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First published online 13 March 2008
doi: 10.1242/dev.020255
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1 Department of Biological Sciences, Faculty of Science, National University of
Singapore, Singapore 117543, Singapore.
2 Temasek Life Sciences Laboratory, 1 Research Link, National University of
Singapore, Singapore 117604, Singapore.
3 Institute of Molecular and Cell Biology, Proteos, Singapore 138673,
Singapore.
* Author for correspondence (e-mail: dbsyuhao{at}nus.edu.sg)
Accepted 22 February 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Flowering time, MADS-box transcription factor, Transcriptional regulation, Chromatin immunoprecipitation, Gibberellin, Arabidopsis
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Balasubramanian et al., 2006;
Blazquez et al., 2003;
Cerdan and Chory, 2003;
Halliday et al., 2003;
Simpson and Dean, 2002). The
photoperiod pathway perceives the light quantity and circadian clock, whereas
the vernalization pathway responds to low temperatures. The autonomous pathway
monitors endogenous cues from specific developmental states, which are
independent of environmental signals. The gibberellin (GA) pathway
particularly regulates flowering in non-inductive short-day conditions. In
addition to these major genetic pathways, the pathways mediating the responses
to various wavelengths of light and temperature alteration above a critical
threshold have also been suggested to affect flowering. An intricate network
of the above pathways promotes floral transition via transcriptional
regulation of several floral pathway integrators including FLOWERING LOCUS
T (FT), SUPPRESSOR OF OVEREXPRESSION OF CO 1
(SOC1; also known as AGL20 - TAIR) and LEAFY
(LFY) (Boss et al.,
2004; Mouradov et al.,
2002; Parcy, 2005;
Simpson and Dean, 2002).
MADS-box genes encode a large family of transcription factors in plants
that share a highly conserved MADS-box domain, which recognizes the
CC(A/T)6GG (CArG) box on target genes for binding
(Riechmann et al., 1996;
Shore and Sharrocks, 1995). In
Arabidopsis, the MADS-box gene family is a major class of regulators
mediating floral transition. AGAMOUS-LIKE 24 (AGL24) is one
of the MADS-box genes found to promote flowering
(Michaels et al., 2003;
Yu et al., 2002).
AGL24 expression is detectable in the vegetative shoot apex and is
upregulated in the inflorescence apex during floral transition. Transgenic
studies of 35S:AGL24 and AGL24 RNA interference lines have
shown that the upregulated level of AGL24 expression corresponds to
the degree of precocious flowering and that the reduction in AGL24
expression is related to the degree of late flowering, suggesting that
AGL24 is a dosage-dependent promoter of flowering.
The expression of AGL24 is barely detectable in the center of
emerging floral meristems and is present in floral reproductive organs at
later stages (Yu et al.,
2004). Overexpression of AGL24 promotes flowering and
transforms floral meristems into inflorescence meristems, indicating that
AGL24 specifically promotes inflorescence identity. Direct repression
of AGL24 and two other flowering time genes, SOC1 and
SHORT VEGETATIVE PHASE (SVP), by the floral meristem
identity gene APETALA1 (AP1), prevents the continuation of
the shoot developmental program, contributing to the specification of floral
meristem identity (Liu et al.,
2007; Yu et al.,
2004). On the other hand, expression of AGL24 and
SVP at an appropriate level in the floral meristem is also required
for regulation of class B and C floral homeotic genes at a high temperature
(Gregis et al., 2006).
Therefore, AGL24 regulates both flowering time and flower
development.
