Planta (2010) 232:743–754
DOI 10.1007/s00425-010-1212-z
O R I G I N A L A R T I CL E
Functional analysis of six drought-inducible promoters
in transgenic rice plants throughout all stages of plant growth
Nari Yi · Youn Shic Kim · Min-Ho Jeong · Se-Jun Oh ·
Jin Seo Jeong · Su-Hyun Park · Harin Jung ·
Yang Do Choi · Ju-Kon Kim
Received: 10 May 2010 / Accepted: 10 June 2010 / Published online: 22 June 2010
© Springer-Verlag 2010
Abstract There are few eYcient promoters for use with
stress-inducible gene expression in plants, and in particular
for monocotyledonous crops. Here, we report the identiWca-
tion of six genes, Rab21, Wsi18, Lea3, Uge1, Dip1, and
R1G1B that were induced by drought stress in rice micro-
array experiments. Gene promoters were linked to the gfp
reporter and their activities were analyzed in transgenic rice
plants throughout all stages of plant growth, from dry seeds
to vegetative tissues to Xowers, both before and after
drought treatments. In fold induction levels, Rab21 and
Wsi18 promoters ranged from 65- and 36-fold in leaves to
1,355- and 492-fold in Xowers, respectively, whereas Lea3
and Uge1 were higher in leaves, but lower in roots and
Xowers, as compared with Rab21 and Wsi18. Dip1 and
R1G1B promoters had higher basal levels of activity under
normal growth conditions in all tissues, resulting in smaller
fold-induction levels than those of the others. In drought
treatment time course, activities of Dip1 and R1G1B
promoters rapidly increased, peaked at 2 h, and remained
Electronic supplementary material The online version of this
article (doi:10.1007/s00425-010-1212-z) contains supplementary
material, which is available to authorized users.
N. Yi · Y. S. Kim · M.-H. Jeong · S.-J. Oh · J. S. Jeong ·
S.-H. Park · H. Jung · J.-K. Kim (&)
School of Biotechnology and Environmental Engineering,
Myongji University, Yongin 449-728, Korea
e-mail: jukon@mju.ac.kr
N. Yi · Y. D. Choi
School of Agricultural Biotechnology,
Seoul National University, Seoul 151-921, Korea
Present Address:
S.-J. Oh
Syngenta Seeds Co., Ltd,
100 Gongpyeong-dong Jongro-gu, Seoul 110-702, Korea
constant until 8 h, while that of Lea3 slowly yet steadily
increased until 8 h. Interestingly, Rab21 activity increased
rapidly and steadily in response to drought stress until
expression peaked at 8 h. Thus, we have isolated and char-
acterized six rice promoters that are all distinct in fold
induction, tissue speciWcity, and induction kinetics under
drought conditions, providing a variety of drought-induc-
ible promoters for crop biotechnology.
Keywords Drought-inducible promoter ·
Transgenic rice · GFP · Vegetative tissues ·
Reproductive tissues · Dry seed
Abbreviations
Rab21 Responsive to ABA protein 21
Wsi18 Water stress inducible protein 18
Lea3 Late embryogenesis abundant protein 3
Uge1 UDP-glucose 4-epimerase
Dip1 Dehydration inducible protein 1
R1G1B Early drought induced protein
NT Non-transgenic
Introduction
Gene regulation can occur during diVerent stages of gene
expression, with the initiation of RNA transcription being
the most important point of control. Various 5’ upstream
gene promoter sequences have been isolated from diVerent
plant species and applied to plant biotechnology. At pres-
ent, the cauliXower mosaic virus (CaMV) 35S promoter is
the most commonly used gene promoter for transgenic reg-
ulation of constitutive expression in plants (Odell et al.
1985). Although the 35S promoter and its derivatives can
drive high levels of transgene expression in both monocot
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and dicot plants (Battraw and Hall 1990; Benfey et al.
1990), their activities are substantially lower in monocots
than in dicots (Gupta et al. 2001; Christensen et al. 1992;
Weeks et al. 1993). Conversely, the activity of monocot-
derived promoters is higher in monocots than in dicots
(McElroy et al. 1991; Cornejo et al. 1993), necessitating the
development of both monocot and dicot promoters.
Several promoters have been evaluated for their ability
to drive the constitutive high expression of transgenes in
monocots. These promoters include Ubi1 from maize (Zea
mays) (Cornejo et al. 1993; Kawalleck et al. 1993) and
Act1 (McElroy et al. 1991; Zhang et al. 1991), RbcS
(Kyozuka et al. 1993; Jang et al. 1999), OsCc1 (Jang et al.
2002) and others (Park et al. 2010) from rice (Oryza
sativa). These promoters were successfully applied to the
development of stress-tolerant transgenic crops. For exam-
ple, the rice Act1 promoter has been used to drive the
expression of the barley group 3 LEA gene HVA1, result-
ing in salt and water stress tolerance (Xu et al. 1996). The
maize Ubi1 promoter was used to drive the biosynthesis of
trehalose in rice (Jang et al. 2003) and the expression of
the transcription factors CBF3/DREB1A, ABF3 (Oh et al.
2005), and HvCBF4 (Oh et al. 2007), resulting in increased
abiotic stress tolerance in rice. The OsCc1 promoter was
eVective in driving the constitutive expression of AP37,
