Arabidopsis transcription factor ELONGATED HYPOCOTYL5 plays a role in the feedback regulation of phytochrome A signaling.
ABSTRACT Phytochrome A (phyA) is the primary photoreceptor responsible for perceiving and mediating various responses to far-red light in Arabidopsis thaliana. FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) are two small plant-specific proteins essential for light-regulated phyA nuclear accumulation and subsequent phyA signaling processes. FHY3 and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) are two transposase-derived transcription factors that directly activate FHY1/FHL transcription and thus mediate subsequent phyA nuclear accumulation and responses. Here, we report that ELONGATED HYPOCOTYL5 (HY5), a well-characterized bZIP transcription factor involved in promoting photomorphogenesis, directly binds ACGT-containing elements a few base pairs away from the FHY3/FAR1 binding sites in the FHY1/FHL promoters. We demonstrate that HY5 physically interacts with FHY3/FAR1 through their respective DNA binding domains and negatively regulates FHY3/FAR1-activated FHY1/FHL expression under far-red light. Together, our data show that HY5 plays a role in negative feedback regulation of phyA signaling by attenuating FHY3/FAR1-activated FHY1/FHL expression, providing a mechanism for fine-tuning phyA signaling homeostasis.
- SourceAvailable from: Zhangjun Fei[show abstract] [hide abstract]
ABSTRACT: Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5) was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.BMC Plant Biology 11/2011; 11:169. · 4.35 Impact Factor
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ABSTRACT: Among the five phytochromes in Arabidopsis thaliana, phytochrome A (phyA) plays a major role in seedling de-etiolation. Until now more then ten positive and some negative components acting downstream of phyA have been identified. However, their site of action and hierarchical relationships are not completely understood yet.Plant signaling & behavior 11/2011; 6(11):1714-9.
Article: Phytochrome Signaling MechanismsThe Arabidopsis Book 08/2011;
Arabidopsis Transcription Factor ELONGATED HYPOCOTYL5
Plays a Role in the Feedback Regulation of
Phytochrome A Signaling
Jigang Li,a,bGang Li,bShumin Gao,b,cCristina Martinez,bGuangming He,a,bZhenzhen Zhou,bXi Huang,b
Jae-Hoon Lee,bHuiyong Zhang,b,1Yunping Shen,a,b,2Haiyang Wang,band Xing Wang Denga,b,3
aPeking-Yale Joint Center for Plant Molecular Genetics and Agro-Biotechnology, National Laboratory of Protein Engineering
and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China
bDepartment of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, Connecticut 06520-8104
cCollege of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
Phytochrome A (phyA) is the primary photoreceptor responsible for perceiving and mediating various responses to far-red
light in Arabidopsis thaliana. FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its homolog FHY1-LIKE (FHL) are two small
plant-specific proteins essential for light-regulated phyA nuclear accumulation and subsequent phyA signaling processes.
FHY3 and its homolog FAR-RED IMPAIRED RESPONSE1 (FAR1) are two transposase-derived transcription factors that
directly activate FHY1/FHL transcription and thus mediate subsequent phyA nuclear accumulation and responses. Here, we
report that ELONGATED HYPOCOTYL5 (HY5), a well-characterized bZIP transcription factor involved in promoting
photomorphogenesis, directly binds ACGT-containing elements a few base pairs away from the FHY3/FAR1 binding sites
in the FHY1/FHL promoters. We demonstrate that HY5 physically interacts with FHY3/FAR1 through their respective DNA
binding domains and negatively regulates FHY3/FAR1-activated FHY1/FHL expression under far-red light. Together, our
data show that HY5 plays a role in negative feedback regulation of phyA signaling by attenuating FHY3/FAR1-activated
FHY1/FHL expression, providing a mechanism for fine-tuning phyA signaling homeostasis.
Phytochromes are red (R)/far-red (FR) light photoreceptors that
play fundamental roles in photoperception of the light environ-
ment and the subsequent adaptation of plant growth and devel-
Deng, 2003; Bae and Choi, 2008). There are five distinct phyto-
chromes in Arabidopsis thaliana, designated phytochrome A
(phyA) to phyE. PhyA is light-labile and is the primary photore-
ceptor responsible for perceiving and mediating various re-
sponses to FR light, whereas phyB-phyE are light-stable, and
phyBisthepredominant phytochromeregulating responses toR
light (Sharrock and Quail, 1989; Somers et al., 1991; Nagatani
et al., 1993; Parks and Quail, 1993; Reed et al., 1993; Whitelam
et al.,1993). Phytochromes are synthesized in the cytosolin their
inactive Pr form. Upon light irradiation, phytochromes are con-
verted from the R light–absorbing Pr forms to the FR light–
absorbing Pfr forms, and the Pfr forms of phytochromes (which
are generally considered to be the biologically active forms) are
translocated from the cytosol into the nucleus, triggering a sig-
naling cascade that alters the expression of target genes and
ultimately leads to the modulation of the biological responses
(Sakamoto and Nagatani, 1996; Kircher et al., 2002; Quail, 2002;
Jiao et al., 2007). Thus, light-regulated translocation of the photo-
receptors from the cytosol into the nucleus is a key event in the
phytochrome signaling cascade.
PhyA nuclear import is rapid and can be induced by either FR
or R light; however, phyB nuclear import is relatively slow, only
occurs in R light, and can be reversed by FR light (Kircher et al.,
1999; Nagatani, 2004; Kevei et al., 2007; Fankhauser and Chen,
2008). The C-terminal half of phyB contains a putative nuclear
localization signal (NLS), which is masked by the N-terminal half
in darkness. Light triggers a conformational change in phyB,
potentially unmasking the NLS and thus allowing its nuclear
import (Chen et al., 2005; Fankhauser and Chen, 2008). By
contrast, phyA does not contain any known NLS. Therefore,
phyA translocation appears to depend on other components.
Recently, it has been shown that two small plant-specific pro-
teins, FAR-RED ELONGATED HYPOCOTYL1 (FHY1) and its
homolog FHY1-LIKE (FHL), are essential for nuclear accumula-
tion of light-activated phyA and subsequent light responses
(Hiltbrunner et al., 2005, 2006; Ro ¨sler et al., 2007). A database
search for FHY1/FHL homologs identified FHY1-like proteins in
numerous plant species, and the only motifs conserved among
1Current address: Department of Biology, University of Virginia,
Charlottesville, VA 22904.
2Current address: Department of Molecular, Cell, and Developmental
Biology, University of California, Los Angeles, CA 90095.
3Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Xing Wang Deng
CSome figures in this article are displayed in color online but in black
and white in the print edition.
WOnline version of this article contains Web-only data.
The Plant Cell, Vol. 22: 3634–3649, November 2010, www.plantcell.org ã 2010 American Society of Plant Biologists
these FHY1-like proteins are the NLS in their N-terminal region
and the phyA binding domain at the C terminus, suggesting that
the phyA nuclear import mechanism discovered in Arabidopsis
might be conserved in higher plants (Genoud et al., 2008). Two
transposase-derived transcription factors, FHY3 and FAR-RED
IMPAIRED RESPONSE1 (FAR1), act together to activate directly
the transcription of FHY1 and FHL, thus indirectly regulating
phyA nuclear accumulation andphyA responses (Linet al.,2007,
Genetic and molecular studies have led to the identification of
numerous signaling intermediates that are either specific for
individual photoreceptors or shared by multiple types of photo-
receptors. Many of these intermediates are transcription factors
or transcriptional regulators (for reviews, see Quail, 2002; Wang
and Deng, 2003; Jiao etal., 2007).Inaddition to FHY3 and FAR1,
a group of basic helix-loop-helix class transcription factors, also
known as PHYTOCHROME INTERACTING FACTORS (PIFs),
including PIF1, PIF3, PIF4, and PIF5, have been shown to bind
photoactivated phytochromes directly and play central roles
in phytochrome signaling networks (Ni et al., 1998; Huq and
Quail, 2002;Kim etal.,2003;Huqetal., 2004;Khannaetal., 2004;
Duek and Fankhauser, 2005; Castillon et al., 2007). Recent
data showed that these PIF proteins accumulate in dark-grown
seedlings and together act as constitutive repressors of pho-
tomorphogenesis, while upon light exposure, photoactivated
phytochromes induce rapid phosphorylation and degradation of
these transcription factors, allowing photomorphogenesis to
begin (Bauer et al., 2004; Al-Sady et al., 2006; Shen et al., 2007;
Leivar et al., 2008; Lorrain et al., 2008; Shen et al., 2008).
