Introduction Soil salinity is one of the most important abiotic stress problems which inhibit growth and reduces productivity of crops including rice, tomato, chili and potato especially
Trang 1jo u r n al h om e p a g e :w w w e l s e v i e r d e / j p l p h
Noppawan Nounjana, Phan Tuan Nghiab, Piyada Theerakulpisuta,∗
a Genomics and Proteomics Research Group for Improvement of Salt-tolerant Rice, Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand
b Key Laboratory of Enzyme and Protein Technology, Faculty of Biology, Hanoi University of Science, Hanoi, Viet Nam
a r t i c l e i n f o
Article history:
Received 17 August 2011
Received in revised form 11 January 2012
Accepted 11 January 2012
Keywords:
Antioxidant enzymes
Proline
Rice
Salt stress
Trehalose
a b s t r a c t
Proline(Pro)andTrehalose(Tre)functionascompatiblesolutesandareupregulatedinplantsunder abioticstress.Theyplayanosmoprotectiveroleinphysiologicalresponses,enablingtheplantstobetter toleratetheadverseeffectsofabioticstress.WeinvestigatedtheeffectofexogenousProandTre(10mM)
inseedlingsofThaiaromaticrice(cv.KDML105;salt-sensitive)duringsaltstressandsubsequent recov-ery.Saltstress(S,NaCl)resultedingrowthreduction,increaseintheNa+/K+ratio,increaseinProleveland up-regulationofProsynthesisgenes(pyrroline-5-carboxylatesynthetase,P5CS;pyrroline-5-carboxylate reductase,P5CR)aswellasaccumulationofhydrogenperoxide(H2O2),increasedactivityof antioxida-tiveenzymes(superoxidedismutase,SOD;peroxidase,POX;ascorbateperoxidase,APX;catalase,CAT) andtranscriptup-regulationofgenesencodingantioxidantenzymes(Cu/ZnSOD,MnSOD,CytAPX,CatC) Undersaltstress,exogenousPro(PS;Pro+NaCl)reducedtheNa+/K+ratio,furtherincreasedendogenous ProandtranscriptlevelsofP5CSandP5CR,butdecreasedtheactivityofthefourantioxidantenzymes Thetranscriptionofgenesencodingseveralantioxidantenzymeswasupregulated.ExogenousTre(TS; Tre+NaCl)alsoreducedtheNa+/K+ratioandstronglydecreasedendogenousPro.TranscriptionofP5CS andP5CRwasupregulated,theactivitiesofSODandPOXdecreased,theactivityofAPXincreasedandthe transcriptionofallantioxidantenzymegenesupregulated.Althoughexogenousosmoprotectantsdidnot alleviategrowthinhibitionduringsaltstress,theyexhibitedapronouncedbeneficialeffectduring recov-eryperiodshowinghigherpercentageofgrowthrecoveryinPS(162.38%)andTS(98.43%)comparedwith
S(3.68%).Duringrecovery,plantstreatedwithPSshowedamuchgreaterreductioninendogenousPro thanNaCl-treated(S)orTre-treatedplants(TS).IncreaseinCATactivitywasmostrelatedtosignificant reductioninH2O2,particularlyinthecaseofPS-treatedplants.AdvantageouseffectsofProwerealso associatedwithincreaseinAPXactivityduringrecovery
© 2012 Elsevier GmbH All rights reserved
Abbreviations: APX, ascorbate peroxidase; C, nutrient solution without Pro/Tre
and NaCl; CAT, catalase; GB, glycinebetaine; GSA, glutamate semialdehyde; H 2 O 2 ,
hydrogen peroxide; KDML105, Oryza sativa cv Khao Dawk Mali 105; P, 10 mM
Pro; P5C, pyrroline-5-carboxylate; P5CDH, pyrroline-5-carboxylate dehydrogenase;
P5CR, pyrroline-5-carboxylate reductase; P5CS, pyrroline-5-carboxylatesynthetase;
PDH, proline dehydrogenase; POX, peroxidase; Pro, proline; PS, 10 mM Pro plus
100 mM NaCl; S, 100 mM NaCl; sqRT-PCR, semi-quantitative reverse
transcriptase-polymerase chain reaction; SOD, superoxide dismutase; T, 10 mM Tre; T6P,
Tre-6-phosphate; TPP, Tre-6-phosphate phosphatase; TPS, Tre-6-phosphate
syn-thase; Tre, trehalose; TS, 10 mM Tre plus 100 mM NaCl.
∗ Corresponding author Tel.: +66 43 342908; fax: +66 43 364169;
mobile: +66 89 6231777.
E-mail addresses: 5150200091@stdmail.kku.ac.th (N Nounjan), phantn@fpt.vn
(P.T Nghia), piythe@kku.ac.th (P Theerakulpisut).
Introduction
Soil salinity is one of the most important abiotic stress problems which inhibit growth and reduces productivity of crops including rice, tomato, chili and potato especially in drier parts of many countries around the globe In Thailand, 62% (6.08×106tons/ha) of rice-growing areas are located in the Northeastern part of the country, but due to water short-age and soil salinity problems arising from the presence of underground salt domes, rice productivity from this area has been relatively low In the year 2010, rice productivity from theNortheastwas2×103tons/hacomparedto3.3×103tons/ha fromthecentralplainwhichhardlyexperienceswatershortage and has nosaline soils(ThaiRice Exporters Association, 2011;
http://www.thairiceexporters.or.th/production.htm)
Saltstressarisesfromthecombinationofosmoticandion toxi-cityeffect(primaryeffect),andoxidativestress(secondaryeffect) 0176-1617/$ – see front matter © 2012 Elsevier GmbH All rights reserved.
