1 Department of Plant Biology, University of Minnesota, St Paul, MN 55108-1095,
USA
2 College of Biological Sciences Imaging Center, University of Minnesota, St
Paul, MN 55108-1095, USA
* Author for correspondence (e-mail: dmarks{at}biosci.cbs.umn.edu)
Accepted 19 August 2003
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SUMMARY |
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Key words: Trichome, bHLH, MYB, GL1, TTG1, GL3, CPC, TRY, WER, Transcription factor, pBridge, Lac OP/I-GFP, Endoreduplication, Endoreplication, Elemental analysis, GFP, Nuclear localization, Interphase chromosomes, Cell fate, Differentiation, Cell cycle, Cytoskeleton, Root hair
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Introduction |
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Genetic analyses have identified three genes that are involved in promoting
trichome initiation: GLABROUS1 (GL1), a R2R3-Myb-type
transcription factor; TRANSPARENT TESTA GLABRA1 (TTG1), a
protein with WD-40 repeats; and GLABRA3 (GL3), a bHLH type
transcription factor (Oppenheimer et al.,
1991; Payne et al.,
2000
; Walker et al.,
1999
). Severe loss-of-function mutations in either GL1 or
TTG1 result in greatly reduced trichome initiation. Mutations in
GL3 moderately reduce trichome initiation and result in smaller
trichomes that have fewer branches
(Koornneef et al., 1982
;
Payne et al., 2000
). The
Arabidopsis genome contains another gene closely related to GL3,
ENHANCER OF GLABRA3 (EGL3). Plants containing mutations in both
GL3 and EGL3 display the greatly reduced trichome initiation
phenotype that is exhibited in gl1 and ttg1 mutants
(Zhang et al., 2003
).
Mutations in EGL3 alone only slightly alter trichome initiation and
development. Unlike gl1 mutants, which only affect trichome
development, ttg1 and gl3 egl3 mutants display additional
defects that include the production of extra root hairs and the loss of both
pigmentation and seed coat mucilage production
(Galway et al., 1994
;
Koornneef, 1981
;
Zhang et al., 2003
).
The expression patterns of GL1 and GL3 have been
characterized. Both genes are diffusely expressed at low levels in fields of
epidermal cells on young leaves, and then expressed at higher levels in early
stage trichomes (Larkin et al.,
1993; Zhang et al.,
2003
). Additional studies have shown that the GL1 and GL3 proteins
probably act in a complex with TTG1. It is thought that a threshold level of
the GL1 GL3 TTG1 activator complex is needed to irreversibly push a cell into
the trichome pathway (Payne et al.,
2000
; Schellmann et al.,
2002
; Szymanski et al.,
2000
). The predicted consequence of reaching the threshold is the
generation of an autoregulatory loop that results in the up-regulation of the
complex genes, as well as genes needed for the initial stages of trichome
differentiation, including TRYPTICHON (TRY). TRY, which encodes a
single-repeat R3 Myb, limits trichome initiation and prevents neighboring
cells from becoming trichomes
(Hülskamp et al., 1994
;
Schellmann et al., 2002
). In
yeast assays, TRY physically interacts with GL3 and may function to reduce the
ability of the GL1 GL3 TTG1 activation complex to activate gene expression in
developing trichomes and those cells that neighbor trichomes
(Zhang et al., 2003
). To
mediate this latter role, it has been proposed that once translated in
developing trichomes, TRY protein diffuses through the connecting
plasmodesmata to neighboring cells
(Schellmann et al., 2002
).
A model similar to the trichome initiation model has been proposed for root
hair (H-cell) development where the WER, TTG1 and GL3/EGL3 proteins form an
initiation complex to promote the N-cell fate (non hair)
(Schiefelbein, 2003;
Zhang et al., 2003
). The
CAPRICE (CPC) gene, which encodes a single-repeat R3 MYB
homologous to TRY and is expressed in N-cells, acts as a negative
regulator of the N-cell fate (Lee and
Schiefelbein, 2002
; Wada et
al., 1997
). CPC protein has been shown to be able to travel to
H-cells and to down-regulate the expression of N-cell genes like WER
in H-cells (Lee and Schiefelbein,
2002
; Wada et al.,
2002
).
