Arsenate impact on the metabolite profile, production, and arsenic loading of xylem sap in cucumbers

23 Pages·2012·2.63 MB·English

by  Jörg Feldmann

ORIGINALRESEARCHARTICLE
published:02April2012
doi:10.3389/fphys.2012.00055
Arsenate impact on the metabolite profile, production, and
arsenic loading of xylem sap in cucumbers (Cucumis
sativus L.)
M.KalleUroic1,PascalSalaün2,AndreaRaab1 andJörgFeldmann1*
1TraceElementSpeciationLaboratory,CollegeofPhysicalSciences–Chemistry,UniversityofAberdeen,Aberdeen,UK
2EarthandOceanScience,UniversityofLiverpool,Liverpool,UK
Editedby: Arsenicuptakeandtranslocationstudiesonxylemsapfocusgenerallyontheconcentra-
AndrewMeharg,Universityof tion and speciation of arsenic in the xylem.Arsenic impact on the xylem sap metabolite
Aberdeen,UK
profile and its production during short term exposure has not been reported in detail.
Reviewedby: To investigate this, cucumbers were grown hydroponically and arsenate (AsV) and DMA
Anne-MarieCarey,Universityof
Glasgow,UK were used for plant treatment for 24h.Total arsenic and arsenic speciation in xylem sap
BrittaPlaner-Friedrich,University wasanalyzedincludingametaboliteprofilingunderAsV stress.Producedxylemsapwas
Bayreuth,Germany quantified and absolute arsenic transported was determined.AsV exposure had a signif-
*Correspondence: icant impact on the metabolite profile of xylem sap. Four m/z values corresponding to
JörgFeldmann,TraceElement fourcompoundswereup-regulated,onecompounddown-regulatedbyAsVexposure.The
SpeciationLaboratory,Collegeof
PhysicalSciences–Chemistry, compounddown-regulatedwasidentifiedtobeisoleucine.Furthermore,AsVexposurehad
MestonWalk,AberdeenAB243UE, asignificantinfluenceonsapproduction,leadingtoareductionofupto96%sapproduc-
UK. tionwhenplantswereexposedto1000μgkg−1 AsV.Nodifferencetocontrolplantswas
e-mail:[email protected] observed when plants were exposed to 1000μgkg−1 DMA.Absolute arsenic amount in
xylem sap was the lowest at highAsV exposure.These results show thatAsV has a sig-
nificant impact on the production and metabolite profile of xylem sap.The physiological
importanceofisoleucineneedsfurtherattention.
Keywords:arsenate,dimethylarsinicacid,isoleucine,metaboliteprofile,principalcomponentanalysis,xylemsap
INTRODUCTION structure(γ-Glu-Cys) -Gly(n=2–11;Schmogeretal.,2000)and
n
Arsenic uptake and translocation in plants and arsenic phyto- arefoundinrootcells.ThesePC-Ascomplexeshavebeenidenti-
toxicity have been a focus since the imminent threat of arsenic fiedinvariousplants,includingRauvolfiaserpentine,H.lanatus,
poisoning in Bangladesh and parts of India (Karim,2000; Hos- P.cretica,Helianthusannuus,andBrassicajuncea(Pickeringetal.,
sain,2006;Heikensetal.,2007)hasbeenrecognized.Millionsof 2000;Schmogeretal.,2000;Raabetal.,2004,2005).
peoplearelivingonarseniccontaminatedgroundwaterandusing Sofar,theAs-PCscomplexationistheonlymechanismknown
it for crop irrigation. Rice is the main crop in this region and whichisresponsibleforthelimitationofinorganicarsenictranslo-
thereforeawidelystudiedtargetplant(MehargandJardine,2003; cationfromrootstoshoots(Schmogeretal.,2000;Raabetal.,2004;
Williamsetal.,2005;Xuetal.,2007;Jianetal.,2008;Careyetal., Liuetal.,2010).
2010).Arsenicloadingintocerealsconsumedbyhumansandfeed- Since arsenic is transported, along with water, macro- and
stockisofinterestbecauseitisasecondarsenicintakepathwayfor micronutrients,inthexylemsap,samplingitalsooffersinforma-
humansaftercontaminateddrinkingwater(Hossain,2006). tionaboutarsenictransportationwithintheplant.Severalstudies
The uptake and translocation kinetics of arsenic are mainly haveshownthatarsenictransportedinxylemsapoccursinsev-
dependent on its concentration and species (Wang et al., 2002; eraldifferentspecies,suchasAsV,AsIII,MMA,andDMA(Kertulis
Ouyang,2005;Raab et al.,2005). The mechanism for uptake of etal.,2005;Mihuczetal.,2005;Meireretal.,2007;Yeetal.,2010;
arsenicinplantsisnotyetfullyunderstood.Butitisassumed,due Czechetal.,2011).Wheninorganicarsenicisfedtoplants,inor-
to chemical similarity, that AsV is taken up via the same trans- ganicarsenicspeciesarefoundinxylem.Itisplantdependentwhat
port system as phosphate (Meharg and Macnair,1992) whereas ratioofAsIIItoAsVcanbefound:Plantschallengedwithmethy-
AsIII as neutral molecule is transported through aquaglycero- latedspecies,containthesearsenicspeciesinthesap.Itisassumed
porinchannels(MehargandJardine,2003).Plantshaveevolved thatAsIIIisloadedintothexylemviatheSitransporterLsi2(Ma
a strategy to detoxify arsenic, which is the accumulation of etal.,2007).FortheloadingofAsVandmethylatedarsenicspecies
arsenicintherootsbycomplexingitwithphytochelatins(PCs). noloadingmechanismcouldbeidentifieduntilnow.
Subsequently AsIII is complexed to PC-As complexes which are Sofar,themainfocusofresearchonarsenicimpactonplantsis
thought not to be transported out of the cells and immobi- onitsuptakeanddetoxificationaswellasthespeciationandcon-
lized in the vacuole of the root cells. Phytochelatins are pep- centrationofarsenicinxylemsap.Nostudyshowstheimpactof
tides derived from glutathione (GSH) and have the general arsenic on the metabolite profile of xylem sap of plants. Recent
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Uroicetal. Arsenateimpactonxylemsap
studies have shown that metabolomics is able to uncover the eachwerechallengedwith100,500,and1000μgkg−1 AsV each,
responseofplantsonmetalstress,suchascadmiumonArabidopsis 1000μgkg−1DMA,andnoarsenic(20plantsintotal)for24h.
thaliana (Ducruixetal.,2008)andcopper,iron,andmanganese
on Brassica rapa (Jahangir et al., 2008). However, these studies PlantsetC
wereperformedonwhole,homogenizedplants. Fivesetsofplantswithfourplantseachweregrownfor48±4days
Sofar,metaboliteprofilinginxylemsapwasdoneastargeted (floweringstage).Fourplantseachwerechallengedwith100,500,
analysisincomparingtheamountofknownsubstances(i.e.,tar- and1000μgkg−1 AsV each,1000μgkg−1 DMA,andnoarsenic
geted metabolomics),such as amino acids,sugars,or carboxylic (20plantsintotal)for24h.
acids in stressed plants against control plants (Callahan et al., Allplantsweregrowninagreenhouseatadjustedtemperature
2009;López-Millánetal.,2009;Larbietal.,2010).Nountargeted (26±2˚C).Humiditywasnotadjustedsincetemperaturecontrol
metabolomicstudyonxylemsapunderstress,letalonemetal(loid) wasdonebyopeningthewindowsandaheatingsystem.
impact(likeAsV),onxylemsapwasdone. ForsetBartificiallightwasusedtoensurea12-hlightperiod.
