Improvement of stress tolerance of wheat and barleyby modulation of expression of DREB ⁄CBF factorsSarah Morran1,2, Omid Eini2, Tatiana Pyvovarenko, Boris Parent, Rohan Singh, Ainur Ismagul, Serik Eliby,Neil Shirley, Peter Langridge and Sergiy Lopato*
Australian Centre for Plant Functional Genomics, University of Adelaide, Urrbrae, Adelaide, SA, Australia
Received 7 March 2010;
revised 18 May 2010;
accepted 28 May 2010.*Correspondence (tel +61 8 830 37 499;
fax +61 8 830 37 102;
email [email protected])1Present address: School of Agriculture,
Food & Wine, University of Adelaide, PMB
1, Glen Osmond, Adelaide, SA 5064,
Australia2These authors contributed equally to the
paper.
GenBank accession numbers of TdDREB2
and TdDREB3L genes are GU785008 and
GU785009, respectively.
Keywords: DREB ⁄ CBF, drought-
inducible promoter, drought and frost
tolerance, LEA ⁄ COR ⁄ DHN genes,
wheat, barley.
SummaryTranscription factors have been shown to control the activity of multiple stress
response genes in a coordinated manner and therefore represent attractive targets
for application in molecular plant breeding. We investigated the possibility of modu-
lating the transcriptional regulation of drought and cold responses in the agricultur-
ally important species, wheat and barley, with a view to increase drought and frost
tolerance. Transgenic wheat and barley plants were generated showing constitutive
(double 35S) and drought-inducible (maize Rab17) expression of the TaDREB2 and
TaDREB3 transcription factors isolated from wheat grain. Transgenic populations
with constitutive over-expression showed slower growth, delayed flowering and
lower grain yields relative to the nontransgenic controls. However, both the TaDREB2
and TaDREB3 transgenic plants showed improved survival under severe drought con-
ditions relative to nontransgenic controls. There were two components to the
drought tolerance: real (activation of drought-stress-inducible genes) and ‘seeming’
(consumption of less water as a result of smaller size and ⁄ or slower growth of trans-
genics compared to controls). The undesired changes in plant development associ-
ated with the ‘seeming’ component of tolerance could be alleviated by using a
drought-inducible promoter. In addition to drought tolerance, both TaDREB2 and
TaDREB3 transgenic plants with constitutive over-expression of the transgene
showed a significant improvement in frost tolerance. The increased expression of
TaDREB2 and TaDREB3 lead to elevated expression in the transgenics of 10 other
CBF ⁄ DREB genes and a large number of stress responsive LEA ⁄ COR ⁄ DHN genes
known to be responsible for the protection of cell from damage and desiccation
under stress.
Introduction
Several families of transcription factors, including
DREB ⁄ CBF, ERF, MYK, MYB, AREB ⁄ ABF, NAC and HDZip,
have been shown to be involved in the regulation of
drought response in plants (Yamaguchi-Shinozaki and
Shinozaki, 2006). These factors bind specific cis-elements
in the promoters of drought-regulated genes. The dehy-
dration-responsive element-binding proteins (DREBs) or
C-repeat-binding proteins (CBFs) were among the first
families of transcription factors responsible for gene regu-
lation under conditions of water deficit to be discovered.
They comprise a group of transcriptional factors with a
single AP2 domain, which is a DNA-binding motif of about
60 amino acids initially identified in the Arabidopsis pro-
tein APETALA2. This group of transcription factors controls
the expression of many stress-inducible genes in plants
(Thomashow et al., 2001; Agarwal et al., 2006; Gao et al.,
2007; Kim, 2007). Many DREB ⁄ CBF factors are themselves
induced by abiotic stresses including drought (Liu et al.,
1998; Nakashima et al., 2000; Tian et al., 2005; Sakuma
et al., 2006; Qin et al., 2007), low temperature (Liu et al.,
1998; Nakashima et al., 2000; Gao et al., 2002; Qin et al.,
2004; Li et al., 2005; Vogel et al., 2005; Oh et al., 2007;
ª 2010 ACPFG
230 Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd
Plant Biotechnology Journal (2011) 9, pp. 230–249 doi: 10.1111/j.1467-7652.2010.00547.x
Qin et al., 2007; Gutha and Reddy, 2008), high salt (Naka-
shima et al., 2000; Dubouzet et al., 2003; Shen et al.,
2003; Tian et al., 2005; Cong et al., 2008; Huang et al.,
2008; Wang et al., 2008; Chen et al., 2009) and extreme
heat (Schramm et al., 2008). Six DREB transcription fac-
tors, including four DREB1 ⁄ CBF and two DREB2 genes
were identified in Arabidopsis. Expression of DREB1 ⁄ CBFs
is induced by drought, salt and cold, whereas expression
of DREB2 factors is induced by drought and salt only.
Since the discovery of the role of DREB ⁄ CBF factors in abi-
otic stress response, several groups have explored their
potential to improve stress tolerance in Arabidopsis (Stock-
inger et al., 1997; Kasuga et al., 1999; Qin et al., 2004;
Saleh et al., 2006; Lin et al., 2008) and crop plants,
including Brassica junceae (Cong et al., 2008), soybean
(Chen et al., 2007), rice (Ito et al., 2006; Oh et al., 2007;
Chen et al., 2008; Wang et al., 2008), wheat (Wang
et al., 2006) and other grasses (Zhao et al., 2007). The
over-expression of some DREB factors, e.g. AtDREB2A,
does not lead to the activation of target genes, and
improvement of plant stress tolerance may require stress-
inducible post-translational activation, such as phosphory-
lation (Liu et al., 1998; Sakuma et al., 2006).
Viral 35S (Guilley et al., 1982; Bevan et al., 1985; Liu
et al., 1998; Sakuma et al., 2006), rice Actin 1 (Xu et al.,
1996; He et al., 2009) and maize polyubiquitin (Christen-
sen et al., 1992) promoters were used in most attempts to
over-express DREB factors constitutively (Hsieh et al.,
2002; Oh et al., 2005; Ito et al., 2006). In most cases,
strong ectopic expression led to different degrees of
growth retardation which subsequently resulted in dwarf-
ism and delayed flowering (Oh et al., 2007). However, a
few exceptions have been reported: over-expression of
OsDREB1F (Wang et al., 2008) and ABF3 (Oh et al., 2005)
had no influence on plant development. It was found that
plants with up-regulated expression of dwarf and delayed-
flowering 1 (DDF1) were deficient in the gibberellin (GA)
biosynthesis pathway and this could cause dwarfism (Mag-
ome et al., 2004). Recently, it was shown that the reason
for the GA deficiency is a strong up-regulation of the
GA2-oxidase 7 gene (GA2ox7) which encodes a G20-GA
deactivation enzyme (Magome et al., 2008).
There were several attempts to overcome the problems
of severe growth retardation by cutting down the duration
of DREB over-expression using stress-inducible promoters.
The first such attempt was undertaken in 1999 (Kasuga
et al., 1999). They used the rd29A promoter which was
later used by several other groups for the expression of
DREB factors in Arabidopsis (Qin et al., 2007; Cong et al.,
2008), tobacco (Kasuga et al., 2004; Saleh et al., 2006),
sugarcane (Wu et al., 2008), maize (Al-Abed et al., 2007),
wheat (Hao et al., 2005; Wang et al., 2006), peanut
(Bhatnagar-Mathur et al., 2007) and tall fescue (Zhao
et al., 2007). Other stress-inducible promoters were also
tested. Tomato plants over-expressing the Arabidopsis
CBF1 gene under the control of barley abrc1 or cor15A
stress-inducible promoters showed normal development
and drought tolerance (Hsieh et al., 2002 [data not shown
in the paper but referred as unpublished]). Constructs,
where barley HVA1s and Dhn8 promoters were cloned
upstream of HsDREB1A, were successfully used to increase
stress tolerance of the turf and forage grass (Paspalum
notatum Flugge) (James et al., 2008).
Tolerance of transgenic plants with elevated levels of
DREB ⁄ CBF transcription factors is at least partially a result
of activation of genes encoding late embryogenesis abun-
dant (LEA) proteins known also as dehydrins (DHNs) and
cold-responsive (COR) proteins (Bartels et al., 1996; Jaglo-
Ottosen et al., 1998; Chen et al., 2003; Kasuga et al.,
2004; Lee et al., 2005; Kobayashi et al., 2008). LEA genes
are active during the maturation of embryos and desicca-
tion of seeds in both embryo and endosperm (Rorat, 2006).
They are also induced by drought, cold and salt stresses in
vegetative tissues (Taji et al., 1999; Neumann, 2008; Tom-
masini et al., 2008). Products of these genes are often
quite hydrophobic and are involved in the direct protection
of the cell from stress by increasing membrane stability,
preventing incorrect folding and processing of proteins and
by other still unclear mechanisms (Sales et al., 2000; Wise,
2003; Goyal et al., 2005; Chakrabortee et al., 2007; Toll-
eter et al., 2007; Tunnacliffe and Wise, 2007). Cold accli-
mation of plants leads to LEA accumulation and increases
frost tolerance (Kume et al., 2005). Over-expression of par-
ticular LEA proteins in some cases leads to an improvement
of abiotic stress tolerance (Brini et al., 2007; Lal et al.,
2008; Dalal et al., 2009). Some stress-inducible LEA pro-
moters, including the promoter of ZmRab17, were found
to be activated by DREB transcription factors (Baker et al.,
1994; Brini et al., 2007; Lal et al., 2008; Dalal et al., 2009).
The Rab17 gene from maize belongs to a group of
LEA ⁄ DHN genes. It is induced by abscisic acid (ABA) and
water deficit (Vilardell et al., 1990). The maize Rab17 pro-
moter has been tested in several heterologous systems. The
promoter was tested in stably transformed tobacco and by
transient expression in rice protoplasts and in both cases
induction of the promoter by drought and ABA treatment
was demonstrated (Vilardell et al., 1991). The activity of the
1.3-kb fragment of the Rab17 promoter fused to the GUS
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 231
coding sequence was analysed in transgenic Arabidopsis
plants and in ABA-deficient and ABA-insensitive mutants of
Arabidopsis. Although the ZmRab17 promoter was active in
the embryo and endosperm during late seed development,
promoter activity decreased during seed germination and
GUS activity was not enhanced by ABA and water deficit in
transgenic plants. This suggests different molecular mecha-
nisms for the Rab17 promoter activation in maize and Ara-
bidopsis (Vilardell et al., 1994). Phylogenetic analysis of
5¢-noncoding regions from the Rab16 ⁄ 17 gene family of
sorghum, maize and rice revealed the absence of some
important cis-elements in the promoters, which could
explain some differences in the expression of Rab17-like
genes in these plants (Buchanan et al., 2004). Although the
activation and mechanisms of regulation of the ZmRab17
promoter were intensively studied, the application of this
promoter for stress-inducible over-expression of genes in
either maize or other crop plants has not been reported.
In this paper, we demonstrate that constitutive over-
expression of two different wheat DREB factors leads to
the substantial improvement of barley capacity to survive
during severe drought and frost stresses. We show that
this improvement is at least partially because of activation
or suppression of the large number of other DREB ⁄ CBF
genes and the consequent cascade of activation of down-
stream stress responsive genes, many of which are known
to be directly involved in the protection of cells from dam-
age caused by dehydration. We also show that the
drought-stress-inducible ZmRab17 promoter, which has
low or no basal expression in wheat in the absence of
stress, is quickly and strongly activated in both wheat and
barley by drought. Transgenic plants with stress-inducible
over-expression showed little or no undesirable develop-
mental traits such as stunted growth, dwarfism, delayed
flowering and smaller spikes, traits which were observed
in plants with constitutive over-expression of DREB factors.
