Biochimica et Biophysica Acta 1819 (2012) 86–96

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Biochimica et Biophysica Acta

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Review

AP2/ERF family transcription factors in plant abiotic stress responses☆

Junya Mizoi a, Kazuo Shinozaki b, Kazuko Yamaguchi-Shinozaki a, c,⁎
a
Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
b
Gene Discovery Research Group, RIKEN Plant Science Center, 1-7-22, Suehirocho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan
c
Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1–1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan

a r t i c l e i n f o a b s t r a c t

Article history: In terrestrial environments, temperature and water conditions are highly variable, and extreme temperatures
Received 29 June 2011 and water conditions affect the survival, growth and reproduction of plants. To protect cells and sustain growth
Received in revised form 8 August 2011 under such conditions of abiotic stress, plants respond to unfavourable changes in their environments in
Accepted 8 August 2011
developmental, physiological and biochemical ways. These responses require expression of stress-responsive
Available online 16 August 2011
genes, which are regulated by a network of transcription factors. The AP2/ERF family is a large family of plant-
Keywords:
specific transcription factors that share a well-conserved DNA-binding domain. This transcription factor family
Abiotic stress includes DRE-binding proteins (DREBs), which activate the expression of abiotic stress-responsive genes via
DRE/CRT specific binding to the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element in their
AP2/ERF family transcription factor promoters. In this review, we discuss the functions of the AP2/ERF-type transcription factors in plant abiotic
DREB1/CBF stress responses, with special emphasis on the regulations and functions of two major types of DREBs,
DREB2 DREB1/CBF and DREB2. In addition, we summarise the involvement of other AP2/ERF-type transcription
factors in abiotic stress responses, which has recently become clear. This article is part of a Special Issue
entitled: Plant gene regulation in response to abiotic stress.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction promoters, cis- and trans-acting elements involved in the transcrip-

Abiotic stress conditions such as drought, high salinity and extreme
temperatures have adverse effects on the growth and production of
land plants. To survive, grow and reproduce in environments that
fluctuate annually and diurnally, land plants have evolved complicated
systems that regulate adaptation in response to stress signals. Many
aspects of adaptation processes, which include developmental,
physiological and biochemical changes, are regulated by stress-
responsive gene expression. Transcription factors play central roles
in gene expression by regulating expression of downstream genes
as trans-acting elements via specific binding to cis-acting elements
in the promoters of target genes. From analyses of stress-responsive
tional responses of stress-responsive genes have been identified [1].
The APETALA 2/ethylene-responsive element binding factor
(AP2/ERF) family is a large group of plant-specific transcription
factors that includes four major subfamilies: the AP2, RAV, ERF and
dehydration-responsive element-binding protein (DREB) subfamilies
[2]. Of these, many stress-inducible DREB subfamily members have
been isolated and characterised. It has been established that they
are major factors involved in plant abiotic stress responses by
regulating gene expression via the cis-acting dehydration-responsive
element/C-repeat (DRE/CRT) element [1]. The involvement of ERF
subfamily members, which bind to the ethylene-responsive element
(ERE), in abiotic stress responses, has also been reported. In this
review, we summarise the function and regulation of DREB and ERF
subfamily members in the abiotic stress responses of plants.

Abbreviations: AP2/ERF, APETALA 2/ERE binding factor; ABA, abscisic acid; ABRE,
ABA-responsive element; bHLH, basic helix-loop-helix; CAMTA, calmodulin-binding
transcription activator; CBF, C-repeat binding factor; CE, coupling element; DREB,
DRE-binding protein; DRE/CRT, dehydration-responsive element/C-repeat; EAR motif,
ERF-associated amphiphilic repression motif; EE, evening element; ERE, ethylene-
responsive element; EST, expressed sequence tag; LEA, late embryogenesis abundant;
HSE, heat shock element; HSF, heat shock transcription factor; GA, gibberellin; ICEr,
induction of CBF expression region; QTL, quantitative trait locus
☆ This article is part of a Special Issue entitled: Plant gene regulation in response to
abiotic stress.
⁎ Corresponding author at: 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: +81
3 5841 8137; fax: +81 3 5841 8136.
E-mail address: akys@mail.ecc.u-tokyo.ac.jp (K. Yamaguchi-Shinozaki).
2. The DRE/CRT cis-acting element is a major target of AP2/ERF
transcription factors in plant abiotic stress responses
The RD29A/COR78/LTI78 gene of Arabidopsis (Arabidopsis thaliana) is
induced by exogenously applied abscisic acid (ABA) and stress stimuli
such as cold, dehydration and high salinity [3]. Stress-induced
expression of the RD29A gene also occurs in mutants deficient in ABA
biosynthesis or signalling [4]. Therefore, the stress-responsiveness of
the RD29A gene is regulated in both ABA-dependent and -independent
ways. Analyses of the RD29A promoter reveal that the ABA-dependent
expression of the RD29A gene is regulated by an ABA-responsive

