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Engineering drought tolerance in plants: discovering andtailoring genes to unlock the futureTaishi Umezawa1,2, Miki Fujita2,3, Yasunari Fujita4,Kazuko Yamaguchi-Shinozaki3,4,5 and Kazuo Shinozaki1,2,3
The ability of plants to tolerate drought conditions is crucial for
agricultural production worldwide. Recent progress has been
made in our understanding of gene expression, transcriptional
regulation and signal transduction in plant responses to
drought. Molecular and genomic analyses have facilitated gene
discovery and enabled genetic engineering using several
functional or regulatory genes to activate specific or broad
pathways related to drought tolerance in plants. Several lines of
evidence have indicated that molecular tailoring of genes has
the potential to overcome a number of limitations in creating
drought-tolerant transgenic plants. Recent studies have
increased our understanding of the regulatory networks
controlling the drought stress response and have led to
practical approaches for engineering drought tolerance in
plants.
Addresses1 Gene Discovery Research Group, RIKEN Plant Science Center,
1-7-22 Suehiro-cho, Yokohama, Kanagawa 203-0045, Japan2 Laboratory of Plant Molecular Biology, RIKEN Tsukuba Institute,
3-1-1 Kouyadai, Tsukuba, Ibaraki 305-0074, Japan3 Core Research for Evolutional Science and Technology (CREST),
Japan Science and Technology Agency (JST), Kawaguchi,
Saitama 332-0012, Japan4 Biological Resources Division, Japan International Research Center for
Agricultural Sciences, 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan5 Laboratory of Plant Molecular Physiology, Graduate School of
Agricultural and Life Sciences, The University of Tokyo, 1-1 Yayoi,
Bunkyo-ku, Tokyo 113-8657, Japan
Corresponding author: Shinozaki, Kazuo (sinozaki@rtc.riken.jp)
Current Opinion in Biotechnology 2006, 17:113–122
This review comes from a themed issue on
Plant biotechnology
Edited by Nam-Hai Chua and Scott V Tingey
Available online 21st February 2006
0958-1669/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2006.02.002
IntroductionGrowing in their natural environment, plants often
encounter unfavorable environmental conditions that
interrupt normal plant growth and productivity. Among
such environmental stresses, drought is one of the greatest
worldwide environmental constraints for agriculture [1].
Therefore, engineering drought tolerance in plants has
huge economical importance. To develop novel strategies
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for this purpose, one of the major research goals is to
understand the molecular mechanisms underlying drought
tolerance in plants. Drought triggers a wide variety of plant
responses, including alterations in gene expression, the
accumulation of metabolites such as the phytohormone
abscisic acid (ABA) or osmotically active compounds, and
the synthesis of specific proteins (e.g. largely hydrophilic
proteins, proteins that function to scavenge oxygen radi-
cals, chaperone proteins, etc. [2]). The basic strategy of
genetic engineering for drought tolerance is to introduce
functional genes that are directly involved in these events.
With the advancement of DNA microarray technology,
which allows high-throughput analysis of differential mes-
senger RNA expression, several hundred stress-induced
genes have been identified as candidate genes for genetic
engineering. Among the genes identified, several were
classified as regulatory genes, such as protein kinases
and transcription factors, in addition to functional genes.
Although the conventional approaches in both plant breed-
ing and physiology are of great importance [3,4], the
genetic engineering of key regulatory genes that govern
a subset of stress-related genes appears to be one of the
most promising strategies for enabling scientists to mini-
mize the deleterious effects associated with drought
(Figure 1). This review focuses on recent advances in
the basic research of drought stress tolerance at the mole-
cular level. In particular, we emphasize genetic engineer-
ing approaches in model plants using recently discovered
or tailored genes related to drought tolerance.