In contrast to wheat, in barley the Rab17 promoter is
leaky and a pleiotropic phenotype can be observed in the
well-watered transgenic plants although developmental
setbacks are much less pronounced than in plants with
strong constitutive overexpression of DREB genes.
Results
Expression of TaDREB2 and TaDREB3 in well-watered
plants and under different stresses
Full length cDNAs of TaDREB2 and TaDREB3 were isolated
in a yeast one-hybrid screen from a library prepared from
unstressed wheat grain using the DRE sequence from
Arabidopsis (TACCGACT) as bait (Lopato et al., 2006). The
phylogenetic relationship to other CBF ⁄ DREB factors from
wheat and barley are shown in Figure 1.
TaDREB2 and TaDREB3 are both expressed in flower
and grain tissues in the absence of stress. However, high
levels of expression of TaDREB2 were also detected in
roots, coleoptiles and embryo of germinating seed (Fig-
ure 2a). In the absence of abiotic stress, expression of
TaDREB2 and TaDREB3 in leaf was very low while expres-
sion of both TaDREB genes was enhanced by drought.
Drought stress induced TaDREB2 more strongly than
TaDREB3 (Figure 2b). However, both genes were only
weakly activated by cold (Figure 2c). Surprisingly, TaDREB2
was found to be strongly activated by wounding in grain
with a lower level of activation by wounding in leaves
(Figure 2d). No induction was seen in plants under salt
stress and weak induction of TaDREB3 by ABA was only in
leaf tissues (data not shown).
Full-length genomic clones, including promoter
sequences, were isolated from the BAC library of Triticum
durum cv. Langdon using full-length cDNAs of TaDREB2
and TaDREB3 as probes. Four BAC clones containing the
same gene (#328 O11, #671 O3, # 741 C12, and #871
Figure 1 Phylogenetic tree shows the relationships of TaDREB2 and
TaDREB3 to DREB factors from wheat, barley and diploid progenitors
of wheat. The tree is based on alignment of complete protein
sequences.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.232
H2) were isolated for DREB2 and one BAC clone
(#1111 G2) for DREB3; genes and promoter regions were
identified and sequenced. The genomic sequences were
designated TdDREB2 and TdDREB3L. The deduced protein
sequence of TdDREB2 was identical to TaDREB2. However,
the sequence of TdDREB3L was slightly different from
TaDREB3. Both genes contain no introns. Analysis of 1972
and 2749 bp long promoter sequences of TdDREB2 and
TdDREB3L, respectively, using PLACE software revealed
potential stress-responsive cis-elements. Both promoters
contain multiple abscisic acid responsive elements (ABREs),
which may be responsible for the drought-inducible
activation of both DREB genes. No DREs ⁄ CRTs or drought-
related MYCR and MYBR elements were identified in the
TdDREB2 promoter. However, it contains one site (CAATT-
ATTG) specific for the class I HDZip transcription factors,
some of which are known to be induced by ABA and
drought (Agalou et al., 2008). The TdDREB2 promoter
region also contains a binding site for the GATA-type zinc
finger protein (AGATCCAA) associated with wounding-
induced activation of some MYB transcription factors (Su-
gimoto et al., 2003). This element can be potentially
responsible for the wounding-inducible activation of
TdDREB2 as no GCC-boxes or other wounding-related
-2468
10121416
0 0.5 1 2 3 8 17Hours
Wheat leaf
TaDREB3
TaDREB2
-
10
20
30
40
50
0 0.5 1 2 3 8 17Hours
Wheat grain
TaDREB3
TaDREB2
Copie
s×
10
3/µ
gR
NA
Copie
s×
10
3/µ
gR
NA
0
5
10
15
20
25
30
Control/W Control/D
TaDREB2
0
0.5
1
1.5
2
2.5
Control/W Control/D
TaDREB3
Copie
s×
10
3/µ
gR
NA
Copie
s×
10
3/µ
gR
NA
-
5
10
15
20
25
30
35
40 TaDREB3
TaDREB2
Copie
s×
10
3/µ
gR
NA
-
100
200
300
400
500
0 1 4 24 48
TaDREB3
Hours
-
50
100
150
200
250
300
0 1 4 24 48
TaDREB2
Hours
Copie
s/µ
gR
NA
Copie
s/µ
gR
NA
(c)
(d)
(b)
(a)
Figure 2 Expression of TaDREB2 and TaD-
REB3 in different wheat tissues and under
different stresses assessed by quantitative
PCR. (a) Expression in different plant tissues:
Emb. (germ.), embryo isolated from germi-
nating seed; Immat. fluor., immature influo-
rescence; Car. 3–5 DAP, caryopsis at
3–5 days after pollination; Emb. 22 DAP,
embryo at 22 DAP; End. 22 DAP, endosperm
at 22 DAP. (b) Expression under well
watered (W) and drought (D) in leaf tissues
of three wheat plants. (c) Expression in leaf
under cold stress (4 �C) at 0, 1, 4, 24, and
48 h. (d) Expression in leaf and grain under
wounding stress at 0, 0.5, 1, 2, 3, 8, and
17 h.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 233
elements were identified in this promoter. In contrast, the
TdDREB3 promoter is rich in DRE ⁄ CRT, MYBR, and MYCR
elements. Unfortunately, mapping of functional cis-ele-
ment(s) was not successful because of the weak activity of
both promoters (data not shown).
Slow development allows plants constitutively
expressing TaDREB2 and TaDREB3 to recover after
drought stress in a controlled environment
The coding regions of TaDREB2 and TaDREB3 were cloned
into the pMDC32 vector under the 2 · 35S promoter
(Curtis and Grossniklaus, 2003). This promoter drives
strong expression in transgenic barley although it is two-
to threefold weaker than the polyubiquitin promoter from
maize (unpublished data). In contrast, in wheat, the
2 · 35S promoter is weak and inefficient for constitutive
over-expression (data not shown). Constitutive expressions
of TaDREB2 and TaDREB3 were, therefore, only analysed
in transformed barley plants.
Eleven and 13 independent transgenic barley lines were
obtained for TaDREB2 and TaDREB3, respectively, using
the Agrobacterium-mediated transformation method (Tin-
gay et al., 1997; Matthews et al., 2001). Southern blot
hybridization indicated that most transgenic T0 lines had
2–6 copies of the transgene. In some plants, several copies
of transgene were either inserted at a single site or situated
very close together as no segregation was seen in four sub-
sequent generations (data not shown). Expression levels of
the transgenes were examined by RNA-blot analysis using
total RNA from leaf tissue. Most T0 lines had high levels of
transgene expression in leaves (Figure 3a). Analysis of
transgenic plants was performed using progeny of T1–T3
generations. As experiments commenced using T1 plants
which were not homozygous and often contained several
copies of transgene, Northern blot hybridization was used
to confirm transgene expression in each plant, and plant
phenotypes were compared with levels of expression
(Figure 3). Untransformed plants and plants with no trans-
gene expression (null segregants) were used as controls.
No significant difference was observed in development and
stress tolerance between these two groups of control
plants. In separate experiments, transgenic plants were
produced with empty transformation vectors. These plants
showed no differences to the other control plants.
Plant constitutively expressing TaDREB2 and TaDREB3
grew more slowly than control plants, produced less tillers
and showed delayed flowering by 2–3 weeks under well-
watered conditions (Table S1). The differences in plant size
(plant height and number of leaves) 4 weeks after sowing,
were associated with levels of transgene expression.
Despite these differences of growth rate, plants with con-
stitutive up-regulation of TaDREB2 displayed a phenotype
similar to that of control plants at flowering (similar height,
architecture and spike size, Figure 3a). In contrast,
TaDREB3 plants only reached about 70% of the size of
control plants at flowering stage (Figure 3b), with shorter
spikes and lower yields in the T1 generation (Table S1).
However, in later generations, the size of spikes returned
to normal. No differences in fertility or grain size were
observed between the transgenic lines and control plants.
Both transgenic populations partially returned to normal
phenotypes in the T2–T3 generations although transcription
levels of the transgene, up-regulation of several down-
stream genes and stress tolerance remained unchanged.
Four-week-old control (C) plants and T1 or T4 generation
transgenic plants were subjected to 18–21 days of drought
stress (the drought survival procedure is described in Experi-
mental procedures). Volumetric water content (VWC) in
soil with the small transgenic plants changed more slowly
than in pots with larger plants (VWC at 5% vs. 3% after
4 days without watering). As a result, control plants
showed signs of stress including loss of turgor, leaf rolling
and loss of chlorophyll much earlier than transgenic plants.
Most transgenic lines showed no signs of stress for at least
2–3 days longer than control plants, some retained turgor
and showed no wilting or other signs of stress for even
longer (Figure 3c). Between 5% and 10% of control plants
were able to recover after re-watering following the 18–
21 days of drought. However, almost 100% plants of
some transgenic lines survived and completely recovered
within 1–2 weeks of re-watering; the smallest plants
showed the quickest recovery. These results suggested that
‘improved’ drought tolerance of transgenic plants with
constitutive over-expression of transgenes may be because
of reduced water consumption resulting from slower
growth and smaller size of transgenic plants. To confirm
this hypothesis, we performed the drought survival test
with two transgenic populations which constitutively over-
express genes encoding HD-Zip class II and PHD-finger pro-
teins that are not up-regulated by drought and have no
relation to drought tolerance but strongly suppress plant
growth in a similar manner to DREB factors (N. Kovalchuk
and S. Lopato, unpublished data). The recovery of such
‘placebo’ plants was also higher than that of control plants
although, survival of the ‘placebo’ transgenic plants under
drought conditions was lower than for transgenic barley
with constitutive up-regulation of TaDREB2 or TaDREB3.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.234
Activation of stress responsive genes by constitutive
expression of TaDREB2 and TaDREB3 in transgenic
barley
One of the largest groups of genes up-regulated by
drought, cold and salt stresses comprises the LEA proteins
(Caramelo and Iusem, 2009). Figure 4a displays the levels
of expression in transgenic and control plants of four dif-
ferent LEA genes from barley: HvDHN8, HvA22,
HvCOR14B, and HvDHN5. A strong up-regulation of all
these LEA genes was observed in three generations for
three independent transgenic lines that constitutively over-
expressed TaDREB3. The strongest up-regulation was
shown for HvCOR14B, which was highly correlated with
Lines 1, 5 & 9Control Control Lines 3, 7 & 12
2×35S-TaDREB2 2×35S-TaDREB3
2×35S-TaDREB2 2×35S-TaDREB3
T1 T1
C
2×35S-TaDREB3 Line 12 WT2×35S-TaDREB3 Line 7 WT
Line 7, sub lines 7–1 to 7–15 Line 12, sub lines 12–1 to 12–15
2×35S-TaDREB2 Line 1 WT 2×35S-TaDREB2 Line 5 WT
Line 1, sub lines 1–1 to 1–16 Line 5, sub lines 5–1 to 5–15
T1 T1
T1 T1
(a)
(b)
(c)
Figure 3 Constitutive expression of TaD-
REB2 and TaDREB3 in barley plants. (a) Con-
firmation of transgene expression in T0
transgenic lines using Northern blot hybrid-
ization; (b) Phenotypes of T1 transgenic
plants at flowering stage in the absence of
stress. Three independent lines are shown
for each of transgenics. (c) Results of
drought test performed using T1 progeny of
transgenic barley plants transformed with
2 · 35S-TaDREB2 and 2 · 35S-TaDREB3
constructs. Stress tolerance of transgenic
plants is well correlated with expression of
the transgenes. Results of Northern blot
hybridization are shown under each picture.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 235
the transgene expression level (Table S2) and the weakest
induction was for HvDHN8. Only mild (about 1.5–2-fold)
up-regulation of HvDHN8 was observed in transgenic bar-
ley that constitutively over-express TaDREB2 (data not
shown).
