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The Plant Cell, Vol. 12, 393–404, March 2000, www.plantcell.org © 2000 American Society of Plant Physiologists
Arabidopsis Ethylene-Responsive Element Binding Factors
Act as Transcriptional Activators or Repressors of
GCC Box–Mediated Gene Expression
Susan Y. Fujimoto,1 Masaru Ohta,1 Akemi Usui, Hideaki Shinshi, and Masaru Ohme-Takagi2
Plant Molecular Biology Laboratory, National Institute of 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 specific 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 different 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 classes based on amino acid identity within the
ERF domain, although all five displayed GCC box–specific binding activity. AtERF1, AtERF2, and AtERF5 functioned as
activators of GCC box–dependent transcription in Arabidopsis leaves. By contrast, AtERF3 and AtERF4 acted as repressors 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 box–mediated gene expression
positively or negatively.
INTRODUCTION
Plants are exposed to a number of conditions that may adversely affect their growth and development. To adjust to
changes in their environment, plants modulate the expression of specific sets of genes (O’Donnell 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; O’Donnell et al., 1996, 1998;
Penninckx et al., 1996, 1998). These forms of stress ultimately induce ethylene biosynthesis (Abeles et al., 1992;
Ecker, 1995; O’Donnell et al., 1996), and ethylene has therefore been suggested to be a mediator of the stress response
(Ecker, 1995; O’Donnell 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-
1 These
authors contributed equally to this work.
whom correspondence should be addressed. E-mail masaru@
nibh.go.jp; fax 81-298-61-6090.
2 To
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 (OhmeTakagi and Shinshi, 1995).
ERE binding factor (ERF) proteins (formerly known as ERE
binding proteins [EREBPs]) were isolated as GCC box binding 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 structure 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 affinity, the dissociation constant (Kd) for the ERF domain–
GCC box interaction typically falling in the picomolar range
(Hao et al., 1998). By contrast, basic leucine zipper proteins
exhibit Kd 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.
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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 Pti6 (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 (Büttner 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 underlined 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.
Sequence alignment of extended family members between
these two domains show ⬍30% amino acid identity, whereas
members of each family exhibit ⬎60% sequence identity
(Büttner and Singh, 1997; Hao et al., 1998; Riechmann and
Meyerowitz, 1998; see Figure 1). The recent report of the solution structure of the ERF domain has contributed direct
evidence that amino acid residues within the ERF domain
that interact directly with the GCC box are not present in the
AP2 domain (see Figure 1). Although detailed studies regarding this aspect have not been reported for the AP2 domain, 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 genome project and expressed sequence tag collections indicate 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/dehydration-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; Büttner 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 ⵑ125 genes
in the Arabidopsis genome encode AP2/ERF proteins
(Riechmann and Meyerowitz, 1998).
Functional Analysis of AtERF Proteins
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 expression 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), supporting the idea that Pto may regulate the expression of defense-related genes by regulating the function of
ERF proteins. AtEBP has been shown to interact with an octopine synthase (ocs) element binding protein (Büttner and
Singh, 1997), suggesting that PR gene expression is regulated via a protein–protein interaction between an ERF protein 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 to
bind specifically to the GCC box (Ohme-Takagi and Shinshi,
1995; Büttner 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 information about sequences encoding ERF proteins in Arabidopsis, but information regarding physiological function and
gene regulation of this family of transcription factors is still
lacking. In this report, we isolated cDNAs encoding Arabidopsis ERF proteins—AtERF proteins—to 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 box–dependent gene expression.
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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 putative nuclear localization signal (Raikhel, 1992). Two cysteine residues flanking the ERF domain (Figure 3) were also
conserved.
AtERF3 and AtERF4 comprise class II and possess substantial 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. However, class II AtERFs possess a similar cDNA structure with
respect to the location of the ERF domain, length of the coding 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 tobacco. 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 regions flanking the ERF domain. In addition, a putative mitogen-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).
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 analyzed. Sequence analysis of the cDNAs demonstrated that
AtERF1 to AtERF5 all contain the conserved ERF domain
and 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 identities 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 tobacco and to Pti4 from tomato (Ohme-Takagi and Shinshi,
Figure 2. AtERF cDNAs.
A schematic representation of the AtERF1 to AtERF5 cDNAs is
shown. The AtERF proteins are grouped into three classes according 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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Figure 3. Comparison of Deduced Amino Acid Sequences of AtERF Proteins.