ELONGATED HYPOCOTYL5 (HY5), aconstitutively nuclear bZIP
transcription factor, has been shown to function as a positive
regulator of photomorphogenic development under a wide
spectrum of wavelengths, including FR, R, blue (B), and UV-B,
by binding directly to the promoters of a large number of light-
responsive genes in vivo (Koornneef et al., 1980; Oyama, et al.,
1997; Osterlund et al., 2000; Ulm et al., 2004; Lee et al., 2007).
Multiple photoreceptors, including phytochromes and crypto-
chromes, promote the accumulation of HY5 under specific light
conditions, possibly by reducing the nuclear abundance of
CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), an E3 ubiq-
uitin ligase targeting HY5 for proteasome-mediated degradation
in darkness (Osterlund and Deng, 1998; Osterlund et al., 2000).
LONG HYPOCOTYL IN FAR-RED1 (HFR1) and LONG AFTER
FAR-RED LIGHT1 (LAF1), atypical basic helix-loop-helix and
R2R3-MYB transcription factors, respectively, were identified as
positive regulators of phyA signaling (Fairchild et al., 2000;
Fankhauser and Chory, 2000; Soh et al., 2000; Ballesteros,
et al., 2001). Recently, it was shown that these two transcription
factors interact with each other, and this interaction stabilizes
both factors and enhances phyA photoresponses (Jang et al.,
FR light (Desnos et al., 2001; Lin et al., 2007), indicating that
FHY1/FHL expression is subject to strict negative feedback
regulation by phyA signaling, which is, however, poorly under-
stood. Here, we show that the bZIP transcription factor HY5 acts
in the feedback downregulation of FHY1/FHL expression by
phyA signaling. We demonstrate that HY5 directly binds ACGT-
containing elements (ACEs) in close proximity to the FHY3/FAR1
binding sites in the FHY1/FHL promoters. HY5 physically inter-
to the FHY1/FHL promoters. Thus, HY5 negatively regulates
FHY1/FHL expression upon FR light exposure by modulating the
activities of the transcriptional activators FHY3 and FAR1. There-
fore, HY5 repression of FHY1/FHL expression likely represents a
key mechanism in the feedback regulation of phyA signaling.
HY5 Directly Binds to the FHY1/FHL Promoters
To investigate whether other transcription factors are involved
in regulating FHY1/FHL expression, we performed yeast one-
hybrid assays to test whether PIF3, HY5, LAF1, and HFR1, four
transcription factors known to act in the phytochrome signaling
pathways, bind to the FHY1 and FHL promoters. Our results
show that of these four proteins, only HY5 binds directly to both
promoters (FHY3 and FAR1 were included as positive controls)
(Figure 1A), suggesting that HY5 may play a role in regulating
As previous research indicated that HY5 binds ACEs
(Chattopadhyay et al., 1998; Lee et al., 2007; Shin et al., 2007),
we analyzed the distribution of ACEs in the FHY1 and FHL
promoters. Interestingly, both promoters were found to harbor
two short regions that contain ACE elements: one region very
close to the stop codons of their respective upstream genes and
the other region located ;150 to 300 bp upstream of their
respective ATG start codons (Figure 1B). We then divided the
FHY1/FHL promoters into four overlapping fragments, desig-
nated A,B, C,and D,in which the B and Dfragments correspond
to the two ACE-containing regions (Figure 1B), and generated
yeast one-hybrid reporter constructs allowing the respective
fragments to drive LacZ reporter gene expression. As expected,
HY5 does not bind to the A or C fragments of either promoter,
consistent with the fact that there are no ACEs in these frag-
promoter, it does not bind to the corresponding fragment of the
FHL promoter (Figure 1C). By contrast, HY5 binds strongly to the
B fragments of both promoters (Figure 1C), suggesting that the
B fragments contain the major HY5 binding sites. As HY5 has
a close homolog in the Arabidopsis genome, known as HY5
HOMOLOG (HYH), we tested whether HYH also binds to the
FHY1 promoter. However, HYH failed to bind either the full-
length or the B fragment of the FHY1 promoter (see Supplemen-
tal Figure 1 online), indicating that these two homologs target
diverse genes. Interestingly, as the B fragments also contain
are able to bind to the B fragments of both promoters as well
(Figure 1C). Collectively, our data show that HY5, FHY3, and
FAR1 bind to the same fragments of the FHY1/FHL promoters.
A recent study using the chromatin immunoprecipitation
(ChIP)-chip approach identified 3894 putative HY5 binding
targets in the Arabidopsis genome (Lee et al., 2007), among
which, FHY1 was listed as a potential HY5 binding target (see
Negative Control of FHY1/FHL Expression 3635
Figure 1. Transcription Factor HY5 Binds to the FHY1 and FHL Promoters in Yeast Cells and in Vivo.
(A) Yeast one-hybrid assays to test whether transcription factors PIF3, HY5, LAF1, and HFR1 bind to the FHY1 and FHL promoters. FHY3 and FAR1
were included as positive controls and empty vector expressing AD domain alone as negative controls. LacZ reporter gene expression (leading to blue
color on the plates containing X-gal) was driven by the FHY1 or FHL promoter in yeast.
(B) Diagram of the promoter fragments of FHY1 and FHL and the sequences of B fragments of the FHY1 and FHL promoters. The exon-intron structure
3636 The Plant Cell
Supplemental Table 1 in Lee et al., 2007). To confirm this data
experimentally and to examine whether HY5 binds to the FHL
promoter in vivo, we performed ChIP assays using wild-type
Arabidopsis seedlings. As HY5 protein accumulates mostly in
light conditions (Osterlund et al., 2000; see Supplemental Figure
2online), wethenexaminedwhetherHY5 bindsto theFHY1/FHL
promoters in continuous FR, R, and B light. Our multiplex PCR
and quantitative PCR (qPCR) both show that HY5 specifically
binds to the FHY1 promoter in vivo in all three monochromatic
light conditions (Figures 1D and 1E), compared with an exon
fragment of FHY1 and the 39 untranslated region of At4g26900, a
negative control used in the previous ChIP-chip study. However,
although our results also show that HY5 binds to the FHL
promoter (Figures 1D and 1F), this binding seems much weaker
than that to FHY1 as our qPCR data show that the recovery rate
of an FHL promoter fragment in the FR ChIP sample is;25-fold
less than that of an FHY1 promoter fragment. Moreover, at least
for the FHL promoter fragment, we observed a higher recovery
rate in the FR sample than in the R and B samples (Figures 1D
and 1F). Taken together, we conclude that HY5 binds to the
FHY1 and FHL promoters in yeast cells and in vivo.