Seven barley CBF ⁄ DREB factors (HvCBF1, HvCBF3,
HvCBF6, HvCBF10A, HvCBF11, HvCBF15 and HvCBF16)
were found to be up-regulated by constitutive expression
of TaDREB3 in all transgenic lines tested and over three
consecutive generations (Figure 4b,c; Table S2). In con-
trast, three CBF ⁄ DREB factors (HvCBF2, HvCBF9, and
HvCBF14) were down-regulated in the same lines. Expres-
sion of these 10 CBF ⁄ DREB factors were affected in
exactly the same way in plants over-expressing TaDREB2
(Figure 4c). However, the magnitude of up- and down-
regulation of particular HvCBFs ⁄ HvDREBs was transgene
dependent (e.g. HvCBF6 is more strongly up-regulated in
TaDREB2 than in TaDREB3 transgenic plants). In addition,
up- or down- regulation levels of four barley CBFs ⁄ DREBs
(HvCBF2, HvCBF14, HvCBF1, and HvCBF6) were generation
dependent and were highest in T2 plants. This was also
the generation where plant development appears to
return to normal. Expression of other CBFs ⁄ DREBs
(HvCBF9, HvCBF10A, HvCBF11, HvCBF3, HvCBF15) was
generation dependent in the case of one transgenic line
but was equally up- or down-regulated in all generations
of other transgenic lines.
Transgenics with TaDREB2 and TaDREB3 over-expression
showed activation of two cellulose synthases, HvCesA1
and HvCesA8, which are involved in the biosynthesis of
0.000
0.001
0.002
0.004
0.008
0.016
0.031
0.063
0.125
0.250
0.500
1.000
2.000
4.000
8.000
16.000
Fold
HvCBF1
HvCBF2
HvCBF3
HvCBF6
HvCBF9
HvCBF10A
HvCBF11
HvCBF14
HvCBF15
HvCBF16
HvCBF23
0.002
0.004
0.008
0.016
0.031
0.063
0.125
0.250
0.500
1.000
2.000
4.000
8.000
16.000
32.000
64.000
128.000
256.000
Fold
HvCBF1
HvCBF2
HvCBF3
HvCBF6
HvCBF9
HvCBF10A
HvCBF11
HvCBF14
HvCBF15
HvCBF16
HvCBF23
2×35S-TaDREB2
2×35S-TaDREB3
(a)
(b)
(c)
L7 L11 L12 L7-11 L11-4 L7-10-4 L7-10-5 L11-4-1 L11-4-3 L12-9-4
L1 L5 L2 L1-19 L1-18 L5-7 L5-6 L1-19-4 L5-7-4 L5-7-6
T0 T1 T2
T0 T1 T2
1
2
4
8
16
32
64
128
256
512
1024
2048
4096
8192
16384
32768
Fold
2×35S-TaDREB3
HvDHN8
HvA22
HvCOR14B
HvDHN5
Figure 4 Expression of stress-related and
stress-inducible genes in transgenic barley
with constitutive expression of TaDREB2 and
TaDREB3 demonstrated by quantitative PCR.
(a) Expression of the transgene and LEA ⁄ -COR ⁄ DHN genes in transgenic barley trans-
formed with 2 · 35S-TaDREB3 construct.
The data for the barley genes is presented
as the ratio of expression in transgenic to
control plants. Massive up-regulation of LEA
genes in T0–T2 transgenic barley plants is
presented in fold increase in expression rela-
tive to control plants. (b and c) Expression of
barley CBF ⁄ DREB factors in transgenic barley
plants with strong constitutive expression of
TaDREB2 (b) and TaDREB3 (c).
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.236
primary and secondary cell walls, respectively, and can be
potentially involved in the recovery after wounding
(Figure S1). HvCesA1 is known to be involved in primary
cell wall biosynthesis and was co-ordinately up-regulated
with the transgene expression level in all tested transgenic
lines (Table S2). However, HvCesA8 up-regulation was
weaker and correlation with transgene up-regulation was
poor (Table S2). Analysis of the expression of several
wounding- and pathogenesis-inducible genes (HvHIR1,
HvPR2_4, HvPR5, HvCAT1, and HvSOD2) gave inconclusive
results (data not shown).
Constitutive up-regulation of TaDREB2 and TaDREB3
leads to improved frost tolerance
Several of the downstream genes up-regulated by over-
expression of TaDREB2 and TaDREB3 are known as cold
inducible or cold related. Consequently, frost tolerance in
transgenic plants with constitutive over-expression of
TaDREB2 and TaDREB3 was assessed.
A frost tolerance test, )6 �C for 12 h, was performed in
a cold ⁄ frost cabinet on 3-week-old seedlings of transgenic
and control barley plants. Under these conditions, all
plants were severely damaged and only about 12% of
control plants were able to recover after 2 weeks at nor-
mal temperatures. Under the same conditions, all the
tested transgenic lines showed increased survival (Table 1;
Figure 5), with survival of more than 50% in sublines of
Line 5 (L5-7-4 and L5-4-2) for TaDREB2 and 45% in the
progeny of Line 11 for TaDREB3 transgenic plants. No dif-
ferences in development of the control plants or trans-
genic plants that survived exposure to frost treatment
were detected after several weeks of recovery relative to
the same lines grown under normal growth conditions. In
an experiment where the minimum temperature was
)4 �C, all control and transgenic plants survived. However,
most of control plants showed extensive damage to
leaves. In contrast, no or very little change was detected
in transgenic plants (Figure 5).
Transgenic barley and wheat plants with drought-
inducible expression of DREB factors
As noted above, constitutive over-expression of TaDREB2
or TaDREB3 led to reduced growth in transgenic barley
plants and this appeared to at least partially account for
the observed drought tolerance phenotype. To decrease or
eliminate undesirable phenotypes, barley and wheat were
transformed with constructs in which the 2 · 35S pro-
moter was exchanged for a 634-bp long fragment of the
drought- and salt-inducible Rab17 promoter from maize
(Vilardell et al., 1990). Twenty independent barley lines
were produced for each construct. For wheat transformed
with pRab17-TaDREB2, 45 independent lines were gener-
ated and for pRab17-TaDREB3 construct, 18. The presence
of the transgene was confirmed by PCR using specific
primers from the Rab17 promoter and nos terminator.
In the case of barley, transgenic plants were generally
slightly smaller than control plants. However, the differ-
ence in size was observed only in some plants with high
levels of basal promoter activity (uninduced) and was less
pronounced than in transgenic plants with constitutive
over-expression of the same genes under 2 · 35S
promoter.
In wheat, plants had a comparable phenotype to con-
trol plants (Figure 6a, before drought) for both trans-
formed genes. Under well-watered conditions and under
moderate water deficit (until 5% of VWC, )0.6 MPa), the
stomatal conductance of the transformed plants were
similar to that of control plants, decreasing from
238 ± 29 to 32 ± 3 mmol ⁄ m2 ⁄ s1 with soil drying. This
resulted in no difference in leaf water status, regardless
to the soil water status, with leaf water potential decreas-
ing only slightly with soil drying, from )0.87 MPa under
well-watered conditions to )1.16 MPa under drought
(Figure 6c).
Table 1 Results of the frost tolerance test
Transgenic line progeny
Number
of tested
plants
Number
of survived
plants
% of
survived
plants
Accuracy
of data†
Exp. 1
pUbi-TaDREB2, L5-7-4 33 18 55 ***
pUbi-TaDREB2, L5-4-2 33 17 52 ***
pUbi-TaDREB2, L1-19-4 30 9 30 *
pUbi-TaDREB3, L12-9-4 24 6 25 n.s.
pUbi-TaDREB3, L7-10-3 21 7 33 *
pUbi-TaDREB3, L11-10-6 18 8 44 **
Control 58 7 12
Exp. 2
pRab17-TaDREB2, L2-5-1 20 7 35 n.s.
pRab17-TaDREB2, L11-2-5 13 7 53 n.s.
pRab17-TaDREB3, L8-1-5 18 10 55 n.s.
pRab17-TaDREB3, L8-4-7 9 5 55 n.s.
Control 23 7 30
†Differences in recovery between transgenic lines and control plants were
tested in a Pearson’s Chi-squared test (n.s., *, **, ***, mean
nonsignificant differences, P-value <0.1, <0.01, <0.001, respectively).
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 237
Drought-inducible expression of DREB factors
increases the plant survival after severe drought
stress
Wheat plants transformed with pRab17-TaDREB2 and
pRab17-TaDREB3 constructs showed no developmental
setbacks during the first 3 weeks after germination. As the
point water was withheld, transgenic wheat plants were
the same or very similar to control plants. Drought recov-
ery experiments were performed using the same conditions
as above but the length of drought was 14 days (over the
last 10 days the VWC in soil was lower than 3%). During
the drought exposure, the behaviour of control and trans-
genic plants was nearly the same: all plants dried at a simi-
lar rate and were showing severe damage at the last day
before re-watering although the transgenic plants
remained marginally greener (Figure 6a). One week after
re-watering, only 14% of control plants had recovered,
whereas between 33% to 100% of the transformed plants
recovered, depending on line (Figure 6a,b). The transgenic
lines showing the strongest recovery from drought stress
tended to be those showing the strongest induction of
expression under drought stress. A larger collection of
transgenic lines then used here would be needed to estab-
lish a clear correlation with expression levels.
Wheat plants transformed with pRab17-TaDREB3
recovered more quickly than plants transformed with
pRab17-TaDREB2, and started to flower 3 weeks after re-
watering; plants transformed with pRab17-TaDREB2
started to flower 3–4 days later than this (Figure 6a). Three
weeks after re-watering, both transgenic populations
looked healthy; they had similar size of spikes and number
of fertile florets compared with well-watered control
plants. Two of 20 control plants survived drought stress
but recovered much more slowly than transgenic plants.
They remained about one-third of normal size when they
started to flower and subsequently produced fewer and
smaller spikes compared to well-watered controls.
Activity of maize rab17 promoter in wheat and
barley
Northern blot and Q-PCR analysis of the expression of
DREB factors under Rab17 promoter were performed
using leaf samples collected 1 day before watering was
stopped, 3 days after plants showed clear signs of wilting
(VWC in soil 2%) and, for some lines, 3 weeks after
re-watering. Both Northern blots (data not shown) and
Q-PCR analysis of barley plants revealed relatively high
basal level of activity of the Rab17 promoter in leaves in
the absence of stress (Figure S2a). Levels of basal activity
differed between independent transgenic lines of barley.
