Transgenic plants for abiotic stress tolerance: current status

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Transgenic plants for abiotic stresstolerance: current statusSyed Sarfraz Hussain a , Hasnain Raza a , Irfan Afzal b & MahmoodAkhtar Kayani ca Institute for Molecular Physiology and Biotechnology of Plants(IMBIO-Bartels), University of Bonn, Bonn, Germanyb Department of Crop Physiology, University of Agriculture,Faisalabad, Pakistanc Department of Biosciences, COMSATS Institute of InformationTechnology (CIIT), Islamabad, PakistanPublished online: 16 Jun 2011.

To cite this article: Syed Sarfraz Hussain , Hasnain Raza , Irfan Afzal & Mahmood Akhtar Kayani(2012) Transgenic plants for abiotic stress tolerance: current status, Archives of Agronomy and SoilScience, 58:7, 693-721, DOI: 10.1080/03650340.2010.540010

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Transgenic plants for abiotic stress tolerance: current status

Syed Sarfraz Hussaina*, Hasnain Razaa, Irfan Afzalb andMahmood Akhtar Kayanic

aInstitute for Molecular Physiology and Biotechnology of Plants (IMBIO-Bartels), Universityof Bonn, Bonn, Germany; bDepartment of Crop Physiology, University of Agriculture,Faisalabad, Pakistan; cDepartment of Biosciences, COMSATS Institute of Information

Technology (CIIT), Islamabad, Pakistan

(Received 7 May 2010; final version received 1 November 2010)

Abiotic stress is one of the primary causes of crop losses worldwide (Bray et al.2000. Responses to abiotic stresses. In: Buchanana BB, Gruissem W, Jones RL,editors. Biochemistry and Molecular Biology of Plants. Rockville (MD):American Society of Plant Physiologists. p. 1158–1249). To cope with thedetrimental effects of stress, plants have evolved many biochemical and molecularmechanisms. One of the well-documented stress responses in plants is accumula-tion of osmolytes during stress. Although their actual roles in plant-stresstolerance remain controversial, these molecules are thought to have positiveeffects on enzyme and membrane integrity, along with adaptive roles in mediatingosmotic adjustment in plants grown under drought conditions. Recent studieshave demonstrated that the manipulation of genes involved in the biosynthesis ofthese osmolytes have improved plant tolerance to drought and salinity in anumber of crops. There is renewed hope of understanding the molecular basis ofosmolyte accumulation under stress and manipulating these processes via geneticengineering. For future work on generating transgenic plants with still higherlevels of tolerance, the new knowledge may be used via guided genetic engineeringof multiple genes to create crop plants with significantly increased productivityunder drought stress. This review surveys the current advances in engineeringabiotic stress-tolerant plants, particularly the genetic engineering of osmolytegenes (osmoprotectants) for imparting drought stress tolerance in plants.

Keywords: osmolytes; drought stress; transgenic plants; metabolic engineering

Introduction: a brief account of effects of drought on plants

Crop plants are affected by a variety of abiotic stresses, but drought represents someof the most significant constraints on agricultural productivity (Capell et al. 2004).Drought is the major abiotic stress severely affecting the distribution andproductivity of crop plants. For example, up to 45% of the world’s agriculturallands, wherein 38% of the world human population resides, are subjected tocontinuous or frequent drought (Hussain et al. 2008). Water is essential at everystage of plant growth from seed germination to plant maturation. A shortage ofwater is the single most important factor that reduces global crop yields, with far-reaching socio-economic implications (Castiglioni et al. 2008). Any degree of water

*Corresponding author. Email: [email protected]

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imbalance may produce deleterious effects on crop growth and development. Plantsexhibit a variety of responses to environmental stresses that enable them to surviveor tolerate adverse conditions. Such plants respond to drought by making changes atthe cellular, metabolic and molecular levels to cope with the stress (Zhu 2001;Shinozaki and Yamaguchi-Shinozaki 2007). Although the general effects of droughton plant growth and development are fairly well known, the primary effects of waterdeficit at the cellular and molecular levels are not well understood (Zhu 2002;Chaitanya et al. 2003; Chaves and Oliveira 2004).

In order to improve the agricultural productivity within the limited landresources, it is imperative to ensure high crop yields against unfavorableenvironmental stresses such as drought. Three main approaches have been used inbreeding drought tolerant varieties: (1) select for high yield potential under optimalconditions; (2) select for maximum yield in target environments; and (3) manipulatethe genetic parameters of drought stress tolerance in the breeding and selectionschemes. Based on these approaches, the success in obtaining tolerant plants viaconventional breeding strategies has been useful in some cases to limited extent.However, in general, the success rate has not been encouraging in the case of droughtstress because of the multigenic nature of the stress. Therefore, researchers feel lessmotivated to employ these approaches, which are also time-consuming andlaborious (Bartels and Hussain 2008).

Currently, there are no economically viable technological means to facilitate cropproduction under stress conditions (Ashraf and Foolad 2007) using breedingmethods. However, the development of crop plants tolerant to drought stress isconsidered a promising approach, which may help satisfy growing food demandsfrom both developing and under-developed countries. By contrast, improvement ofstress tolerance by genetic engineering overcomes the bottlenecks of plant breedingmethods. Transgenic approaches can be used in combination with conventionalbreeding strategies to create crops with enhanced drought tolerance (Capell et al.2004). The current genetic engineering strategies rely on the transfer to the targetplant of one or several genes that are either involved in signaling and/or regulatorypathways, or that encode enzymes present in pathways leading to the synthesis offunctional and structural protectants, such as osmolytes and antioxidants, orpathways that encode stress tolerance-conferring proteins (Wang et al. 2003; Vinocurand Altman 2005; Valliyodan and Nguyen 2006; Bhatnagar-Mathur et al. 2007;Kathuria et al. 2007; Sreenivasulu et al. 2007).

Engineering plants with osmoprotectant pathways

Improving crop resistance to drought stress is a long-standing goal of agriculturalbiotechnology (McCue and Hanson 1990; Jain and Selvaraj 1997). Plants haveevolved various mechanisms to cope with stress conditions and these include theshifts in the physiology of the plant and the expression of stress-associated genes,leading to the formation of a wide variety of low molecular mass metabolitescollectively known as compatible solutes such as proline, glycine betaine, sugars suchas sucrose, trehalose and fructans and sugar alcohols like sorbitol, mannitol,ononitol, pinitol and polyols (Bohnert and Jensen 1996; Rajam et al. 1998; Bohnertand Shen 1999; Kumar et al. 2006). These osmolytes are uniformly neutral withrespect to the perturbation of cellular functions even when present at high

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concentrations (Yancy et al. 1982; Serraj and Sinclair 2002). Accumulation of thesemolecules either actively or passively helps plants to retain water within cells andprotects cellular compartments from injury caused by dehydration or maintainsturgor pressure during water stress (Table 1–5). Moreover, these molecules stabilizethe structure and function of certain macromolecules, signaling functions orinduction of adaptive pathways and scavenge reactive oxygen species (Hasegawaet al. 2000; Chen and Murata 2002). However, the molecular and cellularinteractions of these solutes are not completely understood.