HY5 Binds the ACEs Closest to FBSs in the
We next performed yeast one-hybrid assays to delineate the
exact ACEs that are bound by HY5 in the B fragments of the
FHY1/FHL promoters. As there are two and three ACEs in the B
fragments of the FHY1 and FHL promoters, respectively, a total
of 10 reporter constructs were generated to allow the respective
wild-type subfragments and their corresponding mutants (mut;
in which the respective ACGT was mutated to tttT) to drive LacZ
reporter gene expression in yeast cells (Figure 2A). Our results
show that for the two ACEs in the FHY1 promoter, HY5 binds the
wild-type ACE1, but not its mutant, nor ACE2 in yeast cells
(Figure 2B). We also mutated ACE1 and ACE2 in the context of
is the preferred binding site for HY5. Again, mutation of ACE1
almost abolished binding of HY5 to this promoter fragment,
whereas mutation of ACE2 did not affect binding, confirming
that HY5 primarily binds ACE1 in the B fragment of the FHY1
promoter (Figure 2C).
For the three ACEs in the B fragment of the FHL promoter, our
data show that HY5 does not bind to the wild-type subfragments
of ACE1 or ACE2 (Figure 2B). However, for unknown reasons,
the subfragments containing either wild-type or mutant ACE3
always induced strong LacZ reporter gene expression in yeast
cells (Figure 2B). We therefore performed an electrophoretic
mobility shift assay (EMSA) to test whether HY5 binds to the
ACE3 subfragment in vitro. The ACE1 subfragment of the FHY1
promoter was also included in the assay as a positive control.
Our data show that the GST-HY5 fusion protein (glutathione
S-transferase fused with HY5), but not GST alone, bound the
ACE1 subfragment of the FHY1 promoter and the ACE3 subfrag-
ment of the FHL promoter in vitro (Figure 2D), indicating that
ACE3 is the HY5 binding site in the B fragment of the FHL
Interestingly, we noticed that ACE1 of the FHY1 promoter, and
ACE3 of the FHL promoter, are the respective ACEs that are
closest to FBSs in the FHY1/FHL promoters, and there are only
10 bp or less between these two cis-elements (Figure 1B). We
FAR1 binding sites in the FHY1 and FHL promoters.
HY5 Interacts with FHY3 and FAR1
As HY5 and FHY3/FAR1 bind cis-elements that are so close to
each other in the FHY1/FHL promoters, it was intriguing to
investigate whether these two types of transcription factors
could physically interact with each other. To this end, we first
performed yeast two-hybrid assays using bait vectors express-
ing either the full-length, N-terminal (harboring the COP1 inter-
action motif), or C-terminal (encoding the bZIP domain) domains
of HY5 fused to the LexA DNA binding domain and prey vectors
fusing the activation domain (AD) to the indicated Arabidopsis
proteins (Figures 3A and 3B). Our data show that the full-length
HY5 protein indeed interacts with FHY3 and FAR1, and the bZIP
domain of HY5 is responsible for this interaction (AD-COP1
and AD-HY5 were included as positive controls for the N- and
C-terminal domains of HY5, respectively) (Figure 3B).
Figure 1. (continued).
of FHL was based on the fact that FHL has an alternatively spliced transcript consisting of three exons and encoding a 201–amino acid isoform
(GenBank accession number HM029245), in addition to the reported 181–amino acid FHL protein (Zeidler et al., 2001; Zhou et al., 2005). The genes
upstream of FHY1 and FHL are At2g37680 and At5g02190, respectively. The adenine residue of the respective translational start codon (ATG) was
assigned position +1, and the numbersflanking the sequences ofthe B fragments were counted based on this number. Asterisks indicate ACEs. A,B, C,
and D indicate the corresponding promoter fragments used in yeast one-hybrid assays shown in (C). The black lines indicate the regions amplified by
ChIP-PCR shown in (D). The short green lines depict the location of amplicons used for ChIP-qPCR shown in (E) and (F).
(C) Yeast one-hybrid assays showing that HY5 binds to the B fragments of the FHY1 and FHL promoters. FHY3 and FAR1 were included as positive
controls, and empty vector expressing AD domain alone as negative controls.
(D) Representative results of the ChIP-PCR assays. Chromatin fragments (;500 bp) were prepared from 4-d-old wild-type Arabidopsis seedlings
grown under continuous FR, R, and B light conditions, immunoprecipitated by the polyclonal HY5 antibodies, and the precipitated DNA PCR-amplified
using the primer pairs indicated. Input, PCR reactions using the samples before immunoprecipitation. AB, antibody.
(E) and (F) qPCR analysis of the promoter (b fragments) and exon fragments of FHY1 and FHL in anti-HY5 ChIP assays. The 39 untranslated region of
At4g26900, a negative control used in a previous study (Lee et al., 2007), was used as a negative control in these assays. The ChIP values were
normalized to their respective DNA inputs. Error bars represent SD of triplicate experiments.
Negative Control of FHY1/FHL Expression3637
FHY3 and FAR1 were reported to contain three domains: an
N-terminal C2H2 zinc finger domain, a central putative core
transposase domain, and a C-terminal SWIM zinc finger domain
(Lin et al., 2008). To define which of these domains of FHY3 and
FAR1 interact with HY5, we fused them individually with LexA as
indicated (Figure 3C) and then used these fusions as bait pro-
teins in yeast two-hybrid assays. Unexpectedly, the SWIM zinc
finger domain of FAR1 showed strong trans-activation activity in
yeast cells (Figure 3D). Nevertheless, FHY3N and FAR1N also
showed interaction with AD-HY5, allowing the conclusion that
the C2H2 zinc finger motifs (i.e., the DNA binding domains of
FHY3 and FAR1) may be responsible for interacting with HY5
(Figure 3D). To confirm this interaction further, we performed in
vitro pull-down assays using GST-tagged HY5 and 63His-tagged
N-terminal domains of FHY3 and FAR1. Our data showed that
domains of FHY3 and FAR1 (Figure 3E). Taken together, these
binding domains mediate their interactions.
To substantiate the physical interaction between HY5 and
FHY3 in planta, we conducted firefly luciferase complementa-
tion imaging (LCI) assays (Chen et al., 2008) by transiently ex-
pressing HY5-NLuc and CLuc-FHY3N fusions in Nicotiana
benthamiana leaf cells. As shown in Figure 3F, coexpression of
HY5-NLuc and CLuc-FHY3N led to strong LUC activity. By
contrast, HY5-NLuc or CLuc-FHY3N cotransformed with control
Figure 2. HY5 Binds the ACEs Closest to the FHY3/FAR1 Binding Sites in the FHY1 and FHL Promoters.
(A) Diagram of the wild-type (WT) and mutant (mut) FHY1 and FHL subfragments used to drive LacZ reporter gene expression in yeast one-hybrid
assays and as EMSA probes. Wild-type ACE elements are shown in red, and FBS motifs are shown in blue. Nucleotide substitutions in the mutant
fragments are underlined.
(B) Yeast one-hybrid assays showing that HY5 binds the wild-type ACE1 of the FHY1 promoter but not ACE2 of the FHY1 promoter, or ACE1 or ACE2 of
the FHL promoter.
(C) Yeast one-hybrid assays showing that ACE1 is the primary binding site for HY5 in the B fragment of the FHY1 promoter. The B fragment of the FHY1
promoter (sequence shown in Figure 1B) was mutated to abolish ACE1, ACE2, or both and used to drive LacZ reporter gene expression. In these
assays, the respective ACGT was mutated to AaaT to facilitate mutagenesis reactions.
(D) EMSAs showing that GST-HY5 protein, but not GST by itself, specifically binds to the wild-type FHY1p-ACE1 and FHLp-ACE3 probes. The
respective unlabeled probes (with the same sequence as the biotin-labeled probes) were used as competitors. FP, free probe.
3638The Plant Cell
Figure 3. HY5 Interacts with FHY3 and FAR1.
(A) Schematic diagram of bait proteins (HY5, HY5N, and HY5C fused with LexA DNA binding domains).
(B) HY5C (corresponding to the bZIP domain of HY5) interacts with FHY3 and FAR1 in yeast cells. AD-COP1 and AD-HY5 were included as positive
controls for interactions between HY5N and COP1 and the dimerization of bZIP domains, respectively.