which encodes a transcription factor with an AP2 domain.
AP37 expression conferred tolerance to drought stress in
rice (Oh et al. 2009).
However, transgenic plants with a strong constitutive
expression of functional genes and/or transcription factors
often suVer from undesirable phenotypes. For example,
stress-tolerant transgenic Arabidopsis expressing 35S:
DREB1A displayed growth retardation and severe reduction
in seed production (Liu et al. 1998; Kasuga et al. 1999).
Similarly, a transgenic tomato expressing 35S:CBF1 (Hsieh
et al. 2002a, b), a transgenic rice expressing 35S:Adc
(Capell et al. 1998), and a transgenic tobacco expressing
35S:TPS1 (Romero et al. 1997) exhibited varying degrees
of growth retardation, abnormal development, and reduced
seed production. It is therefore desirable to generate trans-
genic plants that accumulate transgenic products only under
stress conditions. Use of a stress-inducible promoter has
proven to be an alternative strategy for elimination of such
phenotypes induced by constitutive transgene expression.
There were no morphological abnormalities in transgenic
rice plants that are highly tolerant to abiotic stress when an
ABA-inducible promoter was employed to express the
PgNHX1 gene (Verma et al. 2007). The growth retardation
observed in transgenic Arabidopsis expressing 35S:
DREB1A was diminished when the stress-inducible pro-
moter rd29A was used in place of 35S (Kasuga et al. 1999).
The rice stress-inducible promoters LIP9 and OsNAC6
have also been eVectively used to over express the OsNAC6
123
Planta (2010) 232:743–754
gene in transgenic rice, improving stress tolerance without
plant growth retardation (Nakashima et al. 2007).
Several stress-inducible promoters have been isolated
and characterized. These include the kin1 and the cor6.6
promoters from Arabidopsis (Wang et al. 1995), the
Gmhsp17.6-L promoter from soybean (Lee and SchöZ
1996), the Rab16A (RoyChoudhury et al. 2007; Rai et al.
2009) the Wsi18 (Joshee et al. 1998; Xiao and Xue 2001)
promoters from rice, and the Hva1 promoter from barley
(Straub et al. 1994; Xiao and Xue 2001). Despite such
eVorts to functionally characterize stress-inducible promot-
ers, very few of them have been analyzed for their response
to stresses throughout the spectrum of transgenic plant
growth. Examples of such stress-inducible promoters
include rice OsABA2 (Rai et al. 2009) and Arabidopsis
Rd29A (Yamaguchi-Shinozaki and Shinozaki 1993; Pelle-
grineschi et al. 2004), which were shown to be eVective in
all the tissues and stages of transgenic plants. Thus, there is
currently a shortage of eYcient promoters for stress-
inducible gene expression, especially in monocot crops. In
addition, stacking of several transgenes in a single trans-
genic plant requires a battery of diVerent promoters, which
may otherwise undergo homology-dependent gene silenc-
ing which often occurs in transgenic plants with multiple
copies of the same promoter (Lessard et al. 2002; Potenza
et al. 2004).
In our current study, we examined the regulation of six
drought-inducible genes and their promoter activities in
transgenic rice plants under drought conditions. Using Xuo-
rescent microscopy and real-time PCR, promoter activities
were analyzed in transgenic plants throughout all stages of
plant growth, including dry seeds, vegetative tissues, and
Xowers. Each promoter possesses a distinct pattern of fold-
induction, tissue-speciWcity, and kinetics of induction under
drought stress, disclosing a variety of drought-inducible
promoters for crop biotechnology.
Materials and methods
Plant materials
The rice (Oryza sativa L. var. Japonica) plants cv. Nippon-
bare and cv. Nakdong (seeds obtained from National Agro-
biodiversity Center, RDA, Suwon, Korea) were used for
isolation of promoters and plant transformation, respectively.
Rice genomic DNA was prepared from leaves of 14-day-old
Nipponbare plants using the DNAzol plant genomic DNA
isolation reagent (Molecular Research Center, Cincinnati,
OH, USA) according to the manufacturer’s instructions for
use in the isolation of the promoters by genomic PCR. The
T3 generation of three independent lines for each of the pro-
moters was used for detailed analysis.
Planta (2010) 232:743–754
Expression of endogenous genes in rice
The rice 3⬘-tiling microarray (GreenGene Biotech, Yongin,
Korea) analysis was performed as reported previously (Kim
et al. 2009). RNA samples from drought-treated and
untreated leaf tissues of 14-day-old rice (O. sativa L. cv.
Nakdong) plants were used to generate Cy3-labeled com-
plementary DNA probes, which were then hybridized to the
microarray. Each data set was obtained from two biological
repeats. Comparisons of data sets from drought-treated
plants with those from untreated plants led us to select four
genes Wsi18, Lea3, Rab21, and Uge1 for promoter analysis
that were induced more than 20-fold or with spot intensity
over 25,000 in the microarray after drought-stress treat-
ments (Supplementary Table S1). We also included two
