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First published online May 16, 2007
doi: 10.1242/10.1242/dev.005017
1 CNAP, Department of Biology (7), University of York, York YO10 5YW, UK.
2 Department of Biological Sciences and Temasek Life Sciences Laboratory,
National University of Singapore, 117543, Singapore.
* Author for correspondence (e-mail: pb22{at}york.ac.uk)
Accepted 18 March 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Cytokinins, Epidermis, Gibberellins, Transcription factor, Trichomes, Arabidopsis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Achard et al.,
2003; Fu and Harberd,
2003; Peng et al.,
1997; Silverstone et al.,
1998). As another strategy, which is suggested by gene expression
studies, they deploy more specialized regulators that may act further
downstream and control distinct gene networks
(Nemhauser et al., 2006).
Little information is available so far on the nature of such regulators.
It is known that epidermal differentiation is regulated by hormone signals
in plants. In particular, hormone levels affect the density of trichomes,
which are large defensive epidermal structures found on aerial organs in many
plant species (Chien and Sussex,
1996; Gan et al.,
2006; Greenboim-Wainberg et
al., 2005; Kazama et al.,
2004; Perazza et al.,
1998; Telfer et al.,
1997; Traw and Bergelson,
2003). As trichome production is tightly regulated and easily
monitored, it is a robust system for studying how epidermal differentiation is
controlled and the role played by phytohormones in this process. Trichome
initiation in Arabidopsis requires gibberellin signalling, and GA
applications have been shown to cause an increase in trichome density on
leaves and stems (Chien and Sussex,
1996; Gan et al.,
2006; Perazza et al.,
1998; Telfer et al.,
1997). In inflorescence organs, gibberellins act in part through
the transcription factor GLABROUS INFLORESCENCE STEMS (GIS), a positive
regulator of trichome initiation that acts upstream of GLABROUS1 (GL1)
(Gan et al., 2006).
Recent data indicate that cytokinins can also stimulate trichome
initiation, as cytokinin applications to flowering Arabidopsis plants
cause trichome proliferation on flowers. This effect is counteracted by
mutations in SPINDLY, which positively regulates cytokinin signalling
(Greenboim-Wainberg et al.,
2005). The stimulation of trichome initiation by both GA and
cytokinins contrasts with other, conflicting effects of the two hormone
signalling pathways. For example, gibberellin applications inhibit the effect
of cytokinin treatments and block cytokinin signalling
(Ezura and Harberd, 1995;
Greenboim-Wainberg et al.,
2005). Reciprocally, increases in cytokinin levels cause changes
in gene expression that antagonise GA signalling
(Brenner et al., 2005). These
opposing effects have been found to be particularly important in the
maintenance of the shoot meristem
(Jasinski et al., 2005;
Yanai et al., 2005).
In the present study, we have investigated molecular mechanisms through which gibberellin and cytokinin signalling modulate epidermal differentiation during inflorescence development. We report that the integration of cytokinin and gibberellin signalling requires the collective action of GIS and two novel related transcription factors, ZFP8 and GIS2. We show that GIS, ZFP8 and GIS2 are functionally interchangeable activators of trichome production and that the corresponding genes have specialized to play distinct roles in GA and cytokinin responses during development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isolation of novel mutants and construction of multiple mutants
A transgenic line carrying a transposon in the exon of GIS2
(SM_3_32778) was obtained from NASC (catalogue number N119489). Homozygous
gis2 mutants were selected using Basta (40 µm) and by PCR using
gene-specific primers (5'-AATCATCAAGGCAAATCCGCA-3' and
5'-TGCTACCACCACCGACATACG-3'), alone or in combination with a
transposon-specific primer (Spm1:
5'-CTTATTTCAGTAAGAGTGTGGGGTTTTGG-3')
(Tissier et al., 1999).
Zfp8 was identified from the SAIL collection (SALK_045674) and also
obtained from NASC (catalogue number N545674). Homozygous zfp8
mutants were selected on kanamycin (50 µg/ml) and by PCR using
gene-specific primers (5'-TTTGAAAAAGGAGGATTGATGG-3' and
5'-TCGGAGTTATCACCGACGAGC-3') or with a
gene-specific/T-DNA-specific primer combination (T-DNA primer:
5'-GCGTGGACCGCTTGCTGCAACT-3'). gis gis2 and gis2
spy-3 double mutants were selected from F2 populations by double
selection on basta and sulfadiazine (5.2 mg/l) or basta and paclobutrazole
respectively. gis, gis2 and gis gis2 lines in which
ZFP8 is silenced were obtained by transforming a ZFP8 RNAi
construct into these mutants.