In this work,a study of the impact of AsV and DMA on the
PLANTTREATMENTANDSAPSAMPLING
productionandmetaboliteprofileofxylemsapinCucumissativus
All plants were starved on phosphate with phosphate-free
L.ispresented.
Hoaglands solution for the last 2days of growth before arsenic
EXPERIMENTAL treatment.FourplantseachwereexposedtoeitherDMAandAsV
atdifferentconcentrations(700and1400μgkg−1ofAsVforplant
CHEMICALS
Arsenate (AsV, disodium hydrogen heptahydrate) was obtained setAand100,500,and1000μgkg−1 forAsV and1000μgkg−1
fromBDHChemicals,Poole,UK.DMA(sodiumdimethylarsinic forDMAornoarsenicforplantsetsBandC,respectively).Plant
acid)wasobtainedfromStremChemicals,UK.Hoaglandnutrient treatmentalwayslasted24hbeforesampling.
solutionwasmadeupindoubledistilledwater(DDW).Chemi- After 24h all stems were cut approx. 1cm above their root
calswereatleastofanalyticalgrade[KNO ,Ca(NO ) ∗4H O, neck,thefirstdropwaswipedawayandsapwascollectedwitha
3 3 2 2
MgSO , NH NO , KH PO , FeSO ∗ 7 H O, MnCl ∗ 4 H O, micropipette for 1h. Sap samples were weighed and then either
4 4 3 2 4 4 2 2 2
Na B O ∗10H O,ZnSO ∗7H O,CuSO ∗3H O,Na MoO measuredrightafterextractionorstoredat−20˚Cuntilfurther
2 4 7 2 4 2 4 2 2 4
∗2H O(allfromSigmaAldrich,Dorset,UK)]. analysis.ThesapwasanalyzedbyESI–MSforsucroseinorderto
2
testwhetherthexylemwascontaminatedwithphloem.
PLANTGROWTH
Cucumber seeds (C. sativus L. Telegraph improved, B&Q, QUANTIFICATIONANDSPECIATIONOFARSENICINSAP
Aberdeen)weregerminatedinseedlingtraysinVermiculite®until Sapcollectedfromexudationwascentrifugedandeitherdiluted
their two leaf stage, which took approx. 1week. Reaching that, with ultrapure water to make up to 500μL or injected directly
four plants each were transferred into one 3L pot containing intoHPLC–ICP-MS(Agilent1100HPLCcoupledtoAgilent7500c
hydroponicnutrientsolution[Hoaglandsolution:2.5mMKNO , ICP-MS).ExternalcalibrationwasachievedbyinjectionofDMA
2.5mM Ca(NO ) , 1.0mM MgSO , 0.5mM NH NO , 0.5mM3 standardsolutionrangingfrom0.5to100μgkg−1.
3 2 4 4 3
KH PO (adjusted to pH 6.3),0.05mM FeSO ,4.5μM MnCl , Todetectchlorideinterferences,m/zvalues77and82werealso
50μ2M 4Na B O , 0.36μM ZnSO , 0.36μM4CuSO , 0.25μM2 monitored. Indium (50μgkg−1) was infused continuously post
2 4 7 4 4
Na MoO ]. For the first week, half-strength, for the rest of the columnviaaperistalticpumpasinternalstandard.
2 4
timefull-strengthnutrientsolutionwasused.Threedifferentplant
MEASUREMENTOFTHEMETABOLITEPROFILEOFXYLEMSAP
batchesweregrown.PlantsetBwasgrownuntilbeforedeveloping
ThemetaboliteprofilewasdeterminedusingplantsetA.Forsep-
flowersandblooming(floweringstage);plantsetsAandCwere
arationareversed-phasecolumn(C18,EclipseXDB-C18,Agilent,
grown until blooming (flowering stage).A pot with four plants
Agilent USA) was used. Hundred microliter of the sample was
eachwasusedasreplicateforthechallengeexperiments.
injected using a 0.1% formic acid in water – 0.1% formic acid
inmethanolgradientwith1mLmin−1flow.Startingfrom100%
PlantsetA
Threesetsofplantsgrownfor28±3days(floweringstage). water,methanol was added to the eluent after 5min. From 5 to
25min the methanol fraction was increased to 100%. The elu-
(a) Threepotsoffourplantseachwerechallengedwithnoarsenic entwasheldat100%methanolforafurther5min,followedby
(12plantsintotal). re-equilibration.Totalchromatographicruntimewas40min.The
(b) Three pots of four plants each were challenged with eluentflowwasinjectedintoanESI–MS(MSDXCT,AgilentUSA).
710μgkg−1AsV(12plantsintotal)for24h. TheESI–MSwasusedinpositive/negativeswitchmodeandMS2
(c) Three pots of four plants each were challenged with spectra were recorded when signal intensities were higher than
1400μgkg−1AsV(12plantsintotal)for24h. 50,000counts.
Intotal,36plantswereusedinplantsetA. STATISTICALANALYSIS
Thevariationinxylemsapproduction,arsenicamount,andthe
PlantsetB intensities for the selected metabolites was determined by t-test
Five sets of plants grown for 26±3days (before flowering with SigmaPlot 11. Multiple linear regressions were done with
stage). Each set was grown in a separate batch. Four plants Minitab15.
