Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants

22 Pages·2010·1.06 MB·English

by  Sarvajeet Singh Gill

PlantPhysiologyandBiochemistry48(2010)909e930
ContentslistsavailableatScienceDirect
Plant Physiology and Biochemistry
journal homepage: www.elsevier.com/locate/plaphy
Review
Reactive oxygen species and antioxidant machinery in abiotic stress
tolerance in crop plants
Sarvajeet Singh Gill, Narendra Tuteja*
PlantMolecularBiologyGroup,InternationalCentreforGeneticEngineeringandBiotechnology,ArunaAsafAliMarg,NewDelhi110067,India
a r t i c l e i n f o a b s t r a c t
Articlehistory: Variousabioticstressesleadtotheoverproductionofreactiveoxygenspecies(ROS)inplantswhichare
Received13May2010 highlyreactiveandtoxicandcausedamagetoproteins,lipids,carbohydratesandDNAwhichultimately
Accepted28August2010 resultsinoxidativestress.TheROScomprisesbothfreeradical(O2(cid:1)(cid:3),superoxideradicals;OH(cid:1),hydroxyl
Availableonline15September2010 radical; HO2(cid:1), perhydroxy radical and RO(cid:1), alkoxy radicals) and non-radical (molecular) forms (H2O2,
hydrogenperoxideand1O2,singletoxygen).Inchloroplasts,photosystemIandII(PSIandPSII)arethe
Keywords: majorsitesfortheproductionof1O2andO2(cid:1)(cid:3).Inmitochondria,complexI,ubiquinoneandcomplexIIIof
AEnbzioytmicastticreasnsttioolxeirdaannctes electrontransportchain(ETC)arethemajorsitesforthegenerationofO2(cid:1)(cid:3).Theantioxidantdefense
machinery protects plants against oxidative stress damages. Plants possess very efficient enzymatic
Non-enzymaticantioxidants
Oxidativestress (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR;
Reactiveoxygenspecies monodehydroascorbatereductase,MDHAR;dehydroascorbatereductase,DHAR;glutathioneperoxidase,
GPX;guaicolperoxidase,GOPXandglutathione-S-transferase,GST)andnon-enzymatic(ascorbicacid,
ASH; glutathione, GSH; phenolic compounds, alkaloids, non-protein amino acids and a-tocopherols)
antioxidantdefensesystemswhichworkinconcerttocontrolthecascadesofuncontrolledoxidationand
protectplantcellsfromoxidativedamagebyscavengingofROS.ROSalsoinfluencetheexpressionof
anumberofgenesandthereforecontrolthemanyprocesseslikegrowth,cellcycle,programmedcell
death (PCD), abiotic stress responses, pathogen defense, systemic signaling and development. In this
review,wedescribethebiochemistryofROSandtheirproductionsites,andROSscavengingantioxidant
defensemachinery.
(cid:1)2010ElsevierMassonSAS.Allrightsreserved.
1. Introduction produced continuously as byproducts of various metabolic path-
waysthatarelocalizedindifferentcellularcompartmentssuchas
Aboutw2.7billionyearsagomolecularoxygenwasintroduced chloroplast,mitochondriaandperoxisomes[2,3].Inhigherplants
inourenvironmentbytheO2-evolvingphotosyntheticorganisms andalgae,photosynthesistakesplaceinchloroplasts,whichcontain
andROShavebeentheuninvitedcompanionsofaerobiclife[1].The a highly organized thylakoid membrane system that harbours all
O2moleculeisafreeradical,asithastwoimpairedelectronsthat components of the light-capturing photosynthetic apparatus and
havethesamespinquantumnumber.ThisspinrestrictionmakesO2 provides all structural properties for optimal light harvesting.
prefertoacceptitselectronsoneatatime,leadingtothegeneration Oxygen generated in the chloroplasts during photosynthesis can
of the so called ROS, which can damage the cells. ROS are also acceptelectronspassingthroughthephotosystems,thus forming
O2(cid:1)(cid:3). Under steady state conditions, the ROS molecules are scav-
engedbyvariousantioxidativedefensemechanisms[5].Theequi-
libriumbetweentheproductionandthescavengingofROSmaybe
Abbreviations:RO(cid:1),alkoxyradicals;APX,ascorbateperoxidase;ASH,ascorbate; perturbedbyvariousbioticandabioticstressfactorssuchassalinity,
CAT, catalase; Cd, cadmium; DTNB, 5,50-Dithiobis(2-nitrobenzoic acid); GPOX,
UV radiation, drought, heavy metals, temperature extremes,
guaiacolperoxidase;GSH,reducedglutathione;GSSG,oxidizedglutathione;OH(cid:1),
hydroxyl radical; HO2(cid:1), perhydroxy radical; H2O2, hydrogen peroxide; MDA, nutrientdeficiency,airpollution,herbicidesandpathogenattacks.
malondialdehyde;NBT,nitrobluetetrazolium;NPT,non-proteinthiols;PCD,pro- These disturbances in equilibrium lead to sudden increase in
grammed cell death; ROS, reactive oxygen species; 1O2, singlet oxygen; SOD, intracellularlevelsofROSwhichcancausesignificantdamagetocell
Tsurapnesrcorxiipdteiondifsamctuotrass.e; O2(cid:1)(cid:3), superoxide radicals; TBA, thiobarbituric acid; TFs, structures (Fig. 1) and it has been estimated that 1e2% of O2
* Correspondingauthor.Tel.:þ911126742357;fax:þ911126742316. consumption leads to the formation of ROS in plant tissues [6].
E-mailaddresses:[email protected](S.S.Gill),[email protected](N.Tuteja). Throughavarietyofreactions,O2(cid:1)(cid:3)leadstotheformationofH2O2,
0981-9428/$eseefrontmatter(cid:1)2010ElsevierMassonSAS.Allrightsreserved.
doi:10.1016/j.plaphy.2010.08.016
910 S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930
reactingwithacellularmolecule.Stress-inducedROSaccumulation
is counteracted by enzymatic antioxidant systems that include
Equilibrium avarietyofscavengers,suchasSOD,APX,GPX,GST,andCATand
AOX = ROS non-enzymatic low molecular metabolites, such as ASH, GSH,
a-tocopherol, carotenoids and flavonoids [13,15]. In addition,
AOX ROS
prolinecannowbeaddedtoanelitelistofnon-enzymaticantiox-
idants that microbes, animals, and plants need to counteract the
Oxidative stress inhibitoryeffectsofROS[16].Plantstresstolerancemaythereforebe
(excess ROS) improved by the enhancement of in vivo levels of antioxidant
AOX
enzymes.Theabovesaidantioxidantsfoundinalmostallcellular
Antioxidants
ROS compartments, demonstrating the importance of ROS detoxifica-
Oxidants tionforcellularsurvival[13].Now,ithasalsobeenshownthatROS
influence the expression of a number of genes and signal trans-
Fig.1. EquilibriumbetweenAOXandROS. ductionpathwayswhichsuggestthatcellshaveevolvedstrategies
touseROSasbiologicalstimuliandsignalsthatactivateandcontrol
OH(cid:1)andotherROS.TheROScomprisingO2(cid:1)(cid:3),H2O2,1O2,HO2(cid:1)(cid:3),OH(cid:1), bvaercioomusegaepnpeatriecnsttrtehsast-rpelsapnotnsseactpirvoeglyramprsod[u1c7e].RROeScenwthlyi,chitmhaays
ROOH, ROO(cid:1), and RO(cid:1) are highly reactive and toxic and causes
control many different physiological processes such as bioticand
damage to proteins, lipids, carbohydrates, DNA which ultimately
abioticstress-response,pathogendefense andsystemic signaling.
results in cell death (Fig. 2). Accumulation of ROS as a result of
HerewehavecoveredthechemistryofROSandtheirproduction
various environmental stresses is a major cause of loss of crop
sitesandROSscavengingantioxidantdefensemachinery.