To date, tolerance to abiotic stresses has mainly been achieved throughengineering for increased cellular levels of compatible solutes (Flowers 2004; KumarS et al. 2004; Chinnusamy et al. 2005). Another noteworthy point is that increasedlevels of solutes often have enhanced tolerance for drought, salt and cold stress at thesame time (Garg et al. 2002; Jang et al. 2003; Park et al. 2007), implying that geneticengineering by altering osmolytes is a fruitful approach for obtaining combinedtolerance to different abiotic stresses. In fact, simple, one-step transformationstrategies have been designed to increase the accumulation of these osmolytes,especially in plant species, in which these molecules do not accumulate naturally. Inmost cases, the introduction of a single foreign gene into a transgenic plant has led toa moderate increase in tolerance with modest accumulation of osmoprotectants.

In this review, we focus on results published in recent years regarding theinvolvement of osmolytes in improving plant stress tolerance via transgenicapproach.

Amino acid-derived osmoprotectants and their role in drought tolerance

Metabolic acclimation via the accumulation of osmolytes/compatible solutes isregarded as a basic strategy for the protection and survival of plants in extremeenvironments. Accumulation of compatible solutes is ubiquitous in plants and isoften referred to as osmoprotection. In stress-tolerant transgenic plants, manygenes involved in the synthesis of osmoprotectants, such as amino acids (proline),quaternary compounds (glycine betaine) and a variety of sugars and sugaralcohols (mannitol, trehalose and galactinol) that accumulate during stressadjustment, have been used (Chen and Murata 2002; Chaves and Oliveira2004; Vinocur and Altman 2005). Although there are many examples of aminoacid-derived osmoprotectants, to date, metabolic engineering studies have mainlyfocused on increasing proline and betaine accumulation (Holmberg and Bulow1998; McNeil et al. 1999).

Proline

Proline is an osmoprotecting molecule that accumulates in many organisms,including bacteria, fungi and plants, in response to drought and salinity (Kumaret al. 2003; Claussen 2005). Proline is one of the most commonly found compatibleosmolytes in water-stressed plants (Delauney and Verma 1993; Yoshiba et al. 1997).Pyrroline-5-carboxylate synthetase (P5CS) is encoded by a nuclear gene that hasbeen cloned from several different plants, for example, Vigna aconitifolia (Hu et al.1992), Arabidopsis thaliana (Strizhov et al. 1997), Medicago sativa (Ginzberg et al.1998), Lycopersicum esculentum (Fujita et al. 1998), Medicago truncatula (Armen-gaud et al. 2004) and Phaseolus vulgaris (Chen et al. 2009).

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Table

1.

Stress-toleranttransgenic

plants

expressingosm

olytes(proline).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

Proline

P5CS

D1-pyrroline-5-carboxylate

synthase

V.aconitifolia

O.sativa

Drought&

salttolerance

Increasedbiomass

(higher

freshshoot&

rootweight)

IgarashiY

etal.1997;

Zhuet

al1998

P5CS

D1-pyrroline-5-carboxylate

synthase

A.thaliana

A.thaliana

Hypersensitiveto

osm

oticstress

Excessivefree

proline

causedgrowth

arrest

Nanjo

etal.1999

P5CSF129A

MutatedP5CS

V.aconitifolia

N.tabacum

Oxidative&

osm

otic

stress

tolerance

Rem

ovaloffeedback

inhibitionresulted

in2-fold

highprolinethantheplants

expressingP5CS

Honget

al.2000

PDH

Prolinedehydrogenase

A.thaliana

A.thaliana

Low

level

of

osm

otolerance

Novisible

effectongermination

&growth

oftransgenic

plants

Maniet

al.2002

d-OAT

Ornithine-d-

aminotransferase

A.thaliana

N.plumbaginifolia

Osm

oticstress

tolerance

Transgenic

plantshowed

higher

germinationand

increasedbiomass

Roosenset

al.2002

d-OAT

Ornithine-d-

aminotransferase

A.thaliana

O.sativa

Drought&

salt

stress

tolerance

Wuet

al.2003

P5CSF129A

MutatedP5CS

V.acinitifolia

C.sinensis

Osm

oticadjustmentand

droughttolerance

Transgenic

plants

showed

superiorosm

otic

adjustment&

higher

photosynthetic

rate

after

15daysofdrought

Molinariet

al.2004

P5CSF129A

MutatedP5CS

V.aconitifolia

O.sativa

Drought&

salinity

tolerance

Stress-inducible

expression

oftheP5CSshowed

faster

growth

ofshootandroot

resultingin

higher

biomass

Su&

Wu2004

P5CSF129A

MutatedP5CS

V.aconitifolia

G.max

Droughtresistance

Availabilityofhighfree

proline

andmaintenance

ofhighRWC

DeR

ondeet

al.2004

P5CSF129A

MutatedP5CS

A.thaliana

P.hybrida

Droughttolerance

Transgenic

petunia

plants

tolerated

14daysdroughtstress

Yamadaet

al.2005

PDH

Proline

dehydrogenase

A.thaliana

G.max

Drought&

heat

tolerance

Rapid

increase

inprolineresulted

inleast

waterloss

under

droughtstress

Sim

on-Sarkadiet

al.2005

P5CSF129A

MutatedP5CS

V.aconitifolia

N.tabacum

Droughtstress

tolerance

Possible

antioxidantrole

offree

proline

Gubis

etal.2007

P5CSF129A

MutatedP5CS

V.aconitifolia

S.officinarum

Droughtstress

tolerance

Significantincrease

inbiomass

after

12daysofwaterdeficit

Molinariet

al.2007

P5CSF129A

MutatedP5CS

V.aconitifolia

T.aestivum

Droughttolerance

Stressinducible

promoterresulted

inhighprolinecontent

Vendruscolo

etal.2007

(continued)

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Table

1.

(Continued).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

P5CSF129A

MutatedP5CS

V.aconitifolia

P.vulgaris

Droughttolerance

Elevatedfree

prolineresulted

inbetter

adaptationto

waterstress

Chen

JBet

al.2009

P5CSF129A

MutatedP5CS

V.aconitifolia

O.sativa

Saltstress

tolerance

Transgenic

plants

withhighprolinelevel

showed

higher

biomass

&growth

perform

ance

under

salinitystress

KumarV

etal.2010

P5CS

D1-pyrroline-5-

carboxylate

synthase

A.thaliana

C.tinctorius

Nospecificresponse

todroughtstress

Reductionin

leafdry

mass

&chlorophyllcontent

Thippeswamyet

al.2010

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Although the exact function of proline is still under investigation, prolineaccumulation can influence stress tolerance in multiple ways (Szabados and Savoure2009). Proline may serve as a hydroxyl radical scavenger (Smirnoff and Cumbes1989), thus reducing the acidity of the cell (Venekamp et al. 1989). In addition, itmay also function as an osmolyte (Kishor et al. 1995) and molecular chaperone,being able to protect protein integrity and enhance the activities of different enzymes(Iyer and Caplan 1998). Proline biosynthesis from the amino acid glutamate can beachieved by using the P5CS gene from mothbean (Vigna aconitifolia), which encodesa bifunctional enzyme containing the catalytic activities of g-glutamyl kinase andglutamic-g-semialdehyde dehydrogenase (Holmberg and Bulow 1998).