Amino acid sequences grouped by class 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 localization 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 AtERF cDNAs are AB008103 (AtERF1), AB008104 (AtERF2), AB008105
(AtERF3), AB008106 (AtERF4), and AB008107 (AtERF5).
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, provoking 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 error. 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 activity, we overexpressed the entire coding region of each
AtERF as a maltose binding protein (MBP) fusion in Escherichia coli and performed electrophoretic mobility shift assays. As shown in Figure 4, all MBP–AtERF 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 box–specific 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, indicating 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 between 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 oligonucleotide, whereas AtERF3 and AtERF4 were able to bind to oligonucleotides 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.
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 luciferase (LUC)-encoding reporter gene, 4⫻HLS, which contains four copies of the GCC box sequence from the
Arabidopsis 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 Arabidopsis leaves by particle bombardment. LUC activity increased at least 12-fold when the reporter plasmid was
coexpressed with AtERF1, AtERF2, or AtERF5 effector plasmids (Figure 5B). No such increase in LUC activity by the
AtERFs was detected when a LUC reporter plasmid containing a mutated GCC box (ATCCTCC) was used (data not
shown). These data indicate that AtERF1, AtERF2, and
AtERF5 are able to function as GCC box sequence–specific
transactivators in Arabidopsis leaves.
Some genes containing the GCC box in their promoter region are known to be upregulated by ethylene in Arabidopsis (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
box–mediated transcription by the AtERFs. The 4⫻HLS reporter gene and the AtERF effector constructs were introduced 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 construct 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 progressively reducing the transactivation ability of AtERF5
(Figure 5E). Thus, this effect is unlikely to result from nonspecific squelching.
To determine whether class II AtERFs are active repressors that possess an intrinsic repression activity that is distinct from their DNA binding activity, we constructed a
reporter gene (5GAL4GCC) containing two different cis-elements 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
Figure 4. Characterization of AtERF DNA Binding Affinity to the
GCC Box.
(A) GCC box sequences used in this experiment. Underlining and boldface denote the GCC box. GCC, wild type; mGCC, a double mutant.
(B) AtERF possesses GCC box–specific binding activity. Electrophoretic mobility shift assays were performed using MBP–AtERF fusion 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 MBP–AtERF5, respectively.
(C) Sequence of the wild-type GCC box (W) and mutants (1 to 7)
used for the DNA binding assays. The GCC box sequence, AGCCGCC, 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.
5GAL4GCC reporter gene and the effector plasmid containing the GAL4 DNA binding domain fused to the transcriptional activation domain of viral protein 16 (VP16) from
herpes simplex virus (Triezenberg et al., 1988) were cobombarded with or without the AtERF3 effector plasmid (Figure
6). Reporter gene activity increased by a minimum of 20-fold
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when the VP16 effector alone was bombarded. The activation by the VP16 activator was reduced to ⵑ50% when
AtERF3 was coexpressed with the VP16 plasmid (Figure
6B). These results indicate that class II AtERFs are active repressors that can suppress the activity of other transcriptional 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 binding 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 Arabidopsis leaves (Figure 7). mRNAs corresponding to each of the
AtERFs could be detected in air-grown, healthy Arabidopsis
plants, although the basal levels of each AtERF transcript
differed. Because some GCC box–containing genes, such
as that encoding chitinase or PDF1.2, are known to be induced 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 AtERFs. Expression of genes encoding chitinase and PDF1.2 was induced 12 hr after ethylene treatment. Correspondingly,
amounts of AtERF1, AtERF2, and AtERF5 transcripts increased two- to threefold 12 hr after ethylene treatment,
whereas AtERF3 or AtERF4 transcript amounts did not increase. In the ein2 mutant, the expression of the AtERFs
was not induced by ethylene treatment, suggesting that ethylene induction of the AtERFs is regulated under the ethylene signaling pathway, as is the case for the genes that
encode chitinase and PDF1.2 (Figure 7). We observed that
the mRNA levels of all of the AtERFs except AtERF3 were
consistently lower in ein2 plants compared with the wild type.
Next, we investigated whether abiotic stresses such as
wounding, cold, heat, high NaCl concentration, or drought
induced AtERF expression (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. AtERF4 was also induced by cold, high NaCl concentration, and drought stress, whereas AtERF5 expression
Figure 5. AtERFs Can Transactivate or Repress GCC-Mediated
Gene Expression in Arabidopsis Leaves.
(A) Schematic diagram of the reporter and effector plasmids used in
transient 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 LUC gene. 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 tobacco mosaic virus.
