arabidopsis ethylene-responsive element binding factors
TRANSCRIPT
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The Plant Cell, Vol. 12, 393404, March 2000, www.plantcell.org 2000 American Society of Plant Physiologists
Arabidopsis Ethylene-Responsive Element Binding FactorsAct as Transcriptional Activators or Repressors ofGCC BoxMediated Gene Expression
Susan Y. Fujimoto,1 Masaru Ohta,1 Akemi Usui, Hideaki Shinshi, and Masaru Ohme-Takagi2
Plant Molecular Biology Laboratory, National Institute o f Bioscience and Human-Technology, Tsukuba 305-8566, Japan
Ethylene-responsive element binding factors (ERFs) are members of a novel family of transcription factors that are spe-
cific to plants. A highly conserved DNA binding domain known as the ERF domain is the unique feature of this protein
family. To characterize in detail this family of transcription factors, we isolated Arabidopsis cDNAs encoding five differ-
ent ERF proteins (AtERF1 to AtERF5) and analyzed their structure, DNA binding preference, transactivation ability, and
mRNA expression profiles. The isolated AtERFs were placed into three c lasses based on amino acid identity within the
ERF domain, although all five displayed GCC boxspecific binding activity. AtERF1, AtERF2, and AtERF5 functioned as
activators of GCC boxdependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as re-
pressors that downregulated not only basal transcription levels of a reporter gene but also the transactivation activity
of other transcription factors. The AtERF
genes were differentially regulated by ethylene and by abiotic stress conditions,
such as wounding, cold, high salinity, or drought, via ETHYLENE-INSENSITIVE2 (EIN2)dependent or independent
pathways. Cycloheximide, a protein synthesis inhibitor, also induced marked accumulation of AtERF
mRNAs. Thus, we
conclude that AtERFs are factors that respond to extracellular signals to modulate GCC boxmediated gene expression
positively or negatively.
INTRODUCTION
Plants are exposed to a number of conditions that may ad-
versely affect their growth and development. To adjust to
changes in their environment, plants modulate the expres-
sion of specific sets of genes (ODonnell et al., 1996, 1998;
Ishitani et al., 1997; Yamaguchi-Shinozaki and Shinozaki,
1997; Lund et al., 1998; Xiong et al., 1999). A number of
genes, including those that encode pathogenesis-related
(PR) and antifungal proteins, are inducible by various forms
of biotic and abiotic stress, such as pathogen attack,
wounding, UV irradiation, high or low temperature, and
drought, and by the gaseous hormone ethylene (Abeles et
al., 1992; Ecker, 1995; ODonnell et al., 1996, 1998;
Penninckx et al., 1996, 1998). These forms of stress ulti-
mately induce ethylene biosynthesis (Abeles et al., 1992;
Ecker, 1995; ODonnell et al., 1996), and ethylene has there-
fore been suggested to be a mediator of the stress response
(Ecker, 1995; ODonnell et al., 1996; Penninckx et al., 1996).Some defense-related genes that are induced by ethylene
were found to contain a short cis
-acting element known as
the GCC box (AGCCGCC; Ohme-Takagi and Shinshi, 1990).
This sequence was determined to be essential for the ex-
pression of several PR
genes (Sessa et al., 1995; Shinshi et
al., 1995; Sato et al., 1996) and to be the core sequence of
the ethylene-responsive element (ERE) in tobacco (Ohme-
Takagi and Shinshi, 1995).
ERE binding factor (ERF) proteins (formerly known as ERE
binding proteins [EREBPs]) were isolated as GCC box bind-
ing proteins from tobacco (Ohme-Takagi and Shinshi, 1995),
and their expression pattern was investigated in detail
(Suzuki et al., 1998). They contain a highly conserved DNA
binding domain (designated as the ERF domain; Hao et al.,
1998) consisting of 58 or 59 amino acids (Ohme-Takagi and
Shinshi, 1995). Recently, the solution structure of the ERF
domain in complex with the GCC box has been determined
(Allen et al., 1998). The ERF domain is novel both in its struc-
ture and in its mode of DNA recognition. It is composed of a
sheet and an
helix (
-
motif; see Figure 1), the
sheet
interacting monomerically with the target DNA (Allen et al.,1998). The ERF domain binds to the GCC box with high af-
finity, the dissociation constant (
K
d
) for the ERF domain
GCC box interaction typically falling in the picomolar range
(Hao et al., 1998). By contrast, basic leucine zipper proteins
exhibit K
d
values in the nanomolar range (Hurst, 1994).
It has been argued that the ERF domain is closely related
to the AP2 domain (Weigel, 1995; Okamuro et al., 1997).
However, knowledge accumulated thus far has indicated
that the AP2 and ERF domains belong to distinct families.
1
These authors contributed equally to this work.
2
To whom correspondence should be addressed. E-mail [email protected]; fax 81-298-61-6090.
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394 The Plant Cell
Sequence alignment of extended family members between
these two domains show
30% amino acid identity, whereas
members of each family exhibit
60% sequence identity
(Bttner and Singh, 1997; Hao et al., 1998; Riechmann and
Meyerowitz, 1998; see Figure 1). The recent report of the so-
lution structure of the ERF domain has contributed direct
evidence that amino acid residues within the ERF domainthat interact directly with the GCC box are not present in the
AP2 domain (see Figure 1). Although detailed studies re-
garding this aspect have not been reported for the AP2 do-
main, the AP2 domain is likely to possess a mode of DNA
recognition distinct from that of the ERF domain as well as a
different DNA target sequence.