Several studies have used this gene to produce transgenic plants (Table 1) withincreased proline production and subsequent stress tolerance (Kishor et al. 1995;Zhu et al. 1998; Roosens et al. 2002; Sawahel and Hassan 2002; Verdoy et al. 2006).Mani et al. (2002) generated transgenic Arabidopsis containing either sense orantisense proline dehydrogenase (PDH). Transgenic plants carrying sense PDH didnot show significant levels of osmotolerance under stress conditions. However,exogenous application of proline increased the tolerance of sense transgenic plants toosmotic stress. Indeed, transgenic Arabidopsis plants with antisense (Mani et al.2002) or knockout (Nanjo et al. 2003) PDH resulted in increased prolineaccumulation and better growth performance under stress (Simon-Sarkadi et al.2005).

It has also been reported that transgenic petunia plants that overexpress P5CSgene from Arabidopsis and P5CS gene from rice can withstand drought conditionsfor longer than wild-type plants (Yamada et al. 2005). Also, the expression of P5CSresulted in drought tolerance in soybean (DeRonde et al. 2004). Similarly, Chen et al.(2009) noticed a higher transcript level (2.5 times) of PvP5CS gene in common beanunder drought. This showed that P5CS from common bean was a stress-induciblegene regulating the accumulation of proline in plants subjected to drought stress.Transgenic wheat plants expressing P5CS gene from Vigna aconitifolia under thecontrol of a stress-induced promoter complex AIPC resulted in enhanced prolineaccumulation as well as drought tolerance under water deficit for 15 days(Vendruscolo et al. 2007). The results of a series of experiments showed that prolinemight confer drought stress tolerance to transgenic wheat plants by increasing theantioxidant system rather than as osmotic adjustment mediator.

However, P5CS, a rate-limiting enzyme in proline biosynthesis, is subjected tofeedback inhibition by proline, and earlier reports suggested that prolineaccumulation in plants under stress might involve the loss of feedback regulationdue to a conformational change in the P5CS protein (Hong et al. 2000). Therefore,Hong et al. (2000) removed this feedback inhibition by site-directed mutagenesis andthe resulting gene P5CSF129A was recently used in two studies, which clearlydemonstrated the involvement of proline in response to drought and salt (Gubiset al. 2007; Chen et al. 2009). Transgenic tobacco carrying mutagenizedP5CSA129A gene showed no negative effects on plant growth or physiologyand accumulated 17–22-fold more proline than wild-type plants under controlconditions. Similarly, plants also exhibited drought tolerance by further elevatingproline content under drought stress (Gubis et al. 2007). However, despite this,there are only a few reports in which researchers have used this mutated versionof P5CS gene (Hong et al. 2000; Molinari et al. 2004; Bhatnagar-Mathur et al.2009; Kumar et al. 2010).

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Improvement in the drought or salt tolerance of crop plants via engineeringproline metabolism is a current possibility (Vendruscolo et al. 2007; Yamchi et al.2007; Bhatnagar-Mathur et al. 2009; Kumar et al. 2010) and should be exploredmore extensively. However, further research is required to study the feasibility ofengineering stress tolerance in plants via targeted engineering of proline metabolism.

Glycine betaine

Glycine betaine (GB) is a quaternary ammonium compound that occurs in a varietyof plants, animals and microorganisms (Rhodes and Hanson 1993). GB accumulatesin the chloroplasts and plastids of many halotolerant plants and increases thetolerance of such plants to various types of abiotic stresses (Chen and Murata 2008).The physiological role of GB in alleviating osmotic stress was proposed on the basisof its enhanced accumulation in some stressed plants (Wyn Jones 1984; Sakamotoand Murata 2001). This compound is one of several such compatible solutes(Dorffling et al. 1990) that have an osmoprotection function. These solutes are alsoknown to protect proteins and enzyme activities under water deficits (Mohanty et al.2002; Ashraf and Foolad 2007), and even to stabilize membranes during freezing(Zhao et al. 1992; Rhodes and Hanson 1993; Papageorgiou and Murata 1995). Thecryoprotective effect of GB appears to come from its compatibility withmacromolecular structure and function (Xing and Rajashekar 2001). It has beensuggested that glycine betaine can help stabilize the protein tertiary structure andprevent or reverse disruption of the tertiary structure of these proteins caused bynon-compatible (perturbing) solutes. Based upon this observation, it has beensuggested that GB has a chaperone function (Sakamoto and Murata 2000; Chen andMurata 2002).

GB-encoding genes have been cloned from various microorganisms and plants,and transgenic plants of various species have been produced that express one orseveral of these genes (Sakamoto et al. 1998; Nuccio et al. 1998; Huang et al. 2000;McNeil et al. 2000, 2001; Mohanty et al. 2002; Shen et al. 2002). A moderate stresstolerance was noted in some, but not all, such GB-producing transgenic lines, withvariable responses to drought, salinity and freezing (Table 2). In some cases, theexogenous supply of GB helps the engineered compound to improve the stresstolerance of the plant (Quan et al. 2004b) by regulating expression of specific stress-responsive genes and by blocking reactive oxygen species (ROS) accumulationduring stress, for example, chilling stress (Einset et al. 2007; 2008; Einset andConnolly 2009). Waditee et al. (2003) found an alternative biosynthetic pathway ofbetaine from glycine, which is catalyzed by two N-methyltransferase enzymes.Overexpression of these enzymes in Arabidopsis resulted in the accumulation ofsignificant level of GB (Waditee et al. 2005).

Despite the encouraging results regarding the effect of GB accumulation ondrought tolerance in transgenic plants, poor accumulation of GB has been observedin some instances due to a limited choline supply (Nuccio et al. 1998) and impairedimport of choline into the chloroplast (Nuccio et al. 2000). Many agronomicallyimportant crops such as wheat, potato and tomato are non-accumulators of GB andare therefore, potential targets for engineering GB biosynthesis to increase theirstress tolerance via the expression of GB biosynthesis genes. (Bartels and Hussain2008). Shirasawa et al. (2006) reported that transgenic rice plants expressing spinachcholine monooxygenase (CMO) accumulated GB at lower levels than those reported

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Table

2.