This paper presents the characterization of a new allele of GL3. In contrast to previously described gl3 mutants, the trichomes of the new mutant display greatly increased levels of endoreduplication and over expand early during development. This novel contradictory gl3 phenotype has provided a new insight into trichome development.
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Materials and methods |
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Mapping and isolation of sst
Bulk segregation analysis of an F2 population derived from a
cross between Col sst and Ler was used to determine the
general chromosomal location of sst
(Lukowitz et al., 2000). To
more finely map the chromosomal location of sst, DNA samples from 302
sst F2 plants were tested with PhyC, CiW9 and CiW10 SSLP
marker primer pairs (Research Genetices). Data from these reactions placed the
sst locus between PhyC and CiW9 on chromosome 5. Additional SSLP
markers were created utilizing information from the insertion/deletion (InDel)
polymorphism collection generated by Cereon Genomics (now Monsanto Co.)
(Jander et al., 2002
). InDels
454480 and 457742 were ultimately used to limit the sst locus to
either TAC clone K1O13 or P1 clone Myc6. Because the Myc6 clone contained the
candidate GL3 gene, sst was tested for allelism to
GL3.
F2 genotyping of gl3-1, gl3-sst and SALK TDNA
line 77439
The gl3-1 mutation causes a C to T transition at codon 378, which
results in the loss of an AciI site. Using forward
5'-TCAGTACGGAGCCTTTTCCAACAGC-3' and reverse
5'-CTTTAACATTCCTTGTGATGGTGCC-3' primers, a 509 bp genomic fragment
was amplified using the Extract-N-Amp PCR system (Sigma). Digesting the
samples with AciI generated fragments of 174 bp and 73 bp for Col
compared with a 247 bp fragment for gl3-1.
The gl3-sst mutation causes a C to T transition at codon 78 leading to the elimination of a DdeI site. Using forward 5'-GCGAATTCGCCATGGCTACCGGACAAAACAGAAC-3' and reverse 5'-GCGGAAGCTCGTCTGGTGAC-3' primers, a 661 bp fragment was generated by PCR. Digesting the samples with DdeI generated fragments of 460 bp and 89 bp for Col compared with a 549 bp fragment for gl3-sst.
SALK line 77439 contains a T-DNA insert in the third exon of At1g63650. To distinguish between the insertional mutant and wild loci two sets of primers were used. One set of primers (forward 5'-CCCCGGAGGAGCGTTATCCAATGGA-3' and reverse 5'-CCCTTAAGTGACGATAAATACACTCCG-3') was used to detect the wild-type gene and another set of primers (forward 5'-GCGTGGACCGCTTGCTGCAACT-3' and reverse 5'-CCCTTAAGTGACGATAAATACACTCCG-3') was used to detect the T-DNA insertion.
Scanning electron microscopy and x-ray elemental analysis
The plant specimens were frozen in liquid nitrogen and processed with an
Emitech K1150 cryo-preparation system where the specimens were sputter coated
with 20 nm gold and imaged using a cold stage with a Hitachi S3500N scanning
electron microscope. For X-ray elemental analysis, the samples were sputter
coated with 20 nm of nickel and analyzed with the EDAX, Inc. Falcon
System.
Vector construction for the Lac Operator/Repressor-GFP reporter
system
The p928 LacOP construct, which contains 256 repeats of the Lac Operator
sequence, was generated by moving a 12.5 kb fragment containing the repeated
sequence from pSV2-dhfr 8.32 (Robinett et
al., 1996) into the T-DNA vector p928 (gift from Dr Mitra,
University of Nebraska).
