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EnvironmentalandExperimentalBotany54(2005)8–21Effectofvarioussalt–alkalinemixedstressconditionsonsunowerseedlingsandanalysisoftheirstressfactorsDechengShia,,YanminShengbaLifeScienceCollege,NortheastNormalUniversity,Changchun,JilinProvince,ChinabDepartmentofBiology,ChangchunNormalUniversity,Changchun,JilinProvince,ChinaAccepted17May2004AbstractSunowerseedlingsweretreatedunder30differentconditionsofalkalinityandsalinity,whichwereestablishedbymixingNaCl,NaHCO3,Na2SO4,andNa2CO3,atvariousproportions.
Thetreatmentsincludedasaltconcentrationrangeof50–250mmolandpHvaluesfrom7.
12to10.
72.
Severalphysiologicalindicesofseedlingsstressed—includingrelativegrowthrate(RGR),leafarea,electrolyteleakagerate,prolinecontent,citricacidcontent,andcontentsofNa+andK+—weredeterminedtoanalyzethecharacteristicsofthestressesduetothesalt–alkalimixesandtheirmainstressfactors.
Theresultsshowedthatthephysiologicalresponsesofsunowercloselycorrelatednotonlywithsalinity(thetotalconcen-trationofstresssalt)butalsowiththepH(oralkalinity)ofthetreatmentsolution.
RGR,leafarea,andofK+contentdecreasedwithincreasingsalinityandpH.
Electrolyteleakagerate,prolinecontent,citricacidcontent,andNa+contentincreasedwithincreasingsalinityandpH.
ThedeleteriouseffectsofahighpHvalueorsalinityaloneweresignicantlylessthanthoseofhighpHincombinationwithsalinity.
Thisresultsuggestedthatforasalt–alkalimixstress,areciprocalenhancementbetweensaltstressandalkalistresswasacharacteristicfeature.
Thebuffercapacityofthetreatmentsolutionwastakenasastressfactorinordertosimplifythestressfactoranalysis.
Theresultsofthestatisticalanalysisshowedthatforthestressfactorsofthesalt–alkalimixstress,[CO32]and[HCO3]couldbefullyrepresentedbythebuffercapacity;[Na+]couldbefullyrepresentedbysalinity;whereas[SO42]wasnegligible.
Therefore,fourfactors,salinity,buffercapacity,pHand[Cl],couldreectallofstressfactors.
Perfectlinearcorrelationswereobservedbetweenallstrainindicesandthefourstressfactors.
However,theeffectsofthefourstressfactorsonthestrainindicesweresignicantlydifferentinmagnitude.
Buffercapacityandsalinityweredominantfactorsforallstrainindices.
Thus,itisreasonabletoconsiderthesumofsalinityplusbuffercapacityasthestrengthvalueofsalt–alkalimixstress.
Furthermore,therelationshipsbetweendifferentstrainindicesandvariousstressfactorswereshowntobedifferent.
2004PublishedbyElsevierB.
V.
Keywords:Alkalistress;Buffercapacity;pH;Salinity;Saltstress;Salt–alkalimixedstress;Stressfactor;SunowerCorrespondingauthor.
Tel.
:+864315269590;fax:+864315684009.
E-mailaddress:shidc274@nenu.
edu.
cn(D.
Shi).
0098-8472/$–seefrontmatter2004PublishedbyElsevierB.
V.
doi:10.
1016/j.
envexpbot.
2004.
05.
003http://www.
paper.
edu.
cnD.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–2191.
IntroductionSalinitystressisawidespreadenvironmentalprob-lem.
Althoughconsiderableefforthasbeendevotedtosolvethisproblem,twoveryimportantaspectshavebeenneglected,i.
e.
salt–alkalistressandcomplexsaltstress.
Eventhoughtheworld'slandsurfaceoccupiesabout13.
2*109ha,nomorethan7*109haarepo-tentiallyarable,andonly1.
5*109haarecurrentlycultivated.
Ofthecultivatedarea,about0.
34*109ha(23%)aresalineandanother0.
56*109ha(37%)aresodic(Tanji,1990).
Actually,theproblemofsoilalka-linizationduetoNaHCO3andNa2CO3,maybemoreseverethantheproblemofsoilsalinizationcausedbytheneutralsalts,suchasNaClandNa2SO4.
Forex-ample,inthenortheastofChina,alkalinizedgrass-landhasreachedmorethan70%(KawanabeandZhu,1991).
Becausesoilsalinizationandalkalinizationfre-quentlyco-occur,theconditionsinthenaturallysalin-izedandalkalinizedsoilareverycomplex,thetotalsaltcontentsandcompositionofsaltsandthepropor-tionofneutralsaltstoalkalinesaltsmayvaryindif-ferentsoils.
Thus,thestressesimposedbythesesoilmediaonplantscouldbeverycomplexanddifculttoapproachexperimentally.
Naturalsaltstressesaremostlymixedsaltsstresses,andmostofthemcontainbothneutralandalkalinesalts.
Therefore,theproblemsofalkalinestressandsalt–alkalimixedstressoughttoberecognizedandinvestigatedasthoroughlyassaltstress.
Todate,theresearchofsaltstressstillemphasizesNaClasthemainsubject,butitisdeeplydevelop-ingtowardsvariousaspectssuchasNa+metabolism(Serranoetal.
,1999),molecularbiologyofsalt-resistancegenes(Holmstr¨ometal.
,2000;Huangetal.
,2000;Quesadaetal.
,2002),andsaltstresssignaltransduction(DeWaldetal.
,2001),andsoon.
How-ever,thereareonlyafewreportsaboutstressbyalkali.