(C) Schematic diagram of bait proteins (FHY3N, FHY3C1, FHY3C2, FAR1N, FAR1C1, and FAR1C2 fused with LexA DNA binding domains).
(D) Yeast two-hybrid assays showing that the DNA binding domains of FHY3 and FAR1 (FHY3N and FAR1N) interact with HY5.
(E) In vitro pull down of FHY3N and FAR1N with HY5. The 63His-tagged N-terminal fragments of FHY3 and FAR1 pulled down with GST-HY5 or GST
were detected by anti-His antibody. Input, 5% of the purified 63His-tagged target proteins used in pull-down assays.
(F) Luciferase complementation imaging assays showing that HY5 interacts with FHY3N in plant cells. Tobacco leaves were transformed with the
construct pairs HY5-NLuc/CLuc, HY5-NLuc/CLuc-FHY3N, and NLuc/CLuc-FHY3N. The leaves were observed for fluorescence imaging 3 d after the
Negative Control of FHY1/FHL Expression 3639
vectors showed only background levels of LUC activity. These
data support a physical interaction between HY5 and FHY3 in
living plant cells.
HY5 Interferes with FHY3 for Binding to the FHY1 Promoter
As the HY5 binding site is only a few base pairs away from the
FHY3 binding site in the FHY1 promoter, we next investigated
how HY5 affects FHY3 binding to the FHY1 promoter by EMSAs
validated its biochemical activity by showing that it binds to the
wild type but not the FBS-mutated FHY1 probe (Figure 4A). As
GST-HY5 always migrates at a similar position as GST-FHY3N
when they bind to the wild-type FHY1 probes (Figure 4A), we
used GST-tagged HY5C (the amino acids 78 to 168 of HY5)
instead in our assays. GST-HY5C also binds to the wild type but
not the ACE1-mutated FHY1 probe and thus possesses the DNA
binding activity of HY5 (Figure 4A).
We then examined how HY5 affects FHY3 binding to the FHY1
GST protein alone had no effect on GST-FHY3N binding to the
promoter (lanes 4 to 6), increasing amounts of GST-HY5C
protein obviously decreased the binding of GST-FHY3N to the
wild-type FHY1 promoter (lanes 7 to 9). Then, we investigated
how the interaction between HY5 and FHY3 affects FHY3
binding to the promoter using the ACE1-mutated probe to which
HY5 could not bind. Notably, increasing amounts of GST-HY5C
FHY1 promoter even though GST-HY5C was not binding the
probe (Figure 4B, lanes 10 to 12). Therefore, these data suggest
that (1) increasing HY5 binding to the FHY1 promoter simulta-
neously decreases FHY3 binding to the promoter, and (2) the
binding to the FHY1 promoter.
HY5 Negatively Regulates FHY3/FAR1-Activated FHY1/FHL
Transcription in Yeast and Plant Cells
To investigate how HY5 affects FHY3/FAR1-mediated FHY1/
FHL transcription in yeast cells, we introduced another set of
constructs into our yeast one-hybrid system. These pGAD-T7–
based constructs were generated to express AD, HY5, or AD-
HY5 fusion proteins in yeast cells (Figures 5A and 5B). As shown
in Figure 5A, HY5 alone does not activate LacZ reporter gene
expression, although it could bind to the FHY1 promoter (cf.
1 and 2), consistent with the previous report that HY5 lacks the
transcriptional activation domain and is unable to activate tran-
scription in yeast cells (Ang et al., 1998). However, addition of an
activation domain to HY5 (AD-HY5) allows HY5 to activate LacZ
reporter gene expression (Figure 5A, cf. 2 and 3). As mentioned
above (Figures 1C and 2B), the B fragment of the FHL promoter
(possibly due to the ACE3-containing sequence shown in Figure
2A) induced strong background expression of the LacZ reporter
gene (Figure 5B, 1 and 2).
We then examined how HY5 affects FHY3/FAR1-mediated
reporter gene expression driven by the B fragments of the FHY1/
FHL promoters. As shown in Figures 5A and 5B, AD-FHY3 and
Figure 3. (continued).
infiltration. The data shown are representative of three independent experiments, and error bars represent SD of four replicates. The middle panel shows
an immunoblot for proteins isolated from tobacco leaves. Anti- full-length firefly LUC antibodies, which react with both the N- and C-terminal firefly LUC
fragments, were used to detect the fusion proteins. The amount of protein loaded in each lane is indicated by Ponceau S staining of ribulose-1,5-
bisphosphate carboxylase/oxygenase (bottom panel).
Figure 4. HY5 Interferes with FHY3 for Binding to the FHY1 Promoter.
(A) GST-FHY3N, GST-HY5, and GST-HY5C proteins, but not GST by itself, bind to the wild-type (WT) FHY1 promoter but not to the FBS (GST-FHY3N)
and ACE (GST-HY5 and GST-HY5C) mutant (mut) probes. The sequences of FHY1p-ACE1 wild-type and mutant probes are shown in Figure 2A. The
sequence of FHY1p-FBS mut probe is identical to that of the FHY1p-ACE1 wild-type probe except that the FBS motif (CACGCGC) was mutated to
CAttttC. FP, free probe.
(B) Increasing amounts of GST-HY5C protein (lanes 7 to 9), but not GST (lanes 4 to 6), decrease the binding of GST-FHY3N to the wild-type FHY1
promoter. Interaction between HY5C and FHY3N prevents GST-FHY3N from binding to the promoter (lanes 10 to 12). The triangles in lanes 3 and 9
indicate a polymer of HY5C bound to the probe.
3640The Plant Cell
AD-FAR1 robustly activate LacZ reporter gene expression in
yeast cells. However,coexpression of HY5 withAD-FHY3 orAD-
FAR1 significantly decreases b-galactosidase activity (at least
P < 0.05 for each compared group), implying that HY5 negatively
regulates FHY3/FAR1-activated FHY1/FHL expression. More-
over, if an activation domain was added to HY5, the reporter
gene expression was increased (Figures 5A and 5B), indicating
that HY5 does occupy the FHY1/FHL promoters when it is
coexpressed with FHY3/FAR1 in yeast cells.
We conducted a transient transcription assay in Nicotiana
benthamiana leaves to study whether HY5 plays a similar regu-
latory role in plant cells. We generated dual-luciferase reporter
constructs to allow the wild-type or ACE-mutated B fragment of
the FHY1 promoter to drive LUC reporter gene expression
(Figure 5C). As shown in Figure 5D, transiently expressed FHY3
acts as an activator of the FHY1 promoter. HY5 alone, however,
its behavior in yeast cells. Coexpression of HY5 with FHY3
dramatically decreases the reporter gene expression to a simi-
lar level as HY5 alone, and this contrast is especially obvious
when the ACEs in the FHY1p-B promoter fragment are mutated
Together with the EMSA assay data shown in Figure 4, we
conclude that HY5 negatively regulates FHY1/FHL expression
via two mechanisms. First, HY5 binds to the FHY1/FHL pro-
moters, and as the binding sites of HY5 are in close proximity to
those of FHY3/FAR1 in the FHY/FHL promoters, HY5’s occupa-
tion consequently decreases the accessibility of the promoters
to FHY3/FAR1. Second, HY5’s interaction with FHY3/FAR1 may
mechanisms are quite distinct from each other, but both seem
important as HY5 could downregulate FHY1/FHL expression
with or without its binding sites in the FHY1/FHL promoters
provided FHY3/FAR1 are present (Figure 5). In either case, HY5
achieves its regulatory goal by modulating the activities of the
transcriptional activators FHY3 and FAR1.