Hormone treatments
GA3 (Sigma) and BA (6-Benzylamino-Purine, Sigma) were used in all
experiments that involved exogenous GA and cytokinin treatments respectively.
Control and mutant plants were grown on soil until the first three to four
leaves had emerged and sprayed twice a week with GA3, BA or mock solutions
until the plants were ready for analysis. For measuring the effect of GA
applications on gene expression, a minimum of eight mutant and control plants
were grown on soil until young inflorescence shoots had reached a size of 2-3
cm. The plants were then sprayed with either 100 µM GA3, 100 µM BA or a
mock solution and the shoots were harvested 4 hours (GA) or 2 hours (BA) after
treatment for RNA extraction.
Molecular biology
RNA extraction, real-time and semi-quantitative RT-PCR
Plant RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad,
USA) according to the manufacturer's protocol. Pooled tissue samples from at
least eight soil-grown plants were used for RNA extractions. The following
gene-specific primer sequences were used for real-time PCR analysis:
GIS, 5'-TTCATGAACGTCGAATCCTTCTC-3' and
5'-ACGAATGGGTTTAGGGTTCTTATCT-3'; ZFP8,
5'-AAGCCGCCATTATTCGTCTCT-3' and
5'-CTGCGGATAAGTTGTCGGAGTT-3'; GIS2,
5'-ACCGCCAACAAAACCACATT-3' and
5'-CGCGTCGTTGATTTGAACAG-3'; ARR5,
5'-TTGCGTCCCGAGATGTTAGAT-3' and
5'-TGAGTAACCGCTCGATGAACTTC-3'; UBQ10,
5'-GGTTCGTACCTTTGTCCAAGCA-3' and
5'-CCTTCGTTAAACCAAGCTCAGTATC-3'; GL1,
5'-CGACTCTCCACCGTCATTGTT-3' and
5'-TTCTCGTAGATATTTTCTTGTTGATGATG-3'. Q-PCR primer design and
reaction conditions were as previously described
(Gan et al., 2006).
UBQ10 transcripts were used as an internal control for normalizing
expression of the other genes (Gan et al.,
2005).
Semi-quantitative PCR analysis of GIS2 gene expression in gis2 mutants was performed with the same primers that were used to clone the full-length coding sequence of the gene (see below). cDNA template amounts were first adjusted according to UBQ10 product intensities and GIS2 amplification was performed over 41 cycles.
Cloning
For the production of all overexpression and RNAi constructs, sequences
were first inserted into the Gateway entry vector pENTR-1A (Invitrogen),
before recombination into the appropriate destination vector using the Gateway
LR reaction (Invitrogen). All destination vectors were obtained from VIB
(Flanders Interuniversity Institute). pH2GW7 (carrying a hygromycin-resistance
gene) was used as the destination vector for 35S:ZFP8 and
35S:GIS2 constructs; pK7GWIWG2(II) (carrying NPTII) and pB7GWIWG2(II)
(Basta resistance), respectively, for ZFP8-RNAi and
GIS2-RNAi constructs; gene-specific fragments were first
PCR-amplified from inflorescence cDNA (overexpression and RNAi constructs) or
genomic DNA (promoter fusion constructs) using primers containing
SalI and NotI restriction sites. The following primers were
used: ZFP8 overexpression:
5'-TTCCATTGTCGACTCTCCCTGATCTCTCTCTTCC-3' and
5'-TTGCATTGCGGCCGCTTCACCGATCAGCGAGTCT-3'; GIS2
overexpression: 5'-TCAACTGTCGACAGCCATCCAGAGTCATAACCA-3'
and 5'-AAGATAGCGGCCGCGAATGGAACTAGAGGCGTAGA-3'; ZFP8-RNAi
construct: 5'-ACTTGTCGACCACCACATCTACGGCTTCCT-3' and
5'-ATTGCGGCCGCTTGCACATTGGGTTTCATCA-3'; GIS2-RNAi
construct: 5'-ATTCGTCGACTTCAACCTCCATTCAAACG-3' and
5'-AAGCGGCCGCGAATGGAACTAGAGGCGTAGA-3'.
