Regulation and functional analysis of ZmDREB2A in response to drought and heat stresses in Zea mays L.

Abstract

DREB1/CBFs and DREB2s are transcription factors that specifically interact with a cis-acting element, DRE/CRT, which is involved in the expression of genes responsive to cold and drought stress in Arabidopsis thaliana. The function of DREB1/CBFs has been precisely analyzed and it has been found to activate the expression of many genes responsive to cold stress containing a DRE/CRT sequence in their promoters. However, the regulation and function of DREB2-type transcription factors remained to be elucidated. In this research, we report the cloning of a DREB2 homolog from maize, ZmDREB2A, whose transcripts were accumulated by cold, dehydration, salt and heat stresses in maize seedlings. Unlike Arabidopsis DREB2A, ZmDREB2A produced two forms of transcripts, and quantitative real-time PCR analyses demonstrated that only the functional transcription form of ZmDREB2A was significantly induced by stresses. Moreover, the ZmDREB2A protein exhibited considerably high transactivation activity compared with DREB2A in Arabidopsis protoplasts, suggesting that protein modification is not necessary for ZmDREB2A to be active. Constitutive or stress-inducible expression of ZmDREB2A resulted in an improved drought stress tolerance in plants. Microarray analyses of transgenic plants overexpressing ZmDREB2A revealed that in addition to genes encoding late embryogenesis abundant (LEA) proteins, some genes related to heat shock and detoxification were also upregulated. Furthermore, overexpression of ZmDREB2A also enhanced thermotolerance in transgenic plants, implying that ZmDREB2A may play a dual functional role in mediating the expression of genes responsive to both water stress and heat stress.

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Regulation and functional analysis of ZmDREB2A in response
to drought and heat stresses in Zea mays L
Feng Qin1, Masayuki Kakimoto2, Yoh Sakuma1,2, Kyonoshin Maruyama1, Yuriko Osakabe1,2, Lam-Son Phan Tran1, Kazuo
Shinozaki3,4and Kazuko Yamaguchi-Shinozaki1,2,4,*
1Biological Resources Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba,
Ibaraki 305-8686, Japan,
2Laboratory 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,
3RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan, and
4Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 4-1-8 Honcho,
Kawaguchi, Saitama 332-0012, Japan
Received 4 August 2006; revised 2 November 2006; accepted 20 November 2006.
*For correspondence (fax +81 29 838 6643; e-mail kazukoys@jircas.affrc.go.jp).
Summary
DREB1/CBFs and DREB2s are transcription factors that specifically interact with a cis-acting element, DRE/
CRT, which is involved in the expression of genes responsive to cold and drought stress in Arabidopsis
thaliana. The function of DREB1/CBFs has been precisely analyzed and it has been found to activate the
expression of many genes responsive to cold stress containing a DRE/CRT sequence in their promoters.
However, the regulation and function of DREB2-type transcription factors remained to be elucidated. In this
research, we report the cloning of a DREB2 homolog from maize, ZmDREB2A, whose transcripts were
accumulated by cold, dehydration, salt and heat stresses in maize seedlings. Unlike Arabidopsis DREB2A,
ZmDREB2A produced two forms of transcripts, and quantitative real-time PCR analyses demonstrated that
only the functional transcription form of ZmDREB2A was significantly induced by stresses. Moreover, the
ZmDREB2AproteinexhibitedconsiderablyhightransactivationactivitycomparedwithDREB2AinArabidopsis
protoplasts, suggesting that protein modification is not necessary for ZmDREB2A to be active. Constitutive or
stress-inducible expression of ZmDREB2A resulted in an improved drought stress tolerance in plants.
Microarray analyses of transgenic plants overexpressing ZmDREB2A revealed that in addition to genes
encoding late embryogenesis abundant (LEA) proteins, some genes related to heat shock and detoxification
were also upregulated. Furthermore, overexpression of ZmDREB2A also enhanced thermotolerance in
transgenic plants, implying that ZmDREB2A may play a dual functional role in mediating the expression of
genes responsive to both water stress and heat stress.