Stress-toleranttransgenic

plants

expressingosm

olytes(glycinebetaine).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

Glycinebetaine(G

B)

BADH

Betainealdehyde

dehydrogenase

A.hortensis

O.sativa

Improved

growth

&salinitytolerance

10%

transgenic

plants

setseed

in0.5%

NaClsolutionin

greenhouse

Guoet

al.1997

BADH

Betainealdehyde

dehydrogenase

H.vulgare

N.tabacum

Salttolerance

Tolerance

wasjudged

byhigh

biomass

&photosynthetic

activityunder

stress

Nakamura

etal.1997

BADH

Betainealdehyde

dehydrogenase

B.vulgaris

N.tabacum

Droughttolerance

Mainly

dueto

increased

dehydrogenase

activity

Trossatet

al.1997

CHO

Cholinedehydrogenase

E.coli

O.sativa

Drought&

salttolerance

Higher

yield

under

stresses

Takabeet

al.1998

codA

Cholineoxidase

E.coli

A.thaliana,

N.tabacum,

B.napus

Moderate

stress

tolerance

basedonrelative

shootgrowth

Variable

stress

(drought,

salinity,freezing)tolerance

inthreeplantspecies

Huanget

al.2000

betA

Cholinedehydrogenase

E.coli

Z.mays

Droughttolerance

atseedlingstage

Grain

yield

wassignificantly

higher

comparedwith

wild-type

Quanet

al.2004a

BADH

Betainealdehyde

dehydrogenase

A.hortensis

T.repens

Saltstress

tolerance

Elevateddehydrogenase

activity

resultingin

tolerance

Chen

CFet

al.2004

BADH

Betainealdehyde

dehydrogenase

D.carota

D.carota

Saltstress

tolerance

Overexpressionofnative

BADH

inplastids

KumarSet

al.2004

BADH

Betainealdehyde

dehydrogenase

A.hortensis

R.pseudoacacia

Saltstress

tolerance

Stressinducible

highbetaine

accumulation

Xia

etal.2004

GSMTþDMT

Glycinesarcosine

methyltransferase

–A.thaliana

Droughttolerance

Highbetaineaccumulation&

improved

seed

yield

Waditee

etal.2005

betA

Cholinedehydrogenase

E.coli

G.hirsutum

Droughttolerance

GBaccumulationwaspositively

correlatedwithdrought

stress

Lvet

al.2007

codA

Cholineoxidase

E.coli

S.tuberosum

Drought,salt&

oxidativestress

tolerance

Stress-inducible

GB

production

innon-accumulators

resulted

intolerance

Ahmadet

al.2008

CMO

Cholinemonooxygenase

A.hortensis

G.hirsutum

Salttolerance

Seedcottonyield

was

significantlyhigher

intransgenic

plants

ZhangH

etal.2009

codA

Cholineoxidase

A.globiform

isO.sativa

Droughttolerance

Transgenicplantsshowed

better

physiologicalperform

ance

atseedling,vegetative&

reproductivestage

Kathuriaet

al.2009

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by Sakamoto et al. (1998) and Mohanty et al. (2002). The evidence for a limitedinternal choline supply pointed to the need to enhance choline synthesis (Nuccioet al. 1998).

Recently, two independent studies addressed the role of GB in non-accumulatingplants. Lv et al. (2007) investigated the high GB accumulation under stress in fivehomozygous transgenic cotton lines at three development stages. The resultsdemonstrated that transgenic plants accumulated higher GB levels than wild-typeplants either before or after drought stress. Similarly, high GB levels in transgenicplants were found to be positively correlated with drought tolerance. Incontinuation, transgenic potato plants expressing a bacterial choline oxidase (betA) gene under the control of an oxidative stress-inducible SWPA2 promoteraccumulated high levels of GB under drought stress (Ahmad et al. 2008). In additionto high GB levels, transgenic plants also maintained high water content andaccumulated high levels of vegetative biomass compared with non-transgenic (NT)plants under drought. These results suggested that GB not only protects cellmembranes and enzymes under water stress, but is also involved in osmoticadjustment in transgenic plants. By contrast, the effectiveness of stress tolerance alsodepends on the cellular compartment in which GB accumulates, as shown in the caseof transgenic tomato plants in which chloroplastic accumulation is more effectivethan cytosolic accumulation (Park et al. 2007; Ahmad et al. 2008). Similarly, theprotective effects of GB in the reproductive organs of plants have been observed inArabidopsis, maize and tomato under different abiotic stresses (Sakamoto et al. 2000;Park et al. 2004; Quan et al. 2004a, 2004b). This protection might have beenmediated by the high levels of GB that accumulate in reproductive organs as aconsequence of translocation from other organs, such as leaves. These resultsrevealed that the accumulation of GB in reproductive organs can effectively protectplants from reproductive failure under stress.

Sugars and sugar alcohols and their role in drought tolerance

Accumulation of sugar-related compounds, such as sucrose, raffinose, sorbitol andfructans, has been observed repeatedly in response to osmotic stress in a variety oforganisms. Many of these compounds are believed to stabilize the membranes andproteins during dehydration. According to Crowe et al. (1992), sugars can replacethe water molecules and thus stabilize the proteins or membranes in a physical statesimilar to that in the presence of water molecules. An alternative function has beenproposed for the disaccharides sucrose and trehalose, which can form a glass phasein the dry state (Crowe et al. 1998). A glass is a liquid of high viscosity that is capableof slowing down chemical reactions, which could lead to long-term stability in aliving system (Bruni and Leopold 1992).

Trehalose

Accumulation of trehalose, a rare non-reducing sugar, occurs in many bacteria,yeast, fungi and in a few desiccation-tolerant plants, so-called resurrection plants.During desiccation stress, trehalose protects the biological molecules in response todifferent stress conditions, but it does not accumulate to high enough levels in mostplants, probably because of the presence of trehalase activity (Goddijn andSmeekens 1998; Singer and Lindquist 1998; Goddijn and van Dun 1999; Iturriaga

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et al. 2000). Trehalase contributes to trehalose degradation (Paul et al. 2008) and inthe presence of a trehalase inhibitor (validamycin A), trehalose accumulates toseveral fold in concentration (Goddijn and van Dun 1999). Trehalose acts as astorage carbohydrate and possesses the unique feature of reversible water-absorptionthat results in protection of the biological molecules from desiccation-induceddamage; this sugar appears to be superior to other sugars in conferring protection todamage (Rontein et al. 2002).

Trehalose is synthesized in two steps from glucose-6-phosphate and uridinediphosphoglucose, via trehalose-6-phosphate. The first step is catalyzed by trehalosephosphate synthase (TPS), and the second by trehalose-6-phosphatase (TPP)(Bartels and Hussain 2008).