For the production of pGIS promoter fusion constructs, a 1.6 kb GIS promoter fragment was amplified using the primers 5'-ATCTTGGAGCTCGGGGGAATGAGTCAAGAGTTC-3' and 5'-ATCTTGTCTAGAGAGAGATAAAAAGACTGGGCG-3' and substituted for the 35S promoter in pH2GW7 after restriction with SacI and SpeI. The coding sequences of GIS, ZFP8 and GIS2 were then recombined into the modified vector from the same entry vector that was used for the production of overexpression constructs. A modified strategy was taken for pGIS2 constructs: a 1.5 kb GIS2 promoter fragment was amplified using primers 5'-TCCTGAGAGCTCTCTCAAGTTGGCTTCGTGTG-3' and 5'-TTAGTAGCGCTAGCGGTGGTTATGACTCTGGATGG-3', restricted with SacI, then ligated into the vector fragment of pH2GW7 that was first restricted with SpeI, blunt-ended, then cut with SacI. The coding sequences of GIS, ZFP8 and GIS2 were then recombined as above.
All binary vector constructs were introduced into Agrobacterium
strain GV3101 by electroporation. Agrobacterium-mediated
transformation of all Arabidopsis genotypes was performed using the
floral dip method (Clough and Bent,
1998).
In situ hybridization
Non-radioactive in situ hybridization was performed as previously described
(Gan et al., 2006). For
synthesis of the ZFP8 and GIS2 RNA probes, gene-specific
fragments were amplified using the same primers as for generating the RNAi
constructs (see above).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). We observed that applications of 6-benzylaminopurine (BA)
also stimulate trichome production on cauline leaves and stems and that this
effect increases in intensity with successive branches or paraclades
(Fig. 1A). As evidence that
cytokinin is not only limiting, but also required for the production of
trichomes, we found that trichome initiation is markedly decreased on flowers
and on upper inflorescence stems of Arabidopsis plants in which the
gene encoding cytokinin breakdown enzyme CKX2 is overexpressed
(Werner et al., 2003).
Similarly, we found that the flowers of loss-of-function SPY mutant
spy-3 are glabrous (Table
1).
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;
Perazza et al., 1998;
Telfer et al., 1997). We
therefore examined in detail the effects on epidermal differentiation of
applying a wide range of GA3 concentrations to wild-type plants and
GA-deficient ga1-3 mutants. GA applications restored trichome
initiation on normally glabrous ga1-3 flowers, although GA
concentrations exceeding 10 µM caused a decrease, rather than a further
increase, in trichome production - an effect also seen with wild-type plants
(Fig. 2A,B). At GA
concentrations exceeding 100 µM, we also observed a reduction in trichome
production on cauline leaves and branches in ga1-3
(Fig. 2A). These results were
consistent with the inflorescence trichome phenotype of spy-3, in
which GA signalling is increased and cytokinin signalling inhibited
(Table 1)
(Greenboim-Wainberg et al.,
2005). The above observations indicated that gibberellins and cytokinins play changing roles in epidermal differentiation as the inflorescence develops. GA signalling is required throughout development, while the requirement for cytokinins is limited to upper inflorescence organs. Consistently with the antagonistic roles of GA and cytokinins, the growing requirement for cytokinin signalling during inflorescence development is accompanied with an increasingly inhibitory effect of GA.
GIS homologues ZFP8 and GIS2 are required for trichome initiation late in inflorescence development and, in contrast to GIS, are necessary for the cytokinin response
The transcription factor GIS is required for trichome production on
inflorescence organs and modulates the regulation by GA of trichome initiation
(Gan et al., 2006). On the
basis of the observed interplay between gibberellins and cytokinins on
inflorescence trichome initiation, we asked whether GIS also mediates the
cytokinin response. To this end, we tested the effects of cytokinin
applications on trichome production in the gis loss-of-function
mutant. As the overall response of cauline leaves and flowers to cytokinin
treatments was similar in gis and control plants
(Fig. 1A), we concluded that
GIS is not required in this process.
We previously identified two genes, At2g41940 (ZFP8) and
At5g06650, that encode proteins closely similar in sequence to GIS
(Gan et al., 2006;
Tague and Goodman, 1995).