Functional proteins: classical and innovativeapproachesRecently, several successful attempts to engineer drought
tolerance have transferred functional genes that encode
enzymes associated with the synthesis of osmotically
active compounds, transporters, chaperones and reactive
oxygen species (ROS) scavengers, as shown in Table 1
(reviewed in [4,5]). In drought-tolerant transgenic plants,
many genes involved in the synthesis of compatible
solutes — organic compounds such as amino acids (e.g.
proline), quaternary and other amines (e.g. glycine-
betaine and polyamines) and a variety of sugars and sugar
alcohols (e.g. mannitol, trehalose and galactinol) that
accumulate during osmotic adjustment — have been used
to date [4,6]. The most recent advances in this area include
the finding of a novel synthetic pathway for glycine-
betaine, one of the major osmoprotectants, in halophilic
microorganisms [7,8,9��]. Higher levels of glycine-betaine
accumulation were detected in transgenic Arabidopsis
Current Opinion in Biotechnology 2006, 17:113–122
114 Plant biotechnology
Figure 1
Strategies for the genetic engineering of drought tolerance. Molecular and genomic analyses have facilitated the discovery of genes involved
in stress tolerance, which can then be used for genetic engineering. In this approach, a range of genes can be employed, including genes
encoding functional proteins such as transporters and chaperones. For metabolic engineering, enzymatic fusions are commonly used to target
multiple steps. In some cases, signal peptides are added to make proteins work in their correct organellar location. Recent studies have also
shed light on the role of regulatory proteins, such as transcription factors and signaling factors, which have provided novel routes for engineering
drought tolerance. The activity of such regulatory proteins can be controlled through point mutations, partial deletions, post-translational
modifications (e.g. phosphorylation by kinases) or by the addition of activation or repression domains. Genes can also be regulated at the
mRNA level (e.g. upregulated by sense overexpression transgenes and downregulated by antisense or RNAi). The intensity and time-space
pattern of transgene expression can be driven by appropriate promoters (Pro).
plants expressing the betaine synthesis genes of the novel
pathway, which acts via glycine, than in plants expressing a
choline-oxidizing enzyme from the conventional choline-
mediated betaine synthesis pathway [9��]. Interestingly,
although the accumulation of glycine-betaine in these
transgenic plants is still lower than in plants of species
that normally accumulate glycine-betaine under condi-
tions of stress, these transgenic plants showed significant
augmentation of dehydration tolerance. The increased
tolerance could be associated with the protective function
of betaine molecules, which might be involved in the
stabilization of proteins and cell structures and/or in
scavenging of free-radicals, as well as the osmotic effects
mediated by betaine molecules [6].
One of the most important metabolites produced in
response to drought stress is the plant hormone ABA
[10��]. Recently, in addition to a key ABA biosynthesis
gene (NCED3 [11]), a cytochrome P450 CYP707A family
has been identified as ABA 80-hydroxylases, which play a
central role in regulating ABA levels during seed imbibi-
Current Opinion in Biotechnology 2006, 17:113–122
tion and dehydration stress conditions [12�,13�]. It is
possible that these findings could enable us to control
ABA levels under conditions of drought stress and may
ultimately contribute to an advancement in the engineer-
ing of drought tolerance. Indeed, an insertional mutant of
CYP707A3, which was expressed most abundantly among
four CYP707A members under stress conditions, exhib-
ited elevated drought tolerance with a concomitant reduc-
tion of transpiration rate [14].
Not only metabolites, but some stress-responsive proteins
such as the late-embryogenesis-abundant (LEA) class of
proteins, have also been thought to function in the
detoxification and alleviation of cell damage during dehy-
dration [10��]. Overexpression of some LEA genes has
been reported to result in enhanced tolerance to dehy-
dration, although the precise mechanism is still unknown
[4,10��]. Recent biochemical analysis demonstrated that
LEA proteins can prevent protein aggregation induced by
desiccation as well as freezing [15]. Taken together with
recent computational studies [16,17], LEA proteins are
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Engineering drought tolerance Umezawa et al. 115
Table 1
Engineering drought tolerance using functional proteins.
Classification Gene namea Transgenic Origin Expressionc Experimentd Parameters Year Ref.