Multiple studies have linked trehalose to stress tolerance in plants (Table 3). Thegenes encoding the trehalose biosynthesis enzymes from different sources weretransferred into plants to obtain transgenic plants with relatively higher levels oftrehalose. Different groups have generated stress-tolerance plants by transferringmicrobial trehalose biosynthetic genes in tobacco (Holmstrom et al. 1996; Goddijnet al. 1997; Romero et al. 1997; Pilon-Smits et al. 1998; Lee et al. 2003; Han et al.2005; Karim et al. 2007), rice (Garg et al. 2002; Jang et al. 2003), tomato (Cortinaand Culianez-Macia 2005), potato (Goddijn et al. 1997), and Arabidopsis (Karimet al. 2007; Miranda et al. 2007). Tobacco plants overexpressing the yeast trehalosephosphate synthase gene (TPS1) showed significant tolerance to drought. However,the plants displayed reduced growth under non-stress conditions and multiplephenotypic alterations (Holmstrom et al. 1996; Romero et al. 1997). Tobacco plantstransformed with bacterial OtsA (TPS1) genes have a greater ability to retain water,whereas plants with OtsB (TPP) have reduced levels of photosynthesis. Both types oftransgenic tobacco plants accumulate only a small amount of trehalose byconstitutively expressing the genes (Pilon-Smits et al. 1998). Although plants withimproved stress tolerance were obtained (Table 3), no direct correlation wasobserved between trehalose level and stress tolerance (Bartels and Hussain 2008).This discrepancy and the observation that modification of the trehalose levels oftenproduced altered growth and morphogenic phenotypes led to the discovery thattrehalose-6-phosphate, a metabolic intermediate of trehalose synthesis, is a signalingmolecule regulating sugar and starch metabolism, which links these pathways toother metabolic networks like photosynthesis and hormonal homeostasis (Goddijnand van Dun 1999; Paul 2007). Nevertheless, subsequent studies circumvent thisproblem by using a microbial TPS–TPP fusion gene in tobacco under the control ofthe Arabidopsis ribulose-1,5-bisphosphate carboxylase (RuBisCO) promoter. Thiseliminates the growth problem, while retaining the improved drought tolerance(Karim et al. 2007). A new development in this direction has been the over-production of trehalose using stress-inducible or tissue-specific expression of abifunctional trehalose-6-phosphate synthase/phosphatase (TPSP) fusion gene (OtsAand OtsB) in rice transgenic plants (Garg et al. 2002). Such transgenic rice plantsexhibited increased tolerance to drought, salt and low-temperature stresses. Inaddition, these transgenic plants also exhibited improved photosystem function andincreased photosynthetic activity under non-stressed conditions.

Recently, Alfalfa transgenic plants expressing bifunctional TPS–TPP enzymeexhibited tolerance to multiple abiotic stresses, however if the CaMV 35S promoterwas used the plants were moderately stunted. By contrast, expression under RD29Astress-inducible promoter led to large plants with significant increase in biomass

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Table

3.

Stress-toleranttransgenic

plants

expressingosm

olytes(trehalose).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

Trehalose

TPS1

Trehalose-6-phosphate

synthase

S.cerevisiae

N.tabacum

Improved

drought

tolerance

Altered

sugarmetabolism

&regulatory

pathwaysledto

pleiotropic

phenotypes

intransgenic

plants

Romeroet

al.1997

TPS1

Trehalose-6-phosphate

synthase

S.cerevisiae

N.tabacum

Enhanceddrought

tolerance

Afew

transgenic

plants

showed

phenotypic

alterationalongwith

stress

tolerance

ZhaoHW

etal.2000

TPS1

Trehalose-6-phosphate

synthase

S.cerevisiae

S.tuberosum

Droughttolerance

Trehalose

accumulationalsoaffected

plantdevelopment

Yeo

etal.2000

otsA

&otsB

TPS&

TPPfusiongene

E.coli

O.sativa

Drought,salt&

cold

tolerance

Sustained

growth,less

photo-oxidative

damage,

enhancedphotosynthesis

under

variousstresses

Garg

etal.2002

TPSP

Trehalose-6-phosphate

synthase

phosphatase

(TPSþTPP)

E.coli

O.sativa

Drought,salt&

cold

tolerance

Novisible

phenotypic

alterations

despitethehightrehalose

accumulation

Janget

al.2003

TPS1

Trehalose-6-phosphate

synthase

A.thaliana

A.thaliana

Droughttolerance

Transgenic

seedlingsexhibited

glucose

&ABA-insensitivephenotype

Avonce

etal.2004

TSase

Trehalose

synthase

G.frondosa

N.tabacum

Enhanceddrought&

salttolerance

Hightrehalose

accumulationresulted

inenhancedtolerance

&nonegative

effects

ZhangSZ

etal.2005

TSase

Trehalose

synthase

G.frondosa

S.officinarum

Improved

tolerance

toosm

oticstress

Multiple

phenotypic

aberrationswere

observed

intransgenics

WangZZ

etal.2005

TP

Trehalose

phosphorylase

P.sajor-caju

S.officinarum

Droughttolerance

Transgenicplants

did

notwithered

even

after

10daysofwaterdeficit&

also

exhibited

nopleiotropic

effects

Hanet

al.2005

TPS1

Trehalose-6-phosphate

synthase

S.cerevisiae

L.esculentum

Drought,salt&

oxidativestress

tolerance

Sustained

yield

under

both

stress

&non-stressconditions

CortinaandCulianez-

Macia2005

TPS1–TPS2

Bifunctionalgene

S.cerevisiae

N.tabacum

Droughttolerance

Maintenance

ofwaterstatus&

positive

influence

oftrehalose

onroot

development

Karim

etal.2007

TPS1–TPS2

Bifunctionalgene

S.cerevisiae

A.thaliana

Multiple

abiotic

stress

tolerance

Overexpressors

wereglucose

insensitive

&alsoinvolved

incarbohydrate

metabolism

Mirandaet

al.2007

(continued)

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Table

3.

(Continued).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

TPS1

Trehalose-6-phosphate

synthase

A.thaliana

N.tabacum

Droughttolerance

Transgenic

plants

showed

sustained

photosynthesisunder

stress

Alm

eidaet

al.2007

TPS1

Trehalose-6-phosphate

synthase

S.cerevisiae

S.tuberosum

Droughttolerance

Maintenance

ofRWC&

photosynthetic

activity

Stiller

etal.2008

TPP1

Trehalose-6-phosphate

phosphatase

O.sativa

O.sativa

Enhancedsalt&

cold

tolerance

Analysisoftransgenic

plants

suggested

apossible

role

ofTPPin

transcriptionalregulationpathways

under

stress

GeLF

etal.2008

TPS1–TPS2

Bifunctionalgene

S.cerevisiae

M.sativa

Multiple

abiotic

stress

tolerance

Improved

growth

withsignificant

increase

inbiomass

Suarezet

al.2009

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(Suarez et al. 2009). Subsequently, it should also be possible to modulate theincreased trehalose accumulation by downregulating the plant trehalase activity orby expressing the trehalose biosynthetic genes under tissue- or stress-specificregulation (Penna 2003; Stiller et al. 2008). Tobacco chloroplasts transformed with(Saccharomyces cerevisiae) ScTPS1 have generated plants with enhanced droughttolerance without the growth defects observed in nuclear-transformed ScTPS1transgenic plants. In such plants, trehalose accumulation was almost 25-fold higherin chloroplast of the transgenic plants compared with the nuclear transgenic plants(Lee et al. 2003). Tobacco plants transformed with trehalose synthase (TSase) fromGrifola frondosa Fr. showed no growth retardation, but rather enhanced droughtand salt tolerance (Zhang et al. 2005). Similarly, Han et al. (2005) used a differenttype of trehalose biosynthetic gene (trehalose phosphorylase) that bypasses thetrehalose-6-phosphate.