The coding sequence of a modified LacI repressor protein and a C-terminal
SV40 nuclear localization sequence used for the pEGAD LacI construct
originated from a 1276 bp EcoRI-DraI fragment from
p3'SSdimer-Cl-EGFP (Robinett et al.,
1996). This fragment was cloned into the EcoRI and
blunt-ended HindIII sites of the 35S::GFP-containing T-DNA vector
pEGAD (Cutler et al., 2000
) to
create plasmid pEGAD LacI.
Construction, transformation and imaging of GFP-fusions in
Arabidopsis
The promoter regions from MYB5 (forward
5'-GAGCTCTGCTGGAGAAATTCATCCCAA-3' and reverse
5'-ACCGGTCTCCGCCGTCTTCAACAAAGC-3'), ATML1 (forward
5'-GAGCTCGATCCATAGTTTCTAAAATGTGC-3' and reverse
5'-ACCGGTGGATTCAGGGAGTTTCTTTAACC-3'), TRY (forward
5'-AAGCTTGAGCTCGTTAGTTTAATGGGTCGA-3' and reverse
5'-GGATCCACCGGTGATGAAATTTTGAGTTTGG-3') were amplified from
Columbia genomic DNA using JumpStart Taq Redimix (Sigma) and cloned into
pCR2.1 using the Topo-TA cloning system (Invitrogen). Each promoter was then
excised as a SacI-AgeI fragment and was swapped with the 35S
promoter of pEGAD (Cutler et al.,
2000). pEMYB5::GFP-cGL3 was generated by excising the
coding region of GL3 from pD2CD-7 (a gift from Dr Lloyd, University
of Texas) as an EcoRI-BamHI fragment and cloning into the
corresponding sites of pEGAD MYB5 Pro. pEATML1::GFP-cGL1 was
generated by excising the coding region of GL1 from pGL1-A
(a gift from Dr Lloyd) with EcoRI and BglII and cloning it
into the corresponding sites of pEGAD ATML Pro. pETRY::GFP-cTRY and
pEMYB5::GFP-cTRY were generated by excising the coding region of
TRY from pCR-TRY-BD as an EcoRI/BamHI
fragment and cloning it into the appropriate sites of pEGAD TRY Pro
and pEGAD MYB5 Pro, respectively
The binary constructs were introduced into Agrobacterium
tumefaciens C58C1 by electroporation. The Arabidopsis Columbia
strains were transformed with Agrobacterium by dipping and selected
on soil with Basta (Clough and Bent,
1998). GFP flourescence was detected in mature trichomes using a
Nikon Eclipse E800 microscope with a Cool Cam color CCD camera (Cool Camera
Co., Decatur, GA) and Imago Pro Plus version 3.0 software (Media Cybermetics,
Silver Spring, MD).
DAPI staining
Mature leaf tissue from Columbia wild-type, gl3-1 and
gl3-sst was stained with DAPI (4'6-diamidino-2-phenylindole;
Sigma) as previously described (Szymanski
and Marks, 1998). The staining was visualized with a Nikon
Diaphot-200 inverted microscope with DIC optics and a Kodak MDS290 photo
capture system.