additional genes Dip1 and R1G1B that were previously
identiWed to be stress-inducible (Oh et al. 2005).
For RNA extraction, rice seeds were germinated in half-
strength Murashige-Skoog (MS) (Murashige and Skoog
1962) solid medium in a growth chamber in the dark at
28°C for 3 days, followed by light exposure at 28°C for
1 day, transplanted into soil, and grown in a greenhouse
(16 h light/8 h dark cycle) at 28–30°C. Each plant was
grown in a pot Wlled with rice nursery soil (Bio-media,
Kyeongju, Korea) for the indicated days after germination
(DAG) and in the Xowering stage when the panicles were at
<10 cm in size. For drought treatment, whole plants at
diVerent DAG were air dried for 2 h under continuous light
of approximately 1,000 mol photons m¡2s¡1. Total RNA
was extracted from dry seeds, leaves, roots, and Xowers
using the Qiagen RNeasy plant mini kit (Qiagen, Valencia,
CA, USA) according to the manufacturer’s instructions.
For ampliWcation of the corresponding genes, cDNA was
synthesized with random primers using SuperScriptTM III
First-strand synthesis system (Invitrogen, Carlsbad, CA,
USA), and then RT–PCR was carried out with 2 g of
cDNA as templates and gene-speciWc primer pairs designed
with Primer Designer 4 software (Sci-Ed Software, Dur-
ham, NC, USA). These primer pairs are listed in Supple-
mentary Table S2. RT–PCR was performed at 95°C for
10 min, followed by 28 cycles at 95°C for 30 s, 55°C
for 30 s, and 72°C for 1 min, with a Wnal cycle at 4°C for
10 min. The OsUbi (AK121590) gene was used to verify
RNA loading. The ampliWed PCR products were sequenced
to ensure Wdelity.
Plasmid construction and transformation of rice
The promoter regions were ampliWed and isolated by PCR
using promoter-speciWc primer pairs and the 2£ EF-Taq
DNA polymerase pre-mix (SolGent, Seoul, Korea). Geno-
mic PCR was performed at 95°C for 10 min, followed by
30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for
745
1 min, with a Wnal cycle at 4°C for 10 min using 50 ng of
gDNA. The ampliWed DNAs were re-ampliWed using the
adaptor primer pairs. All primer pairs are listed in Supple-
mentary Table S2. The isolated promoters with adaptor
sequences were individually linked to the gfp reporter gene
in a rice transformation vector using site-speciWc recombi-
nation. PCR reactions and in vitro recombination reactions
were carried out according to the manufacturer’s instruc-
tions (Gateway® Cloning Technology, Invitrogen). The rice
transformation vector contained the bar gene under the
control of the cauliXower mosaic virus 35S promoter for
use with herbicide-based plant selection. Vectors were
introduced into Agrobacterium tumefaciens LBA4404 by
tri-parental mating. Embryonic callus from the mature
seeds of rice (O. sativa L. cv. Nakdong) were transformed
by co-cultivation (Hiei et al. 1994), selected with 7 mg/L
phosphinothricin, and used to regenerate transgenic plants
as previously described (Jang et al. 2002).
Genomic Southern-blot analysis
Genomic DNA was prepared as described above from
leaves of 30-day-old transgenic (T3 generation) and control
plants. Five micrograms of genomic DNA was digested
with Cla I, fractionated through a 1% agarose gel and
transferred onto Hybond N+ nylon membrane (Amersham
Pharmacia, Uppsala, Sweden). The nylon membrane was
pre-hybridized in a solution (0.5 M Na2PO4/pH 7.2, 1%
bovine serum albumin, 7% SDS, 100 g/mL denatured
salmon sperm DNA) at 65°C for 1 h, and then hybridized
overnight at 65°C with 32P-labeled probe DNA prepared
using a Random Primer Labeling Kit (Takara, Shiga,
Japan) according to the manufacturer’s instructions. After
hybridization, the membrane was washed once in 2£ SSC
(0.3 M NaCl, 50 mM sodium citrate/pH 7.0) and 0.1% SDS
solution at 65°C for 15 min, once in 1£ SSC and 0.1% SDS
at 65°C for 15 min, once in 0.5£ SSC and 0.1% SDS at
65°C for 15 min, and Wnally washed in 0.1£ SSC and 0.1%
SDS at 65°C for 10 min. The membrane was then exposed
on an intensifying plate and analyzed with a phosphoimage
analyzer (FLA 3000, Fuji, Tokyo, Japan).
Drought treatments of rice plants at vegetative
and reproductive stages
Transgenic and non-transgenic (NT) rice seeds were germi-
nated and grown as described earlier. For real time-PCR,
whole plants at both vegetative and reproductive stages
were air-dried for 2–8 h under continuous light of approxi-
mately 1,000 mol photons m¡2s¡1. For detection of GFP
Xuorescence in leaf and root tissues, 7-day-old plants were
air-dried for 4 h and used for confocal laser scanning
microscopy. For detection of GFP Xuorescence in Xowers,
123
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whole plants in pots at the reproductive stage were starved
of water for 3 days at a time when Xowers were developing
in a 10-cm panicle before pollination in a greenhouse.
Detection of GFP Xuorescence
Detection of GFP Xuorescence in transgenic plants was car-
ried out in comparison with that in non-transgenic control
plants. GFP Xuorescence in leaves and roots was visualized