HY5 Negatively Regulates FHY1/FHL Transcript and FHY1
Protein Levels in Vivo
To confirm the role of HY5 in the regulation of FHY1/FHL ex-
pression in vivo, we compared the FHY1 and FHL transcript
levels in wild-type and hy5 mutant seedlings by RNA gel blot and
qRT-PCR analyses. Our data show thatFHY1 and FHLtranscript
Figure 5. HY5 Negatively Regulates FHY3/FAR1-Activated FHY1/FHL
Transcription in Yeast and Plant Cells.
(A) and (B) Quantification of b-galactosidase activity in yeast cells
harboring the FHY1p-B:LacZ (A) or FHLp-B:LacZ (B) reporter construct
and coexpressing AD/AD-FHY3/AD-FAR1 and AD/HY5/AD-HY5 protein
combinations shown on the left. Error bars represent SD (n = 4).
(C) Structure of the dual-luciferase reporter construct in which the firefly
luciferase (LUC) reporter gene is driven by the wild type or ACE-mutated
(both of the ACGT elements were mutated to AaaT) FHY1-B promoter
fragment. The Renillia luciferase (REN) reporter gene is controlled by the
constitutive 35S promoter. A 105-bp (?101 to +4) NOS minimal promoter
(Puente et al., 1996) was inserted upstream of the LUC coding sequence
to allow the promoter fragment to drive the LUC reporter gene tran-
scription. Cauliflower mosaic virus terminator (Ter) and the T-DNA left
border (LB) and right border (RB) are also indicated.
(D) Relative reporter activity in tobacco cells transiently transformed
with the indicated effector and reporter constructs. FHY3 and HY5
are expressed by the 35S:FHY3 and 35S:HY5 effector plasmids (see
Methods), respectively. Tobacco leaves were kept in white light for 4 d
after infiltration. The relative LUC activities normalized to the REN activity
are shown (LUC/REN). Error bars represent SD (n = 3).
[See online article for color version of this figure.]
Negative Control of FHY1/FHL Expression3641
levels are notably elevated in hy5 mutant seedlings particularly
under continuous FR light (Figures 6A to 6C), indicating that HY5
indeed negatively regulates FHY1 and FHL expression in Arabi-
Previous reports showed that FHY1 and FHL transcript levels
declined rapidly when the dark-grown wild-type seedlings were
exposed to FR light (Desnos et al., 2001; Lin et al., 2007). We
confirmed this pattern of FHY1/FHL downregulation using qRT-
PCR (Figures 6D and 6E). To investigate the role of HY5 in this
process, we examined the expression of FHY1 and FHL in hy5
mutant seedlings subjected to the same dark-to-FR light treat-
ment. Intriguingly, expression of both FHY1 and FHL in hy5
mutants showed a lesser decrease compared with wild-type
plants during the first 1 and 3 h, respectively, and then obviously
increased during subsequent FR light treatment (Figures 6D and
6E), indicating that HY5 does play a major role in downregulating
FHY1/FHL transcript levels in this FR light exposure treatment.
We then examined the levels of HY5 and FHY3 proteins in this
time course to rule out the possibility that HY5 might down-
regulate FHY1/FHL transcript levels indirectly bydownregulating
FHY3 and FAR1 expression, as the expression of FHY3 and
FAR1 displayed a pattern similar to that of FHY1 and FHL in this
process (Lin et al., 2007). Our immunoblot data show that HY5
protein levels increased dramatically and continuously after the
dark-grown wild-type seedlings were transferred to FR light
(Figure 6F). By contrast, FHY3 protein levels showed a mild
increase after 1 h of FR light exposure and then remained
relatively stable in the subsequent FR light treatment, at least
without showing an obvious decrease (Figure 6G). These data
suggest that HY5 directly exerts its regulation on the FHY1 and
FHL promoters, rather than indirectly through regulating expres-
sion of FHY3 and FAR1, although we cannot rule out the
possibility that HY5 may regulate FHY3 and FAR1 transcript
levels as well.
We further examined whether the abundance of FHY1 protein
is correspondingly regulated by HY5. As reported in a recent
study from our group (Shen et al., 2009), our FHY1 antibodies
always recognize two endogenous FHY1 bands in immunoblots.
Interestingly, only the smaller FHY1 band seems to be regulated
by the hy5 mutation in different light conditions (Figure 7). More-
over, the abundance of the smaller FHY1 band was increased in
hy5 mutants not only in continuous FR light but also in R and B
light conditions, suggesting that HY5 may be involved in the
posttranscriptional regulation of FHY1 as well.
We also tested FHY1 protein accumulation in fhy3 far1 and
phyA mutants. Consistent with the previous reports that FHY3
and FAR1 are key positive regulators of FHY1 expression
Figure 6. HY5 Negatively Regulates FHY1 and FHL Transcript Levels in
(A) RNA gel blot analysis showing FHY1 and FHL mRNA levels in 4-d-old
wild-type (WT) and hy5 mutant seedlings grown in darkness (D) or
continuous FR, R, and B light conditions. Ethidium bromide staining
showing rRNA was used as the loading control.
(B) and (C) Real-time qRT-PCR analysis showing FHY1 and FHL tran-
script levels in wild-type and hy5 mutant seedlings. Error bars represent
SD of triplicate experiments. *P < 0.05 and **P < 0.01 (Student’s t test) for
the indicated pair of seedlings.
(D) and (E) Changes in FHY1 and FHL transcript levels in wild-type and
hy5 mutant seedlings grown in darkness for 4 d and then transferred to
FR light for various time periods. The expression levels in dark-grown
seedlings were set as 1. Error bars represent SD of triplicate experiments.
(F) and (G) Immunoblots showing the changes of HY5 (F) and FHY3 (G)
protein levels in wild-type seedlings grown in darkness for 4 d and then
subjected to FR light treatment for various time periods. The mutant
plants (hy5-215 and fhy3-1, respectively) were included as negative
controls for immunoblots. Asterisk in (F) indicates a band cross-reacting
with HY5 antibody. Anti-RPT5 was used as a sample loading control.
3642The Plant Cell
(Desnos et al., 2001; Zhou et al., 2005; Lin et al., 2007), FHY1
protein level is severely attenuated in fhy3 far1 double mutants in
darkness and all light conditions, comparable to that in fhy1
mutants (Figure 7). However, phyA only downregulates FHY1
protein level in light conditions (Figure 7), which might be
achieved by direct phosphorylation of FHY1 by phyA under light
conditions (Shen et al., 2009).
COP1 Positively Regulates FHY1/FHL Transcript Levels
As HY5 was targeted for degradation by COP1 in darkness
(Osterlund et al., 2000), and HY5 acts as a repressor of FHY1/
FHL expression, we next examined whether COP1 positively
regulates FHY1/FHL expression in darkness indirectly via HY5.
To this end, we first confirmed that, as reported previously, HY5
protein level was extraordinarily elevated in cop1 mutants in
darkness (Osterlund et al., 2000; see Supplemental Figure 3
online), while FHY3 protein level was not decreased by the cop1
mutation (data not shown). Then, we examined FHY1 and FHL
transcript levels in dark-grown wild-type and cop1 mutant seed-
lings. Intriguingly, both FHY1 and FHL transcript levels in cop1
mutants were decreased to around 10% of that in the wild type
mediating COP1-regulated FHY1/FHL expression. Consistent
with this finding, our previous study shows that COP1 is essen-
tial for FHY1 protein accumulation in darkness (Shen et al.,
As the other COP/DET/FUS proteins, such as DET1, COP10,
and the subunits of the COP9 signalosome, were shown to be
required for degradation of HY5 in darkness (Osterlund et al.,
2000), it is likely that these COP/DET/FUS proteins may posi-
tively regulate FHY1/FHL expression in darkness as well. Con-
these COP/DET/FUS proteins are required for normal accumu-
lation of FHY1 protein in darkness (Shen et al., 2005).
