Tài liệu Báo cáo khoa học: Four divergent Arabidopsis ethylene-responsive element-binding factor domains bind to a target DNA motif with a universal CG step core recognition and different flanking bases preference pptx

Four divergent Arabidopsis ethylene-responsive
element-binding factor domains bind to a target DNA
motif with a universal CG step core recognition and
different flanking bases preference
Shuo Yang
1
, Shichen Wang
1
, Xiangguo Liu
1
, Ying Yu
1
, Lin Yue
3
, Xiaoping Wang
1
and Dongyun Hao
1,2
1 Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun, China
2 Biotechnology Research Centre, Jilin Academy of Agricultural Sciences (JAAS), Changchun, China
3 School of Physical Education, Northeast Normal University, Changchun, China
Introduction
The ethylene-responsive element-binding factor (ERF)
gene family of transcriptional factors is one of the
largest transcriptional factor gene families in the plant
kingdom [1,2]. The ERF domain was first identified as
a conserved motif of 58–59 amino acids in four DNA-
binding proteins from tobacco and was shown to bind
specifically to a GCC box [3]. After the completion of
the sequencing of the Arabidopsis genome [4], 124
genes were predicted to encode proteins belonging to

the AtERF family [2].
Keywords
CG step; DRE motif; ERF domain;
homology; universal binding characteristic
Correspondence
D. Hao, Biotechnology Research Centre,
Jilin Academy of Agricultural Sciences
(JAAS), Changchun 130033, China
Fax: +86 431 87063080
Tel: +86 431 87063195
E-mail:
(Received 31 August 2009, revised
29 September 2009, accepted 8 October
2009)
doi:10.1111/j.1742-4658.2009.07428.x
The Arabidopsis ethylene-responsive element-binding factor (AtERF) fam-
ily of transcription factors has  120 members, all of which possess a
highly conserved ERF domain. AtERF1, AtERF4, AtEBP and CBF1
are members from different phylogenetic subgroups within the family.
Electrophoretic mobility shift assay analyses revealed that the ERF
domains of these four proteins were capable of binding specifically to
either GCC or dehydration-responsive element (DRE) motifs. In vitro
and in vivo binding assays of the four AtERFs with the DRE motif
showed that the recognition of the CG step was indispensable in all four
of the specific binding reactions, implying that there may be a universal
binding characteristic of various ERF domains binding to a given con-
sensus (e.g. the DRE motif). In addition, the core DNA-binding motifs
preferred by the four AtERFs were identified, and all of these motifs
contained a conserved CG step core. Thus, conserved recognition of the
CG step may be the foundation of the formation of the stable complex

by the ERF domain with the DRE motif, which is probably determined
by the highly conserved residues presented in the DNA contact surface
among the whole AtERF family members. The different preferences at
flanking bases of individual ERF domains, which appear to be attrib-
uted to the subfamily- or subgroup-specific residues, may be essential
discrimination of the target binding motif from various similar sequences
by divergent AtERF domains.
Abbreviations
DBD, DNA binding domain; DRE, dehydration-responsive element; EMSA, electrophoretic mobility shift assay; ERE, ethylene-responsive
element; ERF, ethylene-responsive element-binding factor.
FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS 7177
The AtERF family is further divided into various
subgroups according to the homology of ERF
domains [5,6].
An ERF domain consists of a three-stranded anti-
parallel b-sheet and an a-helix, packed approximately
parallel to the b-sheet, with the seven thoroughly con-
served amino acids (Arg6, Arg8, Trp10, Glu16, Arg18,
Arg26 and Trp28) in the b-sheet contacting uniquely
with the bases of the target DNA at the major groove
(see Fig. 1A) [7]. Phylogenetic analyses of the ERF
domains of all members within the AtERF family
show that the residues Arg6, Glu16 and Trp28 are
completely conserved among all 124 members, whereas
more than 95% of members contain the Arg8, Arg18,
Arg26 residues [6].
From the results of the few AtERFs studied,
however, the conserved ERF domains do not seem to
prefer identical DNA consensus sequences. For ins-
tance, some AtERFs have been shown to bind in vitro