Moreover, their relatedness to GIS suggested that they might play a redundant
role in the control of trichome production. We therefore investigated whether
ZFP8 and At5g06650, which we termed GIS2, play a role in
this process and investigated their possible implication in the cytokinin
response. We first searched public collections for mutants containing
insertions in either of the two genes and identified two lines meeting this
criterion. One line carried a transposon 166 bp upstream of the start codon in
GIS2 and the other a T-DNA in the promoter region of ZFP8,
139 bp upstream of the coding region. We found using RT-PCR that the
expression of either of these genes was strongly downregulated or abolished in
these lines (Fig. 1B). To
further assess possible effects of loss of function, we also produced
transgenic plants in which ZFP8 and GIS2 were silenced by
RNAi, and selected lines in which their expression was significantly
downregulated (Fig. 1B; see
Table S1 in the supplementary material). We then examined the trichome
phenotype of T-DNA and RNAi lines.
We observed that ZFP8 loss of function led to a significant reduction in trichome density on upper cauline leaves and branches (Fig. 3C; Table 2). By contrast, lines in which GIS2 was knocked out showed a very strong decrease in trichome production on flowers (Fig. 3B) but only a small decrease on branches and cauline leaves, which was only noticeable on the third paraclade (Table 2). The phenotype of the different lines was otherwise similar to that of controls. In particular, we did not observe significant differences in trichome production on vegetative organs (see Table S2 in the supplementary material). As a confirmation that the mutant phenotypes were caused by the insertions, we found that silencing either of the genes by RNAi had a similar impact on trichome initiation (see Table S1 in the supplementary material). We were also able to complement the zfp8 and gis2 mutants by expressing the coding regions of the corresponding genes under either native or constitutive promoters (see Figs S1, S2 in the supplementary material).
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GIS, ZFP8 and GIS2 encode functionally equivalent proteins and their effects on trichome initiation are additive
To assess the level of functional redundancy between GIS, ZFP8 and
GIS2, we first examined the effects of overexpressing ZFP8
and GIS2 in a wild-type background. We had found that overexpressing
GIS leads to high levels of trichome initiation on all inflorescence
organs and ectopic trichome formation on floral organs
(Gan et al., 2006). As a first
indication that the activities of GIS, ZFP8 and GIS2 are similar, we found
that 35S:ZFP8 and 35S:GIS2 plants closely resembled
GIS overexpressors. In particular, trichome production on
inflorescence stems and leaves was increased and ectopic trichomes were
visible on flowers, in particular carpels
(Fig. 3D). This phenotype was
visible in more than half of the 30 35S:ZFP8 and 35S:GIS2
transgenic lines that we examined. As in 35S:GIS plants,
overexpression of either of these genes also delayed flowering and caused the
appearance of aerial rosettes (data not shown)
(Gan et al., 2006).
As a second step, we tested whether the coding regions of GIS,
ZFP8 and GIS2 had the ability to complement any of the gis,
zfp8 or gis2 mutants. We first used the 35S promoter to
overexpress the three coding regions in the different mutant backgrounds and
obtained a minimum of 30 transformants with each the overexpression
constructs. We found that, in at least 40% of the transgenic lines,
overexpression of either of the three genes was sufficient to restore trichome
production on cauline leaves, stems or flowers to normal levels or higher in
all of the mutants (examples shown on Fig.
3E and Fig. S2 in the supplementary material). As the effects of
ectopic expression could have masked minor differences in transcription factor
activities, we repeated our complementation experiment of the gis and
gis2 mutants using promoter fragments of GIS (pGIS)
or GIS2 (pGIS2), obtaining a minimum of 40 transgenic lines
for each of the combinations. We first confirmed that pGIS and
pGIS2 had comparable levels of activity to native regulatory
sequences by complementing the gis and gis2 mutants with
pGIS:GIS (Fig. 3F) and
pGIS2:GIS2, respectively (see Fig. S1 in the supplementary material).
Complementation was observed in six pGIS:GIS gis and seven
pGIS2:GIS2 gis2 lines, respectively. We then transformed the
gis mutant with pGIS:ZFP8 and pGIS:GIS2 constructs
and the gis2 mutant using pGIS2:GIS and pGIS2:ZFP8
constructs, generating a minimum of ten transgenic lines with each construct.
Consistently with the results of constitutive overexpression, at least four
independent lines had a trichome phenotype that was indistinguishable from
wild type. Interestingly, while 35S:ZFP8 and 35S:GIS2
constructs could not restore the morphology of gis2 stem trichomes,
which produce supernumerary branches (Fig.
