ContentslistsavailableatSciVerseScienceDirect
Environmental
and
Experimental
Botany
j o u r n al ho m e p age :w w w . e l s e v i e r . c o m / l o c a t e / e n v e x p b o t
Copper
tolerance
strategies
involving
the
root
cell
wall
pectins
in
Silene
paradoxa
L.
Ilaria
Colzi
a,
Miluscia
Arnetoli
a,
Alessia
Gallo
a,
Saer
Doumett
b,
Massimo
Del
Bubba
b,
Sara
Pignattelli
a,
Roberto
Gabbrielli
a,
Cristina
Gonnelli
a,∗aDepartmentofEvolutionaryBiology,UniversitàdiFirenze,viaMicheli1,50121Firenze,Italy bDepartmentofChemistry,UniversitàdiFirenze,viadellaLastruccia,3,50019SestoFiorentino(FI),Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received23June2011
Receivedinrevisedform2December2011 Accepted21December2011 Keywords: Coppertolerance Cellwall Pectin Methylation Root
a
b
s
t
r
a
c
t
Newinsightswereprovidedonthefunctionofrootcellwallpectinconcentrationandmethylationdegree incoppertolerancestudyingcontrastingecotypesofSileneparadoxa.Ametallicolouscoppertolerant populationandanon-metallicoloussensitivepopulationweregrowninhydroponicsandexposedto differentCuSO4 treatmentstoevaluatecopperaccumulationinrelationtopectinconcentrationand
methylationdegreeoftherootcellwall.Inshort-termexposureexperimentsthetolerantpopulation decreasedrootcellwallpectinconcentrationandincreasedtheirmethylationdegree,whilethesensitive populationdidnotrespond.Moreover,apositivecorrelationbetweenrootpectinconcentrationandmetal accumulationinrootapoplastandsymplastwasfound.Inaddition,anegativecorrelationbetweenpectin methylationdegreeandapoplasticcopperconcentrationwerefoundtobenegativelycorrelated.In long-termexposureexperiments,thesensitivepopulationincreasedtheconcentrationofpectinswiththe samemethylationdegreeandconsequentlytheabilityofitsrootcellwalltobindthemetal.Theopposite phenomenonwasshownbythetolerantpopulation.Moreover,pectinmethylationdegreewashigher inthetolerantpopulationinrespecttothesensitiveone,possiblytolimitmetalbindingtotheroot cellwall.Therefore,inthecoppertolerantpopulationofS.paradoxathegenerationofmetal-excluding rootcellwallswassuggestedtobeoneofthefactorsconcurringtoguaranteealowapoplasticcopper accumulationandprobablyalsotolimitsymplasticcopperuptakebytherootcells.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Thecellwallissupposedtoplayaroleinthedefenseresponse ofplantstoheavymetals(Krzesłowska,2011).Infact,somepapers indicatethatthecellwallisrecognizedasoneofthemain compart-mentsforheavymetalaccumulation(e.g.,Wójciketal.,2005;Islam etal.,2007;Meyersetal.,2009)asitcancontainhighamountsof heavymetals(Konnoetal.,2005;Kopittkeetal.,2008).Sorptionof metalsinthecellwallrepresentsanimportantphysiological advan-tage, since it allows theirmetabolic inactivation (Krzesłowska, 2011).
Plantcellwallsareabletobinddivalentandtrivalentmetal cationsduetothepresenceofanumberoffunctionalgroupssuchas COOH, OHand SH(Dronnetetal.,1996;Pellouxetal.,2007). However,theessentialcapacityofthecellwallforbinding diva-lentandtrivalentmetalcationsdependsmainlyontheamountof polysaccharidesrichincarboxylgroups.Thesepolymersarethe pectins,largelyrepresentedbyhomogalacturonans(Dronnetetal.,
∗ Correspondingauthor.Tel.:+390552757384;fax:+39055282358. E-mailaddress:cristina.gonnelli@unifi.it(C.Gonnelli).
1996;Fritz,2007;Pellouxetal.,2007;CaffallandMohnen,2009). Theyaresynthesizedandmethyl-esterifiedintheGolgiapparatus andtransportedintothecellwallwheretheycanbeenzymatically demethylated(Krzesłowska,2011).Demethylationof homogalac-turonansleadstotheformationofpectinswithdifferentdegreesof methyl-esterificationandthusdifferentamountsoffreecarboxyl groups, abletobinddivalentand trivalentmetalions(Dronnet et al.,1996;Fritz, 2007).Therefore, themethyl-esterificationof carboxylicgroupsoftheunitsofgalacturonicaciddeterminesthe negativechargecarriedbypectinandeventuallythequantityof metalitcanbind(Sattelmacher,2001).Infact,forpectinsa nega-tiverelationshipbetweenmethylationdegreeandbindingcapacity ofsomemetals,suchaslead(Khotimchenkoetal.,2007)and alu-minum(Schmohletal.,2000;Mimmoetal.,2009),hasbeenfound. Anyway,adirectcorrelationbetweenrootpectinconcentrationand methylationdegreeandmetalaccumulationinrootapoplastand symplastinplantaisstilllacking.
Theincreaseintheamountofpectins,especiallyinthosehaving alowdegreeofmethyl-esterification,hasbeenregardedasa symp-tomofthedefensestrategyandplantadaptationtoelevatedlevels ofheavymetalsinthesoil(Krzesłowska,2011).Resultsofseveral studieshaveprovidedevidencethatplants,inresponsetotheheavy 0098-8472/$–seefrontmatter © 2011 Elsevier B.V. All rights reserved.
metalstress,willincreasetheircellwallcapacityforelement accu-mulationbyelevatingtheamountofpolysaccharides,especially pectins,whichbindheavymetalions(Krzesłowska,2011).
Thepectinresponsetoheavymetaltreatmenthasbeen identi-fiedas“wise”(Krzesłowska,2011),butitisstillunclearifitcan actuallyaccount for naturally evolvedplanttolerance toheavy metals.Infact,asfarasourknowledgeisconcerned,suchkindof studieshavebeenconductedmainlyonselectedcultivarsofcrop plants.Forexample,anincreaseinpectins,especiallyinthosewith alow degreeof methyl-esterification,wasfoundinresponseto leadinAlliumceparootcells(Wierzbicka,1998),tocadmiumin Linumusitatissimumhypocotyls(Douchicheetal.,2007),inSalix viminalis,(Vollenweideret al.,2006)andin Oryzasativa(Xiong etal., 2009).Withregards totheresponse toaluminum, some evidenceis in accordance andsome otherin contrastwiththe above-mentionedresults. In fact,it wasfoundthat Al-resistant cultivarsofCynodondactylonaccumulatedmorealuminumincell walls than the aluminum-sensitive cultivars (Ramgareeb et al., 2004). An aluminum induced increase in pectins wasdetected inrootsof Cucurbitamaxima (LeVanetal.,1994), Triticum aes-tivum(Tabuchi andMatsumoto,2001;Hossainetal.,2006), Zea mays(SchmohlandHorst,2000;Schmohletal.,2000)andSolanum tuberosum(Schmohlet al.,2000).On theother hand,themost recentstudieshaveshownthatanincreaseinthelevelofpectins withalowdegreeofmethyl-esterificationandbindingAl3+within thecellwallaremorecharacteristicofsensitiveplantcultivarsthan thosethataretoleranttothismetal(Etichaetal.,2005;Amenós etal.,2009;Tolráetal.,2009).