to the ethylene-responsive element (ERE), a GCCGCC
motif designated the GCC motif [3,8–12], to conduct
GCC motif-mediated transcription (activation or repres-
sion) in leaves of Arabidopsis [12]. This ERE was first
reported to be a binding site (referred to as the GCC
box) of a number of tobacco ERF proteins [3] and it
was later presumed to be the target site of many other
ERF proteins [2].
The ERF protein, AtEBP, was also found to protect
the GCC box in a DNase I foot-printing analysis [10].
In contrast, the dehydration-responsive element
(DRE), with the TACCGACAT motif, in the drought-
responsive gene rd29A from Arabidopsis has been
proven to be the recognition site of DRE-binding
proteins, which are transcription factors that have
authentic ERF domains [13] and that are involved in
the induction of rd29A expression by low-temperature
stress. A similar element to DRE, the C-repeat
(TGGCCGAC) has been identified in the cold-induc-
ible gene cor15a and it is reported to function in
cold-response regulation through binding by another
ERF protein, CBF1 [14].
The similarity of these ERF-binding elements and
the high similarity of ERF domains among the mem-
bers of the entire ERF family have led to speculation
that the ERF domains from various subgroups within
the AtERF family recognize a certain binding site with
universal binding characteristic to a conserved core.
The divergent short flanking bases, on the other hand,
allow preference to govern differential recognition. We

have previously demonstrated that various ERF
domains had divergences in their DNA recognition
modes [9], but, to date, additional supporting evidence
has been lacking. Indeed, little is still known regarding
the ways in which these differences are important for
the functionalities of members in the AtERF family,
the majority of which have not yet been studied.
In the present study, we selected four representatives
from different functional subgroups of the AtERF
family and characterized the in vivo and in vitro bind-
ing specificities of the four ERF domains for a
sequence containing the DRE motif. In addition, we
used a random sequence selection method to identify
the core recognition motifs preferred by each of the
four domains. A universal binding characteristic was
revealed, in addition to the individual features of vari-
ous ERF domains involved in recognition of the DRE
A
B
C
Fig. 1. (A) Solution structure of AtERF1–GCC box complex (PDB
code: 1GCC) [7]. The DNA-binding domain is shown in the sche-
matic; DNA is represented by tubes. The b-sheet of the ERF
domain is light blue and the seven conserved amino acid residues
reported to contact DNA bases directly are red; other conserved
amino acid residues that do not directly contact with DNA bases
are blue. (B) The DNA base sequence with position numbering
along the 16 bp fragment of DREwt. The bases in the core ACC-
GAC are in bold and boxed in gray. (C) Sequence alignment of four
ERF domains of AtERF1, AtERF4, AtEBP and CBF1. The secondary

structure scheme is indicated above the sequence. The conserved
amino acid residues that directly contact with DNA bases and the
other conserved amino acid residues that do not directly contact
with DNA bases are in red and blue, respectively.
Arabidopsis ERFs recognize a common CG step core S. Yang et al.
7178 FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS
motif. The results have important implications for
understanding the foundations of recognition of a
given binding site by divergent members of the AtERF
family.
Results and Discussion
The members of the ERF family in Arabidopsis can be
classified into a number of different phylogentic sub-
groups according to the sequence similarity of the
ERF domains [6]. We selected four AtERFs –
AtERF1, AtERF4, AtEBP and CBF1 – as representa-
tives from divergent subgroups (for details, see Figs 1C
and 6), to investigate whether the highly homologous
ERF domains of different AtERFs have universal
binding characteristics for the recognition of a given
consensus sequence (e.g. DRE).
Binding specificity of AtERFs to the GCC and DRE
motifs
Having established that CBF1 can specifically recog-
nize both GCC and DRE motifs [9], the two most
popularly reported ERF-binding sites, we continued to
explore the DNA-binding specificity of the other three
AtERFs. Table 1 shows that all four ERF domains
were capable of binding specifically to the 16 bp frag-
ment containing either the GCC or the DRE motif.