Developmental abnormalities observed in some plants cor-
related with levels of basal promoter activity. No or very
(a)
(c) (d) (e)
(b)
Figure 5 Increased frost tolerance of T2
transgenic barley plants with constitutive
expression of TaDREB2 and TaDREB3. (a and
b) plants after mild frost test ()4 �C) 6 h
(Panel a) and 2 days (Panel b) after treat-
ment; (c–e) plants after severe frost test
()6 �C): (c) before treatment, (d) 1 week
after treatment, (e) 3 weeks after treatment.
Control plants are indicated by red arrow.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.238
little basal activity of the maize Rab17 promoter was
detected in wheat by both Northern blot hybridization
and Q-PCR. However, drought stress quickly and strongly
activated the ZmRab17 promoter (Figures 7a and S2b).
Exogenous DREB expression was limited to the duration
of the stress and the first 1–2 days of recovery after
re-watering. This led to minimal undesired changes in
plant development but was sufficient to confer improved
plant survival after drought stress. In both barley and
wheat, re-watering led to the rapid down-regulation of
Control Line 3 Line 30pRab17-TaDREB2 (T1)
Line 10-2Line 9-10Line 7-7
Two weeks of drought
One week after re-watering
Beforedrought(day 0)
Three weeks after re-watering
pRab17-TaDREB3 (T2)(a)
(b)
0
20
40
60
80
100
120
Sur
vive
d pl
ants
(%
)
pRab17-TaDREB2 pRab17-TaDREB3
(c)
g s (m
mol
/m2 s
)LW
P (
MP
a)
Predawn leaf water potential (MPa)
0 –0.2 –0.4 –0.6 0 –0.2 –0.4 –0.6
0
–0.5
–1.5
–1
300
200
0
100
pRab17-TaDREB2 pRab17-TaDREB3Figure 6 Behaviour of wheat plants with
drought-inducible expression of TaDREB2
and TaDREB3 during a ‘survival’ drought tol-
erance test (a, b) and under moderate water
deficit (c). (a) Pictures of transgenic plants at
different stages of the drought test; (b) Per-
centage of plants that survived for several
independent transgenic lines. (c) Stomatal
conductance and leaf water potential of
mature leaves at midday for two TaDREB2
transformed lines (L2-4-3, blue triangle and
L5-4, blue square), two TaDREB3 trans-
formed lines (L7-7-1, red triangle and L10-2-
2, red square) and control plants (black dots
and lines) measured for three water regimes
(well watered, )0.3 and )0.6 MPa of pre-
dawn leaf water potential).
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 239
the Rab17 promoter, but low levels of transgene tran-
scripts were still detectable 2 weeks after re-watering
(Figure S2b). We have not observed any negative influ-
ence of the low level transgene expression on flowering
time, size, the number and shape of spike and size or
shape of grain.
Barley plants with moderate levels of basal activity of
Rab17 promoter were used in the frost tolerance test as a
model for moderate constitutive expression. No induction
of the Rab17 promoter by cold ⁄ frost temperatures was
detected (Figure S2c). Moderate constitutive over-expres-
sion of both TaDREB2 and TaDREB3 led to considerable
improvement of frost tolerance, which was, however, a
bit lower then improvement in frost tolerance under the
strong 2 · 35S promoter (Table. 1; Figure 5g). On the
other hand, the moderate constitutive expression seen
with Rab17-driven expression in barley significantly
reduced the negative pleiotropic effects on development.
Activation of stress-inducible genes by inducible
over-expression of DREB factors
Expression of nine wheat LEA ⁄ COR ⁄ DHN genes known to
be induced by drought and cold were examined in the
transgenic plants. The expression results were initially used
to determine a ratio of expression levels under drought
stress (time of sampling: 4 days after soil VWC reached
2%) relative to well-watered conditions. These data are
then used to calculate the increase in induction of expres-
sion in transgenic plants relative to induction in control
plants (Figure 7b). This reflects additional induction of
these genes by DREB transgenes relative to induction
solely by drought and potentially related to the effects of
the endogenous DREB genes.
Although induction by the transgene reached 50-fold
for some LEA ⁄ COR ⁄ DHN genes, most genes showed lower
induction. Activation of some LEA ⁄ COR ⁄ DHN genes
appeared to be specific for only one of the transgenes.
For example, the induction of expression of TaRAB17 was
much stronger in TaDREB3 transgenic lines, while induc-
tion of expression of TaWZY2 was stronger in TaDREB2
transgenic plants.
Both constitutive and inducible expression of TaDREB3
in transgenic barley plants lead to specific up-regulation of
cold-inducible HvCOR14B gene. Levels of HvCOR14B cor-
related well with levels of TaDREB3 over-expression
(Table S2). No up-regulation of HvCOR14B was detected
in TaDREB2 transgenic plants (Figure S3).
0
100
200
300
400
500
600
Copie
s×
10
5/g
mR
NA
pRab17-TaDREB2
0
5
10
15
20
25
30
35
Copie
s×
10
5/
gm
RN
A
pRab17-TaDREB3
0
10
20
30
40
50
60
Fold
TaRAB16.5
TaWZY2
TaWlt10
TaWcor18
TaWCS19
TaWcor410
TaRAB18
TaRAB17
TaWcor80
pRab17-TaDREB3pRab17-TaDREB2
(a)
(b)
Figure 7 Expression of the transgene and stress-inducible LEA ⁄ COR ⁄ DHN genes in transgenic wheat plants with inducible over-expression of TaD-
REB2 and TaDREB3. (a) Expression of transgenes under well-watered (W) and drought (D) conditions; (b) up-regulation of stress responsive genes
in transgenic plants expressed as fold up-regulation by drought relative to well watered and normalized against controls.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.240
Discussion
Real and seeming drought tolerance
The grain of cereals has a generally high level of ABA rela-
tive to other plant organs and shows strong induction of
expression of genes that are up-regulated in other plant
tissues only in response to environmental stresses (Ali-
Benali et al., 2005; Sreenivasulu et al., 2006). Some of
these genes, including LEA ⁄ COR ⁄ DHN genes, are probably
involved in the protection of cells during grain maturation
and desiccation and help maintain cells and tissues viability
until germination (Ali-Benali et al., 2005; Rorat, 2006). In
the absence of stress, endogenous TaDREB2 and TaDREB3
were expressed at higher levels in flower and grain tissues,
relative to leaves (Figure 2a). However, transcript levels of
both genes were strongly up-regulated in leaves by
drought and slightly by cold (Figure 2b,c). These patterns
of expression suggest a role for TaDREB2 and TaDREB3 in
protection of plant tissues from desiccation. Therefore,
strong up-regulation of expression of these factors in
wheat and barley may help to increase survival under
water deficit.
One of the first reactions of plants to abiotic stress is to
decrease growth rates (Boyer, 1970). This allows plant to
decrease water consumption and save energy. Constitutive
over-expression of most DREB ⁄ CBF genes tested so far in
transgenic plants leads to stunted growth, mild or strong
dwarfism, slower development and a delay in flowering
time (Kasuga et al., 1999; Kim et al., 2004; Oh et al.,
2007). One of the reasons for the smaller size could be
because of down-regulation of gibberellin deactivating
genes (Magome et al., 2004, 2008). This undesirable agro-
nomic trait, although a natural physiological reaction of
plants to drought, becomes severe in transgenic plants
with strong constitutive over-expression of stress-related
regulatory genes. These negative phenotypes were also
observed in our transgenic plants with constitutive over-
expression of TaDREB2 and TaDREB3 (Table S1). However,
the slow growth of transgenic plants makes it difficult to
compare transgenic with control plants and complicates
analysis of changes in drought tolerance. Even small differ-
ences in plant development lead to errors in the assess-
ment of drought tolerance and imprecise or incorrect
conclusions. However, the analysis of the regulation of
stress-inducible genes (Figure 4) suggested that the
improvement in drought tolerance of plants seen with
constitutive over-expression of TaDREB2 and TaDREB3 is
not simply a reflection of stunted growth but also the
result of increased protection of cells from desiccation on
the molecular level.
Partial normalization of transgenic plant growth and
flowering time became noticeable in the T2 generation
and subsequent generations of transgenic plants. This nor-
malization of development cannot be explained by dimin-
ishing of transcription levels of transgene, as transgene
expression was assessed in each generation and was not
seen to decline. It also seems unlikely that protein modifi-
cation or turnover changed as drought and frost tolerance
as well as levels of induction of some stress-inducible
genes in T1–T4 generations of transgenic plants remained
roughly the same and correlated with transgene expres-
sion. However, generation-dependent changes in gene
expression were observed for several groups of down-
stream genes encoding CBF ⁄ DREB factors (Figure 4b,c)
and some aquaporins (data not shown). For these genes,
expression was down-regulated in T0 plants but returned
to normal in later generations of transgenic plants. This
suggests that plants use alternative regulatory pathways to
normalize the expression of genes which are indirectly reg-
ulated by TaDREB2 and TaDREB3 and might otherwise dis-
turb plant development in the absence of stress. It should
also be noted that the plants selected for further study
were those that showed the strongest expression levels
rather than based on zygocity. Consequently, some of the
variation in overall expression was likely because of selec-
tion for homozygous lines from the T2 generation
onwards.
TaDREB2 and TaDREB3 are frost tolerance genes
Protein alignment of TaDREB2 and TaDREB3 to sequences
of other DREB ⁄ CBF factors from barley and wheat (Fig-
ure 1) revealed that both proteins are close homologues
of CBF factors, which were demonstrated to be involved
in cold-stress response. The TaDREB2 is a close homologue
of CBF7 from barley and from Triticum monoccocum.
Southern blot hybridization to nullisomic-tetrasomic lines
of hexaploid wheat (Sears, 1954) with full-length cDNA of
TaDREB2 as probe revealed that the gene is located on
Group 3 chromosomes of hexaploid wheat and most likely
present as a single copy (data not shown). In contrast,
TmCBF7 was mapped on chromosome 5A at or near the
Fr-A2 locus of T. monoccocum and was previously
described as related to frost tolerance (Vagujfalvi et al.,
2005). TaDREB2 protein has highest sequence conserva-
tion with TINY from Arabidopsis thaliana, which belongs
to the small subfamily of proteins with sequences distinct
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 241
from both DREB1 and DREB2 subfamilies from Arabidopsis
(Sun et al., 2008).
TaDREB3 is a close homologue of TmCBF5 (Figure 1).
The TmCBF5 gene was mapped on chromosome 7A of
T. monoccocum (Miller et al., 2006) and TaDREB3 also
mapped on the Group 7 chromosome of Triticum aes-
tivum and is also most likely a single copy gene (data not
shown). However, the bread wheat orthologues of
TmCBF5, TaCBF5, is differentially expressed in cold accli-
mated frost tolerant and frost sensitive lines relative to
nonacclimated controls and hence remains an interesting
candidate gene for the frost tolerance (Sutton et al.,
2009). It was demonstrated that both TaDREB2 and TaD-
REB3 are weakly up-regulated by cold (Figure 2c).
Strong up-regulation of some of drought ⁄ cold stress-
inducible LEA ⁄ DHN ⁄ COR genes was detected in transgenic
barley plants with constitutive over-expression of TaDREB3
under normal growing conditions (Figure 4). Analysis of
the expression levels of 10 different CBF ⁄ DREB genes,
some of which were mapped in vegetative frost tolerance
QTLs, suggests that activation of LEA ⁄ DHN ⁄ COR genes is
a result of co-operative action of DREB3 and CBFs
(Figure 4). As expected, levels of up-regulation of the
stress-inducible genes in transgenic plants differ between
plants having constitutive or inducible expression of the
transgene: on the whole, up-regulation under the induc-
ible promoter in transgenic barley is lower and some
genes that were up-regulated under constitutive expres-
sion were not up-regulated when the inducible promoter
was used (data not shown). This can be explained by
different spatial patterns of transgene expression under
2 · 35S and Rab17 promoters and the relatively short
period of activity of the Rab17 promoter.