Trang 2theability oftheplanttotakeupwaterthusleading toslower
growth.Secondarily,abuildupoftoxiclevelofNa+ andCl− and
inhibitionofK+uptakeseverelyinhibitsseveralenzymesrequiring
K+ascofactorsleadingtoawholerangeofmetabolicimpairment
(Munnsetal.,2006).Undersalinitystress,anincreaseinthe
biosyn-thesisof compatible solutes suchas Pro (Pro), ectoine, glycine
betaine,sorbitolandTre(Tre)protectscellsagainsthyperosmotic
stress.Thehighconcentrationofcompatiblesolutesisableto
bal-ancetheconcentrationofsaltsoutsidethecellononeside,and
ontheother,tocounteract thehighconcentrationsof Na+ and
Cl−inthevacuole(TürkanandDemiral,2009).Undersalinityand
otherabioticstressesplantscangeneratereactiveoxygenspecies
(ROS)suchassuperoxideanion(O2•−),singletoxygen(1O2)and
hydrogenperoxide(H2O2).TheseROSarestronglyreactivebecause
theycaninteractwithessentialmacromoleculesandmetabolites
causingcellulardamage.Inordertoprotectcellsandtissuefrom
oxidative damage plants must produce non-enzymatic
antioxi-dantssuchasglutathione and ascorbate aswellas antioxidant
enzymesincludingperoxidase(POX;EC1.11.1.7),superoxide
dis-mutase(SOD;EC1.15.1.1),ascorbateperoxidase(APX;EC1.11.1.1)
andcatalase(CAT;EC1.11.1.6)todefendagainstoxidativestress
(Ashraf,2009)
Proisthemostcommonosmolyteaccumulatinginplants in
responsetovariousstressconditions.Itoffersawiderangeof
pro-tectiverolesincludingosmoticadjustment,stabilizerforcellular
structureandreductionofdamagetothephotosynthetic
appara-tus.ThelevelofProaccumulationinplantsvariesfromspeciesto
species.TheimportanceofProinenhancingplantstresstolerance
hasrecently beensubstantiatedthrougha transgenicapproach
TransgenicriceexpressingtheP5CSgenefrommothbeanshowed
anenhancedaccumulationofP5CSmRNAlevel,Procontentand
highertolerancetodroughtandsaltstress(SuandWu,2004)
Treisanon-reducingdisaccharidefoundinmanyorganisms
It is an essential component of the mechanisms that
coordi-natemetabolismwithplantgrowthadaptationanddevelopment
(Paul,2007).Treaccumulationinfluencesthealterationofsugar
metabolism leading to an osmoprotectant effect under stress
(Djilianovetal.,2005).IntransgenicricewhichreceivedtheotsA
andotsBgenes(TPSandTPPinhigherplant)fromEscherichiacoli,
Treaccumulated3–10foldhigherwhencomparedtothewildtype
andoverproductionofTreincreasedtolerancetoabioticstresses
(Gargetal.,2002).Geetal.(2008)demonstratedthatOsTPP1
over-expressioninriceenhancedtolerancetosaltandcoldstress
Exogenousosmoprotectantshavebeenreportedtohave
osmo-protectiverolesinabioticstressresponseandhavebeensuggested
asan alternative approach toimprove cropproductivity under
salineconditions(Nakayamaetal.,2005).Exogenousapplication
ofProhasbeenreportedtoofferbeneficialeffectstoplantsunder
stressconditions(AshrafandFoolad,2007).Forexample,intobacco
undersaltstress,addingexogenousProtocellsuspensionculture
alleviatedtheeffectofsaltstressandincreasedtheactivitiesof
antioxidantenzymes(Hoqueetal.,2007).Moreover,exogenousPro
decreasedproteincarbonylationandenhancedantioxidantdefense
andmethylglyoxaldetoxificationsystems(Hoqueetal.,2008)
Pre-treatmentofmaizewith10mMTrerelievedthedamagingeffects
ofsalinitystressonthemetabolicpathwayssuchasHill-reaction
activity,photosyntheticpigmentsandnucleicacidscontent(Zeid,
2009).Trepretreatmentofwinterwheatprotectedthylakoid
mem-branesfromheatdamage,maintainedcellmembraneintegrityand
reducedROSaccumulationfromheatstress(Luoetal.,2010)
Thaiaromatic rice(cv.KDML105) isa well-known
economi-callyimportantThaicultivarhighlyrecognizedintheinternational
market(knownasThaiHomMaliRice)astheworld’sbestquality
aromaticrice.However,KDML105issensitivetosaltstress,
espe-ciallyduringtheseedlingstage,givinglowyieldandpoorgrain
millingqualitywhenitisgrownundersalinesoils(Gregorioetal., 1997; Summartet al., 2010).The aimof this workwas totest theeffects ofexogenous Proand Treonphysiologicalresponses
inseedlingsofKDML105duringsaltstressandrecoveryperiod Fewreportshaveaddressedtheeffectsoftheseosmoprotectants
onmodificationofphysiologicalresponsesduringsaltstressinrice Thisworkprovidesadditionalinformationontherolesof exoge-nously appliedosmoprotectants in modifyingresponses of rice duringsalinitystressaswellasduringtherecoveryperiod
Materials and methods
Plantmaterialsandtreatments Seedsofrice(OryzasativaL.cv.KDML105)weregerminatedin distilledwaterfor5datroomtemperature(RT),andthen trans-ferredtoplastic chamberscontainingYoshida solution(Yoshida
