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Photoperiodic Regulation of Flowering Time through
Periodic Histone Deacetylation of the Florigen Gene
FT
Xiaofeng Gu
1,2
, Yizhong Wang
1,2
, Yuehui He
1,2,3
*
1 Department of Biological Sciences, National University of Singapore, Singapore, 2 Temasek Life Sciences Laboratory, Singapore, 3 Shanghai Center for Plant Stress
Biology, Chinese Academy of Sciences, Shanghai, China
Abstract
The developmental transition from a vegetative to a reproductive phase (i.e., flowering) is timed by the seasonal cue day
length or photoperiod in many plant species. Through the photoperiod pathway, inductive day lengths trigger the
production of a systemic flowering signal, florigen, to provoke the floral transition. FLOWERING LOCUS T (FT), widely
conserved in angiosperms, is a major component of the mobile florigen. In the long-day plant Arabidopsis, FT expression is
rhythmically activated by the output of the photoperiod pathway CONSTANS (CO), specifically at the end of long days. How
FT expression is modulated at an adequate level in response to the long-day cue to set a proper flowering time remains
unknown. Here, we report a periodic histone deacetylation mechanism for the photoperiodic modulation of FT expression.
We have identified a plant-unique core structural component of an Arabidopsis histone deacetylase (HDAC) complex. In
long days, this component accumulates at dusk, and is recruited by a MADS-domain transcription factor to the FT locus
specifically at the end of the day, leading to periodic histone deacetylation of FT chromatin at dusk. Furthermore, we found
that at the end of long days CO activity not only activates FT expression but also enables HDAC-activity recruitment to FT
chromatin to dampen the level of FT expression, and so prevent precocious flowering in response to the inductive long-day
cue. These results collectively reveal a periodic histone deacetylation mechanism for the day-length control of flowering
time in higher plants.
Citation: Gu X, Wang Y, He Y (2013) Photoperiodic Regulation of Flowering Time through Periodic Histone Deacetylation of the Florigen Gene FT. PLoS Biol 11(9):
e1001649. doi:10.1371/journal.pbio.1001649
Academic Editor: Xuemei Chen, University of California Riverside, United States of America
Received November 28, 2012; Accepted July 24, 2013; Published September 3, 2013
Copyright: ß 2013 Gu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants from the Singapore Ministry of Education (AcRF Tier 2; MOE2009-T2-1-081), the Singapore National Science
Foundation (2010NRF-CRP002-018), and the Temasek Life Sciences Laboratory to YH. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: AFR1, SAP30 FUNCTION-RELATED 1; AFR2, SAP30 FUNCTION-RELATED 2; AGL18, AGAMOUS LIKE 18; BiFC, bimolecular fluorescence
complementation; ChIP, chromatin immunoprecipitation; CO, CONSTANS; EYFP, enhanced yellow fluorescent protein; FT, FLOWERING LOCUS T; GUS, b -
GLUCURONIDASE; HAT, histone acetyltransferase; HDAC, histone deacetylase; LD, long day; qRT-PCR, real-time quantitative PCR; RPD3, Reduced Potassium
Dependency-3; SAM, shoot apical meristem; SAP3 0, Sin3-Associated Polypeptide 30; SBR, Sin3-binding region; SD, standard deviation; , Sin3-Associated
Polypeptide 18 (SAP18); ZT, Zeitgeber time.
* E-mail: dbshy@nus.edu.sg
Introduction
The timing of the developmental transition from a vegetative to
a reproductive phase is critical for reproductive success in
flowering plants. Plants synchronize their timings of floral
transition with changing seasons to flower at a suitable time.
The change in day length or photoperiod is a key seasonal cue,
especially at high latitudes, and is perceived in leaves, leading to
the production of florigen in leaf vasculature [1,2]. Florigen, a
systemic signal, is transmitted through phloem from leaf to the
shoot apical meristem (SAM), leading to flower formation [1–3].
Many plant species respond to day length changes through
photoperiod pathways. According to their photoperiodic respons-
es, plants are classified into long-day plants, i.e., flowering
occurring only when the day becomes longer than a threshold
length or accelerates when the day length increases, short-day
plants whose flowering induced upon the day getting shorter, and
day-neutral plants [4].
FLOWERING LOCUS T (FT), first identified in Arabidopsis
thaliana [5,6], and FT homologs in o ther s pecies are the major
component of the mobile florigen [7–10]. In Arabido psis,a
facultative long-day plant that flowers rapidly under inductive
long days , t he o utput of the p hotoperiod pathway CONSTANS
(CO) activates FT expr ess io n in leaf vein s [2,11]. The circadian
clock sets a high CO mRNA expression in the late aftern oon in
long days, which coincides with light exposure resulting in CO
protein accumulation towa rds the day’s end, becau se light
stabilizes the CO protein [2,4]. T he vasculature-expressed CO
protein promotes FT expression activatio n in the phloem
companion cells specifically at the end of long days, resulting in
rhythmic FT expression [2,4]. During night, CO is rapidly
degraded by proteasomes and FT expression is repressed.
Upon its p roduction in dusk, the FT protein moves from
phloem to SAM to induce flowering. FT and its function as
florigen are conserved in th e flow ering pla nts so far examined;
the level of FT expression at the end of long days in Arabidopsis
plays a primary determining role for when a plant to flower
[1,2]. To date, beside CO, o ther factors (if a ny) that d irectly
regulate FT expression specifically at dusk remain to be
identified.
PLOS Biology | www.plosbiology.org 1 September 2013 | Volume 11 | Issue 9 | e1001649
Histone acetylation and de-acetylation can regulate chromatin
structure and gene expression. Histone acetyltransferases (HATs)
add acetyl groups to core histone tails and typically function to
promote transcription of target loci, whereas histone deacetylases
(HDACs) remove acetyl groups and are often linked with
transcriptional repression [12]. Recent studies of genome-wide
binding sites of mammalian HATs and HDACs reveal that both
are targeted to actively transcribed genes to control their
expression [13]. In addition, rapid and synchronous recruitment
of HATs and HDACs to actively transcribed loci have been
observed in yeast as well [14,15]. At these loci HATs act typically
to promote transcription, whereas HDACs deacetylate acetylated
histones to maintain an adequate level of acetylation and/or reset
the acetylation state of a target locus following transcription [13–
15].
Histone acetylation is involved in plant gene regulation. In
Arabidopsis, functional disruption of HATs such as GCN5 and
AtHAC1, or HDACs including HDA6 and HDA19, leads to
pleiotropic abnormalities in growth and development [16–19]. For
instance, loss of AtHAC1 or HDA6 function causes upregulation of
a potent floral repressor FLOWERING LOCUS C (FLC), resulting in
a delay in flowering [17–19]. Certain HATs (e.g., GCN5) and
HDACs (e.g., HDA6) are involved in genome-wide regulation of
gene expression, as revealed by transcript profiling [16,18],
whereas others may control the expression of only a subset of
loci. How a HAT or HDAC is recruited to its target loci remains
elusive.
HDACs such as the reduced potassium dependency-3 (RPD3)
type are often found in multiprotein co-repressor complexes,
among which the yeast Sin3-HDAC complex has been well
characterized [20,21]. The core components of this complex
typically include the master scaffold protein Sin3, the RPD3
HDAC, and several Sin3-associated structural components
including Sin3-Associated Polypeptide 18 (SAP18) and Sin3-
Associated Polypeptide 30 (SAP30); homologs of these compo-
nents are also found in a mammalian Sin3-HDAC like complex
[20]. This complex does not bind to DNA directly and is often
recruited to specific loci through association with DNA-binding
proteins [21]. In Arabidopsis, there are six Sin3 homologs including
SNL1–SNL6 (SNL for SIN3-LIKE), and four close RPD3 homologs
including HDA19, HDA9, HDA7, and HDA6 [22,23]. In addition,
there is only one SAP18 homolog in Arabidopsis, AtSAP18, that has
been shown to play a role in salt stress response and floral organ
formation [24,25]. The Arabidopsis genome does not encode an
apparent homolog of the yeast SAP30; hence, whether there are
Sin3-RPD3 like co-repressor complexes for histone deacetylation
in Arabidopsis remains to be addressed.
Here we report that in Arabidopsis there are two functional
relatives of the yeast SAP30, named as SAP30 FUNCTION-
RELATED 1 (AFR1) and SAP30 FUNCTION-RELATED 2
(AFR2), acting as part of HDAC complexes (AFR1-HDAC or
AFR2-HDAC) to modulate the acetylation level of FT chromatin
upon FT activation at the end of long days (LDs) (16-h light/8-h
dark). Both AFR1 and AFR2 proteins accumulate at the end of
LDs, and bind to FT chromatin at dusk, but not in the middle of
the day. Moreover, we found that the MADS-domain transcrip-
tion factor AGAMOUS LIKE 18 (AGL18) directly interacts with
AFR1 and AFR2 and recruits these proteins and presumably
AFR1/AFR2-HDAC to FT chromatin specifically at the end of
LDs leading to histone deacetylation upon FT activation. The
output of the photoperiod pathway CO at the end of LDs not only
activates FT expression, but also enables the recruitment of AFRs
to the FT locus to dampen
FT expression and set it at an adequate
level, preventing precocious flowering in response to the day
length cue in Arabidopsis.