productivityworldwide[7e13].ROSaffectmanycellularfunctions
by damaging nucleic acids, oxidizing proteins, and causing lipid
2. ROSchemistry
peroxidation(LPO)[5].ItisimportanttonotethatwhetherROSwill
act as damaging, protective or signaling factors depends on the
It is well established thatorganelles such as chloroplast, mito-
delicateequilibriumbetweenROSproductionandscavengingatthe
chondriaorperoxisomeswithahighlyoxidizingmetabolicactivityor
propersiteandtime[14].ROScandamagecellsaswellasinitiate
withintenserateofelectronflowareamajorsourceofROSinplant
responsessuchasnewgeneexpression.Thecellresponseevokedis
cells.Theabilityofphototrophstoconvertlightintobiologicalenergy
stronglydependentonseveralfactors.Thesubcellularlocationfor
iscriticalforlifeonEarthandthereforephotosynthesizingorganisms
formation of an ROS may be especially important for a highly
are especially at the risk of oxidative damage, because of their
reactiveROS,becauseitdiffusesonlyaveryshortdistancebefore
bioenergeticlifestyleandtheabundanceofthephotosensitizersand
polyunsaturatedfattyacids(PUFA)inthechloroplastenvelope.This
situation leads to oxidative stress. The appearance of O2 in the
Abioticstressessuchas Chloroplast,mitochondria, atmosphere enabled respiratory metabolism and efficient energy
Salt,UV,drought,heavy peroxisomesandother generationsystemswhichuseO2asfinalelectronacceptor,leadto
metals,airpollutantsetc sourcesinplantcell theformationofROSincells[18].Although,atmosphericoxygenis
relatively non-reactive,itcangive risetoROS whichincludeO2(cid:1)(cid:3),
H2O2,OH(cid:1)and1O2[19](Fig.3).Ithasbeenestimatedthat1e2%ofO2
consumed by plants is sidetracked to produce ROS in various
subcellularloci[6].ThesingleelectronreductionofO2resultsinthe
ROS generationoftheO2(cid:1)(cid:3).AtlowpH,dismutationofO2(cid:1)(cid:3)isunavoidable,
(O2(cid:129)−,1O2,OH(cid:129),H2O2etc.) withoneO2(cid:1)(cid:3)givingupitsaddedelectrontoanotherO2(cid:1)(cid:3),andthen
withprotonationresultinginthegenerationofH2O2.Furthermore,
O2(cid:1)(cid:3)canbeprotonatedtoformtheHO2(cid:1).Additionally,inthepresence
oftransitionmetalssuchascopperandiron,furtherreactionstake
place, e.g. through the HabereWeiss mechanism or the Fenton
reactiontogiveupOH(cid:1),whichisthemostreactivechemicalspeciesin
thebiologicalworld.O2(cid:1)(cid:3)canalsoreactwithanotherveryinfluential
signalingfreeradicalspecies,NO(cid:1)togiveupperoxynitrite(OONO(cid:3)).
Oxidative 1O2isanotherformofROSbutherethereisnoadditionofanextra
damage electron to O2; rather an electron is elevated to a higher energy
orbital,therebyfreeingoxygenfromitsspin-restrictedstate.1O2can
beformedbyphotoexcitationofchlorophyllanditsreactionwithO2.
Superoxide Peroxide
radical ion
Singlet oxygen Dioxygen
Cell
Perhydroxyl Hydrogen
death radical peroxide
Fig.2. AbioticstressinducedROSproductionandcelldeath. Fig.3. GenerationofROSbyenergytransfer.
S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930 911
2.1. Superoxideradicals(O2(cid:1)(cid:3)) able to diffuse over considerable distances of several hundred
nanometers(nm).Ithasbeenfoundthat1O2canlastfornearly4ms
It has been well established that ROS appear continuously in H2O and 100 ms in polar solvent. 1O2, an oxidizing agent for
duringphotosynthesisinthechloroplastsbypartialreductionofO2 awiderangeofbiologicalmoleculesandcanreactwithproteins,
molecules or energy transfer to them. The major site of O2(cid:1)(cid:3) pigments,nucleic acids andlipids, andis thought tobethe most
production is the thylakoid membrane-bound primary electron importantspeciesresponsibleforlightinducedlossofPSIIactivity
acceptorofPSI.TheproductionofROSisaninevitableconsequence whichmaytriggercalldeath[26,27].Inaninterestingstudyithas
of aerobic respiration. When the terminal oxidases-cytochrome c beenfoundthataphotosensitizerinbacteriacangenerate1O2upon
oxidase and the alternative oxidase-react with O2, four electrons exposuretolight,whichleadstotheoxidationofproteinsorlipids
aretransferredandH2Oisreleased.However,occasionallyO2can andultimatelybacteriadeath.[28].Recently,ithasbeenreported
react with other ETC components. Here, only one electron is that in optimal growth conditions 1O2 was responsible for more
transferred,and the result is the O2(cid:1)(cid:3),a moderately reactive ROS than80%ofthenonenzymaticLPOinArabidopsisleaftissues[29].
withapproximately2e4msofhalf-life.IthasbeennotedthatO2(cid:1)(cid:3)is Further,thisstudyshowedthat,inArabidopsismutantsfavouring
usuallythefirstROStobegenerated.Inplanttissuesabout1e2%of 1O2production,photooxidativestressledtoadramaticincreaseof
O2 consumption leads tothe generation of O2(cid:1)(cid:3) [20]. The O2(cid:1)(cid:3) is LPOthatprecededcelldeath[29].
produceduponreductionofO2duringelectrontransportalongthe It is well established that 1O2 is efficiently quenched by
noncyclicpathwayintheETCofchloroplastsandothercompart- becarotene,tocopherol or plastoquinone andif not,1O2 can acti-
mentsoftheplantcell.ReductionofO2totheO2(cid:1)(cid:3)canoccurinthe vatetheupregulationofgenes,involvedinthemoleculardefense
ETC at the level of PS I. The generation of O2(cid:1)(cid:3) may trigger the responsesagainstphotooxdativestress[27].Inastudy,theimpact
formation of more reactive ROS like OH(cid:1), and more possibly 1O2 of 1O2, (produced by Rose Bengal, a photosensitizer) on the ATP
[1,21],eachofwhichmaycauseperoxidationtomembranelipids hydrolysisandATP-drivenprotontranslocationactivityofCF1CFo
and cellular weakening. It has been noted that O2(cid:1)(cid:3) can undergo was investigated and found that both activities were reduced
protonation to give up e a strong oxidizing agent, HO2(cid:1) in nega- dramatically within 1 min of exposure. They also showed that
tivelychargedmembranesurfaces,whichdirectlyattackthePUFA oxidizedthylakoidATPsynthasewasmoresusceptibleto1O2than
[22].Furthermore,O2(cid:1)(cid:3)canalsodonateanelectrontoiron(Fe3þ)to CF1CFoinitsreducedstate[30].Furthermore,geneexpressionand
yield a reduced form of iron(Fe2þ) which can then reduce H2O2, growth of phototrophic bacterium Rhodobacter sphaeroides was
produced as a result of SOD led dismutation of O2(cid:1)(cid:3) to OH(cid:1). The monitored in the presence of 1O2 and it was noted that this
reactionsthroughwhichO2(cid:1)(cid:3),H2O2andironrapidlygenerateOH(cid:1) bacteriummountsa transcriptional responseto1O2 thatrequires
iscalledtheHabereWeissreaction,whereasthefinalstepwhich thealternativesfactor,sE[31].Theglobalgeneexpressionanalysis
involvestheoxidationofFe2þbyH2O2isreferredtoastheFenton’s identifiedz180genes(z60operons)whoseRNAlevelsincreased
reaction: (cid:4)3-fold in cells with increased sE activity. It was predicted that
gene products encoded by four newly identified sE-dependent
O2(cid:1)(cid:3)þFe3þ/1O2þFe2þ operonsareinvolvedinstress-responseandthusprotectthecells
from1O2damage,ortheconservationofenergy[31].opdenCamp
2O (cid:1)(cid:3)þ2HþS(cid:1)O!DO þH O Fe3þ etal.[32]studiedtheroleof1O2intheinductionofgeneexpression
2 2 2 2 and used the conditional fluorescent (flu) mutant of Arabidopsis
thataccumulatesthephotosensitizerprotochlorophyllide(Pchlide)
Fe2þþH2O2/Fe3þþOH(cid:3)þOH(cid:1) ðFentonreactionÞ inchloroplastinthedark.Itwasnotedthataftertheexcitationof
tahgaalStiocaarnrapinecchtilhoeertoalpli.gla[hs2tt3)s]idsntudurudincieegddatcghteeivnmeereaptthhiooyntlovsoiyofnlgtOhe2ne(cid:1)s(cid:3)(isMinVan,AdOr2as(cid:1)b(cid:3)uigdpgorepossptis-s adPccifthfievliradetenintbgfyrdoliimgsthittnh,c1otOsge2raioscutgpivesantoeefrdaetbaerydlyOin2s(cid:1)t(cid:3)trhe/Hess2p-Orlae2s.stpIitodwnasanesdgsuiesgnigenesvsotthelvdaettdharianet
thatO2(cid:1)(cid:3)generatedinphotosyntheticallyactivechloroplastsleads 1aOct2ivdaoteesssneovteraacltstprreisms-arreilsypoanssaeptoaxtihnwbauyst [r3a2th].er as a signal that
totheactivationofgenesinvolvedinsignallingpathways.Recently,
inaninterestingworkC3andC4photosynthesisundersalinitywas Recently, other sources of 1O2 production have also been
reported in plants. It has been noted that plants trigger the
studiedanditwasfoundthatAmaranthplants,unlikewheat,were
abletodetoxifytheO2(cid:1)(cid:3)bySODandlow-molecular-weightanti- pasroadumceticohnanoifsmantoifmriecsriosbtaianlceseicnonpdlaanrty-pmatehtaobgoelniteinste(prahcyttiooanlsex[3in3s]).