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Uroicetal. Arsenateimpactonxylemsap
All chromatograms obtained by RP-HPLC–ESI–MS after sap This separation of the 710μgkg−1 samples is also visible in
analysisinpositivemodewereconvertedintobins(binsize1m/z thesapflowresults.Thethree710μgkg−1 samplesinthegroup
and 1min) with ProfileAnalysis 1.0 (Bruker). These bins were with the 1400μgkg−1 samples are from one pot where all four
importedintoPyChem3.05g(Jarvisetal.,2006)forPCA.Briefly, samplesexudedlittlexylemsapduringextractionwhichindicates
chromatograms obtained by theAgilent MSD XCT system were highimpactofAsVstress(seeFigureA4inappendix).Theother
preprocessedbyProfileAnalysisintoamatrixinwhicheachcol- eight710μgkg−1 sampleswerefromplantsexudingamountsof
umnrepresentsasampleandeachrow1m/z/timebin.Thespec- sap similar to the control plants showing low AsV impact. This
ificationswere1mintimeand1m/z massperbin.Therangewas howevercouldbetheeffectofthereducedsapflowandsimplya
definedfrom0to35minandfrom80to1000m/z.Anaverageof concentrationeffect.Thereforeindividualmetabolitesneedtobe
allintensitiesineachbinwasused.ProfileAnalysiscreatedamatrix studied.ThisisachievedbyanalyzingthePCAloadingplot.This
with32,810bins(rows)and32samples(columns)whichhavea plotshowswhichbins(m/zvaluesatrespectiveretentiontime)are
totalof1,049,920datapoints.ThematrixwasexportedasASCII responsiblefortheclusteringinthescoresplot;inthiscasewhich
fileandusedforPCAanalysiswiththestatisticalsoftwarepackage m/z value at what retention time. The loadings plot (FigureA5
PyChem.InPyChem,nodatapretreatmentwasperformed.PCA in appendix) reveals that the m/z values of 132, 203, 219, 253,
wasdoneonrawspectrabycalculating10principalcomponents and312aresignificantlydifferentwithinthe32samples.Forver-
(PC). ificationpurposeallchromatographicpeaksresultingfromthese
m/z values in all chromatograms were integrated. The obtained
IDENTIFICATIONOFXYLEMCONSTITUENTS peakareaswereusedfortestingthestatisticalsignificancebetween
SeparationwasperformedonaC reverse-phasecolumn(C18, eachconditiongroup.Furthermore,peakareasofarandomm/z
18
EclipseXDB-C18,Agilent,AgilentUSA),usinga0.1%formicacid valuewhichwasnotidentifiedinthePCAloadingsplotwerealso
inwater–0.1%formicacidinmethanolgradientwith1mL/min integrated(m/z 233).Thiswasdonetoassessifthedecreasedsap
flow.Startingfrom100%water,methanolwasaddedtotheeluent flow,asdiscussedbefore,hasamajorinfluence.Thisrandomvalue
after7min.From7to25minthemethanolfractionwasincreased (m/z 233)waschosenbecauseitspeakareawaswithinthepeak
to 100%. The eluent was held at 100% methanol for a further areaandretentiontimerangeoftheother,significantlydifferent
5min,followedbyre-equilibrationofthecolumn.Totalchromato- peaks.Figure1showsboxplotsofpeakareasofallm/z valuesof
graphicruntimewas40min.Hundredmicroliterof thesample allthreeconditions.
wasused.TheeluentflowwasinjectedintoahighresolutionESI– Theplotshowsthatm/z 132isdown-regulatedwithincreased
MS (LTQ Orbitrap Discovery; Thermo Fisher Scientific) with a AsV exposurewhilem/z 219,253,312,and203areup-regulated.
resolutionof30,000.Accuratemassesofselectedm/z valueswere Thereisnosignificantdifferencebetweencontrolandchallenged
usedforchemicalformulagenerationwithX-calibur2.0(Thermo plantswithm/z 233.Allboxplotsofthem/z valuesof132,219,
FisherScientific). 253,312,and203showthatthereisalwaysastatisticalsignificant
difference with P<0.01 between the control and 1400μgkg−1
RESULTS samplesandwithoneexceptionalsobetweenthecontrolandthe
ARSENATEIMPACTONTHEMETABOLITEPROFILEOFXYLEMSAP 710μgkg−1 samples.Theexceptionisatm/z 253,whichhasno
Xylem sap collected from plant set A was used for determina- significant difference between control and 710μgkg−1 samples.
tion of the metabolite profile. One plant of the 12 challenged There is no statistical difference between both the control and
with 710μgkg−1 AsV and 3 plants of the 12 challenged with the710μgkg−1 samplesandcontrolandthe1400μgkg−1 sam-
1400μgkg−1AsVdidnotproduceenoughsapforanalysis,there- ples at m/z 233, which shows that the reduced sap flow is not
fore32insteadof36samplesweremeasuredwithRP-HPLC–ESI– thereasonforthestatisticalsignificanceoftheotherm/z values.
MS.Totalionchromatogramsofall32sapsamplesareshownin With the peak areas of m/z 132,219,253,312,and 203 and the
FigureA1inappendix.Thechromatogramshaveagoodretention resultsforsapflowandtotalarsenicconcentration,multiplelinear
timealignment,ascanbeseenatthepeaksofthepositivemode regressionsweredone.Them/z valuesof132,312,and203have
chromatograms at ∼10.5 and 23min, respectively. This enables asignificantinfluenceontheresult,withP<0.05(0.022,0.002,
PCAwithoutfurtherpeakalignmentofthemetaboliteprofileof and<0.001,respectively),whileform/z219and253theP-values
thexylemsap.PC1andPC2combinedexplain86%ofthevaria- were>0.05(0.791and0.726,respectively).Hence,thefollowing
tion[PC1(76%ofvariation)vsPC2(10%ofvariation)],therefore equationcanbeusedtocalculatethetotalarsenicconcentration
onlythesetwoPCswereusedforfurtheranalysis. inxylemsapfromtheamountofthethreebio-molecules:
The PCA scores plot (Figure A2 in appendix) indicates that
thereisaseparationofxylemsapsampledfromplantschallenged cmodel(Assap)=10.502−(0.0000946∗m/z132)
withAsVandwithoutchallenge.However,inordertoassessthesta- − (0.000410∗m/z312)+(0.00202∗m/z203)
tisticalsignificance,aclusterobservationwasdone(seeFigureA3
inappendix).ThesamplesinthePCAscoresplotarecolorcoded Plotting the modeled As concentration of this equation vs the
accordingtotheirclusteringinthedendrogram,showingthat8 actual measured concentration (see Figure 2A) shows that the
of the 710μgkg−1 and 1 of the 1400μgkg−1 samples are clus- resultingcurvehasagoodcorrelation(R2=0.71)consideringthe
tered together with the unchallenged plants whereas 4 of the biologicalvariability.
710μgkg−1 challenged plants are clustered with the remaining Furthermore, multiple linear regressions were used to assess
1400μgkg−1challengedsamples. possiblebiomarkersforsapflow,usingthepeakareasofm/z 132,
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Uroicetal. Arsenateimpactonxylemsap
FIGURE1|Boxplotsofpeakareasofpeaksofm/zvalues132, arsenic,710and1400μgkg−1arsenate.Statisticalsignificance
219,253,312,203,and233.Peakareaswereobtainedfrom accordingto:P>0.05(ns),*P=0.01–0.05,**P=0.001–0.01,
chromatogramsofcucumbersapsampleschallengedwithno ***P<0.001(n=12).