Theresultsoftheabove-mentionedstudieshaveprovided evi-dencethat,inresponsetoheavymetalstress,plantsincreasetheir cellwallcapacityforheavymetalaccumulationbyelevatingthe amountofpolysaccharides,especiallypectins,whichbindheavy metalions.Hence,theavoidanceofmetalionaccumulationinthe cellwallasdescribedforsomeAl-tolerantplantsseemstobemore anexceptionthanarule.
Asforcopper,suchkindofstudiesaremainlyconcernedwith somespeciesofbryophytesandpteridophytesabletoaccumulate thismetal.Itwasrecentlyshownthatcellwallpectinshavean importantroleinCuaccumulationinthefernLygodiumjaponicum (Konno etal.,2005)and theso-called copper-mossScopelophila cataractae (Konno et al., 2010), which accumulate the highest amountofCuintheircellwallpectins.Studiesconcerning copper-inducedremodelingofpolysaccharidesasahigherplantdefense strategytothismetalarelacking.
Recently, we investigated the role of the cell wall in the phenomenon of naturally evolved copper tolerance studying metallicolousand non-metallicolous populationsof Silene para-doxaL.(Colzietal.,2011).Thecoppertolerantpopulationofthis specieswasfoundtorealizeavoidanceatboththesymplastandthe apoplastlevelintheroots,involvingalowmetalbindingcapacity oftherootcellwall.Therefore,thehypothesisthatahigh bind-ingcapacity ofcellwalls mayacttopreventtheentryof toxic metalsintoplantmetabolismconsequentlyconferringtolerance (Ernstetal.,1992)wasnotconfirmed.Ontheotherhand,thecell wallhypothesiswasfromalongtime(seeforexampleVerkleijand Schat,1989)putindoubtbysomeevidenceofalackofcorrelation betweenmetalresistanceandmetalbindingcapacityofcellwall material.Asforapossiblemetalinducedremodelingofthecellwall pectins,ourdata(Colzietal.,2011)suggestedthatinthetolerant populationshort-termcoppertreatmentscoulddecreaserootcell wallpectinconcentrationandincreasepectinmethylationdegree. Therefore,alowcellwallabilitytobindcopperwasproposedto concurinthegenerationofthetolerant/excluderphenotypeofthe S.paradoxacoppertolerantpopulation.
InthisstudywecomparedtwopopulationsofS.paradoxa col-lectedfromacopper-enrichedminesoilandanuncontaminated
one,respectively,evaluating iftherecanbea directcorrelation betweenrootpectinconcentrationandmethylation degreeand metalaccumulationinrootapoplastandsymplast.Furthermore, weinvestigatedifinthetwopopulationslongtermcopper expo-surecandifferentlyaffectthedevelopmentoftherootcellwallin termsofpectinconcentrationandtheirmethylationdegree.
2. Materialsandmethods
2.1. Plantmaterialandexperimentalconditions
Seedsof S.paradoxaL.werecollectedattheFeniceCapanne (FC)mine wasteand at ColleValD’Elsa(CVD) uncontaminated soil (Arnetoli, 2004). Seeds were sown in peat soil and after six weeks seedlings of both populations were transferred to hydroponic culture, in 1-l polyethylene pots (three plants per pot)containingamodifiedhalf-strengthHogland’ssolution com-posedof3mMKNO3,2mMCa(NO3)2,1mMNH4H2PO4,0.50mM MgSO4, 20M Fe(Na)-EDTA, 1M KCl, 25M H3BO3, 2M MnSO4,2MZnSO4,0.1MCuSO4and0.1M(NH4)6Mo7O24in milliQ-water(Millipore,Billerica,MA,USA)bufferedwith2mM 2-morpholinoethanesulfonicacid(MES),pH5.5,adjustedwithKOH (Hoagland and Arnon, 1950). Nutrient solutions were renewed weekly and plants weregrown in a growth chamber (24/16◦C day/night;lightintensity75Em−2s−1,12hd−1;relative humid-ity60–65%).
2.2. Short-termexposuretestwithexcisedroots
Plantsweregrowninhydroponiccultureforoneandfortwo months before the copper treatment. After the period of pre-culture, the intact plants were transferred to a pre-treatment solutioncontaining10mMCaCl2and5mMMESwithpHadjusted to5.5,asindicatedbyIrtelliandNavari-Izzo(2008).30minlater, therootsofS.paradoxaplantletswereexcisedandincubatedfor 30mininatestsolutioncontainingcopper(CuSO4)at0and1mM concentrationsand5mMMESadjustedtopH5.5.Attheendof incubationin testsolutions, sampleswerewashedwith milliQ-water and halfof them were washed in ice-cold (4◦C) PbNO3 (10mM)for30mintoremovetheadsorbedcopperfromtheroot cellwall.
Therootmaterial wasusedfor thedetermination of copper concentrationandforthedeterminationoftotalpectinsandthe correspondingmethylationdegree.
2.3. Long-termexposuretest
Sixweeksoldseedlingsofthetwopopulationsweretransferred tohydroponicculturecontaininganutrientbackgroundsolution (modifiedhalf-strengthHogland’ssolution,describedabove)and aseriesof copper(CuSO4)concentrations(0,5,10 and15M). Plantswerecultivatedinagrowthchamber(24/16◦Cday/night; lightintensity75Em−2s−1,12hd−1;relativehumidity60–65%) fortwomonths,renewingthegrowthsolutionsweekly.
Attheendofincubationintestsolutions,therootlengthwas measured. Some of theroots were excisedand underwent the procedureforthedeterminationofpectinsandtheirmethylation degree.Theotherplantsampleswereusedforthedetermination ofcopperaccumulationinrootsandshoots;halfofrootsamples weredesorbedwithice-cold(4◦C)PbNO3(10mM)for30min. 2.4. Determinationofcopperconcentration
Copper concentrations were determined by digesting oven-driedplantmaterialina5–2(v/v)mixtureofHNO3(Romil,69%) andHClO4(Applichem,70%)in25mlbeakersat120–200◦C,after
whichthevolumewasadjustedto10mlwithmilliQ-water. Cop-perwasdeterminedonanatomicabsorptionspectrophotometer modelAnalyst200(PerkinElmer,Waltham,Massachusetts,USA). Apoplastic copperconcentrationin roots was calculated asthe differencebetweencopperconcentrationinnon-desorbedand des-orbedsamples.
2.5. Pectindetermination
Attheendofincubationintestsolutions,theexcisedrootswere rinsedwithmilliQ-waterandtransferredintoa10mlcentrifuge tube.Absoluteethanol(5ml)wasaddedintothetubesandthe samplesweremixedwithavortexmixerfor5minandcentrifuged at15,000rpmfor5min.Thesupernatantwasdiscardedandthe sampleswereextractedthreemoretimeswiththesameamount ofethanol.Rootsweretransferredtopetridishanddriedfor12h at35◦Cinaconventionaloven.