Sequences made available through the Arabidopsis ge-
nome project and expressed sequence tag collections indi-
cate that numerous plant genes encode proteins that
possess an ERF domain (referred to as ERF proteins in this
work), representing a large, multigene family with many
members in both dicots and monocots. They include the
ERFs from tobacco; Pti4
, Pti5,
and Pti6
from tomato; and
TINY, A. thaliana
ERE binding protein (
AtEBP
), C-repeat/de-
hydration-responsive element (DRE) binding factor1 (
CBF1
),
DRE binding protein1 (
DREB1
), DREB2
, abscisic acid (ABA)insensitive4 (
ABI4
), and ETHYLENE RESPONSIVE FACTOR1
(
ERF1
) from Arabidopsis (Ohme-Takagi and Shinshi, 1995;
Wilson et al., 1996; Bttner and Singh, 1997; Stockinger et
al., 1997; Zhou et al., 1997; Finkelstein et al., 1998; Liu et al.,
1998; Solano et al., 1998). To date, genes encoding ERF
proteins have been found only in higher plants and not in
yeast or other fungi. It has been estimated that 125 genes
in the Arabidopsis genome encode AP2/ERF proteins
(Riechmann and Meyerowitz, 1998).
Figure 1. Alignment of the ERF Domain from Various ERF Proteins.
(A) The amino acid sequences of the ERF domain from various ERF proteins are aligned. They include AtERF1 to AtERF5 (this article); tobacco
ERF1 to ERF4 (Ohme-Takagi and Shinshi, 1995); Pti4, Pti5, and Pt i6 (Zhou et al., 1997); TINY (Wilson et al., 1996); CBF1 (Stockinger et al., 1997);
DREB1 and DREB2 (Liu et al., 1998); Arabidopsis ERF1 (Solano et al., 1998); AtEBP (Bttner and Singh, 1997); and ABI4 (Finkelstein et al., 1998).
The asterisks represent those proteins that were shown to bind to the GCC box. Filled box areas with dots indicate amino acid identities; dashes
indicate gaps introduced to maximize alignment. Numbers at right indicate the amino acid position of the ERF domain in each protein. Amino
acid residues identified by nuclear magnetic resonance analysis (Allen et al., 1998) to interact with nucleotides within the GCC box are under-
lined in the ERF domain consensus sequence. The structure of the ERF domain is shown below the sequence; the bar and black arrows indicate
the sheet and the strands within the sheet, respectively. The cross-hatched box indicates the helix (Allen et al., 1998).(B) Comparison of the ERF domain consensus sequence with the AP2 domain. The consensus amino acid sequence of the ERF domain (ERF
cons.) is compared with the D2 (AP2-D2) and D1 (AP2-D1) domains of APETALA2 (Jofuku et al., 1994). The amino acids with asterisks in the ERF
consensus indicate residues that interact with nucleotides within the GCC box (Allen et al., 1998). Amino acids identical to the ERF consensus
are boxed.
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Functional Analysis of AtERF Proteins 395
Several ERF proteins have been suggested to play a role
in plant growth and development. Enhancement of TINY
gene expression suppressed cell proliferation and resulted
in a tiny phenotype (Wilson et al., 1996). Ectopic expres-
sion of CBF1
or DREB1
in Arabidopsis improved tolerance
to freezing (Jaglo-Ottosen et al., 1998; Liu et al., 1998;Kasuga et al., 1999). Pti4, Pti5, and Pti6 interact with the
product of the Pto
disease resistance gene (Zhou et al.,
1997), support ing the idea that Pto may regulate the expres-
sion of defense-related genes by regulating the function of
ERF proteins. AtEBP has been shown to interact with an oc-
topine synthase (
ocs
) element binding protein (Bttner and
Singh, 1997), suggesting that PR
gene expression is regu-
lated via a proteinprotein interaction between an ERF pro-
tein and elicitor-responsive element binding factors. It has
also been reported that ectopic expression of ERF1
in
transgenic Arabidopsis results in a phenotype similar to that
of the constitutive triple response1
mutant (Solano et al.,
1998).
Although a number of ERF proteins have been shown tobind specifically to the GCC box (Ohme-Takagi and Shinshi,
1995; Bttner and Singh, 1997; Zhou et al., 1997; Solano et
al., 1998), it is not known how ERF proteins possessing the
same or similar binding specificity regulate transcription in
plants. Genome projects are providing a wealth of informa-
tion about sequences encoding ERF proteins in Arabidop-
sis, but information regarding physiological function and
gene regulation of this family of transcription factors is still
lacking. In this report, we isolated cDNAs encoding Arabi-
dopsis ERF proteinsAtERF proteinsto investigate their
role in transcriptional regulation in plants. We show that
each AtERF is a factor that can act as either a transcriptional
activator or a repressor for GCC boxdependent gene ex-
pression.
RESULTS
Isolation and Structure of AtERF cDNAs
We isolated cDNAs encoding AtERF proteins by screening a
leaf cDNA library with the tobacco ERF
cDNAs as probes.
Five complete cDNAs, AtERF1
to AtERF5
, which hybridized
strongly with the tobacco ERF
cDNAs, were further ana-
lyzed. Sequence analysis of the cDNAs demonstrated that
AtERF1 to AtERF5 all contain the conserved ERF domainand that the cDNAs encode proteins of 266, 243, 225, 222,
and 300 amino acids with predicted molecular masses of
28.9, 26.8, 25.2, 23.7, and 33.8 kD, respectively. Based on
amino acid sequence identit ies within the ERF domain, each
AtERF was categorized into one of three classes (Figures 2
and 3). AtERF1 and AtERF2 are 58% identical at the amino
acid level and were grouped into class I. The class I AtERFs
have a high degree of amino acid identity to ERF2 from to-
bacco and to Pti4 from tomato (Ohme-Takagi and Shinshi,
1995; Zhou et al., 1997) not only within the ERF domain but
also within the acidic domain found in the N-terminal region.