and photographed using a confocal laser scanning micro-
scope (Carl Zeiss LSM410 CLSM, Jena, Germany) with a
standard Wlter set at the Korea Basic Science Institute (Dae-
joen, Korea). Pseudocolor, similar to the color observed
with a Xuorescence microscope, was added to the images
by importing data collected in the green and red channels of
the confocal microscope. Sections were taken along the
optical axis and were projected into a single image. GFP
Xuorescence in dry seeds and Xowers was observed using a
research stereomicroscope (SZX9-3122, Olympus, Tokyo,
Japan) equipped with an attachment for Xuorescence obser-
vations. Images were collected using a C5060-ZOOM digi-
tal camera (Olympus). Observations under blue light were
carried out using a speciWc Wlter set (460–480 nm excitation
Wlters, dichroic mirrors of 485 nm and a 495–540 nm bar-
rier Wlter).
Real time-PCR analysis
Total RNA was extracted with transgenic and NT rice
plants using the Qiagen RNeasy plant mini kit (Qiagen)
according to the manufacturer’s instructions. The Super-
ScriptTM III First-strand synthesis system for RT–PCR
(Invitrogen) was used for cDNA synthesis with random
primers. RT–PCR and real-time-PCR analyses were carried
out with 2 g of cDNA using 2£ EF-Taq DNA polymerase
pre-mix (SolGent) and TaqMan® universal PCR master
mix (Applied Biosystems, Foster City, CA, USA), respec-
tively. The gfp- and OsUbi-speciWc probes and primer pairs
for real-time-PCR were designed by Assays-by-DesignSM
Service (Applied Biosystems). These probe sets had more
than 97% PCR eYciency in the standard curve. Thermocy-
cling and Xuorescence detection were performed using a
Stratagene Mx3000p Real-Time PCR machine (Stratagene,
La Jolla, CA, USA). Fluorescence observations of all sam-
ples were taken directly from Stratagene Mx3000p soft-
ware v2.02 (Stratagene). RT–PCR was performed at 95°C
for 10 min, followed by 27–30 cycles of 95°C for 30 s,
55°C for 30 s, and 72°C for 1 min, with a Wnal cycle at 4°C
for 10 min. Real-time-PCR was performed at 50°C for
2 min, 95°C for 10 min followed by 40 cycles of 95°C for
30 s, 60°C for 1 min, according to the manufacturer’s
instructions (Applied Biosystems). Expression levels of
promoter-gfp were normalized to that of OsUbi
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Planta (2010) 232:743–754
(AK121590) gene in real-time-PCR. Primer pairs and
probes used are listed in Supplementary Table S2.
Results
IdentiWcation of six drought-inducible genes
To identify genes that are up-regulated by drought condi-
tions, expression proWling was performed on RNA from
drought-treated rice leaves in comparison with untreated
leaves using the rice 3⬘-tiling microarray. These analyses
identiWed the genes Rab21, Wsi18, Lea3,Uge1, Dip1, and
R1G1B as being induced by drought stress at over 25,000
reading value in the microarray (Supplementary Table S1).
Expression patterns of these six genes were further ana-
lyzed by RT–PCR using total RNA from diVerent tissues at
diVerent stages of plant growth both before and after
drought stress (Fig. 1a). Levels of Rab21, Wsi18, and Lea3
mRNA were very low under non-stressed conditions in all
tissues throughout the developmental stages with the
exception of dry seeds. Their expression levels were
increased dramatically upon exposure to drought condi-
tions. In contrast, mRNA levels of Uge1, Dip1, and R1G1B
were relatively high under non-stressed conditions, result-
ing in relatively lower levels of fold-induction upon expo-
sure to drought conditions.
Approximately 2.0 kb of the region immediately
upstream of a gene translation initiation codon (ATG) was
predicted to be the promoter region for that gene (Fig. 1b).
The predicted promoter regions of the six stress-regulated
genes were ampliWed by PCR using rice (Oryza sativa L.,
cv. Nipponbare) genomic DNA as templates. The PCR
products were then inserted into a rice transformation vec-
tor using site-speciWc recombination (Fig. 1c), generating
constructs with each promoter sequence linked to the
green Xuorescent protein gene gfp. Several independent
transgenic lines were obtained for each gene promoter
using the Agrobacterium-mediated transformation method
and were grown to maturity in a greenhouse. Copy num-
bers and integration events of the transgene in each trans-
genic line were determined by genomic Southern blot
(Fig. 1c). All the lines were independent and most of the
transgenic lines contained a single or two copies of trans-
gene. Four lines out of 18 contained more than 2 copies.
Single copy lines may represent promoter activity more
properly due to less chance of silencing. We included at
least two transgenic lines that contained a single copy per
promoter, with the exception of R1G1B:gfp plants. By sel-
Wng and selecting of germinating transgenic seeds on
phosphinothricin-containing MS media, we obtained T3
seeds and three independent lines for each construct were
chosen for further analysis.
Planta (2010) 232:743–754 747

