improvement of stress tolerance of wheat and barley by...

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;

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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

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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.

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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

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(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.

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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.

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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



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

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Fold

HvCBF1

HvCBF2

HvCBF3

HvCBF6

HvCBF9

HvCBF10A

HvCBF11

HvCBF14

HvCBF15

HvCBF16

HvCBF23

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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

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64

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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



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



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



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



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



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



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



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



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



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.

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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.

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