Previous work with close homologues ⁄ orthologues of
TaDREB2, TaDREB3 and several LEA ⁄ COR ⁄ DHN genes
including Cor14 (Vagujfalvi et al., 2000; Miller et al.,
2006; Ganeshan et al., 2008) suggested a possible
involvement of these genes in cold and frost tolerance.
Furthermore, constitutive expression of TaDREB2 and TaD-
REB3 leads to the constitutive expression of a large num-
ber of genes normally induced by cold stress. The elevated
expression of these genes may reduce the requirement for
cold acclimation of transgenic plants. Consequently, the
survival rates improved for T2 and T3 transgenic barley
seedlings expressing TaDREB2 and TaDREB3 under temper-
atures as low as )6 �C and with very short (several hours)
acclimation (Figure 5). The survival rates of all the lines
tested were higher than for control plants grown under
the same conditions (Table 1).
Strong constitutive expression of transgenes led to neg-
ative developmental phenotypes, therefore weak constitu-
tive expression was also investigated to see if frost
tolerance was still observed. The ZmRab17 promoter was
used as this promoter showed moderate basal level of
expression in barley leaves in the absence of drought
stress (Figure S2a,c). Although this promoter is strongly
induced by drought, it is not induced by cold (Figure S2c)
and, in the absence of drought stress, can be used as a
moderate ‘constitutive’ promoter. Frost survival rates in
barley plants expressing TaDREB2 or TaDREB3 under mod-
erate ZmRab17 promoter were slightly lower than for
plants with the strong 2 · 35S promoter but higher than
for control plants (Table 1). Therefore, the Rab17
promoter from maize could be used together with
TaDREB2 and TaDREB3 to improve both drought and frost
survival rates in barley with minimal changes in plant
development.
Reduction of negative effects on plant development
The promoter of the Rab17 gene from maize (Vilardell
et al., 1990) was used to drive strong drought specific
expression of TaDREB2 and TaDREB3. According to results
of others (Close et al., 1989; Mundy et al., 1990) and
results presented here, the Rab17 promoter has low basal
activity in most plant tissues but is stronger in embryo and
developing endosperm. It is rapidly and strongly induced
by drought but not by cold. Genes similar to the maize
Rab17 were isolated from other plant species, and in
many cases promoter activity was similar (Michel et al.,
1994). ABA-induced activation of the Rab17 promoter
was studied in heterologous systems (in stably transformed
tobacco and by transient expression in rice protoplasts)
and its drought inducibility was initially explained by the
presence of the ABRE in a 100 bp segment of the pro-
moter (Vilardell et al., 1991). Later, several cis-elements
(five putative ABREs and four other sequences) important
for the strong activity were mapped in the promoter (Busk
et al., 1997). Six of these elements were shown to be
important for expression in embryos, whereas only three
elements were responsible for the basal and stress-induc-
ible expression in leaves. Among these elements was a
new GC-rich rab Activator element, CACTGGCCGCCC,
responsible for the low constitutive expression of Rab17 in
maize leaves, and the drought responsive elements (DREs)
(Busk et al., 1997). Finally, it was found that the ZmRab17
promoter is activated by ABA, drought and salt
stress through the single DRE (DRE2). Two AP2 domain
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.242
containing transcription factors from maize, designated
DBF1 and DBF2, were isolated in a yeast one-hybrid screen
using DRE2 as bait. Promoter activation by over-expression
of DBF1 and repression by over-expression of DBF2 were
demonstrated (Kizis and Pages, 2002). ABA appears to
play a role in the regulation of DBF activity, and the ABA-
dependent pathway was suggested as the regulatory
mechanism that acted through the C-repeat ⁄ DRE element
(Kizis and Pages, 2002). The results presented by Kizis and
Pages (2002) suggested that the Rab17 promoter could be
suitable for drought-inducible over-expression of DREB fac-
tors. The promoter is strong, has low basal activity in
maize, induction of the promoter starts within several
hours of the plant sensing water deficit, and its activity
quickly returns to basal level after re-watering. Regulation
of the promoter by DREB factors under stress conditions
could potentially further increase activity of the promoter
in transgenic plants by a feedback loop.
Analysis of the activity of this promoter in barley and
wheat demonstrates that in these plants, the promoter
behaves similarly to maize; under drought stress it is rap-
idly and strongly induced in leaves, whereas cold stress
does not induce expression from the ZmRab17 promoter
(Figure S2c). Different levels of basal activity were
observed in transgenic barley and wheat if DREB factors
were used as transgenes (Figure S2). However, these dif-
ferences were not observed when transcription factors
from other families were over-expressed by this promoter
(data not shown). This difference may be because of dif-
ferences in the ability of TaDREB2 and TaDREB3 to nega-
tively influence expression of genes responsible for the
basal activity of the promoter in barley as opposed to
wheat.
The high basal activity of the Rab17 promoter in barley
relative to wheat still leads to negative phenotypes. How-
ever, these are far milder than the negative phenotypes of
transgenic barley with a strong constitutive promoter. In
contrast, wheat transgenic plants before stress and after
re-watering were difficult to distinguish from control
plants.
Enhanced survival of transgenic wheat plants under
drought stress can be explained by up-regulation of wheat
LEA ⁄ COR ⁄ DHN genes to higher levels that under normal
drought stress. In control plants, most of the tested LEA ⁄ -COR ⁄ DHN genes have low to moderate basal levels of
expression in the absence of stress and are strongly
up-regulated by drought. In transgenic wheat plants under
well-watered conditions, expression of these genes
remained at the same level as in well-watered control
plants. However, under drought, expression levels of the
wheat LEA ⁄ COR ⁄ DHN genes examined were from 1.5- to
50-fold higher than in stressed control plants (Figure 7b).
All genes except TaRAB17 were more strongly up-regu-
lated in TaDREB2 transgenic versus control plants. The
strongest up-regulation was observed for Wcor18,
Wcor80, TaRAB16.5, and TaRAB18. Wlt10 and Wcor410
were weakly up-regulated in both transgenics. These data
and the results of the analysis of stress-inducible genes in
barley indicated qualitative and quantitative differences in
up-regulation of downstream genes by TaDREB2 and
TaDREB3 which probably resulted in differences of the
developmental and stress phenotypes of the transgenic
lines.
Several important conclusions can be made from the
results of this work. Constitutive up-regulation of TaDREB2
and TaDREB3 in transgenic barley plants improves survival
rates under severe drought and frost stresses but this leads
to negative developmental phenotypes. The undesired
changes in plant development can be at least partially pre-
vented by use of weak constitutive and stress-inducible
promoters. The promoter used here had low activity in the
absence of stress was induced by drought stress and
quickly returned to basal levels after re-watering.
The enhanced stress tolerance appeared to result from
the up-regulation of many stress-inducible genes involved
in the protection of cell integrity under severe stress. Inde-
pendent transgenic lines varied in their drought recovery
rates (33%–100%) and this variation appeared to be
related to the level of induction of expression under
drought stress. However, a more extensive study would be
needed to confirm this correlation.
Improved ‘survival’ under severe drought did not pro-
vide any advantage to transgenic compared to control
plants during prolonged growth under water limited con-
ditions. These conditions were sufficient for plant survival
but negatively influence crop yields. Comparison of the
transgenic and control plants that survived the drought
stress showed no improvement in grain yield; in fact the
transgenic showed a slightly reduced yield. However, the
greatly increased plant survival should translate to
improved overall yield under field condition. Clearly, field
evaluation is now necessary to determine the efficacy of
these transgenes.
For vegetative frost tolerance, good results were
obtained by using moderate constitutive expression. Fur-
ther improvement may be achievable by using weak cold-
inducible promoters, with low basal activity and ⁄ or tissue
specificity.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 243
Both TaDREB2 and TaDREB3 strongly regulate many dif-
ferent CBF ⁄ DREB genes from barley. Although the
TaDREB2 and TaDREB3 proteins are structurally very differ-
ent, they appear to regulate similar DREB ⁄ CBF genes. It
seems probable that the same situation applies to other
studies where DREB ⁄ CBF factors were over-expressed. The
stress tolerant phenotypes and regulation of downstream
genes described in multiple ‘DREB’ papers may have
resulted from the simultaneous activation or repression of
other DREB ⁄ CBF factor(s). This may have lead to nonspe-
cific binding and activation of nontarget promoters result-
ing in loss of specificity of transgene action and the strong
negative phenotypes often reported. The use of weak, tis-
sue-specific and stress-inducible promoters should partially
alleviate this problem.
Experimental procedures
Plasmid construction and plant transformation
Full-length cDNAs of TaDREB2 (Acc. DQ353852) and TaDREB3
(Acc. DQ353853) isolated in the Y1H screen from a wheat grain
cDNA library (Lopato et al., 2006) were used as templates. Cod-
ing regions of TaDREB2 and TaDREB3 cDNAs were cloned into: (i)
the pMDC32 vector (Curtis and Grossniklaus, 2003) downstream
of the vector’s duplicated 35S promoter and (ii) a pMDC32 vector
in which the 2 · 35S promoter was excised using HindIII–KpnII
restriction sites and replaced with a 634 bp fragment of the
ZmRab17 promoter (Busk et al., 1997). All four constructs were
transformed into barley (Hordeum vulgare L. cv. Golden Promise)
using Agrobacterium-mediated transformation and the method
developed by Tingay et al. (1997) and modified by Matthews
et al. (2001). Wheat (T. aestivum L. cv. Bobwhite) was trans-
formed using microprojectile bombardment as described by Koval-
chuk et al. (2009). pRab17-TaDREB2-nos and pRab17-TaDREB3-
nos fragments were excised from the respective constructs using
PmeI and BsaXI, gel purified and co-transformed together with
the pUbi-hpt-nos cassette (3676 bps fragment of the vector
plasmid, cut with PmeI–SmaI) into wheat using microprojectile
bombardment.
Isolation and analysis of genomic clones
Genomic sequences and 5¢ upstream regulatory sequences of or-
thologues ⁄ homologues of TaDREB2 and TaDREB3 were isolated
using the procedure described by Kovalchuk et al. (2009) from
the BAC library prepared from T. durum L. cv. Langdon. The full-
length cDNAs of TaDREB2 and TaDREB3 were used as probes.
Four BAC clones with strong hybridization signals were isolated
with TaDREB2 as a probe. All contained the same gene, which is
identical to TaDREB2 and thus was designated TdDREB2. Only
one clone with a strong hybridization signal was selected for TaD-
REB3. It encodes a close homologue of TaDREB3 and this gene
was designated DREB3-like (TdDREB3L). The 1972- and 2749-bp
long promoter sequences were isolated for TdDREB2 and
TdDREB3L, respectively. The promoters were analysed for the
presence of potential stress-related elements using PLACE soft-
ware (http://www.dna.affrc.go.jp/PLACE/signalscan.html) and a
database of plant cis-acting regulatory DNA elements (Higo et al.,
1999).