etal.,1976)undernaturalsunlightinagreenhousefor28d dur-ingwhichthesolutionswererenewedevery4d.Theplantswere thendividedintosixtreatmentgroupsbyadditionofthefollowing solutionsintoYoshidasolutionfor6d asfollows:Yoshida solu-tionwithoutPro/TreandNaCl(C),100mMNaCl(S),10mMPro(P),
10mMProplus100mMNaCl(PS),10mMTre(T)and10mMTre plus100mMNaCl(TS).Theuseof10mMProandTrewasbasedon thepreviousreportofGarciaetal.(1997)andapreliminary exper-imentinourlaboratory(unpublisheddata,2006).Theexperiment wassetupaccordingtoacompletelyrandomizeddesignwith5 replications.After6dtreatmenttheplantswerethenallowedto recoverfor5dbyreplacingthetreatmentsolutionswithYoshida solution.Riceplantswereharvestedtwice;thefirst,afterthe6d stressedperiodandthesecond,afterthe5drecoveryperiod.Plants wereanalyzedforfreshanddryweights,Na+/K+ionconcentration, Proaccumulation,H2O2content,totalprotein,activityof antioxi-dantenzymes(POX,SOD,APXandCAT)andgeneexpression(genes encodingProsynthesisandantioxidantenzymes)
Growthparametersandionconcentration Plantfreshweightwasdeterminedandthentheplantswere driedinahot-airovenat70◦Cfor4–5duntilthedryweightwas sta-bilized.Thedriedplantmaterialsweregroundtofinepowder.Dried samples(0.1g)weresubjectedtochemicalanalysesbydigesting
in10mLofnitricacidat300◦C,5mLperchloricacidat200◦Cand
20mLof6Mhydrochloricacid.TheconcentrationofNaandKions wereanalyzedusinganAtomicAbsorptionSpectrometer(Model GBC932AAA,England)
DeterminationofProandH2O2content ThemethoddescribedbyBatesetal.(1973)wasappliedto quan-tifyPro content.Briefly, leafsamples(0.1g)werehomogenized
in5mLof3%sulfosalicylicacidthenfiltered.TwomLoffiltrate wasmixedwith2mLof ninhydrinreagent(1.25gninhydrinin
30mLglacialaceticacidand20mL6Mphosphoricacid)and2mL
ofglacialaceticacid.Thereactionmixturewasheatedat100◦C for1handthenplacedonicefor20minbeforebeingextracted with4mLoftoluene.Theabsorbanceoftheredchromophorein thetoluenefractionwasmeasuredat520nmandtheamountof prolinewasdeterminedbycomparisonwithastandardcurve.For measurementofH2O2,leaftissues(0.1g)werehomogenizedwith
1mLof0.1% (w/v)trichloroaceticacid(TCA)andcentrifugedat 12,000×gfor15min.Thesupernatant(0.5mL)wasaddedto0.5mL
of10mMpotassium phosphatebuffer(pH7.0)and1mLof1M potassiumiodide.TheabsorbanceofH O wasdeterminedusinga
Trang 3fromastandardcurve(Velikovaetal.,2000)
Determinationoftotalproteinandantioxidantenzymeactivities
Leafsamples(0.5g)werehomogenizedina10mMpotassium
phosphate buffer (pH 7.0) containing 4% polyvinyl pyrrolidone
(PVP),thehomogenateswerecentrifugedat12,000×gat4◦Cfor
15min,andthesupernatantswereimmediatelyusedfor
determi-nationofenzymeactivity.Totalproteinwasdeterminedbythe
Bradfordmethod(Bradford,1976).A20Laliquotofthe
super-natantwasmixedwith980LofBradfordreagent(BioRad)andthe
absorbancewasreadat595nm.Proteinconcentrationwas
quan-tifiedbycomparisonwithastandard curveusingbovineserum
albumin
ForSOD,theactivitywasassayedbyitsabilitytoinhibit
pho-tochemicalreduction ofnitrobluetetrazolium chloride(NBT)at
560nm.Thereactionmixture(3mL)contained50mMpotassium
phosphatebuffer(pH7.8),13mMmethionine,75MNBT,0.1mM
EDTAand0.05mLofenzymeextract.Thereactionstartedwhen
adding2Mriboflavin,themixturewasincubatedunder
fluores-centlampsfor10minthenkeptinthedarktostopthereaction.The
absorbanceofthemixturewasmeasuredat560nm.Thereaction
mixturewithnoenzymedevelopedmaximumcolordueto
maxi-mumrateofreductionofNBT.OneunitofSODwasdeterminedas
theamountofenzymethatinhibits50%NBTphotoreduction.The
activitywasexpressedasunitmin−1mg−1protein(Beuchampand
Fridovich,1971;Dhindsaetal.,1981)
ForthePOXassay,thereactionmixture(3mL)contained10mM
potassiumphosphatebuffer(pH7.0),0.2%ofguaiacoland0.04mL
ofenzymesextract.Thereactionwasthenaddedwith3mMof
H2O2andincubatedatRTfor5min,theabsorbancewasthen
mea-suredat470nm.TheactivityofPOXwascalculatedfromtherate
offormationofguaiacoldehydrogenationproduct(GDHP)using
theextinctioncoefficientof26.6mM−1cm−1,andtheactivitywas
expressed as mol GDHP min−1mg−1 protein (Velikova et al.,
2000)
TheactivityofAPXwasdeterminedusingareactionmixture
(3mL)containing0.5mMascorbicacid,0.1mMEDTAand0.1mL
of enzyme extract The reaction started when adding H2O2 to
a finalconcentrationof 1.5mM.The absorbanceofthemixture
wasmeasuredat290nm.TheAPXactivitywascalculatedusing
theextinctioncoefficientof2.8mM−1cm−1andtheactivitywas
expressedasmolascorbateoxidizedmin−1mg−1protein(Nakano
andAsada,1980)
Theactivity of CATwasassayed in a 3mL reactionmixture
containing10mMpotassiumphosphatebuffer(pH7.0),0.1mLof
enzymeextractand0.035%ofH2O2.TheactivityofCATwas
cal-culatedbased ontherateof disappearanceofH2O2 whichwas