Pectinextractionanddeterminationwerecarriedout accord-ingtoYuetal.(1996)withlittlemodifications.Driedrootmaterial (about10mg)wasweighedintesttubes(sixreplicatesforeach thesis),2ml-aliquots of 98%sulfuric acidand 0.5ml-aliquots of milliQ-waterwere addedand sampleswere stirredin a vortex mixerfor5min.Further0.5ml-aliquotsofmilliQ-waterwereadded dropwiseandvortexmixedfor5min.Thesampleswerefilteredon glasswoolandadjustedto10mlwithmilliQ-waterbeforeuse.
Pectin contents of extracts were analyzed by the m-hydroxydiphenyl method; 1ml of 1:10 diluted extracts was pipettedintoatest tube.6mlofsulfuricacid/tetra-borate solu-tion(0.0125Msodiumtetra-borateinconcentratedsulfuricacid) wereaddedtothesamplesinanicewaterbathandmixed care-fullywithavortexmixer.Tubeswereheatedupinawaterboiling bathfor5minandimmediatelyplacedinicewatertocool.0.1 ml-aliquotsof0.15%m-hydroxydiphenyl(Sigma–Aldrich,St.Louise, MO,USA)wereaddedandthesampleswereincubatedfor15minat roomtemperature.Thecorrespondingblankconsistedofasolution containing1mlofmilliQ-water,6mlofsulfuricacid/tetra-borate solutionand0.1mlofsodiumhydroxide0.5%.
Theabsorbanceofthesampleswasmeasuredat520nmusinga spectrometermodelDR4000,(HachCompany,Loveland,CO,USA). Galacturonicacid (Sigma–Aldrich)was used asa calibration standard(concentrationsrangedfrom10 to100g/ml)andthe roottotalpectinconcentrationwasexpressedasgalacturonicacid equivalents.
2.6. Degreeofmethylationofpectins
RootsofS.paradoxawerepreparedinthesamewayasforpectin determination, by meansof foursuccessive washes with abso-luteethanolfollowedbycentrifugeanddriedfor12hat35◦Cin aconventionaloven.Hydrolysisofmethylatedpectinsand deter-minationoftheresultingmethanolwerecarriedoutaccordingto KlavonsandBennett(1986)withlittlemodifications.About10mg ofdriedrootmaterialwereweighedintesttubes(sixreplicatesfor eachthesis),3mlofsodiumhydroxide(1.5M)wereaddedandthe solutionwasincubatedfor30minatroomtemperature.Thepectin hydrolizateswereneutralized withphosphoricacid(1M)topH 7.0±0.1andthevolumewasadjustedto10mlwithmilliQ-water. Aliquotsof1mlofthissolutionweremixedinatubewith1ml ofalcoholoxidasefromPicciapastoris(1UN/mlpreparedby dilut-ing6.8lofalcoholoxidasewith10mlofmilliQ-water)andthen incubatedat25◦Cfor15min.Afterwards,2mloffluoral-P(0.02M 2,4-pentanedionein2.0Mammoniumacetateand0.05Macetic acid)wereaddedandvortexmixed.Thetubeswereplacedinabath waterat60◦Cfor15minandthencooledatroomtemperature.
Absorbanceswere measuredat412nmagainst ablank con-taining1mlphosphatebufferatpH7and1mlofalcoholoxidase.
Methanolwasusedforthecalibrationcurve(concentrationsranged from0.5to5g/ml).
Methylationdegree(M.D.)wascalculatedwiththefollowing equation:
M.D.=100×molesmolesofoftotalmethanolpectins
Standarddeviationassociatedtothemethylationdegreewas calculatedaccordingtotheerrorpropagationrule.
2.7. Statistics
Measurementsofrootlengthwereperformedontwelve repli-cates,thedeterminationofcopperandpectinconcentrationwas madeonsixreplicates.Eachreplicatewasmeasuredthreetimes.
Statisticalanalysiswascarriedoutwithone-wayandtwo-way ANOVAusingthestatisticalprogramSPSS13.0(SPSSInc.,Chicago, IL,USA). Aposteriori comparisonof individualmeanswas per-formedusingTamhaneposthoctest.
3. Results
3.1. Short-termexposuretestwithexcisedroots
Symplasticandapoplastic copperconcentrations(Table1)in excised roots of control plants were not significantly different betweenone-andtwo-month-oldplantsbothinCVDandinFC population. Regarding the treated samples(1mM CuSO4), cop-peraccumulation,inbothsymplastandapoplast,wassignificantly higherintheyoungerplantsinbothpopulations(p<0.01).
Comparingthetwopopulationsymplasticcopper concentra-tions(Table1), nosignificantdifferenceswereobservedfor the one-month-oldroots,whileinthetwo-month-oldrootsa signifi-cantlyhighercopperaccumulationwasfoundinCVDpopulation (p<0.01).Asfor apoplasticconcentrations (Table1), thecopper concentrationswerealsosignificantlylowerinFCplantletsinboth theone-andtwo-month-oldplantexperiment(p<0.01;p<0.05).
Thepercentagesofapoplasticandsymplasticcopper concentra-tionsinexcisedcoppertreatedrootswerecalculated.CVDshowed significantly(p<0.01)higherpercentageofcopperintheapoplast ofone-month-oldplants(72.3±0.5%)inrespecttothe two-month-oldplants(58.1±1.2%),while inFCanoppositetrendhasbeen observed,beingsuchpercentagesignificantly(p<0.01)higherin the older plants (57.6±2.1% and 66.2±2% for one-month-old plantsandtwo-month-oldplantsrespectively).Betweenthetwo populations,CVDshowedsignificantly(p<0.01)higherpercentage ofapoplasticcopperintheone-month-oldplantsandsignificantly (p<0.01)lowerinthetwo-month-oldplantsinrespecttoFCplants. Total pectin concentrations, methanol concentrations and methylation degrees ofpectins arereported inTable 2.Results showedadecreaseinalltheparametersinvestigatedbetween one-andtwo-month-oldplantexperimentsinboththepopulationsand forbothcontrolandcoppertreatedplants.
Inrespecttocontrolplants,coppertreatedonesdidnotgive anysignificantvariationoftotalpectinconcentrations,methanol concentrationsandmethylationdegreesinCVDpopulation. Con-versely,inFCplantsasignificantdecreaseofpectinconcentrations was observed with copper exposure, both in one- and two-month-oldplants(p<0.01).Moreover,theFCpopulationshowed asignificantincreaseofthemethylationdegreeaftercopper treat-mentinbothone-andtwo-month-oldplantexperiments.
Bycomparingthetwopopulations,intheone-month-oldplant experiment total pectin concentrations of control plants were significantly higherin FC thanin CVDpopulation, while in the treated plants there were no significant differences (Table 2). Thetotalpectinconcentrationsintwo-month-oldplantsresulted
Table1
CopperaccumulationinexcisedrootsoftwopopulationsofS.paradoxa(CVDnon-minepopulationandFCminepopulation)aftercopper(CuSO4)exposurefor30min. Valuesaremeansofsixreplicates±standarderror,significantdifferencesbetweenthemeansappearwithdifferentletters,smallfortheintra-populationandcapitalforthe inter-populationones(*p<0.05;**p<0.01).