Osmolyte metabolism
Fructan SacB Tobacco B. subtilisb CaMV35SP 5–10% PEG (soil) Plant growth 1995
Trehalose TPS1 Tobacco S. cerevisiaeb CaMV35SP Desiccation FW, survivability 1996 e
myo-Inositol IMT1 Tobacco Iceplant CaMV35SP Water withholding Photosynthesis 1997 e,f,g
Proline P5CS Rice Mothbean AIPC-ABA-
inducible
Water withholding Shoot growth 1998 e
Trehalose OtsA, OtsB Tobacco E. colib CaMV35SP Limiting water supply Leaf dry weight
(productivity)
1998 e,g
Fructan SacB Sugar beet B. subtilisb CaMV35SP Limiting water supply Biomass production 1999
Glycine betaine COX Arabidopsis/
canola/
tobacco
A. pascensb CaMV35SP Limiting water supply Shoot growth 2000
Galactinol AtGolS2 Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability 2002 e
Trehalose TPSP
(OtsA+OtsB)
Rice E. colib ABA-inducible/
rbcSP
Water withholding Photosynthesis,
shoot growth
2002 e,g
Trehalose TPSP
(OtsA+OtsB)
Rice E. colib Maize Ubi-1P Water withholding Photosynthesis,
shoot growth
2003 e
Mannitol mtlD Wheat E. colib Maize Ubi-1P Limiting water supply Shoot growth, biomass 2003 e,f,g
Polyamines ADC Rice D. stramoniumb Maize Ubi-1P 20% PEG (soil) Shoot growth 2004 [69]
Polyamines SPDS Arabidopsis C. ficifolia CaMV35SP Water withholding Shoot growth 2004 [70]
Proline P5CS Petunia Arabidopsis, rice CaMV35SP Water withholding Survivability 2005 [71]
Trehalose TPS1 Tomato S. cerevisiaeb CaMV35SP Water withholding Shoot growth 2005 [72]
Glycine betaine GSMT+DMT Arabidopsis, A. halophyticab CaMV35SP Water withholding Photosynthesis,
shoot growth
2005 [9��]
Protective proteins
LEA HVA1 Rice Barley Rice Act-1P Water withholding Plant growth 1996 f,g
LEA HVA1 Wheat Barley Maize Ubi-1P Limiting water supply Plant growth, biomass 2000 f,g
Chaperone BiP Tobacco Soybean CaMV35SP Water withholding Shoot growth, RWC,
photosynthesis
2001 e
Heat shock protein AyHsp17.6A Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability, FW 2001 e,f
LEA HVA1 Rice Barley Rice Act-1P Water withholding Shoot growth, RWC,
water potential
2004 [73]
LEA LEA Chinese cabbage Canola CaMV35SP Water withholding Shoot growth,
survivability
2005 [74]
ROS-scavenging proteins
Detoxification MnSOD Alfalfa N. plumbaginifolia CaMV35SP Water withholding,
field trial
Photosynthesis,
electrolyte leakage,
yield
1996 e,f,g
Lipid peroxide MsALR Tobacco Alfalfa CaMV35SP Water withholding Photosynthesis 2000 e,f,g
NAD+ breakdown PARP Canola – CaMV35SP
(RNAi)
Water withholding FW, shoot growth 2005 [75]
Others
Unknown CDT1 C. plantagenium C. plantagenium Agrobacterium
pg5
Callus Survivability of callus 1997 e
Ion transport AVP1 Arabidopsis Arabidopsis CaMV35SP Water withholding Plant growth, RWC 2001 e,f
ABA biosynthesis AtNCED3 Arabidopsis Arabidopsis CaMV35SP Water withholding Shoot growth 2001 e
Stomata Chl-NADP-ME Tobacco Maize Agrobacterium
MAS
Hydroponic culture,
soil
Stomatal conductance,
plant growth
2002 [76]
ABA catabolism CYP707A3 Arabidopsis Arabidopsis Knockout Water withholding Survivability,
transpiration rate
2005 h
aADC, arginine decarboxylase; ALR, aldose/aldehyde reductase; AVP1, H+-pyrophosphatase; Chl-NADP-ME, chlorophyll-targeting NADP-malic
enzyme; COX, choline oxidase; DMT, dimethylglycine methyltransferase; GolS, galactinol synthase; GSMT, glycine sarcosine; methyltransferase; IMT,
myo-inositol O-methyltransferase; MnSOD, manganese superoxide dismutase; mtlD, mannitol-1-phosphate dehydrogenase; NCED, 9-cis
epoxycarotenoid dioxygenase; OtsA, TPS, trehalose-6-phosphate synthase; OtsB, TPP, trehalose-6-phosphate phosphatase; PARP,
poly(ADP-ribose) polymerase; P5CS, D1-pyrroline-5-carboxylate synthase; SPDS, spermidine synthase; TPS, trehalose-6-phosphate synthase;
TPSP, TPS+TPP. bNon-plant species. cPromoter list and comment for mode of regulation (only in the case of downregulation of target genes).dPEG, polyethylene glycol; FW, fresh weight; RWC, relative water content; Chl, chlorophyll. References are cited in review articles: e[10��]; f[77],g[5]; h[6].
www.sciencedirect.com Current Opinion in Biotechnology 2006, 17:113–122
116 Plant biotechnology
proposed to function as chaperone-like protective mole-
cules and act against cellular damage [4,10��].