1874-9399/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbagrm.2011.08.004
 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96
element (ABRE), whereas ABA-independent RD29A expression is
regulated by a novel cis-acting element, TACCGACAT, which was
named dehydration-responsive element (DRE) [5]. Similar cis-acting
elements, a C-repeat (CRT) and a low-temperature-responsive element
(LTRE), are also found in cold-inducible promoters [6,7]. A/GCCGAC,
which is a core DRE sequence, is shared among these cis-acting elements
and is conserved in promoters of abiotic-stress responsive genes [5,6,8].
Transcription factors that bind to these cis-acting elements DRE-
BINDING PROTEIN 1/C-REPEAT BINDING FACTOR (DREB1/CBF) and
DREB2 were identified in Arabidopsis [9,10]. These proteins belong to
the AP2/ERF family of transcription factors and activate gene transcrip-
tion via specific binding to the DRE/CRT sequence in their promoters
[9,10]. DREB1A/CBF3, DREB1B/CBF1 and DREB1C/CBF2 genes, which lie in
tandem in the Arabidopsis genome, are induced by cold but not by
dehydration or high salinity [10–12]. Overexpression of DREB1s/CBFs
induces the expression of downstream stress-responsive genes and
improves freezing, drought and high salinity tolerance in Arabidopsis
[10,13]. By contrast, DREB2A and DREB2B are not induced by cold but
are induced by dehydration, high salinity and heat shock [10,14,15].
Overexpression of a constitutive active form of DREB2A induces
expression of dehydration- or heat shock-inducible genes and improves
the drought, high salinity and heat shock tolerance of Arabidopsis
[15,16]. These findings indicate that DREB1 and DREB2 are involved
in stress-induced acclimation processes in Arabidopsis by activating
transcription via binding to the DRE/CRT sequences in the promoters
of stress-responsive target genes. To date, many stress-inducible DREBs
have been isolated from numerous plants, including eudicots such as
oilseed rape (Brassica napus) and tomato (Solanum lycopersicum) [17],
monocots such as maize (Zea mays) [18–20], rice (Oryza sativa) [21] and
barley (Hordeum vulgare) [22] and even from a moss (Physcomitrella
patens) [23]. The existence of stress-inducible DREBs in phylogenetically
divergent species indicates the importance of the DRE/CRT sequence
and DREBs in the stress response of land plants.
3. An overview of the AP2/ERF family
The AP2/ERF family is a large group of transcription factors con-
taining AP2/ERF-type DNA binding domains, and family members are
encoded by 145 loci in Arabidopsis [2] and 167 loci in rice [24]. This
domain was first found in the Arabidopsis homeotic gene APETALA 2
[25], and a similar domain was found in tobacco (Nicotiana tabacum)
ethylene-responsive element binding proteins (EREBPs) [26]. These
domains, which consist of approximately 60 residues, are closely
related [27]. AP2/ERF family proteins are plant-specific transcription
factors, and the lowest plant in which an AP2/ERF family protein
has been found is the green alga Chlamydomonas reinhardtii [28].
Sequences that are homologous to the AP2/ERF domain have been
found in bacterial and viral endonucleases, and thus, it has been
hypothesised that the AP2/ERF domain was transferred from a
cyanobacterium by endosymbiosis or from a bacterium or a virus by
other lateral gene transfer events [29]. An NMR-based structural
analysis of the Arabidopsis ERF1 protein in complex with a target DNA
molecule revealed that the AP2/ERF domain contains an N-terminal,
three-strand β-sheet that recognises a target sequence, as well as a
C-terminal α-helix [30].
3.1. Classification and phylogenetic relationships of the AP2/ERF family
transcription factors
Fig. 1 shows phylogenetic relationships of AP2/ERF family tran-
scription factors that belong to the four major subfamilies and that
include family members from Arabidopsis, Selaginella moellendorffii,
Physcomitrella patens and Chlamydomonas reinhardtii, which represent
angiosperms, lycophytes, mosses and green algae, respectively. Each
branch represents a group of homologues that are considered to be
diversified in each plant group.
87
Members of the AP2/ERF family can be divided into three
groups based on overall structure [31]. Members of the AP2 subfamily
(14 members in Arabidopsis) contain double AP2/ERF domains,
members of the RAV subfamily (6 members) contain an AP2/ERF
domain and an additional B3 DNA-binding domain, while the other
members (125 members) contain only a single AP2/ERF domain
[2,31]. The AP2 subfamily in seed plants can be further classified into
the AP2 group and the ANT group [32]. Sakuma et al. [2] analysed
the phylogenetic relationships of the 125 single-AP2/ERF members
in Arabidopsis based on the similarity of their AP2/ERF domains.
Through this analysis, the 125 single-AP2/ERF members were further
classified into three groups: the DREB subfamily (56 members; group
A), the ERF subfamily (65 members; group B) and others (4 members).
The DREB and ERF subfamilies were both divided into six subgroups, A-1
to A-6 and B-1 to B-6, respectively. DREB1/CBFs belong to subgroup A-1,
and DREB2s belong to subgroup A-2. Three of four “other” members are
similar to AP2 subfamily members and seem to have an atypical second
AP2/ERF domain, while the fourth “other” protein (AT4G13040) has a
unique type of AP2/ERF domain. Nakano et al. [33] reported a molecular
phylogenetic and motif analysis of single-AP2/ERF members in
Arabidopsis and rice. Their results are similar to the classification of
Sakuma et al. [2], but Nakano et al. also found that the A-1 and A-4
subgroups and the A-2 and A-3 subgroups share common conserved
motifs, respectively, suggesting their common origins.
The AP2/ERF family transcription factors are plant-specific. This
raises the question of how they evolved in plant history. In the
phylogenetic tree in Fig. 1, P. patens and S. moellendorffii branches are
found in all four of the major subfamilies and in a clade of AT4G13040
orthologues. Moreover, P. patens and S. moellendorffii branches are found
in all subgroups except A-1, B-2 and AP2, suggesting that they were
established in a common ancestor of extant mosses and vascular
plants. The A-1 (DREB1) subgroup is closely related to A-4. P. patens and
S. moellendorffii members in A-4 do not seem to belong to any small
groups in the A-4 subgroup, suggesting that diversification of the A-1
subgroup and these small groups may be a recent event. In fact, we could
not find any gymnosperm expressed sequence tag (EST) clones that
encoded A-1 (DREB1)-type proteins.
The tree also contains four branches of C. reinhardtii. Two branches
are in the ERF subfamily, and the other two (each of two repeats) are in
the AP2 subfamily, suggesting that the single- and double-AP2/ERF
transcription factors already existed before the emergence of land
plants. In contrast, no RAV family gene is found in C. reinhardtii. The
two C. reinhardtii branches in the ERF subfamily could not be further
classified into the six subgroups because they have no obvious similarity
other than the AP2/ERF domain. However, they have a clear signature
of ERF subfamily members in the region corresponding to the second
β-strand, which is an AAEIRD motif [2] (Supplementary Fig. S1). Because
this region is important for recognition of a target sequence [2], it may
indicate that they are able to bind to ERE-like sequences.
3.2. Binding specificities of DREB and ERF subfamily proteins
Detailed analyses of the binding preferences of DREB1A and
DREB2A demonstrate that these proteins have the highest affinity
for the DRE core sequence A/GCCGAC [2]. Furthermore, DREB1A
prefers A/GCCGACNT, and DREB2A prefers ACCGAC [16,34]. These
binding preferences were confirmed by analysing the promoters of
genes upregulated in transgenic plants overexpressing each DREB
[16,34]. In contrast, five ERF subfamily proteins display the greatest
affinity for the GCC-box sequence (AGCCGCC), which is the core
sequence of the ERE [35]. However, several exceptions have also been
reported. TINY, which belongs to subgroup A-4 of the DREB subfamily,
can bind to both the DRE and GCC-box [36] sequences. A DREB2 (A-2)
subgroup protein from barley (HvDRF1) shows a preference for
TT/AACCGCCTT [22]. ABA-INSENSITIVE 4 (ABI4) proteins from maize
and Arabidopsis, which belong to the A-3 subgroup, bind to a coupling
88 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96