Treatment(CuSO4mM) SymplasticCuconcentrations(gg−1d.w.) ApoplasticCuconcentrations(gg−1d.w.)
1month 2months 1month 2months
CVD Control 17.2±0.6aA 18.8±0.7aA 1.3±0.3bA 1.6±0.7bA 1 467±14cB** 290±24dC** 1219±97eB** 403±22cB** FC Control 16.0±0.8aA 13.7±0.9aB** 1.5±0.6bA 2.8±1.3bA 1 445±39cB** 171±29dD** 605±77eC** 336±19fC* Table2
Totalpectinconcentrations,methanolconcentrationsresultingfromhydrolysisofmethylatedpectinsandthecorrespondingmethylationdegreesfoundinexcisedrootsof twopopulationsofS.paradoxa(CVDnon-minepopulationandFCminepopulation)aftercopper(CuSO4)exposurefor30min.Valuesaremeansofsixreplicates±standard
error,significantdifferencesbetweenthemeansappearwithdifferentletters,smallfortheintra-populationandcapitalfortheinter-populationones(*p<0.05;**p<0.01). Treatment(CuSO4mM) Totalpectinconcentrations
(molgalacturonicacid equivalentsg−1d.w.)
Methanolconcentrations (molCH3OHg−1d.w.)
Methylationdegrees(%)
1month 2months 1month 2months 1month 2months
CVD Control 596±44aA 376±15bA 206±35aA 52.5±3.7bA 34.9±5.2aA 14.1±1bA 1 657±39aA 425±25bA 184±25aA 60.8±4bA 28.8±4aA 14.5±0.8bA FC Control 799±38aB** 423±21bA 245±22aA 63.3±4.6bA 30.7±2.2aA 15±0.5bA 1 606±22cA 325±13dB** 223±22aA 57.6±2.6bA 36.8±1.9aB* 18±0.8cB**
significantlylowerintreatedFCplantsascomparedtotreatedCVD
ones.Nosignificantdifferenceswerefoundinmethanol
concentra-tionbetweenthetreatmentandthepopulations.Themethylation
degreesofcoppertreatedplantswerehigherinFCplantsthanin
CVDonesinboththeexperiments(one-month-old:p<0.01;
two-month-old:p<0.05).
3.2. Long-termexposuretest
Basedontherootgrowthresponse,theFCplantsexhibiteda
significantlyhighercoppertolerancethantheCVDplants(p<0.01)
(Fig. 1).In FC plants,there wasnosignificantdecrease in root
Fig.1.RootlengthoftwopopulationsofS.paradoxa(,ColleValD’Elsa non-minepopulation(CVD);,FeniceCapanneminepopulation(FC))(meanoftwelve replicates±standarderror)afterlong-termexposuretoincreasingcopper concen-trations(0,5,10,15MCuSO4)fortwomonths.
growth.Onthecontrary,intheCVDplants,alltheconcentrations producedasignificantdecreaseofrootlength(p<0.01).
Copperconcentrationsinrootsandshootsoflong-termexposed plantsareshowninTable3.Thecopperconcentrationsinroots and shoots of both populations increased proportionally with increasingcopperexposure,exceptforCVDshootsthatshowed a saturation trend. Symplastic copperconcentrations were sig-nificantly lower in FC as compared to CVD at all the external copperconcentrationsused(p<0.05),whileapoplasticcopper con-centrationswereloweronlyatthetwohighestexternal copper concentrationsused(p<0.05).
Regardingthepercentageofapoplasticcopperconcentrations in long-exposure coppertreated roots, FCshowed significantly (p<0.01) higher values at higher copper concentrations (10 CuSO4M –46.6±1.5%; 15CuSO4M–46.7±1.8%)inrespect to thelowest copper concentration(5 CuSO4M – 9.7±2.4%). Similarly,inCVDthepercentageofcopperintheapoplastwas sig-nificantly(p<0.01)lowerin thelowestcopperconcentration(5 CuSO4M–3.7±0.7%)inrespecttohighercopperconcentrations (10CuSO4M– 22.7±0.6%; 15CuSO4M–40.4±1.7%).InFC, comparingwithCVDpopulation,thepercentageofapoplastic cop-perconcentrationwassignificantlyhigherforalltheconcentrations (atleastp<0.05).
Copperconcentrationsinshootsweremuchlowerthaninroots eveniftheyincreasedwithincreasingcoppertreatments(Table3). ShootcopperconcentrationwassignificantlylowerintheFCmine populationcomparedtoCVD(p<0.05),exceptforthehighest cop-pertreatment.
Total pectin concentrations, methanol concentrations and methylationdegreesafterlong-termcopperexposuretreatments, areshowninTable4.Resultsindicatedthatcoppertreatmentsgave aninversetrendfor thetwo population.In FCplants,a signifi-cantdecreasewasobservedatthetwohighestcoppertreatments, whileinCVDpopulationasignificantincreaseoftotalpectin con-centrationswasfoundwiththetwohighestcopperconcentrations (p<0.05)inrespecttocontrol.Significantdifferenceswerefound betweenthetwopopulationsintotalpectinconcentrations,asthey
Table3
CopperaccumulationinrootsandshootsoftwopopulationsofS.paradoxa(CVDnon-minepopulationandFCminepopulation)afterlong-termexposuretoincreasingcopper concentrations(0,5,10,15MCuSO4)fortwomonths.Valuesaremeansofsixreplicates±standarderror,significantdifferences(p<0.05)betweenthemeansappearwith differentletters,capitalfortheinter-populationandsmallfortheintra-populationones.b.d.l.=belowdetectionlimit.
Treatments(CuSO4M) RootsymplasticCuconcentrations(gg−1d.w.) RootapoplasticCuconcentrations(gg−1d.w.) ShootCuconcentrations(gg−1d.w.)
CVD Control 19±1aA b.d.l. 9.9±0.3aA 5 258±34bA 10±5aA 42±2bA 10 839±56cA 246±12bA 97±7cA 15 663±17dA 449±52cA 81±3dA FC Control 26±4aA b.d.l. 7.6±0.5aA 5 121±2.5aB 13±9aA 32±2bB 10 204±8bB 178±10bB 62±3cB 15 404±78cB 354±38cB 96±5dB Table4
Totalpectinconcentrations,methanolconcentrationsresultingfromhydrolysisofmethylatedpectinsandthecorrespondingmethylationdegreesfoundinrootsoftwo populationsofS.paradoxa(CVDnon-minepopulationandFCminepopulation)afterlong-termexposuretoincreasingcopperconcentrations(0,5,10,15MCuSO4)fortwo
months.Valuesaremeansofsixreplicates±standarderror,significantdifferences(p<0.05)betweenthemeansappearwithdifferentletters,smallfortheintra-population andcapitalfortheinter-populationones.