To date, the introduction of trehalose biosynthetic genes into several plants hasimproved their drought tolerance without having any negative effect on plant growthor grain yield (Pilon-Smits et al. 1999; Abebe et al. 2003). Miranda et al. (2007)reported construction and overexpression of the TPS1–TPS2 gene fusion inArabidopsis. Their results demonstrated that the transgenic Arabidopsis plantsshowed a significant increase in drought, freezing, salt and heat tolerance, with nosignificant morphological or growth alterations. Recently, Suzuki et al. (2008)revealed the involvement of yet another TPS, AtTPS5, in plant stress tolerance (heatstress). Although using trehalose to confer protection against multiple abioticstresses could potentially be of great use in improving the abiotic stress tolerance ofstress-sensitive crops, our understanding of how it interacts and acts upon stress isfar from complete. Hence, the regulatory network connected with trehalosemetabolism needs to be studied in detail before genetically engineering trehaloselevels can be used in practice.

Fructans and other carbohydrates

Fructan or polyfructose molecules serve as the main storage carbohydrate in manyplant species. Fructan-accumulating plants are globally distributed in temperateclimate zones, which are characterized by seasonal drought, cold or frost; such plantsare almost absent in the tropical regions (Hendry and Wallace 1993). In plants, thephysiological role of fructans is not clear and constitutes one of the most activeresearch areas involving transgenic fructan production (Livingston et al. 2009).However, fructans are thought to be involved in abiotic stress tolerance by virtue oftheir presence in vacuoles (Pilon-Smits et al. 1995). Fructans are also thought toprotect plants against drought and cold stress (Wiemken et al. 1995); evidence of thiswas derived from transgenic plants (Table 4).

To date, the SacB gene (encoding Bacillus subtilis levansucrase) has been usedmost extensively in such studies. Tobacco plants transformed with a bacterial genefor the fructan biosynthesis were more resistant to polyethylene glycol (PEG)-induced drought stress, as determined by changes in their growth properties andbiomass accumulation (Ebskamp et al. 1994; Pilon-Smits et al. 1995). Thesetransgenic plants performed better than controls under drought conditions,demonstrating rapid growth with increased fresh and dry weight accumulation(Chen and Murata 2002). Similarly, transgenic sugarbeet plants had significantlybetter growth statistics than wild-type plants under drought stress (Pilon-Smits et al.

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Table

4.

Stress-toleranttransgenic

plants

expressingosm

olytes(fructanandother

carbohydrates).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

Fructan&

other

carbohydrates

sacB

Levansucrase,a

fructosyltransferase

B.subtilis

N.tabacum,

B.vulgaris

PEG-induceddrought

tolerance

Highgrowth

rate

accompaniedby

norm

alplantdevelopment

Pilom-Smitset

al.1995,1999

Imt1

Myo-inositol-o-

methyltransferase

E.coli

N.tabacum

Drought&

salttolerance

Restorationofphotosynthesisledto

plantrecoveryafter

stress

Shevelevaet

al.1997

mt1D

Mannitol-1-phosphate

dehydrogenase

E.coli

N.tabacum

Drought&

salinitysensitive

Nocontributionto

sustained

growth

andnorm

alplantgrowth

was

negativelyaffected

Karakaset

al.1997

Stpd1

Sorbitol-6-phosphate

dehydrogenase

M.domestica

N.tabacum

Salt&

droughttolerance

Norm

alplantdevelopmentwasaffected

byhighsorbitol

Shevelevaet

al.1998

mtlD

Mannitol-1-phosphate

dehydrogenase

E.coli

S.melongena

Salt,drought&

chillingtolerance

More

chlorophyllcontents

&norm

al

plantmorphology

Prabhavathiet

al.2002

GolS1

Galactinolsynthase

A.thaliana

A.thaliana

Enhanceddroughttolerance

Transgenic

plants

showed

reduced

transpirationfrom

leaves

Tajiet

al.2002

mt1D

Mannitol-1-phosphate

dehydrogenase

E.coli

T.aestivum

Drought&

salttolerance

Improved

growth

perform

ance

of

transgenic

calli&

highbiomass

Abebeet

al.2003

SST/FFT

Sucrose:sucrose

1-

fructosyltransferase/

fructan:fructan1-

fructosyltransferase

C.scolymus

S.tuberosum

Enhanceddroughttolerance

Prolineproductionmaybeinvolved

inosm

o-regulationunder

stress

Knipp&

Honermeier

2006

Ipk2b

Inositolpolyphosphate

6-/3-kinase

A.thaliana

N.tabacum

Enhanceddrought,salinity&

freezingtolerance

Anti-oxidantenzymes

wereelevatedin

transgenic

plants

Yanget

al.2008

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1999). It is still unclear exactly how the fructans contribute to the stress tolerance ofthe transformed plants. It is speculated that, like other sugars, fructans act asregulators or signal molecules, thus influencing plant metabolism, as scavengers ofROS (Shen et al. 1997). However, it seems to be clear that fructans promote theprocess of root branching, thus increasing the root surface and subsequent wateruptake (Datta 2002).

Drought stress also affects the metabolism of other important carbohydrates inplants. In addition to fructans, the raffinose family oligosaccharides (RFO) such asraffinose, stachyose and galactinol play important roles in drought stress tolerance inplants and seeds (Seki et al. 2007). Seven galactinol synthase (GolS)-related geneshave been characterized in the Arabidopsis genome, but information on the roles ofthese genes in conferring drought stress tolerance to these plants is still missing (Tajiet al. 2002; Valliyodan and Nguyen 2006). Transgenic Arabidopsis plants, whichoverexpress the drought-inducible galactinol synthase genes (AtGolS1/AtGolS2)exhibited enhanced drought tolerance because of the accumulation of galactinol andraffinose molecules (Taji et al. 2002). In addition, DREB1A/CBF3-expressingtransgenic plants showed tolerance to drought and cold owing to their accumulationof more galactinol and raffinose than non-transgenic plants (Taji et al. 2002; Sekiet al. 2007). These studies emphasize the importance of galactinol and raffinose instress tolerance and demonstrate that the modification of drought stress tolerance bymanipulation of these sugars is a viable alternative in plants.