219,253,312,and203andthesapflow.Inthiscase,onlym/z253 stressandismostabundantinxylemsapof controlplants.The
and 203 have a significant influence on the result, or a P-value extracted ion chromatogram shows a distinct peak at 4.5min
≤0.05whichleadstothefollowingequation(seeFigure2B): (FigureA6inappendix).TheaccurateMSforthispeakandthe
fragments of the MS2 are presented in Figure A7 in appendix
Sapflow=3.464−(0.0000218*m/z253)−(0.0000851*m/z203)
andapossiblechemicalformulaisshowninTableA1inappen-
dix.Forthedeterminationof theother4m/z species(203,219,
Theresultsshowthatm/z 203and253couldbeusedaspossible
253,and312)xylemsapofplantschallengedwith∼1400μgkg−1
biomarkersforsapflowunderarsenicstress.
AsV for 24h was used (FigureA8 in appendix). All m/z ranges
Identificationofsignificantm/zvalues (species ±0.5m/z) resulted in one peak each (chromatograms
XylemsapwasmeasuredwiththeLTQOrbitrapDiscoverysystem can be found in the supplement information as well as MS
toidentifythecompoundsfoundinexperimentA). and MS2 spectra; Figures A8–A12). The accurate MS data for
Forthedeterminationofm/z 132,xylemsapofcontrolplants thebasepeakandproposedchemicalformulaearepresentedin
wasinjectedsincethiscompoundisdown-regulatedunderarsenic Table1.
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Uroicetal. Arsenateimpactonxylemsap
These formulae were then used to search for known nat- (see FigureA14 for MS and MS2 of isoleucine) and the match-
ural products in a database1. Only m/z 132 was unequivocally ing MS2 are robust proof that the unknown compound at m/z
identifiedasisoleucine. 132 is isoleucine, which is down-regulated under AsV stress in
Aspikingexperimentwithisoleucinestandardshowedthatthe cucumbers.
unknown peak is increasing with added isoleucine (FigureA13
in appendix). Furthermore, the MS2 of this m/z value is also IMPACTOFARSENICSTRESSONSAPFLOW
matching. The co-elution, matching accurate mass for [M-H]+ Plants of set (A) challenged with increasing AsV concentration
showedreducedsapflow.Thiswasfurtherinvestigatedwithtwo
experiments (plant set B and C) which differ in age (before
1DictionaryofNaturalProductsonDVD,ver.19.1,Taylor&FrancisGroup,2010. flowering and during flowering) using the same AsV and DMA
concentrations.Xylemsapwascollectedfor1hafterdecapitation
of each plant. Sap extracted by exudation was clear and with-
out color. Figure 3 shows sap flows of plant set B and C after
challenge with AsV at 100, 500, and 1000μgkg−1 and DMA at
1000μgkg−1,respectivelycomparedtosapflowofunchallenged
plants.
Theplantsof setB(challengedbeforeflowering)showedsig-
nificantly lower sap flow than those of plant set C (challenged
FIGURE2|Plotofmodeledarsenicconcentrationinsapvsmeasured
arsenicconcentrationinsap(A)andmodeledsapflowcalculatedby
multiplelinearregressionvsmeasuredsapflow(B).
Table1|Accuratemassdeterminationforallfivesignificantspeciesin
xylemsapafterarsenatechallenge.
Species Accurate Retention Proposedchemicalformulae
mass[M-H+] time[min] andmeasurederror(Δppm)
FIGURE3|SapflowofdecapitatedcucumbersofplantsetB
132 132.1018 4.5 C6H14NO2 (−0.872) (Figure3A)andC(Figure3B)afterchallengeofplantswithAsVat100,
203 203.1390 3.4 C7H17N5O2 (+3.267) 500,and1000μgkg−1andDMAat1000μgkg−1for24h.Sapwas
219 219.0970 2.4 C8H15N2O5 (−2.136) caostlleercisteksdiffoarp1phli.cSabtaletisatciccaolrdsiignngifitoc:a*nPce=a0g.a0i1n–s0t.c0o5n,t*ro*lPis=in0d.0ic0a1t–e0d.0b1y,
253 253.1180 11.6 C12H17N2O4 (+3.434) ***P<0.001(t-test;n=4).
312 312.1286 4.6 C11H22NO9 (−1.658)
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Uroicetal. Arsenateimpactonxylemsap
during flowering). Unchallenged plants produce the highest sap WhenplantswerechallengedwithDMA,theytransportedonly
flowwithinonesetofplants.PlantschallengedwithAsV showa DMA in the sap, no other species was detected. When plants
significantdecreaseinsapflowwithincreasedarsenicconcentra- wereexposedto100μgkg−1 AsV,bothinorganicarsenicspecies
tion.Thisaccountsbothforplantswhichhavereachedflowering appearedinthexylemsapof olderplants.AsIII,AsV,andMMA
stageandbeforereachingfloweringstage.HighAsV challengeof werefoundwhenplantswereexposedtoMMAwithMMAbeing
1000μgkg−1AsVinthenutrientsolutionleadsto96%reduction themainfraction(Yeetal.,2010).
of xylemsapproduction.PlantschallengedwithDMAshowno Themaindifferencebetweenyoungerplantsandplantsinthe
significantchangeinsapproduction. flowering stage is the increased AsV and AsIII concentration in
Inordertoassessacorrelationbetweeninorganicarsenicstress xylemsapofolderplants.Youngerplantsseemtobeabletocon-
(i.e., inorganic arsenic concentration in nutrient solution) and trolthexylemloadingofAsV.At500and1000μgkg−1exposure
sap flow, arsenic concentration was determined in all nutrient toAsVonlyAsIIIwasdetectedinthesap,leadingtotheassumption
solutions prior to plant challenge. Figure 4 shows sap flow of thatallAsVwasreducedtoAsIIIinrootsbeforexylemsaploading.
cucumberplantssubjectedtoAsVwithsapflowofcontrolplants Arsenicspeciationandconcentrationaswellasarsenicamount
setto100%.Incontrasttothegraphsthesapflowisplottedagainst inplantsetAshowamainfractionofAsIIIwithasmallerfraction
themeasuredAsVconcentrationinthenutrientsolutionpriorto ofAsV.DMAVwasdetectedintraces(seeFigureA17inappendix).
plant treatment. Both slopes show a linear correlation, with R2
of 0.98forsetBand0.89forsetC.Theslopeof bothgraphsis IMPACTOFARSENICSTRESSONABSOLUTEAMOUNTOFARSENIC
similarandnotsignificantlydifferent,whennormalizedtocon- TRANSPORTEDINXYLEMSAP
trolplants.Hence,thecombinedslopeis−8.8withanR2of0.89 Inordertocalculatetheabsoluteamountofarsenictransportedin
(P<0.001). 1hthesapflowismultipliedwiththetotalarsenicconcentration.