Results
Two Arabidopsis Functional Relatives of the Yeast SAP30
(ScSAP30) Associate with Histone Deacetylases to Form
HDAC Complexes
In an effort to explore whether a Sin3-SAP30-RPD3 co-
repressor-like complex in Arabidopsis exists, we first examined
whether Arabidopsis has any functional relatives of ScSAP30
because it lacks of an apparent homolog of the full-length
ScSAP30. In the C-terminal region of ScSAP30 there is a domain
important for its function: the 30-amino acid Sin3-binding region
(SBR) [26]. We used C-SAP30 to BLAST the Arabidopsis protein
database and found that there are nine proteins containing a
region with a similarity to SBR, among which AT1G75060 and
AT1G19330 are the top two hits (Figure S1A; unpublished data).
We reasoned that if these two proteins are functional relatives of
SAP30, they would directly associate with SNLs, AtSAP18, and/
or RPD3-type HDACs. Yeast two-hybrid assays were performed
to check direct interactions of AT1G75060 or AT1G19330 with
SNL2 (SIN3-LIKE 2), AtSAP18, HDA9, and/or HDA19. Indeed,
these proteins directly interacted with AT1G75060 and
AT1G19330 (Figures 1A, 1C, and S2).
Next, to confirm these interactions in plant cells, we
conducted bimolecular fluorescence complementation (BiFC)
assays in which non-fluorescentN-terminalandC-terminal
fragments of enhanced yell ow fluorescen t protein (EYFP) were
fused to the full-length AT 1G75060 , AT1G 19330, At SAP18, or
HDA19 proteins. When AT 1G75060-cEYFP a nd nEYFP-
AtSAP18 were simultaneously expressed in onion epiderma l
cells, in the nuclei fluorescence was observed (Figure 1B),
demonstrating a direct inte raction of AT1G75060 with
AtSAP18. Similarly, we also ex amined and observed direct
interactions of AT1G19330 with AtSAP18, AT1G19330 with
HDA19, and AT1G75060 with HDA19 in the nuclei of onion
cells(Figure1Band1D).Theseresultssuggestthatboth
Author Summary
The timing of the developmental transition from a
vegetative to a reproductive phase is critically important
for reproductive success in flowering plants. Plants
synchronize the timing of their floral transition with the
changing seasons to flower at a suitable time. The change
in day length, or photoperiod, is a key seasonal cue,
especially at high latitudes. It is through the photoperiod
pathway that day lengths trigger the production of a
systemic flowering signal, florigen, to induce the transition
from vegetative to reproductive growth and flowering.
The FT protein is a major component of the mobile
florigen signal. In the model flowering plant Arabidopsis, FT
mRNA expression is rhythmically activated by the gene
CONSTANS, which is the output of the photoperiod
pathway, specifically at the end of long days. In this study,
we aimed to address how the level of FT expression is
modulated in response to the long-day cue to set the
appropriate flowering time. We show that on long days a
transcription factor recruits histone deacetylase activity to
remove acetyl marks from histones at the FT gene
specifically at dusk, thereby dampening FT mRNA expres-
sion upon its transcriptional activation by CONSTANS. This
sets the correct level of FT expression at the end of long
days, and thus prevents precocious flowering in response
to the long-day cue.
Chromatin-Mediated Day-Length Control of Flowering
PLOS Biology | www.plosbiology.org 2 September 2013 | Volume 11 | Issue 9 | e1001649
AT1G75060 and AT1G1933 0 function like ScSAP30, namely,
as an integral component of Sin3-RPD3 co-repressor like
complexes, although there is little sim ilarity of amino acid
sequence between these proteins and ScSAP30 outside of the C-
terminal SBR (Figure S1A).Hence,thehomologous
AT1G75060 and AT1G19330 proteins were named as AFR1
and AFR2, respectively. In short, our findings collectively
suggest that RPD3-type HDACs and AFRs together with other
structural components such as SNLs and AtSAP18 form Sin3-
SAP30-SAP18-RPD3 like complexes in Arabidopsis.
Figure 1. Direct interactions of AT1G75060 (AFR1) and AT1G19330 (AFR2) with AtSAP18 and HDA19 proteins. (A) Interactions of
AtSAP18 with AT1G75060 and AT1G19330 in yeast. The indicated proteins of full-length were fused with the GAL4-BD or AD domain. Yeast cells
harboring the fusion proteins, BD and/or AD (as indicated), were grown on selective synthetic defined media lacking of Trp, Leu, and His. (B) BiFC
analysis of the interactions of AtSAP18 with AT1G75060 and AT1G19330 in onion epidermal cells. Onion epidermal cells were co-transformed
transiently by a pair of plasmid, as indicated, via biolistic gene bombardment. Yellowish-green signals indicate physical associations of paired proteins
in the nuclei. Blue fluorescence from a DAPI (49,6-diamidino-2-phenylindole) staining indicates a nucleus. Bar = 20
mm. (C) Interactions of HDA19 with
AT1G75060 and AT1G19330 in yeast. The indicated full-length proteins were fused with the GAL4-BD or AD domain. Yeast cells were grown on
selective synthetic defined media lacking of Trp, Leu, and His. (D) BiFC analysis of the interactions of HDA19 with AT1G75060 and AT1G19330 in onion
epidermal cells. Bar = 20
mm.
doi:10.1371/journal.pbio.1001649.g001
Chromatin-Mediated Day-Length Control of Flowering
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AFR1 Acts Additively with AFR2 to Repress the Floral
Transition
To elucidate the biological functions of AFR1 and AFR2,we
identified th eir loss-of-function mutants, and each carries a
transfer DNA (T-DNA)insertion.Inafr1-1 and afr1-2,aT-DNA
is inserted into the first intron of AFR1,resultinginagreat
reduction, but not elimination of AFR1 expression (Figures 2A
and S3A). In both afr2 alleles, full-length AFR2 transcripts were
not detected, but AFR2 transcripts truncated at their 39 ends
were detectable in the afr2-2 mutant, which are predicted to
encode a protein containing the first 149 amino acids of AFR2
(Figure S3B). Grown in LDs, afr1-1 , afr1-2,andafr2-1 mutants
flowered moderately earlier than the wild type (WT) Col, as
revealed by the tot al number of leaves formed prior to flower
formation from the primary SAM of an examined line, the
developmen tal sta ndard f or Arabidopsis flowering-time measure-
ment (Figure 2B an d 2C). In shor t days (8-h lig ht/ 16-h dark), all
afr1 and afr2 mutants flowered earlier than WT (Figure 2D ), and
the early-fl owering phenotypes of afr1-1 and afr2-1 were fully
rescued by compl em entati on with tagged AFR1 and AFR2,
respectively (Figure S4). Of note, afr2-2 is a w eak allele
compared to afr1-1, and it is likely that the truncat ed AFR2
may retain some function in the afr2-2 mutant. We furth er
constructed the a fr1 afr2 double mutant (afr1-1 afr2-1), and
found it flowered earlier than eith er single mutant in both LDs
and short days (Figures 2C, 2D, and S5A). Taken together,
these results suggest that AFR1 and AFR2 act a dditively to
repress the floral transition in Arabidopsis, indicating that the
homologous AFR1 and AFR2 p roteins do not function in one
complex. In addition to early flowering, the afr1 afr2 mutant
exhibited a leaf ph enot ype: longer petiole, narrower leaf blade,
and with a slightly increased leaf initiation rate (Figures 2E an d
S5C), suggesting that AFR1 and AFR2 may also be required for
proper leaf development.
AFR1 and AFR2 Downregulate FT Expression Specifically
at the Day’s End to Delay Flowering in LDs
There are several genetic pathways promoting flowering in
Arabidopsis, including the autonomous, photoperiod, vernalization,
and thermosensory pathways [2,27,28]. To explore in which
genetic pathway AFR1 and AFR2 act to regulate flowering, we
introduced the photoperiod-pathway mutation co or a null mutant
of FVE, a component of the autonomous and thermosensory
pathways, into the afr1 afr2 double mutant [27,28]. The late-
flowering phenotype of fve was partly suppressed by afr1 afr2,
whereas the afr1 afr2 co triple mutant flowered at about the same
time as co (Figure 3A and 3B); thus, the co mutation is epistatic to
afr1 and afr2. Furthermore, we found that ft, like co, is epistatic to
afr1 and afr2 (Figure 3B). These results suggest that AFR1 and
AFR2 act through the CO-FT regulatory module of the photope-
riod pathway to control flowering.