Treatments(CuSO4M) Totalpectinconcentrations(molgalacturonic
acidequivalentsg−1d.w.)
Methanolconcentrations(molCH3OHg−1d.w.) Methylationdegrees(%)
CVD Control 392.3±24.5aA 48.4±4.3aA 12.33±0.8aA 5 469.0±34.0aA 56.5±4.7aA 12.0±0.5aA 10 512.8±62.5bA 70.1±9.4bA 13.7±0.8aA 15 605.6±74.7cA 67.4±11.2abA 11.14±1.2aA FC Control 440.1±23.1aA 71.4±8.1aB 16.2±1.6aB 5 405.4±26.9abA 62.5±5.2aA 15.4±1.3aB 10 306.8±27.6bB 60.8±8.3aA 17.7±1.2aB 15 320.2±28.5bB 58.8±5.5aA 15.9±0.8aB
werelowerinFCpopulationinrespecttoCVDoneatthetwo
high-estconcentrationused.Asformethanolconcentration(Table4),the
onlysignificantdifferencewasobservedinCVDpopulationexposed to10MCuSO4,whereitwassignificantlyhigherascomparedto thecontrolandto5MCuSO4.Themethanolconcentrationdidnot differbetweenthetwopopulations,exceptforthecontrolplants, beingFCmethanol concentrationhigherthan inCVD.Eventhe methylationdegreedidnotchangeincreasingcopper concentra-tionintheculturemedium.Comparingthetwopopulations,the methylationdegreeswerehigherinFCplantsthaninCVDplants (p<0.05).
4. Discussion
Plants with different ages were used to compare copper accumulation in roots containing different concentrations and methylationdegreeofpectins,asthelatterareinsertedintothe wallashighlymethyl-esterifiedpolymerssubsequently deesteri-fiedtovaryingdegreesduringthedevelopmentoftheplantitself (Mohnen,2008).Afterashort-termcoppertreatmentforthe eval-uationofplantresponsestoacutemetalstress(Table1),copper accumulation wassignificantly higher in one-month-old plants thanintwo-month-oldplantsinboththepopulationsandinboth apoplastandsymplast.Atthesametime,thetolerantpopulation showedalowercopperaccumulationinrespecttothesensitive one,forapoplasticcopperstatisticallysignificantinplantsofboth thetwoagesandforsymplasticcopperonlyintheolderplants. Probably,thetimeandtheconcentrationusedwerenotadequate togeneratealsoasignificantdifferenceinsymplasticcopper accu-mulationinyoungerplants.Therefore,theexcludernature,both atthesymplasticand theapoplastic levels,of thecopper toler-antpopulationofS.paradoxa (Gonnellietal.,2001; Colzietal., 2011)wasconfirmedandahighercopperaccumulationinyoung
plantsassessed.Thislatterresultcanbeduetothefactthatyoung roots can be more efficient than older roots at taking up ele-mentsfromthesolution,probablybecauseaproportionofroots becomesmoreandmoreinactiveinuptakefunctionsduring devel-opment.Consequently,therootcopperabsorptionfunctionmight varywiththemorphologyandthearchitectureoftheroot sys-tem,asalreadysuggestedbyPerrigueyetal.(2008)forcadmiumin maize.
Calculating thepercentage ratio betweencopper concentra-tion in apoplast and symplast,an interesting result came out. Inthesensitivepopulation,plantsofbothagesallocatedcopper preferentiallyintheapoplast,buttoagreaterextentin one-month-oldplants.Therefore,inthispopulation,whentherootstructure allowedahighertotalamountofcoppertobetakenup,its alloca-tionwaspreferentiallyapoplastic.Thetolerantpopulationshowed theoppositetrend:evenifthepercentageofapoplasticcopperwas alwayshigherthanthesymplasticone,theplantsthat preferen-tiallyallocatedcopperin theapoplastwerethetwo-month-old ones.Theseplantswerealsotheoneswiththelowertotalamount ofcoppertakenup.Thisresultcouldsuggestaninteresting hypoth-esis:inbothpopulationsrootdevelopedinawaythattendedto diminishcopperaccumulation,butinapopulation-dependentway. Thecoppertolerantpopulationunderwentakindofroot develop-mentdevotedtofavortheexclusionofcopperfromthesymplast, thuspreventingtheonsetofthestressasmetaltolerantplants generallyshow(Hall,2002).Inotherwords,insuchdevelopment thedecreaseof copperaccumulationwasmainlydependant on thedecreaseofthesymplasticcopperconcentration,whereasin thesensitivepopulationsuchdecreasewasmainlydependanton theapoplasticcomponent.Infact,oldertolerantplantsshowedthe actuallowestvalueofsymplasticcopper.Probably,thiscondition maybeachievedalsobythelowestcopperaccumulationdisplayed intheirapoplastandnotonlybyareducedcopperuptakeintothe
rootcells,beingthislatterstrategyalreadydemonstratedincopper tolerantS.vulgaris(VanHoffetal.,2001).
TofindifinS.paradoxatheabove-mentionedresultscouldbe correlatedtoadifferentcompositionofthecellwall,itspectin con-centrationandmethylationdegreewereevaluatedinthedifferent populations(Table2).One-month-oldplantsshowedrootpectin concentrationhigherthantwo-month-oldplants,confirmingthat thepectinconcentrationdecreasesduringcellwallandwholeplant developmentanddifferentiation(Schelleretal.,2007).Notonlythe pectinconcentrationwasdifferentbutalsothemethylationdegree. Infact,inaccordancewiththefactthatpectinsaredeposedhighly methyl-esterified and then are demethylated (Mohnen, 2008), youngplantsshowedamethanolconcentrationandamethylation degreehigherthanolderplants.Oncemore,aconsiderationona differentkindofrootcellwalldevelopmentbetweenthetwo pop-ulationscanbemade.Thecontrolplantsofthetolerantpopulation showedahigherage-dependantdecreaseinpectinconcentration (from 800to 420molgalacturonic acidequivalentsg−1d.w. in tolerantplantsandfrom600to380molgalacturonicacid equiv-alentsg−1d.w.inthesensitiveplants)andalowerage-dependant decreaseofthemethylationdegree(from30%to15%inthe toler-antpopulationandfrom35%to14%inthesensitivepopulation), thusseemingtodeveloprootswithasupposedlowercapacityof bindingmetalsduringplantgrowth.