Plant growth and stress conditions
For phenotypic analysis, plants were grown under glasshouse con-
ditions with an average day and night temperature of 25 and
16 �C, respectively, with the day length extended to 15 h with
supplemental lighting. T1 and T2 generation plants were moni-
tored for changes in growth rate, plant height, heading time,
number of tillers, spike phenotype, grain phenotype and yield.
Null segregants from the transgenic lines and untransformed
plants were used as controls.
Seedlings for the ‘survival’ drought tolerance test were grown
under growth room conditions, with a 16 h day at 24 �C and
night temperatures of 16 �C. Progeny of T1 and T2 generations of
transgenic plants (10 plants per independent transgenic line) were
grown either in 12-cm square pots or as pairs of control and
transgenic plants in 20-cm pots for 3 weeks. The volumetric water
content (VWC) of each pot was monitored at least every second
day during the experiment and plants with the same VWC were
used for comparison and documentation. Three weeks after ger-
mination, water was withheld. Seven to 10 days after the pots
reached 2%–3% VWC and wilting was observed, the plants were
re-watered. Plants were assessed for recovery after 1 and 3 weeks
of re-watering, and stress-tolerant plants were transferred to the
glasshouse for generation of seeds. Leaf samples were collected
from well-watered plants 1 day before withholding water (for all
tested transgenic and several control plants), 3–4 days after the
VWC had reached 2% and transgenic lines with inducible pro-
moter and several control plants showed clear wilting.
To check if differences in stomatal conductance and leaf water
status could explain the differences of recovery after severe
drought, an experiment was carried out at stable soil water con-
tent. Pots (22 and 22 cm in deep) were filled with 4 kg of soil
and sampled for measurement of water content. One plant of
each line (two DREB2 and two DREB3 transformed lines) and one
control plant were sown in each pot. At the two-leaf stage, soil
was dried and maintained at the target soil water content by
watering every 2 days. Three different soil water contents were
tested: well watered (VWC = 16%), VWC = 6% corresponding to
a predawn soil water potential of )0.3 MPa and VWC = 4% cor-
responding to a predawn soil water potential of )0.6 MPa.
Because a VWC of 2.5% corresponded to the wilting point, this
level of water deficit was not tested. Stomatal conductance was
measured at midday, the day after watering with a diffusion
porometer (Decagon SC-1 Leaf Porometer, Pullman, WA, USA) on
nonexpanding leaves. Leaf water potential was measured at the
same time on nonexpanding leaves. Leaves were cut, placed into
a Scholander-type pressure chamber (Soil Moisture Equipment
Corp., Santa Barbara, CA, USA). The pressure at which extruded
sap first began to wet the cut surface was registered as the oppo-
site of leaf water potential.
The most appropriate temperature and length of treatment for
assessing frost tolerance was determined as the treatment that
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.244
killed most of the control barley plants. Seeds were sown in 20-
cm pots and grown in the growth chamber with 12 h light at 23
and 18 �C at night temperatures. Seedlings at the four-leaf stage
(approximately 3 weeks after germination) were transferred to a
cold chamber (BINDER, Tuttlingen, Germany). To protect the plant
roots from frost damage, pots were insulated. Temperature pro-
files differed in the severity and duration of the minimum temper-
ature, which were either )5 �C for 1 h, or )6 �C for 1, 6, 12 and
14 h in preliminary tests performed on control barley plants. In all
experiments, the ice nucleating agent SNOMAX� (Sno-Quip Pty
Ltd, Mittagong, NSW, Australia) was sprayed onto plants as a
2 g ⁄ L solution 2 h before the chamber reached the minimum
temperature. A HOBO data logger (Onset Computing, USA) was
used to control and record temperature, light intensity and
humidity in the chamber. After the frost treatment, plants were
moved back to the growth chamber and maintained under the
same growth conditions as before the frost treatment. The num-
ber of recovered plants was recorded after 24 h of recovery.
Based on these preliminary tests, the minimum temperature of
)6 �C for 12 h was selected to test frost tolerance in the trans-
genic barley plants. Only 10%–20% of control plants survived
and recovered under this regime.
In each experiment, three sublines for each independent trans-
genic line and from each subline progeny of 12 plants were
tested for frost tolerance. Three transgenic plants were grown
along with one control plant in each pot (20 cm). The experiment
was repeated at least three times for each transgenic line. Expres-
sion of the transgene was assessed by Northern blot hybridization
using total RNA from tissues collected shortly before the frost
treatment and when the temperature reached 4 �C (for ZmRab17
promoter only).
Analysis of gene expression
Transgene presence and expression in the T0–T4 generations of
transgenic plants were analysed by either Southern or Northern
blot hybridization as described by Sambrook and Russell (2001) or
by RT-PCR and Q-PCR. Q-PCR analysis was performed using prim-
ers from the coding region and nos terminator for transgenes and
primers from 3¢ untranslated regions of endogenous stress-induc-
ible genes. cDNAs prepared using SuperScript III reverse transcrip-
tase (Invitrogen, Carlsbad, CA, USA) from leaf tissues of control
and transgenic plants collected before and during stress and dur-
ing plant recovery were used as a template. Leaf tissues from sev-
eral independent lines and several consecutive generations of
transgenic plants were used for the analysis of downstream
genes. Each PCR was repeated three times. The Q-PCR procedure
was described by Burton et al. (2008). mRNA copy number for
each tested gene was normalized against four control genes as
described by Burton et al. (2008). Primer details appear in
Table S2. Different tissues of T. aestivum cv. Chinese spring plants
were used for tissue-based analysis of expression of endogenous
TaDREB2 and TaDREB3. Inducibility by drought was analysed in
several independent plants of T. aestivum cv. RAC875. Material
was collected from plants grown under well-watered conditions
and 3–4 days after the plants had started to show signs of
drought stress. For cold inducibility, analysis of two independent,
6-week-old plants of T. aestivum cv. RAC875 were incubated at
4 �C, and leaf material was collected at 0, 1, 4, 24 and 48 h after
plants were transferred to 4 �C. For the wounding experiment,
leaf and 10–15 DAP old grain from two independent plants of
T. aestivum cv. Chinese spring were wounded using a fine metal
brush, and material was sampled at 0, 0.5, 1, 2, 3, 8 and 17 h
after wounding.
Acknowledgements
We thank M. Pallotta and N. Bazanova for the technical
assistance with isolation and characterization of BAC
clones, Dr K. Oldach for providing us with Q-PCR primers
for wounding-inducible genes, and Dr U. Langridge and R.
Hosking for assistance with growing plants. This work was
supported by the Australian Research Council, the Grains
Research and Development Corporation and the Govern-
ment of South Australia and the University of Adelaide.
References
Agalou, A., Purwantomo, S., Overnas, E., Johannesson, H., Zhu,
X., Estiati, A., de Kam, R.J., Engstrom, P., Slamet-Loedin, I.H.,
Zhu, Z., Wang, M., Xiong, L., Meijer, A.H. and Ouwerkerk, P.B.
(2008) A genome-wide survey of HD-Zip genes in rice and
analysis of drought-responsive family members. Plant Mol. Biol.,
66, 87–103.
Agarwal, P.K., Agarwal, P., Reddy, M.K. and Sopory, S.K. (2006)
Role of DREB transcription factors in abiotic and biotic stress
tolerance in plants. Plant Cell Rep., 25, 1263–1274.
Al-Abed, D., Madasamy, P., Talla, R., Goldman, S. and
Rudrabhatla, S. (2007) Genetic engineering of maize with the
Arabidopsis DREB1A ⁄ CBF3 gene using split-seed explants. Crop
Sci., 47, 2390–2402.
Ali-Benali, M.A., Alary, R., Joudrier, P. and Gautier, M.F. (2005)
Comparative expression of five Lea Genes during wheat seed
development and in response to abiotic stresses by real-time
quantitative RT-PCR. Biochim. Biophys. Acta, 1730, 56–65.
Baker, S.S., Wilhelm, K.S. and Thomashow, M.F. (1994) The 5’-
region of Arabidopsis thaliana cor15a has cis-acting elements
that confer cold-, drought- and ABA-regulated gene expression.
Plant Mol. Biol., 24, 701–713.
Bartels, D., Furini, A., Ingram, J. and Salamini, F. (1996)
Responses of plants to dehydration stress: a molecular analysis.
Plant Growth Regul., 20, 111–118.
Bevan, M.W., Mason, S.E. and Goelet, P. (1985) Expression of
tobacco mosaic-virus coat protein by cauliflower mosaic-virus
promoter in plants transformed by Agrobacterium. EMBO J., 4,
1921–1926.
Bhatnagar-Mathur, P., Devi, M.J., Reddy, D.S., Lavanya, M.,
Vadez, V., Serraj, R., Yamaguchi-Shinozaki, K. and Sharma,
K.K. (2007) Stress-inducible expression of At DREB1A in
transgenic peanut (Arachis hypogaea L.) increases transpiration
efficiency under water-limiting conditions. Plant Cell Rep., 26,
2071–2082.
Boyer, J.S. (1970) Leaf enlargement and metabolic rates in corn,
soybean, and sunflower at various leaf water potentials. Plant
Physiol., 46, 233–235.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 245
Brini, F., Hanin, M., Lumbreras, V., Amara, I., Khoudi, H.,
Hassairi, A., Pages, M. and Masmoudi, K. (2007)
Overexpression of wheat dehydrin DHN-5 enhances tolerance
to salt and osmotic stress in Arabidopsis thaliana. Plant Cell
Rep., 26, 2017–2026.
Buchanan, C.D., Klein, P.E. and Mullet, J.E. (2004) Phylogenetic
analysis of 5 ‘-noncoding regions from the ABA-responsive rab
16 ⁄ 17 gene family of sorghum, maize and rice provides insight
into the composition, organization and function of cis-
regulatory modules. Genetics, 168, 1639–1654.
Burton, R.A., Jobling, S.A., Harvey, A.J., Shirley, N.J., Mather,
D.E., Bacic, A. and Fincher, G.B. (2008) The genetics and
transcriptional profiles of the cellulose synthase-like HvCslF gene
family in barley. Plant Physiol., 146, 1821–1833.
Busk, P.K., Jensen, A.B. and Pages, M. (1997) Regulatory
elements in vivo in the promoter of the abscisic acid responsive
gene rab17 from maize. Plant J., 11, 1285–1295.
Caramelo, J.J. and Iusem, N.D. (2009) When cells lose water:
lessons from biophysics and molecular biology. Prog. Biophys.
Mol. Biol., 99, 1–6.
Chakrabortee, S., Boschetti, C., Walton, L.J., Sarkar, S.,
Rubinsztein, D.C. and Tunnacliffe, A. (2007) Hydrophilic protein
associated with desiccation tolerance exhibits broad protein
stabilization function. Proc. Natl Acad. Sci. USA, 104, 18073–
18078.
Chen, J.Q., Dong, Y., Wang, Y.J., Liu, Q., Zhang, J.S. and Chen,
S.Y. (2003) An AP2 ⁄ EREBP-type transcription-factor gene from
rice is cold-inducible and encodes a nuclear-localized protein.
Theor. Appl. Genet., 107, 972–979.
Chen, M., Wang, Q.Y., Cheng, X.G., Xu, Z.S., Li, L.C., Ye, X.G.,
Xia, L.Q. and Ma, Y.Z. (2007) GmDREB2, a soybean DRE-
binding transcription factor, conferred drought and high-salt
tolerance in transgenic plants. Biochem. Biophys. Res.
Commun., 353, 299–305.