followedasadeclineintheabsorbanceat240nmmeasuredat2
and4minaftertheadditionofH2O2.Theactivitywascalculated
usingtheextinctioncoefficientof40mM−1cm−1,andexpressed
asH2O2reducedmin−1mg−1protein(Velikovaetal.,2000)
Geneexpressionanalysis
Total RNA of rice leaf tissues (0.03g) was extracted using
anRNA isolation kit (SVTotal RNAisolation system,Promega)
Contaminated DNA was removed by DNaseI treatment (RQ1
RNase-Free DNase, Promega) Total RNA was quantified by
UV–visspectrophotometer(NanoDrop,ThermoFisherScientific)
Gene expression was analyzed using semiquantitative reverse
transcriptase-polymerasechainreaction(sqRT-PCR).First-strand
cDNAwassynthesizedfrom0.72gtotalRNAbyRevertAidTMFirst
StrandcDNASynthesisKit(Fermentas)using0.5g/Loligo(dT)18
primerat42◦Cfor60min.Thesecondstepamplificationreactions
for expressionanalysisofP5CS,Cu/ZnSOD,MnSOD,CytAPX,CatC and actin were performed using published primer sequences (Kimetal.,2003,2007).PrimersforanalysisofP5CRexpression was designed based on O sativa Japonica Group mRNAunder accession number NM001051928, using GeneFisher software (http://bibiserv.techfak.uni-bielefeld.de).Theforwardandreverse primer sequenceswere5–3 TTCAGCTGTTGGACAAGCAGCAand
5–3GGTTCCTGCCGGGGAAGTGA,respectively,whichamplifieda PCRproductof317bp.ThetotalreactionofPCRwas25L(iTaqTM
DNA polymerase kit), containing 0.2L of cDNA template for each sample Thecycling steps includeda pre-denaturationfor
4min at94◦C, 30–35cyclesfor amplification(denaturation for
40sat94◦C,annealingfor45sat60–62◦C,extensionfor30sat
72◦C)andafinalextensionfor7minat72◦C.ThePCRproducts were separated on a 1.5% (w/v) agarose gel then stained with SYBR®Gold(Invitrogen)and observedonaUV transilluminator TherelativequantificationofmRNAlevelwascalculatedbyusing PhotoCaptMWsoftware,version10(VilberLourmat)
Statisticalanalysis Allresultswerepresentedasmeans±SD.Thesignificanceof differencesbetweenthemeanvalueswasdeterminedbyANOVA ThePvaluesmallerorequalto0.05wasconsideredasstatistically significant
Results
Growthparametersandionconcentration Aftertheplants weretreated for6d,mean freshweightfor saltstressed(S,PSandTS),compatiblesolutetreatments(Pand T)andcontrolplants(C)weredetermined.Nosignificant differ-encesbetweenCandP,SandT,PSandTSwereobserved(Fig.1A) However,plantfreshweightsofbothSandTtreatmentswere sig-nificantlyhigherthanthoseundersaltstressedsuppliedwithPro (PS)andTre(TS).After5drecoveryfromstress,freshweightsofall treatmentgroupsweresignificantlyincreased:83.39%inC,46.22%
inP,93.05%inT,162.38%inPSand98.44%inTS,exceptSwhich showedaslightincreaseof3.68%.Freshweightafterrecoveryofthe
C,PandPSgroupswerehighestfollowedbyT,TSandS,respectively
A similartrendwasobserved forplant dryweight (Fig.1B), thesupplementofProandTredidnotimproveplantdryweight understress.After5d recoveryfromstress,meansofplantdry weightwereincreasedupto81.05%,78.35%and40.78%fornon salt-stressedgroups(C,PandT).Inplantswhichexperiencedsalt treatments,dryweightswereincreased25.33%,78.31%and53.16%
inS,PSandTS,respectively
Sixdaysafterthetreatmentwithosmoprotectants,Na+/K+ratios
inC,PandTweremuchlowerthanthoseinsaltstressedplants(S,
PSandTS)asshowninFig.2.Procausedasmallreduction,whereas TreledtosignificantincreaseinNa+/K+ratioswhencomparedwith thecontrol.Insalt-stressedgroups,supplementationwithPro(PS) andTre(TS)ledtoareductioninNa+/K+ratiocomparedwiththe onestreatedwithonlyNaCl(S),buttheeffectwasmorepronounced withPro.Fivedaysafterrecoveryfromstress,Na+/K+ratiosinS,
PSandTSdecreased,althoughnotsignificantlydifferent,fromthe valuesonday6afterstress
ProaccumulationandgeneexpressionofP5CSandP5CRduring salt-stressandafterrecovery
AsshowninFig.3A,aftertheplantsreceivedthetreatmentsfor
6d,Procontentinalltreatmentgroupsweresignificantlydifferent Generally,plantsundersaltstressaccumulatedahighProlevel PlantstreatedwithbothProplusNaCl(PS)showedthehighestPro content,morethanthosetreatedwithNaCl(S)andTreplusNaCl
Trang 4Fig 1. The effect of NaCl (S), Pro (P), Tre (T), NaCl and Pro (PS) and NaCl and Tre (TS) on fresh (A) and dry (B) weight of rice after 6 d salt-stress treatment (dark bars) and after 5 d recovery (light bars) C, control The values showed means ± SD Different small letters for the dark shaded bars and capital letters for the light shaded bars indicated that the means are significantly different (P ≤ 0.05) The asterisk (*) indicates the significant difference (P ≤ 0.05) in the mean values between 6 d after salt stress and 5 d after recovery.
