1 Botanical Institute, University of Cologne, Gyrhofstrasse 15, 50931 Cologne,
Germany
2 Department of Molecular, Cellular, and Developmental Biology, University of
Michigan, Ann Arbor, MI 48109, USA
3 Department of Botany, UF Genetics Institute, University of Florida, 220
Bartram Hall, PO Box 118526, Gainesville, FL 32611-8526, USA
Author for correspondence (e-mail:
martin.huelskamp{at}uni-koeln.de)
Accepted 19 January 2005
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SUMMARY |
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Key words: Trichomes, Cell shape, Redundancy, GLABRA1 (GL1)
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Introduction |
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A particularly attractive solution to addressing the mechanisms of gene
diversification in evolution is provided by the large family of MYB-related
R2R3 transcription factors in plants (Jin
and Martin, 1999; Stracke et
al., 2001
). It is estimated that this family has rapidly evolved
within the last 500 million years after the divergence of the vascular plants
from the bryophytes (Rabinowicz et al.,
1999
). It has been suggested that the amplification of the R2R3
MYB genes occurred in conjunction with the development of new plant-specific
cellular functions (Martin and Paz-Ares,
1997
) and that the role of MYB genes is to provide plasticity to
plant metabolism and development (Romero
et al., 1998
). This view is supported by the findings that members
of the R2R3 MYB-family are involved in many different biological processes,
including the trichome and root-hair differentiation
(Lee and Schiefelbein, 1999
;
Oppenheimer et al., 1991
),
cell-shape determination (Noda et al.,
1994
), regulation of leaf form
(Waites et al., 1998
), control
of secondary metabolism (Mol et al.,
1998
), pathogen response (Yang
and Klessig, 1996
), drought stress response
(Urao et al., 1993
),
protection from UV radiation (Jin et al.,
2000
) and hormone signalling
(Gubler et al., 1995
).
A particularly well-suited model for studying the diversification of
duplicated genes is the sub-class of the three paralogous R2R3 MYB genes,
WEREWOLF (WER), GLABRA1 (GL1) and
AtMYB23 (MYB23). WER and GL1 act in two different
functionally non-overlapping developmental processes. WER is
important for root-hair development and GL1 for trichome development
on aerial organs (Lee and Schiefelbein,
1999; Oppenheimer et al.,
1991
). WER and GL1 proteins with 57% of sequence identity are
functionally equivalent and changes in the cis-regulatory sequences completely
account for the functional diversification of the two proteins
(Lee and Schiefelbein, 2001
).
Overexpression studies and the analysis of the expression pattern suggested
that MYB23 could have a similar function to GL1
(Kirik et al., 2001
). The
identification of two myb23 mutants enabled us now to study the
function of MYB23 and its functional relationship to GL1.
The myb23 single mutants exhibit reduced trichome branching but no
obvious effect in trichome initiation. The gl1 myb23 double mutants,
however, are devoid of trichomes at the leaf edges, which are not effected in
the gl1 single mutants indicating a functional redundancy of
GL1 and MYB23. Promoter and protein-coding region swap
experiments showed that the two proteins are functionally equivalent with
respect to the regulation of trichome initiation, but not with respect to
trichome branching. This indicates that changes in the regulation of trichome
initiation are evolved at the level of the cis-regulatory regions and
that diversification with respect to the regulation of branching evolved at
the level of cis-regulatory regions, as well as at the level of altered
protein function.
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Materials and methods |
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Microscopy
Trichomes were analyzed on the first two leaves of soil-grown plants.
The histochemical analysis of plants containing the GUS reporter
constructs was performed essentially as described previously
(Vroemen et al., 1996).
The DISCUS software package (Carl H. Hilgers-Technisches Büro,
Königswinter, Germany) was used to measure the fluorescence intensity of
DAPI-stained nuclei. The relative fluorescence units (RFU) were calibrated
with wild type trichome nuclei that were previously reported to have an
average of 32C (Schnittger et al.,
1998; Szymanski and Marks,
1998
). By defining the average fluorescence intensity of the
wild-type trichomes as 32 RFU our RFU values should roughly correspond to C
values (Walker et al., 2000
).
As a control for this procedure, gl3 (16C) and try (64C)
mutants were included.
Molecular biology methods
RNA was isolated from rosettes of 2-week-old plants and subjected to
RT-PCR, which was essentially performed as described previously
(Kirik et al., 2002).