DREB Chlamydomonas reinhardtii

(DREB2) A-3 A-5 subfamily Physcomitrella patens
Selaginella moellendorffii
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Fig. 1. Phylogenetic tree of the AP2/ERF family transcription factors in green plants. The NJ tree was constructed from the amino acid sequences of the AP2/ERF domain
(Supplementary Fig. S1) using the MEGA5 program based on the JTT model. A consensus tree (after 1000 bootstrap samplings) is shown, and support values are indicated on the
sides of important nodes. The scale bar indicates the substitution rate per residue. Peptide sequence information was obtained from Phytozome (Phytozome v7.0, http://www.
phytozome.net/). A branch of Chlamydomonas reinhardtii or Arabidopsis thaliana represents a homologous group conserved in green algae (two species) or angiosperms (21 species).
A branch of Physcomitrella patens or Selaginella moellendorffii represents a homologous group in each plant. The name of each branch indicates its locus identifier or transcript ID in
Phytozome. The names of each subfamily and their subgroups are given according to Sakuma et al. [2] or Shigyo and Ito [32]. R1 and R2 refer to repeat 1 and repeat 2, respectively. For
Arabidopsis branches, classifications of small groups in the DREB and ERF subfamilies are given in parentheses according to Nakano et al. [33]. *, Pp1s83_93V6.1 has a second repeat-
like sequence, but this sequence is too divergent to align with the second repeats of other AP2 subfamily proteins.

element 1 (CE1)-like sequence, CACCG, and a CCAC motif, respectively ance [39]. Transcriptome analyses of DREB1/CBF-overexpressing Arabi-
[37,38]. Such variations in binding sequences may allow complex dopsis revealed that many cold-inducible genes are upregulated in
regulation of gene expression in response to many changes in external these plants [34,40,41]. These potential target genes encode cold-
and internal environments by over 100 members of the AP2/ERF inducible proteins that function in survival at low temperatures,
family of transcription factors. including late embryogenesis abundant (LEA) proteins (i.e., hydrophilic
proteins abundantly expressed during seed maturation and in response
4. DREB1/CBF (A-1) subgroup — major regulator of to cold or dehydration) and enzymes for sugar metabolism and fatty
cold-stress responses acid desaturation. Several transcription factors are also upregulated,
suggesting the existence of transcriptional cascades that act down-
4.1. Cold-inducible DREB1s/CBFs contribute to cold tolerance by regulating stream of DREB1s/CBFs (Fig. 2). Metabolomic analyses demonstrate
many transcriptomic and metabolomic changes that cold stress induces the accumulation of many metabolites, such
as sugars, and a large portion of these metabolites also accumulate
The DREB1 subgroup of Arabidopsis consists of six members [2]. in transgenic Arabidopsis constitutively overexpressing DREB1s/CBFs
Among these, DREB1A/CBF3, DREB1B/CBF1 and DREB1C/CBF2 are rapidly [42,43]. Furthermore, a comparison between Arabidopsis ecotypes
induced in response to cold stress [9–12]. The importance of these differing in freezing tolerance revealed that the expression levels of
three cold-inducible DREB1s/CBFs in cold-stress responses has been well DREB1B/CBF1 and DREB1C/CBF2 are significantly correlated with
established. Transgenic Arabidopsis constitutively overexpressing any freezing tolerance [44]. Differences in the transcriptomes and metabo-
one of these three DREB1s/CBFs display significantly improved tolerance lomes between these ecotypes are also similar to those between wild-
to freezing, drought and high salinity [10,11,13], whereas suppression type and DREB1/CBF transgenic Arabidopsis [44]. Finally, an Arabidopsis
of DREB1A/CBF3 or DREB1B/CBF1 causes a reduction in freezing toler- quantitative trait locus (QTL) for freezing tolerance is associated with
 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96 89

the three tandem-repeated DREB1/CBF genes, where expression levels
of DREB1C/CBF2 seem to be positively correlated with freezing tolerance
[45]. Collectively, these data indicate that the cold-inducible DREB1/CBF
genes play central roles in the survival of Arabidopsis at low tem-
peratures by regulating cold-induced transcriptomic and metabolomic
changes (Fig. 2).
Cold-inducible DREB1/CBF genes have also been isolated from
numerous plant species, such as tomato, oilseed rape, wheat (Triticum
aestivum), rye (Secale cereale) [17], rice [21] and maize [19]. Further,
overexpression of Arabidopsis DREB1/CBF genes improves freezing
tolerance in oilseed rape [17] and confers chilling tolerance in tomato,
tobacco and rice [46–48]. It has been noted that major frost tolerance
QTLs in diploid wheat (Triticum monococcum) and barley map to
DREB1/CBF genes [49–51], and frost tolerance is correlated with the
expression levels of linked DREB1/CBF genes [51]. Thus, the function of
the DREB1/CBF pathway in the regulation of low-temperature stress
responses is widely conserved among angiosperm species.
4.2. Regulation of plant growth by DREB1s/CBFs
In addition to freezing tolerance, constitutive expression of
DREB1/CBF genes causes growth inhibition in Arabidopsis [10,11],
tobacco [47] and rice [48]. Because overexpression of a cold-inducible
DREB1/CBF target transcription factor gene, SALT TOLERANCE ZINC
FINGER (STZ), also causes growth inhibition [52], this gene may be
partially responsible for the growth inhibition. The mechanism of the
growth inhibition caused by DREB1/CBF expression involves down-
regulation of a gibberellin (GA) signalling pathway [53]; expression of
DREB1B/CBF1 reduces the level of active GA by stimulating the expres-
sion of genes for a GA-inactivating enzyme. The reduction in the active
GA content causes accumulation of two DELLA proteins, which are
negative regulators of GA signalling and plant growth. Indeed, over-
expression of DREB1B/CBF1 in mutants of the DELLA proteins does
not cause growth inhibition but does reduce the positive effect of
DREB1B/CBF1 on freezing tolerance. Thus, the regulation of freezing
tolerance and growth by DREB1s/CBFs under low temperatures involves
hormonal actions.
4.3. Regulation at the transcript level is important for DREB1/CBF activity
As described above, DREB1/CBF genes are stress-inducible, and their
overexpression results in transcriptomic and metabolomic changes, as
well as elevated freezing tolerance. Differences in freezing tolerance
between ecotypes or cultivars are correlated with the expression levels
of DREB1/CBF genes. Thus, regulation of their expression level is a major
regulatory mechanism for DREB1/CBF activity. The expression of
DREB1s/CBFs is induced by cold stress, but it is now clear that it is also
regulated by the circadian clock (Fig. 2).