In addition, members of this group possess a region rich in
basic amino acids (P/L-K-K/R-R-R) that could serve as a pu-
tative nuclear localization signal (Raikhel, 1992). Two cys-
teine residues flanking the ERF domain (Figure 3) were alsoconserved.
AtERF3 and AtERF4 comprise class II and possess sub-
stantial amino acid identity within the DNA binding domain
to the tobacco ERF3 protein (Figure 3). The characteristic
features of class II ERFs are that the ERF domain is located
close to the N terminus of the protein and that it consists of
58 amino acids, one residue shorter than that of class I
ERFs. AtERF3 and AtERF4 do not exhibit pronounced amino
acid sequence similarity outside of the ERF domain. How-
ever, class II AtERFs possess a similar cDNA structure with
respect to the location of the ERF domain, length of the cod-
ing region, presence of the C-terminal acidic domain, and
calculated pI.
AtERF5 is the only identified member of the third class,class III, possessing sequence similarity to ERF4 from to-
bacco. This class of ERF
gene has the longest coding region
among members of the ERF proteins isolated thus far.
Acidic domains are located in both the N- and C-terminal re-
gions flanking the ERF domain. In addition, a putative mito-
gen-activated protein (MAP) kinase phosphorylation site
(PXXSPXSP, in which X represents any amino acid; Pearson
and Kemp, 1991) is conserved in the C-terminal region of
class III ERFs (Figure 3).
Figure 2. AtERFcDNAs.
A schematic representation of the AtERF1 to AtERF5 cDNAs is
shown. The AtERF proteins are grouped into three classes accord-
ing to sequence similarity within the ERF domain. Boxes indicate the
open reading frames, starting from the first ATG codon, and lines
show putative untranslated regions. Black boxes indicate the ERF
domain; hatched boxes represent acidic domains. Black boxes with
a black triangle above them represent putative nuclear localization
signals, and plus signs () indicate putative MAP kinase target sites.
Numbers above the line indicate positions of amino acid residues;
numbers below the line refer to nucleotide positions.
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396 The Plant Cell
Database searches show that the nucleotide sequence of
AtERF4
is almost identical to that of expressed sequence
tag T45365, which defines the gene encoding RELATED TO
APETALA2 PROTEIN5 (RAP2.5) (Okamuro et al., 1997), with
the exception of a one-nucleotide insertion within the coding
region. This insertion causes the reading frame to shift, pro-
voking changes in the deduced amino acid sequence of the
two genes. Because the nucleotide sequence of AtERF4
is
identical to a chromosomal sequence (GenBank accession
number AP000413), the extra nucleotide found in RAP2.5
may be attributed to intrinsic variation or to a sequencing er-
ror. Further database analyses revealed that AtERF3
and
AtERF4
are found on chromosomes 1 and 3, respectively.
AtERF2
and AtERF5
are located on chromosome 5,
whereas AtERF1
resides on chromosome 4.
DNA Binding Preference of the AtERFs
To analyze whether the AtERFs have GCC box binding ac-
tivity, we overexpressed the entire coding region of each
AtERF as a maltose binding protein (MBP) fusion in Esche-
richia coli
and performed electrophoretic mobility shift as-
says. As shown in Figure 4, all MBPAtERF fusion proteins
were able to bind to a 16-bp oligonucleotide that contained
the wild-type GCC box sequence (AGCCGCC), whereas
binding activity was abolished when both G residues within
the GCC box were replaced by T residues (ATCCTCC). This
experiment indicated that all AtERFs have GCC boxspe-
cific binding activity.
To investigate the DNA binding preference of each AtERF
in more detail, we generated a series of oligonucleotides in
which each nucleotide within the GCC box was separately
substituted with a T nucleotide; these oligonucleotides were
used to assess the relative binding activities of each AtERF
in comparison to the wild-type GCC box sequence (Figures
4C to 4E). Results showed that any mutations within the
GCC box reduced the binding ability of the AtERFs, indicat-
ing that GCC box is the optimal sequence for AtERF binding
among the mutants investigated. The G residue at position 5
(G-5 in Figure 4C) appears to be essential for the GCC box
sequence because no complex formation was detected be-
tween the AtERF proteins and the oligonucleotide carrying a
mutation at this position. By contrast, positions A-1 and C-6
appeared to be less critical for binding, because all AtERFs
could bind to some degree to the oligonucleotides carrying
mutations at these positions. Substituting T for G-2, C-4, G-5,or C-7 reduced the binding activity of AtERF1, AtERF2, and
AtERF5 to
10% of that on the wild-type GCC oligonucle-
otide, whereas AtERF3 and AtERF4 were able to bind to oli-
gonucleotides mutated at the C-4 and C-7 positions with 30
to 40% efficiency. These data suggest that AtERF1, AtERF2,
and AtERF5 are more sensitive to changes in the GCC box
sequence whereas members of class II AtERFs appear to be
more flexible in target sequence recognition than other
AtERFs.
Figure 3.Comparison of Deduced Amino Acid Sequences of AtERF Proteins.
Amino acid sequences grouped by c lass are aligned and compared with tobacco ERFs. Identity within the same class is indicated by shading.
The filled bar below the sequences represents the ERF domain; AD indicates putative acidic domains. Carets represent putative nuclear local-
ization signals. Plus signs () indicate putative MAP kinase target sites. Dashes indicate gaps used to optimize alignment. The GenBank, DDBJ,
EMBL, and NCBI accession numbers of the nucleotide sequences of the AtERFcDNAs are AB008103 (AtERF1), AB008104 (AtERF2), AB008105
(AtERF3), AB008106 (AtERF4), and AB008107 (AtERF5).