MYB23 gene-specific primers were used for RT-PCR (do-s2,
5'-AGAAGAATGAGAATGACAAGAG; and do-1, 5'-TACGTCAGTTGGTGTTGCGTGGAC).
Amplifications of the translation elongation factor EF1
A4 cDNA (primers
EF1
-UP: 5'-ATGCCCCAGGACATCGTGATTTCAT and EF1
-RP:
5'-TTGGCGGCACCCTTAGCTGGATCA) were used as a control.
The MYB23::cMYB23 was constructed by fusing the 2032 bp 5'
regulatory region (Kirik et al.,
2001) with the MYB23 cDNA and cloning the fusion in the
pGPTV-BAR vector (Becker et al.,
1992
).
The GL1 regulatory sequences used in these experiments are
identical to the ones published previously
(Lee and Schiefelbein, 2001)
and include a 1.4 kb 5'' fragment and a 1.8 kb 3' fragment. The
MYB23 regulatory sequences include a 3.1 kb 5' fragment and a
1.0 kb 3' fragment. Constructs encoding the GL1 and MYB23 proteins
include the entire transcriptional unit of each gene from the start to the
stop codons.
In the GL2::GL3 construct a 2.1 kb 5' GL2
regulatory region (Szymanski et al.,
1998) drives the expression of the full-length GL3 cDNA.
Details of the transgene constructs are available upon request.
In situ RNA hybridization
RNA in situ hybridisation was essentially performed as described previously
(Larkin et al., 1993).
Antisense and sense strand digoxigenin-labelled RNA probes were derived from
the 450 bp 3' fragment of the MYB23 cDNA that includes 180 bp of the
3' UTR. This cDNA region does not comprise the conserved MYB-domain
coding sequence and corresponds to the region with the lowest sequence
similarity to other related MYB genes.
Yeast two-hybrid assay
Fusions with the GAL4 activation domain and GAL4 DNA-binding domain were
performed in the pACT and pAS plasmids (Clontech). TRY, GL3 and N-terminal
truncation of GL3 (GL3-96 aa) were fused to the GAL4 activation domain in the
pACT plasmid. For the GL1 and MYB23 fusions with GAL4 DNA-binding domain in
the pAS vector, we used truncated fragments missing 27 amino acids and 25
amino acids at the C terminus respectively. All used constructs and empty
vectors did not show any self activation in yeasts. The interaction strength
was determined by measuring the activity of the lacZ reporter gene
using the ONPG assay (Clontech).
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Results |
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The MYB23 and GL1 proteins share 63% of identical amino acid residues,
which suggests that these two genes may have redundant or overlapping
functions (Kirik et al.,
2001). Plants that harbour a null gl1-1 mutant allele,
resulted from a deletion of the entire GL1 protein-coding region, are not
completely glabrous, a small number of trichomes develop at the edges of late
rosette leaves and petioles (Koornneef et
al., 1982
; Oppenheimer et al.,
1991
) (Fig. 2A). We
did not find any changes in trichome production at the leaf edges of the
myb23 mutants (data not shown). To test whether MYB23
function at the edges of leaves and petioles is masked by genetic redundancy,
we created the gl1 myb23-2 double mutant. The double mutant plants
were completely glabrous (Fig.
2A). The introduction of the MYB23::MYB23 construct in
the double mutant resulted in a rescue of the leaf edge and petiole trichomes
(Fig. 2B). Thus, the gl1
myb23-2 double mutant uncovers the redundancy of MYB23 and
GL1 in trichome initiation at the edges and petioles of the rosette
leaves.
|
The finding that the above-described MYB23::MYB23 construct
rescues the myb23 mutant phenotype indicates that the 3.1 kb of
5' regulatory sequence and 1 kb of 3' sequence contain all
regulatory sequences necessary for normal MYB23 gene function. For
the GL1 gene, we used 1.4 kb of the 5' regulatory region and
1.8 kb of the 3' region that was previously used in a complementation
construct (Lee and Schiefelbein,
2001). As a reference for the subsequent experiments, the
GL1::GL1 construct and the MYB23::MYB23 constructs were
introduced into the gl1 and the gl1 myb23-2 backgrounds.
Whereas the GL1::GL1 construct rescued completely the gl1
mutant phenotype, the MYB23::MYB23 construct showed no effect in
gl1 mutants but induced leaf edge and petiole trichomes development
in the double mutant (Fig. 2B).