Cold 4.3.1. Transcriptional regulation of DREB1s/CBFs by low temperature

HOS1 signals
Comparison of the promoters of the three cold-inducible
Ub Light DREB1/CBF genes demonstrates that they share six common conserved
Ub
ICE1
motifs, designated Box I through Box VI [12]. Zarka et al. [54] analysed
Circadian clock [Ca2+]cyt the DREB1C/CBF2 promoter and identified Box IV and part of Box VI
Unstable
Night PhyB as essential elements for cold inducibility of this promoter; they
SIZ1 ?
TOC1 (Pfr) designated these elements as Induction of CBF Expression region 1 and 2
CaM ? (ICEr1 and ICEr2), respectively. Doherty et al. [55] compared the
SUMO
CCA1
sequences of the promoters of DREB1C/CBF2 and another cold-inducible
ICE1 PIF7 CAMTA3
Day LHY gene, ZAT12, and found conserved motifs CM1 through CM7. CM2,
Stable/Active which overlaps with ICEr2, is sufficient for cold inducibility. They also
MYB15 found that a MYC-binding motif (CANNTG) within ICEr1 is a positive
regulator of DREB1C/CBF2 expression. An inducer of cbf expression 1
DREB1A DREB1B/C (ice1) mutant was isolated, which exhibits poor cold inducibility of the
Myb Myc EE EE G-box CGCG
(CBF3) (CBF1/2) DREB1A/CBF3 gene [56]. The responsible ICE1 gene encodes a MYC-like
basic helix-loop-helix (bHLH) transcription factor that can bind to
MYC-binding motifs in the DREB1A/CBF3 promoter, including the motif
DREB1A DREB1B/C in Box IV. ICE1 is constitutively expressed in every tissue [56], suggesting
(CBF3) (CBF1/2) activation of the ICE1 protein in response to low-temperature signals.
Active Active
The stability of ICE1, expression of DREB1/CBF genes and freezing
tolerance are negatively regulated via ubiquitination by a RING finger E3
ligase, HOS1 [57,58]. Miura et al. [59] found that sumoylation of ICE1 by
SIZ1, a SUMO E3 ligase, inhibits polyubiquitination of ICE1. Repressed
DRE/CRT Cold-inducible genes
cold-inducible expression of DREB1A/CBF3 and reduced freezing
tolerance in siz1 mutants indicate the importance of sumoylation for
TFs the function of ICE1 and freezing tolerance of Arabidopsis [59]. Ser403
Transcriptomic changes is a key amino acid in ICE1, and its substitution results in enhanced
ubiquitination and transactivation activity, suggesting post-translational
regulation of ICE1 at this residue, though no modifications of this
residue have been identified [60]. Despite accumulating reports on the
Cold-stress responses regulations and functions of ICE1 at low temperature, the effect of the
ice1 mutation on the cold inducibility of DREB1B/CBF1 and DREB1C/CBF2
Fig. 2. Transcriptional regulation of cold-inducible DREB1s/CBFs in Arabidopsis. The is small [56], indicating the involvement of other factors. For instance,
activity of DREB1s/CBFs is mainly regulated at the transcriptional level. The MYB15 was isolated as an ICE-interacting protein. MYB15 binds to
transcription of DREB1s/CBFs is under the control of low-temperature signals and MYC recognition sites in DREB1/CBF promoters. Although MYB15 is a
circadian/light signals. Expressed DREB1/CBF proteins bind to DRE/CRT and activate transcriptional activator, its overexpression and loss of function reduce
transcription of target cold-inducible genes, including transcription factors, and thus
bring about transcriptomic changes, which eventually cause cold stress responses. Ub,
and increase transcript levels of DREB1/CBF genes, respectively [61].
ubiquitin; CaM, calmodulin; Myb, Myb-recognition sequence; MYC, MYC-recognition Doherty et al. [55] identified a calmodulin-binding transcription
sequence; EE, evening element; CGCG, CGCG-Box; TFs, transcription factors. activator (CAMTA) protein, CAMTA3, as a protein that can bind to CM2
90 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96
in the DREB1C/CBF2 promoter. A camta3 mutation results in reduced
transcript levels of DREB1C/CBF2 and DREB1B/CBF1 but not DREB1A/
CBF3. A rapid transient elevation of cytosolic Ca 2+ concentration
occurs upon exposure to cold and is involved in cold-induced gene
expression in Arabidopsis [62,63]. Perturbation of Ca 2+ homeostasis
by mutations in a vacuolar Ca 2+/H + antiporter gene, CALCIUM
EXCHANGER 1 (CAX1), positively affects cold inducibility of CBF/DREB1
and its downstream genes and then enhances acquired freezing
tolerance [64]. Therefore, Ca 2+-regulated proteins such as CAMTA
may act as mediators of calcium signalling in cold responses. Thus, a
portion of the mechanism that positively regulates expression levels
of DREBI/CBF genes at low temperatures has now been elucidated.
However, further studies are needed to understand how cold stress
signals provoke induction of DREB1/CBF genes.
4.3.2. Transcriptional regulation of DREB1s/CBFs by the circadian clock
The transcript levels of DREB1/CBF genes oscillate according to a
circadian rhythm: high after subjective dawn and low in subjective
evening [34,65,66]. In addition, peak transcript levels of DREB1/CBF
genes are high and low when plants are exposed to low temperature
after subjective dawn and subjective evening, respectively, indicating
that cold induction of DREB1/CBF genes is circadian-gated [67].
Kidokoro et al. [68] found that a G-box sequence (CACGTG) in Box V
of the DREB1C/CBF2 promoter is involved in this circadian regulation.
They isolated a bHLH transcription factor, PHYTOCHROME-INTER-
ACTING FACTOR 7 (PIF7), as a key negative regulator that binds to the
G-box sequence. PIF7 interacts with TIMING OF CAB EXPRESSION 1
(TOC1) and PHYTOCHROME B (PhyB), which are components of the
circadian oscillator and a red light photoreceptor, respectively [69],
and these interactions enhance activity of PIF7 as a transcriptional
repressor of the DREB1C/CBF2 promoter. Analysis of a pif7 mutant
revealed that PIF7 represses expression of the DREB1A/CBF3 and
DREB1C/CBF2 genes during subjective night. Dong et al. [70] found
that the promoter regions of the three cold-inducible DREB1/CBF
genes have several evening elements (EEs; AAAATATCT) [65] and
CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) binding sites (AATCT) [71].
These are target sequences for the MYB-related transcription factors
CCA1 and LATE ELONGATED HYPOCOTYL (LHY), which are compo-
nents of the central oscillator [69,72]. They detected binding of CCA1
to DREB1/CBF promoter regions. Furthermore, cold induction of the
DREB1/CBF genes is dramatically reduced in the cca1-11 lhy-21 double
mutant. Therefore, CCA1 and LHY are positive regulators of DREB1/CBF
cold induction. Conversely, Nakamichi et al. [73] demonstrated that in
a triple mutant of PSEUDO RESPONSE REGULATOR 9 (PRR9), PRR7 and
PRR5, which are genes encoding other components of the circadian
clock mechanism, the expression levels of DREB1s/CBFs are constantly
higher than in the wild type, and cold induction of the DREB1/CBF
genes is always effective regardless of when cold treatment begins.
This suggests that these PRRs are negative regulators in the circadian
control of DREB1/CBF expression at ambient temperature and during
cold induction. Collectively, these findings illustrate how the circadian
clock regulates the expression levels of DREB1/CBF genes under warm
and cold conditions. Expression of tomato DREB1/CBF genes also
demonstrates diurnal changes in a pattern similar to that of Arabidopsis
DREB1s/CBFs [74]. Such circadian regulation of DREB1/CBF genes may
allow effective protection of plants against low temperature stresses by
avoiding excess expression of DREB1s/CBFs that can result in negative
effects on plant growth.
4.4. Non-cold-inducible DREB1s/CBFs also function in abiotic stress responses
Arabidopsis has three DREB1/CBF genes other than the three cold-
inducible DREB1s/CBFs: DREB1D/CBF4, DREB1E/DWARF AND DELAYED-
FLOWERING (DDF2) and DREB1F/DDF1 [2,75,76]. These three DREB1/CBF
genes are not cold-inducible. However, DREB1D/CBF4 is dehydration-
and ABA-inducible, and DREB1F/DDF1 is high salinity-inducible
[2,75,76]. Transgenic Arabidopsis overexpressing DREB1D/CBF4 display
growth retardation, expression of cold- and dehydration-inducible
DREB1/CBF target genes, and freezing and drought tolerance [75].
Overexpression of DREB1F/DDF1 results in a dwarf phenotype, expres-
sion of the RD29A gene and improved tolerance to high salinity [76].
DREB1F/DDF1-overexpressing plants display reduced levels of active
GA, while the dwarf phenotype and high-salinity tolerance are restored
by application of active GA. These findings suggest that these non-cold-
inducible DREB1s/CBFs can function in water stress adaptation by
regulating downstream gene sets that are similar to those of the cold-
inducible DREB1s/CBFs.
5. DREB2 subgroup — dual functions in dehydration and heat
shock responses
The DREB2 subgroup is composed of eight members in Arabidopsis
[2] and five in rice [77]. Phylogenetic analyses of DREB2s from
Arabidopsis and rice, as well as related proteins, revealed that the
DREB2 subgroup is closely related to the A-3 subgroup, which con-
tains ABI4, and the DREB2 subgroup can be further divided into
three subtypes that are conserved between Arabidopsis and rice [77].
These findings are supported by our large-scale phylogenetic analysis
using genomic information from 21 angiosperm species (J. Mizoi,
unpublished result). DRE-binding proteins that belong to the DREB2
subgroup have been isolated from a number of other angiosperm
species, including eudicot species such as sunflower (Helianthus
annuus) [78] and grass species such as rice [21,77], wheat [79,80],
barley [22] and maize [20]. Most of the DREB2 genes that have been
reported are responsive to water stress or heat shock, but DREB2
genes from grass species are also responsive to cold stress [77,79,80].
Interestingly, all isolated stress-responsive DREB2s belong to
subtype 1, which contains DREB2A, B, C, E and H of Arabidopsis, as
well as OsDREB2A and OsDREB2B of rice, indicating that subtype 1
represents the major stress-inducible DREB2s. Subtype 2 contains
Arabidopsis DREB2D, DREB2G and rice OsDREB2C, whereas subtype 3
contains Arabidopsis DREB2F and rice OsDREB2E. DREB2 genes in
these two subtypes are not (or only slightly) induced by stress [2,77]
and have not been isolated from other plants by means of cDNA
screening. Although all 21 of the angiosperm species have genes
for these two subtypes (J. Mizoi, unpublished result), their roles are
unknown.
5.1. DREB2s are important transcriptional regulators in water- and high
temperature-stress conditions
In Arabidopsis, DREB2A and DREB2B are induced by dehydration,
high salinity and heat [2,10,14,15] (Fig. 3), while DREB2C is induced
by heat later than DREB2A or DREB2B [81]. DREB2C, DREB2D and
DREB2F are high salinity-inducible and DREB2E is ABA-inducible,
though their expression is very weak [2]. Therefore, DREB2A and
DREB2B seem to be major DREB2s involved in dehydration-
inducible gene expression through DRE/CRT in an ABA-independent
pathway [2,14]. In contrast to DREB1s/CBFs, ectopic overexpression
of DREB2A does not cause significant changes in plant growth,
gene expression or stress tolerance [10], which suggests post-
translational regulation of the DREB2A protein. Sakuma et al. [16]
found that DREB2A has a negative regulatory domain (NRD)
adjacent to the DNA-binding domain, and deletion of NRD converts
DREB2A into a constitutive active form (DREB2A CA). Transgenic
plants constitutively expressing DREB2A CA display stunted growth
and improved tolerance to drought and high salinity but not to
freezing. Many dehydration-responsive genes, such as those
encoding LEA proteins, are induced in these transgenic plants. In
addition, many heat shock-responsive genes are also upregulated,
and indeed, the transgenic plants exhibit enhanced tolerance to
heat shock [15]. Partial reduction of dehydration- or heat shock-
 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96 91