(B) Transactivation of the 4⫻HLS GCC box–LUC 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
AtERF3 effector were co-bombarded with AtERF5 effector (from a
ratio of 1:0 to 1:1; AtERF3 to AtERF5) and the reporter plasmid.
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).
Functional Analysis of AtERF Proteins
399
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-transcriptional regulatory mechanisms may be involved
in AtERF expression.
DISCUSSION
Structural Features
The five AtERFs isolated during the course of this work were
grouped into three classes based on their amino acid
Figure 6. AtERF3 Suppresses Transactivation without Competing
for the Same DNA Binding Site.
(A) Schematic diagram of the reporter and effector plasmids used.
The GAL4 binding site (patterned hatched boxes) and GCC box sequence (filled circles) were fused to a minimal TATA box and the
LUC gene. The AtERF3 effector and VP16 effector plasmid containing 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 competing 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).
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 AtERF expression. Similarly, high temperature stress did not affect
the expression of any AtERF gene.
The induction of the AtERFs by wounding, cold, and drought
stress was observed in ein2 mutants 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 pathway mediated by EIN2 may regulate the salt stress–mediated induction of AtERF3 and AtERF4, even though ethylene
itself did not induce expression of these AtERF genes.
By contrast, treatment of Arabidopsis plants with cycloheximide (CHX), a protein synthesis inhibitor, resulted in the
marked accumulation of AtERF mRNAs, indicating that de
novo protein synthesis may not be required for AtERF expression (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
Figure 7. Induction of AtERF mRNA Expression.
(A) Expression pattern of AtERF mRNAs after exposure to ethylene
and abiotic stress in the wild type (wild) or an ethylene-insensitive
mutant, ein2 (ein2). Total RNA was prepared from Arabidopsis
leaves 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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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 chromosomes. In contrast, members of the CBF/DREB family
most 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 protein–protein interactions.
By contrast with AtERF1 and AtERF2, the class II proteins
AtERF3 and AtERF4 do not share much amino acid sequence identity except for residues within the ERF domain.
Nevertheless, the overall structure and the function of these
two proteins as transcriptional repressors are conserved, indicating 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 box–dependent 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 localization, or transactivation. The fact that a putative site for
MAP kinase–mediated phosphorylation is found in the class
III protein AtERF5, although such sites appear not to be
present in representatives of the other two classes, suggests 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 identified 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 nucleotide within this sequence is essential for AtERF binding
(Figure 4). However, results also showed that each AtERF
possessed a distinct DNA binding preference. AtERF1,
AtERF2, and AtERF5 appear to be most sensitive to the single-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 flexible than other AtERF proteins with respect to their target sequence preferences because they were able to bind to
several mutated oligonucleotides to which other AtERF proteins did not bind. 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 transcriptional 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 sequence 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 mediating the protein–DNA interaction in vitro.
Transcriptional Regulation
We showed that AtERF1, AtERF2, and AtERF5 act as transcriptional activators for GCC box–dependent transcription
in Arabidopsis leaves. Similarly, other ERF proteins, such as
CBF1 and DREB, have been shown to function as transactivators 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 transcription of a reporter gene in ein2 mutant as well as in wildtype leaves in our transient expression system, indicating
that AtERF proteins may not be regulated post-transcriptionally 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 utilizing antagonistic mechanisms for controlling GCC box–dependent
transcription operates in plants. Repression can be categorized 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 a
passive repressor that downregulates the transcriptional activity 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).
Functional Analysis of AtERF Proteins
Our results demonstrate that class II AtERFs utilize an active 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 proposed (Cowell, 1994; Hanna-Rose and Hansen, 1996; Pazin
and Kadonaga, 1997). One is that repressors interfere with
transcriptional activators that interact with the basal transcriptional machinery, such as TFIID. Alternatively, repressors
might recruit co-repressors such as histone deacetylase,
which modifies chromatin structure and prevents other transcriptional activators from binding to their target cis-elements
(Pazin and Kadonaga, 1997). Analyses of factors that interact with the repression domain of AtERF would demonstrate
the mechanism by which class II AtERFs function as transcriptional repressors.
Because all AtERFs can bind to the same target sequence, one point of regulation of transactivation or repression 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 transactivation activities of class I or III AtERFs. As GCC box–mediated
transcription is upregulated in plant cells in response to extracellular signals, mechanisms that downregulate the repression activity of class II AtERFs, such as protein degradation or regulation at the mRNA level, should be operating.
401
addition to its role as an ERE, again implying that the
AtERFs may act as transcription factors for stress-responsive genes.