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Functional Analysis of AtERF Proteins 397
Transcriptional Regulation by the AtERFs
The ability of the AtERFs to regulate transcription in plant
cells was tested by using transient assays (Figure 5). A lu-
ciferase (LUC)-encoding reporter gene, 4
HLS
, which con-
tains four copies of the GCC box sequence from theArabidopsis HOOKLESS1
(
HLS1
) promoter (Lehman et al.,
1996) fused to LUC, and an effector plasmid consisting of
each AtERF
under the control of the cauliflower mosaic virus
(CaMV) 35S promoter (Figure 5A) were delivered to Arabi-
dopsis leaves by particle bombardment. LUC activity in-
creased at least 12-fold when the reporter plasmid was
coexpressed with AtERF1
, AtERF2
, or AtERF5
effector plas-
mids (Figure 5B). No such increase in LUC activity by the
AtERFs was detected when a LUC reporter plasmid contain-
ing a mutated GCC box (ATCCTCC) was used (data not
shown). These data indicate that AtERF1, AtERF2, and
AtERF5 are able to funct ion as GCC box sequencespecific
transactivators in Arabidopsis leaves.
Some genes containing the GCC box in their promoter re-gion are known to be upregulated by ethylene in Arabidop-
sis (Chen and Bleecker, 1995; Lehman et al., 1996; Penninckx
et al., 1996). We investigated whether components of the
ethylene signaling pathway affect the activation of GCC
boxmediated transcription by the AtERFs. The 4
HLS
re-
porter gene and the AtERF
effector constructs were intro-
duced into leaves of the ethylene-insensitive mutant ein2
(Guzman and Ecker, 1990). LUC activity increased when
AtERF1, AtERF2, or AtERF5 were coexpressed in ein2
leaves,
approaching levels observed in the wild type (Figure 5C).
These data suggest that transactivation activity of the
AtERFs is not affected by a mutation in the EIN2 ethylene
signal transducer.
By contrast, AtERF3 and AtERF4 (class II ERFs) did not
activate transcription but appeared to repress reporter gene
activity. Coexpression of AtERF3
or AtERF4
with the 4
HLS
reporter construct resulted in a 50% reduction in the basal
LUC activity. Furthermore, coexpression of AtERF3
, AtERF5
,
and the 4
HLS
reporter resulted in a level of LUC activity
that was close to the levels obtained with the reporter con-
struct alone (Figure 5D), indicating that AtERF3 repressed
not only basal activity of a reporter gene but also the activity
of another transcriptional activator. AtERF3 suppression of
AtERF5 activation was found to be dose dependent, with
the addition of increasing amounts of AtERF3 effector pro-
gressively reducing the transactivation ability of AtERF5
(Figure 5E). Thus, this effect is unlikely to result from non-specific squelching.
To determine whether class II AtERFs are active repres-
sors that possess an intrinsic repression activity that is dis-
tinct from their DNA binding activity, we constructed a
reporter gene (5GAL4GCC) containing two different cis
-ele-
ments in the promoter region, the yeast GAL4 sequence and
the GCC box. The purpose of this construct was to examine
whether a repressor can suppress the activity of an activator
without competition for the same DNA binding site. The
5GAL4GCC reporter gene and the effector p lasmid contain-
ing the GAL4 DNA binding domain fused to the transcrip-
tional activation domain of viral protein 16 (VP16) from
herpes simplex virus (Triezenberg et al., 1988) were cobom-
barded with or without the AtERF3
effector plasmid (Figure
6). Reporter gene activity increased by a minimum of 20-fold
Figure 4. Characterization of AtERF DNA Binding Affinity to the
GCC Box.
(A) GCC box sequences used in this experiment. Underlining and bold-
face denote the GCC box. GCC, wild type; mGCC, a double mutant.
(B) AtERF possesses GCC boxspecific binding activity. Electro-phoretic mobility shift assays were performed using MBPAtERF fu-
sion proteins and a 16-bp wild-type GCC box fragment or a mutated
version. MBP was used alone as a control. Numbers indicate MBP
AtERF1 to MBPAtERF5, respect ively.
(C) Sequence of the wild-type GCC box (W) and mutants (1 to 7)
used for the DNA binding assays. The GCC box sequence, AGC-
CGCC, is underlined. Positions within the GCC box systematically
substituted with a T residue are indicated as A-1, G-2, C-3, C-4, G-5,
C-6, and C-7, respectively. Dashes indicate nucleotides identical to
the wild-type sequence.
(D) Effect of single-base substitutions within the GCC box on DNA
binding activity of the AtERFs. The binding activities of each AtERF
to the mutated versions relative to the wild-type GCC box are shown
graphically. W and the number (1 to 7) indicate the probes shown in (C).
(E) Autoradiographs of only the shifted bands from (D) are shown.
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398 The Plant Cell
when the VP16 effector alone was bombarded. The activa-
tion by the VP16 activator was reduced to 50% when
AtERF3 was coexpressed with the VP16 plasmid (Figure
6B). These results indicate that class II AtERFs are active re-
pressors that can suppress the activity of other transcrip-
tional activators without competing for DNA binding sites.Additionally, AtERF3 and AtERF4 could repress the activity
of a GAL4 reporter gene when fused to the GAL4 DNA bind-
ing domain (data not shown), suggesting the presence of a
discrete repression domain in class II AtERF proteins.