However,therehavebeensomestudiesaboutcalcare-oussoils(Brandetal.
,2002;Nuttalletal.
,2003),al-kalinesoil(Hartungetal.
,2002;YinandShi,1993),alkalinesaltstress(CampbellandNishio,2000;ElandShaddad,1996;ShiandYin,1992,1993),andmixedsaltstress(Shietal.
,1998).
Furthermore,somereportsclearlydemonstratedtheexistenceofalkalistressandshowedthatitismoreseverethansaltstress(ShiandYin,1993;TangandTurner,1999).
Inpreviousstudies,itwasfoundthatalkalisaltstressandneutralsaltstressareactuallytwodistinctkindsofstresses(ShiandYin,1993).
Basedonourresults,alkalinesaltstressisbestcalled"alkalistress,"while"saltstress"onlyincludestheneutralsaltstress.
Theresistancetoalkalistressofsunower(He-lianthusannuusL.
)isstrongerthanthatofothercrops.
Somesunowerbreedsareabletogrowonalkalin-izedsoil.
However,thereareveryfewreportsaboutsunowerresistancetosaltstressoralkalistress(LiuandBaird,2003).
Acultivarofsunowerwasselectedasthematerialtoinvestigatethefeaturesandactingfactorsofsalt–alkalimixedstress.
TheneutralsaltsNaClandNa2SO4andthealkalinesaltsNaHCO3andNa2CO3arethemainsaltcompo-nentsintheextensivealkalinesoilovermuchofnorth-eastChina(GeandLi,1990).
Therefore,mixturesoftheaforementionedsalts,invariousproportions,wereusedtosimulatearangeofmixedsaltandalkalineconditions.
ThirtykindsofthemixedsaltandalkalineconditionswithdifferentsalinitiesandpHvalueswereobtainedtoinvestigatetheeffectofmixedsaltandal-kalinestressesonsunowerseedlingsandtoanalyzethecorrespondingstressfactors.
2.
Materialsandmethods2.
1.
PlantmaterialsH.
annuusL.
cv.
Baikuiza4wasprovidedbytheBaichengAcademyofAgricultureSciences,JilinProvince,Chinaandwasselectedbecauseofitstol-erancetosalt–alkalineconditions.
Baikuiza4seedsweresownin24cmdiameterplasticpotscontainingwashedsand.
Eachpotcontainedsixplants.
Allpotswereplacedoutdoorsavoidingrainfall.
SeedlingsweresufcientlywateredwithHoaglandnutrientsolutionevery2days.
Evaporatedwaterwasreplenishedwithdistilledwateratothertimes.
2.
2.
DesignofsimulatedsaltandalkalineconditionsTwoneutralsalts(NaClandNa2SO4)andtwoalka-linesalts(NaHCO3andNa2CO3)wereselectedbasedonthesaltcomponentsintheextentofsalt–alkalinesoilovernortheastChina(GeandLi,1990).
Thefourselectedsaltsweremixedinvariousproportionsac-10D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–21Table1ThesaltcompositionanditsmolarratioofvarioustreatmentsTreatmentgroupSaltcompositionandmolarproportionsNaClNa2SO4NaHCO3Na2CO3A1100B1210C1991D1111E9119F1199cordingtothetolerabilityoftheBaikuiza4cultivartothesalt–alkalinestressandthevaryingrangesofsalin-ityandpHinthesoil.
Sixtreatmentgroups(labeledasA–F)weresetwithgraduallyincreasingalkalin-ity.
ThesaltcompositionofthesixtreatmentgroupsisshowninTable1.
Alltreatmentgroupshada1:1molarratioofmonovalentsalts(NaCl+NaHCO3)todivalentsalts(Na2SO4+Na2CO3);therefore,iftheindividualmolarconcentrationswerethesamethenthetotalionconcentrationswerethesamethrough-outthetreatments.
Withineachgroup,veconcentra-tiontreatmentswereutilized,namely50,100,150,200and250mmolL1totaling30salt–alkalimixedstresstreatments(labeledasA1,F5)withvaryingsalinityandpH.
2.
3.
StresstreatmentsWhentheseedlingswere4weeksold,theyweresubjectedtostresstreatments.
Seedlingsgrowinguni-formly(in96pots)wereselected,randomlydividedinto32sets,3potsperset.
Eachpotwasconsid-eredasonereplicatewiththreereplicatesperset.
Onesetwasusedasacontrol;asecondsetwasusedforgrowthindexdeterminationatthebeginningoftreatment;andtheremaining30portionsweretreatedwithvariousstresstreatments.
Controlplantsweremaintainedbywateringwithnutrientsolution;plantsunderallthevariousstresstreatmentswerewateredwithnutrientsolutionwithaddedstresssaltasthetreatmentsolution.
Stresstreatmentswereperformedaround4–5p.
m.
,bywateringthoroughlytreatedplantswith1000mloftreatmentsolutionperpot,inthreeportions.
Theamountofevaporatedwaterwasdeter-minedbyweightandreplenishedwithdistilledwaterdaily.
2.
4.
StainindicesmeasurementsAllplantswereharvestedcarefullyafter7daysoftreatment,washedwithtapwaterrst,thenwithdis-tilledwater.
Waterremainingonthesurfaceoftheplantswasblottedwithlterpaper.
Rootsandshootswereseparatedineachplant.
Eighteenplantsfromeachtreatment(sixperreplicate)weresampledtodeterminestrainindices.
2.
4.
1.
GrowthmeasurementThefreshweights(fr.
wt.
)ofshootandrootwereweigheddirectly.
Thethirdleafbladefromeachshootbottomwastakentodetermineleafareawithanareameter(model1671-VHA).