Construction, transformation, and analysis of yeast constructs
pGL3-AD was constructed by cloning an
EcoRI/BamHI fragment containing the GL3 coding
region from pD2DC-7 [(Payne et al.,
2000) a gift from Dr Lloyd] into the EcoRI and
BglII sites of pGAD424 (Clontech). psst-AD was generated by
swapping a 702 bp BamHI/BglII fragment from
pCR-sst-N with the corresponding fragment from pGL3-AD. The
GL3 coding region from the gl3-sst mutant
(pCR-sst-N) was generated by PCR amplifying a 1.08 kp fragment from
gl3-sst cDNA using the primers, forward
5'-GCGAATTCGCCATGGCTACCGGACAAAACAGAAC-3' and reverse
5'-CGGATCAAGAACGTTGTCGATGTG-3'. The fragment was cloned into
pCR2.1 (Invitrogen) and the presence of only the gl3-sst specific C
to T change at positon 235 was confirmed by DNA sequencing. The coding regions
for GL1, TTG1 and TRY used to make GAL4 binding domain
fusions originated from pGL1-B, pWS10 and pCR-TRY-BD. Both
pGL1-B and pWS10 were gifts from Dr Lloyd
(Payne et al., 2000
), whereas
pCR-TRY-BD was a PCR-generated TRY coding region from
Columbia cDNA (forward 5'-GAATTCGCCATGGATAACACTGACCGTCGT-3' and
reverse 5'-GGATCCCTAGGAAGGATAGATAGAAAAGCG-3') that was cloned into
pCR2.1 (Invitrogen) and verified by DNA sequencing. The pGL1-BD,
pTTG1-BD and pTRY-BD were generated by cloning a 0.62 kb
EcoRI/PstI fragment, 1 kb EcoRI/SalI
fragment and 0.32 kb EcoRI/BamHI fragment, respectively,
into the corresponding sites of pBridge (Clontech).
The construct used for the GL1-TRY competition assay, pGL1-BD/TRY-free, was generated by cloning a 0.32 kb NotI/BamHI fragment from pCR-TRY-FR into the NotI and BglII sites of pGL1-BD. pCR-TRY-FR was generated by cloning a PCR-generated TRY coding region from Columbia cDNA (forward 5'-GCGGCCGCAGCCATGGATAACACTGACCG-3' and reverse 5'-GGATCCCTAGGAAGGATAGATAGAAAAGCG-3') into pCR2.1 (Invitrogen).
The appropriate pGAD424- and pBridge-based constructs were transformed
sequentially into the yeast strain Y190 using electroporation. The cells were
selected on plates containing SD synthetic medium (2% glucose, 1x yeast
nitrogen base) lacking first only Leu, then Leu and Trp. Liquid cultures of SD
synthetic medium lacking Leu and Trp were used to measure ß-galactosidase
(ß-gal) activity (Ausubel et al.,
1995). The cells were grown to an OD600=0.7-1.0,
pelleted by centrifugation and suspended in z-buffer (60 mM
Na2HPO4, 40 mM NaH2PO4, 10 mM KCl,
1 mM MgSO4, 50 mM ß-mercaptoethanol, pH 7.0). The cells were
permeabilized by adding a final concentration of 0.005% SDS and 3.5%
chloroform (v/v). ONPG (o-nitrophenyl-D-galactopyranoside; Sigma) was added as
a substrate. After incubation at 30°C, the reaction was stopped with
sodium carbonate and measured for activity with an OD420. Units of
ß-gal activity was determine by the equation
U=1000x[OD420]/time (in seconds) x volume (in ml)
x [OD600]. For each comparison, multiple independent yeast
isolates (n=4-8) were tested multiple times (n=3-6).
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Results |
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The sst mutation was mapped to a chromosomal region containing the
candidate gene GL3. To determine if sst represented a new
allele of GL3, sst was crossed with the gl3-1 mutant. The
F1 plants had a distinct phenotype as shown in
Fig. 1L. The trichomes on these
plants tended to be less branched than wild-type trichomes, but the branches
were much larger in diameter than gl3-1 mutant trichomes
(Fig. 1K). These results
suggested that sst is an allele of GL3. To obtain molecular
evidence that sst was a new GL3 allele, the GL3
locus in the sst mutant was isolated by PCR and sequenced. The
GL3 locus of sst contained a single base change of C to T in
codon 78, which converts Leu to Phe. As a final indicator that sst
was a new allele of GL3, a green fluorescent protein (GFP)-tagged
version of the wild-type GL3 coding region was moved into
sst plants (see Materials and methods). This gene fusion was placed
under the control of the Arabidopsis MYB5 gene promoter, which has
previously been shown to drive gene expression in trichomes
(Li et al., 1996). This
construct rescued the phenotype resulting from both sst and
gl3-1 mutations (Fig.
