Awaiting better times: A quiescence response and adventitious root primordia formation prolong survival under cadmium stress in Tetradenia riparia (Hochst.) Codd

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Awaiting

better

times:

A

quiescence

response

and

adventitious

root

primordia

formation

prolong

survival

under

cadmium

stress

in

Tetradenia

riparia

(Hochst.)

Codd

Nadia

Bazihizina

a

,

Cosimo

Taiti

a

,

Nelson

Serre

a

,

Chiara

Nocci

a,b

,

Francesco

Spinelli

a

,

Werther

Guidi

Nissim

a

,

Elisa

Azzarello

a

,

Lucia

Marti

a

,

Mirvat

Redwan

a

,

Cristina

Gonnelli

b

,

Stefano

Mancuso

a,

*

a_LINV_–_Department_of_Agrifood_Production_and_{Environmental}_Sciences_–_Università_degli_Studi_di_Firenze,_Viale_delle_Idee_30,₅₀₀₁₉_Sesto_F.no,_Florence,

Italy

b

DepartmentofBiology,UniversitàdegliStudidiFirenze,viaMicheli1,50121Florence,Italy

ARTICLE INFO Articlehistory:

Received4February2016

Receivedinrevisedform28April2016 Accepted7May2016

Availableonline10May2016 Keyword: Adventitiousroots Gasexchange Heavymetals Histone Quiescence Recovery ABSTRACT

Plantssurvivestressfulconditionsbyemployingthreemainstrategies:escape,tolerance,andsurvival.

ExperimentswereconductedtopinpointtheresponsestrategyadoptedbyTetradeniaripariatocadmium

(Cd)stressbymonitoringseveralphysiologicalparametersandplants’abilitytorecoveroncethestress

receded.Cadmium(30and150mM)stronglyaffectedshootandrootgrowthinadosedependentmanner,

withalmostcompleteinhibitionofshootgrowth,stomatalopeningandCO2assimilationinplants

exposedto150mMCdSO4.IndependentlyoftheCdSO4concentrations,plantsexcludedCdfromthe

aboveground tissues, with concentrations in shoots remaining around 0.1mmolg1 dry mass.

Furthermore, Cd stresswas associated withadecline inmethylationof Lys4ofhistone H3, likely

associatedwiththetransitionfromanactivetoaquiescentstate.Surprisingly,Cdalsoinducedthe

initiationofrootprimordiainT.ripariastems,which,onceplacedinaCd-freemedia,quickly(24h)

developedintoadventitiousroots,whichwerelikelythedrivingfactoroftherapidresumptionofleaf

elongationandphotosyntheticactivity,whichincreasedalmost20-foldoverthe3weeksofrecovery.

ThereforeT.ripariaabilitytosurviveCdstresswasmediatedbyquiescence,whichassociatedwithan

excluderstrategyandstemrootprimordiaformation,enabledrapidresumptionofgrowthunderCd-free

conditions.

ã2016ElsevierB.V.Allrightsreserved.

1.Introduction

Cadmium(Cd) is a highly toxictrace pollutantfor humans, animalsandplants,anditspresenceintheenvironmenthasbeen widelyrecognizedasaseriousconcernsincethe1960s(Panetal., 2010). Cadmium is naturally present in soils, however recent advancesinindustryandagriculturestimulateincreased deposi-tionofmetalsintheenvironmentandmorethan90%ofCdinsoils

is currently from anthropogenic sources (Pan et al., 2010). Particularly,highlevelscanbefoundwhenagriculturalexpedients are used, such as contaminated irrigation water (Caldas and Machado, 2004). It has now been demonstrated that Cd has cytotoxic,mutagenicand/orcarcinogeniceffectsinanimalandhas beenclassiﬁedasahumancarcinogen(Waalkes,2003).Giventhat plants caneasilyabsorbCd,theyareanimportantsourceofCd exposureforhumansthroughthefoodchain(Uenoetal.,2009; Luxetal., 2011a,b;Gallegoet al.,2012; UraguchiandFujiwara, 2012,2013;Clemensetal.,2013).Thereforeintensiveresearches havebeenperformedtounderstandthelevelsofCdaccumulation inthedifferentplantorgansandtheeffectsofthisheavymetalon plantphysiology(Luxetal.,2011a,b;Clemensetal.,2013;Uraguchi andFujiwara,2013).Inplants,Cdisanon-essentialelementand hasbeenfoundtoinducecomplexchangesinplantsatgenetic, biochemical and physiological levels that ultimately result to substantialgrowthreductions(Sandalioetal.,2001;Cosioetal.,

Abbreviations:Ci,intercellularCO2concentration;ETR,photosyntheticelectron

transportrate;Fm,maximumﬂuorescenceyield;Fs,steadystateﬂuorescenceyield;

Fv/Fm, maximumquantumyield;gs,stomatalconductance;H3-K4,lysine4of

histoneH3; NPQ, non-photochemicalquenching;PFDa, absorbedphoton ﬂux density;Pn,netphotosyntheticrate;PSII,photosystemII;FPSII,actualquantum

yield;RWC,relativewatercontent. * Correspondingauthor.