Theresultofleafareawasexpressedincm2piece1.
Relativegrowthrate(RGR)wasdeterminedasdescribedinKingsburyetal.
(1984)usingthefollowingformula:RGR=lnbiomassatendoftreatmentlnbiomassatstartoftreatmentdurationoftreatment(days).
Onegramoffreshleafwastakenfromeachpottodeterminetherateofelectrolyteleakage.
Otherfreshsampleswereoven-driedat80Cfor15min,thenvacuum-driedat40Ctoconstantweight,thenthedryweight(drywt.
)wasmeasured.
Theresultofbiomasswasexpressedingdrywt.
2.
4.
2.
OrganicandinorganicsolutesdeterminationsDrysampleswerehomogenizedbypowdering.
TwohundredmilligramsofdryshootsamplesweretakentodetermineK+andNa+contentsbyamephotometry(WangandZhao,1995),and100mgofdryshootsam-pleswereusedtomeasureprolinecontentaccordingtoZhuetal.
(1983).
Thecontentofcitricacidwasdeter-minedbythepentabromoacetonemethodwithmodi-cations(Shietal.
,2002).
TheresultsofK+andNa+contentswereexpressedinmmolg1drywt.
,prolineinmolg1drywt.
,andcitricacidinmmolg1drywt.
2.
4.
3.
MembranepermeabilitydeterminationMembranepermeabilitycanbereectedbytherateofelectrolyteleakage(REL).
RELwasdeter-minedasdescribedbyLuttsetal.
(1996).
Freshleafdiscs(1g)weretakenfromeachpot,andwashedD.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–2111threetimeswithdeionizedwatertoremovesurface-adheredelectrolytes.
Leafdiscsweredividedequallyandplacedintotwoclosedvialscontaining20mlofdeionizedwater.
Oneofthevialswasincubatedat25Conarotaryshakerfor3h,andthentheelec-tricalconductivityofthesolution(EC1)wasdeter-minedwithaconductivitygauge.
Theothervialwasautoclavedat120Cfor20minandelectricalcon-ductivityofthesolution(EC2)wasdeterminedaf-terequilibrationto25C.
RELcanbedenedasfollows:REL(%)=EC1EC2*100.
2.
5.
AnalysisofstressfactorsItiscommonlythoughtthatsaltstressinvolvesbothosmoticeffectsandspecicioneffects,theformeroneschieydependingonsaltconcentration.
Foral-kalinestress,besidesthesetwokindsofeffects,thereseemstobeahigh-pHeffect.
Preliminaryresults(Shietal.
,1998)showthataspecialresponseofplantstoalkalinestressistheadjustmentoftheirinternalandexternalpHandthatthebuffercapacityofthetreatmentsolutionisanimportantfactorforchang-ingpH.
Therefore,thestressfactorsofasalt–alkalimixedstressshouldinvolvetotalsaltconcentration,variousionconcentrations,pHvalues,andbuffercapacity.
Thetotalsaltconcentrationandtheconcentrationofeachionsuchas[Na+],[Cl],[SO42],[HCO3]and[CO32]invarioustreatmentsolutionswerecal-culatedbasedonthecompositionofthesolutions.
ThepHvaluesofvarioustreatmentsolutionsweredeter-minedwithadigitalpHmeter.
ThebuffercapacitywasdeterminedusingthemethodofShietal.
(1998)withbuffercapacitydenedasthemillimolaramountofH+neededtodropthepHof1LoftreatmentsolutiontothesamepHasthecontrolbytitrationwithHCl.
2.
6.
StatisticaldataanalysisAlldataobtainedweretheaverageofthreerepli-cas.
Statisticalanalysisontwo-wayvarianceanalysis(ANOVA),correlationcoefcient,andmultivariatere-gressionwasperformedusingMicrosoftExcel.
3.
Results3.
1.
SalinityandpHcoveragewithvarioustreatmentsolutionsFig.
1showsthatthepHvaluesincreasegradu-allyfromgroupAtogroupF.
Inaddition,withinthesametreatmentgroup,pHvaluesincreasewithincreas-ingtotalsaltconcentration.
TherangeofpHvaluesisgreateramonggroupsthanwithinagroup.
ForNa+,themaintoxicion,theconcentrationsusedwere75,150,225,300,and375mmol,correspondingtothevesaltconcentrationsinatreatmentgroup.
Insum,30salt–alkalineconditionswithdifferentsalinityandpHvalueswereestablished.
Thesalinitycoveragewasfrom50to250mmol;[Na+]coveragewasfrom75to375mmol;pHcoveragewasfrom7.
12to10.
72.
Becauseofthesaltcomponent,salinityandpHval-uesinthe30simulatedsalt–alkalineconditionsaresimilartotheconditionsinnaturalsalt–alkalinesoil.
Thesesimulatedsalt–alkalineconditionsreproducedFig.
1.
SalinityandpHofvarioustreatments.
ThevaluesofpHaremeansofthreereplicates.
(A)NaCl:Na2SO4:NaHCO3:Na2CO3=1:1:0:0,pH7.
12–7.
25;(B)NaCl:Na2SO4:NaHCO3:Na2CO3=1:2:1:0,pH7.
91–8.
20;(C)NaCl:Na2SO4:NaHCO3:Na2CO3=1:9:9:1,pH8.
47–8.
83;(D)NaCl:Na2SO4:NaHCO3:Na2CO3=1:1:1:1,pH9.
41–9.
88;(E)NaCl:Na2SO4:NaHCO3:Na2CO3=9:1:1:9,pH10.
18–10.
46;(F)NaCl:Na2SO4:NaHCO3:Na2CO3=1:1:9:9,pH10.