oxidantamarathineandreducedtheintensityofLPO.Acompen-
The occurrence of phenalenone chromophores inphytoalexins of
satoryrelationbetweenSODactivityandamaranthinecontentin
plants originally nonphototoxic which suggests that these plants
amaranthleavesundersaltstresshasalsobeennoted[24].
respond to pathogen attacks by biosynthesizing 1O2 photosensi-
tizers.Furthermore,somespeciesconstitutivelyproducedifferent
2.2. Singletoxygen(1O2) types of secondary metabolite with photosensitizing properties
that make use of 1O2 to increase their efficacy as antimicrobial
Singletoxygen,O2(1Dg)or1O2isthefirstexcitedelectronicstate agents[33].
ofO2and, is anunusualROS becauseitis notrelatedtoelectron
transfer to O2. Insufficient energy dissipation during photosyn- 2.3. Hydrogenperoxide(H2O2)
thesiscanleadtoformationofchlorophyll(Chl)tripletstate.The
Chltripletstatecanreactwith3O2togiveuptheveryreactive1O2. TheunivalentreductionofO2(cid:1)(cid:3)producesH2O2.H2O2ismoder-
Ithasbeenfoundthattheformationof1O2duringphotosynthesis atelyreactiveandhasrelativelylonghalf-life(1ms)whereas,other
hasapowerfuldamagingeffectonPSIandPSIIaswell asonthe ROSsuchasO2(cid:1)(cid:3),OH(cid:1)and1O2,havemuchshorterhalf-life(2e4ms)
wholephotosyntheticmachinery.Further,variousabioticstresses [6].IthasbeenwellestablishedthatexcessofH2O2intheplantcells
suchassalinity,droughtetc.leadtoclosingofstomataandresulted leads to the occurrence of oxidative stress. H2O2 may inactivate
lowintercellularCO2concentrationinthechloroplastfavoursthe enzymes by oxidizing their thiol groups. Tewari et al. [34] noted
formationof1O2.Thelifetimeof1O2inacellhasbeenmeasuredto significantly high accumulation of H2O2 in the middle portion of
beapproximately3ms[25]andinthistime,afractionof1O2maybe trichomes in Cu-deficient leaves of Morus alba cv. Kanva 2 in
912 S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930
comparisonwiththeplantsgrownunderCu-excess.Furthermore, generation[44].Inthedarknessthemitochondriaappeartobethe
theactivitiesofvariousantioxidantenzymessuchasSOD,CAT,APX main ROS producers. It has been estimated that 1e5% of the O2
andGRincreasedinbothCu-deficientandCu-excessplants.Itwas consumptionofisolatedmitochondriaresultsinROSproduction[45].
suggestedthatCudeficiencyaggravatesoxidativestresscondition
through excessive ROS production which disturbed the redox 3.1. Mitochondria
couple,whereas,excessofCudamagedtheroots,acceleratedthe
rate of senescence in the older leaves, induced antioxidant Plant mitochondria as “energy factories” are believed to be
responses and disturbed the cellular redox environment in the a major site of ROS production such as H2O2 as well as the ROS
youngleavesofmulberryplants[34].CdinduceddecreaseinCalead targets[46].Itdifferssignificantlyfromtheiranimalcounterparts,
to Cu/Zn SOD down-regulation which resulted in the over- withspecificETCcomponentsandfunctionsinprocessessuchas
productionoftheH2O2andO2(cid:1)(cid:3)inPisumsativum[35].Chloroplast photorespiration.Thecellularenvironmentofplantmitochondria
isoneofthesourceofROSbecauseofitsphotoactivenature.Ithas isalsodistinctivebecauseofthepresenceofphotosynthesis,which
beenfoundthatchlorophyllase1(encodedbyAtCLH1)ofA.thaliana createsanO2andcarbohydrate(sucrose,glucoseandfructose)rich
showed quick induction after tissue damage caused by Erwinia environment[47].ThemitochondrialETCharbourselectronswith
carotovora (bacterial necrotroph) or by Alternaria brassicicola sufficientfreeenergytodirectlyreduceO2whichisconsideredthe
(fungulnecrotroph).ItwasfoundthatE.carotovoraunderhighlight unavoidable primary source of mitochondrial ROS generation,
resulted in elevated levels of H2O2 in AtCLH1 silenced plants. anecessaryaccompanimenttoaerobicrespiration[48].However,
Interestingly, downregulation of AtCLH1 resulted in increased ROS production in mitochondria takes place under normal respi-
susceptibility to A. brassicicola, resistance to which requires ratoryconditionsbutcanbeenhancedinresponsetovariousbiotic
jasmonatesignaling[36]. and abiotic stress conditions. Complex I and III of mitochondrial
H2O2playsadualroleinplants:atlowconcentrations,itactsas ETCaretheverywellknownsitesofO2(cid:1)(cid:3)production.Inaqueous
asignalmoleculeinvolvedinacclimatorysignalingtriggeringtoler- solution, O2(cid:1)(cid:3) is moderately reactive, but this O2(cid:1)(cid:3) can further
ancetovariousbioticandabioticstressesand,athighconcentrations, reduced by SOD dismutation to H2O2 [37,45,49,50]. It has been
itleadstoPCD[37].H2O2hasalsobeenshowntoactasakeyregulator estimatedthatabout1e5%ofmitochondrialO2consumptionleads
inabroadrangeofphysiologicalprocesses,suchassenescence[38], toH2O2production[45].ThisH2O2canreactwithreducedFe2þand
photorespirationandphotosynthesis[39],stomatalmovement[40], Cuþ to produce highly toxic OH(cid:1), and these uncharged OH(cid:1) can
cellcycle[15]andgrowthanddevelopment[41].H2O2isstartingto penetrate membranes and leave the mitochondrion [48,50,51].