Previous studies on the role of AGL24 in flowering time control
have revealed that AGL24 and SOC1 affect expression of each
other (Michaels et al., 2003;
Yu et al., 2002), implying
that these two MADS-box transcription factors might directly or indirectly
interact to mediate flowering. However, AGL24 and SOC1 are
differently regulated during floral transition in several aspects. First,
although AGL24 expression is regulated by vernalization, it is
independent of FLOWERING LOCUS C (FLC), a potent repressor
of flowering (Michaels et al.,
2003). By contrast, FLC represses SOC1
expression in the meristem and also delays SOC1 expression by
repressing FT, which encodes a protein acting as a long-distance
floral signal moving from the leaf to the meristem
(Corbesier et al., 2007;
Hepworth et al., 2002;
Searle et al., 2006). Second,
in the photoperiod pathway, AGL24 is affected by CONSTANS
(CO), but not by FT (Yu
et al., 2002), whereas SOC1 is mainly regulated by
FT and indirectly by CO via an unknown DNA-binding factor
(Hepworth et al., 2002;
Lee et al., 2000;
Samach et al., 2000). Lastly,
alteration of AGL24 activity determines flowering time partially
independently of SOC1, and vice versa, indicating that they can
promote flowering in independent pathways
(Michaels et al., 2003;
Yu et al., 2002). These
observations suggest that AGL24 perceives flowering signals that are
different from those integrated by SOC1. Therefore, what the exact
relationship is between AGL24 and SOC1 and how they interact
to affect flowering are essential questions for understanding the integration
of flowering signals.
In this study we established and applied a functional estradiol-inducible AGL24 system in combination with microarray analysis to identify AGL24-induced genes including SOC1. We provide evidence that AGL24 and SOC1 directly regulate mutual transcription to integrate flowering signals from several genetic pathways, including the GA pathway. This direct interaction confers a positive-feedback regulation of the expression of AGL24 and SOC1 to a quantitative threshold required for the transition from vegetative to reproductive growth.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Plasmid construction and plant transformation
For the construction of pER22-AGL24, the AGL24 cDNA was
amplified with primers (restriction sites underlined) AGL24-F1-XhoI
(5'-CCGCTCGAGGTAGTGTAAGGAGAGATCTGG-3') and
AGL24-R1-ApaI
(5'-ATGGGCCCTTCCCAAGATGGAAGCCCAA-3'). The digested PCR
products were cloned into the pER22 vector. The pER8 vector
(Zuo et al., 2000) was cut
with ApaI and SpeI, the cohesive ends filled in, and
self-ligated to produce pER22.
To construct 35S:AGL24-6HA, the AGL24 cDNA was amplified
with primers AGL24-F1-XhoI and AGL24-R1-ApaI. The digested
PCR products were cloned into the pGreen-35S-6HA vector to obtain an in-frame
fusion of AGL24-6HA under the control of the 35S promoter. The
pGreen-35S-6HA vector was generated by cloning six repetitive HA epitopes into
the SpeI site of pGreen-35S (Yu
et al., 2004).
To construct 35S:SOC1-9myc, the SOC1 cDNA was amplified with primers SOC1-F1-XhoI (5'-CCGCTCGAGTAGCCAATCGGGAAATTAACTA-3') and SOC1-R1-XmaI (5'-CGCCCGGGCTTTCTTGAAGAACAAGGTAAC-3'). The digested PCR products were cloned into the pGreen-35S-9myc vector to obtain an in-frame fusion of SOC1-9myc under the control of the 35S promoter. The pGreen-35S-9myc vector was generated by cloning nine repetitive myc epitopes into the SpeI site of pGreen-35S.
To construct ProSOC1:GUS, the 2.0 kb SOC1
5' upstream sequence (Fig.
4C) was amplified with the primers SOC1-P4-XmaI
(5'-AACCCGGGATCGTATTTACTAGTGGTATACG-3') and
SOC1-R2-XmaI
(5'-AACCCGGGATCTTCTTCTTTAGTTAATTTCCC-3'). The digested
PCR products were cloned into the pHY107 vector
(Liu et al., 2007). This
construct was mutagenized to produce the mutated AGL24 binding site
(Fig. 4C) using the QuikChange
II XL Site-Directed Mutagenesis Kit (Stratagene).
To construct ProAGL24:GUS, the 4.7 kb AGL24 genomic sequence (Fig. 5F) was amplified with primers AGL24-P1-PstI (5'-AACTGCAGTCGTTCCTTATAGCGGTGGAT-3') and AGL24-P4-SpeI (5'-GGACTAGTTTCCCAAGATGGAAGCCTAACCAAC-3'). The digested PCR products were cloned into pHY107. This construct was mutagenized to produce the mutated sites of M-2003 and M-2039 (Fig. 5F).