Next, we examined CO expression upon loss of AFR1 and AFR2
function at the end of LDs (ZT16, 16 h after light on or dawn;
Zeitgeber time [ZT]), because CO is highly expressed to activate
FT expression at this time point. CO expression remained
unchanged in the afr1 afr2 double mutant compared to WT
(Figure 3C); additionally, loss of CO function didn’t affect AFR1 or
AFR2 expression (Figure S6). We further examined FT expression
in afr1, afr2, and afr1 afr2 seedlings at the end of LDs. FT transcript
levels were increased moderately in afr1 and afr2 single mutants
and in the double mutant (Figure 3D), consistent with that AFR1
and AFR2 act additively to repress flowering. In addition, FT
expression was slightly upregulated in afr1 afr2 in short days,
though its transcript levels were very low in both WT and afr1 afr2
plants (Figure S7). Taken together, these results show that
AFR1
and AFR2 function additively to downregulate FT expression and
so delay flowering. We further measured FT transcript levels in co
and afr1 afr2 co seedlings at ZT16, and found that co completely
suppressed FT up-regulation in the afr1 afr2 background
(Figure 3D). Hence, CO is required for FT downregulation at
ZT16 by AFR1 and AFR2.
FT expression is activated by CO specifically at the end of LDs,
and repressed at other times of the day, resulting in rhythmic FT
mRNA expression at dusk in LDs. Several histone modifiers
including the histone H3 lysine-27 (H3K27) methytransferase CLF
and the PKDM7B H3 lysine-4 demethylase (also known as JMJ14
or AtJMJ4), are required for continuous FT repression along day/
night cycles [29–32]. We explored whether AFR1 and AFR2 could
repress FT expression in other times of LDs beside ZT16. Upon
loss of AFR1 and AFR2 function, FT expression in LDs was not
affected from ZT0 through ZT14, but upregulated only from
ZT14 to ZT20 (Figures 3E and S8A). Thus, in LDs AFR1 and
AFR2 downregulate FT expression specifically in the presence of
CO activity at the day’s end, to delay flowering.
AFR1 and AFR2 Downregulate FT Expression in Leaf
Vasculature
Day length is perceived in leaves, leading to FT activation
specifically in the phloem companion cells of leaf veins [1,2].
We asked whether AFR1 and AFR2 are expressed in leaf veins to
downregulate FT expression.Tothisend,genomicAFR1 and
AFR2 fragments (including 59 promote r plus part of the coding
region of each gene), were fused to the b-GLUCURONIDASE
(GUS)geneinframeandthefusionswereintroducedinto
Arabidopsis by transformation. Histochemica l staining revealed
that both AFR1 and AFR2 were strongly expressed in leaf
vasculature in addition to root tips and shoot apices (Figure 4A).
To examine in which tissues AFR1 and AFR2 downregulate FT
expression, we introduced afr1-1 and afr2-1 into an FT-GUS
reporter line [33], and found that the GUS expression directed
by a FT prom oter was upregulated only in l eaf veins (Figure 4A).
Thus, AFR1 and AFR2 downregulate FT expression in the leaf
vasculature. Next, we examined the subcellular localization of
both AFR1 and AFR2 proteins by fusing them with green
fluorescence protein (GFP). Upon their expression in Arabi dopsis
roots, these fusion proteins were specifically localized i n the
nuclei (Figure 4B), consistentwiththatAFR1andAFR2actas
part of nuclear AFR1/AFR2-HDAC complexes in leaf veins to
downregulate FT expression.
AFR1 and AFR2 Proteins Accumulate and Directly Interact
with the FT locus at the End of LDs
We hypothesized that the downregulation of FT expression by
AFR1 and AFR2 in the presence of CO activity at the end of LDs
may be attributed to AFR-HDAC binding to FT chromatin only
at this time in LDs. To test this hypothesis, we first examined the
diurnal expression patterns of AFR1 and AFR2 in LDs, and found
that the expression of both genes peaked at the day’s end
(Figure 4C). Next, using the functional AFR1:HA-expressing line
and the functional AFR2:FLAG-expressing line (driven by their
native promoters; see Figure S4), we measured the abundance of
both proteins every 4 h over a 24-h LD cycle. The levels of AFR1
and AFR2 proteins changed diurnally in LDs, and both proteins
accumulated at the end of the day (Figures 4D and S9). This
indicates that AFR1/AFR2-HDAC may accumulate at the end of
LDs.
Chromatin-Mediated Day-Length Control of Flowering
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Figure 2. Phenotypes of
afr1
,
afr2
, and
afr1 afr2
mutants. (A) AFR1 and AFR2 gene structures. Exons are represented by black boxes, and arrows
indicate transcription start sites (TSS); triangles for T-DNA insertion sites. (B) afr1, afr2, and afr1 afr2 mutants grown in LDs. The arrow indicates a main
bolt with flowers. (C) Flowering times of the indicated genotypes grown in LDs. 19–23 plants were scored for each line. Double asterisks indicate
statistically significant differences in the means between Col (WT) and the indicated mutants, as revealed by two-tailed Student’s t test (**, p,0.01).
Bars indicate SD (for standard deviation). (D) Flowering times of the indicated genotypes grown in short days. 11–15 plants were scored for each line.
Double asterisks indicate statistically significant differences in the means between Col and the indicated mutants. (E) Leaf phenotype of the afr1 afr2
double mutant grown in LDs.
doi:10.1371/journal.pbio.1001649.g002
Chromatin-Mediated Day-Length Control of Flowering
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ft
afr1 afr2 ft
afr1 afr2
WT
Total leaf number at flowering
fve
afr1 afr2 fve
afr1 afr2
Col (WT)
AB
D
E
0.5
1.0
1.5
2.0
2.5
WT
afr1-1
afr1-2
afr2-1
afr2-2
afr1-1 afr2-1
Fold changes of FT expression
(relative to WT)
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0 4 8 1214161820 24ZT (h)
WT
afr1 afr2
FT transcript levels (FT /UBQ10)
0
10
20
30
40
50
Total leaf number at flowering
0
10
20
30
40
50
60
**
**
0
0.003
0.006
0.009
co afr1-1 afr2-1
co
co
afr1 afr2 co
C
0
0.2
0.4
0.6
0.8
1.0
1.2
Fold changes of CO expression
(relative to WT)
WT afr1 afr2
Chromatin-Mediated Day-Length Control of Flowering
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We further explored whether AFR1 could bind to FT
chromatin, and if so, at what time of the day. Chromatin
immunoprecipitation (ChIP) assays were performed using the
AFR1:HA-expressing seedlings. We found that at the end of LDs
(ZT16), AFR1 bound to the proximal FT promoter region, but not
in the first exon or the first intron (Figures 4E and S10A). Unlike at
ZT16, AFR1 was not enriched at all in the FT locus in the middle
of the day (ZT8) (Figure 4E). Moreover, using ChIP with the
AFR2:FLAG-expressing seedlings, we uncovered that AFR2, like
AFR1, bound to the proximal FT promoter region at ZT16, but
not at ZT8 (Figures 4F and S10B). The bindings of AFR1 and
AFR2 to FT chromatin at the end of LDs coincide well with that
both proteins accumulate at this time. Taken together, these
results suggest that in LDs AFR1/AFR2-HDAC binds to FT
chromatin specifically at the day’s end to modulate FT expression
in the presence of CO activity.
AFR1 and AFR2 Mediate Periodic Histone Deacetylation
at FT at the End of LDs
The recruitment of AFR1 and AFR2 to FT chromatin at the
day’s end, may cause periodic histone deacetylation in LDs. To
test this, we examined steady-state levels of histone H3 acetylation
of FT chromatin in WT and afr1 afr2 seedlings (and rosette leaves)
at ZT8 and ZT16. Loss of AFR1 and AFR2 function led to an
increase of steady-state level of acetylated H3 in the proximal FT
promoter region, but not in the first exon or the first intron at
ZT16, whereas at ZT8 there was no noticeable change in all three
regions examined (Figures 5 and S11). This is consistent with the
binding of AFR1 and AFR2 to FT chromatin in the proximal
promoter at the end, but not the middle of LDs (Figure 4E).
Together, these results show that AFR1 and AFR2 (presumably as
part of AFR1/AFR2-HDAC) mediate histone deacetylation at the
FT locus to downregulate its expression at the end of LDs.
Interestingly, there is no apparent increase of the steady-state
acetylation levels of FT chromatin in WT at ZT16 relative to ZT8,
although FT expression is activated at ZT16 (Figure 5; note that
the relative acetylation level of each sample was normalized to the
WT at ZT8). Therefore, there must be active histone acetylation
by HAT(s) on FT chromatin to ‘‘counteract’’ the AFR-mediated
histone deacetylation at the end of LDs.
The Transcription Factor AGL18 recruits AFRs to the FT
Locus Specifically at the End of LDs
In yeast SAP30 is involved in the recruitment of SAP30-Sin3-
RPD3 co-repressor complex to a target locus [21]. We hypoth-
esized that transcription factors may directly interact with AFR1
and AFR2 to recruit AFR1/AFR2-HDAC to the FT locus at the
end of LDs. Previous studies have revealed that several MADS-
domain transcription factors including FLC, SHORT VEGETA-
TIVE PHASE (SVP), AGL18, and AGAMOUS LIKE 15
(AGL15), are involved in FT repression in the vasculature to
inhibit flowering [28,34,35]. Using a candidate gene approach, we
found that the AGL18 protein directly interacted with AFR1 and
AFR2 in the nuclei of onion epidermal cells, as revealed by the
BiFC experiments (Figures 6A and S12A). AGL18, like AFR1 and
AFR2, are also expressed in leaf veins [35]. To further confirm the
in vivo association of AGL18 with AFR1, we conducted co-
immunoprecipitation experiments using the seedlings expressing
AFR1:HA and a functional AGL18:FLAG (Figure S13A), and
found that anti-HA (recognizing AFR1:HA) immunoprecipitated
AGL18:FLAG from the seedlings (Figure 6B). Thus, indeed,
AGL18 directly interacts with AFR1 and presumably AFR1-
HDAC in Arabidopsis.