HY5 RepressionofFHY1Expression Requires thePresence
of FHY3 and FAR1
As discussed above, since HY5 lacks transcriptional repression
activity in yeast and plant cells (Figure 5), it seems that HY5
represses FHY1/FHL expression by modulating the activities of
their transcriptional activators FHY3 and FAR1. To test this
hypothesis in vivo, we generated hy5 fhy3 far1 triple mutants to
mutate both the positive regulators FHY3/FAR1 and the negative
regulator HY5 of FHY1/FHL expression (see Supplemental Fig-
ure 4 online). We selected two independent triple mutant lines
and confirmed that all three loci are homozygous in both lines
(Figure 9A). qRT-PCR data show that in hy5 fhy3 far1 triple
mutants, FHY1 transcript levels are similar to those in fhy3 far1
double mutants (Figure 9B), suggesting that HY5 repression of
FHY1 expression requires the presence of its transcriptional
activators FHY3 and FAR1 in vivo.
FHY1 and FHL are two small proteins (202 and 201 amino acids,
respectively) in Arabidopsis that were found to have homologs in
both monocot and dicot plant species (Genoud et al., 2008). It
Figure 7. HY5 Negatively Regulates FHY1 Protein Levels.
FHY1 protein levels in 4-d-old fhy1, wild-type (WT), hy5, fhy3 far1, and
phyA seedlings grown in darkness (D) or continuous FR, R, and B light
conditions. The two arrowheads indicate two endogenous FHY1 bands
in immunoblots recognized by our FHY1 antibodies (Shen et al., 2009).
Anti-RPT5 was used as a sample loading control.
Figure 8. COP1 Positively Regulates FHY1 and FHL Transcript Levels in
Real-time qRT-PCR analysis showing that FHY1 (A) and FHL (B) tran-
script levels were markedly decreased in dark-grown cop1 mutants
compared with wild-type (WT) plants. Error bars represent SD of triplicate
Negative Control of FHY1/FHL Expression3643
was shown that FHY1 and FHL are required for nuclear accu-
mulation of phyA since phyA is localized only in the cytosol of
fhy1 fhl double mutants (Hiltbrunner et al., 2006; Ro ¨sler et al.,
2007). This finding was greatly extended by a recent report that
FHY3 and FAR1, two transposase-derived transcription factors,
are the key activators of FHY1/FHL transcription and thus
indirectly regulate phyA nuclear accumulation and phyA signal-
ing (Lin et al., 2007). However, the mechanism by which FHY1/
FHL expression is downregulated by the feedback regulation of
phyA signaling remains unclear, although previous reports indi-
cate that expression of FHY1/FHL, as well as FHY3/FAR1, are
under this control (Desnos et al., 2001; Lin et al., 2007).
In this study, we show that the well-characterized Arabidopsis
bZIP transcription factor HY5 directly represses FHY1/FHL ex-
pression in FR light, and, interestingly, this action of HY5 is
accomplished by modulating the activities of the transcriptional
activators FHY3 and FAR1. Because FHY1 and FHL are the key
positive regulators of phyA signaling, the consequence of this
action of HY5 may potentially attenuate phyA signaling. How-
ever, HY5 has been genetically defined as a positive regulator of
phyA signaling, as mutations in HY5 cause a defect in the
inhibition of hypocotyl elongation in continuous FR light (Oyama
et al., 1997; Ang et al., 1998; see Supplemental Figure 4 online).
In fact, our results may not be contradictory to the previous
findings because this role of HY5 in downregulating FHY1/FHL
transcript levels serves mainly in the feedback process of phyA
signaling (i.e., phyA signaling has already been triggered and,
thus, FHY1/FHL transcripts are not required at high levels), as it
was shown that the accumulation of HY5 protein in FR light also
requires phyA (Osterlund et al., 2000). Thus, once phyA is
imported into the nucleus by FHY1 and FHL, phyA triggers a
signaling cascade, and one consequence of this cascade is the
accumulation of HY5, which acts to promote photomorphogen-
esisand downregulateFHY1/FHL transcriptlevels simultaneously
(Figure 10). Therefore, our data suggest that HY5 plays dual roles
in phyA signaling.
Figure 9. HY5 Repression of FHY1 Expression Requires the Presence of
FHY3 and FAR1.
(A) Detection of HY5 and FHY3 proteins in 4-d-old wild-type (Col and
No-0 ecotypes), fhy3 far1, hy5, and two independent homozygous lines
of hy5 fhy3 far1 triple mutant plants grown under continuous FR light.
Anti-RPT5 was used as a sample loading control. The far1-2 mutation in
the two lines of hy5 fhy3 far1 triple mutants was confirmed by directly
sequencing the PCR products that contain the mutation (data not shown).
(B) Real-time qRT-PCR analysis showing FHY1 transcript levels in 4-d-
old wild-type (Col and No-0 ecotypes), fhy1 fhl, fhy3 far1, phyA, hy5, and
two independent homozygous lines of hy5 fhy3 far1 triple mutant plants
grown under continuous FR light. Error bars represent SD of triplicate
Figure 10. A Working Model Depicting How HY5 Functions in the
Feedback Regulation of phyA Signaling.
In the absence of light, FHY3 and FAR1 induce the expression of FHY1
and FHL in anticipation of the upcoming light signal. Accumulation of
FHY1 and FHL proteins in dark-grown seedlings may serve to ensure
rapid and sufficient phyA nuclear accumulation upon FR light exposure
to jump start phyA signaling events in the nucleus. Upon light exposure,
the Pfr form of phyA is imported into the nucleus by FHY1/FHL and thus
triggers phyA signaling leading to multiple light responses, including the
reduction of COP1 in the nucleus and accumulation of HY5 (Osterlund
and Deng, 1998; Osterlund et al., 2000), and feedback regulation of FHY3
and FAR1 transcript levels (Lin et al., 2007). HY5 plays dual roles in phyA
signaling: promoting photomorphogenesis and downregulating FHY1/
FHL transcript levels by modulating the activities of the transcriptional
activators FHY3 and FAR1. FHY3 and FHY1 (indicated by larger letters)
are the more predominant players in the phyA signaling process com-
pared with their respective homologs FAR1 and FHL. Pr, R-absorbing
form of phyA (inactive); Pfr, FR-absorbing form of phyA (active). Arrow,
positive regulation; bar, negative regulation.
3644The Plant Cell
Because FHY1 and FHL regulate phyA nuclear accumulation,
and HY5 negatively regulates FHY1 (and possibly FHL) protein
levels (Figure 7), it is reasonable to propose that HY5 might
negatively regulate phyA nuclear accumulation. However, we
failed to detect any difference in nuclear phyA levels between FR-
grown wild-type and hy5 mutant seedlings in our nuclear fraction-
one molecular event after phyA is imported into the nucleus by
FHY1/FHL is the degradation of the photoreceptor (Seo et al.,
excess phyA proteins imported into the nucleus by the increased
levels of FHY1/FHL proteins may be degraded rapidly. Second,
overaccumulation of FHY1/FHL may affect mainly the kinetics of
phyA nuclear accumulation, rather than the steady state levels of
nuclear phyA, whereas nuclear fractionation assays only roughly
detect the steady state levels of nuclear phyA. So the question
whether HY5 regulates phyA nuclear accumulation will require
further investigation and better techniques.
We show that HY5 acts as a repressor of FHY1/FHL expres-
sion. However, HY5 itself does not show any transcriptional
activity in yeast and plant cells (Figure 5). Our data suggest that
FHY3/FAR1, two activators of FHY1/FHL expression. This con-
clusion isalso supported by the examination of FHY1 expression
in hy5 fhy3 far1 triple mutants (Figure 9). In another report, HY5
was shown to be necessary for normal circadian expression of
the Lhcb genes via interaction with CCA1 (Andronis et al., 2008).