(TS).TheamountofProinPSwas2.0foldthatofSand4.2fold
thatofTS.InthegroupswithoutNaCl(C,PandT),theapplication
ofPro(P)dramaticallyenhancedProaccumulationinplants(7.9
foldcomparedwiththecontroland11.4foldcomparedwithplants
suppliedwithTre).TheexpressionlevelsofP5CSandP5CRgenes
(Fig.3B)inresponsetoalltreatments(exceptPforP5CR)followed
similarpatternsofchangeinProaccumulation(Fig.3A)
Afterrecoveryfromsaltstress,Proaccumulationwasmarkedly
decreased,exceptintheplantstreatedwithTS.Thereductionwas
verymarkedwithexogenousProtreatmentsi.e.12.29and11.08
foldinPandPScomparedto1.69and2.43inCandS.Incontrast,
exogenousTredidnotaffectProcontentinTandcausedaslight
increaseinTS(1.17foldincrease).ThetranscriptlevelsofP5CSinS,
P,PSandTdecreasedinaccordancewiththereductioninPro
con-tent.ThelevelofP5CSexpressioninTS,however,wasnotconsistent
withthechangeinProcontent.IncontrasttoP5CS,theexpression
levelofP5CRforS,PandPSincreasedwhichdidnotcoincidewith
thereductioninPro
Antioxidativedefenseandexpressionofgenesencoding
antioxidantenzymesduringsalt-stressandafterrecovery
TheeffectsofsaltstressandexogenousProandTreonthe
activ-ityofantioxidantenzymesduringsalt-stressandafterrecovery
Fig 2.The effect of NaCl (S), Pro (P), Tre (T), NaCl and Pro (PS) and NaCl and Tre
(TS) on ratio between Na + and K + in rice leaves after 6 d salt-stress treatment (dark
bars) and after 5 d recovery (light bars) C, control The values showed means ± SD.
Different small letters for the dark shaded bars and capital letters for the light shaded
bars indicated that the means are significantly different (P ≤ 0.05) The asterisk (*)
indicates the significant difference (P ≤ 0.05) in the mean values between 6 d after
Fig 3.The effect of NaCl (S), Pro (P), Tre (T), NaCl and Pro (PS) and NaCl and Tre (TS) on Pro content and expression of Pro synthesis genes (A) Free Pro content in rice leaves after 6 d of salt treatment (dark bars) and after 5 d of recovery (light bars) The values showed means ± SD Different small letters for the dark bars and capital letters for the light bars indicated that the means are significantly different (P ≤ 0.05) The asterisk (*) indicates the significant difference (P ≤ 0.05) in the mean values between 6 d after salt stress and 5 d after recovery (B) and (C) Expression, using sqRT-PCR, of P5CS and P5CR, in rice leaves on day 6 after salt stress and day
5 after recovery The product of RT-PCR of rice actin gene was used as a loading control The histogram shows relative abundance of cDNA from P5CS or P5CR after normalization with the actin signal The experiment was repeated at least three times.
Trang 5Fig 4.The effect of NaCl (S), Pro (P), Tre (T), NaCl and Pro (PS) and NaCl and Tre (TS) on (A) H 2 O 2 content, (B) POX activity, (C) SOD activity, (D) APX activity and (E) CAT activity in rice leaves after 6 d salt-stress treatment (dark bars) and after 5 d recovery (light bars) C, control The values showed means ± SD Different small letters for the dark bars and capital letters for the light bars indicated that the means are significantly different (P ≤ 0.05) The asterisk (*) indicates the significant difference (P ≤ 0.05) in the mean values between 6 d after salt stress and 5 d after recovery.
areshowninFig.4.Undersaltstress,theproductionofH2O2was
significantlyincreased.TheH2O2 contentinriceplantssupplied
withexogenous Pro(PS)and Tre(TS)were2 and 1.5fold that
of stressed plants without osmoprotectants (S) Among groups
withoutNaCl(C,PandT),theplantssuppliedwithTrealone(T)
producedthehighestH2O2content.Duringrecoveryperiod,H2O2
contentdecreasedinplantspreviouslytreatedwithNaCl(37.39%
inS,34.94%inPSand21.14%inTS).ForCandP,theH2O2content
remained the same Conversely, significant reduction of H2O2
contentwasobservedinT
TheSODandPOXactivitiesshowedsimilarpatternsofresponse
after the6d salt stress period Under salt stress conditions (S,
PS and TS), SOD and POX activities were markedly enhanced
when compared to groups with only exogenous
osmoprotec-tants (P and T) The highest SOD and POX activities were
observed in plants exposed to NaCl (S) In non-NaCl groups,
theplants added withPro alone (P) expressedthelowest SOD
activity The lowest POX activity was observed in rice treated
with either exogenous Pro (P) or Tre (T) During the
recov-ery period, activities of these enzymes significantly decreased
in almost all treatments, but in the case of plants previously
supplied with Pro (P), theSOD and POX activitiessignificantly increased
TheactivitiesofAPXand CATpresentedsimilargeneral pat-ternsofresponsestosaltandosmoprotectants.Underthesaltstress treatment,KDML105plantsexternallysuppliedwithTre(TS)had thehighestAPXactivity[1.8foldcomparedtoplantsreceivingNaCl only(S)and2.6foldcomparedwithplantssuppliedwithPro(PS) respectively].Inresponsetosaltstress,CATactivitywas signifi-cantlyincreasedinplantssuppliedwithNaClonly(S)and NaCl plusTre(TS),whiletheactivityinPSplantswasthelowest ForplantsreceivingosmoprotectantswithoutNaCl;Pro(P)had
noeffectonAPXactivitycomparedtothecontrol(C),whereasTre (T)significantlysuppressedtheactivityofthisenzyme.Exogenous Pro(P)orTre(T)hadnoeffectonCATactivity.Aftertheplants wereallowedtorecoverfromsaltstressfor5d,theactivitiesof APXandCATincreasedinalltreatments(exceptforthecaseofAPX activityinS).PlantspreviouslysuppliedwithProundersaltstress (PS)showedextremelyhighAPXandCATactivities.APXactivity
inPSincreased5foldcomparedtoSand1.4foldcomparedtoTS Likewise,CATactivityinPSincreased1.8foldcomparedtoSand 1.9foldcomparedtoTS
Trang 6Fig 5.Expression of genes encoding antioxidative enzymes (Cu/ZnSOD, MnSOD, CytAPX and CatC) monitored in rice leaves after 6 d of salt treatment (A) and after 5 d of recovery (B) The product of RT-PCR of rice actin gene was used as loading control The histogram shows relative abundance of cDNA from each gene after normalization with the actin signal The experiment was repeated at least three times.
