Arabidopsis Ethylene-Responsive Element Binding Factors

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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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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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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).


9/12


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.


10/12

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.


11/12


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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Arabidopsis Ethylene-Responsive Element Binding Factors