beacceptedasasecondmessengerforsignalsgeneratedbymeansof PeroxidationofmitochondrialmembranePUFAisinitiatedbythe
ROSbecauseofitsrelativelylonglifeandhighpermeabilityacross abstractionofahydrogenatombyROS,especiallybyOH(cid:1).Thisleads
membranes[37].Inaninterestingstudytheresponseofpre-treated to the formation of cytotoxic lipid aldehydes, alkenals, and
citrusrootswithH2O2(10mMfor8h)orsodiumnitroprusside(SNP; hydroxyalkenals, such as the much-studied 4-hydroxy-2-nonenal
100mMfor48h)wasinvestigatedtoknowtheantioxidantdefense andmalondialdehyde(MDA).Onceformed,LPOproductscancause
responses in citrus leaves grown in the absence or presence of cellulardamagebyreactingwithproteins,otherlipids,andnucleic
150mMNaClfor16d[42].ItwasnotedthatH2O2andSNPincreased acids.Keyoxylipinsandsmaller,lipid-derivedreactiveelectrophile
theactivitiesofleafSOD,CAT,APXandGRalongwiththeinductionof speciesmayalsobeproducedfromLPO[52].Inaninterestingwork
related-isoform(s) under non-NaCl-stress conditions. Salinity microscopic observations were done to monitor in vivo the
inducedreductionintheASHredoxstatewaspartiallypreventedby behaviour of mitochondria, as well as the production and locali-
H2O2andSNPpre-treatments,ontheothersidetheGSHredoxstate zationofROSduringprotoplastPCDinducedbyUV-C[53].Itwas
was increased by SNP under normal and NaCl-stress conditions. notedthatUV-CexposureinducesquickappearanceofROSinthe
Moreover,NaCl-dependentproteinoxidationwastotallyreversedby protoplasts, which was restricted in chloroplasts and the mito-
pre-treatmentswithH2O2andSNP[42]. chondria.Itwassuggestedthatthemitochondrialtransmembrane
potential loss and the changes in distribution and mobility of
2.4. Hydroxylradicals(OH(cid:1)) mitochondria,aswellastheproductionofROSplayimportantroles
duringUV-inducedplantPCD[53].
Particularly, OH(cid:1), are among the most highly reactive ROS Itiswellknownthatabioticstressesstronglyaffecttheplant
known.Inthepresenceofsuitabletransitionalmetals,especiallyFe, cellbioenergetics.PlantmitochondriamaycontrolROSgeneration
OH(cid:1) canalsobe produced fromO2(cid:1)(cid:3) and H2O2 at neutral pH and bymeansofenergy-dissipatingsystems.Therefore,mitochondria
ambient temperatures by the iron-catalyzed, O2(cid:1)(cid:3)-driven Fenton may play a central role in cell adaptation to abiotic stresses,
reaction. whichareknowntoinduceoxidativestressatcellularlevel.Ithas
beenfoundthattheenergy-dissipatingsystemsofdurumwheat
H O þO (cid:1)(cid:3)/Fe2þ;Fe3þ OH(cid:3)þO þOH(cid:1) mitochondria diminish mitochondrial ROS production. ROS
2 2 2 2 induced activation of the ATP-sensitive plant mitochondrial
TheseOH(cid:1)arethoughttobelargelyresponsibleformediating potassiumchannel(PmitoKATP)andtheplantuncouplingprotein
oxygentoxicityinvivo.OH(cid:1)canpotentiallyreactwithallbiological (PUCP)hasbeenfoundinthemitochondriaofcontrolandhyper
moleculeslikeDNA,proteins,lipids,andalmostanyconstituentof osmotic stressed seedlings, in turn, dissipate the mitochondrial
cellsandduetotheabsenceofanyenzymaticmechanismforthe membrane potential and reduce the chances of large-scale ROS
elimination of this highly reactive ROS, excess production of OH(cid:1) production [54]. To investigate the effect of ROS on plant mito-
ultimatelyleadstocelldeath[43]. chondria,Pastoreetal.[55]usedtheROSproducingsystemcon-
sistingofxanthineplusxanthineoxidaseontherateofmembrane
3. ROSproductionindifferentorganelles potential(DJ)generationduetoeithersuccinateorNADHaddi-
tiontodurumwheatmitochondriaandshowedthattheearlyROS
Photosynthesizing plants are especially at the risk of oxidative productioninhibitsthesuccinatedependent,butnottheNADH-
damage,becauseoftheiroxygenicconditionsandtheabundanceof dependent,DJgenerationandO2uptake.Itwasfoundthatearly
thephotosensitizersandPUFAinthechloroplastenvelope.Inlight generation of ROS can affect plant mitochondria by impairing
the chloroplasts and peroxisomes are the main source of ROS metabolitetransport,thuspreventingfurthersubstrateoxidation,
S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930 913
DJgenerationandconsequentlarge-scaleROSproduction[55].A by decreasing the amount of Rubisco, oxygen-evolving enhancer
Nicotiana sylvestris mitochondrial mutant was used to study the and PSII stability factor proteins. Finally it was found that the
role of plant mitochondria in the regulation of cellular redox symptomsobservedinpealeavescouldbeduetoanimbalancein
homeostasisandstressresistance[56]anditwasnotedthatthe antioxidantsystemsaswellastoanincreasedgenerationofROSin
cytoplasmic male-sterile mutant (CMSII) impaired in complex I chloroplasts, induced probably by a disturbance of the electron
functionand displayedenhancednonphosphorylatingrotenone- transport chain, suggesting that chloroplasts can be a source of
insensitive [NAD(P)H dehydrogenases] and cyanide-insensitive oxidativestressduringviraldiseasedevelopment[65].
(alternative oxidase) respiration which was not associated with
increasedoxidativestress.ThelossofcomplexIfunctionreveals 3.3. Peroxisomes
effective antioxidant crosstalk and acclimation between the
mitochondriaandotherorganelles tomaintainwhole cellredox Peroxisomesaresmall,usuallysphericalmicrobodiesbounded
balance.Thisreorchestrationofthecellularantioxidativesystem by a single lipid bilayer membrane. Peroxisomes are subcellular
wasassociatedwithhighertolerancetoozoneandtobaccomosaic organelles with an essentially oxidative type of metabolism and
virus[56]. are probably the major sites of intracellular ROS production. Like
mitochondriaandchloroplasts,peroxisomesproduceO2(cid:1)(cid:3)radicals
3.2. Chloroplasts as a consequence of their normal metabolism. Two sites of O2(cid:1)(cid:3)
generationareestablishedinperoxisomes[66].Firstoneisinthe
Inhigherplantsandalgae,photosynthesistakesplaceinchlo- organelle matrix, where xanthine oxidase (XOD) catalyzes the
roplasts, which contain a highly organized thylakoid membrane oxidationofxanthineandhypoxanthinetouricacid[67].Second
system that harbours all components of the light-capturing site is in the peroxisome membranes dependent on NAD(P)H
photosyntheticapparatusandprovidesallstructuralpropertiesfor where a small ETC is composed of a flavoprotein NADH and
optimallightharvesting[4].Oxygengeneratedinthechloroplasts cytochrome b, and here O2(cid:1)(cid:3) is produced by the peroxisome
during photosynthesis can accept electrons passing through the ETC. MDHAR participates in O2(cid:1)(cid:3) production by peroxisome
photosystems,thusresultsintheformationofO2(cid:1)(cid:3).Therefore,the membranes [66]. The main metabolic processes responsible for
presence of ROS producing centres such astriplet Chl, ETC in PSI the generation of H2O2 in different types of peroxisomes are the
andPSIImakechloroplastsamajorsiteofROS(O2(cid:1)(cid:3),1O2andH2O2) photorespiratory glycolate oxidase reaction, the fatty acid
production.Variousabioticstressessuchasexcesslight,drought, b-oxidation, the enzymatic reaction of flavin oxidases, and the
saltstressandCO2limitingconditionsenhancetheproductionof disproportionationofO2(cid:1)(cid:3)radicals[2,66,68].Recently,ithasbeen
ROSin chloroplasts.Normally, theelectronflowfromtheexcited demonstratedthatNO(cid:1)radicalsarealsoproducedinperoxisomes.