TheexpressionpatternsofgenesencodingenzymesCu/ZnSOD,
MnSOD,CytAPXandCatCareshowninFig.5.At6dafterstress,the
expressionofCu/ZnSOD,MnSOD,CytAPXandCatCinalltreatments
withthepresenceofsalts(S,PSandTS)weremarkedlyenhanced
comparedtothecontrol.ForplantstreatedwithProwithout
salt-stress(P),theexpressionofallgeneswassuppressed.Tre
supple-ment,ontheotherhand,resultedingeneup-regulation,especially
Cu/ZnSOD,inbothunstressed(T)andsalt-stressed(TS)conditions
Aftertheplants wereallowedtorecoverfor5d,plants
pre-viouslystressedwithsalt (S)showeda slightrepression inthe
expressionof Cu/ZnSODand CytAPX,but smallup-regulation of
MnSODandCatC.Theexpressionofthesegenesinnon-stressed
plantspreviouslysuppliedwithPro(P)wasaslowasduringstress
TheexpressionlevelofallgenesinthePSgroupwashighlysimilar
tothatofS.ForplantspreviouslytreatedwithTrewithout(T)and
withsalt(TS),theexpressionofallgeneswasconsiderablyreduced
Discussion
EffectsofexogenousProandTreonfreshanddryweights,and
Na+/K+ratio
Sodium chloride in the nutrient solution inhibited growth
resultinginthereductioninfreshand dryweightsofKDML105
seedlings Adding Pro and Treinto salinenutrient solution did notpresentanybeneficialeffectsongrowth.Yamadaetal.(2005)
reportedthatexogenousPro(0,5,10and50mM)stronglyinhibited growthandacceleratedleafsenescenceofArabidopsisandpetunia ThetoxicityofProwasfoundtobemediatedbyP5Caccumulation
intheProdegradationpathway(Hellmannetal.,2000).Ourresults correspondwiththesefindings.Plantgrowthinthesolutionwith combinedNaClandPro(PS)wassuppressedpresumablynotonly
byNaClstressbutalsobyPro
TheeffectofTreongrowthinhibitionwasfoundinKDML105 plantsfedwithTrealone(T).SeedlingsofArabidopsisculturedin
MSmediumsupplementedwith100mMTrefailedtodevelop pri-maryleavesandprimaryroots(Aghdasietal.,2010).Schluepmann
etal.(2004)summarizedthatexogenouslysuppliedTreresultedin T6Paccumulationwhichisagrowthinhibitor.Althoughexogenous ProandTredidnotclearlyshowprotectiverolesduringthe salt-stressperiod,theyobviouslyfurnishedtheplantswithenhanced abilitytorecoverascompared withstressedplantswithoutthe osmoprotectants
Amongthemostcommoneffectsofsalinityisgrowthinhibition
byaccumulationofNa+andreductioninK+uptakeandtheratio
ofNa+toK+showedaninverserelationshipwithgrowth(Gregorio andSenadhira,1993).ExogenousProshowedhigherabilitythan TreinalleviatingtheinhibitoryeffectofsaltbyreducingNa+uptake
Trang 7resultinginlowervaluesofNa+/K+ (Fig.2).Similareffectswere
observedbySobahanetal.(2009)thatexogenousProandGB
sup-pressedNa+ uptakeandaccumulationwhileK+contentwasnot
affectedresultinginlowerNa+/K+ratioinriceplants.Inaddition,
themitigatingeffectsofexogenousProandGBonNa+/K+ratiowere
observedinsalt-sensitivefreshmarkettomato(Heuer,2003)
EffectsofexogenousProandTreonProaccumulationand
expressionP5CSandP5CRgenes
Theeffects Proand TreonPro accumulationandexpression
ofgenesintheProsynthesispathway(P5CSandP5CR)by
sqRT-PCRwereinvestigatedafter6dsaltstressand5drecovery.Plants
treatedwithNaCl(S)accumulated5.53foldhigherProthanthe
control.Luttsetal.(1999)demonstratedthatthesalt-sensitiverice
accumulatedhigherlevelsofNa+ andProthanthesalt-resistant
rice and concluded that accumulationof Pro is related to
salt-stressinjury.ThesefindingsareconsistentwithVaidyanathanetal
(2003)andTheerakulpisutetal.(2005)thatsalt-sensitiverice
cul-tivarsshowedhighergrowthinhibitionandaccumulatedgreater
amountsofPro thanthetolerantonesandconcludedthat high
Prolevelsinsensitivecultivarsdidnotaffordmuchprotection.The
resultsfromthepresentstudyindicatedthatover-accumulationof
Proinriceseedlingsdidnotofferplantprotectionbutwasoneof
theconsequencesofmetabolicperturbationtriggeredbysaltstress
ElevatedamountsofProinplantstreatedwithexogenousPro
alone(P) andNaClplusexogenous Pro(PS)compared withthe
StreatmentcanprobablybeattributedtopassiveProuptakeas
suggestedbyHeuer(2003)inhydroponicallygrowntomatoplants
treatedwith1and10mMPro.Similarresultswerealsoobserved
byHuangetal.(2009)whensalt-sensitivecucumberplantswere
sprayedwith25mMPro.Conversely,supplementswithTre
neg-ativelyaffectedProamountsinbothunstressedandsalt-stressed
conditionsresultinginasignificantreductioninPro.Exogenous
TrealsoreducedProaccumulationintwomaizecultivarsunder
drought stresswhile increasing biomass production, improving
plant water relations and some key photosynthetic attributes
(AliandAshraf,2011).Consideringprofoundmitigatingeffectsof
exogenousTreonrice(Garciaetal.,1997)andprotectiverolesof