2). These results demonstrated that sst represented a new
GL3 allele, which hereafter will be called gl3-sst.
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The DNA content of mature Col trichomes ranges from 16C to 64C
(Hülskamp et al., 1994;
Melaragno et al., 1993
;
Szymanski and Marks, 1998
).
The analysis of images of DAPI-stained gl3-sst trichome nuclei
indicated that the gl3-sst trichomes contain much more DNA than
wild-type or gl3-1 trichomes (compare
Fig. 4A, B and C, captured at
the same magnification and exposure settings). Interestingly, the DAPI-stained
gl3-sst mutant nuclei appeared to be composed of numerous lobes
collapsed upon one another. To observe the nuclei in living plants, Col and
gl3-sst plants expressing the nuclear-localized N7 GFP fusion were
used for confocal analysis (Cutler et al.,
2000
) (see Materials and methods). This analysis showed that
wild-type N7 nuclei were single spheres
(Fig. 4D), whereas the
gl3-sst N7 nuclei were composed of a series of
interconnected lobes (Fig.
4E,F).
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The gl3-sst mutation alters GL3 interaction with GL1, TTG1
and TRY
The mutation responsible for gl3-sst allele is in a region that
encodes a protein domain which mediates interaction with GL1 and TRY proteins
(Payne et al., 2000;
Zhang et al., 2003
). To
determine if the mutation affected protein-protein interactions, the yeast
two-hybrid system was used to compare the interactions of gl3-sst and GL3
proteins with GL1, TTG1 and TRY. To study the interactions, the coding regions
of the GL3 and gl3-sst were fused to the activation domain
(AD) of GAL4, whereas GL1, TRY and TTG1 were fused to the
binding domain (BD) of GAL4. Yeast containing either empty pGAD424 (AD) or
pBridge (BD) vectors in conjunction with any of the corresponding trichome
protein fusions did not exhibit significant ß-gal activity (data not
shown), whereas yeast containing either sst-AD or GL3-AD and GL1-BD, TRY-BD or
TTG1-BD exhibited ß-gal activity (Fig.
6A-C). However, there was a significant difference in activity
level between the strains containing either sst-AD or GL3-AD. Yeast isolates
containing GL1-BD and GL3-AD exhibited approximately fourfold higher levels of
ß-gal activity than isolates containing GL1-BD and sst-AD
(Fig. 6A). Yeast containing
TTG1-BD and GL3-AD exhibited approximately a twofold higher level of
ß-gal activity than isolates containing TTG1-BD and sst-AD
(Fig. 6B). Finally, it was
found that TRY-BD interacted equally well with either GL3-AD or sst-AD
(Fig. 6C).
|
To corroborate the yeast interaction data, the cellular location of the
GL3, GL1 and TRY proteins was ascertained. This analysis made use of the
GFP-GL3 fusion construct that was used to rescue the gl3-1
and gl3-sst mutants. In addition, GFP fusions with GL1 (GFP-GL1) and
TRY (GFP-TRY) were generated (see Materials and methods). The L1
layer-specific promoter from ATML1, which is upregulated in
developing trichomes, was used to drive the expression of GFP-GL1 and the
TRY promoter was used to express the GFP-TRY fusion
(Schellmann et al., 2002;
Sessions et al., 1999
). Both
of these constructs were able to rescue their respective mutants (see
supplemental Fig. S3 at
http://dev.biologists.org/supplemental/).
Strong gl1 mutants typically lack all adaxial leaf surface trichomes.