For the complementation test, the AGL24 genomic fragment was
amplified with primers AGL24-P1-PstI and AGL24-p-R-XbaI
(5'-CCTCTAGATCATTCCCAAGATGGAAGCC-3'), and the
SOC1 genomic fragment was amplified with primers
SOC1-P4-XmaI and SOC1-p-R-XbaI
(5'-CCTCTAGATCACTTTCTTGAAGAACAAGG-3'). The digested PCR products
were cloned into pHY105 (Liu et al.,
2007). The constructs containing the mutated forms of the genomic
AGL24 and SOC1 fragments were generated using the QuikChange
II XL Site-Directed Mutagenesis Kit.
For the complementation test, the relevant constructs were introduced into
agl24-1 or soc1-2, whereas other constructs were introduced
into wild-type Columbia plants using the Agrobacterium-mediated
floral dip method (Clough and Bent,
1998). Except for transgenic plants with the pER22-AGL24
construct that were selected on MS medium (Sigma) supplemented with
hygromycin, transgenic plants with other constructs were selected by
Basta.
β-estradiol induction of pER22-AGL24
To observe the phenotype of pER22-AGL24 plants upon
β-estradiol induction, they were grown on solid MS medium supplemented
with 1% sucrose at 22°C under long-day conditions before applying various
treatments. Once we started the treatment, 10 µM β-estradiol was
applied and replaced every 2 days. For examining the induction of
AGL24 by estradiol, the seedlings at different developmental stages
grown on solid MS medium were transferred into liquid MS medium supplemented
with 10 µM β-estradiol. These seedlings were incubated in the liquid
medium with gentle shaking for 1 to 24 hours. Mock treatment of transgenic
plants was also performed for the above experiments in which the solvent
dimethyl sulfoxide substituted for β-estradiol.
Microarray analysis
Isolation of total RNA, cDNA synthesis, cRNA labeling with the IVT Labeling
Kit, and hybridization on the Arabidopsis ATH1 genome arrays were
performed following the manufacturer's instructions (Affymetrix). Two
biological replicates were tested for each treatment. The Affymetrix
microarray suite software package (MAS 5.0) was used to scan and obtain
signals. MAS-generated data files (.CEL files) were used as the input for
preprocessing using the software package RMA to summarize probe sets and
normalize signal intensities (Bolstad et
al., 2003). Further analysis and filtering was performed using
GeneSpring (Agilent). All samples were normalized per chip to the fiftieth
percentile and per gene to median signals. For the Affymetrix flags, we
filtered on `present' value to appear in at least one sample. This reduced
22,746 total probe sets to 15,690 probe sets. The minimum expression value was
set to 0.5 (log scale). Confidence in replicates was tested using standard
deviation test with GeneSpring's default cross-gene error model turned on. The
filter for P-values was set to 0.01. One-color data with deviation
from one as an error model gave an average base/proportional of 34.94. First,
we compared the transcriptomes in pER22-AGL24 induced by estradiol relative to
mock-treatment. Second, we compared the transcriptomes in estradiol-induced
pER22-AGL24 relative to those in estradiol-induced wild-type seedlings. Only
genes showing consistently altered expression (fold change 
1.1) in these
two comparisons were chosen as putative AGL24-regulated genes. The
complete microarray data set is available as the accession number GSM6954 in
the Gene Expression Omnibus
(http://www.ncbi.nlm.nih.gov/geo).
ChIP assay
About 300 mg of 9-day-old 35S:AGL24-6HA and 35S:SOC1-9myc
seedlings were fixed at 4°C for 40 minutes in 1% formaldehyde under
vacuum. Fixed tissues were homogenized, and chromatin was isolated and
sonicated to produce DNA fragments shorter than 500 bp. The solubilized
chromatin was incubated with anti-HA agarose beads (Sigma) for 90 minutes at
4°C or used as an input control. Beads were washed five times with IP
buffer (50 mM HEPES, pH 7.5, 150 mM KCl, 5 mM MgCl2, 10 µM
ZnSO4, 1% Triton X-100, 0.05% SDS), and then incubated with elution
buffer (50 mM Tris, pH 8.0, 1% SDS, 10 mM EDTA) for 30 minutes at 65°C.