Keywords: transcription factor, DRE/CRT, ZmDREB2A, drought tolerance, heat stress.
Introduction
Plant growth and productivity are affected by various abiotic
stresses such as heat, cold, drought and high salinity, and
plants must respond and adapt to these stresses in order to
survive. Exposure to these stresses induces various bio-
chemical and physiological changes in the process of
acquiring stress tolerance. A number of genes have been
described that respond to these stresses at the transcrip-
tional level (Bartels and Sunkar, 2005; Thomashow, 1999;
Yamaguchi-Shinozaki and Shinozaki, 2006; Zhu, 2002). The
cis- and trans-acting factors involved in the expression of
stress-responsive genes have been extensively analyzed as
a means to elucidate the molecular mechanisms of gene
expression in response to the stresses of cold, drought and
high-salinity (Yamaguchi-Shinozaki and Shinozaki, 2005).
The dehydration-responsive element (DRE) containing the
core sequence A/GCCGAC was identified as a cis-acting
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promoter element which regulates gene expression in re-
sponse to drought, high salinity and cold stresses in Ara-
bidopsis (Yamaguchi-Shinozaki and Shinozaki, 1994). A
similar motif was identified as the CRT (C-repeat) and LTRE
(low-temperature-responsive element) in cold-inducible
genes (Baker et al., 1994; Jiang et al., 1996). Complementary
DNAs encoding DRE-binding proteins, CBF/DREB1s and
DREB2s, have been isolated (Liu et al., 1998; Stockinger
et al., 1997) and their corresponding gene products showed
significant sequence similarity to the conserved DNA-bind-
ing domain found in ERF/AP2 proteins. Functional analyses
ofDREB1/CBFtranscriptionfactors haveestablishedthemas
important components of the cold stress response in Ara-
bidopsis (Jaglo-Ottosen et al., 1998; Liu et al., 1998). In
Arabidopsis DREB1/CBF genes are induced by cold stress
and theirgene productsactivatethe expressionof >40 genes
in the DREB1/CBF regulon (Fowler and Thomashow, 2002;
Maruyama et al., 2004). The expression of this regulon re-
sults in an improved tolerance not only to freezing, but also
to drought and high salinity. DREB1/CBF orthologs have
been reported andshown to function in cold stresstolerance
from various species, such as Brassica napus, tomato, bar-
ley, maize, rice, rye, and wheat (Choi et al., 2002; Dubouzet
et al., 2003; Gao et al., 2002; Jaglo et al., 2001; Qin et al.,
2004; Skinner et al., 2005; Xue, 2002).
Although DREB1/CBF and DREB2 share a high homology
in the ERF/AP2 DNA-binding domain and can bind the same
DRE core sequence (A/GCCGAC), these two proteins differ-
entially transmit cold or dehydration stress. Expression of
the DREB1/CBF genes is induced to high levels specifically
in response to a low-temperature stimulus. In contrast,
Arabidopsis DREB2A is gradually induced by dehydration
and high salinity stresses over 24 h, but hardly responds to
cold stress (Liu et al., 1998; Sakuma et al., 2002). Multiple
amino acid sequence alignment of DREB1 and DREB2
proteins shows that the sequences of PKK/RPAGRxKF-
xETRHP and DSAWR, which are located upstream and
downstream of the DNA-binding domain, are conserved in
DREB1-type transcription factors. These sequences are
designated as DREB1/CBF ‘signature sequences’ (Jaglo
et al., 2001) and are absent in DREB2-type proteins. How-
ever, a serine- and threonine-rich region adjacent to the
DNA-binding domain is found to be unique to the DREB2A
protein. Overexpression of DREB2A in transgenic plants
does not activate downstream genes under normal growth
conditions, which implies that post-translational regulation
may be involved in its activation (Liu et al., 1998). Recently,
a negative regulatory domain was identified in the central
region of DREB2A and deletion of this region transforms
DREB2A to a constitutive active form (DREB2A CA; Sakuma
et al., 2006a,b). Transgenic Arabidopsis overproducing
DREB2A CA showed increased expression of many stress-
responsive genes and improved tolerance to drought stress
(Sakuma et al., 2006a).