(a) Dry
20 DAG 30 DAG 60 DAG (b) ATG(+92)
seed Leaf Root Leaf Root Leaf Root Flower 1557bp +1
Rab21 CHR11
Drought - + - + - + - + - + - + - + +63
Rab21 ATG(+129)
1887bp +1
Wsi18 Wsi18 CHR1
+105
Lea3 ATG(+101)
1963bp +1
Uge1 Lea3 CHR5
+64
Dip1 ATG(+86)
1890bp +1
R1G1B Uge1 CHR5
+47
OsUbi
ATG(+120)
1967bp +1
Dip1 CHR2
variable +108

ATG(+145)
(c) BR
ClaI
BL 1799bp +1
R1G1B CHR5
+94
Promoter gfp TPINII P 35S bar TNOS
attB1 attB2
MAR

Probe (0.57 kb)

Rab21 Wsi18 Lea3 Uge1 Dip1 R1G1B

M NT #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3

kb
8.5 
6.5 

4.3 
3.7 

2.3 
1.9 

1.4 

0.7 
1 1 M 2 1 1 1 1 M 1 1 1 2 1 1 M 2 M (copy number)

Fig. 1 Expression and genomic structures of six drought-inducible
genes. a Expression of six drought-inducible genes in response to
drought stress in rice plants. Total RNA was extracted from diVerent
tissues of non-transgenic (NT) rice plants both before and after 2 h of
exposure to drought conditions. Dry seeds, leaves, and roots collected
at the indicated days after germination (DAG) and Xowers collected be-
fore pollination were used for RNA extraction. The rice ubiquitin gene
(OsUbi) was used to verify RNA loading. plus and minus denotes
plants with or without drought-stress treatments. b Genomic structures
of six drought-inducible genes. Bold lines indicate the 5⬘ upstream re-
gion, Wlled boxes indicate exons, and lines between the boxes indicate
introns. The length of all boxes and lines were drawn to scale. The tran-
scriptional start site and the translational start codon are marked with
+1 and ATG, respectively. c Schematic representation of the rice
transformation vector (up) and genomic Southern-blot analysis of
promoter:gfp transgenic plants (down). Rice promoters were linked to
the gfp reporter gene by site-speciWc recombination. Expression of the
gfp reporter gene is regulated by the promoter and the potato Pin II 3⬘
region (TpinII). The bacterial phosphinothricin acetyl transferase gene
(bar), a selectable marker for rice transformation, was fused between
the CaMV35S promoter (P35S) and the nopaline synthase polyadenyl-
ation region (Tnos). MAR is the 5⬘-matrix attachment region from the
chicken lysozyme gene (Oh et al. 2005). Genomic DNA from leaves of
three (#1–3) independent lines of each promoter:gfp transgenic plants
were digested with Cla I, and hybridized with a 0.57 kb MAR-speciWc
probe. The names of the promoters denote the transgenic plants ana-
lyzed. Copy numbers of transgene for each line are shown below the
blots. M multiple (>2) copies, NT Genomic DNA from a non-transgenic
plant, M the DNA molecular size marker

Spatial and temporal expression patterns Xuorescence driven by the Uge1 and Dip1 promoters was
of drought-inducible promoters barely detectable in the embryos, while the activity of
R1G1B was detectable both in embryos and in aleurone lay-
To examine promoter activity in the whole plant at diVerent ers. No GFP Xuorescence was detected in the hulls of any
developmental stages, GFP Xuorescence from promoter:gfp of the transgenic seeds (Fig. 2a).
transgenic plants was visually detected with a Xuorescence To investigate drought-induced expression of gfp in veg-
microscope under normal and drought conditions. Images etative tissues, transgenic and NT control plants were air-
of non-transgenic (NT) tissues were used as negative con- dried at the bench for 4 h (Fig. 2b, c). We used a confocal
trols. In dry seeds, Rab21, Wsi18, and Lea3 promoters laser scanning microscope for detection of GFP Xuores-
directed high levels of gfp expression in the whole grain, cence in green leaves and roots in order both to avoid
especially in the embryo and in the aleurone layers. GFP interference from chlorophyll red Xuorescence in leaves

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748 Planta (2010) 232:743–754

(a) Vertical Horizontal (b) Normal Drought

Light GFP Light GFP
Red Green Merged Red Green Merged
Al Al
E
NT S V
Em Em
NT
M

Rab21
Rab21

Wsi18
Wsi18

Lea3
Lea3

Uge1 Uge1

Dip1 Dip1

R1G1B R1G1B

Fig. 2 GFP Xuorescence of transgenic and non-transgenic (NT) rice
plants. a GFP Xuorescence in dry seeds. GFP was detected in hand-cut
vertical and horizontal sections of dry seeds using a Xuorescence
microscope (Olympus, Tokyo, Japan). Al aleurone layer, E embryo,
Em endosperm. Bars = 1 mm. b GFP Xuorescence in leaves. GFP Xuo-
rescence was detected in leaves of 7-day-old plants both before (nor-
mal) and after (drought) treatment with drought stress by a confocal
laser scanning microscope. The names of the promoters denote the
transgenic plants analyzed. Red red channel, Green green channel,
Merged, merged channel, M mesophyll cell, S stomata, V vascular bun-
dle sheath. Bars = 80 m. c GFP Xuorescence in roots. GFP Xuores-
cence was detected as described in b. RA root apex, RC root cap,
E epidermis, V vascular cylinder. Bars = 100 m. d GFP Xuorescence
in Xowers. GFP was detected in a whole spikelet (left panel) and Xoral
organs inside of a spikelet (right panel) at panicles 10 cm in size. Plants
in pots were starved of water in a greenhouse for 3 days. An anther,
F Wlament, Lm lemma, Lo lodicule, O ovary, P palea. Bars = 1 mm

