Engineering drought tolerance in plants_ discovering and tailoring genes to unlock the future

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 ([email protected])

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

www.sciencedirect.com

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


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-


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


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


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


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,



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


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



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


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].



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


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.



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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Engineering drought tolerance in plants_ discovering and tailoring genes to unlock the future

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