Thegraphshowsthatalthoughtheabsolutesapflowisdiffer- Thefollowingequationwasused:
ingbyafactorof8–10betweenthetwosets,therelativesapflow (cid:2) (cid:3) (cid:2) (cid:3)
reductionissimilarinbothplantsets.TheimpactofAsV onthe Arsenic transported ngh−1 =Sapflow gh−1
(cid:2) (cid:3)
sapflowisindependentoftheplantsdevelopmentalstage. ×Arsenicconcentration ngg−1
Thisisthefirsttimethatalinearcorrelationisobservedbetween
thearsenicconcentrationplantsaresubjectedtoandthesapflow. Withthisformulatheactualamountofarsenictransportedinthe
xylemwithin1hiscalculated.
ARSENICCONCENTRATIONANDSPECIATIONINXYLEMSAP Figure5showstheamountofarsenicinsapsamplesofplant
Sap samples extracted from plants grown hydroponically and setBandC.Whenplantsarechallengedwith1000μgkg−1DMA,
extracted via exudation were injected for arsenic determina- thetotalarsenicamountishighestinbothplantsets.Incontrast,
tion and speciation into anion-exchange HPLC–ICP-MS. Chro- the amount of arsenic transported is significantly smaller when
matogramsofxylemsapofplantsetBandCcanbefoundinthe plantsaresubjectedto1000μgkg−1AsV.
supplementinformationandshowonlytheoccurrenceof DMA Inyoungerplants(plantsetB),theamountoftotalarsenicin
andtheinorganicarsenicspecies(FiguresA15andA16inappen- xylemsapissignificantlyreducedwithincreasingAsVconcentra-
dices). Table2 shows the concentration of those arsenic species tioninthenutrientsolution.
found in xylem sap after arsenic challenge with AsV and DMA, Older plants (plant set C) show a different pattern,with the
respectively. plants challenged with ∼500μgkg−1 AsV having the highest
arsenic amount with 46±30ngh−1.Arsenic amounts of plants
challengedwith∼100and∼1000μgkg−1arelower,with5.6±1.4
and14.0±1.7ngh−1,respectively.However,thereisnostatistical
significantdifferencebetweenthearsenicamountsofplantschal-
lengedwithinorganicarsenic(P>0.05).Incomparisontoplants
challengedwithDMA,thereisadifferencetoallAsVcontaminated
plants(P<0.05).
Youngerplantsbeforethefloweringstageinthisstudywereable
torestrictarsenicloadingintothexylemathighAsV concentra-
tionsinthenutrientsolution.Thisandthereducedsapfloware
leadingtoasignificantlyreducedarsenicamountcomparedtolow
AsVexposure.
In contrast, older plants are not able to restrict AsV loading
intothexylemeffectivelywhentreatedwithincreasingAsV con-
centration.Eventhereducedsapflowcannotcompensateforthe
increasedarsenicconcentrationinthesap,leadingtostatistically
similar arsenic amounts at different concentrations. High AsV
FIGURE4|Xylemsapflownormalizedagainstsapofcontrolplants concentrations in the nutrient solution and low phosphate sta-
flowvsarsenateconcentrationinnutrientsolutionbeforechallenge.
tus will lead to highly efficient uptake of As into the root cells.
Sapflowofcontrolplantssetto100%.
Thisresultinghighinfluxmaysaturatethearsenatereductase,an
FrontiersinPhysiology|PlantPhysiology April2012|Volume3|Article55|6
Uroicetal. Arsenateimpactonxylemsap
Table2|ArsenicconcentrationandspeciationofsapsamplesofplantsetBandplantsetCafterchallengewith1000μgkg−1DMA,100,500,
and1000μgkg−1AsVfor24h.
Arsenic c(As)innutrientsol1(μg/kg) c(DMA)2insap(μg/kg) c(MMA)2insap(μg/kg) c(AsIII)insap(μg/kg) c(AsV)insap(μg/kg)
species
BDMA 1117 97.3±15.5 n.d. n.d. n.d.
BAsV 66 n.d. n.d. 3.8±2.4 13.5±5.8
567 n.d. n.d. 1.5±0.4 n.d.
909 Traces n.d. 9.9±4.6 n.d.
CDMA 759 60.3±3.6 n.d. n.d. n.d.
CAsV 125 n.d. n.d. 2.7±0.1 1.0±0.1
554 n.d. n.d. 13.2±0.6 23.4±8.6
759 Traces n.d. 23.2±4.8 47.7±1.1
(n=4).traces:belowLOQof1.5μg/kg(10σ),n.d.:belowLODof0.5μg/kg(3σ).SelectedHPLC–ICP-MSchromatogramsareshowninFiguresA15andA16in
appendicesinthesupplement.
1Beforetreatment;2expressedasμgAs/kg.
betweentheseredoxspecies.WhileAsIII isefficientlycomplexed
(Raabetal.,2005;Bluemleinetal.,2008)withphytochelatins,this
isresultinginhigherAsVloadingintothexylem.
Plants treated with high concentrations of DMA do neither
showadecreasedarsenicamountnoradecreasedsapflow.This
lack of response indicates the non-existence of toxicity or the
absenceofaspecificdefenseresponsebytheplant.
DISCUSSION
Thismetabolitestudyisthefirstavailablewhichisfocusedonthe
impactofarsenicstressontheorganiccompositionofxylemsap.
Sofar,onlyiron-deficientplantswereusedfortargetedmetabolite
analysis. The concentration of organic anions in xylem sap was
monitorediniron-deficientLycopersiconesculentumL.(tomato),
whichresultedinasignificantchangeoftheorganicanioncompo-
sition(López-Millánetal.,2009).Thiseffectwasalsomonitored
inxylemsapofiron-deficientBetavulgaris(sugarbeet;Larbietal.,
2010).
In this study, fivem/z values were identified as being up- or
down-regulatedunderarsenatestress,correspondingtofivepeaks
intheLC-MSchromatogramsandthereforetofivecompounds.
Fourofthecompoundswereup-regulated,onedown-regulated.
Thisup-ordown-regulationwasnotduetothereducedsapflow
inducedbyarsenatestressasshownbytheintegrationof aran-
domm/zvalue(m/z233)whichwasunaffectedbyAsVtreatment.
Threeofthesem/z valuescanbeusedaspotentialbiomarkersfor
eithertotalarsenicconcentrationorsapflow.
Identificationofthedown-regulatedcompoundwasachieved
forisoleucine(m/z 132).Thedatafromthemetaboliteprofiling
study and the identification part show that under arsenic stress
isoleucineisdown-regulatedinthexylemsapofcucumbers.