Because of the significance of gene regulation under
dehydration and high salinity stress, multiple research
efforts have been initiated to isolate drought- and osmotic
stress-inducibletranscriptionfactorsinotherspecies.Inrice,
Oryza sativa L., one homolog, named OsDREB2A, was
identified as a DREB2-type protein. Similar to Arabidopsis
DREB2A, OsDREB2A was gradually induced by dehydration
and high salinity stresses, but hardly increased under cold
stress(Dubouzetet al.,2003).Inwheat,TriticumaestivumL.,
the TaDREB1 gene was found to be induced by cold, salinity
and drought and was classified into the DREB2-type tran-
scription factors by phylogeneticanalysis(Shen et al., 2003).
A barley, Hordeum vulgare L., gene for the DREB2-type
protein, HvDRF1, was reported to accumulate under drought
and salt stresses and was involved in ABA-mediated gene
regulation (Xue and Loveridge, 2004). However, the function
of these genes in plants under stress conditions is still
unclear.
In the field, heat stress and water deficit often occur in a
parallel manner, especially in tropical areas. The effects of
these stresses may cause oxidative damage of cellular
components or result in the misfolding or denaturation of
cellular proteins. Heat shock proteins (HSPs) are synthesized
and accumulated in the heat shock response and are
correlated with thermotolerance in the plant (Li and Werb,
1982). Heat shock proteins act as molecular chaperones by
maintaining the homeostasis of protein folding and thus
help to maintain the metabolic and structural integrity of
cells (Sung and Guy, 2003; Vierling, 1991; Wang et al., 2004).
Heat shock transcription factors (HSFs) have been found to
primarily control the expression of HSP genes through the
binding of the conserved heat-shock element (HSE) in the
promoter region of these genes (Wu, 1995).
We previously isolated a DREB1-type transcription factor
from maize (Zea mays; ZmDREB1A) by yeast one-hybrid
screening (Qin et al., 2004). Analysis of gene expression,
phylogenetic studies and functional characterization of
ZmDREB1A in transgenic plants demonstrated that it en-
codes a typical DREB1-type protein in maize. In the current
study, we were interested to determine if a dehydration and
salt stress signal transduction pathway mediated by DREB2-
type transcription factor(s) also exists in this agriculturally
important plant. We isolated a DREB2-type transcription
factor ZmDREB2A from maize and identified its two tran-
scription forms, ZmDREB2A-L and ZmDREB2A-S. Overex-
pression of ZmDREB2A in Arabidopsis resulted in an
enhanced tolerance to drought stress. Microarray analyses
of the 35S:ZmDREB2A transgenic plants revealed the upreg-
ulation of genes not only encoding late embryogenesis
abundant(LEA)proteins,butalsogenesrelatedtoheatshock
stress, detoxification and seed maturation. Overexpression
ofZmDREB2Aalsoresultedinthermotoleranceintransgenic
plants,suggestingthatZmDREB2Ahasadualfunctioninthe
expression of genes responsive to water and heat stress.
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Results
Isolation of ZmDREB2A and identification of its two
transcription forms
To determine whether DREB2-type transcription factors
exist in maize, we performed a TBLASTN search with the
amino acid sequence of the ERF/AP2 DNA-binding domain
of Arabidopsis DREB2A. As a result, a new maize sequence
was identified and found to potentially encode an ERF/AP2
DNA-binding domain (accession no. AY108198; http://
www.ncbi.nlm.nih.gov/BLAST/Blast.cgi). Translation of the
DNA according to the coding frame of the potential DNA-
binding domain produced a putative protein containing 274
amino acid residues. Using a gene-specific primer pair
designed from the AY108198 nucleotide sequence, we
successfully obtained a 1034-bp fragment containing a
coding sequence and a polyadenylation signal by using
cold-, heat-, dehydration- or salt-treated maize cDNA sam-
ples.MultiplesequencealignmentwithDREB2A, OsDREB2A
and otherDREB2 proteins found that the putativeprotein did
not contain a full DNA-binding domain and nuclear local-
ization signal (NLS) which is normally found in the N-ter-
minal of DREB proteins.