Normal condition Heat Drought

Basal expression HsfA1

ABA-independent
DREB2A HSE ? DREB2A
ABA

ABA-dependent
DREB2A DREB2A
Inactive/Unstable Inactive/Unstable

Ub ? P AREB/
DREB2A
Ub HSF DREB2A ABF
? ?
DRIP1/2 Active/Stable

Ubiquitination

HSE DRE DRE ABRE

Degradation
Heat- DRE DRE Drought-
inducible inducible
genes genes

Transcriptomic changes Transcriptomic changes

Heat-stress responses Drought-stress responses

Fig. 3. Transcriptional and post-translational regulation of DREB2A in Arabidopsis. The activity of DREB2A is regulated at both transcriptional and post-translational levels. Under
normal conditions, the DREB2A protein expressed at a basal level is degraded via the ubiquitin-proteasome pathway. The transcription of the DREB2A gene is activated independently
by heat shock or dehydration. The expressed DREB2A protein is further stabilised and/or activated by stress signals. Although the mechanism for this post-translational regulation is
unclear, protein modifications may be involved in this process. DREB2A activates expression of target genes in a stress-specific manner via DRE/CRT sequences in the promoters.
Heat- and drought-inducible genes' promoters often contain heat shock elements (HSEs) and ABA-responsive elements (ABREs), respectively. Interaction between DRE/CRT and
these cis-elements has been suggested. Such interactions may involve interactions between transcription factors.