enhancedproductionofTreintransgenicrice(Geetal.,2008),it
maybepresumedinthiscasethatosmoprotectiveeffectsofTre
reducedtheneedforplantstoaccumulatePro
TranscriptionlevelsofbothPro-synthesizinggenes,P5CSand
P5CR,werestronglyinducedundersaltstress(S)coincidingwith
severalfoldincreaseinPro(Fig.3B).Severalauthorsdetermined
thattheexpressionoftheP5CSgeneisrelatedtoProaccumulation
undersaltstressinrice(Hienetal.,2003;Kimetal.,2007).Although
P5CRdoesnotcatalyzearatelimitingstep,P5CRgeneexpression
is up-regulatedundersalt stressin someplantspecies suchas
soybean(DelauneyandVerma,1990)andArabidopsis(Verbruggen
etal.,1993),whilethedataregardingexpressionofP5CRinriceare
scanty
Proapplicationtoplantswithoutsaltstress(P)showed
consid-erablyhighercontentofProbutP5CStranscriptswereonlyslightly
induced,andthatofP5CRwasclearlysuppressed.ExogenousPro
intheabsenceofNaCldidnothavenegativeeffectsongrowthand
Na+/K+ratio,andaslighteffectonH2O2accumulation.Ontheother
hand,acombinationofProandsalt(PS)exacerbatedtheeffectof
saltleadingtoexcessiveProaccumulation(Fig.3A)inassociation
withenhancedexpressionofP5CSandP5CR,plantweightreduction
(Fig.1)andH2O2over-accumulation(Fig.4A)comparedtoplants
stressedwithNaClalone(S).DespitetheessentialfunctionsofPro
inprimarymetabolismandstressresponse,excessiveamountsof
ProcausedtheP5Ctoincreasetoatoxiclevelandalsoelevated
theflowofelectronsthroughthemitochondrialelectrontransport
chainleadingtoconcomitantgenerationofROS(Milleretal.,2009)
Afterrecoveryfromsaltstress,markeddecreaseinPro accu-mulationwasobservedinalltreatmentsexceptTS.Thisdeclinein Prowasalsofoundincotton(Paridaetal.,2008)andwas asso-ciatedwithadown-regulationinthetranscriptionlevelofP5CS ThisresultwassimilartothatofPengetal.(1996)whofoundthat AtP5CStranscriptlevelsinArabidopsisdeclinedduringtherecovery fromsalinitystress.Ontheotherhand,theexpressionlevelofP5CR wasupregulatedinalmostalltreatments.However,up-regulation
ofP5CRwasnotrelatedtoProaccumulation.Theresultsregarding P5CRexpression,wereinlinewiththoseofTrovatoetal.(2008)
that declinein Proafter saltstressrecovery wasnotrelated to P5CRexpressionbutinsteadcloselyassociatedwithup-regulation
ofPDH(Milleretal.,2005)
EffectofexogenousProandTreonH2O2,antioxidantenzymes activityandexpressionofrelatedgenes
Increase in H2O2 content in response to salt stress in rice waspreviouslyreported(LinandKao,2001;Vaidyanathanetal.,
2003).SOD activity considerably increased under salt stress to convertO2•− toH2O2 whichisconsequentlydetoxifiedbyPOX, APX and CAT Despite the considerable increase in activity of thefourantioxidativeenzymes(SOD,POX,APXandCAT),H2O2
content was significantly higher in S compared to C (Fig 4) Although a regulatedamount of increased ROS in response to abiotic stress plays an essential role in adjusting the cellular redoxstateandregulatorygeneexpressionassociatedwithstress responsestooptimizedefenseandsurvival,excessiveROSdueto imbalancebetweenthedetoxificationprocessandROSgeneration ultimatelyleadstocellulardamageandgrowthinhibition(Shao
etal.,2008)
ExogenousProplusNaCl(PS)ledtoloweractivityofSOD,POX, APXandCATinplantscomparedtoScorrespondingtoadramatic riseinH2O2inPS.Thesuppressionofantioxidativeenzyme activ-ityunderstressconditionsbyanexternalsupplyofProwasalso foundforSODin Salviaofficinalis underUV-Bstress(Radyukina
etal.,2011)andforSOD andCATingrapevineunderoxidative stress(Ozdenetal.,2009).Incucumberundersaltstress supple-mentedwithexogenousPro,therewasadeclineinSODactivity butanincreaseinPOX(Huangetal.,2009).AddingTretoplants withoutsaltstress(T)significantlyreducedtheactivityofPOXand APX,relatingtomuchhigherlevelofH2O2comparedtoC Expo-sureofTrecombinedwithNaCl(TS)didnotaffectCATactivity, markedlyreducedtheactivitiesofSODandPOXbutenhancedthat
ofAPX.However,increasedactivityofAPXalonewasnotenough
toefficientlyremoveH2O2resultinginasignificantlyhigherlevel
ofH2O2inTSthanS.Incontrast,AliandAshraf(2011)foundthat foliarapplicationofTresignificantlyincreasedPOXandCAT SaltstressinducesexpressionofCu/ZnSODandMnSODto sim-ilar extents Thisis in agreement with Kaminaka et al (1999)
who presented thatMnSOD and Cu/ZnSODgenes werestrongly enhancedbydroughtandsalinity.Yu-zhuoetal.(2008)illustrated thatCu/ZnSODisafirstcellulardefenseenzymetoscavengeO2 •−
andaccountedformostofthetotalSODactivity.Inwheat,Sairam