AGL18 acts redundantly with its homolog AGL15 to repress
flowering [35]. We further found that AFR1 directly interacted
with AGL15 in yea st and pla nt ce lls (Figure S12B a nd S12C).
Of note, it has been previously shown that AGL15 could
interact moderately with the conserved HDAC domain of
HDA19 in yeast cells [36], which can directly associate with
AFR1 and AFR2 (Figure 1C and 1D). Next, we examined the
genetic interaction of afr1 afr2 wit h agl15 agl18 in flowering-time
regulation. Grown in LDs, the afr1 afr2 agl15 agl18 quadruple
mutant flowered at a similar time to either double mutant
(Figure 6 C), suggesting that these four genes act in the same
flowering-regul atory pat hway. Fu rther FT expression analysis
revealed that FT transcript levels (at ZT16) in both afr1 afr2 and
agl15 ag l18 seedlings increased to about 2-fold of the WT level,
and that in the quadruple, FT levels were slightly higher than
either double mutant (Figure 6D). This slight increase is not
surprising given that neither double mutant may not be null as
there are low levels of full-length AFR1 ,andAGL15 and AGL18
transcripts in afr1 and agl15 agl18, respectively (Figures S3 and
S14). Take n to geth er, these data are in line w ith that the DNA-
binding AGL15 and A GL18 may act to recruit AFR1 and
AFR2 to downregulate FT expression.
To explore the role of AGL18 for AFR1 recruitment, first we
examined whet he r and when AGL18 could directly interact
with the FT locus. Using ChIP with the AGL18:FLAG-
expressing seedlings, we found that at the end of LDs
AGL18:FLAG, like AFR1, was apparently enriched in the
proximal FT promoter region, in addition to a slight enrichment
in the first exon (Figure 6E). In the middle of LDs, AGL18 was
not associated with FT chroma tin (Figure 6E). Thus , AGL18
directly interacts with the FT locus specifically at the end o f
LDs. Next, we investigate d w hether AGL18 is r equire d f or
AFR1 recruitment to FT chromatin at the end of LDs . The line
expressing the functional AFR1:HA (in afr1-1 background) was
crossed to agl15 agl18 , and th e seedlings of
afr1 AFR1:HA and
agl15 agl18 afr1 AFR 1:HA harvested at ZT16 were subjected to
ChIP assays wit h anti-HA. We found that loss o f AGL15 and
AGL18 functio n nearly eliminated AFR1 binding to FT
chromatin at the end of LDs (Figure7).Theseresults,together
with th e direct interaction s of A GL18 and AGL15 with AFR1
and AFR2, led us to infer that these two MADS-domain
transcription factors recruit AFRs (presumably AFR-HDAC) to
the FT locus specifically at theendofLDstodampenFT
expression in the presence of CO activity.
Figure 3.
AFR1
acts additively with
AFR2
to downregulate
FT
expression specifically at the day’s end in LDs. (A,B) Flowering times of the
indicated genotypes grown in LDs. 11–17 plants were scored for each line. The afr1 afr2 double mutant is afr1-1 afr2-1. Double asterisks indicate
statistically significant differences in the means between the indicated genotypes, as revealed by two-tailed Student’s t test (**, p,0.01). Bars indicate
SD. (C,D) Relative CO (C) and FT transcript levels (D) in the seedlings of indicated genotypes at the end of LDs (ZT16), quantified by qRT-PCR. The
transcript levels were normalized first to the endogenous control UBQ10, and relative fold changes to WT are presented (note that the FT transcript
level in co was less than 1% of that in WT). Bars indicate SD of triplicate measurements. One of two biological repeats with similar results is shown. (E)
FT mRNA levels in Col and afr1 afr2 seedlings over a 24-h LD cycle, as quantified by qRT-PCR. FT transcript levels were normalized to UBQ10; bars
indicate SD of triplicate measurements. A biological repeat of this analysis is included as Figure S8A. White and dark bars below the x-axis indicate
light and dark periods, respectively.
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Chromatin-Mediated Day-Length Control of Flowering
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CO Activity Is Required for the AFR1 Recruitment to FT
Chromatin at the End of LDs
As we showed earlier, CO is required for AFR1- and AFR2-
mediated FT downregulation at the end of LDs. We reasoned that
the CO activity at the FT locus at the end of LDs may be required
for AGL18/AGL15 recruiting AFRs to FT. To test this, first we
crossed the AFR1:HA-expressing line (in afr1-1)toco and created
co afr1 AFR1:HA. Next, we carried out ChIP assays with the
AFR1:HA-expressing seedlings harvested at ZT16, and found that
CO, indeed, is required for AFR1 binding to FT chromatin at
ZT16 (Figure 7). Upon its accumulation towards dusk in LDs, CO
directly binds to the FT proximal promoter to promote its
expression [37]. This raises a possibility that CO might directly
interact with AGL18 and/or AFRs to recruit these proteins to the
FT proximal promoter. Using the yeast-two-hybrid approach we
found that CO didn’t directly interact with AGL18, AFR1, or
AFR2 (Figure S15); hence, we conclude that the CO protein is not
directly involved in the recruitment of AFRs to FT chromatin.
Taken together, these results suggest that the CO-mediated
transcriptional activation of FT at the end of LDs enables or
gates the recruitment of AFR-HDAC by AGL18/AGL15 to the
FT locus, leading to the dampening of FT expression and so
preventing precocious flowering in response to the day-length cue.
Discussion
In this study, we have revealed that the plant-unique AFR1 and
AFR2 mediate periodic histone deacetylation on the rhythmically
expressed florigen gene FT to dampen its expression specifically at
dusk in LDs in Arabidopsis. The output of the photoperiod pathway
CO at the end of LDs not only activates FT expression, but also
enables the recruitment of AFR-HDAC to the FT locus to dampen
FT expression and set it at an adequate level, conferring a proper
flowering time in response to the day length cue. The MADS-
domain transcription factor AGL18 directly interacts with the
AFR1 and AFR2 proteins that accumulate at dusk, and recruits
AFR1 (presumably AFR1/AFR2-HDAC) to the FT locus at the
end of LDs to modulate FT expression. Our findings collectively
uncover a temporal chromatin mechanism, namely periodic
histone deacetylation, for the day-length regulation of flowering
time in higher plants.
Dynamic Cycles of Histone Acetylation and Deacetylation
at the FT Locus
The histone acetylation mark added by HATs can be rapidly
removed by HDACs. Both HATs and HDACs have been found to
associate with actively transcribed genes in yeast and human, and
are often synchronously recruited to certain target loci, leading to
dynamic cycles of acetylation and deacetylation [13–15]. HATs
can bind transiently to and add acetyl groups to certain loci where
HDACs transiently bind to erase the acetylation mark, resulting in
constant acetylation and de-acetylation cycles at these loci [13–
15]. Our study reveals that at the FT locus, accompanying
rhythmic CO-mediated FT activation at the end of LDs, AFR1
and AFR2 (presumably AFR-HDAC) are recruited to the actively
transcribed FT chromatin for histone deacetylation. Given that
disruption of AFR1 and AFR2 function leads to an increase of
steady-state level of acetylation on FT chromatin at the end of
LDs, there must be a HAT (or HATs) to add acetyl groups,
suggesting that constant cycles of acetylation and de-acetylation
occur at the FT locus upon its activation by CO at dusk. AFR1/
AFR2-HDAC is expected to act antagonistically to a HAT to
regulate FT expression. Of note, the HAT(s) has not been
identified yet.
The dynamic histone acetylation and deacetylation cycles in the
proximal FT promoter may result from transient binding of HATs
and AFR1/AFR2-HDAC to FT chromatin. Consistent with the
transient or unstable AFR1-HDAC binding, our ChIP analysis
shows that AFR1 is moderately enriched at the FT promoter
(Figures 4E and 7). The increase of steady-state acetylation level at
FT and FT upregulation upon disruption of AFR1/AFR2
Figure 4. Analyses of AFR1 and AFR2 expression patterns and their bindings to
FT
chromatin. (A) Spatial expression patterns of AFR1-
GUS, AFR2-GUS, and FT-GUS. LD-grown Col seedlings or rosette leaves were stained for 6 h except for AFR1-GUS staining with 8.5 h. Arrows indicate
stained veins. (B) Nuclear localization of the AFR1:GFP and AFR2:GFP fusion proteins in Arabidopsis root cells. Scale bars are 50
mm. The blue DAPI
staining indicates nuclei. (C) AFR1 and AFR2 mRNA levels in Col (WT) seedlings over a 24-h LD cycle. The mRNA levels were normalized to UBQ10; bars
indicate SD of triplicate measurements. A biological repeat of this analysis is included as Figure S8B. White and dark bars below the x-axis indicate
light and dark periods, respectively. (D) AFR1:HA and AFR2:FLAG protein levels in Col seedlings over a 24-h LD cycle. Total proteins loaded in a
duplicated SDS-PAGE gel were stained with Coomassie Blue, serving as loading controls. (E) ChIP analysis of AFR1:HA enrichment at the FT locus.