The equilibrium dissociation constants (K
d
)of
AtERF1, AtERF4 and AtEBP for binding to the DRE
motif were within the level of typical monomeric
interaction, although the binding activities were in gen-
eral lower than those for binding to the GCC motif.
CBF1 appeared to bind to the DRE motif more
strongly than to the GCC motif, implying CBF1 may
prefer the DRE motif over the GCC motif. To further
confirm if these variations in binding affinity were
caused by binding instability as a result of nonspecific
interference, rather than the alternation of a binding
site, we carried out the competition binding assay
using a nonspecific competitor poly[dA-dT].poly[dA-
dT] in an electrophoretic mobility shift assay (EMSA).
Figure 2 shows that most of the AtERFs exhibited
similar stability in binding to either the GCC or the
DRE motif. The most remarkable feature arising from
the competition binding assay was the consistency of
the binding preference of the AtERFs with the EMSA
analysis. The three AtERFs, AtERF1 AtERF4 and
AtEBP, with higher sequence similarity to each other
than to CBF1, had similar binding preferences in
comparison with CBF1.
Verification of the binding characteristics of the
selected AtERFs with the DRE motif
To verify the detailed binding characteristics of the
four different AtERFs to a given consensus sequence
DRE, EMSAs were carried out with the DRE motif

and its mutants possessing single T substitutions (see
Fig. 3). Each base in the DRE motif from T5 to C11
was replaced with a T, except that T5 was replaced by
A, and the binding free energy changes (DDG) were
obtained from quantitative titration analysis. Figure 3
shows that AtERF1 and AtERF4 exhibited the highest
specific interactions at C7, C8, G9 or C11, because the
Table 1. Binding activities of the selected AtERFs to GCC and
DRE motif-containing sequences. Four ERF domains were tested
for binding to the 16 bp DRE or GCC motif-containing sequences
using quantitative EMSA, as described in Materials and methods.
K
d
values are represented as the mean of three replicates ± stan-
dard deviation. The K
d
value for nonspecific binding was estimated
to be  1 l
M or higher.
ERF fragments GCCwt (n
M) DREwt (nM)
AtERF1-f 0.17 ± 0.04 2.02 ± 1.44
AtERF4-f 0.38 ± 0.24 31.7 ± 14.3
AtEBP-f 0.34 ± 0.17 1.25 ± 0.62
CBF1-f 5.63 ± 0.61 1.46 ± 0.99
Fig. 2. Competition binding assay of the ERF–DNA complex. The
binding complex of the ERFs and their binding DNAs were incu-
bated together with 0, 0.001, 0.01, 0.1, 1.0 and 10 lg poly[dA-
dT].poly[dA-dT] in a 10 lL volume and analysed by EMSA, as
described in the Materials and methods.

S. Yang et al. Arabidopsis ERFs recognize a common CG step core
FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS 7179
base substitution at that position caused the greatest
decline in binding activity. AtEBP requested C8, G9
and C11 most frequently and with the moderate
requirements of C7. As for CBF1, the prerequisite
bases appeared to be C8 and G9, whereas the other
bases within the binding motif were only moderately
required to varying extents. In the four reactions, bases
C8 and G9 in the DRE motif were absolutely
requested by all AtERFs for specific binding, indicat-
ing that the recognition of the CG step was conserved
by various AtERFs and may be the universal binding
characteristic of different AtERFs in recognition with
the DRE motif. In addition, bases C7 and C11 within
the motif were required to different extents by AtERFs
from the divergent phylogentic subgroups, implying
that the recognition of these bases was the individual
feature of distinct AtERFs binding to the DRE motif.
In vivo DNA binding specificity of AtERFs by the
reporter–effector transient assay
To confirm if these binding specificities of AtERFs
observed in vitro were also capable of regulating the
DRE-mediated transcription within plant tissue, repor-
ter effect cotransformation assays were carried out. An
effector plasmid possessing the coding region of the
full-length AtERF1, AtEBP or CBF1 genes driven by
the CaMV 35S promoter, together with the luciferase
reporter gene containing four tandem copies of either
the DRE motif or its mutants at the upstream regula-