3E) (Gan et al.,
2006), pGIS:ZFP8 gis and pGIS:GIS2 gis plants
(like pGIS:GIS gis plants) produced normal trichomes
(Fig. 3F). Possibly, the timing
and localization of pGIS-driven expression were more appropriate for
restoring a normal trichome developmental programme than 35S-driven
expression. We also found that expression of pGIS2:GIS and
pGIS2:ZFP8 constructs in the gis2 mutant restored trichome
production on flowers (see Fig. S1 in the supplementary material). The results
of these cross-complementation experiments provided strong evidence that GIS,
ZFP8 and GIS2 have largely equivalent activities, at least in the control of
trichome production.
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GIS2 and GL1 are cytokinin-inducible, but not GIS or ZFP8
To start defining how cytokinins might modulate ZFP8 and GIS2 action and
ultimately trichome initiation, we measured the expression of ZFP8,
GIS2 and GL1 in wild-type inflorescence organs in response to
increases in cytokinin signalling. For this analysis, developing inflorescence
organs were harvested 2 hours after BA treatment. The strong increase in the
expression of primary response gene ARR5 indicated that hormone
applications led to a significant increase in cytokinin signalling. They also
caused a significant elevation in GIS2 and GL1 transcript
levels. This response was abolished in the spy-3 mutant, an
indication that the transcriptional effect was specifically due to variations
in cytokinin signalling and required SPY
(Fig. 4). By contrast, this
treatment had little effect on ZFP8 expression and, consistently with
the cytokinin response of gis mutant plants, no effect on
GIS transcript levels. These observations indicated that, in line
with the contrasting sensitivities of gis, zfp8 and gis2 to
cytokinin applications, GIS, ZFP8 and GIS2 are also
differentially responsive to increases in cytokinin signalling. They also
suggested that the induction of trichome initiation by cytokinins proceeds in
part through the transcriptional activation of GIS2 and
GL1.
|
To determine whether increasing transcript levels of these redundant genes can be sufficient for saturating the response, we examined the effect of GA applications on 35S:GIS2 overexpressors. We found that GA applications had no effect on trichome production on lower cauline leaves and branches, where they normally stimulate initiation, or on flowers, where they are normally inhibitory (see Fig. S4 in the supplementary material). These observations suggested that the control of trichome initiation by gibberellins in Arabidopsis inflorescence organs requires the transcriptional regulation of GIS, ZFP8 and/or GIS2.
On the basis these results, we examined whether exogenous GA treatments
affect ZFP8 and GIS2 gene expression. We found that GA
applications to ga1-3 mutants strongly induced the expression of
ZFP8 and GIS2, as we had previously observed with
GIS and GL1 (Fig.
4) (Gan et al.,
2006). This result indicated that, although they differ from
GIS in their response to cytokinins, ZFP8 and GIS2
are similarly inducible by GA.
GIS2 and ZFP8 act downstream of SPY and upstream of GL1
To further investigate the genetic control of trichome initiation by
cytokinins, we first examined genetic interactions between GIS2 and
SPY. As GIS2 loss of function strongly inhibits trichome initiation
on sepals and spy-3 flowers are glabrous
(Fig. 5A), we first
overexpressed GIS2 in the spy-3 background. We found that
trichome initiation was restored on flowers of 35S:GIS2 spy-3 plants
(Fig. 5B), a result that was
also obtained when ZFP8 was overexpressed in spy-3 (see Fig.
S3 in the supplementary material). We also produced gis2 spy-3 double
mutants and found that their trichome production was most similar to
gis2 on branches and cauline leaves but that they produced glabrous
flowers (Table 3).
|
). We found that both 35S:GIS2 gl1 and
35S:GIS2 gl3 lines were glabrous, like gl1 and gl3
(Fig. 5C). Conversely, as we
had seen in the case of gis mutants, overexpressing in gis2
the maize R gene, which is functionally equivalent to GL3,
restored trichome initiation on flowers and increased trichome production on
branches (Fig. 5D). Similarly,
R overexpression increased trichome density on cauline leaves in
zfp8 (see Fig. S3 in the supplementary material), but ZFP8
overexpression did not induce trichome production on gl1 or
gl3 plants (data not shown). We also found that GL1
expression was increased in inflorescence organs of 35S:GIS2 plants
but decreased in gis2 mutants, in particular late in development
(Fig. 5E). The results of these experiments suggested that GIS2 is a positive regulator of GL1 expression and that both ZFP8 and GIS2 act downstream of SPY but upstream of the trichome initiation complex.