Subsequently six maize expressed sequence tag (EST)
and mRNA sequences were identified in the UniGene
database which potentially covered the 5¢-end of this gene.
Using a newly designed primer, in combination with the
original AY108198 reverse primer, two different sized tran-
scripts for this gene were amplified. These transcripts were
subsequentlynamed ZmDREB2A-L
AB218833) and ZmDREB2A-S (accession no. AB218832)
according to their length. Both transcripts shared a 100%
identity over a 1283-bp long nucleic acid sequence even on
the 5¢- and 3¢-untranslated regions (UTRs). These transcripts
onlydifferedby53additionalbasepairswhichwerefoundin
ZmDREB2A-L and lacking in ZmDREB2A-S (Figure 2c).
ZmDREB2A-S was 1283 bp in length, encoding 318 amino
acids, with a potential NLS and a typical ERF/AP2 DNA-
binding domain (Figure 1a). In contrast, ZmDREB2A-L was
1336 bplong.Becauseofaprematureterminationcausedby
the 53-bp insertion and a frame shift ZmDREB2A-L encoded
only89aminoacids.Thisshortpeptidechainstoppedbefore
the ERF/AP2 DNA-binding domain and probably rendered it
as a non-functional transcription form. Sequence alignment
also showed that ZmDREB2A shared a homology not only
within its DNA-binding domain but also in its C-terminal
region with OsDREB2B (AK099221), HvDRF1 (AY223807) and
PgDREB2A (AY829439) proteins (Figure 1a, dotted line).
Thus, we named the protein encoded by ZmDREB2A-S as
ZmDREB2A.Asexpected,phylogeneticstudyoftheERF/AP2
proteins according to the previous classification of this
superfamily (Dubouzet et al., 2003; Qin et al., 2004; Sakuma
et al., 2002) assigned this protein to the DREB2 subgroup
(accession no.
(Figure 1b). Two additional AP2/ERF transcription factors,
DBF1 and DBF2, cloned in maize (Kizis and Pages, 2002),
were classified to the A-4 and A-6 subgroup in DREB group
proteins and share a high homology with RAP2.4 and TINY
proteins, respectively.
Stress-inducible expression profiles of ZmDREB2A
The expression pattern of ZmDREB2A was examined in
maize seedlings treated by cold (4?C), dehydration, salt
(250 mM NaCl) and heat (42?C) stresses. Firstly, RNA gel blot
analysis was used to study accumulation of ZmDREB2A
mRNA in 7-day-old maize leaves, stems and roots, respect-
ively. An accumulation of the ZmDREB2A mRNA was ob-
served after 5 h of cold treatment in maize leaf, stem and
root tissues. A relatively stronger induction was observed in
root tissues during the cold treatment. Under dehydration
stress, a rapid induction of ZmDREB2A was observed after
10 min which then remained unchanged in leaf tissue. In
root tissue, ZmDREB2A expression was detected under
normal conditions and was gradually upregulated. Under
salt stress, ZmDREB2A was remarkably induced in root tis-
sues up to 24 h, but in leaves and stems it was only slightly
induced. Notably, a significant and transient induction of
ZmDREB2A was observed with heat stress treatments in
maize leaf, stem and root tissues. Subsequent to this
induction, its mRNA rapidly became undetectable in stems
10 min after the induction and it gradually decreased in leaf
and root tissues (Figure 2a). Exogenous application of ABA
to maize seedlings did not result in the induction of
ZmDREB2A (data not shown). Due to the small size differ-
ence of only 53 bp, these two fragments could not be clearly
separated and distinguished by RNA gel blot analysis.
In order to verify the two kinds of transcription forms
under stress conditions, a common pair of primers was
designed according to the flanking sequence of the 53-bp
insertion. These primers were then used in reverse tran-
scription PCR (RT-PCR) with stress-treated maize cDNA
templates. As shown in Figure 2(b), two kinds of transcript
could be clearly identified under both heat and salt stresses.