47–10.
72.
12D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–21Table2Resultoftwo-wayvarianceanalysis(ANOVA)ofsalinity(S)andtreatmentgroup(T)forthestrainindexesselectedDependentvariableIndependentvariableSTRGR(day1)41.
9422.
55Leafarea(cm2piece1)44.
8025.
06Electrolyteleakagerate(%)13.
6432.
65Prolinecontent(molg1drywt.
)9.
5934.
495Citricacidcontent(mmolg1drywt.
)31.
5616.
81Na+content(mmolg1drywt.
)43.
2620.
22K+content(mmolg1drywt.
)36.
8446.
79NumbersrepresentFat5%level.
P8.
8).
Theseresultsdemonstratethatalkalistressalsocancauseheavyaccumulationofprolineandthatthephysiologicalfunctionsoftheprolineaccumulatedinsunowerundersaltandalkalimixedstressmaynotbejustbehaveasanosmolyteandprotectantbutmayalsohaveotherrolesrelatedtoalkalistress.
Thecitratecontentsofallgroupsincreasedwhensalinitywasaugmented;concomitantly,theextentofcitratecontentincreasetendedtobehigherwithin-creasingalkalinity.
Asitoccurswithproline,citratecontentincreasedsharplywhenbothsalinityandalka-linitywerehigh.
14D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–21TheNa+contentsofthealltreatmentgroupsin-creasedwithincrementingsalinity,thisbasicallywasinagreementwiththeresultsfromprevioussaltstressexperiments(ShiandYin,1992,1993)(Fig.
4).
ThedegreeofNa+increasetendedtobehigherwithin-creasingalkalinity.
Conversely,theK+contentofthesixgroupsdecreasedwithincreasingsalinity,andtheextentofdecreasediminishedwithincreasingalkalin-ity(Fig.
4).
Fig.
4.
Effectsofvarioussaltandalkalimixedstressesonthecontentsofproline,citricacid,Na+,K+intheshootsofsunower.
Four-week-oldseedlingswerestressedwithmixedsaltsfor7days.
Thevaluesaremeansofthreereplicates.
(A)NaCl:Na2SO4:NaHCO3:Na2CO3=1:1:0:0,pH7.
12–7.
25;(B)NaCl:Na2SO4:NaHCO3:Na2CO3=1:2:1:0,pH7.
91–8.
20;(C)NaCl:Na2SO4:NaHCO3:Na2CO3=1:9:9:1,pH8.
47–8.
83;(D)NaCl:Na2SO4:NaHCO3:Na2CO3=1:1:1:1,pH9.
41–9.
88;(E)NaCl:Na2SO4:NaHCO3:Na2CO3=9:1:1:9,pH10.
18–10.
46;(F)NaCl:Na2SO4:NaHCO3:Na2CO3=1:1:9:9,pH10.
47–10.
72.
3.
3.
Analysisoftheactingfactorsofsalt–alkalimixedstresses3.
3.
1.
Dataofstressfactorsforvariousmixedsalt–alkalistresstreatmentsAllplantsintheF4andF5groupsdiedafterstresstreatmentsurelybecausethestressstrengthswereovertheirtolerability.
Thedataofstressfactorsfortheother28treatmentscanbeseeninTable3.
D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–2115Table3StressfactorsforvarioustreatmentsTreatmentSalinity(mmolL1)Buffercapacity([H+],mmol)pH[Cl](mmolL1)[SO42](mmolL1)[CO32](mmolL1)[HCO3](mmolL1)A1500.
017.
12252500B15087.
9112.
525012.
5C15019.
88.
472.
522.
52.
522.
5D15028.
99.
4112.
512.
512.
512.
5E15043.
910.
222.
52.
522.
52.
5F15050.
210.
52.
52.
522.
522.
5A21000.
027.
14505000B210017.
28.
012550025C210038.
78.
62545545D210057.
89.
4925252525E210082.
210.
3455455F2100106.
410.
5554545A31500.
037.
18757500B315026.
88.
0937.
575037.
5C315058.
18.
717.
567.
57.
567.
5D315086.
89.
6237.
537.
537.
537.
5E3150123.
410.
467.
57.
567.
57.
5F3150159.
610.
67.
57.
567.
567.
5A42000.
047.
2110010000B420033.
58.
1650100050C4200768.
810901090D4200113.
19.
7550505050E420016710.
590109010A52500.
057.
2512512500B525038.
68.
262.
5125062.
5C525097.
38.
8312.
5112.
512.
5112.
5D52501519.
8862.
562.
562.
562.
5E5250196.
510.
5112.
512.
5112.
512.
5BuffercapacityandpHdataweremeasuredexperimentallyandthemeanofthreereplicatesisreported;othervalueswerecalculated.
3.
3.
2.
CorrelativitybetweenstressfactorsandstrainindicesCorrelationcoefcientsbetweenstressfactorsandstrainindiceswerecalculatedinordertostudytheirrelationshipandtheresultsareshowninTable4.
Allthecorrelationcoefcientsbetweenvestressfactors(pH,buffercapacity,salinity,[Na+],and[CO32]),andsevenstrainindiceswerestatisticallysignicant.
Buffercapacityshowedthehighesttotalcorrelationstrengthamongstressfactorsfollowedby[CO32],salinity,and[Na+].
Forthesevenstrainindices,theirfactorwiththehighestcorrelationwasbuffercapac-ity.
Thus,alkalinesaltshaveaprofoundeffectonstrain.
Thecorrelativitybetween[SO42]andthevariousstrainindiceswasthelowestamongallthestressfac-torsandnoneofitscorrelationcoefcientsreachedalevelofsignicance.