FIGURE5|Arsenicamountofsapsamplesofdecapitatedcucumbers So far, isoleucine has not been found to be involved in the
inexperimentB(Figure5A)andC(Figure5B)afterchallengeofplants plantresponsetowardarsenic.InArabidopsisthaliana,isoleucine
withnoarsenic,DMAat1000μgkg−1andAsVat100,500,and is part of the γ-glutamyl-cycle along with all other 20 α-amino
1000μgkg−1for24h.Sapwascollectedfor1h(n=4).Statisticalsimilarity
inonegraphisindicatedbyletterabovebar. acids. This cycle is thought to be used by plants to transport
aminoacidinter-andintracellularsincetheγ-glutamyl-enzyme
ispartof thecellmembrane.Interestingly,thiscycleisbasedon
enzymewhichreducesAsVtoAsIII.Theeffectmightbesmallbut thetransformationofglutathione,whichisalsotheinitialbioth-
aspecies-specifictransportintothexylemmaychangetheratio iolinthephytochelatinsynthesis(leadingtoAsIII-PCcomplexes).
www.frontiersin.org April2012|Volume3|Article55|7
Uroicetal. Arsenateimpactonxylemsap
Furthermore, the genes encoding the enzymes that convert the nutrientsolutionislinearforyoungerplants,whichhavenotyet
aminoacid(e.g.,isoleucine)andglutathioneintoa5-l-glutamyl- reachedfloweringstageandolderplantswhichhavereachedflow-
l-aminoacidand l-cysteineglycinearecloselyrelatedtothePC eringstage.ThismightbeanotherreasonforAsVtoxicitybecause
encodinggenes(Ohkama-Ohtsuetal.,2009;Destroetal.,2011).It reducedsapflowcouldleadtolessnutrientsandwaterintakeand
mightbethatisoleucineisinvolvedintheglutathioneproduction thereforetolowerbiomassproduction.However,itisyetunclear
andisthereforeconsumedunderarsenicstress. whatmechanismsareinvolvedinlowersapproduction.
Furthermore, isoleucine is a substrate for a plant stress hor- Thedecreasedsapflowleadsfurthermoretoadecreasedarsenic
mone.InArabidopsisthaliana,(−)-jasmonoyl-l-isoleucineissyn- amountwithintheplant.Evenwithincreasedtotalarseniccon-
thesized from jasmonic acid and l-isoleucine when the plant centrationinthexylem,thedecreasedsapflowisthesignificant
is attacked by an herbivore, wounded by mechanical means or factor.Itisinterestingthatthehighestsapamountmonitoredin
infected by a pathogen (Svoboda and Boland, 2010; Sato et al., plantsetAwasatthelowestconcentrationinthenutrientsolu-
2011). It might be thatAsV contamination is also causing stress tion.Theremightbeathresholdconcentrationwhichtriggersthe
in the roots of cucumbers leading to an increased formation of reductionofsapflow.
(−)-jasmonoyl-l-isoleucineandadepletionofl-isoleucineinthe ThedecreaseinarsenicamountunderAsVstresscomparedto
rootsandsubsequentlyinthesap. methylatedarseniccouldbeduetotworeasons:
Furthermore, isoleucine along with valine and leucine was
decreasedinratplasmaandredbloodcellswhenratswerefedwith • the plants have a mechanism to shut down sap production
AsV(Kilroe-SmithandMcLoughlin,1979).Isoleucine-containing in order to avoid arsenic contamination in the xylem and
enzymeswerealsoidentifiedasplayingaroleinthearsenicmethy- throughouttheplant,or
lation in humans, which are up-regulated under arsenic stress • theconcentrationofAsV isalreadycausingtoxicitysymptoms
(Woodetal.,2006).However,nofunctionforisoleucineinxylem leadingtoadecreaseincellfunctionality.
sapunderarsenatestresshassofarbeendescribedintheliterature.
Arsenicconcentrationandspeciationinxylemsapwasfound Usually,thetotalarsenicconcentrationinleafsandfruitsarehigher
tobeconsistentwithotherstudies.PlantschallengedwithDMA inplantsgrownonhighlycontaminatedsoilornutrientsolution
didtransportonlyDMAinthexylem.PlantschallengedwithAsV (Abedinetal.,2002).However,itseemsasiftranslocationfactors
transportedbothinorganicarsenicspeciesinthexylem,whichis from roots to shoots are higher when plants are grown on less
consistent with previous studies (Pickering et al., 2000; Mihucz contaminatedsoilornutrientsolutionaspresentedlater(Caetano
et al., 2005; Xu et al., 2007). There are probably several factors etal.,2008).Theyhaveinvestigatedthemetalretentioninbelow-
governing the loading of AsV into the xylem,including theAsV groundandabovegroundbiomassofsaltmarshplantscompared
reductioncapacityoftheroots.Inthisstudy,xylemsapofplants tothemetalcompositioninthesurroundingmarsh.Forarsenic
inthefloweringstatecontainsbothinorganicarsenicspecies.The theyfoundthatplantsgrownonsoilwiththehighestAsconcen-
in vivo reduction of AsV is thought to be done by the enzyme trationhadalsothehighestAsconcentrationinthebelowground
ACR2,whichhasshowninvitro AsV reducingcapacity(Bleeker biomass. Interestingly, those plants had a significantly lower As
et al., 2006; Ellis et al., 2006). Here, plants which have reached concentrationintheabovegroundbiomassthanplantsgrownon
floweringstagehadasignificantlyhigherconcentrationofAsVin soilwithlowerAsconcentration.Thismeansthatalargereduc-
thexylemthanyoungerplants.Thisleadstotheassumptionthat tionof availablearsenicinthesoilhasonlyasmalleffectonthe
arsenic concentration in the above ground biomass. In another
• arsenatereductasewasnotabletoquantitativelyreduceAsVto studyitwasrevealedthatof13differentplantsgrownon3differ-
AsIIIinplantsatthefloweringstagewithhighxylemsapflow entsoilswithdifferentarsenicconcentrationsinthesurfacewater,
• theoverallarsenicuptakeissignificantlyhigherinolderplants. theplantsgrownontheleastcontaminatedsoilhadthehighest
arsenictranslocationfactorfromroottoshoot(Zandsalimietal.,
During sap extraction a new arsenic stress effect was observed. 2011).Thesameeffectwasobservedinarecentstudywherethe
Withincreasinginorganicarsenicstress(i.e.,increasinginorganic highesttranslocationfactorswereobservedinplantsgrownonthe
arsenic concentration in the nutrient solution) a decreased sap lowestbio-availablearsenicfractioninthreedifferentsoils(Baig
flowwasmonitored.Thiswasfirstmentionedinarecentpublica- etal.,2011).
tionwherecastorbeanschallengedwith3.75and7.5mgkg−1AsV Theseresultssuggestthehypothesisthatthereisaconcentra-
did not exudate xylem sap (Ye et al., 2010). In another recent tionthresholdatwhichplantsreactwiththeirdefensemechanisms
hydroponic study this effect was studied more closely, reveal- (phytochelatin synthesis/xylem sap reduction). However, these
ing that xylem sap exudation of cucumbers was significantly resultsraisethequestionwhetherconcentrationdataobtainedby
reducedathigharseniclevels(7.5mgkg−1)inthenutrientsolu- thisxylemsapsamplingtechniqueisvalidforinter-comparison
tion (Czech et al., 2011). Furthermore, leaf transpiration and (control–challengedplants).Thesignificantreductionofsappro-
biomassproductionweresignificantlyinhibited. ductioncouldhaveasignificantinfluenceonthearsenicloading.