(Zhou et al. 2005) and to see GFP expression in diVerent
cell types. Activity of all six promoters was overall
enhanced in whole plants with distinct patterns under
drought conditions. For example, in Rab21:gfp, Wsi18:gfp,
Lea3:gfp, and Uge1:gfp plants, elevation of GFP Xuores-
cence was evident in mesophyll and vascular bundle sheath
cells, but not in guard cells upon exposure to drought con-
ditions. In contrast, in Dip1:gfp and R1G1B:gfp plants, GFP
Xuorescence was increased in all leaf cell types including
guard cells upon exposure to drought conditions (Fig. 2b).
Increase in GFP Xuorescence was also evident in root apex,
root cap, and elongating regions of all the promoter:gfp
plants with diVerent intensities under drought conditions.
Under normal growth conditions, all vegetative transgenic tis-
sues did not show any GFP Xuorescence, with the exceptions
of Dip1:gfp and R1G1B:gfp plants. In particular, Dip1:gfp
plants showed brighter Xuorescence in stomata, mesophyll
cells in leaves(Fig. 2b), and root apex, root cap in roots
(Fig. 2c). R1G1B:gfp plants also showed GFP Xuorescence
in leaves such as stomata and mesophyll cells (Fig. 2b). We
also examined GFP expression in transgenic spikelets
(Xowers) at the stage of meiosis (Fig. 2d). As was observed
in the vegetative tissues, under normal growth conditions,
background GFP Xuorescence was also observed in
Dip1:gfp and R1G1B:gfp Xowers. Following drought stress
for 3 days, all transgenic Xowers showed high level of
GFP Xuorescence in all Xoral organs, with the exception
of Uge1:gfp Xowers. Overall, activities of the six pro-
moters were signiWcantly enhanced by exposure to drought
conditions.

123
Planta (2010) 232:743–754 749

Fig. 2 continued (c) Normal Drought

Light Green Merged Light Green Merged
RA V
NT E
RC

Rab21

Wsi18

Lea3

Uge1

Dip1

R1G1B

(d) Normal Drought Normal Drought

Light GFP Light GFP Light GFP Light GFP

An F
NT Lm
P
Lo

Rab21

Wsi18

Lea3

Uge1

Dip1

R1G1B

123
750 Planta (2010) 232:743–754

non-stressed
Leaf non-stressed Root drought-stressed
500 drought-stressed 1800
20 DAG 20 DAG
1600
400 1400
1200
300
1000
200 800
600
Relative mRNA level

100 400
200
0 * 0 *
450 300
60 DAG 240
60 DAG
180
120
300

40
150
20

0 * 0 *
#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3
Rab21 Wsi18 Lea3 Uge1 Dip1 R1G1B Rab21 Wsi18 Lea3 Uge1 Dip1 R1G1B

2500 Flower 12
Dry seed

Relative mRNA level

Relative mRNA level

10
2000
8
1500
6
1000
4
500 2
*
0 * 0
#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3

Rab21 Wsi18 Lea3 Uge1 Dip1 R1G1B Rab21 Wsi18 Lea3 Uge1 Dip1 R1G1B

Fig. 3 Relative gfp mRNA levels in drought-stressed transgenic
plants. Levels of gfp mRNA in diVerent tissues of non-stressed and
stressed-transgenic plants were measured using real-time PCR. Total
RNA was extracted from the leaves and roots of three (#1–3) indepen-
dent T3 lines of transgenic plants at the indicated days after germina-
tion (DAG) both before and after 2 h of exposure to drought
conditions. Also extracted was total RNA from dry seeds and panicles
10 cm in size from three (#1–3) independent T3 transgenic plant lines
before and after 3 days of exposure to drought conditions. All levels
were normalized with respect to the internal control gene OsUbi
(AK121590) and were plotted relative to the level of gfp mRNA in
Wsi18:gfp plants marked with asterisks. Data bars represent the
mean § SD of triplicate measurements

Quantitative analysis of promoter activity
To compare levels of promoter activities induced by
drought conditions, gfp mRNA levels were measured in
diVerent tissues of three independent T3 transgenic lines at
various developmental stages by the quantitative real-time
PCR. Total RNA was extracted from dry seeds, leaves,
roots, and Xowers at the indicated days after germination
(DAG) and in the presence or absence of drought stress. In
leaves, roots, and Xowers, gfp mRNA levels were largely
increased in all of the transgenic plants upon exposure to
drought conditions (Fig. 3). Fold-induction levels, ratios of
the amount of gfp mRNA in drought-treated over untreated
conditions, were varied widely depending on the speciWc
promoter and plant tissue (Table 1). For example, fold
induction levels for Rab21 and Wsi18 promoters ranged
from 65- and 36-fold in leaves to 1,355- and 492-fold in
Xowers, respectively. Fold induction levels of Lea3 and
Uge1 promoters were higher in leaves but lower in roots
and Xowers than was observed for Rab21 and Wsi18. Dip1
and R1G1B promoters had higher basal levels of activity
under normal growth conditions in all tissues, resulting in
smaller levels of fold induction than those of the other pro-
moters. In Xowers, the fold induction levels were much
higher in Rab21and Wsi18 than in others. In dry seeds
(Fig. 3), promoter activity levels were higher in Rab21,
Wsi18, and Lea3 than in Uge1, Dip1, and R1G1B, which
was consistent with expression of the respective endoge-
nous gene (Fig. 1a). The low levels of the Uge1, Dip1, and
R1G1B promoter activities in dry seeds were due in part to
the lack of gfp expression in endosperm, as demonstrated in
Fig. 2a. Copy numbers and expression levels appeared not
to be directly correlated. For example, expression levels of
line #3 of Lea3 promoter that contained multicopies were
lower in roots yet higher in leaves of 60 DAG plants than
the other lines of Lea3 that contained a single copy (Fig. 3).
To determine the range of promoter activity in several
independent transgenic lines, we measured gfp mRNA levels