Amounts of the immunoprecipitated genomic fragments were measured by qPCR, and normalized first to the endogenous control TUBULIN8 (TUB8).
The fold enrichment of AFR1:HA in each examined region (at each time point) was calculated by dividing the TUB8-normalized amount of examined
region from the AFR1:HA-expressing line, by that of WT (without AFR1:HA) at each time point. Error bars indicate SD of triplicate quantifications
(technical replicates). A biological repeat of this analysis is presented as Figure S10A. (F) ChIP analysis of AFR2:FLAG enrichment at the FT locus. The
fold enrichments of AFR2:FLAG were calculated in a way similar to those of AFR1:HA. Error bars indicate SD of triplicate quantifications. A biological
repeat of this analysis is presented as Figure S10B.
doi:10.1371/journal.pbio.1001649.g004
0
0.3
0.6
0.9
1.2
1.5
1.8
Col (WT)
afr1 afr2
FT-P
Relative fold enrichment
2.1
ZT8 ZT16
FT-E1
ZT8 ZT16
FT-I1
ZT8 ZT16
Figure 5. ChIP analysis of levels of acetylated histone H3 in Col
and
afr1 afr2
rosette leaves. Amounts of the immunoprecipitated
genomic fragments were quantified by qPCR. The fold enrichments
were calculated as follows: for each examined FT region, the amount of
DNA fragments from WT or afr1 afr2 at each time point (ZT8 or ZT16)
was first normalized to the constitutively expressed TUBULIN2 (TUB2)in
each sample, and subsequently, the TUB2-normalized values for the afr1
afr2 at ZT8, the afr1 afr2 at ZT16, or the WT at ZT16 were divided by the
value for the WT at ZT8 to obtain fold enrichments. Shown are the
means and SD of two ChIP experiments. An analysis of H3 acetylation
on FT chromatin in Col and afr1 afr2 seedlings is presented in Figure
S11.
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Chromatin-Mediated Day-Length Control of Flowering
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Figure 6. AGL18 directly interacts with AFR1 and binds to
FT
chromatin at the day’s end in LDs. (A) BiFC analysis of the interaction of
AGL18 with AFR1 in onion epidermal cells. Yellowish-green signals indicate the physical association of AGL18 with AFR1 in the nuclei (indicated by
the blue fluorescence from DAPI). Bar = 20
mm. (B) Co-immunoprecipitation of AFR1 with AGL18 in Arabidopsis seedlings. Total protein extracts from
F
1
seedlings of the doubly hemizygous AGL18:FLAG and AFR1:HA, were immunoprecipitated with anti-HA agarose; subsequently, the precipitates
were analyzed by western blotting with anti-FLAG (recognizing AGL18:FLAG) and anti-HA (recognizing AFR1:HA). (C) Flowering times of the indicated
genotypes grown in LDs. 12–16 plants were scored for each line. Double asterisks indicate a statistically significant difference in the means betwee n
Col and agl15 agl18, as revealed by two-tailed Student’s t test (**, p,0.01). Bars indicate SD. (D) Relative FT transcript levels in the seedlings of
indicated genotypes at ZT16, quantified by qRT-PCR. The transcript levels were first normalized to UBQ10, and relative fold changes to Col are
presented. Bars indicate SD of triplicate measurements. One of two biological repeats with similar results is shown. (E) ChIP analysis of AGL18:FLAG
enrichment at the FT locus. Amounts of immunoprecipitated genomic fragments were measured by qPCR, and normalized first to the endogenous
control TUB8. The fold enrichment of AGL18:FLAG in each examined region (at each time point) was calculated by dividing the TUB8-normalized
amount of examined region from the AGL18:FLAG-expressing line, by that of WT (without AGL18:FLAG) at each time point. Error bars indicate SD of
triplicate measurements. A biological repeat of this analysis is presented as Figure S13B.
doi:10.1371/journal.pbio.1001649.g006
Chromatin-Mediated Day-Length Control of Flowering
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function, indicate that at the end of LDs histone acetylation
promotes FT transcription, whereas histone deacetylation execut-
ed by AFR-HDAC acts to downregulate FT expression. It is very
likely that when FT chromatin is actively transcribed upon
transcriptional activation by CO at the end of LDs, AFR-HDAC
may remove acetyl groups following each cycle of HAT activities
associated with FT transcription to reset the acetylation state of FT
chromatin, and functions to dampen FT expression at dusk. The
AFR-HDAC activities at dusk may also serve to reset FT
chromatin rapidly to a silent state with a low level of acetylation
at night. Consistent with this notion, we have observed that upon
loss of AFR1 and AFR2 function, it takes several hours longer to
reset FT expression to a silent state at night (Figures 3E and S8A).
Interestingly, the steady-state acetylation levels of FT chroma-
tin, as measured by ChIP, remain largely unchanged from the
middle to the end of LDs (Figure 5), although FT expression is
switched on at dusk. Not surprisingly, this may reflect the dynamic
cycles of histone acetylation and deacetylation upon FT expression
induction by CO. In the budding yeast, during transcriptional
induction of ARG1 (encoding an argininosuccinate synthase), the
Gcn5 HAT and several HDACs are synchronously recruited to
ARG1 chromatin, and these opposing activities result in no
noticeable changes of steady-state acetylation levels at the ARG1
locus, as measured by ChIP [14]. A similar situation has been
observed during the transcriptional induction of several heat-
responsive genes by heat shock in yeast [15].
Role of CO in Gating the Recruitment of AFR1/AFR2-
HDAC to FT Chromatin at the End of LDs
In this study we have revealed that the MADS-domain
transcription factor AGL18 and presumably AGL15 act to recruit
AFR1/AFR2-HDAC to the FT locus at the end of LDs. AGL18 is
well expressed from ZT8 to ZT16 (unpublished data), but the
AGL18 protein binds to FT chromatin (in the proximal promoter)
only at ZT16. This indicates that the chromatin state of the FT
locus may play a role for the AGL18 binding to it; for instance, the
state may determine whether a cis-element such as a CArG motif,
recognized typically by a MADS domain, in FT promoter is
accessible to AGL18.
We found that the output of the photoperiod pathway CO is
required for, but not directly involved in the recruitment of AFR1
to FT chromatin at dusk. The CO protein directly binds to FT
proximal promoter and also associates with NY-F transcriptional
factors that bind to FT distal promoter, to promote FT expression
specifically at the end of LDs [37–40]. Given the indirect role of
CO for AFR1/AFR2 recruitment, we infer that at the end of LDs
the binding of CO to the FT proximal promoter and/or
consequent transcriptional activation of FT expression enable
AGL18 recruiting AFRs to the FT locus. It is likely that CO
activity may cause a change in FT chromatin state so that the cis-
element(s) such as a CArG motif or its variant in the FT promoter
become accessible by AGL18. In short, in the photoperiodic
flowering regulation CO plays a dual role: activating FT expression
and enabling AFR-HDAC-mediated FT downregulation to set a
proper level of FT expression at the end of LDs.
The accumulation of both AFR1 and AFR2 at the end of LDs
indicates that AFR1/AFR2-HDAC may accumulate at dusk,
which may enable or enhance their recruitment by AGL18 to the
FT proximal promoter. CO activity not only enables AGL18-
AFR-HDAC binding to FT chromatin to dampen FT expression,
but may also facilitate dynamic recruitment of a HAT to the FT
locus to promote its expression given the dynamic nature of
histone acetylation and deacetylation on FT chromatin at dusk
(Figure 8). The opposing activities of HAT and AFR-HDAC at the
end of LDs conceivably modulate the acetylation dynamics of FT
chromatin and set FT expression at an adequate level at the right
time.
Role of AFR-HDAC for Photoperiodic Flowering-Time
Regulation
We show he re that AFRs act in the photoperiod pathwa y to
dampen FT expression in response to inductive long days and so
prevent precocious flowering in Arabidopsis.Interestingly,onlya
moderate early-flow ering phenot ype ha s been observe d in the
afr1 afr2 mutant in which AFR1 is still expressed at a low level
(Figure S3A). It is likely that a complete loss of function of AFR1
and AFR2 may cause a strong early-flowering phenotype. In
short days, the CO protein does not a ccumulate and thus FT is
expressed at a very low level [2]. We have observed that in short
days FT expression is relatively high at ZT4 and ZT20-ZT24 in
WT, and that loss of AFR1 and AFR2 function leads t o a
moderate FT upregulation specifically at these time points
(Figure S7B). In addition, it has been shown that AGL18 als o
moderately represses FT expression in short days [ 35]. Taken
together, these findings suggest that A GL18-AFR-HDAC acts to
downregulate FT expression to prevent early f lowering in non-
inductive short days as well, conferring an accurate photope-
riodic response.