To test whether MYB23 protein is functionally equivalent to the GL1 protein,
we transformed gl1 mutant and the gl1 myb23-2 double mutant
with the GL1::MYB23 and the MYB23::GL1 constructs. The
GL1::MYB23 gl1 and GL1::MYB23 gl1 myb23-2 transgenic plants
showed a wild-type trichome number and pattern
(Fig. 2B), indicating that the
MYB23 protein posses the same biochemical activities necessary for trichome
initiation as the GL1 protein. No effect of the MYB23:GL1 construct
was found in gl1 plants but gl1 myb23-2 plants were rescued
back to the gl1 phenotype (Fig.
2B). Together, these data demonstrate that during trichome
initiation the GL1 and MYB23 proteins are functionally interchangeable and
difference in function of these genes during trichome patterning is due to
differences in the transcriptional regulation.
Diversification of the MYB23 and GL1 genes with respect to branch regulation occurs at both the transcriptional regulation level and protein function
Reduced trichome branching of the myb23 mutants demonstrates that
MYB23 function is necessary for the proper trichome cell
morphogenesis. Can GL1 protein substitute this morphogenetic function of the
MYB23 protein? We tested this by introducing the MYB23::GL1 construct
into myb23-1 plants. The resulting transgenic plants showed only weak
rescue of the branching phenotype (Table
1). As MYB23::MYB23 showed full rescue of trichome
branching it is conceivable that GL1 protein is less active than MYB23 in
promoting trichome branching.
To study the importance of the transcriptional regulation in functional specification of the GL1 and MYB23 genes, we introduced the GL1::MYB23 construct into the myb23-1 mutant background. These plants displayed no rescue of trichome branching (Table 1), indicating that transcriptional control is pertinent for the regulation of branching by MYB23. Taken together, the functional diversification of the MYB23 and GL1 genes with respect to branching occurs at the level of both the transcriptional regulation and protein function.
Differences in the transcriptional regulation of MYB23 and GL1 during leaf development
Although the initial studies of the expression of MYB23 suggested
that it is expressed similarly as GL1
(Kirik et al., 2001), the
above-described studies suggest differences in transcriptional regulation
during early stages of trichome development.
To follow the expression of GL1 and MYB23 during trichome
development, we used GL1::GUS and MYB23::GUS reporter lines
(Kirik et al., 2001;
Larkin et al., 1993
). The
GL1::GUS reporter line was shown previously to adequately reflect the
transcription of the GL1 gene
(Larkin et al., 1993
). For
MYB23, we narrowed the relevant promoter region down to a 1.9 kb
region that was previously used to analyze the expression of the
AtMYB23 gene (Kirik et al.,
2001
). We used this fragment to drive the expression of the
MYB23 cDNA and found that this fragment is sufficient to rescue the
myb23 mutant phenotype in the gl1 myb23 background (data not
shown). This shows that the MYB23::GUS reporter provides a pertinent
proxy of the MYB23 gene expression.
A comparison of the reporter expression MYB23::GUS and
GL1::GUS in wild type revealed marked differences in the spatial and
temporal pattern. Initially, GL1::GUS is expressed ubiquitously in
the young leaf primordia and becomes more prominent in developing trichome
cells (Fig. 3B)
(Larkin et al., 1993). This
expression ceases when trichomes begin to initiate branches. By contrast,
using the same staining conditions we did not find a ubiquitous expression of
MYB23::GUS in young leaf primordia. Expression is confined to
developing trichome cells where it persists at high levels throughout all
stages of trichome development (Fig.
3B).
|
Comparable results were obtained using the respective promoter::GUS fusions. The ubiquitous expression of the GL1::GUS reporter is not affected in ttg1 and gl1 mutants (Fig. 3B). Expression of both the GL1::GUS and MYB23::GUS reporters was not affected in the gl3 mutant (data not shown), suggesting that reduced expression of MYB23 detected by RT-PCR is due to reduced number of trichomes in this mutant. As MYB23 is not initially ubiquitously expressed, we focused on the rare trichomes that occasionally formed at the leaf edges of gl1 and ttg1 mutants. In the gl1 mutant, both reporter constructs were active in trichomes. Rare trichomes in the ttg1 mutant showed expression of the GL1::GUS but not MYB23::GUS reporter, indicating that MYB23 gene transcription depends on TTG1 activity, whereas the GL1 gene transcription is not TTG1 dependent.