Therefore,theeffectof[SO42]onstrainindicescouldbeoverlooked.
Twoofthesevencorrelationcoefcientsbetween[Cl]andthesevenstrainindiceswerestatisticallysignicantandthere-foretheimpactof[Cl]shouldnotbeneglected.
ForthecorrelativityamongvariousstressfactorsnotlistedinTable4,therewasafullpositivecorrelativitybetweensalinityand[Na+](r=1),andthecorrelationcoefcientsofsalinityand[Na+]withthesevenstrainindiceswerethesame.
Therefore,salinitycouldeffec-tivelyrepresent[Na+].
Buffercapacitywasdependenton[CO32]and[HCO3].
Iftheexpression2[CO32]+[HCO3]isusedtorepresentthetotalstrengthofalkalinesalt,thecorrelationcoefcientbetweenitandbuffercapacityisestimatedas0.
9943.
Thus,[CO32]and[HCO3]couldbefullyrepresentedbythebuffercapacity.
16D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–21Table4CorrelationcoefcientsbetweenstressfactorsandstrainindicesStressfactorStrainindexRGRLeafareaCitratecontentNa+contentK+contentProlinecontentElectrolyteleakagerateMeanabsolutevaluepH0.
56990.
63320.
57330.
56070.
76660.
51810.
82190.
6348Salinity0.
74810.
72650.
73980.
75070.
55640.
61100.
41690.
6499Buffercapacity0.
88690.
91330.
88250.
88170.
92290.
85300.
95430.
8992[Na+]0.
74810.
72650.
73980.
75070.
55640.
61100.
41690.
6499[Cl]0.
3166(NS)0.
3403(NS)0.
42840.
44160.
1801(NS)0.
2112(NS)0.
2353(NS)0.
3076[SO42]0.
0745(NS)0.
0171(NS)0.
0276(NS)0.
0387(NS)0.
1467(NS)0.
0321(NS)0.
3633(NS)0.
1000[HCO3]0.
51380.
46070.
3734(NS)0.
3709(NS)0.
44700.
47490.
2176(NS)0.
4083[CO32]0.
75510.
80340.
80510.
80360.
81810.
73470.
93450.
8078NS=notsignicant.
r0.
05=0.
374,r0.
01=0.
487,n=28.
Correlationsignicantat0.
05levelofprobability.
Correlationsignicantat0.
01levelofprobability.
3.
3.
3.
MultivariateregressionanalysisbetweenmainstressfactorsandvariousstrainindicesAccordingtoaboveanalysisdata,[SO42]couldbeneglected,osmosisand[Na+]couldberepresentedbysalinity,and[HCO3]and[CO32]couldberep-resentedbybuffercapacity.
Thus,thefourmainstressfactors,buffercapacity,salinity,pH,and[Cl],mightbeenoughtorepresentallthestressfactorsinvolved.
Thesefourmainstressfactorsweretakenasinde-pendentvariableswherex1=buffercapacity,x2=salin-ity,x3=pH,andx4=[Cl];andthestrainindicesweretakenasdependentvariables,withY=RGRandsoon.
MultivarianceregressionanalysiswasperformedforeachstrainindexusingtheformulaY=a+b1x1+b2x2+b3x3+b4x4.
Theimportanceofeachstressfactorwascomparedaccordingtotheirstandardizedregres-sioncoefcients(b)andthesignicanceofregressionwasestimatedbythesquareoftotalcorrelationcoef-cient(R2).
TheresultsofregressionareshowninTable5.
ItshowedthattheR2valueswerelargerthan0.
9,ex-ceptforproline(0.
8437),indicatingahighlinearcor-relativitybetweeneachoneofthestrainindicesandthefourstressfactors.
Theimportanceofthefourfac-torswasclearlyshownbycomparingthevaluesofb(Table5).
Amongtheabsolutevaluesofthefourb,thoseofbuffercapacity(b1)werethehighestforallofthestrainindices,exceptleafarea,seeminglyindi-catingthatbuffercapacitywasadominantfactorforallstrainindexes.
Furthermore,theabsolutevaluesofb2weregreaterthanthoseofb3andb4,exceptforelectrolyteleakagerateandproline;therefore,salinitywasanotherdominantfactorbesidesbuffercapacity.
Moreover,thesignicancesofpHand[Cl]werede-terminedbyanalyzingb3andb4.
pHwasanimportantfactorforelectrolyteleakagerate,proline,andK+con-tent,butwaslessimportantandcouldbeneglectedforotherstrainindices.
Thefactor[Cl]wasnegligibleforallstressindexes.
Insum,buffercapacityandsalinitywerebothdominantfactors,pHwaslessimportant,and[Cl]wastheleastimportantone.
4.
Discussion4.
1.
Simulationofmixedsalt–alkaliconditionsFoursalts(NaCl,NaHCO3,Na2SO4,andNa2CO3)weremixedatvariousproportionstosimulatecom-D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–2117Table5ResultsofmultipleregressionbetweeneachstrainindexandfourfactorsYRegressionparametersb1b2b3b4ab1b2b3b4R2RGR0.
378000.
270541.
362000.
12075121.
170.
625620.
558230.
048090.
120750.
9609Leafarea0.
065510.
055251.
314360.
0085946.
8950.
510330.
536580.
218440.
000230.
9802Electrolyteleakagerate0.
201890.
004774.
117600.
0823514.
6360.
679540.
02002;0.
295670.
170870.
9401Prolinecontent0.
137630.
022342.
423750.
0331520.
0211.
124030.
227450.
422310.
166900.
8437Citratecontent5.
08*1042.