Chen, J.Q., Meng, X.P., Zhang, Y., Xia, M. and Wang, X.P. (2008)
Over-expression of OsDREB genes lead to enhanced drought
tolerance in rice. Biotechnol. Lett., 30, 2191–2198.
Chen, J.H., Xia, X.L. and Yin, W.W. (2009) Expression profiling and
functional characterization of a DREB2-type gene from Populus
euphratica. Biochem. Biophys. Res. Commun., 378, 483–487.
Christensen, A.H., Sharrock, R.A. and Quail, P.H. (1992) Maize
polyubiquitin genes: structure, thermal perturbation of
expression and transcript splicing, and promoter activity
following transfer to protoplasts by electroporation. Plant Mol.
Biol., 18, 675–689.
Close, T.J., Kortt, A.A. and Chandler, P.M. (1989) A cDNA-based
comparison of dehydration-induced proteins (dehydrins) in
barley and corn. Plant Mol. Biol., 13, 95–108.
Cong, L., Zheng, H.C., Zhang, Y.X. and Chai, T.Y. (2008)
Arabidopsis DREB1A confers high salinity tolerance and
regulates the expression of GA dioxygenases in Tobacco. Plant
Sci., 174, 156–164.
Curtis, M.D. and Grossniklaus, U. (2003) A gateway cloning
vector set for high-throughput functional analysis of genes in
planta. Plant Physiol., 133, 462–469.
Dalal, M., Tayal, D., Chinnusamy, V. and Bansal, K.C. (2009)
Abiotic stress and ABA-inducible Group 4 LEA from Brassica
napus plays a key role in salt and drought tolerance.
J. Biotechnol., 139, 137–145.
Dubouzet, J.G., Sakuma, Y., Ito, Y., Kasuga, M., Dubouzet, E.G.,
Miura, S., Seki, M., Shinozaki, K. and Yamaguchi-Shinozaki, K.
(2003) OsDREB genes in rice, Oryza sativa L., encode
transcription activators that function in drought-, high-salt- and
cold-responsive gene expression. Plant J., 33, 751–763.
Ganeshan, S., Vitamvas, P., Fowler, D.B. and Chibbar, R.N. (2008)
Quantitative expression analysis of selected COR genes reveals
their differential expression in leaf and crown tissues of wheat
(Triticum aestivum L.) during an extended low temperature
acclimation regimen. J. Exp. Bot., 59, 2393–2402.
Gao, M.J., Allard, G., Byass, L., Flanagan, A.M. and Singh, J.
(2002) Regulation and characterization of four CBF transcription
factors from Brassica napus. Plant Mol. Biol., 49, 459–471.
Gao, J.P., Chao, D.Y. and Lin, H.X. (2007) Understanding abiotic
stress tolerance mechanisms: recent studies on stress response
in rice. J. Integr. Plant Biol., 49, 742–750.
Goyal, K., Walton, L.J. and Tunnacliffe, A. (2005) LEA proteins
prevent protein aggregation due to water stress. Biochem. J.,
388, 151–157.
Guilley, H., Dudley, R.K., Jonard, G., Balazs, E. and Richards, K.E.
(1982) Transcription of cauliflower mosaic-virus: detection of
promoter sequences, and characterization of transcripts. Cell,
30, 763–773.
Gutha, L.R. and Reddy, A.R. (2008) Rice DREB1B promoter shows
distinct stress-specific responses, and the overexpression of
cDNA in tobacco confers improved abiotic and biotic stress
tolerance. Plant Mol. Biol., 68, 533–555.
Hao, X., Chen, M., Xu, H., Gao, S., Chen, X., Li, L., Du, L., Ye, X.
and Ma, Y. (2005) Obtaining of transgenic wheats with GH-
DREB gene and their physiological index analysis on drought
tolerance. Southwest China J. Agric. Sci., 18, 616–620.
He, C., Lin, Z., McElroy, D. and Wu, R. (2009) Identification of a
rice Actin2 gene regulatory region for high-level expression of
transgenes in monocots. Plant Biotechnol. J., 7, 227–239.
Higo, K., Ugawa, Y., Iwamoto, M. and Korenaga, T. (1999) Plant
cis-acting regulatory DNA elements (PLACE) database: 1999.
Nucleic Acids Res., 27, 297–300.
Hsieh, T.H., Lee, J.T., Charng, Y.Y. and Chan, M.T. (2002)
Tomato plants ectopically expressing Arabidopsis CBF1 show
enhanced resistance to water deficit stress. Plant Physiol., 130,
618–626.
Huang, B., Jin, L.G. and Liu, J.Y. (2008) Identification and
characterization of the novel gene GhDBP2 encoding a DRE-
binding protein from cotton (Gossypium hirsutum). J. Plant
Physiol., 165, 214–223.
Ito, T., Ito, Y., Maruyama, K., Hiratsu, K., Ohme-Takagi, M.,
Shinozaki, K. and Yamaguchi-Shinozaki, K. (2006) Functional
analysis of a rice transcription factor OsDREB1F involved in
environmental stress-inducible gene expression. Plant Cell
Physiol., 47, S212.
Jaglo-Ottosen, K.R., Gilmour, S.J., Zarka, D.G., Schabenberger, O.
and Thomashow, M.F. (1998) Arabidopsis CBF1 overexpression
induces COR genes and enhances freezing tolerance. Science,
280, 104–106.
James, V.A., Neibaur, I. and Altpeter, F. (2008) Stress inducible
expression of the DREB1A transcription factor from xeric,
Hordeum spontaneum L. in turf and forage grass (Paspalum
notatum Flugge) enhances abiotic stress tolerance. Transgenic
Res., 17, 93–104.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.246
Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K. and
Shinozaki, K. (1999) Improving plant drought, salt, and freezing
tolerance by gene transfer of a single stress-inducible
transcription factor. Nat. Biotechnol., 17, 287–291.
Kasuga, M., Miura, S., Shinozaki, K. and Yamaguchi-Shinozaki, K.
(2004) A combination of the Arabidopsis DREB1A gene and
stress-inducible rd29A promoter improved drought- and low-
temperature stress tolerance in tobacco by gene transfer. Plant
Cell Physiol., 45, 346–350.
Kim, J. (2007) Perception, transduction, and networks in cold
signaling. J. Plant Biol., 50, 139–147.
Kim, H.J., Hyun, Y., Park, J.Y., Park, M.J., Park, M.K., Kim, M.D.,
Kim, H.J., Lee, M.H., Moon, J., Lee, I. and Kim, J. (2004) A
genetic link between cold responses and flowering time
through FVE in Arabidopsis thaliana. Nat. Genet., 36, 167–171.
Kizis, D. and Pages, M. (2002) Maize DRE-binding proteins DBF1
and DBF2 are involved in rab17 regulation through the
drought-responsive element in an ABA-dependent pathway.
Plant J., 30, 679–689.
Kobayashi, F., Ishibashi, M. and Takumi, S. (2008) Transcriptional
activation of Cor ⁄ Lea genes and increase in abiotic stress
tolerance through expression of a wheat DREB2 homolog in
transgenic tobacco. Transgenic Res., 17, 755–767.
Kovalchuk, N., Smith, J., Pallotta, M., Singh, R., Ismagul, A., Eliby,
S., Bazanova, N., Milligan, A.S., Hrmova, M., Langridge, P. and
Lopato, S. (2009) Characterization of the wheat endosperm
transfer cell-specific protein TaPR60. Plant Mol. Biol., 71, 81–
98.
Kume, S., Kobayashi, F., Ishibashi, M., Ohno, R., Nakamura, C.
and Takumi, S. (2005) Differential and coordinated expression
of Cbf and Cor ⁄ Lea genes during long-term cold acclimation in
two wheat cultivars showing distinct levels of freezing
tolerance. Genes Genet. Syst., 80, 185–197.
Lal, S., Gulyani, V. and Khurana, P. (2008) Overexpression of
HVA1 gene from barley generates tolerance to salinity and
water stress in transgenic mulberry (Morus indica). Transgenic
Res., 17, 651–663.
Lee, S.C., Lee, M.Y., Kim, S.J., Jun, S.H., An, G. and Kim, S.R.
(2005) Characterization of an abiotic stress-inducible dehydrin
gene, OsDhn1, in rice (Oryza sativa L.). Mol. Cells, 19, 212–
218.
Li, X.P., Tian, A.G., Luo, G.Z., Gong, Z.Z., Zhang, J.S. and Chen,
S.Y. (2005) Soybean DRE-binding transcription factors that are
responsive to abiotic stresses. Theor. Appl. Genet., 110, 1355–
1362.
Lin, R.C., Park, H.J. and Wang, H.Y. (2008) Role of Arabidopsis
RAP2.4 in regulating light- and ethylene-mediated
developmental processes and drought stress tolerance. Mol.
Plant, 1, 42–57.
Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-
Shinozaki, K. and Shinozaki, K. (1998) Two transcription
factors, DREB1 and DREB2, with an EREBP ⁄ AP2 DNA binding
domain separate two cellular signal transduction pathways in
drought- and low-temperature-responsive gene expression,
respectively, in Arabidopsis. Plant Cell, 10, 1391–1406.
Lopato, S., Bazanova, N., Morran, S., Milligan, A.S., Shirley, N.
and Langridge, P. (2006) Isolation of plant transcription factors
using a modified yeast one-hybrid system. Plant Methods, 2,
3–17.
Magome, H., Yamaguchi, S., Hanada, A., Kamiya, Y. and Oda, K.
(2004) dwarf and delayed-flowering 1, a novel Arabidopsis
mutant deficient in gibberellin biosynthesis because of
overexpression of a putative AP2 transcription factor. Plant J.,
37, 720–729.
Magome, H., Yamaguchi, S., Hanada, A., Kamiya, Y. and Oda, K.
(2008) The DDF1 transcriptional activator upregulates
expression of a gibberellin-deactivating gene, GA2ox7, under
high-salinity stress in Arabidopsis. Plant J., 56, 613–626.
Matthews, P.R., Wang, M.B., Waterhouse, P.M., Thornton, S.,
Fieg, S.J., Gubler, F. and Jacobsen, J.V. (2001) Marker gene
elimination from transgenic barley, using co-transformation
with adjacent ‘twin T-DNAs’ on a standard Agrobacterium
transformation vector. Mol. Breed., 7, 195–202.
Michel, D., Furini, A., Salamini, F. and Bartels, D. (1994) Structure
and regulation of an ABA-responcive and desiccation-
responcive gene from the resurrection plant Craterostigma
plantagineum. Plant Mol. Biol., 24, 549–560.
Miller, A.K., Galiba, G. and Dubcovsky, J. (2006) A cluster of 11
CBF transcription factors is located at the frost tolerance locus
Fr-Am2 in Triticum monococcum. Mol. Genet. Genomics, 275,
193–203.
Mundy, J., Yamaguchi-Shinozaki, K. and Chua, N.H. (1990)
Nuclear proteins bind conserved elements in the abscisic acid-
responsive promoter of a rice rab gene. Proc. Natl Acad. Sci.
USA, 87, 1406–1410.
Nakashima, K., Shinwari, Z.K., Sakuma, Y., Seki, M., Miura, S.,
Shinozaki, K. and Yamaguchi-Shinozaki, K. (2000) Organization
and expression of two Arabidopsis DREB2 genes encoding DRE-
binding proteins involved in dehydration- and high-salinity-
responsive gene expression. Plant Mol. Biol., 42, 657–665.