Consistent with these findings, a recent study demonstrates that
HY5 binding to the target gene promoters is not sufficient for
transcriptional regulation, implying that HY5 may need other
cofactors to regulate target gene expression (Lee et al., 2007).
Moreover, a transcription factor protein microarray study dis-
covered 20 transcription factor candidates thatmay interactwith
HY5 (Gong et al., 2008). Thus, the discovery of more HY5-
interacting cofactors and elucidation of more regulatory modes
of HY5 may help in understanding how HY5 implements its
hierarchical role in promoting photomorphogenesis.
In summary, our work reveals an interesting new role of HY5 in
the feedback regulation of phyA signaling. Taken together with
achieved by at least four distinct mechanisms: negative regula-
tion of FHY1/FHL expression by HY5 shown in this study, FHY3/
FAR1 repression by phyA signaling (Lin et al., 2007), direct
phosphorylation of FHY1 by phyA and subsequent FHY1 deg-
radation (Shen et al., 2005, 2009), and phosphorylation and
subsequent degradation of phyA itself (Saijo et al., 2008). The
multiple layers of feedback regulation imply a complicated and
to respond quickly, appropriately, and precisely to their dynamic
Plant Materials and Growth Conditions
The wild-type Arabidopsis thaliana used in this study is of the Columbia
(Col) ecotype, unless otherwise indicated. The phyA-211 (Reed et al.,
1994), fhy1-3 (Zeidler et al., 2001), fhy1-3 fhl-1 (Ro ¨sler et al., 2007), hy5-
215 (Oyama et al., 1997), fhy3-1 (Whitelam et al., 1993), and cop1-4
(McNellis et al., 1994) mutants are of the Col ecotype, and the fhy3-4
(Wang and Deng, 2002) and fhy3-4 far1-2 (Lin et al., 2007) mutants are of
the No-0 ecotype and have been described previously. The hy5 fhy3 far1
triple mutant was constructed by crossing hy5-215 and fhy3-4 far1-2
mutants. The mutation hy5-ks50 (Oyama et al., 1997) was introduced into
the Col background by genetic backcrossing (Lee et al., 2007). The
growth conditions and light sources were as described previously (Shen
et al., 2005).
The FHY1p:LacZ and FHLp:LacZ reporter constructs were described
previously (Lin et al., 2007). To generate FHY1p-A:LacZ, FHY1p-B:LacZ,
FHY1p-C:LacZ, FHY1p-D:LacZ, FHLp-A:LacZ, FHLp-B:LacZ, FHLp-C:
LacZ, and FHLp-D:LacZ reporter constructs, the promoter fragments
were amplified by PCR using FHY1p:LacZ and FHLp:LacZ constructs as
the templates and the respective pairs of primers (see Supplemental
Table 1online), andthenclonedintotheEcoRI-XhoIsitesofthepLacZi2m
vector (Lin et al., 2007), respectively. To generate various LacZ reporter
genes driven by the wild-type and mutant subfragments of the FHY1 and
two complementary oligo primers with an EcoRI site overhang at the 59
end and an XhoI site overhang at the 39 end (see Supplemental Table
1 online). The oligo primers were annealed, and the double-stranded
oligonucleotides were ligated into the EcoRI-XhoI sites of the pLacZi2m
vector, producing FHY1p-ACE1WT:LacZ, FHY1p-ACE2WT:LacZ, FHLp-
ACE1WT:LacZ, FHLp-ACE2WT:LacZ, FHLp-ACE3WT:LacZ, FHY1p-
ACE1mut:LacZ, FHY1p-ACE2mut:LacZ, FHLp-ACE1mut:LacZ, FHLp-
ACE2mut:LacZ, and FHLp-ACE3mut:LacZ, respectively.
the FHY1p-B:LacZ reporter plasmid (described above) was used as the
template using the QuikChange site-directed mutagenesis kit (Strata-
the manufacturer’s instructions. To generate the FHY1p-B(ACEm):LacZ
reporter constructin which both ofthe ACGT elements were mutated into
AaaT, two rounds of mutagenesis reactions were performed, with each
round introducing one mutation into the promoter.
The AD-FHY3, AD-FAR1, and AD-COP1 constructs were described
AD-HY5, AD-LAF1, AD-HFR1, and AD-HYH, the full-length coding se-
quencesofPIF3, HY5,LAF1,HFR1,andHYH wereamplified byPCRwith
cloned into the EcoRI-XhoI sites of the pB42AD vector (Clontech),
respectively. To generate pGAD-T7 constructs for expressing HY5 and
AD-HY5 fusion proteins in yeast cells, the full-length coding sequence of
HY5 was amplified by PCR with the respective pairs of primers (see
Supplemental Table 1 online) and then cloned into the KpnI-XhoI and
EcoRI-XhoI sites of the pGAD-T7 vector (Clontech), respectively.
The LexA-HY5, LexA-HY5N, and LexA-HY5C constructs were de-
scribed previously (Ang et al., 1998). To generate LexA-FHY3N, LexA-
FHY3C1, and LexA-FHY3C2, the fragments were amplified by PCR with
cloned into the BamHI-SalI sites of the pLexA vector (Clontech), respec-
tively. To generate LexA-FAR1N, LexA-FAR1C1, and LexA-FAR1C2, the
respective PCR fragments were cloned into the BamHI-XhoI sites of the
pLexA vector, respectively.
The GST-HY5 and GST-FHY3N constructs were described previously
(Ang et al., 1998; Lin et al., 2007). To generate GST-HY5C, the PCR
fragment was cloned into the BamHI-XhoI sites of the pGEX-4T-1 vector
the PCR fragments were cloned into the BamHI-SalI and BamHI-XhoI
sites of the pET-28a vector (Novagen), respectively.
Negative Control of FHY1/FHL Expression 3645
To generate HY5-NLuc, a BamHI-SalI PCR fragment of full-length HY5
was cloned into the corresponding sites of the vector 35S:NLuc (Chen
et al., 2008). To generate CLuc-FHY3N, a KpnI-SalI PCR fragment of
FHY3N (first 260 amino acids) was cloned into the corresponding sites of
the vector 35S:CLuc (Chen et al., 2008).
To generate the 35S:FHY3 vector, a BamHI-SalI fragment containing
the full-length coding sequence of FHY3 was released from LexA-FHY3
the BamHI-SalI sites of the pSPYNE-35S vector (Walter et al., 2004). To
generate the 35S:HY5 vector, a BamHI-SalI PCR fragment of full-length
HY5 was cloned into the corresponding sites of the pSPYNE-35S vector.
To generate the FHY1p-B:LUC and FHY1p-B(ACEm):LUC reporter
constructs, a PstI-BamHI PCR fragment containing the 105 bp (2101 to
+4) NOS minimal promoter was amplified from NOS101-GUS (Puente
et al., 1996) and inserted into the corresponding sites of pGreenII 0800-
LUC (Hellens et al., 2005), resulting in miniPro:LUC vector. Then, the B
fragment of the FHY1 promoter was amplified by PCR using FHY1p-B:
LacZ and FHY1p-B(ACEm):LacZ constructs (described above) as the
templates and cloned into the KpnI-XhoI sites of miniPro:LUC to produce
the FHY1p-B:LUC and FHY1p-B(ACEm):LUC vectors, respectively.
listed in Supplemental Table 1 online, and all of the constructs were
confirmed by sequencing prior to usage in various assays.
For yeast one-hybrid assays, plasmids for AD fusions were cotrans-
formed with the LacZ reporter genes driven by various FHY1 and FHL
promoter fragments into the yeast strain EGY48; for yeast two-hybrid
assays, the respective combinations of LexA and AD fusion plasmids
were cotransformed into the yeast strain EGY48, which already contains
the reporter plasmid p8op:LacZ (Clontech). Transformants were grown
on proper dropout plates containing X-gal (5-bromo-4-chloro-3-indolyl-
and liquid assay were conducted as described in the Yeast Protocols
For anti-FHY3 immunoblots, Arabidopsis seedlings were ground to a fine
powder and total proteins were eluted in 23 SDS loading buffer. For all
the other immunoblots, Arabidopsis seedlings were homogenized in an
extraction buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10
mM MgCl2, 0.1% Tween 20, 1 mM PMSF, 40 mM MG132, and 13
complete protease inhibitor cocktail (Roche). Immunoblotting was per-
formed as previously described (Shen et al., 2005).