CO and FT are widely conserved among angiosperms from the
monocotyledonous rice grass to the dicotyledonous poplar tree
[1,2]. In the plants so far examined, the function of FT or FT
homologs as a major inducer of flowering (florigen) is largely
conserved, and the CO-FT regulatory module underlying the
photoperiodic flowering-time control in the dicotyledonous LD
plant Arabidopsis, also plays a central role for flowering-time control
in the monocotyledonous short-day plant rice [2]. Homologs of the
core components of AFR-HDAC including AFR1/AFR2, At-
SAP18, RPD3-type HDACs, and SNLs are highly conserved in
0
0.3
0.6
0.9
1.2
1.5
1.8
2.1
AFR1:HA
AFR1:HA agl15 agl18
AFR1:HA co
CK (Col)
Relative AFR1:HA fold enrichment
Figure 7. AFR1 binding to
FT
chromatin at the end of LDs
requires
CO
,
AGL15
, and
AGL18
. Seedlings of afr1 AFR1:HA, agl15
agl18 afr1 AFR1:HA, co afr1 AFR1:HA, and WT (negative CK) were
harvested at ZT16 and subjected to ChIP assays with anti-HA. The fold
enrichments of AFR1:HA in FT-P (a proximal promoter region) in the
AFR1:HA-expressing lines over CK, are presented. Error bars indicate SD
of triplicate measurements. A biological repeat of this analysis is
presented as Figure S10C.
doi:10.1371/journal.pbio.1001649.g007
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PLOS Biology | www.plosbiology.org 11 September 2013 | Volume 11 | Issue 9 | e1001649
angiosperms (Figure S1B) [22,23,25]. These findings collectively
indicate that the temporal chromatin mechanism of periodic
histone deacetylation underlying FT expression control in
Arabidopsis may modulate the expression of FT homologs in other
plant species and so confer an optimal flowering time in response
to photoperiodic changes.
Materials and Methods
Plant Materials and Growth Conditions
The co, ft, fve, agl15-3, and agl18-1 were described previously
[6,27,35,41]. afr1-1 (Salk_110828), afr1-2 (Salk_059944), afr2-1
(Salk_032653), and afr2-2 (CS842844) were obtained from the
Arabidopsis Biological Resource Center and directly used in this
study. Plants were grown in long days (16-h light/8-h dark) or
short days (8-h light/16-h dark) under cool white fluorescent light
at 22uC [41].
Yeast Two-Hybrid Assay
The Matchmaker GAL4 Two-Hybrid System 3 (Clontech)
was adapted for this assay. Full-length coding sequences for
AFR1, AFR2, AtSAP18, HDA9, HDA19, and SNL2 were
cloned into the pGADT7 and/or pGBKT7 vectors, which were
subsequently introduced into the yeast strain AH109 according
to the manufacturer’s instructions (Clontech). Yeast cells were
spotted on selective media lacking of leucine, tryptophan, and
histidine.
BiFC Assay
The full-length coding sequences for AFR1, AFR2, AtSAP18,
HDA19, AGL15, and AGL18 were fused in frame with either the
coding sequence for an N-terminal EYFP fragment in the pSAT1A-
nEYFP-N1/pSAT1-nEYFPC1 vectors and/or for a C-terminal
EYFP fragment in the pSAT1AcEYFP-N1/pSAT1-cEYFP-C1-B
vectors [42]. Plasmid pairs were used to transiently transform
onion epidermal cells via bombardment by the Helium Biolistic
Gene Transformation System (Bio-Rad). Within 24 h following
the bombardment, the EYFP fluorescence emitted from the onion
cells was imaged with a Zeiss LSM 5 EXCITER upright laser
scanning confocal microscopy (Zeiss) [43].
RNA Analysis by Real-Time Quantitative PCR
Total RNAs were extracted from aerial parts of 10- to 11-d-old
seedlings grown in long days (at various time points) or 21-d-old
seedling grown in short day using the RNeasy Plus Mini Kit
(Qiagen) according to the manufacturer’s instructions. cDNAs
were reverse transcribed from the total RNAs. Real-time
quantitative PCR (qRT-PCR) was performed on an ABI Prism
7900HT sequence detection system using a SYBR green PCR
master mix; PCR was conducted as follows: 50uC (2 min), 95uC
(10 min), and 40 cycles of 95uC (15 s) and 60uC (60 s). Each
sample was quantified at least in triplicate, and the constitutively
expressed UBQ10 (its expression is not influenced by day-night
cycles) [44], was used for normalization. Ratio of the transcript
level of a gene of interest to that of UBQ10 is calculated as 2
2DCt
[DC
T
=C
T
(gene of interest) 2 C
T
(UBQ10)]. The primer pair for
CO amplification has been described previously [44], and primer
pairs for AFR1, AFR2, FT, and UBQ10 amplifications are specified
in Table S1.
Plasmid Construction
To construct AFR1-GUS and AFR2-GUS, genomic fragments of
3.0-kb AFR1 (1.0-kb 59 promoter plus 2.0-kb genomic coding
sequence) and 3.9-kb AFR2 (1.2-kb 59 promoter plus 2.7-kb
genomic coding sequence) were inserted upstream of GUS in the
pMDC162 vector [45] via gateway technology (Invitrogen). For
AFR1 and AFR2 subcellular localization, the full-length AFR1 and
AFR2 coding sequences (except the stop codons) were inserted
between the 35S promoter and GFP in the pMDC85-GFP vector
[45] via gateway technology (AFR1 and AFR2 are in frame with
GFP).
To generate the pAFR1-AFR1:HA plasmid, a 3.0-kb AFR1
genomic fragment (1.0-kb 59 promoter plus the 2.0-kb full-length
genomic coding sequence except the stop codon) was first fused
with a 3xHA tag, and cloned into the pHGW vector [46] via
gateway technology. For pAFR2-AFR2:FLAG construction, a 3.9-
kb AFR2 genomic fragment (1.2-kb 59 promoter plus the 2.7-kb
full-length genomic coding sequence except the stop codon) was
first fused with a 3xFLAG tag, and cloned into pHGW. To construct
p35S-AGL18:FLAG, the full-length AGL18 coding sequence without
the stop codon (772 bp) was first fused with a 3xFLAG tag, and the
AGL18:FLAG fusion was subsequently placed downstream of the
35S promoter in the pB2GW7 vector [46] via that gateway
technology. The sequences of primers used for plasmid construc-
tion are specified in Table S1.
Co-immunoprecipitation
Co-immunoprecipitation experiments were carried out as
previously described [43]. Briefly, total proteins were extracted
from 10-d-old seedlings and immunoprecipitated with anti-HA
M2 affinity gel (Sigma, catalog number A2220). AGL18:FLAG in
Figure 8. A working model for control of
FT
expression by the
dynamic cycles of histone acetylation and deacetylation at the
end of LDs. The coincidence of high CO mRNA expression with light
exposure at the day’s end leads to the CO protein accumulation
towards dusk. CO directly binds to the FT proximal promoter, and CO
activity at the FT locus may change the chromatin state and enables/
gates AGL18 (and presumably AGL15) binding to the FT proximal
promoter. AGL18 recruits AFR1/AFR2-HDAC to FT chromatin at dusk. In
addition, the CO activity may also enable the recruitment of a HAT to FT
chromatin. The opposing activities of HAT and AFR-HDAC on FT
chromatin at the end of LDs conceivably modulate the acetylation
dynamics of FT chromatin and set FT expression at an adequate level at
dusk. At night, CO is rapidly degraded by proteasomes, which prevents
the actions of HAT and AFR-HDAC on FT chromatin, resulting in a
‘‘silent’’ chromatin state. In early day, FT chromatin remains ‘silent’ due
to lack of the CO protein. Day and night are indicated with white and
gray shadings, respectively.
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the immunoprecipitates was detected by western blotting with
anti-FLAG (Sigma, catalog number A8592).
Chromatin Immunoprecipitation
ChIP experiments were carried out as described previously with
minor modifications [47]. Briefly, total chromatin was extracted
from 10-d-old seedlings or the first pair of rosette leaves from 13-d-
old seedlings grown in LDs, and immunoprecipitated with Rabbit
polyclonal anti- acetylated histone H3 (with acetyl
K9+K14+K18+K23+K27; abcam, catalog number ab47915),
anti-HA (Sigma, catalog number H6908), or anti-FLAG (Sigma,
catalog number F7425). Quantitative PCR was conducted to
measure the amounts of FT and the constitutively expressed TUB2
and TUB8 fragments on an ABI Prism 7900HT sequence
detection system using a SYBR Green PCR master mix. The
primers used are specified in Table S1.
Supporting Information
Figure S1 Alignments of AFR1 and AFR2 relatives.
Numbers refer to amino acid residues. Identical residues among
these proteins are shaded black, whereas similar residues are
shaded gray. (A) Alignment of the Arabidopsis AFR1 (AtAFR1) and
AFR2 (AtAFR2) with the yeast Sap30 (ScSap30). The conserved
residues in the putative SBRs are indicated by asterisks (*). (B)
Alignment of AtAFR1 with its homologs from other plants
including Populus trichocarpa (PtSFR), Picea sitchensis (PsSFR), and
Physcomitrella patens (PpSFR). The GenBank accession numbers for
PtSFR, PsSFR, and PpSFR are XP_002302473.1, ADE76839.1,
and XP_001766227.1, respectively.