Expression of the AtERF mRNAs
The expression of the AtERF
genes was investigated by
RNA gel blot analysis with total RNA isolated from Arabidop-
sis leaves (Figure 7). mRNAs corresponding to each of the
AtERF
s could be detected in air-grown, healthy Arabidopsis
plants, although the basal levels of each AtERF
transcript
differed. Because some GCC boxcontaining genes, suchas that encoding chitinase or PDF1.2, are known to be in-
duced by ethylene (Chen and Bleecker, 1995; Penninckx et
al., 1996), we exposed Arabidopsis plants to ethylene for 1,
6, and 12 hr and monitored expression of the AtERF
s. Ex-
pression of genes encoding chitinase and PDF1.2 was in-
duced 12 hr after ethylene treatment. Correspondingly,
amounts of AtERF1
, AtERF2
, and AtERF5 transcripts in-
creased two- to threefold 12 hr after ethylene treatment,
whereas AtERF3 or AtERF4 transcript amounts did not in-
crease. In the ein2 mutant, the expression of the AtERFs
was not induced by ethylene treatment, suggesting that eth-
ylene induction of the AtERFs is regulated under the ethyl-
ene signaling pathway, as is the case for the genes that
encode chitinase and PDF1.2 (Figure 7). We observed thatthe mRNA levels of all of the AtERFs except AtERF3were
consistently lower in ein2plants compared with the wild type.
Next, we investigated whether abiotic stresses such as
wounding, cold, heat, high NaCl concentration, or drought
induced AtERFexpression (Figure 7). In these experiments,
plants were exposed to stress conditions for only 6 hr to avoid
possible secondary effects. Wounding induced marked
accumulation of mRNAs for all of the AtERFs except
AtERF3. AtERF4was also induced by cold, high NaCl con-
centration, and drought stress, whereas AtERF5expression
Figure 5. AtERFs Can Transactivate or Repress GCC-Mediated
Gene Expression in Arabidopsis Leaves.
(A) Schematic diagram of the reporter and effector plasmids used intransient assays. A region of the Arabidopsis HLS1 gene, which
contains the GCC box (filled circles), was fused to a minimal TATA
box and a firefly LUCgene. Effector plasmids were under the control
of the CaMV 35S promoter. Nos denotes the terminator signal of the
gene for nopaline synthase. indicates translational enhancer of to-
bacco mosaic virus.
(B) Transactivation of the 4HLS GCC boxLUC reporter gene by
AtERF1, AtERF2, and AtERF5.
(C) Transactivation of the reporter gene by AtERF1, AtERF2, and
AtERF5 in the ethylene-insensitive mutant ein2.
(D) Repression of reporter gene activity by AtERF3 and AtERF4 and
suppression of AtERF5-mediated transactivation by AtERF3.
(E) Dependence of repression on the amount of AtERF3 effector
plasmid. Activation of the reporter gene by AtERF5 is reduced by
AtERF3 in a dose-dependent manner. Different concentrations of
AtERF3effector were co-bombarded with AtERF5effector (from a
ratio of 1:0 to 1:1; AtERF3to AtERF5) and the reporter plasmid.
Values shown are averages of results from three independent exper-
iments. Error bars indicate standard deviation. All LUC activities are
expressed relative to the reporter construct alone (value set at 1).
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Functional Analysis of AtERF Proteins 399
was upregulated by cold stress. AtERF3 expression was
moderately induced by salinity and drought stress. Cold and
drought stresses are known to promote ABA biosynthesis
(Zeevaart and Creelman, 1988; Chandler and Robertson,
1994); however, exogenous ABA did not induce AtERFex-
pression. Similarly, high temperature stress did not affect
the expression of any AtERFgene.
The induction of the AtERFs by wounding, cold, and drought
stress was observed in ein2mutants as well as in wild-type
plants, although the fold induction was slightly lower in ein2
than it was in the wild type. In contrast, the induction of
AtERF3 and AtERF4 by high salinity was not observed in
ein2 (Figure 7), indicating that the ethylene signaling path-
way mediated by EIN2 may regulate the salt stressmedi-ated induction of AtERF3and AtERF4, even though ethylene
itself did not induce expression of these AtERFgenes.
By contrast, treatment of Arabidopsis plants with cyclo-
heximide (CHX), a protein synthesis inhibitor, resulted in the
marked accumulation of AtERFmRNAs, indicating that de
novo protein synthesis may not be required for AtERFex-
pression (Figure 7). By 6 hr after CHX treatment, the mRNA
levels of AtERF1, AtERF2, AtERF4, and AtERF5 increased
20- to 100-fold compared with the control, whereas AtERF3
mRNA increased only approximately fivefold. The observed
accumulation of mRNA after CHX treatment may be due to
the inhibition of de novo production of labile transcriptional
repressors and/or mRNA-degrading enzymes (Berberich
and Kusano, 1997) and suggests that transcriptional or
post- transcript ional regulatory mechanisms may be involvedin AtERFexpression.
DISCUSSION
Structural Features
The five AtERFs isolated during the course of this work were
grouped into three classes based on their amino acidFigure 6. AtERF3 Suppresses Transactivation without Competing
for the Same DNA Binding Site.
(A) Schematic diagram of the reporter and effector p lasmids used.
The GAL4 binding site (patterned hatched boxes) and GCC box se-quence (filled circles) were fused to a minimal TATA box and the
LUC gene. The AtERF3effector and VP16 effector plasmid contain-
ing the GAL4 DNA binding domain (GAL4DBD) fused upstream of
the VP16 activation domain (VP16 AD) were under the control of the
CaMV 35S promoter. Nos denotes the terminator signal of the gene
for nopaline synthase. indicates translational enhancer of tobacco
mosaic virus.
(B) AtERF3 represses VP16-mediated transactivation without com-
peting for the same DNA binding site. Values shown are averages of
results from three independent experiments. Error bars indicate
standard deviation. All LUC activities are expressed relative to the
reporter construct alone (value set at 1).
Figure 7. Induction of AtERF mRNA Expression.
(A) Expression pattern of AtERFmRNAs after exposure to ethylene
and abiotic stress in the wild type (wild) or an ethylene-insensitive
mutant, ein2 (ein2). Total RNA was prepared from Arabidopsisleaves treated with ethylene gas for 0, 1, 6, and 12 hr, respectively,
or treated with cold (Co), heat (H), NaCl (Na), drought (D), abscisic
acid (A), CHX (Cx), or wounded (W) for 6 hr. C, the control for CHX
treatment; EtBr, ethidium bromide staining.