The supernatant was collected and co-immunoprecipitated DNA was recovered
according to a published protocol (Wang et
al., 2002). An unrelated DNA sequence from the ACTIN2/7
(ACTIN) gene that is constitutively expressed in Arabidopsis
was used as an internal control for normalization
(Johnson et al., 2002). Primer
sequences used for the ChIP enrichment test are listed in
Table 1. All ChIP assays were
repeated at least twice and representative data are presented. For
identification of the precise binding sites of AGL24 and SOC1, DNA enrichment
was evaluated by real-time quantitative PCR in triplicate. Relative enrichment
of each fragment was calculated first by normalizing the amount of a target
DNA fragment against the ACTIN fragment, and then by normalizing the
value for transgenic plants against the value for wild type as a negative
control using the following equation: 2(CtTransgenic
Input-CtTransgenic ChIP)/2(CtWT Input-CtWT
ChIP).
|
). The
relative fold change was eventually calculated based on both Ct value and
primer efficiency according to a published protocol
(Pfaffl, 2001).
Semi-quantitative PCR was performed as previously described
(Yu et al., 2002). Primer
sequences used for gene expression analyses are listed in
Table 2.
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; Liu et al.,
2007). |
RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
To test the dose response of the XVE inducible system, we examined the
time-course of AGL24 expression in seedlings from a selected
transgenic pER22-AGL24 line at different developmental stages (3, 6,
9, 12 and 15 days after germination) after they were transferred into
Murashige and Skoog (MS) liquid medium supplemented with 10 µM
β-estradiol. The XVE system proved to be a potent and reliable inducible
system, as pER22-AGL24 plants demonstrated consistent induction of
AGL24 expression irrespective of the developmental stage of the
tested seedlings (data not shown). Fig.
1A shows an example of induction of AGL24 expression in
transgenic pER22-AGL24 seedlings at 9 days after germination, in
which AGL24 induction nearly reached a maximal level after 8 hours of
β-estradiol treatment and remained saturated thereafter.
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SOC1 is induced by AGL24
We then chose 9-day-old pER22-AGL24 seedlings at the floral
transitional stage to investigate the change in transcriptomes responding to
the induced AGL24 expression. As AGL24 induction reached a
steady maximal level 8 hours after β-estradiol treatment
(Fig. 1A), we collected
seedlings at this time point for microarray analyses. Statistical analysis of
the microarray data revealed 97 AGL24-downregulated genes and 87
AGL24-upregulated genes (see Table S1 in the supplementary material),
among which SOC1, a flowering pathway integrator, was one of the
genes activated by AGL24.
In pER22-AGL24 seedlings treated with estradiol, AGL24
expression was continuously induced, whereas SOC1 expression was
gradually upregulated up to 12 hours of induction, after which it was
dramatically increased (Fig.
2A). This result, together with a previous observation that
overexpression of AGL24 affected SOC1 expression in
FLC-dependent and late flowering backgrounds
(Michaels et al., 2003),
indicates that AGL24 affects SOC1 expression under certain
conditions. In wild-type plants grown in soil, AGL24 expression was
increased at 7 days after germination and was dramatically upregulated during
floral transition, which was marked by significantly increased AP1
expression from 9 days after germination
(Fig. 2C,
Fig. 5A). SOC1
expression was gradually elevated in wild-type seedlings after germination and
significantly increased from 9 days after germination, whereas its
upregulation was delayed in agl24-1 during floral transition
(Fig. 2B). SOC1
expression was much more elevated in 35S:AGL24 than in wild-type
seedlings after 9 days post-germination
(Fig. 2B). We further dissected
developing agl24-1 and wild-type seedlings to detect the change in
SOC1 expression in the leaf (cotyledon and rosette leaf) and the
aerial part without leaf, including the shoot apex and young leaf primordia
(Fig. 2D). SOC1
expression was slightly altered in the leaf of agl24-1, whereas its
expression in the aerial part without leaf of agl24-1 was
significantly reduced. In situ hybridization further revealed the reduced
SOC1 expression mainly at the shoot apex of agl24-1 during
floral transition (Fig. 2E).