Thus, unraveling the mechanisms underlying the action
of metabolites and proteins involved in drought tolerance
could facilitate the creation of stress-tolerant plants. In
addition, the recent progress of metabolome analysis
should also contribute to the discovery of novel stress
resistance systems that can be utilized for stress engineer-
ing [18].
Transcription factors for regulonbiotechnologyTranscriptome analyses using microarray technology,
together with conventional approaches, have revealed
that dozens of transcription factors (TFs) are involved
in the plant response to drought stress [4,10��]. Most of
these TFs fall into several large TF families, such as AP2/
ERF, bZIP, NAC, MYB, MYC, Cys2His2 zinc-finger and
WRKY. The expression of TFs regulates the expression
of downstream target genes that are involved in the
drought stress response and tolerance. Recent progress
in TF study (Table 2), which has led to the engineering of
drought tolerance using both transcriptional activators
and repressors, is discussed below.
Transcriptional activators that upregulate stress-respon-
sive genes have been utilized to produce drought-tolerant
transgenic plants. In addition to the enhancement of
drought tolerance, overexpression of the DREB1/CBF3
(dehydration-responsive element binding protein/CRT-
binding factor) TF in Arabidopsis controlled many stress-
inducible target genes [19–21] and increased tolerance to
freezing and high salt exposure [22]. In a more recent
example, transgenic plants expressing a drought-respon-
sive AP2-type TF, SHN1-3 or WXP1, induced several
wax-related genes and resulted in enhanced cuticular wax
accumulation and increased drought tolerance [23,24].
Thus, it has been shown that the overexpression of some
drought-responsive transcription factors leads to the
expression of downstream genes and the enhancement
of drought tolerance (see [25] for a review).
By contrast, overexpression of AREB1 (ABA-responsive
element binding protein; ABF2) and DREB2A does not
result in the significant induction of downstream genes
nor cause any phenotypes related to drought tolerance
[26,27��]. There are two possible explanations for this
observation. Firstly, downstream target genes governed
by the TFs might not be directly involved in drought
tolerance or, secondly, some modification could be
required after transcription to induce the expression of
downstream target genes. In the latter case, an altered
form of AREB1 has been recently reported that over-
comes this problem [27��]. Although overexpression of
the intact AREB1 gene on its own is not sufficient to
induce the expression of downstream genes, transgenic
Current Opinion in Biotechnology 2006, 17:113–122
plants that express a constitutively active form of AREB1,
created by deleting the regulatory sequence linking the
activation and DNA-binding domains of the protein, did
confer drought tolerance [27��]. In combination with loss-
of-function approaches, the downstream genes of
AREB1, which encode LEA proteins and regulatory
genes, were identified by microarray analysis [27��]. This
is consistent with recent findings that a DNA-binding
domain alone is sufficient to confer target gene specificity
of a native TF [28]. Overexpression of DREB2A carrying
a small internal deletion of 30 amino acids induces the
expression of downstream genes under untreated condi-
tions and enhances drought tolerance (Y Sakuma et al.,personal communication). Interestingly, the deleted
region contains a transcriptional inhibitory region with
a PEST sequence, which is generally known to play a role
in the degradation of the protein, although the actual
inhibitory mechanism of DREB2A is still unknown (Y
Sakuma et al., personal communication).
In addition to the deletion of inhibitory regions, the use of
point mutations is an important strategy to produce active
forms of transcriptional activators for engineering drought
tolerance. A point mutation mimicking the phosphoryla-
tion of a rice bZIP transcription factor, TRAB1, from
serine to aspartic acid at a phosphorylation site, signifi-
cantly increased the level of transcriptional activation in
the absence of the inducer ABA in a rice protoplast
transient assay [29]. Recently, transgenic plants expres-
sing a phosphorylated active form of AREB1 with multi-
site mutations also resulted in the induction of many
ABA-responsive genes without exogenous ABA applica-
tion [30]. These data suggest that TFs rendered consti-
tutively active by point mutations could have the
potential to contribute to the enhancement of drought
tolerance by gene transfer.