responsive gene expression in dreb2a mutants indicates that
DREB2A plays important roles in gene expression in response to
dehydration and heat shock [15] (Fig. 3.).
5.2. Post-translational regulation of DREB2A activity
Post-translational regulation of DREB2A minimally involves stabi-
lisation of the protein (Fig. 3). The NRD in DREB2A is a Ser/Thr-rich
hydrophilic sequence and meets the criteria for being a PEST sequence,
which is a sequence motif found in unstable proteins from eukaryotes
[82,83]. Under non-stressful conditions, green fluorescent protein
(GFP)-fused DREB2A CA accumulates more stably in the nucleus
compared with GFP-fused wild-type DREB2A [16]. Qin et al. [84]
isolated DREB2A-INTERACTING PROTEINS 1 and 2 (DRIP1 and DRIP2),
which are homologous RING E3 ligases that interact with DREB2A in
the nucleus and can mediate ubiquitination of DREB2A. Overexpres-
sion of DRIP1 delays dehydration-responsive expression of genes
downstream of DREB2A. In contrast, dehydration responsiveness of
genes downstream of DREB2A is significantly enhanced in a drip1 drip2
double mutant. In addition, stable accumulation of the GFP-fused wild-
type DREB2A protein in the nucleus occurs even under non-stressful
conditions in the drip1 drip2 double mutant background or under
treatment with a proteasome inhibitor. Therefore, it is hypothesised
that DRIP1 and DRIP2 act as negative regulators of dehydration-
responsive gene expression by facilitating ubiquitination and degra-
dation of DREB2A (Fig. 3). However, it is not clear whether the amount
of the DREB2A protein itself is the key factor in activating transcription
of downstream genes or whether further activation of DREB2A is
required.
Phosphorylation has been suggested as another post-translational
regulatory mechanism for PgDREB2A, which is a DREB2 subgroup
protein isolated from a grass plant, pearl millet (Pennisetum glaucum)
92 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96
[85]. A whole-cell extract of pearl millet can phosphorylate a recom-
binant PgDREB2A protein expressed in Escherichia coli. The phosphor-
ylation, which seems to occur at Thr residue(s), reduced binding affinity
of the PgDREB2A protein to a DRE/CRT sequence. Although the phos-
phorylation site has not been specified, this finding suggests the
possibility of phosphorylation as a post-translational regulatory mech-
anism for DREB2s.
5.3. Post-transcriptional regulation of DREB2s in grass species by
alternative splicing
In grass plants, DREB2 orthologues are regulated by alternative
splicing. Stress-inducible (subtype 1) DREB2s in grass plants are divided
into two clades: OsDREB2A orthologues and OsDREB2B orthologues
[77]. Regulation by alternative splicing was found for genes in the latter
clade in barley (HvDRF1) [22], wheat (Wdreb2) [80], maize (ZmDREB2A)
[20] and rice (OsDREB2B) [77]. These genes have two types of tran-
scripts: one is an inactive transcript that has a stop codon before the
DNA-binding domain, and the other is an active transcript that can
encode a full-length protein. Generally, inactive transcripts are dom-
inantly expressed under non-stressful conditions. Active transcripts
accumulate in response to stress signals in higher induction ratios than
do inactive transcripts. The alternative splicing of DREB2 genes in grass
species may allow rapid stress responses by skipping the activation
steps of the DREB2 promoter or by providing a pool of premature DREB2
mRNAs.
5.4. DREB2 is a component of a transcriptional cascade in heat-shock
responses
In Arabidopsis, DREB2A is an element of a transcriptional cascade
in heat-shock responses. DREB2A is rapidly expressed under heat
shock and induces many heat shock-responsive genes, such as
transcription factors and molecular chaperones [15]. Knockout
mutants of DREB2A display reduced expression of these genes and
are sensitive to heat shock [15]. One of the important target genes of
DREB2A is HEAT SHOCK TRANSCRIPTION FACTOR A3 (HsfA3), a member
of the HSF family, which is composed of 21 members in Arabidopsis
[86]. Overexpression of HsfA3 induces many heat-shock-responsive
genes, whereas disruption of the HsfA3 gene results in reduced
induction of heat shock-responsive genes and impaired heat shock
tolerance [87,88]. Recently, it has been reported that induction of
DREB2A is exclusively regulated by a group of HsfA1 genes [89] (Fig. 3).
It is now thought that heat shock provokes activation of HsfA1
proteins, which induce transcription of DREB2A and another impor-
tant transcription factor gene, HsfA2 [90]. DREB2A then activates the
promoter of HsfA3, and the expressed HsfA3 protein further induces
downstream genes (Fig. 3). Because the active transcript of rice
OsDREB2B displays an expression pattern similar to that of DREB2A
under heat shock [77] and several HSFs in rice have DRE/CRT in their
upstream regions, a similar transcriptional cascade may be widely
distributed in angiosperms.
5.5. Function of DREB2s in combination-stress conditions
Drought and high temperature are closely related stress condi-
tions in natural environments and often occur in combination [91].
The combination of drought and high temperature affects the
physiology of plants more severely than single stresses [91–93].
Physiological, transcriptomic and metabolomic analyses in tobacco
and Arabidopsis have shown that plant responses to a combination
of drought and high temperature partially overlap with, but are
distinct from, those against a single stress [92,93]. DREB2A is induced
by drought or heat shock alone but also by a combination of drought
and heat shock [93], suggesting that DREB2 genes play a role in
regulating gene expression under various combinations of drought
and heat stresses.
6. Functional relationships between DREB1s/CBFs, DREB2s and other
transcription factors
6.1. Differences in binding specificity of DREB1A/CBF3 and DREB2A may
be a key for stress-specific expression of DRE/CRT-regulated genes in
Arabidopsis
In Arabidopsis, overexpression of DREB1/CBF genes improves
tolerance to dehydration and freezing [10,19,21,94]. In contrast, over-
expression of DREB2 genes significantly enhances tolerance to dehy-
dration but not to freezing [16,20]. Maruyama et al. [43] demonstrated
that genes downstream of DREB1A/CBF3 and DREB2A CA partially
overlap with each other. However, genes of several functions are more
specific to each DREB: genes that encode enzymes for carbohydrate
metabolism are specific to DREB1A, while those encoding molecular