(B) Expression patterns of the genes encoding PDF1.2 (top) and
chitinase (bottom) in wild-type (Wild) and ein2 (ein2) Arabidopsis
plants treated with ethylene for 0, 1, 6, and 12 hr.
Ten micrograms of RNA was loaded and hybridized with the cDNA
encoding AtERF, chitinase (Chen and Bleecker, 1995), or PDF1.2
(Penninckx et al., 1996).
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400 The Plant Cell
sequence identities within the ERF domain, although other
regions of AtERF1 and AtERF2 also share extensive amino
acid similarity. Nevertheless, AtERF1 and AtERF2 are not
thought to have evolved simply by gene duplication because
the genes that encode them are located on different chro-
mosomes. In contrast, members of the CBF/DREB familymost likely arose after gene duplication events because the
proteins are highly similar. Moreover, the corresponding
genes are closely linked and have the same transcriptional
orientation (Gilmour et al., 1998; Shinwari et al., 1998;
Medina et al., 1999). AtERF1 and AtERF2 share high amino
acid sequence identities with Pti4, which interacts with the
product of the Pto disease resistance gene (Zhou et al.,
1997). It is possible, then, that class I AtERFs are involved in
pathogen signal transduction in Arabidopsis via proteinpro-
tein interactions.
By contrast with AtERF1 and AtERF2, the class II proteins
AtERF3 and AtERF4 do not share much amino acid se-
quence identity except for residues within the ERF domain.
Nevertheless, the overall structure and the function of thesetwo proteins as transcriptional repressors are conserved, in-
dicating that AtERFs in this class may have diverged from a
single ancestral origin.
The involvement of protein kinases has been reported in
ethylene signal transduction and in the transactivation of
GCC boxdependent transcription (Kieber et al., 1993; Raz
and Fluhr, 1993; Sessa et al., 1996; Suzuki et al., 1998).
Therefore, phosphorylation may regulate some aspects of
AtERF function, such as DNA binding activity, nuclear local-
ization, or transactivation. The fact that a putative site for
MAP kinasemediated phosphorylation is found in the class
III protein AtERF5, although such sites appear not to be
present in representatives of the other two classes, sug-
gests that phosphorylation may play an important role in the
function of class III ERFs.
AtERFs Are GCC Box Binding Factors
Each of the ERFs isolated in this study displayed GCC box
specific binding activity, and a number of previously identi-
fied ERF proteins have also been shown to possess GCC
box binding activity (see Figure 1). Binding assays revealed
that the GCC box (AGCCGCC) appears to be the optimal
target site for the AtERF proteins and that the G-5 nucle-
otide within this sequence is essential for AtERF binding
(Figure 4). However, results also showed that each AtERFpossessed a distinct DNA binding preference. AtERF1,
AtERF2, and AtERF5 appear to be most sensitive to the sin-
gle-nucleotide substitutions within the GCC box sequence
because these AtERF proteins were hardly able to bind to
five of the seven single mutated oligonucleotides that were
tested.
By contrast, AtERF3 and AtERF4 appear to be more flexi-
ble than other AtERF proteins with respect to their target se-
quence preferences because they were able to bind to
several mutated oligonucleotides to which other AtERF pro-
teins did not b ind. The binding flexibility of the class II AtERF
proteins implies that these proteins might interact with
genes with which other classes of AtERF proteins cannot.
Nevertheless, it is important to analyze whether the DNA
binding activities obtained in vitro correlate with the tran-scriptional activities of the AtERFs in vivo.
It is difficult to explain at this stage where differences in
binding preferences between class II AtERFs and other
AtERFs might arise because the amino acid residues that
have been shown to interact with DNA (Allen et al., 1998) are
conserved in all ERF domains (Figure 1). The slight se-
quence variations across the ERF domains may result in
conformational changes that affect DNA binding specificity;
however, although we used the entire AtERF coding region
in our DNA binding assays, our results are consistent with
those in which only the minimum DNA binding domains
were tested (Hao et al., 1998). This suggests that the regions
flanking the DNA binding domain are not critical for mediat-
ing the proteinDNA interaction in vitro.
Transcriptional Regulation
We showed that AtERF1, AtERF2, and AtERF5 act as tran-
scriptional activators for GCC boxdependent transcription
in Arabidopsis leaves. Similarly, other ERF proteins, such as
CBF1 and DREB, have been shown to funct ion as transacti-
vators in Arabidopsis or yeast (Stockinger et al., 1997; Liu et
al., 1998). Additionally, ERF1 (Solano et al., 1998) has been
shown to activate a subset of ethylene-inducible genes,
such as PDF1.2 and the gene encoding chitinase, when
overexpressed. The AtERFs were capable of activating tran-
scription of a reporter gene in ein2mutant as well as in wild-
type leaves in our transient expression system, indicating
that AtERF proteins may not be regulated post-transcrip-
tionally by the ethylene signaling pathway. Similar results
have been reported in transgenic plants overexpressing
ERF1 (Solano et al., 1998).
One of our most interesting findings was that AtERF3 and
AtERF4 can act as transcriptional repressors in Arabidopsis
leaves. This suggests that a dynamic system ut ilizing antag-
onistic mechanisms for controlling GCC boxdependent
transcription operates in plants. Repression can be catego-
rized broadly into two modes: passive and active (Cowell,
1994; Hanna-Rose and Hansen, 1996). In plants, the maize
Dof2 protein (Yanagisawa and Sheen, 1998) appears to be apassive repressor that downregulates the transcriptional ac-
tivity of Dof1 by competing for the same DNA binding site; it
does not possess intrinsic repressive activity. On the other
hand, active repression involves obstruction of transcription
initiation by proteins that contain intrinsic repression activity,
as distinct from DNA binding activity (Cowell et al., 1992;
Cowell, 1994; Hanna-Rose and Hansen, 1996). In plants,
soybean G box Binding Factor2 is thought to be an active
repressor (Liu et al., 1997).