photosystem centers is directed to NADPþ, which is reduced to Plant peroxisomes also play a significant role in photomorpho-
NADPH.ItthenenterstheCalvincycleandreducesthefinalelec- genesisdegradationofbranchedaminoacids,biosynthesisof the
tronacceptor,CO2.InsituationsofoverloadingoftheETC,apartof planthormonesjasmonicacidandauxin,andtheproductionand
theelectronflowisdivertedfromferredoxintoO2,reducingitto glycinebetaine[69].Theexistenceofregulatoryproteinslikeheat
O2(cid:1)(cid:3)viaMehler reaction [57,58].Laterstudies haverevealed that shockproteins,kinases,andphosphataseshasalsobeensuggested
theacceptorsideofETCinPSIIalsoprovidessides(QA,QB)with inperoxisomes[70,71].
electron leakage to O2 producing O2(cid:1)(cid:3) [59]. 1O2 is a natural In one hand increased production of H2O2 and O2(cid:1)(cid:3) in the
byproduct of photosynthesis, mainly formed at PS II even under peroxisomesleadtooxidativedamageandpossiblycelldeath[66]
low-light conditions [30]. On the external, “stromal” membrane butontheotherhandithasalsobeenshownthatsmalllevelsof
surface, O2(cid:1)(cid:3) is spontaneously dismutated to H2O2 by CuZn-SOD H2O2andO2(cid:1)(cid:3)workassignalmoleculeswhichmediatepathogen-
[59]. Therefore, chloroplasts are also a major source for ROS inducedPCDinplants[72,73].Therefore,ithasbeensuggestedthat
production. peroxisomesshouldbeconsideredascellularcompartmentswith
Recent researches have linked chloroplast-produced ROS with the capacity to generate and release important signal molecules
thehypersensitive response [60].Chloroplast-produced ROShave suchasO2(cid:1)(cid:3),H2O2andNO(cid:1)intothecytosol,whichcancontributeto
been shown to be capable of transmitting the spread of wound- a more integrated communication system among cell compart-
inducedPCDthroughmaizetissue[61].Theexpressionofanimal ments[67]. Rodriguez-Serrano et al.[35]studied theperoxisome
anti-apoptotic Bcl-2 family members in transgenic tobacco has movementinArabidopsislineexpressingtheGFP-SKLpeptidetar-
revealedtheinvolvementofchloroplastinoxidativestress-induced geted to peroxisomes and observed peroxisome-associated fluo-
PCD [62]. It has been shown that in A. thaliana cell suspension rescenceinallplanttissues.Furthermore,itwasalsonotedthatthe
cultures the cells containwell-developed, functional chloroplasts plantstreatedwith100mMCdCl2producedasignificantincreasein
whengrowninthelight,butnotwhengrowninthedarkandcan speed,whichwasdependentonendogenousROSandCa2þbutwas
beusedasmodelsystemstostudyPCD.Inastudytreatmentwith notrelatedtoactincytoskeletonmodifications[35].
antioxidantoflight-grownculturesresultedinincreasedapoptotic
like-PCDinductionwhichsuggeststheinvolvementofchloroplast- 3.4. OthersourcesofROSgenerationinplants
producedROSapoptoticlike-PCDregulation.Ithasbeensuggested
that chloroplasts canplaya significant role in apoptotic like-PCD OtherimportantsourcesofROSproductioninplantsthathave
regulation[63].ChillinginducedaccumulationofROSincucumber receivedlittle attention aredetoxification reactionscatalysed by
resulted in decreased net photosynthetic rate and cytochrome cytochrome P450 in cytoplasm and endoplasmic reticulum [74].
respiratorypathway.Meanwhile,chillingresultedinanenhance- ROS are also generated at plasma membrane level or extracel-
ment of the protective mechanisms such as thermal dissipation, lularlyinapoplastinplants.pH-dependentcellwall-peroxidases,
alternativerespiratorypathway,andROSscavengingmechanisms germin-like oxalate oxidases and amine oxidases have been
(SODsandAPXs)inchloroplastsandmitochondria[64].Ithasalso proposed as a source of H2O2 in apoplast of plant cells [75]. pH
been found that plum pox virus (PPV) infection produced an dependent cell-wall peroxidases are activated by alkaline pH,
alteration in the pea chloroplast ultrastructure, giving rise to which,inthepresenceofareductantproducesH2O2.Alkalization
dilatedthylakoids,anincreaseinthenumberofplastoglobuliand ofapoplastuponelicitorrecognitionprecedestheoxidativeburst
adecreasedamountofstarchcontent.PPVinfectionalsoeffectPSII and productionof H2O2 byapH-dependentcellwall peroxidase
914 S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930
has been proposed as an alternative way of ROS production Propagationstep
duringbioticstress [75].
R(cid:1)þO /ROO(cid:1)
2
ðLipidPeroxyradicalÞ
4. ROSandcellbiochemistry ROO(cid:1)þRH/ROOHþR(cid:1)
ROOH/RO(cid:1) Epoxides;hydroperoxides;glycol;aldehydes
4.1. Lipidperoxidation(LPO)
Terminationstep
Theperoxidationoflipidsisconsideredasthemostdamaging
process known to occur in every living organism. Membrane
damage is sometimes taken as a single parameter to determine R(cid:1)þR(cid:1)/RþR
the level of lipid destruction under various stresses. Now, it has ðFattyaciddimerÞ
been recognized that during LPO, products are formed from R(cid:1)þROO(cid:1)/ROOR
polyunsaturated precursors that include small hydrocarbon ðPeroxidebridgeddimerÞ
fragments such as ketones, MDA, etc and compounds related to ROO(cid:1)þROO(cid:1)/ROORþO
2
them [76]. Some of these compounds react with thiobarbituric ðPeroxidebridgeddimerÞ
acid (TBA) to form coloured products called thiobarbituric acid
reactive substances (TBARS) [77]. LPO, in both cellular and IthasbeenfoundthatthePUFAs(linoleicacid(18:2)andlino-
organelle membranes, takes place when above-threshold ROS lenicacid(18:3))are particularlysusceptibletoattackto1O2 and
levels are reached, thereby not only directly affecting normal HO(cid:1), giving rise to complex mixtures of lipid hydroperoxides.
cellular functioning, but also aggravating the oxidative stress Increased PUFA peroxidation decreases the fluidity of the
through production of lipid-derived radicals [78]. The overall membrane, increases leakiness and causes secondary damage to
process of LPO involved three distinct stages: initiation, membraneproteins[81].Severalaldehydes suchas4-hydroxy-2-
progression and termination steps. Initiation step involves tran- nonenal(HNE)andMDA,aswellashydroxylandketofattyacids,
sition metal complexes, especially those of Fe and Cu. However, areformedasaresultofPUFAperoxidation(Fig.4).Thealdehyde
O2(cid:1)(cid:3)andH2O2arecapableofinitiatingthereactionsbutasOH(cid:1)is breakdownproductscanformconjugateswithDNAandproteins.
sufficiently reactive, the initiation of LPO in a membrane is Aldehydesformedinthemitochondriamaybeinvolvedincausing
initiated by the abstraction of a hydrogen atom, in an unsatu- cytoplasmicmalesterilityinmaizebecausearestorergeneinthis
rated fatty acyl chain of a polyunsaturated fatty acid (PUFA) speciesencodesamitochondrialaldehydedehydrogenase[81].