Comparingthechangesinducedbytheshort-timecopper treat-mentbetweenthepopulations,dataconfirmedtheresultsobtained by Colzi et al. (2011). In the tolerant populationcopper treat-mentprovokedasignificantdecreaseintotalpectinconcentrations, whereas the statistically significant difference found in pectin methylationdegreebetweenthetwopopulationsdependedmainly onthepectindecreaseobservedinthetolerantpopulation,rather thanonanincreaseoftheirmethylation.Thus,inthepresenceof copper,thetolerantpopulationofS.paradoxaseemedtobeableto reducethemetalbindingsitesofthecellwalland,asaconsequence, theapoplastcopperconcentration.Infact,thestructuralandionic characteristicsoftheapoplastcanaffecttheioncompositionofthe mediumthatbathesthecellmembrane(GrignonandSentenac, 1991)andthustheamountofmetalthatentersthecell.The dif-ferencesfoundinpectincompositionandmethylationdegreethat generatedapoplastswithdifferentcationexchangecapacityofthe root(Sattelmacher,2001)couldbeoneofthefactorsthatconcurto alowercopperaccumulationalsointhesymplast.So,atleastfor short-timeexposureexperiments,inS.paradoxatheremodelingof polysaccharidesforcoppertolerancestrategydidnotseeminfavor ofanincreaseofpectinsandadecreaseofmethylationdegree,as foundforselectedcultivarsofcropplantsortheso-calledcopper mosses(asreviewedin Krzesłowska(2011))andidentified asa “wise”response.
Comparingresultsoncopperaccumulationandpectins,a pos-itive correlation can be found, that is the higher the pectin concentration,thehigher thecopperaccumulationfor boththe apoplastandthesymplast.Untilnow,adirectcorrelationbetween rootpectinconcentrationandmetalaccumulationinrootapoplast andsymplastinplantahasnotbeenfound.Correlationsbetween differentmethylation degrees and copper accumulationcan be drawn comparing only treated one-month-old plants, as they displayednotstatisticallydifferentpectinconcentration.In this condition,S.paradoxatolerantpopulationplantsshowedahigher methylationdegreeandalowerapoplasticcopperconcentration, thussuggestinganegativecorrelationbetweenthesetwo parame-tersinvivo,asitwasforexamplesuggestedbyMimmoetal.(2009) studyingaluminumsorptioninextractedpectins.
Long-termexposureexperimentswereperformedtoevaluate ifthepresence ofdifferentcopperconcentrationin theculture mediumcouldaffectdifferentlyinthetwopopulationsthe devel-opmentofrootcellwalls,intermsoftheirpectincompositionand
methylationdegree.Theconcentrationsusedinthatexperiment allowedanormalplantdevelopmentforthetolerantpopulation andanimpaireddevelopmentforthesensitiveone(Fig.1).Asfar asmetalaccumulationisconcerned(Table3),bothapoplasticand symplasticcopperconcentrationsincreasedwithincreasingmetal concentrationintheculturemedium.Onlyatthehigher concen-trationused,thesymplasticcopperaccumulationdidnotfollow thistrendinthesensitivepopulation.Thisconditioncouldjustbe theresultofcoppertoxicity,impairingeverycellularfunction,ion uptakeincluded.Comparingthepopulations,eveninthiskindof experiment,thetolerantpopulationconfirmeditsexcludernature, forbothcopperaccumulationinthesymplastandintheapoplast, inrespecttothesensitivepopulation.Thepercentageofcopper allocationinrootsymplastandapoplastwascomparedbetween thepopulationsexcludingthelowestconcentrationused,asit gen-eratedaverylowaccumulationofcopperintheapoplast,toofar fromaresultthatcanbereliablydiscussed.Resultsindicatedthat, increasingcoppermediumconcentration,thetolerantpopulation didnotchangeitsallocationpattern probablyasaconsequence ofanefficientandactiveexclusionfromboththecompartments atthesametime.Increasingcoppermediumconcentration,the sensitivepopulationtendedtoallocatethemetalpreferentiallyin theapoplast.Whateverthereasonofthesensitivepopulationwas tosignificantlyallocatemorecopperinthiscompartment,itdid notsucceedinprotectingtheplantfromcoppertoxicity. Further-more,thisbehaviorcouldalsobeoneofthecausesofthelower rootelongationdisplayedbythesensitivepopulation,as,besides inhibitingmeristematiccelldivisions,metalsboundtothecellwall contributetoitsstiffening,thusbeingoneofthemaincausesof plantgrowthinhibitionobservedunderheavymetalstress(Hall, 2002;Patraetal.,2004;Poschenriederetal.,2008).
Theexcluderphenotypewasrealizedalsoforshootcopper accu-mulationasthetolerantpopulationshowedthelowervalues.Only atthehighestconcentrationused,thesensitivepopulationshowed a lower shoot copperaccumulation. Thisresult couldprobably derivefromanimpairingoftheroottoshoottransportsystemdue toatoohighcoppertoxicity.
Thedifferencesfoundincopper-inducedchanges inrootcell wallcomposition (Table4)couldconcur to explain the above-mentioneddata.ThemostintriguingresultwasthatinthetwoS. paradoxapopulationscopper-inducedresponseswereboth oppo-siteandatdifferentdegrees,thesensitivepopulationshowingthe widestresponse,probablyjustbecauseitwasthemore copper-stressed.Particularly,thetolerantpopulationdecreasedtheroot pectinconcentration,eveniftoalowextent,whereasastatistically significantincreaseintotalpectinconcentrationinthesensitive populationwasfoundfollowingtheincreaseofcopperinthe cul-turemedium. Toourknowledge, this wasthefirst time that a similarresultwasfoundstudyingnaturallyevolvedtolerant popu-lationofanexcluderplant(fordetailedreferencesonplantcellwall responsestoheavymetalsseeforexampleKrzesłowska(2011)).
Regardingthemethanolconcentration,itincreasedinthe sen-sitivepopulation,evenifnotalwaysinasignificantwayforallthe concentrationsused.Thisincreasewasproportionaltotheincrease inthetotalpectinconcentration,sothatthemethylationdegree didnotchange.Inthetolerantpopulation,thepresenceofcopper intheculturemediumdecreasedthemethanolconcentration,even ifinanon-significantwayinrespecttothecontrol,thusnot lead-ingtoanyvariationinthemethylationdegree.Therefore,inthe presenceofincreasingcopperconcentrations,thesensitive popu-lationseemedtorespondbyincreasingtheabilityoftherootcell walltobindthemetalrisingtheconcentrationofpectinswiththe samemethylationdegree.Theoppositebehaviorwasshownbythe tolerantpopulation.Thisresponsecouldbefundamentalto guar-anteealowerapoplasticcopperconcentration,thatcouldbeone ofthefactorsconcurringalsotolimitcopperuptakebytheroot
cells.Moreover,otherimportantresultsregardedthemethylation degree,thatwasalwayshigherinthetolerantpopulationinrespect tothesensitiveone,possiblytolowertheabilityoftherootcellwall tobindcopper.Thatsituationwasfoundeveninthecontrolplants, inwhich,inaddition,alsothemethanolconcentrationwashigher, probablytoconstitutivelyallowtheformationofrootcellwalls withalowerabilitytobindanymetal.