Here,thiseffectwasanalyzedinmoredetail.Decreasedsapflow
with increased inorganic arsenic stress is consistent for all plant ACKNOWLEDGMENTS
experiments shown in this study. Reduced sap production was We thank Dr. Sabine Freitag for her help with PCA and criti-
notmonitoredwithhighconcentrationsofdimethylatedarsenic cally reading this manuscript. This study was funded by EPSRC
species.Thecorrelationbetweensapflowvs.AsVconcentrationin (EP/E061303).
FrontiersinPhysiology|PlantPhysiology April2012|Volume3|Article55|8
Uroicetal. Arsenateimpactonxylemsap
REFERENCES Ellis, D. R., Gumaelius, L., Indriolo, Meharg,A.A.,andJardine,L.(2003). of jasmonoyl-isoleucine conjugate.
Abedin, M. J., Cotter-Howells, J., and E.,Pickering,I.J.,Banks,J.A.,and Arsenite transport into paddy rice Phytochemistry71,1445–1449.
Meharg, A. A. (2002). Arsenic Salt,D.E.(2006).Anovelarsenate (Oryza sativa) roots. New Phytol. Wang, J., Zhao, F. J., Meharg, A. A.,
uptake and accumulation in rice reductasefromthearsenichyperac- 157,39–44. Raab,A.,Feldmann,J.,andMcgrath,
(OryzasativaL.)irrigatedwithcon- cumulatingfernPterisvittata.Plant Meharg, A. A., and Macnair, M. R. S.P.(2002).Mechanismsofarsenic
taminated water. Plant Soil 240, Physiol.141,1544–1554. (1992). Suppression of the high hyperaccumulationinPterisvittata.
311–319. Heikens, A., Panaullah, G. M., and affinity phosphate uptake system: Uptake kinetics, interactions with
Baig, J. A., Kazi, T. G., Shah, A. Q., Meharg, A. A. (2007). Arsenic amechanismof arsenatetolerance phosphate, and arsenic speciation.
Afridi,H.I.,Kandhro,G.A.,Khan, behaviour from groundwater and inHolcuslanatusL.J.Exp.Bot.43, PlantPhysiol.130,1552–1561.
S., Kolachi, N. F., Wadhwa, S. K., soiltocrops:impactsonagriculture 519–524. Williams,P.N.,Price,A.H.,Raab,A.,
Shah,F.,Arain,M. B.,and Jamali, and food safety. Rev. Environ. Meirer, F., Pepponi, G., Streli, C., Hossain, S. A., Feldmann, J., and
M.K.(2011).Evaluationofarsenic Contam.Toxicol.189,43–87. Wobrauschek, P., Mihucz, V. G., Meharg,A.A.(2005).Variationin
levels in grain crops samples,irri- Hossain,M.F.(2006).Arseniccontami- Zaíray, G., Czech, V., Broekaert, arsenic speciation and concentra-
gatedbytubewellandcanalwater. nationinBangladesh–anoverview. J. A. C., Fittschen, U. E. A., and tioninpaddyricerelatedtodietary
FoodChem.Toxicol.49,265–270. Agric.Ecosyst.Environ.113,1–16. Falkenberg,G. (2007). Application exposure.Environ.Sci.Technol.39,
Bleeker,P.M.,Hakvoort,H.W.J.,Bliek, Jahangir,M.,Abdel-Farid,I. B.,Choi, of synchrotron-radiation-induced 5531–5540.
M.,Souer,E.,andSchat,H.(2006). Y. H., and Verpoorte, R. (2008). TXRF-XANESforarsenicspeciation Wood, T. C., Salavagionne, O. E.,
Enhanced arsenate reduction by a Metal ion-inducing metabolite in cucumber (Cucumis sativus L.) Mukherjee, B., Wang, L., Klumpp,
CDC25-like tyrosine phosphatase accumulation in Brassica rapa. J. xylem sap. X-Ray Spectrom. 36, A. F., Thomae, B. A., Eckloff, B.
explains increased phytochelatin PlantPhysiol.165,1429–1437. 408–412. W., Schaid, D. J., Wieben, E. D.,
accumulation in arsenate-tolerant Jarvis,R.M.,Broadhurst,D.,Johnson, Mihucz, V. G., Tatár, E., Virág, I., and Weinshilboum, R. M. (2006).
Holcuslanatus.PlantJ.45,917–929. H.,O’Boyle,N.M.,andGoodacre, Cseh, E., Fodor, F., and Záray, G. Human arsenic methyltransferase
Bluemlein,K.,Raab,A.,Meharg,A.A., R. (2006). PYCHEM: a multivari- (2005).Arsenicspeciationinxylem (AS3MT) pharmacogenetics:
Charnock,J. M.,and Feldmann,J. ate analysis package for python. sap of cucumber (Cucumis sativus gene resequencing and functional
(2008).Canwetrustmassspectrom- Bioinformatics22,2565–2566. L.). Anal. Bioanal. Chem. 383, genomicsstudies.J.Biol.Chem.281,
etry for determination of arsenic Jian,F.M.,Yamaji,N.,Mitani,N.,Xu,X. 461–466. 7364–7373.
peptides in plants: comparison of Y.,Su,Y.H.,Mcgrath,S.P.,andZhao, Ohkama-Ohtsu, N., Fukuyama, K., Xu,X.Y.,Mcgrath,S.P.,andZhao,F.J.
LC-ICP-MS and LC-ES-MS/ICP- F.J.(2008).Transportersofarsenite and Oliver, D. J. (2009). Roles of (2007).Rapidreductionofarsenate
MSwithXANES/EXAFSinanalysis inriceandtheirroleinarsenicaccu- γ-glutamyl transpeptidase and in the medium mediated by plant
ofThunbergiaalata.Anal.Bioanal. mulation in rice grain. Proc. Natl. γ-glutamylcyclotransferaseinglu- roots.NewPhytol.176,590–599.