As recent genomics technologies have shown that various
types of TFs are involved in the response to drought
stress in plants, the creation of active forms of transcrip-
tional activators is an attractive approach for the produc-
tion of drought-tolerant plants. Along similar lines, an
excellent review recently provided valuable insight into
the creation of plants overexpressing TFs and considered
how they can be used to analyse TF function [31��].
Transcriptional repressors that downregulate gene
expression under conditions of stress have also been used
to engineer drought tolerance. AtMYB60, an R2R3-MYBtranscriptional repressor in Arabidopsis, functions in the
regulation of stomatal movements. This gene is specifi-
cally expressed in guard cells and its expression is nega-
tively regulated during drought stress [32]. A null, T-
DNA insertion mutation in AtMYB60 results in the con-
stitutive reduction of stomatal opening and minimizes
wilting under water stress conditions. Interestingly, the
atmyb60-1 mutation results in guard-cell-specific defects,
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Engineering drought tolerance Umezawa et al. 117
Table 2
Engineering drought tolerance using transcription factors.
Classificationa Gene nameb Transgenic Origin Expressionc Experiment Parametersd Year Ref.
AP2/ERF family
DREB1/CBF DREB1A/CBF3 Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability 1998 e,f
DREB1/CBF DREB1A/CBF3 Arabidopsis Arabidopsis Arabidopsis
RD29AP
Water withholding Survivability 1999 e,f
DREB1/CBF DREB1B/CBF1 Tomato Arabidopsis CaMV35SP Water withholding Plant growth 2002 e,f,g
DREB1/CBF CBF4 Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability 2002 e,f
DREB1/CBF ZmDREB1A Arabidopsis Maize CaMV35SP Desiccation Electrolyte leakage 2004 [78]
DREB1/CBF DREB1C/CBF2 Arabidopsis Arabidopsis Knock out Desiccation FW 2004 [79]
AP2/ERF SHN1/WIN1 Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability 2004 [23]
DREB1/CBF DREB1A/CBF3 Wheat Arabidopsis Arabidopsis
RD29AP
Water withholding Plant growth 2004 [80]
DREB1/CBF DREB1A/CBF3 Tobacco Arabidopsis Arabidopsis
RD29AP
Water withholding Survivability 2004 [33]
DREB1/CBF DREB1A/CBF3 Rice Arabidopsis Maize Ubi-1P Water withholding Photosynthesis,
survivability
2005 [81]
AP2/ERF WXP1 Alfalfa M. truncatula CaMV35SP Water withholding Survivability 2005 [24]
DREB2 DREB2A (active
form with internal
deletion)
Arabidopsis Arabidopsis CaMV35SP,
Arabidopsis
RD29AP
Water withholding Survivability 2005 h
Basic leucine-zipper (bZIP) protein
bZIP ABF3, AREB2/ABF4 Arabidopsis Arabidopsis CaMV35SP Water withholding Plant growth,
survivability
2002 e,f
bZIP AREB1/ABF2 Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability 2004 [82]
bZIP ABF3 Rice Arabidopsis Maize Ubi-1P Water withholding Photosynthesis,
survivability
2005 [81]
bZIP AREB1/ABF2
(active form with
internal deletion)
Arabidopsis Arabidopsis CaMV35SP Water withholding,
dehydration
Survivability 2005 [27��]
bZIP AREB1/ABF2
(phosphorylated
active form)
Arabidopsis Arabidopsis CaMV35SP Dehydration Survivability 2005 [30]
MYB/MYC
MYB, MYC AtMYC2, AtMYB2 Arabidopsis Arabidopsis CaMV35SP Mannitol treatment Electrolyte leakage 2002 e,f
MYB CpMYB10 Arabidopsis C. plantagineum CaMV35SP Water withholding Survivability 2004 e
R2R3-MYB AtMYB60 Arabidopsis Arabidopsis Knock out Water withholding Plant growth, RWC 2005 [32]
Zinc-finger protein
Cys2His2-type ZPT2-3 Petunia Petunia CaMV35SP Dehydration Survivability 2003 e
Cys2His2-type CAZFP1 Arabidopsis Pepper CaMV35SP Water withholding Survivability,
Chl content
2004 [39]
Cys2His2-type STZ Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability,
electrolyte leakage
2004 [38]
Others
NAC ANAC019/055/
072
Arabidopsis Arabidopsis CaMV35SP Water withholding Survivability 2004 [83]
aAP2/ERF, APETALA2/ethylene-response factor; DREB1/CBF, dehydration-responsive element binding protein/C-repeat binding factor;
NAC, NAM/ATAF/CUC. bCAZFP1, Capsicum annuum zinc-finger protein 1; SHN1/WIN1, shine1/wax inducer 1; STZ, salt-tolerance zinc
finger protein; WXP1, wax production 1. cFW, fresh weight; RWC, relative water content; Chl, chlorophyll. d Promoter list and comment for
mode of regulation (only in the case of downregulation of target genes). References are cited in review articles: e[10��]; f[77], g[5]; h Y Sakuma,
et al. unpublished.