123
Planta (2010) 232:743–754 751

Table 1 Fold induction levels of gfp mRNA

20 DAG 60 DAG

Leaf Root Leaf Root Flower

Rab21 109.9 § 9.4 100.1 § 134.0 65.8 § 53.5 87.7 § 43.8 1,355.5 § 1187.3
Wsi18 53.1 § 28.6 325.5 § 189.8 36.7 § 12.8 237.2 § 205.6 492.2 § 82.2
Lea3 330.4 § 407.8 68.2 § 4.2 170.3 § 7.4 3.0 § 0.5 38.3 § 13.2
Uge1 287.5 § 31.3 9.3 § 6.4 46.9 § 14.2 16.9 § 7.2 2.3 § 0.1
Dip1 14.1 § 5.6 2.5 § 1.2 29.8 § 6.5 16.0 § 2.4 3.2 § 0.1
R1G1B 1.5 § 0.8 5.7 § 3.8 5.0 § 2.7 8.8 § 3.6 9.1 § 3.3
Levels of promoter-driven gfp mRNAs in drought-stressed transgenic plant lines were compared with those in non-stressed transgenic plants. Data
are the mean § SD of the fold induction levels in three independent transgenic lines

Fig. 4 Promoter activity in (a) 140 (b) 1200

diVerent transgenic lines over a 120 Rab21
time course of drought treat-
Wsi18
ment. a Promoter activities in
1000 Lea3
the leaves (30 DAG) of several Uge1
(4–18) independent transgenic 60 Dip1
lines treated with drought for R1G1B

Relative mRNA level

2 h. Levels of gfp mRNA were 800
measured using real-time PCR
and plotted relative to the gfp 50
mRNA levels in Wsi18:gfp 600
Relative mRNA level

plants marked with an arrow.

b Changes in promoter activities
40
over a time course of drought
treatments (0, 2, 4, and 8 h). 400
Levels of gfp mRNA were mea-
sured using real-time PCR per-
30
formed on total RNA from 200
leaves (30 DAG) of transgenic
plants. Levels were normalized
with respect to the internal con-
20
trol gene OsUbi (AK121590) (h)
0 2 4 6 8
and were plotted relative to the
level of gfp mRNA in non-
stressed Wsi18:gfp plants. Data 10
bars represent the mean of § SD
level of triplicate measurements

by the real-time PCR method using RNA from leaves
exposed to drought conditions (Fig. 4a). For these analyses,
4–18 transgenic lines were employed per promoter. As
shown in Fig. 4a, promoter activity levels varied largely
from 1.5- to 63-fold, depending on the transgenic plant
lines with the same promoter. We also investigated relative
promoter activity under prolonged drought conditions. We
performed real-time PCR using RNA from leaves exposed
to drought conditions over a time course (Fig. 4b). In
Dip1:gfp and R1G1B:gfp plants, gfp mRNA levels rapidly
increased in response to drought stress, peaked at 2 h, and
then remained relatively constant until 8 h. In Wsi18:gfp
plants, gfp mRNA levels gradually increased until 4 h and
then decreased slowly until 8 h. In Lea3:gfp plants, gfp
mRNA levels gradually increased up to 8 h. Interestingly,
in Rab21:gfp plants, gfp mRNA levels increased rapidly
and steadily in response to drought stress until 8 h, reaching
the highest level of any of the transgenic lines. Overall, our
results demonstrated that these six promoters are all distinct
in response to drought conditions in terms of fold induc-
tion, tissue speciWcity, and kinetics of induction.
Discussion
In this study, we quantitatively analyzed the activity of six
diVerent drought-inducible promoters through the entire
growth cycle of transgenic rice plants. These promoters