86*1043.
93*1051.
12*1040.
01470.
604220.
428310.
100710.
082940.
9432Na+content0.
008180.
004310.
036750.
001880.
42080.
638160.
419340.
061150.
090580.
9518K+content0.
001810.
001420.
045840.
000521.
64530.
445760.
435980.
398460.
079550.
9616Note:Y=a+b1x1+b2x2+b3x3+b4x4,wherex1=buffercapacity,x2=salinity,x3=pH,x4=[Cl],b1–b4=standardizedregressioncoefcientscorrespondingx1–x4andR2=thesquareoftotalcorrelationcoefcient.
plexsalt–alkalineconditions.
Asaresult,30kindsofsalt–alkalineconditionswithdifferentsalinityandpHvalueswereestablished.
Theresultsshowedthatthesimulated30treatmentconditionsevenlycoveredvarioussalt–alkalineconditionsinarangeoftotalsaltconcentrationfrom50to250mMandpHfrom7.
12to10.
72.
Thestressingconditionsandinterfer-encefactorsareverycomplexandunrestrainableinnaturalsalt–alkalinesoils,andthisgreatlylimitsin-vestigationsoncomplexsalt–alkalinestress.
However,throughthemethodsusedinthisworkwesuccessfullyreproducedthecomplexsalt–alkalineconditionsunderarticialconditionsandmadetheresearchofcomplexsalt–alkalinestresspossible.
Thesesalt–alkalinecondi-tionscreatedbymixingfoursaltsweredifferentfromthepreviousworksofsaltstress(Cheeseman,1988)andalkalistress(ShiandYin,1992,1993)whichin-volvedbothsalinityandalkalinityandproducedanewsalt–alkalimixedstress.
Byutilizingthisnewmethod,researchonplantsaltstresscouldbefurtherexpandedapproachedinacloserwaytonaturalsoilconditions.
4.
2.
Responsesofsunowerseedlingstosalt–alkalimixedstressBaikuiza4,thesunowercultivarusedinthiswork,isrelativelytoleranttosalt;asaconsequence,thegrowthparametersofplantstreatedwithlowsaltcon-centrationsolutionswerebetterthanthecontrol(A1inFig.
2).
However,bothRGRandleafareadecreasedwithincreasingsalinityandalkalinityunderallotherstressconditions(Fig.
2),especiallyathighsalinityandhighalkalinity,treatmentsinwhichgrowthwasinhib-itedsointenselythatalltheplantsdied(F4andF5).
Itisgenerallyconsideredthatsaltstressinhibitsplantgrowthbywaterdeciencyandiontoxicityamongotherfactors(deLacerdaetal.
,2003;Marcum,1999;Ghoulametal.
,2002;Soussietal.
,1998),butinsalt-tolerantspecies,plantgrowthisonlymoderatelyin-hibited,orevenstimulated,bysaltstress(Crameretal.
,1986;Marcum,1999).
TheresultsoftheplantsetA1conrmedthatthesunowervarietyusedwasarelativelysalt-tolerantone.
ChangesinRGRareaconsequenceofsaltstresseffectsinintactplants.
ItcanbeseeninFig.
2thatthegrowthinhibitioneffectofalkalinesaltstresswasstrongerthanthatofneutralsaltstressatthesamesaltconcentration(cf.
A3andF3inFig.
2).
Thiswasin18D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–21agreementwiththeresultsofwheatgrowingincal-careoussoil(Nuttalletal.
,2003),onion(Sharmaetal.
,2001),eucalyptus(Jamesetal.
,2002),pea(ElandShaddad,1996),amongothers.
Therefore,ourresultsindicatedthathighpHandionimbalancearoundrhizo-spherecausedbyalkalinesalt(CampbellandNishio,2000;Shietal.
,1998)werealsomainfactorsinin-hibitingplantgrowth,andthismightberelatedtotheeffectsofhighpHaroundtherootsonthetransportofABA(Degenhardtetal.
,2000).
However,theeffectsofhighpHwerecloselyrelatedtosaltconcentration,i.
e.
theeffectsofhighpHwereclearlyenhancedwithincreasingsalinity(cf.
D1–D5inFig.
2).
Therefore,thepeculiar—andevident—featureofasalt–alkalimixedstressistheirreciprocalenhancementofitsindividualcomponents(salinityandalkalinity).
Thepermeabilityoftheplasmamembraneisanevi-dentindexthatreectsthedegreeofstress-inducedin-jurytoplants(HongandLin,1996;SurjusandDurand,1996).
Electrolyteleakageratedata(Fig.
3)showedthatthepermeabilityofthecellmembraneofsunowerseedlingswasnotonlyincreasedwithrisingsalinitybutalsowithrisingalkalinity.
Thus,fromthestress-causedinjuryonplasmamembrane,itcanbeseenthatarecip-rocalenhancementbetweensaltstressandalkalistresswasanevidentfeatureofthesalt–alkalinemixedstress.
Theprimaryphysiologicalresponseofplantstosaltorosmosisstressistoundergoosmoticadjustmentsbytwoprocesses:accumulationofionsinthevacuoleandsynthesisofcompatiblesolutesinthecytosol.
Thechangesofsolutesintheshootsofsunowerseedlingsundersalt–alkalimixedstress(Fig.
4)correspondedtoplantresponsestosaltstress,namely,increasesofNa+,proline,andcitricacidcontentsandadecreaseofK+contentwithincreasingstress.
Nevertheless,salt–alkalinemixedstresswasdistinctfromsaltstressinsomeparticularaspects:First,prolineaccumulatedundersaltordroughtstressisusuallyconsideredasanorganic-compatibleosmolyteandaprotectingagentfortheactivityofin-tracellularmacromolecule(Tang,1989);prolineaccu-mulationiscloselyrelatedtoosmoticeffects.