with no apparent deleterious affects upon other develop-
mental and physiological processes [32]. This is very
important when considering the yield of agricultural
crops, because most transgenic plants that constitutively
express TFs show growth retardation and alterations in
basic metabolism [4]. Although such undesirable traits
can be improved to some extent through the use of stress-
inducible promoters that regulate the expression of TFs
[22,33], the engineering of a stomatal response as a means
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to reduce water loss is an attractive approach to confer
drought tolerance in crops [34].
Several plant zinc-finger transcriptional repressors carry
repression domains and are involved in the downregula-
tion of gene expression. A repression domain, SRDX,
which consists of only 12 amino acids derived from the
EAR motif of SUPERMAN, a TFIIIA-type zinc finger
repressor, was recently shown to be useful for determining
Current Opinion in Biotechnology 2006, 17:113–122
118 Plant biotechnology
the role of stress-responsive TFs, which often have func-
tionally redundant homologs [27��,35��,36��]. Overexpres-
sion of a chimeric repressor comprising a TF fused to the
SRDX domain dominantly suppressed the expression of
target genes, even in the presence of redundant TFs
[27��,35��,36��,37]. The repression domain SRDX is use-
ful for identifying the role of TFs through the creation of a
dominant loss-of-function mutant. Constitutive expres-
sion of a Cys2/His2-type zinc-finger transcriptional repres-
sor, STZ, which was upregulated by dehydration, high-
salt, cold stress and ABA treatment, increased tolerance to
drought stress [38]. Transgenic Arabidopsis plants expres-
sing an STZ ortholog (CAZFP1), which normally func-
tions as a transcriptional repressor in yeast, also showed
tolerance to drought stress and exhibited resistance against
bacterial infection [39]. Overall, in addition to the con-
ventional overexpression of TFs in regulon biotechnology,
the modification of TFs might one day allow us to create
TF-modified plants that confer novel traits.
Engineering drought tolerance usingsignaling factorsUpstream of TFs, various signal transduction systems
function in abiotic stress responses, involving protein
phosphorylation and/or dephosphorylation, phospholipid
metabolism, calcium sensing, protein degradation and so
on (for reviews, see [4,10��,40�]). Although these complex
signaling processes are not yet fully understood, several
genes encoding signaling factors that function in the
drought response have been identified [25,41,42]. Some
of these factors are currently available for engineering
drought tolerance (Table 3).
Table 3
Engineering drought tolerance using signaling factors.
Classificationa Gene nameb Transgenic Origin Expressionc
Protein kinases
CDPK OsCDPK7 Rice Rice CaMV35SP
GSK3/Shaggy AtGSK1 Arabidopsis Arabidopsis CaMV35SP
MAPKKK NPK1 Maize Tobacco CaMV35SP
SnRK2 SRK2C Arabidopsis Arabidopsis CaMV35SP
Others
Calcium sensor CBL1 Arabidopsis Arabidopsis Agrobacteri
MAS
14-3-3 Protein GF14l Cotton Cotton CaMV35SP
CC-NBS-LRR ADR1 Arabidopsis Arabidopsis CaMV35SP
Farnesyl-
transferase
ERA1 Arabidopsis,
canola
Arabidopsis CaMV35SP/
RD29AP
(antisense)
aCC-NBS-LRR, coiled-coil motif – nucleotide-binding site – leucine-rich rep
GSK3/Shaggy-like protein kinase; MAPKKK, mitogen-activated protein k
enhanced response to ABA 1; bNPK1, Nicotiana protein kinase 1. cPro
downregulation of target genes). dFv/Fm, variable fluorescence/maximum fl
Current Opinion in Biotechnology 2006, 17:113–122
One of the merits for the manipulation of signaling factors
is that they can control a broad range of downstream
events, which results in superior tolerance for multiple
aspects. For example, a tobacco mitogen-activated pro-
tein kinase kinase kinase (MAPKKK), NPK1, which was
truncated for constitutive activation, activated an oxida-
tive signal cascade and led to cold, heat, salinity and
drought tolerance in transgenic plants [43,44]. Conver-
sely, as shown in the case of farnesyltransferases, suppres-
sion of signaling factors can also effectively enhance
tolerance to abiotic stress. In the regulation of stomatal
closure by ABA, a b subunit of farnesyltransferase ERA1
functions as a negative regulator of ABA signaling [45,46].