Under cold or dehydration stress ZmDREB2A-S was less
induced than ZmDREB2A-L. Furthermore, we used quanti-
tative real-time RT-PCR with two pairs of primers specific for
either ZmDREB2A-S or ZmDREB2A-L in order to clarify the
expression pattern of these two transcripts under different
stresses (Figure 2c). Under cold stress, both kinds of tran-
scripts displayed a similar pattern of induction, but ZmDRE-
B2A-L appeared to be more abundant and increased to
greater levels than ZmDREB2A-S. Under drought stress,
both kinds of transcript appeared to be slightly increased.
When the plants were treated by NaCl, both ZmDREB2A-L
and ZmDREB2A-S were significantly upregulated to an
equivalent amount. Upon exposure to a 42?C stimulus, both
types of transcript displayed a transient induction pattern.
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However, ZmDREB2A-S increased to a higher level within
10 min of the treatment (Figure 2d). Taken together, under
normal conditions ZmDREB2A-L was present but ZmDRE-
B2A-S transcriptswere always undetectable. The expression
of ZmDREB2A-S was only induced subsequent to stress
treatments. Furthermore, the induction ratios of ZmDRE-
B2A-S were always higher than ZmDREB2A-L under all
stresses (Figure 2e). Quantitative analysis revealed that
under heat or salt stress the amount of ZmDREB2A-S
transcript was increased >100-fold compared with the non-
stressed condition. On the other hand, the highest induction
ratio of ZmDREB2A-L was obtained under cold or salt stress
andwasincreasedbyonlyabout10-fold.Thesedataseemto
indicate that splicing mechanisms play an important role in
regulating ZmDREB2A gene activity under stress conditions.
ZmDREB2A functions as a transcriptional activator and the
region of amino acids 235–272 is important for its activation
Since only ZmDREB2A-S could be translated into a potential
functional protein, in order to determine whether the
ZmDREB2A protein was capable of transactivating DRE-
dependent transcription in plant cells, we performed trans-
activation assays using Arabidopsis T87 protoplasts. A
b-glucuronidase (GUS) gene driven by the trimeric 75-bp
fragments containing the DRE sequence was used as a
reporter (Figure 3a; Dubouzet et al., 2003). As expected,
transfection with ZmDREB2A-L could not activate GUS gene
expression because it did not encode a functional DRE-
binding protein. In contrast, the relative GUS/LUC activity
was distinctly upregulated when ZmDREB2A-S, DREB2A
(DREB2A-wt), or the constitutive active form of DREB2A
(DREB2A-CA), were transfected in T87 protoplasts. Further-
more, the ZmDREB2A protein exhibited a rather high
transactivation activity, which led to a 40.46-fold increase of
GUS/LUC activity in comparison with the vector-only trans-
fection control. In a parallel experiment, DREB2A-wt and
DREB2A-CAproteinresultedin5.73-and22.34-foldincreased
GUS/LUC activity, respectively (Figure 3b). These data sug-
gested that ZmDREB2A functioned as a stronger transacti-
vator than Arabidopsis DREB2A.
Figure 1. Sequence alignment of ZmDREB2A and DREB2 proteins from
different species and phylogenetic analysis of the AP2/ERF DNA-binding
domain of ZmDREB2A in the AP2/ERF supertranscription factor family.
(a) The entire protein sequences for DREB2A (AB007790), DREB2B
(NM111939), OsDREB2A (AF300971), OsDREB2B (AK099221), ZmDRBE2A
(AB218832), HvDRF1.1/1.3 (AY223807) and PgDREB2A (DQ227697) were
aligned by the CLUSTERX program. The conserved amino acid residues
amongallsequencesarehighlightedwithablackor greybackground; nuclear
localization signals, as predicted by PSORT (http://psort.nibb.ac.jp/) are double
underlined; ERF/AP2 DNA-binding domains are underlined; and a conserved
region inthe potential activation domain among monocotDRBE2A proteinsis
indicated by a dashed line.