chaperones are specific to DREB2A CA [43]. Metabolite profiling
revealed that metabolite accumulation patterns are similar between
DREB1A-overexpressing plants and cold-treated plants, as well as be-
tween DREB2A CA-overexpressing plants and dehydration-treated
plants [43], suggesting that these differences in metabolite accumula-
tion are responsible for the difference in stress tolerance. Maruyama
et al. [34] and Sakuma et al. [16] demonstrated that DREB1A and
DREB2A have preferences for A/GCCGACNT and ACCGAC, respectively,
both in vitro and in planta. Because freezing of plants induces cellular
dehydration, it is reasonable that several protection systems that are
common between cold- and drought-stress responses, such as LEA
proteins, are regulated via a common DRE/CRT (e.g., ACCGACNT) and
induced by cold- and dehydration-stress signals. Conversely, genes
that function in specific stress conditions may be regulated by specific
DRE/CRT sequences. Such slight differences in or around DRE/CRTs,
together with regulation by a number of DREB subfamily genes, may
improve the fitness of plants against a variety of conditions in natural
environments.
6.2. Functional interactions of DREB1s/CBFs and DREB2s with other
transcription factors
DRE/CRT interacts with other cis-acting elements, and DREBs interact
with other transcription factors (Fig. 3). In the RD29A promoter, DRE/CRT
has a positive relationship with ABRE and functions as a coupling
element of ABRE [95]. Transactivation of an RD29A promoter fragment
that contains both DRE/CRT and ABRE is synergistically enhanced by co-
expression of DREB1A/CBF3 or DREB2A with ABRE-BINDING PROTEIN
1/ABRE-BINDING FACTOR 2 (AREB1/ABF2) or AREB2/ABF4 [95]. The
DREB1A, DREB2A and DREB2C proteins specifically interact with the
AREB1/ABF2 and AREB2/ABF4 proteins in yeast and/or in vitro [96].
Similarly, DRE/CRT and heat shock elements (HSEs) cooperatively
function in the promoter of a small heat shock protein in developing
sunflower embryos [78]. A DREB2-type protein, HaDREB2, and an
HSF, HaHSFA9, bind to this promoter and mediate synergistic
transactivation. HaDREB2 and HaHSFA9 interact in vitro, where the
AP2/ERF domain of HaDREB2 and the C-terminal region of HaHSFA9
containing the activation domain, respectively, were identified as
interacting regions for each protein.
7. Functions of other DREB subfamily members
Although DREB1s/CBFs and DREB2s are well characterised as major
positive regulators of stress responses, other DREB subgroup genes have
also been reported to be stress-responsive and/or confer stress tolerance
in transgenic plants. Here, we briefly summarise the current under-
standing of these genes.
 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96
7.1. ABI4 (A-3) subgroup — related to the DREB2 subgroup but with
distinct functions
The A-3 subgroup consists of ABI4 and its orthologues. Although
A-3 members are related to DREB2s [33,77], they have specialised
functions in ABA, sugar and retrograde signalling [37,38,97,98]. In
contrast, an abi4 mutant displays salt insensitivity in the germination
and seedling stages [99]. As described above, reported binding
sequences are distinct to DRE/CRT [37,38]. Thus, A-3 genes may be
involved in stress response via a pathway different from the DREB2
pathway.
7.2. A-4 subgroup — related to the DREB1 subgroup but with unclear
function(s)
The A-4 subgroup is related to DREB1s/CBFs. According to Nakano
et al. [33], DREB1s/CBFs and A-4 share a conserved motif and are
included in the same group. Generally, A-4 genes do not have high
stress inducibility [18,100]. Overexpression of the TINY [101] or
HARDY [100] gene results in stunted growth and thick leaves.
Overexpression of HARDY also changes the structure and architecture
of roots and confers drought tolerance. TINY binds to both DRE/CRT
and GCC-box motifs [36]. The functions of A-4 genes are not clear, but
they may play several roles in configuring stress responses.
7.3. A-5 subgroup — including transcriptional repressors that act
downstream of DREB1s/CBFs and DREB2s
The A-5 subgroup contains stress-inducible genes in Arabidopsis
[34,41,102], cotton (Gossypium hirsutum) [103] and soybean (Glycine
max) [104,105]. Interestingly, this subgroup contains a clade of
proteins that have a functional ERF-associated amphiphilic repression
(EAR) motif [106] at their C-terminus [33,103,104]. Moreover, genes
encoding these EAR motif-containing proteins are upregulated in
transgenic plants overexpressing DREB1A/CBF3 or DREB2A CA [15,34].
Overexpression of these genes results in reduced expression of
DREB1/CBF and DREB2 target genes under cold and dehydration,
respectively [102,107]. Conversely, mutations in one of genes
encoding these EAR motif-containing proteins (RELATED TO AP2.1;
RAP2.1) result in increased expression of these target genes and
in improved tolerance to drought and freezing [107]. Thus, it is
possible that EAR motif-containing A-5 subgroup proteins function in
negative feedback regulation of the DREB1/CBF and DREB2 pathways.
Another interesting gene in the A-5 subgroup is PpDBF1, which is
from the moss Physcomitrella patens [23]. PpDBF1 is weakly induced
by high salinity, dehydration, cold, salicylic acid and hydrogen peroxide
but is strongly induced by ABA. PpDBF1 can bind to DRE/CRT, and its
overexpression induces expression of stress-responsive genes and
reduces sensitivity to high salinity or high osmolality in transgenic
tobacco. These results suggest that the regulation of expression of
stress-responsive genes by DREBs via DRE/CRT was established in
ancient land plants.
7.4. A-6 subgroup — involved in stress responses in ways different from
those of DREB1s/CBFs and DREB2s
The A-6 subgroup also contains stress-inducible genes. RAP2.4 and
RAP2.4B (AT1G22190) are expressed in response to dehydration, high
salinity and heat [108,109]. RAP2.4 and RAP2.4B are also responsive to
cold and heat shocks, respectively. Microarray analyses of a RAP2.4
overexpressor and a double mutant of RAP2.4 and RAP2.4B identified
multiple aquaporin genes as their targets, suggesting their role in
water homeostasis [109]. RAP2.4A (AT1G36030) was identified
as a protein that binds to a redox state-responsive promoter of
an anti-oxidant enzyme, 2-Cys peroxiredoxin [110]. The binding
sequence in this promoter is a CE3-like sequence, CACGCGATTC
93
[110], while RAP2.4 binds to both the DRE/CRT and GCC-box [108].