ZFP8 and GIS2 are differentially expressed in inflorescence organs
To determine whether the expression pattern of the genes in the plant are
consistent with their roles in trichome initiation and hormone signalling, we
examined the distribution of ZFP8 and GIS2 transcripts in
different tissues using quantitative RT-PCR and in the inflorescence by in
situ hybridization. ZFP8 and GIS2 were found to be expressed
at early stages of inflorescence development but at different levels in
different inflorescence organs. Specifically, ZFP8 was most expressed
in cauline leaves (Fig. 6B,D,E)
and GIS2 in the primary and secondary meristems and in developing
flowers (Fig. 6A,C,E). We also
found that GIS2 was expressed at increasing levels in successive
cauline leaves (Fig. 6E). These
patterns of expression were consistent with the phenotype of gis2,
which is most pronounced in later cauline leaves and flowers, and with the
cauline leaf phenotype of zfp8. They were also consistent with the
patterns of hormone response during inflorescence development and the relative
influences of ZFP8 and GIS2 on this process.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Integration of hormone signalling by transcription factors
Our findings show that related transcription factors play different roles
in regulating the influence of different hormone signals on epidermal
differentiation in Arabidopsis. The roles of GIS, ZFP8 and GIS2
appear to be mainly in the modulation of specific epidermal responses to
cytokinins and gibberellins, as the triple mutant does not show a general
hormone response phenotype. In this respect, the GIS clade differs from
regulators such as the DELLA transcription factors, which play a general role
in modulating growth and differentiation in response to a variety of hormone
signals (Achard et al., 2006;
Achard et al., 2003;
Fu and Harberd, 2003). In a
further contrast with GIS, ZFP8 and GIS2, the DELLA proteins are
post-transcriptionally regulated (Dill et
al., 2004; Sasaki et al.,
2003; Silverstone et al.,
2001), and the relative influence of the different members of the
clade may depend more on their abundance in a particular tissue than on their
specific responsiveness to hormone signals
(Sun and Gubler, 2004). In
mainly modulating hormone responses, the GIS clade proteins also differ from
the KNOX-type transcription factors that regulate gibberellin and cytokinin
signalling in the meristem, as the latter act upstream, rather than
downstream, of the hormone biosynthesis pathways
(Jasinski et al., 2005;
Yanai et al., 2005). The same
holds true of FUSCA 3, which has been proposed to regulate gibberellin and
abscisic acid biosynthesis in the embryo
(Gazzarrini et al., 2004). The
role of the GIS clade therefore highlights the central role played by
transcription factors not only upstream but also downstream of hormone
production.
Functional specialization of GIS, ZFP8 and GIS2
The GIS, ZFP8 and GIS2 genes encode functionally
equivalent proteins but have diverged in their response to phytohormones and
in their role during inflorescence development. The functional specialization
of genes encoding paralogous transcription factors appears to be an important
mechanism through which plants regulate similar differentiation programs at
different stages in development. For example, MYB transcription factor
WEREWOLF 1 (WER) is functionally equivalent to GL1; however, WER is
expressed in the root, where it regulates epidermal hair development, whereas
GL1 regulates trichome development in the shoot
(Lee and Schiefelbein, 2001).
Similarly, GL1 and MYB23 are functionally interchangeable, but MYB23
and GL1 have distinct although overlapping expression domains during
leaf development (Kirik et al.,
2005).
|
) and both
the roles and inducibility of the genes in response to cytokinins are clearly
different. Rather, our data suggest that GIS, ZFP8 and GIS2
have diverged in their responses to both developmental and hormonal signals
and that these signals are not strictly interdependent.
|
). The
heterochronic phenotypes of 35S:ZFP8 and 35S:GIS2 lines
could indicate that ZFP8 and GIS2 also play a role in phase change, although
these phenotypes may just reflect the fact that the three transcription
factors have similar activities. Because of the localized nature of the
zfp8 and gis2 phenotypes, it seems unlikely that ZFP8 and
GIS2 play a significant role in phase change. As gis ZFP8-R gis2
mutants go through normal phase transition, such a role, if it exits, is not
just masked by genetic redundancy between GIS, ZFP8 and GIS2. Elucidating how
increases in GIS activity influence phase change therefore awaits further
investigation. In conclusion, our results illustrate the importance of functional specialization within a plant transcription factor family in the control of cellular differentiation and in response to various hormonal and developmental cues. A better understanding of how developmental and hormonal signals are integrated in plants is likely to emerge as our knowledge of transcription factor function becomes more complete. Progress in this field will also benefit from system biology approaches that integrate the analysis of gene networks with the precise mapping of hormone influence within the plant.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/11/2073/DC1
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ACKNOWLEDGMENTS |
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