residue, mainly by OH(cid:1). In an aerobic environment, oxygen will It has also been noted that plants exposed to various abiotic
add tothe fattyacid at the carbon-centered lipid radical to give stresses exhibit an increase in LPO due to the generation of ROS.
rise to a ROO(cid:1). Once initiated, ROO(cid:1) can further propagate the TreatmentwithCdsignificantlyincreasedtheaccumulationoflipid
peroxidationchainreactionbyabstractingahydrogenatomfrom peroxidesindifferentplants[82e87].KhanandPanda[88]studied
adjacentPUFAsidechains.Theresultinglipidhydroperoxidecan thecultivarresponseofOryzasativaundersaltstressandfoundthatit
easily decompose into several reactive species including: lipid increased the LPO in both cvs. of rice but its level was higher in
alkoxyl radicals, aldehydes (malonyldialdehyde), alkanes, lipid Begunbitchi than Lunishree and they correlated the higher free
epoxides,andalcohols[79,80].Asingleinitiationeventthushas radicals scavenging capacityand more efficient protection mecha-
thepotentialtogeneratemultipleperoxidemoleculesbyachain nism of Lunishree against salt stress with lower level of LPO in
reaction. The overall effects of LPO are to decrease membrane comparisontoBegunbitchi.Kukrejaetal.[89]notedmarkedincrease
fluidity; make it easier for phospholipids to exchange between inLPOinCicerarietinumrootsundersalinitystress.Similarincreasein
the two halves of the bilayer; increase the leakiness of the MDAcontenthasalsobeennotedinC.arietinumL.cv.Gokce[90].It
membranetosubstancesthatdonotnormallycrossitotherthan has also been reported that water stress increased the LPO,
through specific channels and damage membrane proteins, membrane injury index, H2O2 and OH(cid:1) production in leaves of
inactivatingreceptors,enzymes,andion channels. stressed Phaleolus vulgarisplants [91]. Liu et al. [92]reportedthat
transgenic tobacco plants overexpressing VTE1 (catalyzes the
Initiationstep penultimatestepoftocopherolsynthesis)fromArabidopsisexposed
todroughtconditionsshoweddecreasedLPO,electrolyteleakageand
RHþOH(cid:1) /R(cid:1)þH2O H2O2 content in comparison to wild type plants. Simova-Stoilova
ðLipidÞ ðLipidAlkylradicalÞ etal.[93]reportedthattheweakeningofmembraneintegrityand
HC ROS O O
3
(CH)COOH
27
Linolenicacid Malondialdehyde
OH
ROS O
H C H C
11 5 (CH)COOH 11 5
27
Linoleicacid 4-Hydroxy-2-nonenal (HNE)
Fig.4. PUFAoxidation.
S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930 915
oxidativedamagetolipidsweremorepronouncedinthesensitive directoxidationofaminoacidsidechains(e.g.prolineandarginine
varietiesunderfielddroughtconditionsinwheatplants.Panetal. tog-glutamylsemialdehyde,lysinetoaminoadipicsemialdehyde,
[94] also reported increase in MDA content in liquorice seedlings andthreoninetoaminoketobutyrate)[100](Fig.5).Whateverthe
(GlycyrrhizauralensisFisch)undersaltanddroughtstress.Agarwal locationofROSsynthesisandaction,ROSarelikelytotargetproteins
[95] reported that UV B irradiated Cassia auriculata L. seedlings thatcontainsulfur-containingaminoacidsandthiolgroups.Cysand
showed oxidative damage with an increase in MDA and H2O2 Metarequitereactiveespeciallywith1O2andOH(cid:1).Activatedoxygen
content. The response of five cherry tomato varieties to oxidative canabstractanHatomfromcysteineresiduestoformathiylradical
stress under moderate water deficit was investigated [96]. It was that will cross-link to a second thiyl radical to form disulphide
suggestedthatLPOasaimportantdeterminantphysiologicalprocess bridges [101]. Alternatively, oxygen can add onto a methionine
inselectingtomatoplantstoleranttowaterstress[96].Recently,the residuetoformmethioninesulphoxidederivatives[102].
radishPHGPxgeneintroducedintoayeastPHGPx-deletionmutant Ithasbeenfoundthatvariousstressesleadtothecarbonyla-
wasreportedtosignificantlyrescuethegrowthoftherecombinant tion of proteins in tissues. Carbonylation of storage proteins has
cellexposedtolinolenicacid,indicatingasimilarroletotheyeast beennotedindryArabidopsisseedsbutcarbonylationofanumber
PHGPx3geneinprotectionofmembraneagainstLPO[97]. ofotherproteinsincreasedstronglyduringseedgermination[99].
Bartolietal.[103]foundthatproteincarbonylationwashigherin
4.2. Proteinoxidation themitochondriathaninchloroplastsandperoxisomesinwheat
leaveswhichsuggestthatthemitochondriaaremoresusceptible
Proteinoxidationisdefinedascovalentmodificationofaprotein to oxidative damage. A number of carbonylated proteins in
induced by ROS or byproducts of oxidative stress. Most types of a soluble fraction from green rice leaf mitochondria have been
protein oxidations are essentially irreversible, whereas, a few identified [104]. Cd2þ treatment raised the carbonylation level
involvingsulfur-containingaminoacidsarereversible[98].Protein from4to5.6nmol/mgproteininpeaplants[105].Thecarbonyl-
carbonylation is widelyused markerof protein oxidation [81,99]. ation level increases from 6.9 to 16.3 nmol/mg of peroxisomal
TheoxidationofanumberofproteinaminoacidsparticularlyArg, proteinasaresultofCd2þtreatmentoftheintactplantthatcanbe
His, Lys, Pro, Thr and Trp give free carbonyl groups which may due to the higher local ROS concentration in the peroxisomes
inhibitoraltertheiractivitiesandincreasesusceptibilitytowards [105]. Proteins can be damaged in oxidative conditions by their
proteolytic attack [81]. Protein carbonylation may occur due to reactionswithLPOproducts,suchas4-hydroxy-2-nonenal(HNE).
Fig.5. Proteinoxidation.
916 S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930
reported that OH(cid:1) is the most reactive and cause damage to all
components of the DNA molecule, damaging both the purine and
pyrimidine bases and also the deoxyribose backbone [110], 1O2
primarilyattacksguanine,andH2O2andO2(cid:1)(cid:3)don’treactatall[111]
(Fig. 6). ROS is capable of inducing damage to almost all cellular
macromolecules including DNA which includes base deletion,
pyrimidinedimers,cross-links,strandbreaksandbasemodification,
such as alkylation and oxidation [112,113]. DNA damage results in
variousphysiologicaleffects,suchasreducedproteinsynthesis,cell
membrane destruction and damage to photosynthetic proteins,
whichaffectsgrowthanddevelopmentofthewholeorganism[114].