E-mailaddress:stefano.mancuso@uniﬁ.it(S.Mancuso).

http://dx.doi.org/10.1016/j.envexpbot.2016.05.006

0098-8472/ã2016ElsevierB.V.Allrightsreserved.

ContentslistsavailableatScienceDirect

Environmental

and

Experimental

Botany

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2006;Lopez-Millan etal.,2009;Gallegoetal.,2012).Cadmium altersawidearrayofprocessesingrowingplants,amongstwhich plantwaterrelations(Haag-Kerweretal.,1999;Perfus-Barbeoch etal.,2002;Lefevreetal.,2014),leafgasexchanges(Barylaetal., 2001;Küpperetal.,2007;Heetal.,2011;Kiefferetal.,2009;Wang etal.,2009;Parmaretal.,2013),carbohydrateandionhomeostasis (Besson-Bardet al.,2009;Wu etal.,2012;Heetal., 2015), and oxidativestatus(Heetal.,2011,2013a,b).

Plants survivestressful conditions byemploying three main strategies:escape (i.e. escaping the stress, e.g., short life cycle limited to the wet season in Mediterranean environments), toleranceand survival.Thetolerance strategyinvolvesdifferent mechanismsthat enable the plantto tolerate and grow in the presence of the stress(Lawlor, 2013). By contrast,the survival strategyisaformofstressresistancebywhichcells,tissues,and organs can cease growing in presence of the stress, while maintainingkeycellularfunctions,whichenablethemtorecover rapidly,withminimal damage, oncethe stresshasreceded (cf.

Lawlor, 2013). This kind of survival has also been termed quiescence,andexamplescanbefoundinplantsexposedtolong periodsofdroughtandsalinity(Granotetal.,2009;Julkowskaand Testerink,2015)orinfloodedplants(Bailey-SerresandVoesenek, 2008;Colmeretal.,2009;Luoetal.,2011;Bailey-Serresetal.,2012; SasidharanandVoesenek,2013).Thisinherentflexibilityofplant inresponsetoa changingenvironment hasbeenrelated tothe abilityoftheepigeneticstatustoalterrapidlyandreversibly(Luo etal.,2012).In responsetoexternalsignals (e.g.,stress), plants activate specific signaling pathways that alter physiological reactionsandtranscriptionratesofrespondinggenes.Chromatin status, which is governed by pattern and quantity of histone modificationsthatalterDNA–histoneinteractionandaccessibility oftranscriptionfactors,hasbeenimplicatedintheregulationof eukaryoticgeneactivity(ChinnusamyandZhu,2009;Kimetal., 2008).Inparticulartheprimarylevelcontrollinggeneexpression isthestructureofchromatin,andmodificationsofthehistoneH3 atlysine4(H3-K4)havebeenassociatedwiththetransitionfrom quiescentintoactivestateastheydictateeithergenesilencingor geneactivation(Tsujietal.,2006;Granotetal.,2009).

Plants have formed the basis of traditional medicine for thousands of years and, while in developing countries a large proportionofthepopulationreliesheavilyonmedicinalplantsto meetprimaryhealthcareneeds,increasedscientiﬁcinterestand consumer demand have boosted their use also in developed countriesin thepastdecades(Gurib-Fakim,2006;Annanetal., 2013;Barthwaletal.,2008).Therefore,inparalleltotheincreased useofmedicinalplants,concernsregardingthesafetyoftheiruse haverisenastheyhavebeenrecognizedasanimportantsourceof heavymetaltoxicitytobothhumansandanimalsandaprolonged consumption of such medicinal plants may be detrimental to health (Dwivedi and Dey,2002; Haider etal., 2004;Luxet al., 2011a,b).Indeed,despiteplantsdonotrequireCdforgrowthor reproduction,thebioaccumulationindexofCdinplantsexceeds thatofallothertraceelements(Grantetal.,1998),and,duetoits hightoxicity,theWorldHealthOrganizationhassetthemaximum permissiblelimitofCdinmedicinalplantsto0.3

m

gg1drymass (WorldHealthOrganization,1998).