Inthisexperiment,itisevidentthatprolinecontentincreasednotonlywithrisingsalinity,butalsowhenalkalinityincreasedatthesamesaltconcentration(Fig.
4).
Thissuggeststhattheinductionofprolinesynthesisisre-latednotonlytochangesinwaterpotentialand[Na+],butalsotohighpH.
Ourresultsindicatethatthephysi-ologicalfunctionofaccumulatedproline,inadditiontobeingandosmolyteandprotectingagent,couldbeaninvolvementininjuryduetoalkalinestress,anaspectthatoughttobeinvestigatedfurther.
Second,ithasbeenfoundrecentlythatcitricacidaccumulationisrelatedtoaluminumtoxicity(Lietal.
,2000),phosphorusdeciency(Neumannetal.
,1999),andseveralothersadverseconditions.
However,theaccumulationofcitricacidundersaltstressdependsonthechemicalpropertiesofthestress-inducingsalt.
Citricacidaccumulationismoderateorabsentunderneutralsaltstress(Francoiseetal.
,1991;Shietal.
,2002),butitisheavywhenthestressisduetoalka-linesalts(Shietal.
,2002).
Theresultsofourexper-iments(Fig.
4)provedagainthatalkalistressclearlyaffectsonorganicacidmetabolisminplants.
Thecon-tentofcitricacidintheshootsofstressedsunowerseedlingsincreasedwithincreasingsalinityandalka-linity(Fig.
4).
Citricacidisasmallorganicmoleculethatcertainlymayfunctionasanosmolyte.
However,previousworkhasalsoshownthatthedistributionofcitricacidaccumulatedunderalkalinestressinplantswasverydifferentfromthatofproline,i.
e.
whileofprolineaccumulationoccurredmainlyintheyoungertissueofalkalistressedplants,citricacidaccumulatedmainlyinoldertissueandwasassociatedwiththedis-tributionofNa+(Shietal.
,2002).
Thissuggestedthatthefunctionoftheaccumulatedcitricacidwasdiffer-entfromprolineandmainlyrelatedtopHadjustmentinthecell.
Thisisstillunclearandfurtherinvestigationisrequired.
Third,themetabolismofNa+andK+isanim-portantcomponentofsaltstress(Cheeseman,1988).
Usually,Na+increasesandK+decreasesinplantsstressedbysalt(deLacerdaetal.
,2003).
Theresultsofsalt–alkalinemixedstressinthisexperiment,though,showedthat[Na+]increasedand[K+]decreasednotonlywithincreasingsalinitybutalsowithrisingal-kalinity(cf.
Fig.
4),aphenomenonperhapsrelatedtoplasmamembranebeingdestroyedmoreseverelybyalkalinestress.
Thesechangesin[Na+]and[K+]areareectionofareciprocalenhancementbetweensaltstressandalkalistress,whichwasthepeculiarfeatureofsalt–alkalimixedstress.
Moreover,[Na+]and[K+]changesalsoreectedtheeffectsofsalt–alkalimixedstressonthemetabolismofNa+andK+andindicatethatthephysiologicalresponsesofplanttosalt–alkalimixedstressweremorecomplexthanthatofsaltstressD.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–2119alone.
Thisisanaspectthatshouldberesearchedmoredeeply.
4.
3.
SaltstressandalkalistresssynergismAccordingtoourresults,weconcludedthatthemixedsalt–alkalinestressnotonlycausedstressduetobothsaltandalkali,but,additionally,itdisplayedaninteractionbetweensaltstressandalkalistress.
Iftotalsaltconcentration(salinity)istakenasameasureofthestrengthofthesaltstressandpH(fromgroupAtogroupF)istakenasthestrengthofthealkalistress,asyner-gismbetweensalinityandpHcanbefound(Figs.
2–4).
Thestresstosunowerseedlingsduetosalinityasasingularfactor(cf.
groupsA1–A5)orhighpHasasin-gularfactor(groupsE1andF1approachedthecases)wassmallerthanthatofsalinitycoupledwithhighpH(cf.
D5andE5).
Usually,neutralsaltstressorsaltstressingeneralinvolvesosmoticeffects,whichdependonsaltconcen-tration,andspecicioneffects(Crameretal.
,1986;Cheeseman,1988).
Ontheotherhand,alkalinesaltstressoralkalistressalone,inadditiontoosmoticandspecicioneffects,alsoincludeshigh-pHeffects(ShiandYin,1993).
Themechanismofsalinitytoleranceinplantsmainlyinvolvestwoprocesses:one,osmo-sisadjustment,includesionaccumulationandsyn-thesisofcompatiblesolutes,amongotherphenom-ena;theother,iontoxicavoidance,includesspecicionmetabolismandcompartmentationoftoxicions(Cheeseman,1988),etc.
Althoughthemechanismoftolerancetoalkalinityinplantsisstillunclear,apHadjustmentprocessispossiblyinvolved(ShiandYin,1993),additionallytotheabovetwoprocesses.
Theosmosiseffectsandiontoxiceffectsdependonstresssaltconcentration,whilepHeffectsdependonbuffercapacity,whichinturniscloselyrelatedtoboththealkalinityandconcentrationofthestresssalt.
Inotherwords,thehigherthealkalinityandtheconcentrationthegreaterthebuffercapacity,anditismoredifcultforplantstoadjust.
Perhapsthisisthecauseofthesynergismbetweensaltstressandalkalinestress.