The expression of the GFP-GL1 construct in the gl1
background restored the development of wild-type-like trichomes. try
mutants typically have trichomes that are extra branched, and 10-15% of
try trichomes develop next to one another. The GFP-TRY
construct was able to rescue both of these phenotypes. As shown in
Fig. 7A-I, all of the fusion
proteins can be clearly detected in the trichome nuclei. The filter set used
to collect the images limits the fluorescence emission to the green range. At
the higher gain and exposure setting on the CCD camera, the primarily red
chlorophyll autofluorescence was still detected in the green range. This
background autofluoresecence was detected in the mesophyll cells of all
samples at the higher gain and exposure settings. In addition, a low
background of green autofluorescence was emitted from control Col wild-type
trichome cell walls at the higher settings. Importantly, Col wild-type
trichome nuclei lacked detectable fluorescence
(Fig. 7J-L). These results
place TRY, GL3 and GL1 proteins in the trichome nuclei, where they could
interact as observed in the yeast system.
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Discussion |
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The gl3-sst mutant is phenotypically distinct from the other
loss-of-function gl3 mutants. The trichomes on previous gl3
mutants typically had more slender stalks and fewer branches than wild-type
trichomes (Koornneef et al.,
1982; Payne et al.,
2000
). In contrast, the gl3-sst trichomes have a swollen
stalk and vary widely in branch number. Another difference is in the degree to
which the nuclear DNA of gl3-1 and gl3-sst trichomes
undergoes endoreduplication. Wild-type Col trichome nuclei typically contain a
range between 16C and 64C with an average of 32C, while gl3-1
trichomes average 16C (Hülskamp et
al., 1994
; Melaragno et al.,
1993
; Szymanski and Marks,
1998
). While the exact levels of nuclear DNA were not determined,
it was clear from the intensity of DAPI staining and the increased nuclear
size that gl3-sst trichomes exhibited higher than wild-type levels of
endoreduplication.
The EGL3 gene, located on chromosome one, is closely related to
GL3 (Payne et al.,
2000; Zhang et al.,
2003
). To determine if this gene has a role in the
gl3-sst phenotype, the genetic interactions between an EGL3
T-DNA mutant and both gl3-1 and gl3-sst were studied. As
previously reported, it was found that the trichomes that develop on
gl3-1 mutants required EGL3 function
(Zhang et al., 2003
).
Furthermore, it was found that gl3-sst plants produced trichomes in
the absence of a functional EGL3 gene. This showed that the
gl3-sst allele is functional.
Some gl3-sst trichome abnormalities may be due to
cytoskeletal alterations
Many of the mutant gl3-sst trichome defects could be due to
alterations in the microtubule cytoskeleton. For example, the swollen
appearance of early stage gl3-sst trichomes is similar to trichomes
on wild-type plants treated with microtubule destabilizing agents such as
oryzalin (Mathur and Chua,
2000; Szymanski et al.,
1999
). Likewise, the abnormally lobed structure of
gl3-sst trichomes nuclei is similar to nuclei in plants treated with
antimicrotubule drugs. In this case, the lobate nuclear structure is thought
to result from spindle microtubule disassembly, which results in a disruption
of karyokinesis.
gl3-sst trichomes exhibit cell wall defects
The majority of the cell walls of the mature gl3-sst leaf
trichomes had a `glassy' appearance when viewed by stereoscopic microscopy.
SEM analysis revealed that the walls lacked the papillae that are normally
associated with wild-type trichomes. This suggested that most gl3-sst
trichomes failed to undergo a final maturation step. To further analyze these
differences, the elemental composition of the trichome cell wall was
ascertained. Compared to wild-type trichomes, the gl3-sst
trichomes showed two forms of cell wall immaturity. In gl3-sst
trichomes with a papillate surface, magnesium, which was found throughout the
cell wall of wild-type trichome, and phosphorus, which was localized within
the wild-type trichome papillae, were both absent. In papillae-less
gl3-sst trichomes, calcium, in addition to magnesium and
phosphorus, was missing from the cell wall, which suggested that the
deposition of these elements play an important role in trichome cell wall
maturation.