Tetradenia riparia (Hochst.) Codd belongs to the Lamiaceae family and is a widespread aromatic shrub with a height of approximately1–3moccurringthroughouteastern andtropical Africa(Gazimet al.,2014). T. riparia occupiesa wide range of ecologicallycontrastinghabitatsbutisoftenfoundonhillsidesand riverbanks(Gairolaetal.,2009).ThefamilyLamiaceaeisrichin aromaticspecies,whichareusedasculinaryherbs,folkmedicines andfragrantscents(Gairolaetal.,2009),andinparticularT.riparia isawell-knownherbalmedicineandhastraditionallybeenusedin thetreatmentofvariousillnessesincludingcoughs,dropsy,fever,

andmalaria(Gairolaetal.,2009;Gazimetal.,2011).Despitebeing widelyused,todatevirtuallynothingisknownaboutitsresponse toabioticstressanditspotentialandsafeuseinmarginalsoils(i.e. inareaswithapossiblepollutionofsoilsbyheavymetals)hasyet tobeevaluated.Henceforth,withthepresentstudyweaimedto evaluatethephysiologicalresponsesofT.ripariatotheadditionof Cdinthegrowingmedia.Therefore,followingtheobservationthat T.ripariaexhibitedthesurvivalstrategyresponse,wetestedthe following hypotheses: (i) the quiescent response and the prolonged survival of T. riparia to Cd stress is associated with low Cd concentrations in shoot tissues; (ii) the Cd-induced quiescence enables plants to recover rapidly, with minimal damage,oncethestresshasreceded.

2.Materialsandmethods 2.1.Plantmaterial

Rootedplantswereestablishedfromcuttings(3–4internodes) takenfromT.ripariaplantsgrowninanaturallylitglasshousefor 1yearinstandardpottingmix.Thecuttingswerepropagatedby inserting them in aerated water for 3–4 weeks in the same glasshouse (with average day/night temperatures during the experimentalperiodof35/25Candanaveragehumidityof76% inthetwoexperiments).Rootedcuttingswerethentransferredto anaeratednutrientsolution,whichcontained:3mMKNO3,2mM Ca(NO3)2,1mMNH4H2PO4,0.50mMMgSO4,40

m

MFe(Na)-EDTA, 1

m

MKCl,25

m

MH3BO3,2

m

MMnSO4,2

m

MZnSO4,0.1

m

MCuSO4, and0.1

m

M(NH4)6Mo7O24,andbufferedwith1mMMES.ThepHof the solution was adjusted to 5.8 using KOH. Solutions were changedweekly.Twomonthsaftertransferringthecuttingstothe aeratednutrientsolution,plantswereselectedforshootandroot uniformity, and CdSO4 was added to the nutrient solutions to obtaintherequiredﬁnalconcentrations.

2.2.PhysiologicalresponsesofT.ripariatodifferentconcentrationsof CdSO4(Expt1)

Experiment1(Expt1)consistedofthreetreatmentswithﬁve replicatesinacompletelyrandomizedblockdesign,whereplants were exposed to increasing Cd concentrations: 0

m

M CdSO4, consideredasthecontroltreatment,and30and150

m

MCdSO4. Plantswerethenharvested5weeksafterimposingthetreatments. 2.3.RecoveryofT.ripariafollowingCdstress(Expt2)

In thesecond experiment (Expt2)plants weregrown for5 weekswithasolutioncontaining150

m

MCdSO4andsubsequently were transferred in aerated water to assess recovery in the following3weeks(i.e.,onlytherootsystemandthebasalportion ofthestembathingintheCd-freemedia).Inaddition,asinthe_ﬁrst experimentweobservedthatCdstronglyinducedtheformationof rootprimordiainthetopinternodesofthestems,cuttings(withall leavesremovedexceptthepairofleavesonthetopinternode)from thetop3–4internodesofthestemsofcontrolandtreatedplants wereinsertedinaeratedwatertoassessleafandrootformationfor thefollowing3weeks.

2.4.Leafandrootelongationandleafsampling(Expt1and2) In Expt 1, leaf length was measuredevery 2–4days for the durationoftheexperimenttodetermineitsextensionduringthe treatmentperiod.Atdifferenttimepoints(1,7,14and35days), youngfullyexpandedleaves(oneforeachplantinalltreatments) were collected between 11.00h and 11.30h for subsequent analyses of ions,total soluble sugars and leaf pigments.These

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leaftissuesweresnap-frozeninliquidN2,storedat80C, freeze-dried,andthen storedat20_C._In _addition,_the_relative_water content(RWC)of theleaveswas measured usingthefollowing formulaRWC=(FMDM)/(TMDM)100,whereFMisleaffresh mass,DMisleafdrymassaftertheleavesweredriedat65Cfor3 d,andTMistheturgidmassafterleavesweresoakedinwaterfor 4hatroomtemperature.