tory region, was coexpressed into Arabidopsis leaves by
particle bombardment. Figure 4 shows that these three
AtERFs were able to transactivate the transcription of
a gene carrying the wild-type DRE motif (DREwt),
which was represented by an increase in luciferase
activity of about four- to seven-fold over the control.
No luciferase activity was detected when any of the
three ERF effectors was cotransformed with a reporter
carrying DREt1, in which the C8 was replaced by
T. Although AtERF1 did not activate transcription of
the reporter gene carrying either DREt2 or DREt3,
the coexpressions of AtEBP and CBF1 activated tran-
scription of DREt3 reporter genes to varying degrees.
As AtERF4 was a repressor, an extra effector in which
the activation domain of viral protein 16 was fused to
the yeast GAL4 DNA binding domain (DBD) and
then coexpressed with the AtERF4 effector was used
to test the in vivo binding specificity of AtERF4. The
reporter gene containing multicopies of the GAL4
binding sequence was inserted into the existing lucifer-
ase reporter next to the four tandem DRE motifs and
the transcription suppression by AtERF4 was assayed.
Figure 5 shows that AtERF4 suppressed viral protein
16 activation by more than 50% when cotransformed
with the reporter carrying DREwt, whereas no repres-
sion was detected with a reporter having mutant DRE
motifs in which C8, G9 or C11 were replaced by T.
0
1
2

3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
0
1
2
3
4
5
AtERF1
AtERF4
AtEBP
CBF1
5 6 7 8 9 10 11
ΔΔG (Kcal·mol
–1
) ΔΔG (Kcal·mol
–1

) ΔΔG (Kcal·mol
–1
) ΔΔG (Kcal·mol
–1
)
Fig. 3. Effect of single base substitutions on the relative binding
free energy change (DDG) in the binding of the four ERF domains
to the DRE motif. The DNA sequence shown at the bottom is the
DRE motif in which each base was substituted individually one by
one as illustrated. The solid bars indicate the increase in DDG
caused by the base substitution at the corresponding position. Posi-
tive DDG represents a decreased binding activity; a 10-fold
decrease in binding activity increased DDG by  1.3 kcalÆmol
)1
.
Arabidopsis ERFs recognize a common CG step core S. Yang et al.
7180 FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS
These observations were consistent with the findings in
the in vitro single base substitution binding assays: the
substitution at C8 or G9 abolished the specific recogni-
tion of the DRE motif by all four of the AtERFs.
Random binding site selection reveals the
binding characteristics of divergent AtERFs
to the DRE motif
To clarify the possible existence of the moderately
divergent binding motifs of the four AtERFs from the
divergent phylogenetic subgroups, randomized oligonu-
cleotide selection was performed. The resulting binding
motif of hexamers selected by these four ERF domains
is shown in Table 2. AtERF1 seemed to prefer the

hexamer GCCGCC motif, which is consistent with the
results from previous studies [7,8]. Although the
AtERF4 required a relatively relaxed G or A at posi-
tion 2 of the hexamer G ⁄ aCCGCC, AtEBP selected a
binding motif of hexamer GCCGCC. The selected
motif of CBF1, AA ⁄ cCGAC, appears to agree with a
previous report [14]. Although each ERF domain
showed different binding preferences, all of the binding
sites selected by the AtERFs from the four subgroups
possessed a common CG core in the centre and a con-
served C at the last position (position 7). These moder-
ately divergent bases existed in the other positions
within the binding motifs, discriminating the members
from different subgroups.
The solution structure of the complex formed by the
ERF domain of AtERF1 with the GCC box (1GCC)
shows that two categories of residues within the domain
are considered to be important for specific DNA bind-
ing: one consists of the residues in the b-sheet directly
contacting the DNA bases; and the other is made up of
the numerous Ala residues in the a-helix and the hydro-
phobic residues with larger side chains in the b-sheet (in
particular Phe13, Phe32, Val27 and Ile17), which
appears to determine the geometry of the a-helix rela-
tive to the b-sheet [3–5,7–9,17]. A multiple alignment of
Arabidopsis ERF domains (Fig. 6) shows that a series
of residues (e.g. Gly4, Arg6, Arg8, Gly11, Glu16, Ile17,
Arg18, Arg26, Trp28, Leu29, Gly30, Ala38, Ala39,
Asp43 and Asn57) were almost absolutely conserved
among all members of the ERF family. Most of these