Neumann, P.M. (2008) Coping mechanisms for crop plants in
drought-prone environments. Ann. Bot., 101, 901–907.
Oh, S.J., Song, S.I., Kim, Y.S., Jang, H.J., Kim, S.Y., Kim, M., Kim,
Y.K., Nahm, B.H. and Kim, J.K. (2005) Arabidopsis
CBF3 ⁄ DREB1A and ABF3 in transgenic rice increased tolerance
to abiotic stress without stunting growth. Plant Physiol., 138,
341–351.
Oh, S.J., Kwon, C.W., Choi, D.W., Song, S.I. and Kim, J.K. (2007)
Expression of barley HvCBF4 enhances tolerance to abiotic
stress in transgenic rice. Plant Biotechnol. J., 5, 646–656.
Qin, F., Sakuma, Y., Li, J., Liu, Q., Li, Y.Q., Shinozaki, K. and
Yamagushi-Shinozaki, K.Y. (2004) Cloning and functional
analysis of a novel DREB1 ⁄ CBF transcription factor involved in
cold-responsive gene expression in Zea mays L. Plant Cell
Physiol., 45, 1042–1052.
Qin, Q.L., Liu, J.G., Zhang, Z., Peng, R.H., Xiong, A.S., Yao, Q.H.
and Chen, J.M. (2007) Isolation, optimization, and functional
analysis of the cDNA encoding transcription factor OsDREB1B in
Oryza Sativa L. Mol. Breed., 19, 329–340.
Rorat, T. (2006) Plant dehydrins—tissue location, structure and
function. Cell. Mol. Biol. Lett, 11, 536–556.
Sakuma, Y., Maruyama, K., Osakabe, Y., Qin, F., Sek, i.M.,
Shinozaki, K. and Yamaguchi-Shinozaki, K. (2006) Functional
analysis of an Arabidopsis transcription factor, DREB2A,
involved in drought-responsive gene expression. Plant Cell, 18,
1292–1309.
Saleh, A., Lumbreras, V., Lopez, C., Dominguez-Puigjaner, E.,
Kizis, D. and Pages, M. (2006) Maize DBF1-interactor protein 1
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 247
containing an R3H domain is a potential regulator of DBF1
activity in stress responses. Plant J., 46, 747–757.
Sales, M.P., Gerhardt, I.R., Grossi-de-Sa, M.F. and Xavier, J.
(2000) Do legume storage proteins play a role in defending
seeds against bruchids? Plant Physiol., 124, 515–522.
Sambrook, J. and Russell, D. (2001) Molecular Cloning: A
Laboratory Manual, 3rd edn. Cold Spring Harbour, New York:
Cold Spring Harbour Laboratory Press.
Schramm, F., Larkindale, J., Kiehlmann, E., Ganguli, A., Englich,
G., Vierling, E. and von Koskull-Doring, P. (2008) A cascade of
transcription factor DREB2A and heat stress transcription factor
HsfA3 regulates the heat stress response of Arabidopsis. Plant
J., 53, 264–274.
Sears, E.R. (1954) The Aneuploids of common wheat. Mo. Agric.
Exp. Sta. Res. Bull., 572, 1–58.
Shen, Y.G., Zhang, W.K., Yan, D.Q., Du, B.X., Zhang, J.S., Liu, Q.
and Chen, S.Y. (2003) Characterization of a DRE-binding
transcription factor from a halophyte Atriplex hortensis. Theor.
Appl. Genet., 107, 155–161.
Sreenivasulu, N., Radchuk, V., Strickert, M., Miersch, O.,
Weschke, W. and Wobus, U. (2006) Gene expression patterns
reveal tissue-specific signaling networks controlling
programmed cell death and ABA- regulated maturation in
developing barley seeds. Plant J., 47, 310–327.
Stockinger, E.J., Gilmour, S.J. and Thomashow, M.F. (1997)
Arabidopsis thaliana CBF1 encodes an AP2 domain-containing
transcriptional activator that binds to the C-repeat ⁄ DRE, a cis-
acting DNA regulatory element that stimulates transcription in
response to low temperature and water deficit. Proc. Natl
Acad. Sci. USA, 94, 1035–1040.
Sugimoto, K., Takeda, S. and Hirochika, H. (2003) Transcriptional
activation mediated by binding of a plant GATA-type zinc
finger protein AGP1 to the AG-motif (AGATCCAA) of the
wound-inducible Myb gene NtMyb2. Plant J., 36, 550–564.
Sun, S., Yu, J.P., Chen, F., Zhao, T.J., Fang, X.H., Li, Y.Q. and Sui,
S.F. (2008) TINY, a dehydration-responsive element (DRE)-
binding protein-like transcription factor connecting the DRE-
and ethylene-responsive element-mediated signaling pathways
in Arabidopsis. J. Biol. Chem., 283, 6261–6271.
Sutton, F., Chen, D.G., Ge, X. and Kenefick, D. (2009) Cbf genes
of the Fr-A2 allele are differentially regulated between long-
term cold acclimated crown tissue of freeze-resistant
and—susceptible, winter wheat mutant lines. BMC Plant Biol.,
9, 34–42.
Taji, T., Seki, M., Yamaguchi-Shinozaki, K., Kamada, H., Giraudat,
J. and Shinozaki, K. (1999) Mapping of 25 drought-inducible
genes, RD and ERD, in Arabidopsis thaliana. Plant Cell Physiol.,
40, 119–123.
Thomashow, M.F., Gilmour, S.J., Stockinger, E.J., Jaglo-Ottosen,
K.R. and Zarka, D.G. (2001) Role of the Arabidopsis CBF
transcriptional activators in cold acclimation. Physiol. Plant, 112,
171–175.
Tian, X.H., Li, X.P., Zhou, H.L., Zhang, J.S., Gong, Z.Z. and Chen,
S.Y. (2005) OsDREB4 genes in rice encode AP2-containing
proteins that bind specifically to the dehydration-responsive
element. J. Integr. Plant Biol., 47, 467–476.
Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M.B., Thornton,
S. and Brettell, R. (1997) Agrobacterium tumefaciens-mediated
barley transformation. Plant J., 11, 1369–1376.
Tolleter, D., Jaquinod, M., Mangavel, C., Passirani, C., Saulnier,
P., Manon, S., Teyssier, E., Payet, N., Avelange-Macherel, M.H.
and Macherel, D. (2007) Structure and function of a
mitochondrial late embryogenesis abundant protein are
revealed by desiccation. Plant Cell, 19, 1580–1589.
Tommasini, L., Svensson, J.T., Rodriguez, E.M., Wahid, A.,
Malatrasi, M., Kato, K., Wanamaker, S., Resnik, J. and Close,
T.J. (2008) Dehydrin gene expression provides an indicator of
low temperature and drought stress: transcriptome-based
analysis of Barley (Hordeum vulgare L.). Funct. Integr.
Genomics, 8, 387–405.
Tunnacliffe, A. and Wise, M.J. (2007) The continuing conundrum
of the LEA proteins. Naturwissenschaften, 94, 791–812.
Vagujfalvi, A., Crosatti, C., Galiba, G., Dubcovsky, J. and
Cattivelli, L. (2000) Two loci on wheat chromosome 5A
regulate the differential cold-dependent expression of the
cor14b gene in frost-tolerant and frost-sensitive genotypes.
Mol. Gen. Genet., 263, 194–200.
Vagujfalvi, A., Aprile, A., Miller, A., Dubcovsky, J., Delugu, G.,
Galiba, G. and Cattivelli, L. (2005) The expression of several Cbf
genes at the Fr-A2 locus is linked to frost resistance in wheat.
Mol. Genet. Genomics, 274, 506–514.
Vilardell, J., Goday, A., Freire, M.A., Torrent, M., Martinez, M.C.,
Torne, J.M. and Pages, M. (1990) Gene sequence,
developmental expression, and protein phosphorylation of
Rab17 in maize. Plant Mol. Biol., 14, 423–432.
Vilardell, J., Mundy, J., Stilling, B., Leroux, B., Pla, M., Freyssinet,
G. and Pages, M. (1991) Regulation of the maize Rab17 gene
promoter in transgenic heterologous systems. Plant Mol. Biol.,
17, 985–993.
Vilardell, J., Martinezzapater, J.M., Goday, A., Arenas, C. and
Pages, M. (1994) Regulation of Rab17 gene promoter in
transgenic Arabidopsis wild-type, ABA-deficient and ABA-
insensitive mutants. Plant Mol. Biol., 24, 561–569.
Vogel, J.T., Zarka, D.G., Van Buskirk, H.A., Fowler, S.G. and
Thomashow, M.F. (2005) Roles of the CBF2 and ZAT12
transcription factors in configuring the low temperature
transcriptome of Arabidopsis. Plant J., 41, 195–211.
Wang, P.-R., Deng, X.-J., Gao, X.-L., Chen, J., Wan, J., Jiang, H.
and Xu, Z.-J. (2006) Progress in the study on DREB transcription
factor. Yi Chuan, 28, 369–374.
Wang, Q.Y., Guan, Y.C., Wu, Y.R., Chen, H.L., Chen, F. and
Chu, C.C. (2008) Overexpression of a rice OsDREB1F gene
increases salt, drought, and low temperature tolerance in both
Arabidopsis and rice. Plant Mol. Biol., 67, 589–602.
Wise, M.J. (2003) LEAping to conclusions: a computational
reanalysis of late embryogenesis abundant proteins and their
possible roles. BMC Bioinformatics, 4, 52–71.
Wu, Y., Zhou, H., Que, Y.X., Chen, R.K. and Zhang, M.Q. (2008)
Cloning and identification of promoter Prd29A and its application
in sugarcane drought resistance. Sugar Technol., 10, 36–41.
Xu, D., Duan, X., Wang, B., Hong, B., Ho, T. and Wu, R. (1996)
Expression of a late embryogenesis abundant protein gene,
HVA1, from barley confers tolerance to water deficit and salt
stress in transgenic rice. Plant Physiol., 110, 249–257.
Yamaguchi-Shinozaki, K. and Shinozaki, K. (2006) Transcriptional
regulatory networks in cellular responses and tolerance to
dehydration and cold stresses. Ann. Rev. Plant Biol., 57, 781–
803.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Sarah Morran et al.248
Zhao, J.S., Ren, W., Zhi, D.Y., Wang, L. and Xia, G.M. (2007)
Arabidopsis DREB1A ⁄ CBF3 bestowed transgenic tall fescue
increased tolerance to drought stress. Plant Cell Rep., 26,
1521–1528.
Supporting information
Additional Supporting information may be found in the
online version of this article:
Figure S1 Up-regulation of cellulose synthases in trans-
genic barley plants with constitutive overexpression of
TaDREB2.
Figure S2 Activity of maize Rab17 promoter in transgenic
wheat and barley under drought or cold stress.
Figure S3 Strong and specific up-regulation of HvCOR14B
by TaDREB3 in transgenic barley plants.
Table S1 Phenotype traits of transgenic barley plants with
constitutive overexpression of DREB factors.
Table S2 Correlation of expression levels of transgenes
(TaDREB2 and TaDREB3) and potential downstream genes
(left column) intransgenic barley plants with constitutive
overexpression of DREB factors.
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting materials sup-
plied by the authors. Any queries (other than missing
material) should be directed to the corresponding author
for the article.
ª 2010 ACPFG
Plant Biotechnology Journal ª 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 9, 230–249
Improvement of stress tolerance of wheat and barley 249