(b) A phylogenetic tree of the ERF/AP2 domains was constructed by
CLUSTARX. The scale indicates branch lengths. A-1 to A-6 indicate subgroups
proposed by Sakuma et al. (2002). The accession number of each appended
protein is: LeCBF1 (AY034473), BNCBF7 (AF499032), BNCBF17 (AF499034),
GhDREB1A(AY321150),HvCBF1 (AF418204),ZmDREB1A(AF045481), HvCBF2
(AF442489), BCBF1 (AF298230), HvDRF1.1 (AY223807), ScCBF1 (AF370730),
BCBF3 (AF298231), TaDREB1 (AF303376), DBF1 (AF493800), DBF2 (AF493799),
Glossy15 (U41466) and ids1 (AF048900). Genes belonging to the DREB
subgroup are bold-faced; branches for genes from other monocot plants are
designated in green. ZmDREB1A and ZmDREB2A were classified into the A-1
and A-2 subgroup, respectively. DBF1 (AF493800) and DBF2 (AF493799) from
maize classified to the A-4 and A-6 subgroup and are underlined.
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We performed mutational transactivation domain analy-
sis for two reasons. First, we aimed to identify which region
brought a high transactivation activity to the ZmDREB2A
protein. We also wanted to determine whether the removal
of any internal region would confer a higher transactivation
activity to the ZmDREB2A protein. A series of C-terminal
deletions of ZmDREB2A, ZmDREB2A (amino acids (aa)
1–272), ZmDREB2A (aa 1–253), ZmDREB2A (aa 1–208),
ZmDREB2A (aa 1–191) and ZmDREB2A (aa 1–141) were
constructed as effectors. As a result, removal of the
zC-terminal end of the protein from aa 272 to aa 318
significantly reduced GUS/LUC activity. Further deletion
from the C-terminal end to aa 236 completely abolished the
ZmDREB2A transactivation capability by reducing GUS/LUC
activity to 1.49-fold. As a positive control, a parallel experi-
ment was conducted with ZmDREB2A protein and it always
showed a high transactivation activity (Figure 3c). These
data suggested that the C-terminal region from aa 236 to the
C-terminal end was necessary for ZmDREB2A activity. In
order to further localize its activation domain, an additional
series of internal deletion fragments of ZmDREB2A was
generated and constructed as effectors. Removal of the
region of aa 236 to 272 greatly reduced its GUS/LUC activity.
On the other hand, none of the other internal deletions
Figure 2. Expression analysis of ZmDREB2A under various stress conditions.
(a) Seven-day-old maize seedlings were treated by cold (4?C), heat (42?C), dehydration or NaCl (250 mM) for the time course indicated above each line. Ethidium
bromide-stained total rRNAs were shown to indicate equal loading of samples.
(b) Seven-day-old maize seedlings treated by cold (4?C, 24 h), heat (42?C, 10 min), dehydration (1 h) and NaCl (250 mM, 24 h) were used for RNA preparation. As a
negative control, dH2O was used as template in reverse transcription and PCR. Plasmids containing ZmDREB2A-L or ZmDREB2A-S were amplified by the same
primers to indicate the length of the two kinds of transcripts.
(c) A schematic diagram to indicate the position of two pairs of sequence-specific primers designed accordingly for ZmDREB2A-L and ZmDREB2A-S.
(d) Root samples from 7-day-old maize seedlings treated by cold (4?C), heat (42?C), dehydration or NaCl (250 mM), for indicated time courses, were used for
quantitative real-time RT-PCR analysis. The relative amount of two kinds of transcripts was calculated, when the amount of ZmDREB2A-L was defined as 1.0. (Bars
indicate the standard error from three individual experiments.)
(e) Root samples from 7-day-old maize seedlings treated by cold (4?C), heat (42?C), dehydration or NaCl (250 mM), for the indicated time courses, were used for
quantitative real-time RT-PCR analysis. The induction ratios of two kinds of transcripts were calculated, when the amount of each transcript under control condition
was defined as 1.0 respectively. Bars indicate the standard error from three individual experiments.
58 Qin et al.
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