residues are present in the b-sheet, especially Arg6,
Arg8, Glu16, Arg18, Arg26 and Trp28 (Fig. 1A), which
are reported to contact directly with DNA, suggesting
that the conformation of a partial DNA contact surface
may be conserved among various ERF domains, which
result in the conserved recognition of the CG step in
the DRE motif by all four of the different AtERFs.
On the other hand, some other residues reported to
determine the geometry of the a-helix relative to the
b-sheet were not as conserved as these other residues,
but instead were subfamily or subgroup specific, e.g.
the Ile17 in almost all of the ERF family (V17 in
CBF1), V27 in ERF subfamily (Ile27 or Leu27 in the
DRE-binding protein subfamily) and Tyr42 in the
major ERF family (His42 in the CBF1 and TINY sub-
group) (Fig. 6). However, these subfamily- or group-
specific residues seem not to be involved in the direct
base contact, which may affect the local conformation
of the interface by the determination of the geometry
of the a-helix relative to the b-sheet. It seems that the
Reporters
Effectors
4 x DRE
TATA
LUC Nos
Ω
CaMV-35S
Nos
ERF
Relative luciferase activity

0
2
4
6
8
10
DREwt
0
2
4
6
8
10
DREt2
DREt1
DREt3
Binding motifs:
l
o r
t
n o C
1 F B C
1
F
R E t A
P B E t A
l
o r t
n
o C

1
F B C
1 F R E t A
P B E t A
Fig. 4. AtERF1, AtEBP and CBF1 activate the transcription of the
luciferase reporter gene driven by the DRE motif and its mutants.
The luciferase reporter gene contains four copies of the cis-acting
binding motif, DREwt, DREt1, DREt2 or DREt3, which are high-
lighted and underlined. The effector was constructed with a full
length of ERF cDNA that was controlled under the CaMV 35S pro-
moter following a translation enhancer (X) from tobacco mosaic
virus. These effectors induce transactivation of the reporter gene.
The control in the transient assay was the same as the experi-
ments without the addition of an effector. The results are shown
as relative luciferase activity per control.
S. Yang et al. Arabidopsis ERFs recognize a common CG step core
FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS 7181
flanking positions, as well as the CG step core in the
DNA motif, were required to varying extents by diver-
gent ERF domains, and may be determined by these
subfamily- or group-specific residues.
The biological function in DNA binding of individ-
ual ERF domains is apparently determined by the
primary structures of the divergent DBD and a phylo-
genetic classification of the ERF family may partly
reflect the features in DNA binding of a certain popu-
lation of ERF domains. The observations acquired in
the present study imply that the divergent ERF
domains from various groups of the family bind to a
given consensus sequence by conserved recognition of

a CG step core as the universal binding characteristic.
This may be the foundation of the formation of a sta-
ble ERF–DNA complex and the different flanking
position preferences by individual ERF domains may
be crucial for the precise regulation of their own target
genes by various ERFs.
Materials and methods
Preparation of ERF domain-containing proteins
The coding region of the ERF domain of CBF1 (Uni-
ProtKB: P93835) (amino acids 47–142), which contains 10
and 38 amino acids in the N- and C-terminal regions,
respectively, was prepared as described previously [9]. The
Fig. 5. AtERF4 suppresses the transcription of the luciferase repor-
ter gene driven by the DRE motif and it mutants. A multicopy of
the GAL4 binding sequence was inserted into the DRE:luciferase
reporter next to the 4· DRE motif. An extra effector was con-
structed carrying the coding sequences of the activation domain of
viral protein 16 and the yeast GAL4 DBD. The reporter and two
effectors in a ratio of 1 : 1 : 1 were cotransformed into plant tissue;
the remainder was the same as in Fig. 4.
Table 2. Selection of binding sites from a random oligonucleotide
pool by ERFs. Selections were performed using a 60 bp oligonu-
cleotide containing a randomized site of 10 bp. The selected
sequences were aligned computationally and the appearance of a
base at each position in a motif was presented as a percentage fre-
quency of all four kinds of base. The base with a frequency higher
than 50% (bold) was defined as the selected site. If the second
highest frequency base showed not less than half the highest fre-
quency (marked with an asterisk), it was defined as the second
possible site and is presented in lower case letter.