Nev-ertheless,thephenomenonofsalinityenhancingtheharmofhighpHiscomplexandisrelatedtothemech-anismofalkalinestressresistanceinsunower,anddeservesfurtherinvestigation.
Ourresultsprovedoncemoretherelaxingactionofneutralizationtothealkalistress(Shi,1995;Yanetal.
,2000):loweringthesoil'ssalinityorpHbasedonitssaltcomponentscanreducesalt–alkalinesoil'sharmtotheplant.
EspeciallyforbarrenlandwithhighpH,therecoveryofvegetationispossibletobeachievedbydecreasingitspH.
4.
4.
Salinityandbuffercapacitywerethedominantfactorsofmixedsaltandalkalistress4.
4.
1.
ActionfactorsofmixedsaltandalkalistressOneofthefactorsthatcausesthecomplexityofnaturalsaltandalkalimixedenvironmentalconditionsisthatthecomponents,proportions,andcontentsofcontainedsaltsaresovaried.
Differentsaltshavedif-ferentchemicalpropertiesandtheiractionsonplantarealsodifferent.
Theeffectiveactionsofmixedsalts,especiallyneutralsaltsmixedwithalkalinesalts,aremuchmorecomplexthanthoseofasingularneutralsalt(LiuandZhu,1997)orasingularalkalinesalt(ElandShaddad,1996;ShiandYin,1993;Shietal.
,2002).
Ingeneral,thestressfactorsoftheneutralsaltNaClaremainlytheioneffectsofNa+asadominantinjur-ingionandtheosmoticeffectsoflowwaterpotentialcausedbyhighsaltconcentration(Cheeseman,1988;Crameretal.
,1986),butforthealkalinesaltNa2CO3thereisanaddedstressfactor,namelyhighpH(ShiandYin,1993;Shietal.
,1998,2002).
However,whenvariousneutralsaltandalkalicsaltaremixedtogether,theeffectsofthemixedsaltsaremorethanjustasim-plecombinationoftheseparateeffectsfromthetwokindsofsaltsduetotheinteractionsbetweendiffer-entionsandsoon.
Thus,theeffectsofsaltandal-kalimixedstressshouldbeexperimentallyanalyzedindepth.
Thenovelconceptofbuffercapacityiskeyinsimplifyingstressfactoranalysisofthe30simulatedsalt–alkalineconditions.
Buffercapacitycouldrepre-senttheconcentrationandproportionnotonlyofthetwoalkalinesaltsbutalsoofthecarbonateandhy-drocarbonateanions.
Accordingtoabovecorrelationanalysisdata,[SO42]couldbeneglected,osmosisand[Na+]couldberepresentedbysalinity,and[HCO3]and[CO32]couldberepresentedbybuffercapac-ity.
Thus,thefourfactors,buffercapacity,salinity,pHand[Cl],couldbasicallyrepresentalltheeffectorsofmixedsaltandalkalistress(Table4).
Theresultsofsta-tisticalanalysis(Tables4and5)provedconvincinglythattheintroductionoftheconceptofbuffercapacity20D.
Shi,Y.
Sheng/EnvironmentalandExperimentalBotany54(2005)8–21iscorrectandsignicantinbringingtolightthemech-anismofalkalistressonplant.
4.
4.
2.
RelationshipbetweenvariousstressfactorandstrainindexThemagnitudesofdifferentstressfactorsonsev-eralstrainindiceswerefoundtobedifferentbecauseofthevaryingaccruingmechanismsinplants.
Fromtheregressionanalysisresultsofsevenstrainindices,showninTable5,itwasevidentthatbothbuffercapacityandsalinityweredominantandindispens-ablefactors.
Thesignicanceofbuffercapacitywasmuchgreaterthansalinityforsixofthesevenin-dices,andonlyslightlysmallerforoneindex(leafarea).
Thus,buffercapacitywasaveryimportantstrengthindexofmixedsalt–alkalinestress,whereas[Cl]andpHwerelesssignicantandevennegli-gibleinsomecases.
Theresultsofregressionalsoshowedthatthedegreesofimpactofdifferentstressfactorsonseveralstrainindiceswerenotequalandthatthedifferencemightberelatedtoboththephysi-ologicalmechanismsoftheplant'sresponsestostressandthephysiologicalprocessesassociatedwithstraindevelopment.
4.
4.
3.
Idealstrengthindexofmixedsalt–alkalinestressItwasveryimportanttodetermineproperstrengthindicesinresearchingstress.
Saltconcentration,[Na+],orspecicconductancemightbeusedtorepresentthestrengthofsaltstress(Tanji,1990);whereasbufferca-pacityorpHmightbeusedtorepresentalkalinestressstrength(Shietal.
,1998);butformixedsalt–alkalinestress,noneoftheseindicescouldcompletelyreectthestressstrength.
AccordingtotheresultsinTable5,itwasreasonabletoconsidersalinityplusbuffercapacityasthestrengthindexofmixedsalt–alkalinestress.
Af-tercomparingtheresultsofindividuallytakingsalinity,pH,orbuffercapacityasthestrengthofstress(Shietal.
,1998),itwasevidentthatusingsalinityplusbuffercapacityasthestrengthindexofmixedsalt–alkalinestresswasamorereasonableapproach.
Itisverydifculttoobjectivelyestimatethepoten-tialdegreeofinjurytoplantforasalt–alkalinizedsoil,especiallyforsoilswithhighpHvalues.
Accordingtothisconclusion,studiesusingsyntheticconditionscombiningsalinityandbuffercapacityofsoilshouldconstituteanewmodeltosolvethisproblem.
AcknowledgementSupportedbytheNationalNaturalScienceFounda-tionofChina(30270139,30070545).
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