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Functional Analysis of AtERF Proteins 401
Our results demonstrate that class II AtERFs utilize an ac-
tive repression mechanism because they suppress the
transactivation activity of other transcription factors without
competing for the same DNA binding site (Figure 6). Several
possible mechanisms of active repression have been pro-
posed (Cowell, 1994; Hanna-Rose and Hansen, 1996; Pazinand Kadonaga, 1997). One is that repressors interfere with
transcriptional activators that interact with the basal transcrip-
tional machinery, such as TFIID. Alternatively, repressors
might recruit co-repressors such as histone deacetylase,
which modifies chromatin structure and prevents other tran-
scriptional activators from binding to their target cis-elements
(Pazin and Kadonaga, 1997). Analyses of factors that inter-
act with the repression domain of AtERF would demonstrate
the mechanism by which class II AtERFs function as tran-
scriptional repressors.
Because all AtERFs can bind to the same target se-
quence, one point of regulation of transactivation or repres-
sion lies in DNA binding affinity. Because lower amounts of
AtERF3 effector can suppress transactivation by AtERF5(Figure 5E) and because the dissociation constant (DNA
binding affinity) of the class II ERF domain to the GCC box
sequence was comparable to that of the class I or class III
ERF domains (Hao et al., 1998; D. Hao and M. Ohme-Takagi,
unpublished results), the repression activity displayed by
class II AtERFs appears to be stronger than the transactiva-
tion act ivities of c lass I or III AtERFs. As GCC boxmediated
transcription is upregulated in plant cells in response to ex-
tracellular signals, mechanisms that downregulate the re-
pression activity of class II AtERFs, such as protein degra-
dation or regulation at the mRNA level, should be operating.
AtERF Proteins Are Stress SignalResponsive Factors
AtERF genes respond differently not only to ethylene but
also to various forms of abiotic stress (Figure 7). AtERF1,
AtERF2, and AtERF5were induced by ethylene 12 hr after
treatment, whereas the ethylene response of ERF1 is ob-
served within 2 hr (Solano et al., 1998). Thus, the AtERFs
may function later in the induction of ethylene-inducible
genes. In contrast, responses of AtERF genes to abiotic
stress occur much more quickly than do those to ethylene,
suggesting that the AtERFs are involved in regulating re-
sponses to abiotic stresses in addition to ethylene. This idea
is supported by our recent database survey (M. Ohta, S.
Fujimoto, and M. Ohme-Takagi, unpublished results). Thissurvey indicates that several stress-inducible genes, such
as ARSK1 and a dehydrin gene, both of which are induced by
ABA, NaCl, cold, and/or wounding (Hwang and Goodman,
1995; Rouse et al., 1996), possess the GCC box sequence
in their 5 upstream regions. Moreover, the GCC box has
been recently shown to act as an elicitor-responsive ele-
ment in tobacco cells (Yamamoto et al., 1999). These find-
ings suggest that the GCC box may act as a cis-regulatory
element for biotic and abiotic stress signal transduction in
addition to its role as an ERE, again implying that the
AtERFs may act as transcription factors for stress-respon-
sive genes.
Induction of the AtERFs by wounding, cold, and drought
appears to be independent from the ethylene signaling path-
way because responses to these abiotic stresses were ob-served in ein2 plants. By contrast, induction of the genes
encoding AtERF3 and AtERF4 by high salinity stress seems
to be regulated by the ethylene signaling pathway mediated
by EIN2, although the expression of these class II AtERFs is
not induced by ethylene. Thus, AtERFexpression appears
to be controlled by a complex pathway that is independent
from or dependent on ethylene signal transduction.
A model for the regulation of GCC boxdependent tran-
scription mediated by ERF proteins is shown in Figure 8.
Several possible roles for ERF proteins in the regulation of
GCC boxdependent transcription are presented. As the
AtERF1 to AtERF5genes are expressed in response to differ-
ent stimuli, the AtERF proteins probably function as signal-
and sequence-specific transcription factors by activating or
Figure 8. A Model for GCC BoxMediated Stress SignalDepen-
dent Transcription by ERF Proteins in Arabidopsis.
After reception of a stress signal by plants, ERFgenes may be up-
regulated via an ethylene-dependent pathway or an ethylene-inde-
pendent pathway. ERFgenes such as ERF1 (Solano et al., 1998) are
induced by ethylene and can interact with GCC boxcontaining
stress response genes. The AtERFs are able to interact with the
GCC box sequence in an ethylene-independent manner. AtERF1,
AtERF2, or AtERF5 may activate a specific subset of GCC boxcon-
taining genes, whereas AtERF3 and AtERF4 may repress the ex-
pression of these genes. These data suggest that the ERF proteins
may act as factors responsive to extracellular signals and that they
are involved in regulating a subset of GCC boxcontaining stress re-
sponse genes.
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402 The Plant Cell
repressing the expression of GCC boxcontaining stress-
responsive genes dependent on or independent of the ethyl-
ene signal. Other proteins, such as ERF1 (Solano et al., 1998),
probably act as activators of GCC boxcontaining stress-
and/or ethylene-responsive genes by positively modulating
gene expression in response to external or internal ethylene.This model suggests that the AtERFs are regulated differen-
tially at both the transcriptional and post-transcriptional levels
and that subsets of GCC boxcontaining genes may be regu-
lated by different ERF proteins.