To verify the expression pattern of the MYB23::GUS reporter, we
localized MYB23 transcript by in situ hybridization
(Fig. 4). We detected a strong
hybridization signal in developing trichomes of young leaf primordia
(Fig. 4A) that was also
persistent in the older trichomes (Fig.
4B). In contrast to the GL1 mRNA in situ localization
reported by Larkin et al. (Larkin et al.,
1993), we did not find any epidermis-specific accumulation of the
MYB23 transcript, which is not confined to developing trichome
precursors.
|
In gl2-4AA mutants, the majority of trichomes fail to grow out and only trichomes at the leaf edges develop and branch. In the gl2-4AA myb23-2 double mutant, leaf edge trichomes do not grow out (Fig. 5H,I). Thus myb23 mutants enhance the morphogenesis defect in the gl2 mutant. Conversely, overexpression of MYB23 in trichomes using strong GL2 promoter partially rescued the gl2 mutant phenotype (Fig. 5J-M). This result was surprising as GL2 is thought to act downstream of GL1 and therefore was expected to also act downstream of MYB23. Both observations together suggest that MYB23 has a function in trichome cell morphogenesis at the same genetic level as GL2.
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To further elucidate the role of MYB23 and GL3 in
trichome branch formation, we overexpressed the two genes under the control of
the GL2 promoter, which drives a strong trichome-specific expression
in leaves (Szymanski et al.,
1998). Plants transformed with the GL2::GL3 construct
exhibited enlarged, strongly over-branched trichomes with up to 10 branches
(Fig. 5G;
Table 1). The trichome nuclear
size was drastically increased (Fig.
5G) with a ploidy level of on average 110C, suggesting that those
nuclei have undergone six endoreduplication cycles, two more than wild-type
trichome nuclei (Fig. 6).
Arabidopsis plants transformed with the GL2::MYB23 construct did not exhibit any changes in trichome size, branching and DNA endoreduplication level (average is 31.6 C; n=66), indicating that the wild-type level of MYB23 is not a limiting factor for endoreduplication cycles and branch initiation in wild-type plants. To test whether the inhibitory activity of TRY may suppress the possible effect of the GL2::MYB23 construct, we introduced this construct in the try mutant background. Overexpression of MYB23 under the GL2 promoter in try background resulted in ectopic trichome development but did not have any significant effect on the trichome branching and DNA content (try GL2::MYB23: 57C, n=64; try: 64C, n=65; Student's t-test: P=0.16).
Taken together, our data show that MYB23 is required for proper branch formation but, in contrast to GL3, increased MYB23 levels do not trigger additional branch formation. This suggests that the MYB23 concentration in trichomes is not limiting for induction of trichome branches.
Protein-protein interactions of MYB23 with other trichome patterning proteins
The functional equivalence of the GL1 and MYB23 proteins during trichome
initiation suggests that these proteins have similar biochemical properties.
The GL1 protein directly interacts with GL3, GL3 binds to the WD-40 protein
TTG1 but GL1 does not interact with TTG1. It has been suggested that these
three proteins form the activator complex
(Payne et al., 2000;
Szymanski et al., 2000
). To
test whether MYB23 interacts with the GL3 protein, we made fusion constructs
of the GL3 cDNA with the GAL4 activation domain (AD) and
MYB23 cDNA with the GAL4 DNA binding domain (DB). Yeast two hybrid
assays revealed an interaction between GL3 and MYB23
(Fig. 7) but not with TTG1
(data not shown). Payne et al. (Payne et
al., 2000
) demonstrated that GL3 interacts with GL1 through the
N-terminal end of the GL3 protein. We found that the truncation of 96 amino
acids from the N-terminal end of the GL3 protein also abolished the
interaction between GL3 and MYB23 (Fig.
7). These results suggest that MYB23 interacts with the same GL3
protein domain as GL1.
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Discussion |
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Functional specification of paralogous genes provides a valuable
experimental system with which to study the molecular mechanisms of gene
diversification. In this study, we have investigated the functional
diversification of MYB23 and GL1. They share a sequence
identity of 63% over the entire protein and 92% in the actual MYB domain.