InrespecttothepaperofKrzesłowska(2011)andtherootcell wallresponsethereproposed,withtheonlyexceptionofsomeof thestudiesonaluminum,S.paradoxatolerantplantsseemedto per-formtheoppositestrategy,thatwasrestrictingtheimmobilization ofcopperintherootcellwall.Inouropinion,boththeresponsescan be“wise”andadoptedbyplants,dependingonthelevelof toler-ancethatneedstobereached.Thetolerancelevelofmetallophytes isundoubtedlyhigherthantheoneofcropspeciesselected cul-tivars,astheformerliveinverystringentenvironmentsrealizing adaptivemetaltolerancestrategiesextremelyeffectiveinthe pro-tectionfromhighheavymetalstress.Indeed,alsocropplantscan becharacterizedbydifferentlevelsofconstitutivemetaltolerance, justhavingsomevariation intheirmetalhomeostaticnetwork. However,thisfeaturecannotbesufficienttoenablethemtocope withextremelyhighmetalconcentrationsintheenvironment,as metallophytesdo.Bothquantitativeandqualitative reasonscan beproduced:aninadequatevariationlevelofmetaltoleranceor theidentificationofaresponsethatrepresentedjustanaccidental mechanismallowingsomecultivarstoachieveadifferentlevelof tolerance.Moreover,anotherconsiderationdeservestobemade: asimpletolerancestrategy,basedonthecellwallworkinglikea “filter”immobilizingelementstoexcludethemfromthesymplast, wouldhavescarcesuccess.Althoughtherootcellwallisdirectlyin contactwithmetalsinthesoilsolution,adsorptionontothecell wallmustbeoflimitedcapacity and thushavea limitedeffect onmetalactivityatthesurfaceoftheplasmamembrane. There-fore,itishardtoexplainmetaltolerancewithsuchamechanism (Ernstetal.,1992).Moreover,arootcellwallwithahighcapacity ofmetalbindingwillbeunfavorableforgrowinginametalliferous soil,asthehighlevelofrootmetalbindingthatwouldberealized willinhibittherootgrowthitself promotingcellwallstiffening (Etichaetal.,2005;Yangetal.,2008).Therefore,thedecreasein pectinlevelsoperatedbytheminepopulationcanbereally con-sideredasoneoftheadaptationstonaturallyrealizeexclusionfor livingincoppercontaminatedenvironments.Thisassumptionisin accordancewithsomerecentresultsobtainedforaluminum(Eticha etal.,2005;Amenósetal.,2009;Tolráetal.,2009).Ontheother hand,aconfirmationofsuchhypothesiscanbeindirectlyprovided bythebehaviorofcopperhyperaccumulatorplantsthatisexactly theotherwayround,suchastheirtolerancestrategy: hyperaccu-mulatorshavehighmetallevelsintheircellwallsasreviewedin Krzesłowska(2011).
5. Conclusions
Exclusionofcoppershowedbythemetallicolouspopulationof S.paradoxaseemedtoberealizedthroughafineremodelingof someoftherootcellwallpolysaccharides.Pectinsconcentrations andtheirmethylationdegreechangedindifferentwaysinthe sen-sitiveandthetolerantpopulationsinbothshort-andlong-term exposureexperiments.Thecommonfeaturewasthatthetolerant populationalwaysseemedtogeneraterootcellwallswithalow abilityofbindingcopperthroughadecreaseofpectin concentra-tionandanincreaseoftheirmethylationdegree.Thisphysiological adjustmentconcurredtogeneratemetal-excludingrootcellwalls inthetolerantpopulationofS.paradoxa.Thisconditionresultedin aloweraccumulationofcopperintherootapoplastandprobably
wasalsooneofthefactorsconcurringtothelimitationofcopper accumulationinthesymplast.
Therefore,themetallophyterootresponsetoexcessivemetal concentrations is still far to be exhaustively elucidated and is undoubtedlyfundamentaltounravelsomephysiologicalstrategies concurringtometaltolerance. Furthermore,information onthis topiccanbeoffundamentalimportancealsotooptimizethe pos-sibleuseofmetallicolousplantsinphytoremediationtechniques.
Acknowledgments
ThisstudywasfundedbytheUniversityofFirenze(Fondidi Ateneo2008–2009)andbytheMIUR(Cofinanziamento2008).
References
Amenós,M.,Corrales,I.,Poschenrieder,C.,Illeˇs,P.,Baluˇska,F.,Barcelo,J.,2009. Dif-ferenteffectsofaluminumontheactincytoskeletonandBrefeldinA-sensitive vesiclerecyclinginrootapexcellsoftwomaizevarietiesdifferinginroot elon-gationrateandaluminumtolerance.PlantCellPhysiol.50,528–540. Arnetoli,M.,2004.Tossicitàetolleranzaall’arsenicoinduepopolazionidiSilene
paradoxaL.BachelorThesis.UniversitàdiFirenze,Italy.
Caffall,K.H.,Mohnen,D.,2009.Thestructure,function,andbiosynthesisofplantcell wallpecticpolysaccharides.Carbohydr.Res.344,1879–1900.
Colzi,I.,Doumett,S.,DelBubba,N.,Fornaini,J.,Arnetoli,M.,Gabbrielli,R.,Gonnelli, C.,2011.Ontheroleofthecellwallinthephenomenonofcoppertolerancein SileneparadoxaL.Environ.Exp.Bot.72,77–83.
Douchiche,O.,Rihouey,C.,Schaumann,A.,Driouich,A.,Morvan,C.,2007. Cadmium-inducedalterationsofthestructuralfeaturesofpectinsinflaxhypocotyl.Planta 225,1301–1312.
Dronnet,V.M.,Renard,C.M.G.C.,Axelos,M.A.V.,Thibault,J.F.,1996.Heavymetals bindingbypectins:selectivity,quantificationandcharacterization.Carbohydr. Polym.30,253–263.
Ernst,W.H.O.,Verkleij,J.A.C.,Schat,H.,1992.Metaltoleranceinplants.ActaBot. Neerl.43,229–248.
Eticha,D.,Stass,D.A.,Horst,J.W.,2005.Cell-wallpectinanditsdegreeof methyla-tioninthemaizeroot-apex:significanceforgenotypicdifferencesinaluminium resistance.PlantCellEnviron.28,1410–1420.
Fritz,E.,2007.Measurementofcationexchangecapacity(CEC)ofplantcellwalls byX-raymicroanalysis(EDX)inthetransmissionelectronmicroscope.Microsc. Microanal.13,233–244.
Gonnelli,C.,Galardi,F.,Gabbrielli,R.,2001.Nickelandcoppertoleranceandtoxicity inthreeTuscanpopulationsofSileneparadoxa.Physiol.Plant.113,507–514. Grignon,C.,Sentenac,H.,1991.pHandionicconditionsintheapoplast.Annu.Rev.
PlantPhysiol.42,103–128.
Hall,L.J.,2002.Cellularmechanismforheavymetaldetoxificationandtolerance.J. Exp.Bot.53,1–11.
Hoagland,D.R.,Arnon,D.I.,1950.Thewater-culturemethodforgrowingplants withoutsoil.Calif.Agric.Exp.Stn.Circ.347,1–39.
Hossain,A.K.M.Z.,Koyama,H.,Hara,T.,2006.Growthandcellwallpropertiesoftwo wheatcultivarsdifferingintheirsensitivitytoaluminiumstress.J.PlantPhysiol. 163,39–47.
Islam,E.,Yang,X.,Li,T.,Liu,D.,Jin,X.,Meng,F.,2007.EffectofPbtoxicityonroot mor-phology,physiologyandultrastructureinthetwoecotypesofElsholtziaargyi.J. Hazard.Mater.147,806–816.