The activity and DNA-binding affinity of the RAP2.4A protein is
regulated by redox state. A rap2.4a mutant displays slightly lower
expression of redox-regulated nuclear genes encoding chloroplast
proteins and higher sensitivity to high light than the wild type. These
findings suggest that A-6 proteins function in stress responses,
but their target genes are different from those of DREB1s/CBFs and
DREB2s.
8. ERF subfamily members in abiotic stress responses
Submergence is a stress condition that inhibits the gas exchange
of plants, thereby impairing photosynthesis and respiration. The process
of acclimation to submergence includes the synthesis of ethylene [111].
Submergence 1A (Sub1A) is a rice ERF subfamily B-2 subgroup protein
that was identified by cloning Sub1, a major QTL for submergence
tolerance [112,113]. A Sub1A allele from a submergence-tolerant
cultivar confers tolerance of submergence to an intolerant cultivar.
Sub1A is responsive to submergence and ethylene, and it negatively
regulates cell elongation and carbohydrate consumption [112,113].
Recently, Sub1A was shown to regulate the expression of other AP2/ERF
family transcription factors during submergence [114]. Multiple genes
in the B-1 and B-2 subgroups of the ERF subfamily, and in the A-1
(DREB1/CBF) subgroup of the DREB subfamily, are upregulated by
Sub1A, while a B-2 subgroup gene is down-regulated. In deepwater rice,
SNORKEL1 (SK1) and SK2, which are related to Sub1A, play central roles
in ethylene-responsive internode elongation in response to submer-
gence [115]. Thus, the Sub1A and SK proteins regulate plant elongation
in opposite ways to tolerate flash or long-term flooding. In Arabidopsis,
the importance of a B-2 subgroup gene, RAP2.2, in hypoxia has been
demonstrated [116]. RAP2.2 is induced by hypoxia in an ethylene-
dependent manner. Overexpression and knocking-out of the RAP2.2
gene results in improved and impaired survival under hypoxia,
respectively. Four transcription factors are downstream of RAP2.2:
two (ERF71 and ERF73) are subgroup B-2 genes, and one (ERF4) is a
subgroup B-6 gene. These findings in rice and Arabidopsis indicate
that a transcriptional cascade of the B-2 subgroup and its downstream
AP2/ERF genes plays a central role in survival under hypoxia or sub-
mergence conditions.
Although ERF subfamily transcription factors are generally consid-
ered to be mediators of ethylene-related responses, they include
members that respond to abiotic stresses, such as drought and high
salinity [35,117], and can confer tolerance to these stresses by over-
expression in transgenic plants [117]. Genome-wide expression anal-
yses of AP2/ERF family genes in poplar (Populus trichocarpa) [118],
soybean [119], tomato [120] and rice [24] reveal that many ERF sub-
family genes are also induced by low or high temperature, dehydra-
tion or high salinity. Although the functions of abiotic stress-inducible
ERF subfamily genes in abiotic stress responses are largely unknown,
they are expected to be involved in gene regulation under stress
conditions in both ethylene-dependent and -independent ways.
Recently, it was reported that osmotic stress causes cell-cycle arrest,
and ethylene signalling is important for this process [121]. When
Arabidopsis plants are exposed to mild (non-lethal) osmotic stress,
ethylene production is facilitated and ethylene-signalling-related genes,
including a number of ERF genes (B-1 subgroup: ERF11; B-3 subgroup:
ERF1, ERF2, ERF5 and ERF6), are induced. Although genes downstream
of these ERFs in this process are unknown, this observation suggests
one of the important roles of ERFs in an aspect of plant adaptation to
abiotic stress conditions.
9. Conclusion and perspective
To date, many AP2/ERF family transcription factors from various
plant species have been shown to be involved in abiotic stress
responses. Namely, the DREB1/CBF and DREB2 subgroups play central
94 J. Mizoi et al. / Biochimica et Biophysica Acta 1819 (2012) 86–96
roles in the acquisition of stress tolerance by regulating the expression
of gene sets via DRE/CRT sequences in promoters of stress-inducible
genes. The functions of other AP2/ERF family transcription factors in
stress responses have also become clear. These transcription factors
exhibit a variety of stress-related inducibilities, tissue specificities,
DNA-binding specificities and transactivation activities. Thus, AP2/ERF
family transcription factors, by cross-talking with each other, are
likely to regulate the developmental, physiological and biochemical
responses of plants to a variety of environmental stress conditions,
including those occurring in combination with other abiotic and biotic
stresses.
The activity of DREB1s/CBFs is mainly regulated at the transcriptional
level. In Arabidopsis, expression of three cold-inducible DREB1s/CBFs is
under the control of low temperature and circadian signals. Transcrip-
tion factors that are involved in transcriptional control of these genes
have been isolated, but further investigation of their regulation and
functional interactions, together with identification of new factors, is
needed to understand how plants respond to low temperature signals.
In contrast, the activity of DREB2s is regulated by multiple steps.
In addition to transcriptional activation, alternative splicing is important
in grass species, whereas post-translational stabilisation and activation
are required in Arabidopsis. It is necessary to clarify the mechanisms
of these regulatory steps to understand how DRE/CRT-mediated
gene expression is regulated under water and heat stress conditions.
Furthermore, interactions between DRE/CRT and other cis-acting
elements, as well as interactions between AP2/ERF transcription factors
and other proteins, will be important research subjects.
Supplementary materials related to this article can be found online
at doi:10.1016/j.bbagrm.2011.08.004.
Acknowledgement
We thank Dr. Satoshi Kidokoro for critical comments on this
manuscript and Dr. Kyonoshin Maruyama for suggestions in preparing
the phylogenetic tree. This work is supported by a Grant-in-Aid for
Young Scientists (B) (21780314), a Grant-in-Aid for Scientific Research
on Innovative Areas (22119004), the Targeted Proteins Research
Program (TPRP) of the Ministry of Education, Culture, Sports, Science,
and Technology (MEXT) of Japan and the Program for the Promotion of
Basic Research Activities for Innovative Biosciences (BRAIN) of Japan.
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