123
752
showed diVerent patterns of relative activity in diVerent tis-
sues and/or in diVerent stages of growth under drought con-
ditions. For example, in response to drought stress, Rab21
promoter was similarly high in fold induction levels in all
tissues, the highest being in Xowers, while the fold-induc-
tion level for Wsi18 was highest in roots and that for Lea3
was highest in Xowers and leaves. The Uge1, Dip1, and
R1G1B promoters exhibited higher activity in leaves than
in other tissues. Most stress-inducible promoters reported
so far have been analyzed in the vegetative tissues of young
seedlings, especially in leaves (Wang et al. 1995; Xiao and
Xue 2001) or in seed aleurone layers (Straub et al. 1994; Su
et al. 1998). Crop grain yields have been reported to be
highly sensitive to drought stress at the Xowering stage
(Wopereis et al. 1996). This observation may result from
repressed development of the panicle and/or spikelet meri-
stem under drought conditions, resulting in a reduction in
the number of panicles per plant and/or the number of spik-
elets per panicle (O’Toole and Namuco 1983; Boonjung
and Fukai 1996; Asch et al. 2005; Kim et al. 2009). Nearly
half of all reported pollination or fertilization-related genes
appear to be regulated by dehydration (Lan et al. 2005).
It has also been reported that root growth is an important factor
related to rice plant adaptation to drought stress (Price et al.
1997; Nemoto et al. 1998; Matsui and Singh 2003). There-
fore, it is desirable to analyze promoters in Xowers and
roots as well as in leaves at the reproductive stages.
Rab21and Wsi18 promoters showed high fold-induction
levels following drought stress, ranging from 36- to 1355-
fold in both vegetative and reproductive tissues. In particu-
lar, Wsi18 and Rab21 promoters were predominantly higher
in fold-induction levels in roots and Xowers, respectively,
than the levels for the other promoters. Conversely, Lea3,
Uge1, Dip1, and R1G1B promoters exhibited relatively
lower fold-induction levels following drought stress, espe-
cially in roots and Xowers.
Consistent with our results, the 800-bp Rab21 promoter
was previously reported to be induced in the leaves, roots,
and Xowers of rice plants following desiccation treatments
(Ono et al. 1996). These results were, however, determined
using histochemical analysis of GUS staining and levels of
fold-induction following drought stress were not reported.
Unexpectedly, the 358-bp Rab21 promoter was shown to
have high constitutive expression levels under normal
growth conditions (Rai et al. 2009). The discrepancy
observed in the two independent studies might be due to the
use of diVerent size of the promoter. The activity of the
Wsi18j promoter, a homolog of our Wsi18 with the same
sequence at the ABRE2 motif as is found in ABRC3, was
examined by transient expression analysis using 4-day-old
barley seedlings (Xiao and Xue 2001). The study did not
include results on the Wsi18j promoter activity in diVerent
tissues or at various stages under stress conditions.
123
Planta (2010) 232:743–754
As shown in Fig. 4a, levels of promoter activity in trans-
genic lines with the same promoter under drought condi-
tions varied from 1.5- to 63-fold. This variance may result
from diVerent loci of transgene integration in the genome
(Dean et al. 1988; Blundy et al. 1991; Mlynárová et al.
1994). Peach and Velten (1991) reported that the activity of
a reporter gene driven by the mas promoter, a mannopine
synthetase promoter from Agrobacterium, was variable up
to 175-fold among individual transformants. Such a large
variation was also observed in studies of plant stress-induc-
ible promoters (Wang et al. 1995; Rai et al. 2009), suggest-
ing the importance of the position eVect on promoter
activities.
Here, we investigated changes in activities of the six
promoters under prolonged drought conditions up to 8 h of
drought stress. The Dip1 promoter responded rapidly to
drought stress, reaching a maximum level following 2 h of
drought, whereas the Rab21 promoter responded steadily,
reaching a higher level of expression than that of Dip1 after
4 h and until 8 h of exposure. These changes in promoter
activity in leaves under drought conditions are, to some
extent, in agreement with previous observations of the
expression of the respective endogenous genes (Rabbani
et al. 2003).
Rab21 and Dip1 belong to the group 2 Lea gene family/
dehydrins (RoyChoudhury et al. 2007), while Wsi18 and
Lea3 belong to the Group 3 Lea family (Takahashi et al.
1994; Chen and Chen 1996). The Dip1promoter, a typical
member of the Group 2 Lea gene family, showed high lev-
els of basal activity in rice plant roots and Xowers. The
Rab21 promoter, in contrast, is more similar in activity to
that of the group 3 Lea family than to that of the Dip1pro-
moter. Rab21 promoter activity was high in roots and Xow-
ers following drought stress, with low levels of background
activity. The Wsi18 promoter showed higher activity in
roots than in leaves, whereas the Lea3 promoter was exhib-
ited the opposite pattern. Such a diVerence in promoter
activities may reXect diVerent functions of the genes in the
response of plants to drought stress.
In the current study, we characterized six diVerent
drought-inducible promoters in transgenic rice plants
throughout all stages of plant growth. These promoters
exhibited distinct patterns of fold induction, tissue speciWc-
ity, and patterns of induction under drought stress. As a
result, we have identiWed a variety of drought-inducible
promoters for use in crop biotechnology. We found Rab21
and Wsi18 to be the most recommendable promoters for use
in transgene expression in monocot crops under prolonged
drought conditions. These promoters drive transgene
expression at very low levels under normal conditions, yet
increase the level dramatically upon exposure to drought
conditions in the reproductive as well as in the vegetative tis-
sues. Use of alternative promoters with similar characteristics
Planta (2010) 232:743–754
is essential for the stacking of several transgenes in order to
avoid the homology-dependent gene silencing that often
occurs in transgenic plants with multiple copies of the same
promoter. Our promoters may also be useful for transgene
expression in other monocot crops such as maize, barley,
and wheat.
Acknowledgments This work was supported by the Ministry of
Education, Science and Technology, Korea, through the Crop Func-
tional Genomics Center (CG2111 to J-KK) and by the Rural Develop-
ment Administration through the Biogreen21 Program (PJ0007149 to
J-KK).
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