Antisense downregulation of the a or b subunits of
farnesyltransferase enhances response to ABA and
drought tolerance of canola plants [47�].
An additional advantage of engineering signaling factors
is that they can control the signal output involved in stress
resistance. In many cases, signaling factors are activated
or inactivated in response to various stress conditions. For
example, an SNF1-related protein kinase 2 (SnRK2) was
shown to be activated by ABA or osmotic stress, suggest-
ing that SnRK2 might be important for signal transduc-
tion in the stress response [48–51]. There are ten
members of the SnRK2 family in Arabidopsis (SRK2A–
J/SnRK2.1–10) and ten members identified in the rice
genome (SAPK1–10); almost all SnRK2s showed specific
activation by ABA or hyperosmolarity [52,53]. The most
crucial finding in SnRK2 function was established in
guard cells: that is, fava bean ABA-activated protein
kinase (AAPK) was essential for ABA-dependent stomatal
Experiments Parametersd Year Ref.
Water withholding Shoot growth, Fv/Fm,
wilting, gene expression
2000 e,f
Water withholding Survivability 2001 e
Limiting water supply Leaf number, kernel
yield
2004 [44]
Water withholding Survivability, gene
expression
2004 [57��]
um Water withholding Survivability, gene
expression
2003 [59]
Limiting water supply Senescence, Chl content,
photosynthesis
2004 [84]
Water withholding Survivability, gene
expression
2004 e
Water withholding,
field test
Survivability, water loss,
seed yield, oil content
2005 [47�]
eat domains; CDPK, calcium-dependent protein kinase; GSK3/Shaggy,
inase kinase kinase. bADR1, activated disease resistance 1; ERA1,
moter list and comment for mode of regulation (only in the case of
uorescence. References are cited in review articles: e[10��]; f[5].
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Engineering drought tolerance Umezawa et al. 119
closure [50]. In Arabidopsis, the physiological function of
SRK2E/OST1 (SnRK2.6), an ortholog of AAPK, has been
confirmed in planta: srk2e or ost1 mutants showed hyper-
sensitivity to drought because of their lack of transpira-
tional control [54–56]. These data suggested that SnRK2
serves as a central hub to mediate ABA signaling in guard
cells. In addition, one of the Arabidopsis SnRK2s, SRK2C
(SnRK2.8), is an osmotic stress-activated protein kinase
and its overexpression significantly improves drought
tolerance in Arabidopsis [57��]. Under normal conditions,
the activity of overexpressed SRK2C in complex with
green fluorescent protein (SRK2C–GFP) was difficult to
detect, despite considerable amounts of mRNA. By con-
trast, SRK2C–GFP was dramatically activated after
drought stress, resulting in the enhancement of transcrip-
tional events and drought tolerance in Arabidopsis. These
data support the supposition that stress-dependent acti-
vation or deactivation of signal components might func-
tion as a molecular switch for the biotechnological
manipulation of the stress response in plants.
