First published online 13 March 2008
doi: 10.1242/dev.020255
Development 135, 1481-1491 (2008)
Published by The Company of Biologists 2008
Direct interaction of AGL24 and SOC1 integrates flowering signals in Arabidopsis
Chang Liu1,2,
Hongyan Chen1,2,
Hong Ling Er1,
Hui Meng Soo3,
Prakash P. Kumar1,2,
Jin-Hua Han1,
Yih Cherng Liou1 and
Hao Yu1,2,*
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.
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Fig. 1. Generation of a functional estradiol-inducible AGL24 expression
system. (A) Induction of AGL24 expression in 9-day-old
pER22-AGL24 Arabidopsis seedlings mock-treated (M) or treated with 10
µM β-estradiol (E) for 0, 1, 2, 4, 8, 12 or 24 hours. TUB2
expression was used as a control. (B) The estradiol-inducible
AGL24 system is biologically functional. The pER22-AGL24
plants (right) initially treated with β-estradiol at 9 days after
germination show earlier flowering than mock-treated plants (left). (C)
Upregulation of AGL24 during floral transition is sufficient to
promote flowering. β-estradiol treatment did not affect the flowering of
wild-type plants, whereas initial treatment of pER22-AGL24 with
β-estradiol before or at the floral transitional stage (3, 6 or 9 days
after germination) accelerated flowering.
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Fig. 2. SOC1 expression is upregulated by AGL24 during floral
transition. (A) Induced expression of AGL24 (left) and
SOC1 (right) in 9-day-old pER22-AGL24 Arabidopsis seedlings
treated with β-estradiol or mock-treated for 0, 2, 4, 8, 12 and 24 hours.
(B,C) Relative temporal expression of SOC1 (B) and
AP1 (C) in developing seedlings with different genetic background
under long-day conditions. (D) Relative temporal expression of
SOC1 in the aerial part without leaf and leaf of agl24-1 and
wild-type seedlings. Transcript levels in A-D were determined by quantitative
real-time PCR analyses of three independently collected samples. Results were
normalized against the expression of TUB2. Error bars indicate s.d.
(E) In situ localization of SOC1 at the shot apex of
11-day-old agl24-1 and wild-type seedlings. For the purpose of
comparing signals, sections of these plants were placed on the same slides for
hybridization and detection. Scale bars: 25 µm.
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Fig. 3. Generation of functional 35S:AGL24-6HA and
35S:SOC1-9myc transgenic lines. (A) 35S:AGL24-6HA
and 35S:AGL24 Arabidopsis plants show early flowering under long-day
conditions. (B) 35S:SOC1-9myc and 35S:SOC1 plants
show early flowering under long-day conditions. (C) An ectopic
secondary flower (arrow) is observed in a 35S:AGL24-6HA flower.
(D) Flowering time of generated transgenic lines under long-day
conditions. Number of rosette leaves represents flowering time. Values
representing the mean±s.d. were scored from at least 20 plants of each
genotype.
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Fig. 4. AGL24 directly regulates SOC1. (A)
Schematic of the Arabidopsis SOC1 genomic region. Black boxes, exons;
white boxes, introns and upstream regions. Bent arrows denote translation
start sites and stop codons. Arrowheads indicate the sites containing either
single mismatch or perfect match with the consensus binding sequence (CArG
box) of MADS-domain proteins. Ten PCR fragments corresponding to the DNA
sequences near these CArG boxes were designed for ChIP analysis. (B)
ChIP enrichment test by quantitative real-time PCR shows the binding of
AGL24-6HA to the region near the number 6 fragment. (C) Schematic of
the ProSOC1:GUS construct. The native CArG box within the
number 6 fragment identified in B was mutated as indicated. (D) GUS
staining of ProSOC1:GUS plants. Representative GUS
staining of 12-day-old transformants containing
ProSOC1:GUS and its mutated form is shown in the upper
panels. Representative lines were crossed with 35S:AGL24, and GUS
staining of 10-day-old F1 plants is shown in the lower panels. (E)
Distribution of relative GUS staining intensity in the transformants
containing ProSOC1:GUS and its mutated construct.