METHODS
Plant Material and Treatments
Wild-type (Arabidopsis thalianaecotype C24) and ein2 plants were
grown in a 1:1 mixture of Soil Mix (Sakata Co., Kyoto, Japan) and
vermiculite in growth chambers at 22C with a 16-hr light/dark cyclefor 20 to 30 days. Treatments were performed at 22C unless other-
wise noted and were as follows: ethyleneplants were placed in an
ethylene flow chamber (100 ppm C2H4, at 5 mL/min); abscisic acid
(ABA)0.1 mM ABA was sprayed on plants; temperature stress
plants were incubated at either 37 or 4C; droughtwhole plants
were placed on filter paper and allowed to desiccate; high salt
plants in pots were placed directly into a container of 300 mM NaCl;
woundingrosette leaves were detached from the plant, cut into
5-mm strips, and floated on 50 mM phosphate buffer, pH 7.0; and
cycloheximide (CHX)the root systems of whole plants were
washed gently with water to remove soil and then the plants were in-
cubated in 10 g/mL CHX in 0.1% (v/v) DMSO or 0.1% (v/v) DMSO
as a control. Aerial tissues were excised after treatment and immedi-
ately frozen in liquid nitrogen.
cDNA Screening
An Arabidopsis gt10 cDNA library prepared from air-grown plant
leaves was screened with the tobacco ethylene-responsive element
binding factor (ERF) cDNA coding regions as probes by using stan-
dard procedures (Sambrook et al., 1989).
Electrophoretic Mobility Shift Assay
The coding region of each AtERF was cloned into the pMAL vector
(New England Biolabs, Beverly, MA) and expressed in Escherichia
coliBL21 cells. Maltose binding protein (MBP)AtERF fusion proteins
were purified using amylose resin, as specified by the manufacturer.
Both strands of the following oligonucleotides were synthesized:
wild- type GCC box (W: CATAAGAGCCGCCACT) and double mutant
GCC box (mGCC: CATAAGATCCTCCACT). In addition, each single
mutant GCC box oligonucleotide in which each of the nucleotides in
the GCC box was systematically replaced with T (Figure 4C; Hao et
al., 1998) was synthesized. Electrophoretic mobility shift assays were
performed in a 10-L volume that contained 0.1 g of MBPAtERF
fusion protein, 1 g of poly(dA-dT), 4 fmol of a 16-bp oligonucleotide
end-labeled with phosphorus-32, and DNA binding buffer (25 mM
Hepes-KOH, pH 7.5, 5% [v/v] glycerol, 40 mM KCl, 0.5 mM EDTA,
and 1 mM DTT). After a 15-min incubation at room temperature, the
reaction mixture was subjected to 5% nondenaturing PAGE in 0.25
TBE (1 TBE is 22.5 mM Tris-borate, pH 8.0, and 0.25 mM EDTA).
Binding was quantified on a BAS-2000 system (Fuji, Tokyo, Japan).
Transient Expression
For a reporter gene construct, a 29-bp region containing the GCC
box sequence from the Arabidopsis HLS1 gene (ATGAGTTAACGCA-
GACATAGCCGCCATTT) was multimerized four times, placed up-
stream of the minimal 42 to 8 TATA box from the cauliflower
mosaic virus (CaMV) 35S promoter, and fused transcriptionally to the
firefly luciferase (LUC) gene (Promega). For the reporter construct
with two cis-elements, the GAL4 binding site repeated in tandem five
times and the GCC box sequence (GATCAGCCGCCGGATC) multi-
merized four times were placed upstream of the reporter gene
containing the minimal TATA box. For effector plasmids, the -gluc-
uronidase (GUS) gene in pBI221 (Clonetech, Palo Alto, CA) was re-
placed by the coding region of each AtERF. The tobacco mosaic
virus sequence (Gallie et al., 1987) was inserted downstream of t heCaMV 35S promoter in pBI221 to enhance the efficiency of transla-
tion. For the viral protein 16 (VP16) activator plasmid, the GAL4 DNA
binding domain (amino acid positions 1 to 147; Ma and Ptashne,
1987) was fused upstream of the VP16 activation domain (positions
413 to 490; Triezenberg et al., 1988).
Transient assays were performed by using the particle gun bom-
bardment method, as follows: 510 g of 1-m gold biolistic particles
(Bio-Rad) were coated with 1.6 g of reporter plasmid and a total
amount of 1.2 g of effector p lasmid or blank plasmid. As a reference,
the Renilla LUCgene (Promega) under the control of the CaMV 35S
promoter was used at a reference genereporter gene ratio of 1:5.
Bombardment was done with a Bio-Rad PDS-1000/He machine. After
bombardment, the samples were floated on 50 mM phosphate buffer,
pH 7.0, incubated in a plant growth chamber at 22C for 6 hr, frozen in
liquid nitrogen, and quantified for LUC activity.
RNA Analyses
Total RNA was extracted as described previously (Fukuda et al.,
1991). Aliquots of 10 g of total RNA were denatured, separated on
1.2% formaldehydeagarose gels, transferred to GeneScreen Plus
(NEN Life Science Products, Boston, MA), and allowed to hybridize
with a 32P-labeled probe. The intensity of the hybridization signal was
analyzed by using the BAS-2000 system (Fuji). Equal loading of sam-
ples was confirmed by visualization of rRNA after ethidium bromide
staining.
ACKNOWLEDGMENTS
We are grateful to Dr. Kazuko Yamaguchi-Shinozaki for the Arabi-
dopsis cDNA library and to the Arabidopsis Biological Resource
Center (Ohio State University, Columbus) for the C-24 and ein2Ara-
bidopsis seed. We also thank Kyoko Matsui for technical assistance.
S.Y.F. and M.O. were recipients of Science and Technology Agency
Fellowships from the Japan Science and Technology Corporation.
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Functional Analysis of AtERF Proteins 403
S.Y.F. would also like to thank the National Science Foundation for
its support.
Received December 3, 1999; accepted January 5, 2000.
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