DNAdamagecanresulteitherinarrestorinductionoftranscription,
induction of signal transduction pathways, replication errors, cell
membranedestructionandgenomicinstability[114,115].Themajor
typeofDNAdamagecausedbyexposuretoUV-Bistheformationof
dimersbetweenadjacentpyrimidines,i.e.,UVphotoproductsconsist
primarily of cyclobutane pyrimidine dimmers (CPDs) and 6-4PPs
Fig.6. DNAoxidation.
dimers[108].LPOandDNAdamageindividuallyhavebeenconsid-
eredmajordeterminantsofseedviabilityloss.Recently,ithasbeen
shownthatLPOinleavesandrootsofViciafabaincreasedwiththe
Treatment of mitochondria with 4-hydroxy-2-nonenal (HNE) or
addition of arsenate, indicated oxidative stress [116]. A numberof
paraquat(whichcausessuperoxideformationinchloroplastsand
mechanisms are available for repairing DNA damage both in the
mitochondria) or cold or drought treatment of plants leads
nucleusandinthemitochondria.Theseincludedirectreversalofthe
to formation of a covalent HNE-derived adduct of the lipoic
damage, replacement of the base, and replacement of the whole
acid moiety of several mitochondrial enzymes, including Gly
nucleotide[108,112].
decarboxylase (an enzyme in the photorespiratory pathway),
2-oxoglutarate dehydrogenase (a TCA cycle enzyme), and pyru-
vatedecarboxylase[106,107]. 5. ROSscavengingantioxidantdefensemechanism
Exposure of plants to unfavourable environmental conditions
4.3. DNAdamage such as temperature extremes, heavy metals, drought, water
availability, air pollutants, nutrient deficiency, or salt stress can
Though the plant genome isverystablebut its DNA might get increase the production of ROS e.g.,1O2, O2(cid:1)(cid:3), H2O2 and OH(cid:1). To
damaged due to the exposure to biotic and abiotic stress factors protectthemselvesagainstthesetoxicoxygenintermediates,plant
whichmightdamagetheDNA,andtherebyexertsgenotoxicstress cellsanditsorganelleslikechloroplast,mitochondriaandperoxi-
[108].EndogenouslygenerateddamagetoDNAisknownas‘‘spon- somes employ antioxidant defense systems. A great deal of
taneous DNA damage” which is produced by reactive metabolites researchhasestablishedthattheinductionofthecellularantioxi-
(OH(cid:1), O2(cid:1)(cid:3) and NO(cid:1)). High levels of ROS can cause damage to cell dantmachineryisimportantforprotectionagainstvariousstresses
structures, nucleic acids, lipids and proteins [109]. It has been [10,12,13,117] (Fig. 7). The components of antioxidant defence
Fig.7. ROSandantioxidantsdefensemechanism.
S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930 917
systemareenzymaticandnon-enzymaticantioxidants.Enzymatic Table2
antioxidants include SOD, CAT, APX, MDHAR, DHAR and GR and DifferentSODs,theirlocationincellorganelles.
non-enzymatic antioxidants are GSH, AA (both water soluble), SODisozymes Location Resistantto Sensitiveto
carotenoidsandtocopherols(lipidsoluble)[13,15,117]. Fe-SOD Chloroplast KCN H2O2
Mn-SOD Mitochondriaand KCNandH2O2 e
Peroxisomes
5.1. ROSscavengingenzymaticantioxidants Cu/Zn-SOD Chloroplast e H2O2andKCN
andCytosol
5.1.1. Superoxidedismutase(SOD)
Metalloenzyme SOD is the most effective intracellular enzy-
maticantioxidantwhichisubiquitousinallaerobicorganismsand undersaltstresswasobserved.Panetal.[94]studiedtheeffectof
inallsubcellularcompartmentspronetoROSmediatedoxidative salt and drought stress on Glycyrrhiza uralensis Fisch and found
stress. It is well established that various environmental stresses significantly increased SOD activity but an additional MnSOD
oftenleadtotheincreasedgenerationofROS,where,SODhasbeen isoenzymewasdetectedunderonlysaltstress.Moreover,increased
proposedtobeimportantinplantstresstoleranceandprovidethe SOD activity has also been detected following Cd treatment in
first line of defense against the toxic effects of elevated levels of Hordeum vulgare [126], A. thaliana [127], O. sativa [128], Triticum
ROS.TheSODsremoveO2(cid:1)(cid:3)bycatalyzingitsdismutation,oneO2(cid:1)(cid:3) aestivum[129],Brassicajuncea[82];Vignamungo[87],C.arietinum
being reduced to H2O2 and another oxidized to O2 (Table 1). It [130].IncreaseinSODactivityfollowingdroughtstresswasnoted
removesO2(cid:1)(cid:3)andhencedecreasestheriskofOH(cid:1)formationviathe inthreecultivarsofP.vulgaris[91],Alternantheraphiloxeroides[131]
metal catalyzed HabereWeiss-type reaction. This reaction has andO.sativa[132].WangandLi[133]studiedtheeffectwaterstress
a10,000foldfasterratethanspontaneousdismutation.SODsare ontheactivitiesoftotalleafSODandchloroplastSODinTrifolium
classified by their metal cofactors into three known types: the repens L. and reported significant higher increase in SOD activity
copper/zinc (Cu/Zn-SOD), the manganese (Mn-SOD) and the iron underwaterstress.Simonovicovaetal.[134]reportedincreasein
(Fe-SOD), which are localized in different cellular compartments SODactivityinH.vulgareL.cv.AlforroottipsunderAlstressat72h.
[7]. InA. thaliana genome, three FeSOD genes (FSD1, FSD2 and Yangetal.[135]showedthecombinedeffectofdroughtandlow
FSD3), three Cu/ZnSOD genes (CSD1, CSD2 and CSD3), and one light in Picea asperata Mast. seedlings grown at two watering
MnSODgene(MSD1)havebeenreported[118].TheactivityofSOD regimesi.e.,well-watered,100%offieldcapacityanddrought,30%
isozymescanbedetectedbynegativestainingandidentifiedonthe offieldcapacityandlightavailabilities(HL,100%offullsunlightand
basisoftheirsensitivitytoKCNandH2O2.TheMn-SODisresistant low light, 15% of full sunlight) and found that under high light
to both inhibitors; Cu/Zn-SOD is sensitive to both inhibitors condition, drought significantly increased the SOD activity in
whereas; Fe-SOD is resistant to KCN and sensitive to H2O2. The comparisontolowlight.InaninterestingstudyRossaetal.[136]
subcellular distribution of these isozymes is also distinctive. The studied the light regulation of SOD in red alga Gracilariopsis ten-
Mn-SOD is found in the mitochondria of eukaryotic cells and in uifrons and they found that the blue light wavelength exerted
peroxisomes [119]; some Cu/Zn-SOD isozymes are found in the agreaterinductionofSODactivitythanotherspecificwavelengths.
cytosolic fractions, and also in chloroplasts of higher plants [66]. Agarwal[95]reportedthatUVB(7.5and15.0kJm(cid:3)2)irradiation
TheFe-SODisozymes,oftennotdetectedinplants[120]areusually showed significant increase in SOD activity in C. auriculata
associatedwiththechloroplastcompartmentwhenpresent[121] L. seedlings. Li et al. [137] reported significant increase in SOD
(Table 2). The prokaryotic Mn-SOD and Fe-SOD, and the eukary- activity in two cultivars of Brassica compestris under Cu stress. A
otic Cu/Zn-SOD enzymes are dimers, whereas Mn-SOD of mito- generalinductioninSODactivityinAnabaenadoliolumunderNaCl
chondria are tetramers. Peroxisomes and glyoxysomes of Citrillus andCu2þstresshasalsobeenreported[138].
vulgaris have been shown to contain both Cu/Zn- and Mn-SOD Therehavebeenmanyreportsoftheproductionofabioticstress
activity [122], but there are no reports of extracellular SOD toleranttransgenicplantsoverexpressingdifferentSODs(Table3).
enzymesinplants.AllformsofSODarenuclear-encodedandtar- Transgenic rice plants overexpressing OsMT1a demonstrated
geted to their respective subcellular compartments by an amino enhanced drought tolerance [139]. Protoplasts with Mn-SOD
terminal targeting sequence. Several forms of SOD have been overexpressionshowedlessoxidativedamage,higherH2O2content
clonedfromavarietyofplants[123].TheupregulationofSODsis andsignificantincreaseinSODandGRactivitiesunderphotooxi-
implicatedincombatingoxidativestresscausedduetobioticand dative stress [140]. Overexpression of a Mn-SOD in transgenic
abioticstressandhaveacriticalroleinthesurvivalofplantsunder Arabidopsis plants also showed increased salt tolerance [141].
stresses environment. Significant increase in SOD activity under Furthermore, they showed that Mn-SOD activity as well as the
saltstresshasbeenobservedinvariousplantsviz.mulberry[124], activities of Cu/Zn-SOD, Fe-SOD, CAT and POD was significantly
C.arietinum[89]andLycopersiconesculentum[125].Eyidoganand higher in transgenic Arabidopsis plants than control [141].