Polyols

The accumulation of sugar alcohol/polyols is related to drought and salinity stresstolerance in many plant species (Bohnert and Jensen 1996). Some plants (Tobaccoand Arabidopsis) are non-accumulators of these compounds (e.g. mannitol),therefore, accumulation of these sugar alcohols such as mannitol (Tarczynskiet al. 1992; Thomas et al. 1995; Shen et al. 1997; Karakas et al. 1997), D-ononitol(Vernon et al. 1993; Sheveleva et al. 1997), inositol (Smart and Flores 1997) andsorbitol (Tao et al. 1995; Sheveleva et al. 1998) constitute another class ofcompounds with osmoprotective activities that have been the target of molecularengineering studies in plants (Table 4). However, it should be kept in mind thattransgenic Arabidopsis plants accumulating inositol did not show any significantdifference in stress tolerance, but the transgenic plants accumulating mannitol, D-ononitol and sorbitol had enhanced tolerance to drought or salt stress. Transgenictobacco plants expressing IMT1 gene coding for the myo-inositol-o-methyltransfer-ase enzyme were more drought and salt tolerant than wild-type tobacco plants(Sheveleva et al. 1997). Similarly, inositol phosphates (IPs) have been implicated inimportant roles in stress signaling. Many genes encoding inositol polyphosphatekinases have been identified but their physiological functions have not been fullyunderstood. Recently, Yang et al. (2008) showed that constitutive expression ofArabidopsis IPK2b gene in transgenic tobacco enhanced its tolerance towardsvarious abiotic stresses including drought, salt and freezing stresses. Although theprecise mode of action of AtIPK2b in plant responses to stress remains elusive, theseresults provided direct evidence that tolerance to various abiotic stresses can beachieved by alteration of AtIPK2b expression in transgenic plants. In addition,mannitol-producing tobacco plants exhibited increased tolerance to high salinity.Mannitol-expressing seeds of Arabidopsis were able to germinate in media with high

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concentrations of NaCl, compared with control seeds, which ceased to germinate ata much lower NaCl concentration (Thomas et al. 1995). Expression of the MT1Denzyme in the chloroplasts of the tobacco plants confers enhanced resistance tooxidative stress induced by the presence of methyl viologen (Shen et al. 1997). Suchresistance was attributed to an increased capacity to scavenge the hydroxyl radicals.Similarly, when the MT1D gene was engineered in wheat, it improved tolerance todrought and salinity stress in wheat primarily through osmotic adjustment (Abebeet al. 2003). However, the amount of accumulated mannitol was too small to protectagainst stress through osmotic adjustment. Therefore, the osmoprotective role ofthese compounds cannot be attributed solely to mere osmotic adjustment; they mayalso protect plants against damage by scavenging ROS and thus maintaining proteinstructure and function (Wang et al. 2003).

Studies involving the transfer of genes involved in the biosynthetic pathway ofpolyols were performed during early attempts to produce stress-tolerant plants.Unfortunately, engineering some of these compounds had a number of undesirableeffects. One such problem was observed when a new pathway for sorbitol synthesiswas engineered in tobacco plants, which resulted in growth defects and tissuenecrosis (Sheveleva et al. 1998). This study is a good example of a case in which acompetitive or antagonistic effect is elicited between the transgene activity and thehost plant metabolism. A reasonable explanation for the negative effects of polyolsin transgenic plants could be that a competition is set up for one or the same productbetween the host and the transgene.

Another case in which a positive effect is generated is that of the engineering of anew pathway for D-ononitol accumulation in the tobacco plants. In this case, D-ononitol production has been engineered to be stress dependent and thetransformants recovered faster than wild-type plants after rewatering (Shevelevaet al. 1997). These studies highlight the importance of conducting the metabolicanalyses of the transgenic stressed plants, in order to predict the in vivo pathwayfluxes and the pool sizes of the intermediate compounds (Nuccio et al. 1999). Oncesuch data has been obtained it could be incorporated into the metabolic models andcan be used to estimate the impact on other pathways when a transgene is insertedinto the plant of interest.

Polyamines and their role in drought tolerance

In plants the polyamines (PA), mainly diamine putrescine (Put), triamine spermidine(Spd) and tetraamine spermine (Spm), are the major components. These polyaminesare not only involved in the fundamental cellular processes, for example cell division,morphogenesis, chromatin organization, mRNA translation, ribosome biogenesis,cell proliferation, secondary metabolism, senescene and programmed cell dealth(Igarashi and Kashiwagi 2000; Thomas T and Thomas TJ 2001; Davies 2004; Kuehnand Phillips 2005; Kumar et al. 2006), but also play a pivotal role in plant responsesto abiotic and biotic stresses (Rajam 1997; Bouchereau et al. 1999; Galston 2001;Walters 2003; Ma et al. 2005; Bhattacharya and Rajam 2006; Kumar et al. 2006; Liuet al. 2006; Yamaguchi et al. 2007). A general phenomenon observed is that the PAscan alter their titers in response to various types of environmental stresses such aswater stress (Capell et al. 2004; Kasukabe et al. 2004; Ma et al. 2005), low and hightemperatures (Song et al. 2002; Hummel et al. 2004; Imai et al. 2004) and salinity(Maiale et al. 2004; Roy et al. 2005; Liu et al. 2006). Genes encoding these PA

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biosynthetic enzymes have been isolated from a variety of plant species (Malmberget al. 1998; Bagni and Tassoni 2001). A recent study with Arabidopsis mutant plantshas demonstrated that the cellular polyamine content often changes in response toabiotic stresses; however, its physiological relevance is unknown. It was alsoobserved that the PA biosynthesis is enhanced in drought-stressed plants(Bouchereau et al. 1999) and Spm content also increases, which suggests a role forthe Spm in drought stress tolerance (Yamaguchi et al. 2007). In the model plantArabidopsis, all of the pathway genes have been identified, these are: ADC-encodinggenes (ADC1 and ADC2, Watson and Malmberg 1996; Watson et al. 1997); theagmatine iminohydrolase gene (Janowitz et al. 2003); the N-carbamoylputrescineamidohydrolase gene and four genes for AdoMetDC (Franceschetti et al. 2001; Ge Cet al. 2006); two genes (SPDS1 and SPDS2) of the Spd synthase enzyme (Hanzawaet al. 2002); and two genes (ACL5 and SPMS) of the Spm synthase enzyme(Hanzawa et al. 1997, 2000). It has been reported that overexpression of PAbiosynthetic genes like the ADC (Capell et al. 1998, 2004; Roy and Wu 2001), ODC(Kumria and Rajam 2002), SAMDC (Roy and Wu 2002; Waie and Rajam 2003) andSPD SYN (Franceschetti et al. 2004; Kasukabe et al. 2004, 2006) in plants like rice,tobacco, Arabidopsis and sweet potato, has resulted in increased tolerance to variousabiotic stresses.