etal.(2005)showedthattheactivitiesofCu/ZnSODandMnSOD werestrongly enhanced and responsible forsalt tolerance The activityofFeSOD,ontheotherhand,wasextremelylow, insen-sitivetosalt stressanddidnot contributetothescavengingof salinityinducedROS.ItwasfoundthattheexpressionofaFeSOD geneencodingchloroplast-specificSODisoformfromKDML105in saltstresstreatmentwasverylow(datanotshown).Exogenous ProorTrecombinedwithNaClalsoup-regulatedtheexpression
of Cu/ZnSODandMnSOD but intheabsenceof NaClexogenous ProstronglysuppressedwhereasTreenhancedtheexpressionof Cu/ZnSODandMnSOD
Trang 8The increase in CytAPX transcription and APX activity of
KDML105inresponsetosaltstresscorrespondedwiththatfound
byHongetal.(2007)fortheOsAPX8geneinriceinresponseto
NaCl.Theactivityof CATintheKDML105 leafincreased
corre-spondingtoanelevationoftranscriptlevelofCatC;theleaf-specific
isoform,asearlierdetectedinricebyKimetal.(2007).Similartothe
effectsonexpressionofCu/ZnSODandMnSODdescribed,external
supplyofProorTreundersaltstressalsoup-regulatedthe
expres-sionofCytAPXandCatCbutintheabsenceofNaClexogenousPro
suppressedwhereasTreenhancedtheexpressionofCytAPXand
CatC
Notably,5dafterrecoveryfromstress,thedeclineinH2O2 in
alltreatmentgroups,exceptP,wasrelateddirectlytosignificant
increaseinCATactivity.However,SODandPOXactivityinall
treat-mentgroupsdecreased appreciably,exceptinthecaseofplants
previouslytreatedwithProwithoutNaCl(P).Patternofchanges
inAPXactivitywassimilartothatofCATactivity(exceptforS)
ThissuggestedthatthereductionofH2O2afterthestressdepended
largelyontheincreasedCATandAPXactivities.Thisagreedwith
thefindingsbyLeeetal.(2001)thatCATactivityincreased
cor-respondingtothereductioninH2O2whereasSOD,POXandAPX
activitiesdecreasedduringrecoveryfromsaltstressinrice
Inter-estingly,plantswhichpreviouslyexperiencedbothProandNaCl
(PS),showedextremely highactivityof APXand CAT.Afterthe
plantswererelievedfromstress,thetranscriptlevelsofCu/ZnSOD,
MnSOD,CytAPXandCatCinalltreatmentgroupsweregenerally
down-regulatedor remainedstablecompared withthecontrol
PlantspreviouslytreatedwithProwithoutNaCl(P)stillshoweda
lowlevelofgeneexpression.Onthecontrary,whileplants
receiv-ingTretreatmentalone(T)showedastrongup-regulationofgene
expressionduringstress,theydisplayedanotabledeclinein
tran-scriptlevelsofallgenesafterrecovery
Inthisstudy,thechangesintranscriptlevelsofgenes
encod-ingantioxidantenzymesinmostcasesdidnotcoincidewiththe
increase/decreaseinactivitiesofthecorrespondingenzymes
Sim-ilarly,Hernándezetal.(2000)showedthatlong-termsaltstress
inducedtranscriptlevelsofantioxidantenzymesgenes,but this
inductionwasnotcorrelatedwiththecorrespondingchangesin
theenzymeactivities.Thisdiscrepancymayresultfromahigher
turnoveroftheseenzymesand/oranincreaseoftheirinactivation
byH2O2(Scandalios,1993)
Thedataobtainedinthisworkwasinconsistentwiththe
find-ingsofHoqueetal.(2007)andGerdakanehet al.(2010)which
reportedthatexogenousProshowedprotectiverolesin
alleviat-ingsaltstress.Thismaybebecauseinthispresentstudytherice
plantswerehydroponicallygrownundernaturalconditions,while
thecellculturesystemswereusedintheearlierreports.Garciaetal
(1997)alsofoundthepositiverolesofexogenousTreonreversing
theadverseeffectsofsaltstressinricegrowninthetissueculture
system.However,ourresultsshowedthatexogenousapplication
ofProandTrepromotedastrongerabilityofplantstorecoverfrom
stress
Therolesandmechanismsofactionofexogenous
osmoprotec-tantsarecomplexandremaincontroversial.Thebeneficialeffects
of external supply of osmoprotectants vary dependingon
sev-eralconditionsincludingplantspecies,developmentalstages,the
severityanddurationofsaltstress.Theeffectivenessofthe
osmo-protectantsalsodependsonwhethertheyareappliedpriortoor
duringstress,methodsofapplicationandtheconcentrationsofthe
osmoprotectants.AvailablereportsinriceinvolvedtheuseofGB
andPro,thereforeitisworthexploringtheeffectsofother
osmo-protectantssuchasTre,sorbitolandectoine.Commercialprospects
ofenhancingstresstolerancein ricebyexogenous
osmoprotec-tantswarrantsfurtherin-depthresearchinthisareatogainabetter
understandingofthemechanismofactionwhichcanbeapplicable
toimprovecropproductioninstressfulenvironments
Acknowledgements
ThisworkwasfinanciallysupportedbytheTRFMasterResearch Grant(WII-MRG525S056),KhonKaenUniversityResearchGrantto theGenomicsandProteomicsResearchGroupforImprovementof Salt-tolerantRice,andtheGraduateSchool,KhonKaenUniversity
WewishtothankProf.Dr.DonaldC.Slack,theUniversityofArizona, Tuscon,Arizona,USA,forproofreadingthemanuscript
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