Thus, AGL24 mainly upregulates SOC1 at the shoot apex during
floral transition, which is in accordance with the observation that
upregulation of AGL24 in floral transition is responsible for
accelerating flowering (Fig.
1C).
AGL24-6HA binds directly to the SOC1 promoter
To examine whether AGL24 directly controls SOC1 transcription, we
performed ChIP assays using a functional transgenic line expressing an
AGL24-6HA fusion protein driven by the CaMV 35S promoter. By examining the
phenotypes and genetic segregation ratios, we isolated one transgenic line
containing a single insertion of the 35S:AGL24-6HA transgene, which
showed comparable flowering time to 35S:AGL24
(Fig. 3A,D). A notable floral
phenotype relevant to AGL24 function in promoting inflorescence
identity is the generation of secondary flowers from a primary floral meristem
when AGL24 is overexpressed (Yu
et al., 2004), a phenotype which was also observed in the selected
35S:AGL24-6HA plant (Fig.
3C). These observations suggest that the fusion protein of
AGL24-6HA retains the same biological function as AGL24.
We scanned the SOC1 genomic sequence for CArG motifs with a maximum one nucleotide mismatch, and designed ten pairs of primers near the identified motifs for measurement of DNA enrichment by quantitative real-time PCR (Fig. 4A). The number 6 genomic fragment (-1260 to -1133, relative to the translation start site) containing one CArG motif showed the strongest enrichment of around 6-fold (Fig. 4B), suggesting that AGL24-6HA binds directly to this site in vivo.
|
|
). Based on this construct, we created another
reporter gene cassette in which the putative AGL24 binding site was mutated
(Fig. 4C). Among 24 independent
lines of transformants harboring ProSOC1:GUS, 20 lines
displayed strong GUS staining during floral transition
(Fig. 4D,E), whereas among 18
lines of the transformants harboring the construct with the mutated AGL24
binding site, 11 lines displayed intermediate GUS staining
(Fig. 4D,E). It is noteworthy
that the difference in GUS staining conferred by
ProSOC1:GUS and its mutated form was most apparent at the
shoot apex. These observations, which are consistent with the different
expression of SOC1 at the shoot apex of wild-type and
agl24-1 seedlings, demonstrate that the tested AGL24 binding site is
responsible for upregulating SOC1 expression at the shoot apex during
floral transition. We further crossed the transformants harboring
ProSOC1:GUS and its mutated construct with
35S:AGL24, and examined the change in GUS staining in response to the
increased AGL24 activity. In the 35S:AGL24 background, GUS
staining of both ProSOC1:GUS and its mutated form slightly
increased in the leaf compared with that in wild-type plants
(Fig. 4D). By contrast, GUS
staining of ProSOC1:GUS at the shoot apex of
35S:AGL24 during floral transition increased compared with that in
the wild-type background (Fig.
4D), whereas staining of the mutated construct remained lower at
the shoot apex of 35S:AGL24 than in wild type
(Fig. 4D). Thus, mutation of
the AGL24 binding site almost completely abolishes upregulation of
SOC1 by AGL24 at the shoot apex, corroborating that AGL24
specifically binds to this site to promote SOC1 expression at the
shoot apex during floral transition.
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SOC1-9myc binds directly to the AGL24 and LFY promoters
Since AGL24 expression is also affected by SOC1
(Michaels et al., 2003;
Yu et al., 2002), we
quantitatively examined the effect of SOC1 on AGL24
expression. AGL24 expression was increased in wild-type seedlings
from 5 days after germination, whereas its upregulation was delayed in
soc1-2 (Fig. 5A). In
35S:SOC1, AGL24 expression was high in seedlings 3 and 5 days after
germination, and reduced thereafter (Fig.