Induction of the AtERFs by wounding, cold, and drought
appears to be independent from the ethylene signaling pathway because responses to these abiotic stresses were observed 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, AtERF expression 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 box–dependent transcription mediated by ERF proteins is shown in Figure 8.
Several possible roles for ERF proteins in the regulation of
GCC box–dependent transcription are presented. As the
AtERF1 to AtERF5 genes are expressed in response to different stimuli, the AtERF proteins probably function as signaland sequence-specific transcription factors by activating or
AtERF Proteins Are Stress Signal–Responsive Factors
AtERF genes respond differently not only to ethylene but
also to various forms of abiotic stress (Figure 7). AtERF1,
AtERF2, and AtERF5 were induced by ethylene 12 hr after
treatment, whereas the ethylene response of ERF1 is observed 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 responses 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). This
survey 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 element in tobacco cells (Yamamoto et al., 1999). These findings suggest that the GCC box may act as a cis-regulatory
element for biotic and abiotic stress signal transduction in
Figure 8. A Model for GCC Box–Mediated Stress Signal—Dependent Transcription by ERF Proteins in Arabidopsis.
After reception of a stress signal by plants, ERF genes may be upregulated via an ethylene-dependent pathway or an ethylene-independent pathway. ERF genes such as ERF1 (Solano et al., 1998) are
induced by ethylene and can interact with GCC box–containing
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 box–containing genes, whereas AtERF3 and AtERF4 may repress the expression 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 box–containing stress response genes.
402
The Plant Cell
repressing the expression of GCC box–containing stressresponsive genes dependent on or independent of the ethylene signal. Other proteins, such as ERF1 (Solano et al., 1998),
probably act as activators of GCC box–containing stressand/or ethylene-responsive genes by positively modulating
gene expression in response to external or internal ethylene.
This model suggests that the AtERFs are regulated differentially at both the transcriptional and post-transcriptional levels
and that subsets of GCC box–containing genes may be regulated by different ERF proteins.
METHODS
Plant Material and Treatments
Wild-type (Arabidopsis thaliana ecotype C24) and ein2 plants were
grown in a 1:1 mixture of Soil Mix (Sakata Co., Kyoto, Japan) and
vermiculite in growth chambers at 22⬚C with a 16-hr light/dark cycle
for 20 to 30 days. Treatments were performed at 22⬚C unless otherwise noted and were as follows: ethylene—plants 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 4⬚C; drought—whole 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;
wounding—rosette leaves were detached from the plant, cut into
ⵑ5-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 incubated 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 immediately frozen in liquid nitrogen.
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 (ATGAGTTAACGCAGACATAGCCGCCATTT) was multimerized four times, placed upstream 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) multimerized four times were placed upstream of the reporter gene
containing the minimal TATA box. For effector plasmids, the ␤-glucuronidase (GUS) gene in pBI221 (Clonetech, Palo Alto, CA) was replaced by the coding region of each AtERF. The tobacco mosaic
virus ⍀ sequence (Gallie et al., 1987) was inserted downstream of the
CaMV 35S promoter in pBI221 to enhance the efficiency of translation. 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 bombardment 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 plasmid or blank plasmid. As a reference,
the Renilla LUC gene (Promega) under the control of the CaMV 35S
promoter was used at a reference gene–reporter 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 22⬚C for 6 hr, frozen in
liquid nitrogen, and quantified for LUC activity.
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 standard 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
coli BL21 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 MBP–AtERF
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,
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% formaldehyde–agarose 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 samples was confirmed by visualization of rRNA after ethidium bromide
staining.
ACKNOWLEDGMENTS
We are grateful to Dr. Kazuko Yamaguchi-Shinozaki for the Arabidopsis cDNA library and to the Arabidopsis Biological Resource
Center (Ohio State University, Columbus) for the C-24 and ein2 Arabidopsis 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.
Functional Analysis of AtERF Proteins
403
S.Y.F. would also like to thank the National Science Foundation for
its support.
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GCC box recognition by the DNA-binding domain of ethyleneresponsive element-binding factor (ERF domain) in plants. J. Biol.
Chem. 273, 26857–26861.
Received December 3, 1999; accepted January 5, 2000.
Hurst, H.C. (1994). Protein Profile: Transcription Factors 1: bZip
Proteins. (London: Academic Press).
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Repressors of GCC Box−Mediated Gene Expression
Susan Y. Fujimoto, Masaru Ohta, Akemi Usui, Hideaki Shinshi and Masaru Ohme-Takagi
Plant Cell 2000;12;393-404
DOI 10.1105/tpc.12.3.393
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