Role of cell cycle control in the gl3-sst phenotype
Extra endoreduplication occurred in gl3-sst trichome nuclei. In
both Col wild-type and gl3-sst trichomes, the Lac OP/I-GFP reporter
experiments suggested that the chromatids of endoreduplicated chromosomes are
tightly associated with one another. Comparable results have also been
reported using a similar version of the Lac OP/I-GFP system to study the
nature of chromosomes in pavements cells
(Kato and Lam, 2001;
Kato and Lam, 2003
). However,
Kato and Lam (Kato and Lam,
2003
) reported the presence of multiple Lac I-GFP binding sites in
pavement cells, which is contradictory to the idea that the sister chromatids
are associated with one another as in polytene chromosomes. In the present
study, only single binding sites were observed in most nuclei, whether from
trichome, pavement, or guard cells. Overall our results suggest that the
chromatids of endoreduplicated chromosomes are highly associated with one
another (or are polytene). Furthermore, the results provided evidence that the
highly lobed gl3-sst nuclei contain single sets of chromosomes that
have not undergone karyokinesis.
The mutation in gl3-sst highlights the importance of
protein-protein interaction in trichome development
It was previously shown that GL1 and TTG1 can interact with GL3 in yeast
two-hybrid assays (Payne et al.,
2000). Thus, the interactions between either GL1 or TTG1 with
either GL3 or gl3-sst were compared. The location of the mutation in gl3-sst
in the 78th codon predicted that gl3-sst might display an aberrant interaction
with GL1, and indeed, this result was seen. The mutation resulted in a 75%
reduction in the interaction between gl3-sst and GL1, based upon reproducible
quantitative beta-gal assays. In addition, it was found that the gl3-sst
interaction with TTG1 was reduced by 50%. Since GL1, GL3 and TTG1 proteins
have been proposed to form an activating complex that regulates genes needed
for trichome development, it is likely that the gl3-sst phenotype is
due to a reduction in this complex.
A model based on the dynamic interactions between GL1, GL3 and TTG1
activating complex has previously been presented
(Szymanski et al., 2000). The
model proposed that the loss of trichomes on plants that overexpress the CPC
gene were due to the ability of CPC to inhibit the physical interaction
between GL3 and GL1. Because it has subsequently been shown that TRY
encodes a CPC-like protein, the model was modified to propose that TRY
functions to inhibit the physical interaction between GL3 and GL1
(Marks and Esch, 2003
). In
this paper, evidence is presented that supports this model. Competitive yeast
interaction assays demonstrated that TRY can interact with GL3 and that this
interaction can prevent the GL3-GL1 interaction. Interestingly, the mutation
in gl3-sst does not influence the interaction between gl3-sst and
TRY.
TRY has been found to be expressed in young trichomes and it has
been predicted that TRY protein can migrate from initiating trichomes into
neighboring cells (Schellmann et al.,
2002). Given the interaction data described, it is likely TRY
would be able to disrupt any residual GL1 GL3 complexes in neighboring cells
and that may be the mechanism by which TRY acts to inhibit the initiation of
neighboring trichomes.
Further support for TRY movement comes from the analysis of CPC.
It has recently been shown that CPC protein is produced in the non root hair
cells and can move into the adjacent files of cells that become root hairs
(Wada et al., 2002). In the
root, CPC is probably inhibiting the formation of a complex between WER and
GL3/EGL3. Direct movement of TRY could not be detected in this study, because
the level of fluorescence from the GFP-TRY fusion was below the background
autofluorescence emitted by chlorophyll in the mesophyll. Given the expression
pattern of TRY and the recent CPC findings, the movement of
TRY into cells neighboring developing trichomes would not be unexpected.