CBL/SCaBP (calcineurin B-like protein/SOS3-like cal-
cium-binding protein) and its interacting partner CIPK/
PKS, which is identical to SnRK3, form a complex that is
known to be an important regulator of signal transduction
in the presense of ABA and under conditions of drought,
salt or cold stress [58,59,60�,61�,62,63]. CBL1 is a member
of the CBL/SCaBP family of proteins which are induced
by cold, NaCl, ABA and hyperosmotic stress. Plants
overexpressing CBL1 exhibited elevated drought and
salt tolerance and upregulated expression of stress-
responsive genes [59]. Although SnRK3 seems to be
important for engineering drought tolerance, it is not a
simple case. The manipulation of SnRK3 for drought
stress is complicated because its kinase activity is regu-
lated by a Ca2+-dependent interaction of CBL/SCaBP
proteins which sense internal or external stimuli. As
demonstrated in the case of SOS2 or PKS18 kinases,
mutations in the activation loop of the catalytic domain
or deletion of the FISL motif, which is essential for CBL/
SCaBP interactions, enable the production of a constitu-
tively active form of SnRK3 [64]. It is thought that this
technology will prove useful for the application of SnRK3
to genetic engineering.
Although recent progress in this field has identified
several signaling factors related to the drought stress-
response in plants, these findings also highlight the
complexity of the signal transduction network with
cross-talk, feedback and physical interactions delivering
appropriate signals to suitable targets at the correct time.
As with the yeast ‘scaffold’ hypothesis, signaling proteins
often have multiple signal outputs, an occurrence that
explains the different functions of the MAPKKK, Ste11p
[65,66]. For example, AtMPK6 functions in at least two
different MAPK cascades, MKK2-MPK6 and MKK4/
MKK5-MPK6, and each combination can transmit or
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transduce different signals from cold or salt stress, and
bacterial pathogens, respectively [67]. Further functional
or biochemical analyses will be required to gain a pre-
cise understanding of the functions of various signaling
factors in plants, which can then be used to promote
the engineering of drought tolerance using signaling
factors.
ConclusionsWe have identified several factors related to the plant
response to drought stress and many of these factors have
already been shown to be effective for engineering
drought tolerance in model plants (Tables 1–3). Such
demonstrated success under experimental conditions has
encouraged the use of this strategy to engineer drought
tolerance in crop species (Figure 1). Since the application
of functional genomics approaches, the identification of
candidate genes related to drought response has
increased, thus fueling this approach for genetic engi-
neering. In future, the combination of transcriptomic,
proteomic or metabolomic analyses will also be useful
for gene discovery in the engineering of drought toler-
ance. As well as gene discovery, technical advances in
applied research are also important for engineering
drought-tolerant plants. Transformation methodologies
should be developed, adapted and applied to many more
plant species, because currently we are only able to
produce transgenic plants with a limited selection of plant
species. An effective expression system, including suita-
ble promoters, will also be required for each plant species,
because constitutive promoters such as the CaMV35Spromoter are not always functional or can have negative
effects on plant growth or development [22,68��].
Recent studies have shed light not only on structural
proteins, such as metabolic enzymes and LEA proteins,
but also on regulatory proteins such as TFs and signaling
factors that control a broad range of stress responses.
Significant cross-talk and inter-connections are involved
in stress signaling, such as positive and negative regula-
tors or trans- and cis-acting factors [25,42,68��]. Never-
theless, systematic approaches with molecular and
genomic analyses — mainly using Arabidopsis or other
model plants — will facilitate the resolution of such
complex networks and lead to the discovery of additional
stress factors or mechanisms. Furthermore, it is antici-
pated that several areas, such as post-transcriptional reg-
ulation involving protein modification, protein
degradation and RNA metabolism, will emerge and
assume importance in the future. Such basic studies will
allow us to create novel approaches for the molecular
tailoring of genes (e.g. modulation of the activity of target
proteins with deletions, mutational or conformational
changes as shown in the case of AREB1 or DREB2A).
It is also hoped that these approaches will allow us to fine-
tune drought tolerance according to the time and circum-
stances for the onset of this environmental constraint.
Current Opinion in Biotechnology 2006, 17:113–122
120 Plant biotechnology
AcknowledgementsThis work was supported in part by a grant for Genome Research fromRIKEN, the Program for Promotion of Basic Research Activities forInnovative Biosciences from the Bio-Oriented Technology ResearchAdvancement Institution of Japan (BRAIN), the Special CoordinationFund of the Japan Science and Technology Agency (JST), and aGrant-in-Aid from the Ministry of Education, Culture, Sports, Scienceand Technology of Japan (MEXT) to KS and KY-S. This work was alsosupported in part by Grant-in-Aids for Scientific Research for YoungScientists (B) 14760075 and 16770043 from MEXT to TU.
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