Proteins
Selection
position
Frequency (%)
AC G T
Deduced
consensus
AtERF1 1 38.5 15.4 30.8 15.4 N
2 0.0 11.5 88.5 0.0 G
3 0.0 80.8 15.4 3.8 C
4 11.5 76.9 7.8 3.8 C
5 0.0 0.0 100 0.0 G
6 15.4 69.2 3.8 11.5 C
7 11.5 80.8 3.8 3.8 C
AtERF4 1 29.6 33.3 22.2 14.8 N
2 25.9 11.1 51.8 11.1 G ⁄ a*
3 3.7 77.8 14.8 3.7 C
4 22.2 66.7 3.7 7.4 C
5 0.0 0.0 100 0.0 G
6 7.4 81.5 11.1 0.0 C
7 7.4 74.1 18.5 0.0 C
AtEBP 1 15.4 57.7 23.1 3.8 C
2 0.0 3.8 96.2 0.0 G
3 3.8 92.3 0.0 3.8 C
4 0.0 92.3 0.0 7.7 C
5 0.0 0.0 100.0 0.0 G
6 11.5 84.6 3.8 0.0 C
7 0.0 100.0 0.0 0.0 C
CBF1 1 20.0 40.0 16.0 24.0 N
2 36.0 20.0 36.0 8.0 V

3 8.0 64.0 16.0 12.0 C
4 8.0 60.0 20.0 12.0 C
5 0.0 4.0 88.0 8.0 G
6 56.0 32.0 8.0 4.0 A ⁄ c
7 4.0 68.0 24.0 4.0 C
Arabidopsis ERFs recognize a common CG step core S. Yang
et al.
7182 FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS
Fig. 6. Sequence alignment of ERF domains of members of the Arabidopsis ERF family. All ERF domain sequences were aligned and classi-
fied according to the results from the phylogenetic tree. The names of the ERF domains are represented by their gene locus numbers
except that the names of the four domains used in this study are represented by the transcriptional factor names. The secondary structure
indicated above the sequence and the seven conserved amino acid residues reported to contact DNA bases directly [7] are in red; other
conserved amino acid residues that do not directly contact DNA bases are in blue.
S. Yang et al. Arabidopsis ERFs recognize a common CG step core
FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS 7183
ERF domains of AtERF1 (UniProtKB: O80337), AtERF4
(UniProtKB: O80340) and AtEBP (UniProtKB: P42736)
with 10 and 8 amino acids in the terminal regions, respec-
tively, were prepared according to the previous work of Hao
et al. [8]. The PCR products were then cloned into the
pET16b plasmid (Novagen, Merck, Darmstadt, Germany)
Fig. 6. (Continued ).
Arabidopsis ERFs recognize a common CG step core S. Yang et al.
7184 FEBS Journal 276 (2009) 7177–7186 ª 2009 The Authors Journal compilation ª 2009 FEBS
and the corresponding proteins were expressed in
BL21(DE3) pLysS (Merck) Escherichia coli cells and puri-
fied using a His-Trap his-tagged protein purification kit
(Amersham Pharmacia Biotech, Uppsala, Sweden). The pro-
tein concentrations were determined using the bicinchoninic
acid protein assay kit (Pierce, Chester, UK) and further

