taken together,
these results support a working model in which TCL1 controls trichome
patterning by suppressing the expression of GL1
(Fig. 8).
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; Wang et al.,
2004). As a mock control, we used rabbit preimmune sera, and we
did not detect any specific PCR products using the same sets of primers
(Fig. 7E). Therefore, we
conclude that GL1 is indeed a target gene of TCL1. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Wada et al.,
1997; Schellmann et al.,
2002; Esch et al.,
2004; Kirik et al.,
2004a; Kirik et al.,
2004b), acts as a negative regulator for trichome initiation.
First, the dominant mutant tcl1-1D isolated from an activation-tagged
population displayed a glabrous phenotype, owing to TCL1
overexpression (Fig. 1,
Table 1). Second, ectopic
overexpression of TCL1 under the control of the CaMV 35S
promoter in wild-type plants resulted in the loss of trichomes
(Fig. 1,
Table 1). Finally, a
loss-of-function mutation in TCL1 conferred ectopic trichome
formation on the inflorescence stems and pedicels (Figs
3,
4,
5). We provide genetic evidence
that TCL1 is the major player of the single-repeat R3 MYB proteins in
regulating trichome formation on the inflorescence stems and pedicels (Figs
4,
5), but it does not affect root
epidermal cell fate (Fig. 1,
and see Table S1 in the supplementary material). Furthermore, we provide
evidence that TCL1 negatively regulates trichome patterning in a novel manner
by directly suppressing the expression of GL1 (Figs
6,
7).
TCL1 is a major negative regulator for trichome patterning on the inflorescence stems and pedicels
In wild-type plants, trichome initiation decreases on successive internodes
with only very few or no trichomes on the internode subtending the site of the
first flower on the main inflorescence stem
(Gan et al., 2006), and no
trichomes were found on the main inflorescence stem above the site of the
first flower (Figs 3,
4). In the tcl1-1
mutant, the number and patterning of trichomes on leaves were
indistinguishable from those of wild type
(Table 1). However, the number
of trichomes was significantly increased in the internodes of tcl1-1
mutants, and trichomes were also formed beyond the site of the first flower
(Figs 3,
4). Loss-of-function of
TCL1 also results in trichome formation on pedicels, which normally
do not bear any trichomes (Figs
3,
4,
5). Because these trichome
phenotypes on inflorescence stems and pedicels have not been reported in
mutants of other members of the single-repeat R3 MYB transcription factor
family, our data support the notion that TCL1 is the predominant member of
this family for the regulation of trichome formation on these organs.
Although TCL1 functions as a trichome initiation repressor, as do TRY, CPC,
ETC1 and ETC2 (Hülskamp et al.,
1994; Wada et al.,
1997; Schellmann et al.,
2002; Esch et al.,
2004; Kirik et al.,
2004a; Kirik et al.,
2004b), our results suggest that TCL1 might have a unique
biochemical role in regulating epidermal cell specification because TCL1 does
not appear to affect root hair formation and patterning. The
position-dependent specification of root epidermal cells is not altered in
either the gain- or loss-of-function mutants of TCL1
(Fig. 1, and see Table S1 in
the supplementary material), whereas overexpression of the other single-repeat
R3 MYBs tested, including CPC, TRY, ETC1 and ETC2, induces ectopic root hair
cells (Wada et al., 1997;
Schellmann et al., 2002;
Kirik et al., 2004a;
Kirik et al., 2004b). These
results imply significant differences in the biochemical properties of these
single-repeat R3 MYBs. Our results from genetic analysis of double and triple
mutants among tcl1-1, cpc and try, suggested that TCL1 and
CPC, but not TRY, have a synergistic effect on trichome formation on
inflorescence epidermis (Figs
4,
5). These findings are
consistent with the promoter-swap assays. We found that the expression of
TCL1 under the control of the CPC promoter in a cpc
mutant background (PCPC:HA-TCL1/cpc) could partially
rescue the root hair phenotype of the cpc mutant while simultaneously
repressing trichome initiation on leaves (see Fig. S1 in the supplementary
material). On the other hand, trichome clusters, a phenotypic trait of the
try mutant (Esch et al.,
2003), were still present on the leaves of plants expressing
TCL1 under the control of the TRY promoter in a try
mutant background (PTRY:HA-TCL1/try), although their
frequency was significantly reduced (see Fig. S1 the in supplementary
material). These results suggest that the TCL1 protein is not fully
interchangeable with TRY in controlling trichome patterning on rosette leaves,
and that TCL1 is functionally similar, but not identical, to CPC in the
developmental context of regulation of epidermal cell specification. These
results support the notion that the functional specificity of TCL1 largely
derives from the protein coding sequence, rather than from its promoter
activity.
The phenotype of ectopic trichome formations on the inflorescence stems
observed in tcl1-1 mutants has also been reported for plants
overexpressing GIS (Gan et al.,
2006). GIS encodes a transcription factor of the C2H2
family, and acts upstream of the trichome initiation complex
(Gan et al., 2006). Recently,
it has been found that GIS and two other C2H2 transcription factors, ZFP8 and
GIS2, play partially redundant and essential roles in inflorescence trichome
initiation and are regulated by the plant hormones, gibberellins and
cytokinins (Gan et al., 2007).
It is unclear whether TCL1 can work together with GIS to regulate trichome
formation on inflorescence epidermis.