Chem.390,1739–1751. Acad.Sci.U.S.A.105,9931–9935. tathioneandglutathione-conjugate Ye, W. L., Wood, B. A., Stroud, J. L.,
Caetano,M.,Vale,C.,Cesário,R.,and Karim, M. M. (2000). Arsenic in metabolisminplants.Adv.Bot.Res. Andralojc,P.J.,Raab,A.,Mcgrath,
Fonseca, N. (2008). Evidence for groundwaterandhealthproblemsin 52,87–113. S. P., Feldmann, J., and Zhao, F.
preferential depths of metal reten- Bangladesh.WaterRes.34,304–310. Ouyang, Y. (2005). Phytoextraction: J. (2010). Arsenic speciation in
tion in roots of salt marsh plants. Kertulis,G.M.,Ma,L.Q.,Macdonald, simulatinguptakeandtranslocation phloem and xylem exudates of
Sci.TotalEnviron.390,466–474. G.E.,Chen,R.,Winefordner,J.D., ofarsenicinasoil-plantsystem.Int. castor bean. Plant Physiol. 154,
Callahan,D.L.,DeSouza,D.,Bacic,A., andCai,Y.(2005).Arsenicspecia- J.Phytoremediation7,3–17. 1505–1513.
and Roessner, U. (2009). Profiling tionandtransportinPterisvittata Pickering,I. J.,Prince,R. C.,George, Zandsalimi,S.,Karimi,N.,andKohan-
of polar metabolites in biological L.andtheeffectsonphosphorusin M.J.,Smith,R.D.,George,G.N., del, A. (2011). Arsenic in soil,
extracts using diamond hydride- thexylemsap.Environ.Exp.Bot.54, and Salt, D. E. (2000). Reduction vegetationandwaterof acontam-
based aqueous normal phase 239–247. and coordination of arsenic in inated region. Int. J. Environ. Sci.
chromatography. J. Sep. Sci. 32, Kilroe-Smith, T. A., and McLoughlin, Indianmustard.PlantPhysiol.122, Technol.8,331–338.
2273–2280. J.L.(1979).Theeffectsof sodium 1171–1177.
Carey,A.M.,Scheckel,K.G.,Lombi,E., arsenateonthefreeaminoacidlevels Raab,A.,Feldmann,J.,andMeharg,A. Conflict of Interest Statement: The
Newville,M.,Choi,Y.,Norton,G.J., inerythrocytesandplasmainrats. A. (2004). The nature of arsenic- authors declare that the research was
Charnock,J.M.,Feldmann,J.,Price, Chem.Biol.Interact.28,161–170. phytochelatin complexes in Holcus conducted in the absence of any
A. H., and Meharg, A. A. (2010). Larbi,A.,Morales,F.,Abadía,A.,and lanatus and Pteris cretica. Plant commercial or financial relationships
Grainunloadingof arsenicspecies Abadía,J. (2010). Changes in iron Physiol.134,1113–1122. thatcouldbeconstruedasapotential
inrice.PlantPhysiol.152,309–319. andorganicacidconcentrationsin Raab, A., Schat, H., Meharg, A. A., conflictofinterest.
Czech,V.,Cseh,E.,andFodor,F.(2011). xylem sap and apoplastic fluid of and Feldmann, J. (2005). Uptake,
Arsenate induces water stress. J. iron-deficient Beta vulgaris plants translocationandtransformationof Received: 18 January 2012; accepted:
PlantNutr.34,60–70. inresponsetoironresupply.J.Plant arsenate and arsenite in sunflower 27February2012;publishedonline:02
Destro,T.,Prasad,D.,Martignago,D., Physiol.167,255–260. (Helianthus annuus): formation of April2012.
LlisoBernet,I.,Trentin,A.R.,Renu, Liu, W. J., Wood, B. A., Raab, A., arsenic-phytochelatin complexes Citation:Uroic MK,SalaünP,RaabA
I. K., Ferretti, M., and Masi, A. Mcgrath,S.P.,Zhao,F.J.,andFeld- during exposure to high arsenic andFeldmannJ(2012)Arsenateimpact
(2011). Compensatory expression mann, J. (2010). Complexation of concentrations. New Phytol. 168, onthemetaboliteprofile,production,and
and substrate inducibility of γ- arsenitewithphytochelatinsreduces 551–558. arsenicloadingofxylemsapincucum-
glutamyltransferaseGGT2isoform arsenite efflux and translocation Sato, C., Aikawa, K., Sugiyama, bers(CucumissativusL.).Front.Physio.
inArabidopsisthaliana.J.Exp.Bot. fromrootstoshootsinArabidopsis. S., Nabeta, K., Masuta, C., and 3:55.doi:10.3389/fphys.2012.00055
62,805–814. PlantPhysiol.152,2211–2221. Matsuura, H. (2011). Distal ThisarticlewassubmittedtoFrontiersin
Ducruix, C., Vailhen, D., Werner, López-Millán, A. F., Morales, F., transport of exogenously applied PlantPhysiology,aspecialtyofFrontiers
E., Fievet, J. B., Bourguignon, J., Gogorcena, Y., Abadía, A., and jasmonoyl-isoleucine with wound- inPhysiology.
Tabet,J.C.,Ezan,E.,andJunot,C. Abadía, J. (2009). Metabolic ing stress. Plant Cell Physiol. 52, Copyright©2012Uroic,Salaün,Raab
(2008). Metabolomic investigation responses in iron deficient tomato 509–517. and Feldmann. This is an open-access
oftheresponseofthemodelplant plants.J.PlantPhysiol.166,375–384. Schmoger,M.E.V.,Oven,M.,andGrill, article distributed under the terms of
Arabidopsis thaliana to cadmium Ma,J.F.,Yamaji,N.,Mitani,N.,Tamai, E.(2000).Detoxificationofarsenic theCreativeCommonsAttributionNon
exposure: evaluation of data pre- K., Konishi, S., Fujiwara, T., Kat- by phytochelatins in plants. Plant CommercialLicense,whichpermitsnon-
treatment methods for further suhara,M.,andYano,M.(2007).An Physiol.122,793–801. commercialuse,distribution,andrepro-
statisticalanalyses.Chemom.Intell. effluxtransporterofsiliconinrice. Svoboda, J., and Boland, W. (2010). duction in other forums, provided the
Lab.Syst.91,67–77. Nature448,209–212. Plant defense elicitors: analogues originalauthorsandsourcearecredited.
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Uroicetal. Arsenateimpactonxylemsap
APPENDIX
FIGUREA1|RP-HPLC-ESI-MStotalionchromatogramsofall32sapsamplesfromplantsetA.
6e+7
C-4
4e+7
C-4
C-5
%) 2e+7
(10 C-4 C-5 C-3 5-4 10-410-3
2 C-5
C C-4
P 0 C-3 10-310-5
C-3 5-45-4
C-3C-5 5-35-3 51-03-5 101-03-3 10-4
10-4
5-5 5-5
55--55
-2e+7
5-3
-4e+7
-6.0e+7 -4.0e+7 -2.0e+7 0.0 2.0e+7 4.0e+7 6.0e+7 8.0e+7 1.0e+8 1.2e+8
PC1 (76 %)
FIGUREA2|PCAscoreplotofPC1vsPC2oftheHPLC-ESI-MSobtainedfromplantsetA.Graynumbers:GroupA,blacknumbers:groupBaccordingto
clusteranalysis.Lettersandnumbersarecodedas:10–3:10=arsenateconcentrationinnutrientsolution(1000μgkg−1);3=potnumber,therewerefourplants
inonepot.
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