(EPS)
Figure S2 Direct interactions of AT1G75060 (AFR1) and
AT1G19330 (AFR2) with SNL2 and HDA9 proteins in
yeast. The indicated full-length proteins were fused with the
GAL4-BD or AD domains. Yeast cells harboring the fusion
proteins, BD and/or AD (as indicated), were grown on the
selective synthetic defined media lacking of Trp (W), Leu (L), and
His (H). (A,B) Interactions of SNL2 with AFR1 and AFR2 in
yeast. (C,D) Interactions of HDA9 with AFR1 and AFR2 in yeast.
(TIF)
Figure S3 Analysis of AFR1 and AFR2 expression in afr1
and afr2 seedlings by semiquantitative RT-PCR. The
constitutively expressed TUB2 served as a control. (A) Analysis of
the full-length AFR1 and AFR2 transcripts. (B) Analysis of 59-or39-
truncated AFR2 transcripts in afr2 mutants.
(EPS)
Figure S4 Rescue of afr1 and afr2 mutants by pAFR1-
AFR1:HA and pAFR2-AFR2:FLAG, respectively. Plants
were grown in short days, and total leaf number for each line
(9–11 plants per line) was scored. Bars indicate SD. (A) Flowering
times of afr1-1 and a transgenic line of pAFR1-AFR1:HA in afr1-1
(native AFR1 fused with 3x HA; single-locus T
3
homozygotes). (B)
Flowering times of afr2-1 and a transgenic line of pAFR2-
AFR2:FLAG in afr2-1 (native AFR2 fused with 3x FLAG; single-
locus T
3
homozygotes).
(EPS)
Figure S5 Analysis of flowering time and leaf initiation
rate of afr1 afr2 in LDs. 12 plants were scored for each line;
bars for SD. (A) Days to flowering of Col and afr1 afr2. (B) Total
leaf numbers at flowering of Col and afr1 afr2. (C) Leaf initiation
rates of Col and afr1 afr2 plants.
(EPS)
Figure S6 Relative AFR1 and AFR2 transcript levels in
Col and co seedlings at the end of LDs, quantified by
qRT-PCR. The transcript levels were normalized first to the
endogenous control UBQ10, and relative fold changes to Col are
presented. Bars indicate SD of triplicate measurements.
(EPS)
Figure S7 AFR1 and AFR2 moderately repress FT
expression in short days. ( A) Flowering tim es o f the
indicated lines grown in short days. Total leaf number for each
line (8–14 plan ts per line) was scored. Error bars in dicate SD.
(B) FT m RNA levels in Col and afr1 afr2 seedlings over a 24-h
short-day cycle. T otal RNAs were extracted from 21-d old
seedlings every 4 h and quantified by qRT-PCR. Bars indicate
SD of three measurements.
(EPS)
Figure S8 Biological repeats of FT, AFR1, and AFR2
expression analyses. (A) A biological repeat of the FT
expression analysis presented in Figure 3E. (B) A biological repeat
of the AFR1 and AFR2 expression analysis presented in Figure 4C.
(EPS)
Figure S9 Relative levels of AFR1:HA and AFR2:FLAG
proteins over a 24-h LD cycle. The proteins in respective 10-
to 11-d-old seedlings were analyzed by western blotting. The
intensities of protein bands were quantified by the ImageJ 1.44j
program, and relative levels to ZT0 are presented. Note that the
time of the day has a significant effect on the levels of both AFR1
and AFR2 proteins in the seedlings, as revealed by single-factor
ANOVA tests (for AFR1, p,0.01; for AFR2, p,0.05). (A) Relative
AFR1:HA levels over a 24-h LD cycle. Average values of three
biological repeats are presented; bars for SD. (B) Relative
AFR2:FLAG levels over a 24-h LD cycle. Average values of four
biological repeats are presented; bars for SD.
(EPS)
Figure S10 Biological repeats of the ChIP assays on
AFR1 and AFR2 binding to FT chromatin. (A) A biological
repeat of the ChIP analysis presented in Figure 4E. (B) A biological
repeat of the ChIP analysis presented in Figure 4F. (C) A biological
repeat of the ChIP analysis presented in Figure 7.
(EPS)
Figure S11 ChIP analysis of the histone H3 acetylation
state of FT chromatin in Col and afr1 afr2 seedlings
grown in LDs. Amounts of the immunoprecipitated genomic
DNA was quantified and normalized first to the constitutively
expressed TUB2. Subsequently, the fold enrichments were
calculated by dividing the levels of acetylated H3 in afr1 afr2 (at
ZT8 or ZT16) or the WT at ZT16, by the level in the WT at ZT8.
Shown are the means and SD of two ChIP experiments.
(EPS)
Figure S12 Direct interactions of AGL18 with AFR2 and
AGL15 with AFR1. (A) BiFC analysis of the interaction of
AGL18 with AFR2 in onion epidermal cells. Yellowish-green
signals indicate the binding of AGL18 with AFR2 in the nuclei
(indicated by the blue fluorescence from DAPI). Bar = 20
mm. (B)
AFR1 interacts with AGL15, but not FLC, in yeast cells. The full-
length AFR1, and full-length FLC and the 208-aa AGL15
(without MADS domain) were fused with the GAL4-AD and
BD domains, respectively. Yeast cells were grown on the selective
synthetic defined media lacking of W, L, and H or lacking of W
and L. Of note, FLC directly interacts with SVP and binds to FT
chromatin to repress FT expression [48], and serves as a negative
control in this experiment. (C) BiFC analysis of the interaction of
Chromatin-Mediated Day-Length Control of Flowering
PLOS Biology | www.plosbiology.org 13 September 2013 | Volume 11 | Issue 9 | e1001649
AGL15 with AFR1 in onion epidermal cells. The full-length
AGL15 and AFR1 were fused with nEYFP and cEYFP fragments,
respectively. Yellowish-green signals indicate the binding of
AGL15 with AFR1 in the nuclei. Bar = 20
mm.
(TIF)
Figure S13 Characterization of the p35S-AGL18:FLAG
line and a biological repeat of ChIP analysis of AGL18
binding to FT chromatin. (A) Flowering times of the p35S-
AGL18:FLAG line (T
3
; in the Col background) grown in LDs.
Overexpression of AGL18 causes moderate late flowering [35].
Double asterisk indicates a statistically significant difference in the
means between Col and the transgenic line, as revealed by two-
tailed Student’s t test (**, p,0.01). Bars indicate SD. (B) A
biological repeat of ChIP analysis of AGL18 binding to FT
chromatin presented in Figure 6E.
(EPS)
Figure S14 Analysis of AGL15 and AGL18 expression in
Col and agl15 agl18 seedlings by RT-PCR. The constitu-
tively expressed TUB2 served as a control.
(EPS)
Figure S15 Examination of CO interaction with AFR1,
AFR2, or AGL18 by the yeast-two-hybrid assay. The full-
length CO was fused with GAL4-AD, whereas the full-length
AFR1, AFR2, and AGL18 were fused with GAL4-BD. Yeast cells
were grown on the selective synthetic defined media lacking of W,
L, and H or lacking of W and L.
(TIF)
Table S1 List of primers used in this study.
(DOCX)
Acknowledgments
We thank Donna E. Fernandez for generously providing agl15 and agl18
seeds, Stanton Gevin for providing the BiFC vectors, and Flanders
Interuniversity Institute for Biotechnology (Belgium) for providing the
pHGW and pB2GW7 vectors.
Author Contributions
The author(s) have made the following declarations about their
contributions: Conceived the research: YH. Designed the experiments:
XG YW. Performed the experiments: XG YW. Analyzed the data: XG
YW YH. Wrote the paper: YH.
References
1. Wigge PA (2011) FT, a mobile developmental signal in plants. Curr Biol 21:
R374–378.
2. Turck F, Fornara F, Coupland G (2008) Regulation and identity of florigen:
FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol 59: 573–
594.
3. Kobayashi Y, Weigel D (2007) Move on up, it’s time for change–mobile signals
controlling photoperiod-dependent flowering. Genes Dev 21: 2371–2384.
4. Imaizumi T, Kay S (2006) Photoperiodic control of flowering: not only by
coincidence. Trends Plant Sci 11: 550–558 .
5. Kobayashi Y, Kaya H, Goto K, Iwabuchi M, Araki T (1999) A pair of related
genes with antagonistic roles in mediating flowering signals. Science 286: 1960–
1962.
6. Kardailsky I, Shukla V, Ahn J, Dagenais N, Christensen S, et al. (1999)
Activation tagging of the floral inducer FT. Science 286: 1962–1965.
7. Corbesier L, Vincent C, Jang S, Fornara F, Fan Q, et al. (2007) FT protein
movement contributes to long-distance signaling in floral induction of Arabidopsis.
Science 316: 1030–1033.
8. Tamaki S, Matsuo S, Wong H, Yokoi S, Shimamoto K (2007) Hd3a protein is a
mobile flowering signal in rice. Science 316: 1033–1036.