Although TCL1 is a major negative regulator for trichome patterning on the inflorescence stems and pedicels, it is possible that TCL1 might also have a role in leaf epidermal development. For example, TCL1 transcript can be detected in the leaf (Fig. 6C), and overexpression of TCL1 represses trichome formation (Fig. 1). A higher-order combination of mutations in single-repeat R3 MYBs might help clarify the exact role of TCL1 in leaf trichome formation.
A possible molecular mechanism for the action of TCL1 in the regulation of trichome patterning
Previous analyses in yeast two-hybrid assays have demonstrated that TRY,
CPC, ETC1 and ETC2, can interact with GL3 to limit the interaction between GL1
and GL3 (Payne et al., 2000;
Esch et al., 2003;
Esch et al., 2004;
Kirik et al., 2004b;
Zimmermann et al., 2004), thus
inhibiting the formation of an activator complex between TTG1, GL1 and
GL3/EGL3, that is required for the activation of GL2 transcription
(Schiefelbein, 2003). Our
results showed that GL2 expression was reduced in plants
overexpressing TCL1 (Fig.
6), which could be explained if TCL1 operates by the same
mechanism. However, overexpression of TCL1 also suppressed the
expression of GL1 (Fig.
6). The suppression of GL1 by TCL1 appears to be specific
to this member of the activator complex, because the expression levels of the
other two components in the activator complex, TTG1 and GL3,
were largely unaffected by overexpression of TCL1
(Fig. 6). One possible scenario
is that TCL1 directly suppresses the expression of GL1 and, thereby,
indirectly causes a reduction in GL2 expression
(Fig. 8). This possibility is
supported by the elevated levels of GL1 and GL2 transcripts
in the developing inflorescence of tcl1-1 mutants
(Fig. 6). Furthermore, the
notion that GL1 might be a potential target gene for TCL1 was
supported by the observation that plants overexpressing a TCL1-VP16 fusion
protein, shown to be a transactivator in the protoplast transfection assay,
have elevated expression levels of GL1 (but largely unchanged levels
of TTG1 and GL3) and display phenotypes similar to plants
overexpressing GL1 (Fig.
7). An increase in GL1 level in response to TCL1-VP16
overexpression presumably alters the titration balance among components of the
GL1-GL3-TTG1 activator complex, thereby disrupting efficient formation of the
activator complex and, subsequently, its transcriptional activity. Finally,
our ChIP assay demonstrates that TCL1 can be recruited to the cis-acting
regulatory elements of GL1, suggesting that GL1 is indeed a
target gene of TCL1. A study on GL1 promoter activity and GL1 protein
localization at a subcellular level in a tcl1 mutant background might
strengthen the proposed model, and is worth further investigation.
Little is known about the regulation of GL1 expression. Here we
provide evidence that TCL1 might act to directly control GL1
expression. It is possible that, in addition, TCL1 competes with GL1 for
binding to GL3, so as to inhibit the formation of the activator complex, as
suggested for the other single-repeat R3 MYB transcription factors. It is
worth noting that TCL1 has the amino acid signature [D/E]Lx2[R/K]x3Lx6Lx3R
(Zimmermann et al., 2004) that
is required for interacting with R/B-like bHLH transcription factors
(Fig. 2C). TCL1 also has the
conserved amino acids in the MYB domain
(Kurata et al., 2005) that are
crucial for cell-to-cell movement (Fig.
2C). Using transgenic plants expressing a TCL1-GFP fusion protein,
we found that the TCL1-GFP can be detected in epidermal cells
(Fig. 3D). Using transgenic
plants expressing a TCL1 promoter-GUS fusion construct
(PTCL1:GUS), we found that PTCL1:GUS
was widely expressed (see Fig. S2 in the supplementary material). A detailed
study on TCL1 mRNA expression pattern and TCL1 protein subcellular
localization might help clarify the cell-to-cell movement ability of TCL1.
TCL1 is unique among the single-repeat R3 MYB proteins in that
overexpression of TCL1 does not affect root epidermis cell fate, which does
not depend on GL1 function. However, we found that overexpression of
CPC could also dramatically suppress the expression of GL1 (see Fig.
S3 in the supplementary material). Therefore, it is possible that other
single-repeat R3 MYBs, such as CPC, can also function in a similar manner to
TCL1 to directly suppress the transcription of GL1 and regulate
trichome patterning. It is worth noting that a similar mechanism has been
proposed for root hair patterning in which CPC (single-repeat MYB) negatively
regulates WER (R2R3 MYB) expression in hair-forming cells
(Lee and Schiefelbein, 2002).
WER has been proposed to be functionally equivalent to GL1
(Lee and Schiefelbein,
2001).
In summary, we identified TCL1 as a previously unknown member of the single-repeat R3 MYB transcription factor family. We provide evidence that TCL1 is a negative regulator of trichome initiation and patterning, and that TCL1 has a specific role in regulating epidermal cell specification on inflorescence stems and pedicles. This provides new insight into the way that organ-specific regulation of epidermal patterning might be achieved using a common mechanism and related transcription factor molecules. Furthermore, we show that TCL1 is likely to negatively regulate trichome formation in a novel manner, by directly suppressing the expression of GL1 (Fig. 8). This suggests the existence of a novel regulatory loop in trichome patterning, and offers a fine-tuning mechanism for the interaction between the negative regulators and the activator complex.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/21/3873/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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