Primary antibodies used in this study include anti-FHY1 (Shen et al.,
2005), anti-HY5 (Osterlund et al., 2000),anti-FHY3(Saijo et al.,2008), and
anti-RPT5 (Kwok et al., 1999) antibodies.
for 4 d were used for ChIP assays following the procedure described
previously (Lee et al., 2007). Briefly, 2 g of seedlings grown under the
indicated light conditions were first cross-linked with 1% formaldehyde
under vacuum. The samples were ground to powder in liquid nitrogen,
and the chromatin complexes were isolated and sonicated and then
incubated with polyclonal HY5 antibodies (Osterlund et al., 2000). The
primers in Supplemental Table 1 online. Real-time qPCR analysis was
performed using the respective pair of primers and Power SYBR Green
PCR Master Mix (Applied Biosystems) with a Bio-Rad CFX96 real-time
PCR detection system. PCR reactions were performed in triplicate for
each sample, and the ChIP values were normalized to their respective
DNA input values.
Preparation of Recombinant Proteins
All constructs were transformed into Escherichia coli BL21 (DE3) cells
that were treated with isopropyl-b-D-thiogalactoside to induce fusion
protein expression. The GST fusion proteins were purified with Glu-
tathione Sepharose 4B beads (Amersham Biosciences), and the
63His-fusion proteins were purified with nickel-nitrilotriacetic acid
EMSAs were performed using biotin-labeled probes and the Lightshift
Chemiluminescent EMSA kit (Pierce). The sequences of the comple-
mentary oligonucleotides used to generate the biotin-labeled and
unlabeled probes are shown in Supplemental Table 1 online. Briefly,
0.5 mg of GST or GST fusion proteins were incubated together with
biotin-labeled probes in 20-mL reaction mixtures containing 10 mM
Tris-HCl, 150 mM KCl, 1 mM DTT, 50 ng/mL poly (dI-dC), 2.5% glycerol,
0.05% Nonidet P-40, 100 mM ZnCl2, and 0.5 mg/mL BSA for 20 min at
room temperature and separated on 6% native polyacrylamide gels in
Tris-glycine buffer. For the competition assays shown in Figure 4B, 0.5,
1, and 2 mg of GST or GST-HY5C proteins were used, respectively. The
labeled probes were detected according to the instructions provided
with the EMSA kit.
RNA Gel Blot Analysis and Real-Time qRT-PCR
Total RNA was extracted from Arabidopsis seedlings using the RNeasy
plant mini kit (Qiagen). For RNA gel blot analysis, 15 mg of total RNA were
loaded per lane and blotting was performed as described previously
(Martı ´nez et al., 2004). Fragments of FHY1 and FHL used for probe
labeling were generated by PCR, and the primers are shown in Supple-
mental Table 1 online.
For real-time qRT-PCR, cDNAs were synthesized from 2 mg total RNA
using SuperScript II first-strand cDNA synthesis system (Invitrogen)
according to the manufacturer’s instructions. Real-time PCR was per-
formed as described above. PCR reactions were performed in triplicate
for each sample, and the expression levels were normalized to that of a
In Vitro Pull-Down Assay
For in vitro binding, 2 mg of purified recombinant bait proteins (GST-HY5
and GST) and 2 mg of prey proteins (63His-FHY3N and 63His-FAR1N)
were added to 1 mL of binding buffer containing 50 mM Tris-HCl, pH 7.5,
100 mM NaCl, and 0.6% Triton X-100. After incubation at 48C for 2 h,
Glutathione Sepharose 4B beads (Amersham Biosciences) were then
added and incubated for a further 1 h. After washing three times with the
binding buffer, pulled-down proteins were eluted in 23 SDS loading
buffer at 958C for 10 min, separated on 10% SDS-PAGE gels, and
detected by immunoblotting using anti-His antibody (Qiagen).
Transient LCI assays in Nicotiana benthamiana were performed as
described previously (Chen et al., 2008). Briefly, Agrobacterium tumefa-
ciens (strain GV2260) bacteria containing indicated constructs were
infiltrated into young but fully expanded leaves of the 7-week-old N.
benthamiana plants using a needleless syringe. After infiltration, plants
weregrown under 16-hlight/dark for 3d, andluciferasesignalswerethen
3646The Plant Cell
viewed in an IVIS Spectrum imaging system (Caliper LifeSciences) and
quantified with the Living Image 4.0 software. To confirm the expression
of the NLuc and CLuc fusion proteins, total protein was extracted from
equal amounts of tobacco leaves and subjected to immunoblot analysis
N- and C-terminal firefly LUC fragments. The amount of protein loaded in
each lane is indicated by Ponceau S staining of ribulose-1,5-bisphos-
Transient Transcription Dual-Luciferase Assay
Transient dual-luciferase assay in N. benthamiana was performed as
described previously (Hellens et al., 2005). After infiltration, plants were
left under continuous white light for 4 d, and then leaf samples were
collected.Firefly luciferaseand Renillia luciferase wereassayed using the
dualluciferaseassayreagents (Promega) andwereperformed essentially
as previously described (Liu et al., 2008). Briefly, leaf discs (1 to 2 cm in
diameter) were excised, ground in liquid nitrogen, and homogenized in
100 mL of the Passive Lysis Buffer. Eight microliters of this crude extract
was mixedwith40 mLofLuciferase AssayBuffer, andthe fireflyluciferase
(LUC) activity was measured using a GLOMAX 20/20 luminometer
(Promega). Forty microliters of Stop and Glow Buffer was then added to
the reaction, and the Renillia luciferase (REN) activity was measured.
Three biological repeats were measured for each sample.
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under the following accession
numbers: FHY1 (At2g37678), FHL (At5g02200), FHY3 (At3g22170), FAR1
(At4g15090), HY5 (At5g11260), PIF3 (At1g09530), LAF1 (At4g25560),
HFR1 (At1g02340), COP1 (At2g32950), and PHYA (At1g09570).
The following materials are available in the online version of this article.
Supplemental Figure 1. HYH Does Not Bind to the FHY1 Promoter.
Supplemental Figure 2. HY5 Protein Levels in Wild-Type Seedlings
Grown in Different Light Conditions.
Supplemental Figure 3. HY5 Protein Levels in Wild-Type and cop1
Mutant Seedlings in Darkness.
Supplemental Figure 4. Phenotypes of the Various Arabidopsis
Mutants Defective in phyA Signaling.
Supplemental Table 1. Summary of Primers Used in This Study.
We thank Mathias Zeidler for fhy1-3 and fhy1-3 fhl-1 seeds, Jian-Min
Zhou for 35S:NLuc and 35S:CLuc plasmids, Chentao Lin for pGreenII
0800-LUC plasmid, and Tian Xu for kindly sharing their imaging system.
We also thank Shangwei Zhong and Jeffery Q. Shen for their sugges-
tions on the project and William Terzaghi and Hongwei Guo for critical
comments on the manuscript. This work was supported by a National
Institutes of Health grant (GM47850) to X.W.D. and in part by a National
Science Foundation award (MCB-1004808) to H.W. Studies conducted
at Peking University were supported by grants from the Ministry of
Science and Technology of China (2009DFB30030) and the Ministry of
Agriculture of China (2009ZX08012-021B).
Received April 13, 2010; revised October 28, 2010; accepted November
5, 2010; published November 19, 2010.
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