MYB23 and GL1 are located on chromosomes III and V in the
regions that originated from chromosomal duplication
(The Arabidopsis Genome Initiative,
2000). It is therefore likely that the two genes are derived from
gene duplication. Our genetic analysis revealed that MYB23 and
GL1 have a partially redundant function with respect to trichome
initiation, and that MYB23 has an additional role in the regulation
of trichome branching. The analysis of their functional specification by
promoter and protein-coding region swap experiments, expression studies and
protein-protein interaction studies gave insights in the functional
diversification of the two genes.
Functional divergence of cis-regulatory sequences of GL1 and MYB23
In contrast to GL1 and WER genes, which have completely
non-overlapping expression domains and unrelated functions
(Lee and Schiefelbein, 2001),
GL1 and MYB23 are both expressed in trichomes and regulate
their development. Our swapping experiments, however, revealed that their
actual regulation is significantly different and not interchangable. In this
study, we have not specifically addressed the regulatory role of introns,
which have recently been shown to play a role in GL1 regulation
(Wang et al., 2004
). However,
as GL1::MYB23 and GL1::GL1 or MYB23::MYB23 and
MYB23::GL1 constructs rescued the corresponding mutants equally well,
it is unlikely that the coding region and the introns carry any information
relevant for the regulatory differences between GL1 and
MYB23.
A comparison of their temporal and spatial expression revealed two
differences. First, GL1 but not MYB23 is initially expressed
ubiquitously in young leaves; and second, MYB23 is expressed
throughout all stages of trichome development, whereas GL1 expression
ceases long before trichome development is completed. Both aspects can be
correlated with their respective functions. The initial ubiquitous expression
of GL1 is thought to be important for early pattern formation
(Hulskamp, 2004;
Larkin et al., 2003
). It is
postulated that the initial ubiquitous expression of GL1 and other
positive regulators of trichome initiation triggers a patterning mechanism
that leads to a regular pattern of differentiated cells in which the positive
regulators, including GL1 are upregulated. Consistent with the
finding that MYB23 is not expressed in these initial stages,
MYB23 has only a subtle role in trichome initiation and the
expression of GL1 under the MYB23 promoter cannot rescue
gl1 mutants.
In addition, the extended expression of MYB23 in developing trichomes is consistent with the primary role of MYB23 gene in trichome branching. This extended expression of MYB23 is functionally relevant, as trichome branching in myb23 mutants is not rescued when MYB23 is expressed under the control of the GL1 promoter.
Some of these regulatory changes can be correlated with different responses to known trichome patterning genes. Although GL1 expression is independent from GL3, GL1 and TTG1, the expression of MYB23 in leaf trichomes is regulated by TTG1 but not by GL3 and GL1.
Functional divergence of GL1 and MYB23 proteins
Despite the relative low sequence identity of GL1 and MYB23, the two
proteins can functionally replace each other during trichome initiation.
Trichome initiation on the whole leaf surface was rescued, with the
GL1 promoter driving either MYB23 or GL1 protein; trichome initiation
at the leaf edges and petioles of the gl1 myb23 double mutant was
rescued with the MYB23 promoter driving either GL1 or MYB23
proteins.
In accordance with the ability of the MYB23 and GL1 proteins to rescue the trichome initiation phenotype of each mutant, yeast two-hybrid assay showed that MYB23 protein interactions with known trichome patterning proteins are similar to those found with GL1. Therefore it came as a surprise that protein functions are not fully conserved during trichome branching. When GL1 is expressed under the MYB23 promoter, trichome branching was not completely rescued, indicating that GL1 cannot fully replace the protein function of MYB23 in this developmental context. This raises the question of whether the regulation of trichome branching by MYB23 involves the same downstream genes as during trichome patterning. Although the current data do not allow the determination of this, some observations suggest that this is the case. Notably, mutations in GL3 and TTG1 not only lead to patterning defects but also to reduced branching; mutations in TRY, a gene that inhibits trichome initiation, also result in increased branching. A conceivable scenario is that, similar as postulated in the context of pattern formation, TTG1 and GL3 form a branch-promoting complex together with MYB23 and that TRY counteracts this by competing with MYB23 for the binding with GL3. One possible explanation for the difference between GL1 and MYB23 proteins in this context is that MYB23 protein may endow the trimeric complex with higher activity in the promotion of cell growth and DNA endoreduplication.
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ACKNOWLEDGMENTS |
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Footnotes |
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