5A). AP1 expression was notably higher in
35S:SOC1 than in wild-type seedlings and its expression in
35S:SOC1 5 days after germination was almost comparable with that in
wild-type seedlings 11 days after germination
(Fig. 5B). As AGL24
expression is repressed by induced AP1 activity
(Yu et al., 2004),
AGL24 expression in 35S:SOC1 may reflect a combined effect
of repression of AGL24 by AP1 and promotion of
AGL24 by overexpression of SOC1.
We also dissected developing soc1-2 and wild-type seedlings to detect the change in AGL24 expression in the leaf and aerial part without leaf (Fig. 5C). In wild-type seedlings, AGL24 expression in the leaf was much lower than that in the aerial part without leaf (data not shown). Compared with its expression in wild-type tissues, AGL24 expression only slightly decreased in the leaf of soc1-2, whereas its expression in the aerial part without leaf of soc1-2 was significantly reduced during floral transition. Thus, SOC1 upregulates AGL24 mainly at the shoot apex during floral transition.
|
Using the same ChIP approach, we tested whether SOC1-9myc and AGL24-6HA could bind directly to the genomic sequences of two floral meristem identity genes, AP1 and LFY. Our results showed that only one fragment near a CArG motif in the LFY promoter was enriched by anti-myc antibody in SOC1-9myc plants (Fig. 6), suggesting that SOC1-9myc binds directly to the LFY promoter in vivo. In addition, we found that SOC1-9myc and AGL24-6HA did not bind directly to their own genomic sequences (see Fig. S1 in the supplementary material).
|
). The
GUS expression in this line is similar to that of endogenous
AGL24 expression. Based on this ProAGL24:GUS
construct, we generated two reporter gene cassettes, M-2003 and M-2039, in
which one or other of two CArG motifs within the number 1 genomic fragment
were mutated (Fig. 5F). Among
17 independent lines of the transformants bearing the M-2003 mutation
(Fig. 5G,H), 13 lines exhibited
strong GUS staining, which was comparable with that conferred by the
ProAGL24:GUS construct. By contrast, the majority of 20
independent lines of the transformants bearing the M-2039 mutation exhibited
intermediate or weak GUS staining (Fig.
5G,H). The difference in the GUS staining of wild-type, M-2003 and
M-2039 plants was most apparent at the shoot apex. These results, together
with differential expression of AGL24 in soc1-2 and
wild-type plants, strongly suggest that SOC1 mainly binds to the CArG motif of
M-2039 to upregulate AGL24 expression at the shoot apex during floral
transition.
We further crossed the transformants harboring
ProAGL24:GUS and its mutated construct M-2039 with
35S:SOC1, and examined the change in GUS staining in response to the
increased SOC1 activity. As 35S:SOC1 showed very early
flowering (Lee et al., 2000;
Samach et al., 2000) and
AGL24 was only upregulated at early developmental stages of
35S:SOC1 (Fig. 5A), we
compared GUS staining in 4-day-old seedlings. GUS staining of 4-day-old
ProAGL24:GUS and M-2039 seedlings did not reveal any
difference in wild-type background (Fig.
5I), which was consistent with unaltered AGL24 expression
in soc1-2 and wild-type seedlings at a similar developmental stage
(Fig. 5A). However, GUS
staining of ProAGL24:GUS at the shoot apex and hypocotyl
of 35S:SOC1 was increased compared with that in the wild-type
background, whereas staining of M-2039 remained the same in 35S:SOC1
as in wild type (Fig. 5I).
Thus, mutation of the SOC1 binding site indeed compromises upregulation of
AGL24 in young seedlings.
To confirm that the revealed SOC1 binding site is essential for AGL24 function in flowering, agl24-1 was transformed with either a genomic AGL24 construct or its derived construct with the M-2003 or M-2039 mutation. The average flowering time of agl24-1 mutants transformed with the AGL24 genomic construct, which comprised 2.23 kb of 5' upstream sequence (Fig. 5F) and the full gene coding region plus introns, was around 11.9 rosette leaves (Fig. 5J). This was comparable to the average flowering time of agl24-1 mutants transformed with the M-2003 mutation (12.2 rosette leaves), but was earlier than that of agl24-1 mutants transformed with the M-2039 mutation (14.4 rosette leaves) (Fig. 5J). These results substantiate that the SOC1 binding site at M-2039 is important for AGL24 function in promoting flowering.