Irtelli,B.,Navari-Izzo,F.,2008.UptakekineticsofdifferentarsenicspeciesbyBrassica carinata.PlantSoil303,105–113.
Khotimchenko,M.,Kovalev,V.,Khotimchenko,Y.,2007.Equilibriumstudiesof sorptionoflead(II)ionsbydifferentpectincompounds.J.Hazard.Mater.149, 693–699.
Klavons,J.A.,Bennett,R.D.,1986.Determinationofmethanolusingalcoholoxidase anditsapplicationtomethylestercontentofpectins.J.Agric.FoodChem.34, 597–599.
Konno,H.,Nakato,T.,Nakashima,S.,Katoh,K.,2005.Lygodiumjaponicumfern accu-mulatescopperinthecellwallpectin.J.Exp.Bot.56,1923–1931.
Konno,H.,Nakashima,S.,Katoh,K.,2010.Metal-tolerantScopelophilacataractae mossaccumulatescopperinthecellwallpectinofprotonemataunder copper-enrichedconditions.J.PlantPhysiol.167,358–364.
Kopittke,P.M.,Asher, C.J.,Blamey, F.P.,Auchterlonie,G.J., Guo,Y.N., Menzies, N.W.,2008.LocalizationandchemicalspeciationofPbinrootsofsignalgrass (Brachiariadecumbens)andRhodesgrass(Chlorisgayana).Environ.Sci.Technol. 42,4595–4599.
Krzesłowska,M.,2011.Thecellwallinplantcellresponsetotracemetals: polysac-charideremodelinganditsroleindefensestrategy.ActaPhysiol.Plant.33, 35–51.
LeVan,H.,Kuraishi,S.,Sakurai,N.,1994.Aluminium-inducedrapidrootinhibition andchangesincell-wallcomponentsofsquashseedlings.PlantPhysiol.106, 971–976.
Meyers,D.E.,Kopittke,P.M.,Auchterlonie,G.J.,Webb,R.I.,2009.Characterizationof leadprecipitatefollowinguptakebyrootsofBrassicajuncea.Environ.Toxicol. Chem.28,250–255.
Mimmo,T.,Marzadori,C.,Gessa,C.E.,2009.Doesthedegreeofpectinesterification influencealuminiumsorptionbytherootapoplast?PlantSoil314,159–168. Mohnen,D.,2008.Pectinstructureandbiosynthesis.Curr.Opin.PlantBiol.11,
266–277.
Patra,M.,Bhowmik,N.,Bandopadhyay,B.,Sharma,A.,2004.Comparisonofmercury, leadandarsenicwithrespecttogenotoxiceffectsonplantsystemsandthe developmentofgenetictolerance.Environ.Exp.Bot.52,199–223.
Pelloux, J., Rustérucci, C., Mellerowicz, E.J., 2007. New insight into pectin methylesterasestructureandfunction.TrendsPlantSci.12,267–277. Perriguey,J.,Sterckeman,T.,Morel,J.L.,2008.Effectofrhizosphereandplant-related
factorsonthecadmiumuptakebymaize(ZeamaysL.).Environ.Exp.Bot.63, 333–341.
Poschenrieder,C.,Gunsé,B.,Corrales,I.,Barceló,J.,2008.Aglanceintoaluminium toxicityandresistanceinplants.Sci.TotalEnviron.400,356–368.
Ramgareeb,S.,Cooke,J.A.,Watt,M.P.,2004.Responsesofmeristematiccalluscellsof twoCynodondactylongenotypestoaluminium.J.PlantPhysiol.161,1245–1258. Sattelmacher,B.,2001.Theapoplastanditssignificanceforplantmineralnutrition.
NewPhytol.149,167–192.
Scheller,H.V.,KrügerJensen,J.,OxenbøllSørensen,S.,Harholt,J.,Geshi,N.,2007. Biosynthesisofpectin.Physiol.Plant.129,283–295.
Schmohl,N.,Horst,W.J.,2000.Cellwallpectincontentmodulatesaluminium sen-sitivityofZeamays(L.)cellgrowninsuspensionculture.PlantCellEnviron.23, 735–742.
Schmohl,N.,Pilling,J.,Fisahn,J.,Horst,W.J.,2000.Pectinmethylesterasemodulates aluminiumsensitivityinZeamaysandSolanumtuberosum.Physiol.Plant.109, 419–427.
Tabuchi,A.,Matsumoto,H.,2001.Changesincell-wallpropertiesofwheat(Triticum aestivum)rootsduringaluminiuminducedgrowthinhibition.Physiol.Plant. 112,353–358.
Tolrá,R.,Barcelo,J.,Poschenrieder,C.H.,2009.Constitutiveandaluminium-induced patternsofphenoliccompoundsintwomaizevarietiesdifferinginaluminium tolerance.J.Inorg.Biochem.103,1486–1490.
VanHoff,N.A.L.M.,Koevoets,P.L.M.,Hakvoort,H.W.J.,TenBookum,W.M.,Schat, H.,Verkleij,J.A.C.,Ernst,W.H.O.,2001.EnhancedATP-dependentcopperefflux acrosstherootcellplasmamembraneincopper-tolerantSilenevulgaris.Plant Physiol.113,225–232.
Verkleij,J.A.C.,Schat,H.,1989.Mechanismsofmetaltoleranceinhigherplants.In: Shaw,A.J.(Ed.),HeavyMetalToleranceinPlants:EvolutionaryAspects.CRC Press,BocaRatonFlorida,pp.179–194.
Vollenweider,P.,Cosio,C.,Günthardt-Goerg,M.S.,Keller,C.,2006.Localizationand effectsofcadmiuminleavesofacadmiumtolerantwillow(SalixviminalisL.). PartII.Microlocalizationandcellulareffectsofcadmium.Environ.Exp.Bot.58, 25–40.
Wierzbicka,M.,1998.LeadintheapoplastofAlliumcepaL.roottips:ultrastructural studies.PlantSci.133,105–119.
Wójcik,M.,Vangronsveld,J.,D’Haen,J.,Tukiendorf,A.,2005.Cadmiumtolerancein Thlaspicaerulescens.II.LocalizationofcadmiuminThlaspicaerulescens.Environ. Exp.Bot.53,163–171.
Xiong,J.,An,L.,Lu,H.,Zhu,C.,2009.Exogenousnitricoxideenhancescadmium toleranceofricebyincreasingpectinandhemicellulosescontentsinrootcell wall.Planta230,755–765.
Yang,J.L.,Li,Y.Y.,Zhang,Y.J.,Zhang,S.S.,Wu,Y.R.,Wu,P.,Zheng,S.J.,2008.Cellwall polysaccharidesarespecificallyinvolvedintheexclusionofaluminiumfromthe ricerootapex.Physiol.Plant.146,602–611.
Yu,L.,Reitmeier,C.A.,Love,M.H.,1996.Strawberrytextureandpectincontentas affectedbyelectronbeamirradiation.J.FoodSci.61,844–846.