Threshold model for trichome initiation
It has been posited that a cell enters the trichome pathway only after a
critical threshold concentration of GL1 GL3 TTG1 complex has accumulated
(Szymanski et al., 2000). Once
the threshold is reached, it is predicted that an auto regulatory loop is
generated that results in the up-regulation of the complex genes. Over time,
as the level of the activator increases, it is predicted that different genes
needed for trichome differentiation would be induced. In order for trichome
initiation to proceed, it is predicted that genes needed to positively
influence trichome development would be expressed first and then genes needed
either to modulate or inhibit trichome development would be expressed later.
Genes activated early during trichome initiation, when the activator complex
would be relatively low, would be induced because they have a higher affinity
for the complex than genes that are activated later.
In terms of this threshold model it is interesting to note that different
promoters can be used to functionally drive the activator genes. For example,
it may seem surprising that the MYB5::cGL3 construct could rescue the
gl3-1 mutant trichome phenotype. However, the MYB5 promoter
is upregulated in developing trichomes and gl3-1 mutants do initiate
trichomes, albeit undersized ones (Li et
al., 1996). Thus, GL3 protein would be expressed in the trichomes
of gl3-1 plants containing the MYB5::cGL3 construct. Given
that native MYB5 expression increases as trichomes develop, it is
probable that the levels of GL3 protein would increase. This increase would
allow the sequential activation of positive and negative regulators of
trichome development as predicted by the threshold model. The ability of the
ATML1::cGL1 construct to rescue the gl1 mutant appears to be
further complicated by the fact that gl1 mutants do not initiate
trichomes. The ATML1 promoter is expressed at a low level in all the
epidermal cells of the developing leaf and in situ analyses have shown that
ATML1 expression is enhanced in young developing trichomes
(Sessions et al., 1999
). In
terms of the threshold model it is possible that the ATML1 promoter
provides a threshold level of GL1 protein in all the epidermal cells. However,
only the few cells that achieve the threshold level of GL3 enter the trichome
pathway. Once a cell enters the pathway the levels of ATML1
expression would be enhanced and the level of GL1 protein would increase,
again allowing the sequential activation of genes as predicted by the
threshold model.
Trichome development in gl3-sst provides some additional evidence for the threshold model. This support comes from two aspects of the gl3-sst phenotype, which may seem paradoxical. First, there are fewer trichomes on the leaves of gl3-sst plants, which could indicate increased trichome inhibition. In contrast, there are more trichome clusters, which suggest that trichome inhibition is relaxed. Given the reduced interaction between gl3-sst and both GL1 and TTG1, the reduction in trichome number could be the result of fewer cells reaching a critical concentration of activation complex. The reduced interaction between gl3-sst and GL1 or TTG1 could also result in increased trichome clustering if the inhibition signal emitted from gl3-sst trichomes was reduced. This latter possibility is supported by the observation that increased TRY expression in gl3-sst can prevent trichome clustering. Other aspects of the gl3-sst phenotype such as excess expansion and extra endoreduplication could be due to the unregulated activation of genes needed to mediate these cellular phenomenon during early trichome development. However, levels of activator complex needed to stimulate the expression of genes that either modulate or inhibit these early processes may not be reached in gl3-sst trichomes. Layered on top of this relatively simple threshold model, there is probably an added level of complexity. The activator complex possibly does not consist of just GL1, GL3/EGL3 and TTG1, but contains additional components as well. It is more likely that gene regulation in developing trichomes is controlled by both the quantity of activator complex and its composition. Thus, some aspects of the gl3-sst phenotype could result from aberrant ratios of complexes containing or lacking GL1, TTG1 or other components.
Perspectives
While the pleiotropic nature of the gl3-sst mutant trichomes may
appear confusing, this very feature provides an excellent resource for
studying all aspects of trichome development. These mutants are deficient in
trichome initiation, trichome cell patterning, cell expansion,
endoreduplication and maturation. Through future genetic screens for
suppressors and enhancers of the various components of the phenotype and
through detailed expression profiling of the mutant, the understanding of many
processes involved in cellular differentiation will be advanced.
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ACKNOWLEDGMENTS |
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Footnotes |
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