Interaction of AGL24 and SOC1 mediates the effect of gibberellins on flowering
Previous studies have revealed that the expression of AGL24 and
SOC1 is differently controlled by the photoperiod, autonomous and
vernalization pathways (Michaels et al.,
2003; Yu et al.,
2002). Although it has been shown that GA could affect the
expression of AGL24 and SOC1
(Lee et al., 2000;
Moon et al., 2003;
Yu et al., 2002), it remains
elusive how the GA pathway regulates their expression. We examined the
expression of both genes in the wild-type and mutant seedlings grown under
short-day conditions. In the wild-type seedlings, the expression of
AGL24 and SOC1 gradually increased under mock treatment and
their expression was upregulated upon GA treatment
(Fig. 7A,B), confirming that
both genes are targets of the GA pathway
(Lee et al., 2000;
Moon et al., 2003;
Yu et al., 2002). In
agl24-1 and soc1-2, the respective upregulation of
SOC1 and AGL24 was nearly abolished upon GA treatment
(Fig. 7A,B). This suggests that
upregulation of SOC1 and AGL24 in response to GA is mediated
by AGL24 and SOC1, respectively. Under long-day conditions,
GA treatment did not promote flowering in wild type or mutants, indicating
that signals from other flowering genetic pathways play major roles in
regulating flowering time (Fig.
7C). During our experimental period, soc1-2 agl24-1 did
not flower under short-day conditions without GA treatment, which was
significantly different from the flowering phenotype exhibited by either of
the single mutants (Fig. 7D).
Upon GA treatment, flowering of wild type, soc1-2 and
agl24-1 was accelerated, whereas soc1-2 agl24-1 still
flowered extremely late (Fig.
7D). These observations suggest that SOC1 and
AGL24 upregulate each other in response to GA and synergistically
determine flowering time under short-day conditions.
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|
DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Wigge et al., 2005;
Wilson et al., 2005). In this
study, we used an estradiol-inducible gene expression system in combination
with microarray analysis to identify genes induced by the flowering promoter
AGL24 and identified SOC1 as one of these induced genes. At
the vegetative phase, SOC1 expression remains low and is almost
unaffected by altered AGL24 activity, whereas upregulation of
SOC1 expression at the shoot apex during floral transition is highly
dependent on AGL24 activity (Fig.
2). ChIP assay revealed that AGL24-6HA can bind to the regulatory
sequence of SOC1, and mutagenesis of the AGL24-6HA binding site
reduces SOC1 expression at the shoot apex
(Fig. 4), demonstrating that
AGL24 directly regulates SOC1 transcription specifically at
the shoot apex during floral transition. These results, together with the
observations that AGL24 is significantly upregulated during floral
transition and that induced AGL24 expression during floral transition
is sufficient to promote flowering (Fig.
1C, Fig. 5A),
suggest that direct upregulation of SOC1 by increased AGL24
expression is an important molecular event during floral transition. On the
other hand, several pieces of evidence have also shown that AGL24
expression at the shoot apex is directly upregulated by SOC1
(Fig. 5), suggesting that
AGL24 and SOC1 regulate each other to provide
positive-feedback control of their expression at the shoot apex during floral
transition.
In soc1 and agl24 mutants, changes in AGL24 and
SOC1 expression, respectively, still affect flowering time, implying
that they might regulate different genes involved in flowering
(Michaels et al., 2003;
Yu et al., 2002). Our ChIP
assay revealed that the LFY genomic sequence is only bound by
SOC1-9myc, and not by AGL24-6HA (Fig.
6). This confirms that AGL24 and SOC1 control
distinct genes, while they directly regulate each other.