9. Mathieu J, Warthmann N, Kuttner F, Schmid M (2007) Export of FT protein
from phloem companion cells is sufficient for floral induction in Arabidopsis. Curr
Biol 17: 1055–1060.
10. Jaeger K, Wigge P (2007) FT protein acts as a long-range signal in Arabidopsis.
Curr Biol 17: 1050–1054.
11. Putterill J, Robson F, Lee K, Simon R, Coupland G (1995) The CONSTANS
gene of Arabidopsis promotes flowering and encodes a protein showing similarities
to zinc finger transcription factors. Cell 80: 847–857.
12. Berger S (2007) The complex language of chromatin re gulation during
transcription. Nature 447: 407–412.
13. Wang Z, Zang C, Cui K, Schones D, Barski A, et al. (2009) Genome-wide
mapping of HATs and HDACs reveals distinct functions in active and inactive
genes. Cell 138: 1019–1031.
14. Govind C, Zhang F, Qiu H, Hofmeyer K, Hinnebusch A (2007) Gcn5 promotes
acetylation, eviction, and methylation of nucleosomes in transcribed coding
regions. Mol Cell 25: 31–42.
15. Kremer S, Gross D (2009) SAGA and Rpd3 chromatin modification complexes
dynamically regulate heat shock gene structure and expression. J Biol Chem 284:
32914–32931.
16. Servet C, Conde e Silva N, Zhou D (2010) Histone acetyltransferase AtGCN5/
HAG1 is a versatile regulator of developmental and inducible gene expression in
Arabidopsis. Mol Plant 3: 670–677
17. Deng W, Liu C, Pei Y, Deng X, Niu L, et al. (2007) Involvement of the histone
acetyltransferase AtHAC1 in the regulation of flowering time via repression of
FLOWERING LOCUS C in Arabidopsis. Plant Physiol 143: 1660–1668.
18. Yu C, Liu X, Luo M, Chen C, Lin X, et al. (2011) HISTONE
DEACETYLASE6 interacts with FLOWERING LOCUS D and regulates
flowering in Arabidopsis. Plant Physiol 156: 173–184.
19. Han S, Song J, Noh Y, Noh B (2007) Role of plant CBP/p300-like genes in the
regulation of flowering time. Plant J 49: 103–114.
20. Ahringer J (2000) NuRD and SIN3 histone deacetylase complexes in
development. Trends Genet 16: 351–356.
21. Grzenda A, Lomberk G, Zhang JS, Urrutia R (2009) Sin3: master scaffold and
transcriptional corepressor. Biochim Biophys Acta 1789: 443–450.
22. Pandey R, Muller A, Napoli C, Selinger D, Pikaard C, et al. (2002) Analysis of
histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana
suggests functional diversification of chromatin modification among multicellular
eukaryotes. Nucleic Acids Res 30: 5036–5055.
23. Bowen A, Gonzalez D, Mullins J, Bhatt A, Martinez A, et al. (2010) PAH-
domain-specific interactions of the Arabidopsis transcription coregulator SIN3-
LIKE1 (SNL1) with TELOMERE-BINDING PROTEIN 1 and ALWAYS
EARLY2 Myb-DNA binding factors. J Mol Biol 395: 937–949.
24. Liu C, Xi W, Shen L, Tan C, Yu H (2009) Regulation of floral patterning by
flowering time genes. Dev Cell 16: 711–722.
25. Song C, Galbraith D (2006) AtSAP18, an orthologue of human SAP18, is
involved in the regulation of salt stress and mediates transcriptional repression in
Arabidopsis. Plant Mol Biol 60: 241–257.
26. Xie T, He Y, Korkeamaki H, Zhang Y, Imhoff R, et al. (2011) S tructure of the
30-kDa Sin3-associated protein (SAP30) in complex with the mammalian Sin3A
corepressor and its role in nucleic acid binding. J Biol Chem 286: 27814–27824.
27. Ausin I, Alonso-Blanco C, Jarillo J, Ruiz-Garcia L, Martinez-Zapater J (2004)
Regulation of flowering time by FVE, a retinoblastoma-associated protein. Nat
Genet 36: 162–166.
28. LeeJ,YooS,ParkS,HwangI,LeeJ,etal.(2007)RoleofSVP in the control
of flowering time by ambient temperature in Arabidopsis. Genes D ev 2 1: 39 7–
402.
29. Jiang D, Wang Y, Wang Y, He Y (2008) Repression of FLOWERING LOCUS C
and FLOWERING LOCUS T by the Arabidopsis Polycomb repressive complex 2
components. PLoS ONE 3: e3404.
30. Lu F, Cui X, Zhang S, Liu C, Cao X (2010) JMJ14 is an H3K4 demethylase
regulating flowering time in Arabidopsis. Cell Res 20: 387–390.
31. Jeong J, Song H, Ko J, Jeong Y, Kwon Y, et al. (2009) Repression of
FLOWERING LOCUS T chromatin by functionally redundant histone H3 lysine
4 demethylases in Arabidopsis. PLoS One 4: e8033. doi:10.1371/journal.-
pone.0008033
32. Yang W, Jiang D, Jiang J, He Y (2010) A plant-specific histone H3 lysine 4
demethylase represses the floral transition in Arabidopsis. Plant J 62: 663–673.
33. Takada S, Goto K (2003) TERMINAL FLOWER 2,anArabidopsis homolog of
HETEROCHROMATIN PROTEIN1, counteracts the activation of FLOWERING
LOCUS T by CONSTANS in the vascular tissues of leaves to regulate flowering
time. Plant Cell 15: 2856–2865.
34. Searle I, He Y, Turck F, Vincent C, Fornara F, et al. (2006) The transcription
factor FLC confers a flowering response to vernalization by repressing meristem
competence and systemic signaling in Arabidopsis. Genes Dev 20: 898–912.
35. Adamczyk B, Lehti-Shiu M, Fernandez D (2007) The MADS domain factors
AGL15 and AGL18 act redundantly as repressors of the floral transition in
Arabidopsis. Plant J 50: 1007–1019.
36. Hill K, Wang H, Perry S (2008) A transcriptional repression motif in the MADS
factor AGL15 is involved in recruitment of histone deacetylase complex
components. Plant J 53: 172–185.
37. Tiwari S, Shen Y, Chang H, Hou Y, Harris A, et al. (2010) The flowering time
regulator CONSTANS is recruited to the FLOWERING LOCUS T promoter via
a unique cis-element. New Phytol 187: 57–66.
Chromatin-Mediated Day-Length Control of Flowering
PLOS Biology | www.plosbiology.org 14 September 2013 | Volume 11 | Issue 9 | e1001649
38. Kumimoto R, Zhang Y, Siefers N, Holt B (2010) NF-YC3, NF-YC4 and NF-YC9
are required for CONSTANS-mediated, photoperiod-dependent flowering in
Arabidopsis thaliana. Plant J 63: 379–391.
39. Wenkel S, Turck F, Singer K, Gissot L, Le Gourrierec J, et al. (2006)
CONSTANS and the CCAAT box binding complex share a functionally
important domain and interact to regulate flowering of Arabidopsis. Plant Cell 18:
2971–2984.
40. Blackman B, Michaels S (2010) Does CONSTAN S act as a transcription factor
or as a co-activator? The answer may be–yes. New Phytol 187: 1–3.
41. Michaels S, Amasino R (2001) Loss of FLOWERING LOCUS C activity
eliminates the late-flowering phenotype of FRIGIDA and autonomous pathway
mutations but not responsiveness to vernalization. Plant Cell 13: 935–941.
42. Lee L, Fang M, Kuang L, Gelvin S (2008) Vectors for multi-color bimolecular
fluorescence complementation to investigate protein-protein interactions in
living plant cells. Plant Methods 4: 24.
43. Jiang D, Kong N, Gu X, Li Z, He Y (2011) Arabidopsis COMPASS-like
complexes mediate histone H3 lysine-4 trimethylation to control floral transition
and plant development. PLoS Genet 7: e1001330. doi:10.1371/journal.-
pone.0008033
44. Castillejo C, Pelaz S (2008) The balance between CONSTANS and
TEMPRANILLO activities determines FT expression to trigger flowering. Curr
Biol 18: 1338–1343.
45. Curtis M, Grossniklaus U (2003) A gateway cloning vector set for high-
throughput functional analysis of genes in planta. Plant Physiol 133: 462–469.
46. Karimi M, De Meyer B, Hilson P (2005) Modular cloning in plant cells. Trends
Plant Sci 10: 103–105.
47. Johnson L, Cao X, Jacobsen S (2002) Interplay between two epigenetic marks:
DNA methylation and histone H3 lysine 9 methylation. Curr Biol 12: 1360–1367.
48. Li D, Liu C, Shen L, Wu Y, Chen H, et al. (2008) A repressor complex governs
the integration of flowering signals in Arabidopsis. Dev Cell 15: 110–120.
Chromatin-Mediated Day-Length Control of Flowering
PLOS Biology | www.plosbiology.org 15 September 2013 | Volume 11 | Issue 9 | e1001649