confirmed using the method of Gill and von Hippel [18].
EMSA
Two 16 bp fragments, EREwt (5¢-CATAAGAGCCGCC
ACT-3¢) and DREwt (5¢-ATACT
ACCGACATGAG-3¢)
(for DNA base sequence and position numbering of
DREwt, see Fig. 1B), from the promoter region of the
tobacco Gln2 gene [3] and the Arabidopsis rd29A gene [19],
respectively, were prepared, together with their mutants, by
synthesizing both stands. The EMSA, binding titration
analysis and the calculation of the binding free energy
change (DDG) were performed as described previously [8,9].
Binding competition assay
The binding condition and buffers used in the competition
assay were the same as used in the quantitative DNA-bind-
ing assay described above. The radioisotope-labelled DNA
probe was first mixed with the binding protein at a concen-
tration corresponding to its K
d
. After allowing it to com-
plex for 5 min at room temperature, the mixture was
distributed into aliquots, to which a poly.[d(A-T)].poly[dA-
dT] (Amersham Pharmacia Biotech) gradient of 0.001–
10 lg was added to a final volume of 10 lL of each
aliquot. After incubation for a further 10 min, the contents
were loaded on to an 8% nondenaturing PAGE and visual-
ized as for EMSA.
Construction of the reporter and effector genes
For the reporter gene constructs, see Fig. 4. The detailed
dual-luciferase reporter transient assay was performed as

described previously [9].
Selection of the DNA-binding site
A 60 bp single-stranded DNA RDM10, with 10 random-
ized oligonucleotides in the center, i.e. CTGTCAGTGAT
GCATATGAACGAATN
10
AATCAACGACATTAGGATC
CTTAGC was synthesized. A 100 ng sample of RDM10
was radiolabelled during synthesis of double-stranded DNA
using [
32
P]dATP[aP] with the E. coli Klenow fragment
(New England Biolabs, Ipswich, MA, USA). The selections
were performed after incubation with the individual ERF
domains (25–100 ng) followed by EMSA. Briefly, each
binding reaction was carried out in a 10 lL binding buffer
[25 mm Hepes-KOH (pH 7.5), 40 mm KCl, 0.1 mm EDTA,
0.1 mgÆmL
)1
BSA, 10% glycerol and 1 lg double-stranded
poly(dI–dC)] and 25–100 ng of individual ERF domain.
The bound oligonucleotides were gel purified, extracted with
phenol ⁄ chloroform and precipitated with ethanol. The puri-
fied DNAs were radiolabelled during amplification by PCR
using 5¢ and 3¢ primers in the presence of [
32
P]dATP[aP].
This product was used for the next round of selection follow-
ing the same protocol. After seven cycles of selection, the
retarded DNA band of the final selection was cut off, puri-

fied and then cloned into the pUC119 plasmid (New England
Biolabs). Plasmid DNAs from  40–50 independent insert-
containing colonies were prepared and the insert fragments
were sequenced. At least 25 of the resulting quality sequences
containing the randomized 10 bp oligonucleotides were
aligned computationally using clustal x [20]. The frequency
of each nucleotide appearing in the aligned position of the
selected sequences was calculated, leading to the establish-
ment of the selected binding site.
Phylogenetic analysis
The amino acid sequences of all AtERFs were downloaded
from the Database of Arabidopsis Transcription Factors
(DATF) () [21]. The sequences of
all ERF domains were extracted in bulk by a manual pro-
gram using Perl script. The sequence alignment was gener-
ated using clustal x: Gap at 10; Gap Extension at 0.2;
Delay Divergent Sequence at 10%; Negative Matrix Off
and Protein Weight Matrix of BLOSUM Series [20].
Acknowledgements
The experiments were carried out at the National Insti-
tute of Advanced Industrial Science and Technology,
Japan. DH was a recipient of a fellowship from the
former Agency of Industrial Science and Technology,
MITI, Japan, and of an STA fellowship from the
Science and Technology Agency of Japan. This study
was also supported partially by a grant issued by the
National Natural Science Foundation of China (grant
no. 30470159 ⁄ C01020304) and the National High-
Technology Research and Development Program
(‘863’ Program) of China (grant no. 2007AA10Z110).

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