A significant aspect of the mutual interaction between AGL24 and
SOC1 is the integration of flowering signals from several genetic
pathways (Fig. 8). The
vernalization pathway regulates flowering through at least several different
regulators. In a FLC-independent pathway, vernalization regulates the
expression of at least two genes, AGL24
(Michaels et al., 2003;
Yu et al., 2002) and
AGL19 (Schonrock et al.,
2006). In a FLC-dependent pathway, FLC plays a
dual role in directly repressing SOC1 transcription in the meristem
and indirectly delaying SOC1 expression by repression of FT,
a systemic signal required for the activation of SOC1, in the leaf
(Hepworth et al., 2002;
Searle et al., 2006). Several
recent studies have provided in vitro and in vivo data showing that FLC binds
to a CArG box at the SOC1 5' promoter
(Helliwell et al., 2006;
Hepworth et al., 2002;
Searle et al., 2006).
Nevertheless, vernalization can still upregulate SOC1 expression in
flc mutants under short-day conditions, indicating that SOC1
is also regulated in a FLC-independent way
(Moon et al., 2003). This can
be partly explained by direct regulation of SOC1 by
AGL24.
The autonomous pathway promotes flowering by repressing FLC
(Michaels and Amasino, 2001)
and thus affecting SOC1 expression. Although AGL24
expression is not affected by FLC, its expression is significantly
reduced in several mutants in the autonomous pathway, such as fve,
fpa and fca (Michaels et
al., 2003; Yu et al.,
2002), suggesting that the autonomous pathway also upregulates
AGL24 in a FLC-independent way. Since FLC and AGL24 bind to
distinct sites of the SOC1 promoter region, it will be interesting to
further elucidate the SOC1 transcription complex, in which AGL24 may compete
with FLC in response to the signals from vernalization and autonomous
pathways.
In the photoperiod pathway, SOC1 is mainly regulated by
FT and indirectly by CO via other unknown DNA-binding
factor(s) (Hepworth et al.,
2002; Lee et al.,
2000; Samach et al.,
2000; Yoo et al.,
2005), whereas AGL24 is affected by the activity of
CO, but not of FT (Yu et
al., 2002). Although FT has been suggested as a major
output of CO (Samach et al.,
2000; Wigge et al.,
2005; Yoo et al.,
2005), FT integrates other floral signals irrespective of
CO. For example, FLC directly represses FT in the
leaf, thus affecting its activation of SOC1
(Helliwell et al., 2006;
Searle et al., 2006). In
addition, thermal induction of flowering by elevated growth temperature is
also mediated by FT
(Balasubramanian et al., 2006).
Thus, positive regulation of SOC1 by FT is only partially
controlled by the photoperiod pathway. It is likely that direct regulation of
SOC1 by AGL24, which is regulated by CO, provides
an alternative channel to enhance the effect of the photoperiod pathway on
SOC1 expression.
|
). Removal
of FLC repression only derepresses SOC1 expression and is
not sufficient to activate SOC1 under short-day conditions,
suggesting that GA activation of SOC1 needs positive regulator(s)
(Moon et al., 2003).
AGL24 is a possible regulator of SOC1 in the GA pathway
because SOC1 and AGL24 upregulate each other in response to
GA, and loss of either gene compromises the effect of GA on the promotion of
another gene (Fig. 7). In
addition, flowering of overexpression of SOC1 under short-day
conditions is partially delayed in the GA-deficient mutant ga1-3
(Moon et al., 2003),
indicating that GA regulates other target(s) in addition to SOC1. Our
results have identified that AGL24 is another major target of the GA
pathway as soc1-2 agl24-1 double mutants do not flower under
short-day conditions without GA treatment
(Fig. 7D). Taken together,
direct interaction of AGL24 and SOC1 allows a synergistic
integration of environmental and endogenous signals from several upstream
genetic pathways to promote flowering (Fig.
8). Overall, the results presented here show that AGL24 and SOC1 directly upregulate each other at the shoot apex during floral transition. This integrates flowering signals perceived by these two regulators and provides positive-feedback regulation of their own expression to a quantitative threshold required for the transition of the shoot apical meristem from a vegetative to a reproductive state. Direct cross-regulation between AGL24 and SOC1 represents a novel regulatory mode for the transcription factors involved in the control of flowering time and further investigation of their target genes would provide a better understanding of the subtle regulatory hierarchy of floral transition.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1481/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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