There have been several reports on creating transgenic plants harboring thepolyamine biosynthetic genes in an attempt to enhance stress tolerance (Table 5).For example, in a recent study, transgenic rice plants expressing the Daturastramonium ADC (arginine decarboxylase) gene produced higher levels of Put underdrought stress than wild-type plants, which led to higher levels of Spd and Spm andimproved the drought tolerance, owing to which the transgenic plants exhibited lesschlorophyll loss and leaf curling than wild-type plants (Capell et al. 2004; Peremartiet al. 2009). Another example is introduction of the Tritordeum SAMDC (S-adenosylmethionine decarboxylase) gene into the rice plant, leading to a three- tofour-fold increase in the Spd and Spm levels in transformed plants, which then hadnormal growth and development, even under NaCl stress (Roy and Wu 2002). Inanother example, transgenic tobacco plants overexpressing a human SAMDC genehad higher Spd and Put levels and exhibited tolerance to drought and salt stress(Waie and Rajam 2003). Recently, Peremarti et al. (2009) generated transgenic riceplants constitutively expressing heterologous SAMDC gene from Datura stramoniumto distinguish between the roles of Put and the higher polyamines Spd andSpm. Both transgenic and wild-type plants showed identical symptoms whenexposed to drought stress, but transgenic plants recovered much more quickly onrewatering.

In another set of experiments, ADC-expressing transgenic rice plants producedhigher levels of Put, Spd and Spm and exhibited drought tolerance. These resultsconfirmed the involvement of polyamines in drought stress and further attributedindividual roles to Put, Spd and Spm. Interestingly, introduction of a singlepolyamine biosynthesis gene has been shown to confer tolerance to multiple stresses.Examples of these are when Kasukabe et al. (2004, 2006) and Wi et al. (2006)reported that the overexpression of the SPDS (spermidine synthase) from Curcurbitaficifolia in Arabidopsis, sweet potato (Ipomoea batatas) or tobacco enhanced thetolerance of these plants to drought, chilling, freezing, salinity and oxidative stress.Similarly, Prabhavathi and Rajam (2007) have demonstrated that the transgeniceggplant harboring the oat ADC gene exhibited increased tolerance to drought,

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Table

5.

Stress-toleranttransgenic

plants

expressingosm

olytes(polyamines).

Gene

Molecularfunction

Source

Transform

edplant

Perform

ance

of

transgenic

plant

Additionalnotes

References

Polyamines

SAMDC

S-adenosylm

ethionine

decaboxylase

H.sapiens

N.tabacum

Drought,salinity&

biotic

stress

resistance

Itisdiffi

cultto

pinpointwhether

the

tolerance

wasdueto

increasedPut

orSpdorboth

Waie

&Rajam

2003

SPDS

Spermidinesynthase

C.ficifolia

A.thaliana

Multiple

abioticstress

tolerance

Involved

insignalingpathwayswhich

ledto

tolerance

Kasukabeet

al.2004

ADC

Argininedecarboxylase

D.stramonium

O.sativa

Droughttolerance

Itshowed

thatpolyaminemetabolism

hadstrongconnectionwithdrought

stress

Capellet

al.2004

SAMDC

S-adenosylm

ethionine

decaboxylase

D.caryophyllus

N.tabacum

Multiple

abioticstress

tolerance

Norm

alplantdevelopmentwithhigher

photosynthetic

rate

&increasedseed

rate

Wiet

al.2006

ADC

Argininedecarboxylase

A.thaliana

S.melongena

Tolerance

tomultiple

abioticstresses

IncreasedADC

activityledto

higher

polyamineaccumulation

Prabhavathi&

Rajam

2007

SAMDC

S-adenosylm

ethionine

decaboxylase

D.stramonium

O.sativa

Droughttolerance

Transgenic

plants

showed

arapid

recoveryfrom

droughton

rewatering

Perem

artiet

al.2009

SAMDC

S-adenosylm

ethionine

decaboxylase

S.cerevisiae

L.esculentum

Hightemperature

tolerance

Enhancedantioxidantactivity,

protectionofmem

branelipid

peroxidationwereprobably

responsible

fortolerance

Chenget

al.2009

ADC

Argininedecarboxylase

A.thaliana

A.thaliana

Droughttolerance

Transgenicplantsexhibited

reductionin

transpirationrate

andstomatal

conductance

Alcazaret

al.2010

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salinity, low and high temperature, heavy metal and were also found to be resistanceagainst the fungal wilt disease.

Conclusion

Abiotic stress is one of the primary causes of crop losses worldwide. Crop yieldunder abiotic stress depends not only on the mere survival of plants under stressconditions, but also on the phenological and developmental plasticity of plants.Food security is unsustainable without dramatic yield increases in marginalenvironments, especially drought- and salinity-prone areas (Bartels and Hussain2008; Gill and Tuteja 2010). Drought stress affects practically every aspect of plantgrowth and metabolism. Much progress has been made in unraveling the complexstress-response mechanisms, particularly in the identification of several crucialfactors related to the plant response to drought stress. Model plants like Arabidopsisand tobacco have been mostly used to raise transgenic plants with various genes, butonce their role has been established, stress tolerance could be conferred toeconomically important plants using these genes. It is these types of studies,addressing the function and potentiality of the stress-responsive genes using thetransgenic approach, that we have reviewed here. In future, it is expected that otherapproaches, in particular gene knockouts and RNAi techniques, will further aid inour understanding of the role of various other unknown genes.

Recent progress in the field of genomics, proteomics and metabolomics providesample opportunities for the isolation and characterization of novel drought-responsive genes and pathways. The evolutionary conservation of the stress responsehas shown that functionally analogous stress-associated determinants exist in bothunicellular and multicellular organisms. Therefore, elucidation of the components ofcomplex stress-responsive pathways in simpler systems like bacteria and yeast mayhelp in finding overlapping mechanisms in higher plants. So, coordinated efforts areneeded to understand the illusive genes and the mechanisms of drought tolerance.

A variety of genes has been employed to generate transgenic plants thataccumulate osmoprotectants and are tolerant to drought and other abiotic stresses.It may, however, be noted that these successes reflect the performance of plantsunder controlled greenhouse conditions. In fact, no scientific reports on extensivefield tests have yet been published. Only field trials can show which approaches arepromising for the further development of varieties able to meet economical demands.However, transgenic plants often accumulate low or insufficient osmolytes toimprove plant stress tolerance. Further research is currently underway to improvethe efficiency of this approach by taking into account factors like substrateavailability and metabolic fluxes in transgenic plants, which may result in highaccumulation of these osmolytes. Hence, future studies should involve both areductionist approach, to understand the molecular basis of stress tolerance, and anintegrative approach towards analyzing the benefits of stress-tolerant transgenicplants.

Acknowledgements

We thank Dr Bujun Shi of ACPFG (Australia) for critical reading and English correction ofthe manuscript. SSH is supported by an Endeavour Research Fellowship from Department ofEducation, Employment and Work Relation (DEEWR), Australia.

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