Oz [90] noted three SOD activity bands (MnSOD, FeSOD and Cu/Zn-SOD overexpressing transgenic tobacco plants showed
Cu/ZnSOD) in C. arietinum under salt stress. Furthermore, signifi- multiple stress tolerance [142]. Overexpression of Mn-SOD in
cant increasein the activities ofCu/ZnSOD and MnSODisozymes transformedL.esculentumplantsalsoshowedenhancedtolerance
Table1
MajorROSscavengingantioxidantenzymes.
Enzymaticantioxidants Enzymecode Reactionscatalyzed
Superoxidedismutase(SOD) EC1.15.1.1 O2(cid:1)(cid:3)þO2(cid:1)(cid:3)þ2Hþ/2H2O2þO2
Catalase(CAT) EC1.11.1.6 H2O2/H2Oþ1/2O2
Ascorbateperoxidase(APX) EC1.11.1.11 H2O2þAA/2H2OþDHA
Guaicolperoxidase(GPX) EC1.11.1.7 H2O2þGSH/H2OþGSSG
Monodehydroascorbatereductase(MDHAR) EC1.6.5.4 MDHAþNAD(P)H/AAþNAD(P)þ
Dehydroascorbatereductase(DHAR) EC1.8.5.1 DHAþ2GSH/AAþGSSG
Glutathionereductase(GR) EC1.6.4.2 GSSGþNAD(P)H/2GSHþNAD(P)þ
918 S.S.Gill,N.Tuteja/PlantPhysiologyandBiochemistry48(2010)909e930
Table3
ROSscavengingenzymaticandnon-enzymaticantioxidantsandtheirroleintransgenicplantsforabioticstresstolerance.
Gene Source Targettransgenic Responseintransgenicplants Reference
Superoxidedismutase(SOD)
Cu/ZnSOD OryzasativaL. Nicotianatabacum Enhancedtolerancetosalt,water,PEGstressesand [142]
enhancementinchloroplastantioxidantsystem
Cu/ZnSOD Avicenniamarina OryzasativaPusaBasmati-1 TransgenicplantsweremoretoleranttoMVmediated [275]
oxidativestress,salinitystressanddroughtstress
MnSOD Nicotianaplumbaginifolia Triticumaestivumcv.Oasisprotoplast Photooxidativestresstolerance,loweroxidativedamage, [140]
higherH2O2andsignificantincreaseinSODandGRactivities
MnSOD Tamarixandrossowii PopulusdavidianaxP.bolleana Salttolerance,8-23foldincreaseinrelativeweightgainsof [276]
thetransgenicplantsandincreaseinSODactivity
MnSOD Arabidopsis ArabidopsisecotypeColumbia Salttolerance,IncreasedMn-SOD,Cu/Zn-SOD,Fe-SOD,CAT [141]
andPODundersaltstress
MnSODþAPX Nicotianatabacum FestucaarundinaceaSchreb. MV,H2O2,andCu,CdandAstolerance,LowTBARS,ion [144]
cv.Kentucky-31 leakageandchlorophylldegradationandincreaseinDOS
andAPXactivity
MnSODþCAT Escherichiacoli BrassicacampestrisL.ssp.pekinensis ResistancetoSO2,increaseintheactivitiesofSOD, [161]
cv.TropicalPride) CAT,GRandAPX
MnSODþFeSOD Nicotianaplumbaginifolia MedicagosativaL. Mildwaterstresstolerancewithhighphotosyntheticactivity [277]
andArabidopsisthaliana
Catalase(CAT)
CAT TriticumaestivumL. OryzasativaL.cv.Yuukara Lowtempraturestresstoleranceduetoeffective [278]
orMatsumae detoxificationofH2O2byCAT
CAT3 Brassicajuncea Nicotianatabacum Cdstresstolerance,betterseedlinggrowthandlongerroots [159]
katE Escherichiacoli Nicotianatabacum‘Xanthi’ katEtransgeneincreasedtheresistanceofthechloroplast’s [279]
translationalmachinerytosaltstressbyscavenging
hydrogenperoxide
Ascorbateperoxidase(APX)
cAPX Pisumsativum Lycopersiconesculentumcv. EnhancedtolerancetoUV-B,heat,droughtandchilling [280,281]
ZhongshuNo.5 stresses,increaseinAPXactivity
APX3 Arabidopsisthaliana Nicotianatabacumcv.Xanthi Waterdeficittolerancewithhigherphotosynthesis [282]
APX1 HordeumvulgareL. Arabidopsisthaliana SalttoleranceduetohigherAPX,SOD,CATandGRand [283]
lowH2O2andMDAcontent
swpa4 Ipomoeabatatas Nicotianatabacum ResistancetovariousstressesMV,H2O2,NaCl,Mannitol [284]
andtoP.parasiticanicotianae.swpa4functionasapositive
defensesignalintheH2O2-regulatedstressresponse
andtransgenicplantsshowed50-foldhigher
PODspecificactivity
Glutathionereductase(GR)
GR Escherichiacoli Triticumaestivum,cv.Oasisprotoplast HigherGSHcontentandGSH/GSHþGSSGratiothan [140]
control,noincreaseinSODandGRactivities
GR Arabidopsisthaliana GossypiumhirsutumL.cv.Coker312 Chillingstresstoleranceandphotoprotection [285]
ecotypeColumbia
GR GossypiumhirsutumL. Nooxidativestresstolerance [286]
GR Arabidopsisthaliana GossypiumhirsutumL.cv.Coker312 Nochillingstresstolerance [287]
ecotypeColumbia
Monodehydroascorbatereductase(MDAR)
MDAR1 Arabidopsisthaliana Nicotianatabacum Ozone,saltandPEGstresstoleranceduetohigherMDAR [184]
ecotypeColumbia activityandhigherlevelofreducedAsA
Dehydroascorbatereductase(DHAR)
DHAR Arabidopsisthaliana Nicotianatabacum DroughtandsalttolerancewithhigherDHARactivityand [184]
reducedAsAcontent
DHAR Arabidopsisthaliana Nicotianatabacum OzoneanddroughttolerancewithhigherDHARactivityand [187]
reducedAsAcontent
DHAR Oryzasativa ArabidopsisthalianaL. SalttoleranceduetoslightincreaseinDHARactivityand [186]
(ecotypeWassilewskija) totalascorbate
DHAR Human Nicotianatabacumcv.Xanthi TolerancetoMV,H2O2,lowtemperatureandNaClstress [288]
GlutathioneS-transferase(GST)
parB Nicotianatabacum Arabidopsisthalianaecotype Nowhole-plantsaltresistancedespiteantioxidantactivity [289]
Landsbergerecta
Nt107 Nicotianatabacum GossypiumhirsutumL. Notolerancetosalinity,chilling,orherbicidesand [199]
noincreaseinantioxidantactivity
GST Suaedasalsa Oryzasativacv.ZhonghuaNo.11 SaltandparaquatstresstoleranceduetoGST,CAT [290]
andSODactivity
NtPoxparB Nicotianatabacum Arabidopsisthaliana ProtectagainstAltoxicityandoxidativestress [291]
NtPoxparBAtPox NicotianatabacumL. ArabidopsisecotypeLandsberg ProtectagainstAltoxicityandoxidativestress [292]
andArabidopsis
GSTþGPX Nicotianatabacum NicotianatabacumL.cv.XanthiNN Increasedthermalorsalt-stresstoleranceduetoglutathione [197]
andascorbatecontent
Glutathioneperoxidase(GPX)
GPX Chlamydomonas Nicotianatabacumcv.Xanthi ToleranttoMVundermoderatelightintensity,chilling [293]
stressunderhighlightintensityorsaltstressduetolowMDA
andhighphotosynthesisandantioxidativesystem