(F) Distribution of flowering time in T1 transgenic plants carrying the
wild-type SOC1 gene and its mutated form in the soc1-2
mutant background.
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Fig. 5. SOC1 directly regulates AGL24. (A,B)
Relative temporal expression of AGL24 (A) and AP1 (B) in
developing Arabidopsis seedlings of different genetic background
under long-day conditions. (C) Relative temporal expression of
AGL24 in the aerial part without leaf and leaf of soc1-2 and
wild-type seedlings. Transcript levels in A-C were determined by quantitative
real-time PCR analyses of three independently collected samples. Results were
normalized against the expression of TUB2. Error bars indicate s.d.
(D) Schematic of the AGL24 genomic region. Arrowheads indicate
the sites containing either single mismatch or perfect match with the
consensus binding sequence (CArG box) of MADS-domain proteins. Four PCR
fragments corresponding to the DNA sequences near these CArG boxes were
designed for ChIP analysis. (E) ChIP enrichment test shows the binding
of SOC1-9myc to the region near the number 1 fragment indicated in D.
(F) Schematic of the ProAGL24:GUS construct. Two
native CArG boxes within the number 1 fragment identified in D and E were
mutated as indicated. (G) Representative GUS staining in 12-day-old
transformants containing ProAGL24:GUS and its derived
constructs with the mutated CArG boxes (M-2003 and M-2039). (H)
Distribution of relative GUS staining intensity in the transformants
containing M-2003 and M-2039. (I) GUS staining of
ProAGL24:GUS and M-2039 in the wild-type (left) and
35S:SOC1 (right) background. Representative lines of transformants
containing ProAGL24:GUS and M-2039 were crossed with
35S:SOC1, and GUS staining of 4-day-old F1 plants is shown on the
right. (J) Distribution of flowering time in T1 transgenic plants
carrying the wild-type AGL24 gene and its mutated forms (M-2003 and
M-2039) in the agl24-1 mutant background.
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Fig. 6. ChIP analysis of the binding of AGL24-6HA and SOC1-9myc to the
AP1 and LFY genomic regions. (A) Schematic of the
Arabidopsis AP1 and LFY genomic regions. Arrowheads indicate
the sites containing either single mismatch or perfect match with the
consensus binding sequence (CArG box) of MADS-domain proteins. The hatched
boxes represent the DNA fragments near CArG box(es) amplified in ChIP assays.
(B) ChIP enrichment test shows the binding of SOC1-9myc to the
LFY genomic region.
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Fig. 7. Gibberellin (GA) regulates flowering time through independently
controlling AGL24 and SOC1. (A) Temporal
expression of SOC1 in wild-type and agl24-1 Arabidopsis
seedlings with or without GA treatment under short-day conditions. (B)
Temporal expression of AGL24 in wild-type and soc1-2
seedlings with or without GA treatment under short-day conditions. Time points
on the x-axis indicate the time of collection of plant materials
after first GA treatment. Transcript levels in A and B were determined by
quantitative real-time PCR analyses of three independently collected samples.
Results were normalized against the expression of TUB2. Error bars
indicate s.d. (C) Flowering time of soc1-2 and
agl24-1 mutants with or without GA treatment under long-day
conditions. (D) Flowering time of soc1-2 and agl24-1
mutants with or without GA treatment under short-day conditions. Number of
total leaves represents flowering time in C and D. Values representing the
mean±s.d. were scored from at least 20 plants of each genotype.
Asterisk indicates that flowering was not observed in soc1-2 agl24-1
under short-day conditions without GA treatment.
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Fig. 8. Direct interaction between AGL24 and SOC1 mediates the
integration of flowering signals in Arabidopsis. AGL24
and SOC1 directly regulate mutual mRNA expression at the shoot apex.
This cross-regulation integrates flowering signals from four genetic pathways
to promote the floral transition from vegetative to reproductive development.
Arrows and bars represent promotion and repression effects, respectively.
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