InExpt 2,rootnumber,rootand leaflengthweremeasured every 2–4days for the entire duration of the experiment to determinetheirextensionduringthetreatmentperiod.

2.5.Leafgasexchangeparameters(Expt1and2)

Atdifferenttime points in bothexperiments, net photosyn-theticrate andstomatalconductance werecalculated onyoung fully expanded leaves in each treatment, using the open gas exchangesystemLi-6400(LiCorInc.,Lincoln,NE,USA)asdescribed inBazihizinaetal.,2015.Brieﬂyleafgasexchangemeasurements weretakenonfourplantsfromeachtreatmentatambientrelative humidity(50–60%),ambientCO2,ﬂowrate of400

m

mols1,leaf chamber temperature at 30C and photosynthetically active radiationof300

m

molm2s1.

InExpt1,usingtheintegratedﬂuorescencechamberhead (Li-6400-40;Li-CorInc.) oftheopengasexchange system Li-6400 (LiCor Inc., Lincoln, NE, USA) chlorophyll ﬂuorescence was measuredonthe same leaves usedfor gas exchange measure-ments.Maximumquantumyield(Fv/Fm)wasdeterminedaftera 30mindarkacclimationofselectedleavesusingadarkleafclip. Actual quantum yield (

F

PSII) was measured on light adapted-leavesandwascalculatedas

F

PSII=(Fm0F_s)/F_m0,whichmeasures

theproportionofthelightabsorbedbychlorophyllassociatedwith PSIIthatisusedinphotochemistry(MaxwellandJohnson2000). Fluorescence parameters were also used to calculate the non-photochemicalquenching(NPQ=(FmFm0)/F_m0;Redondo-Gomez etal.,2006),whichislinearlyrelatedtoheatdissipation(Maxwell andJohnson2000).ETRwascalculatedasETR=

F

PSIIPFDa0.5, wherePFDaisabsorbedlightin

m

molphotonm2s1and0.5isa factorthataccountsforthepartitioningofenergybetweenPSIIand PSI(MaxwellandJohnson2000).

2.6.Plantharvest(Expt1)

In Expt 1, plants were sampled at the beginning of the experiment and 5 weeks after applyingthe treatments for the determinationofshootandrootfreshanddrymass.Rootswere carefullywashedthreetimesandeach plantwasseparatedinto roots,leavesandstemsandtheirfreshmasswasrecorded.Tissue sampleswerethenoven-driedat60C for1weektodetermine theirdrymass.

2.7.Leafpigmentanalyses(Expt1)

Cold100% methanol was added to10–20mg of theground freeze-driedtissuesandthesampleswereincubatedindarkness, onice.After30min,sampleswerecentrifugedat9300gfor10min at4C,thesupernatantswereremovedandtheirabsorbancewas determinedat470,665.2and652.4nm,usingaTecanInﬁnite200 Spectrophotometer (Männedorf, Switzerland). Total chlorophyll andcarotenoidconcentrationswerecalculatedusingtheequations fromWellburn(1994).

2.8.DeterminationofKandCdconcentrationsinplants(Expt1) Potassiumconcentrationsindry planttissueswereobtained afterdigestingofgroundtissuesin0.5MHNO3byshakingvialsfor 48h in dark at 25C. Diluted extracts were analysed for K

concentrations(DigiﬂameDV710,NTLaboratory).Cd concentra-tionsindryplanttissueswereobtainedfollowingadigestionina mixtureofconcentratedHNO3andHClO4(2:1v.v.,Sigma-Aldrich, Italy)usingadigester(VELPScientiﬁca,Italy).Cdconcentrations werethendeterminedwithaninductivelycoupledplasma-optical emissionspectrometer(ICP-OES,OPTIMA2000DV,PerkinElmer, USA). The ICP analytical standard (AA/ICP calibration/check standardsforenvironmentalanalyses,1gL1)forCdwassupplied bySigma-Aldrich.

2.9.Acidextractionofproteinsandimmunoblotting(Expt1) Leavesof5weekstreatedplantsofT.ripariaweregroundusing a T25 Ultra-Turrax (Ika Labortechnik) in an extraction buffer (100mMTris–HClpH7,5,2mM EDTA5mM 2-Mercaptoethanol 2mMPhenylmethylsulfonyl ﬂuoride,HCl 0.25M) supplemented with a protease inhibitor cocktail (Sigma-Aldrich St. Louis,MO product number P9599). The acid soluble proteins were TCA-Acetonprecipitatedandproteinconcentrationestimatedwiththe BradfordAssay(Bio-RadHerculesCAproductnumber500-0006). Acidsolubleproteins(20

m

g)wereseparatedin15%SDS-PAGEand stained with Comassie brilliant blue R (Merck Darmstadt) or blotted on nitrocellulose paper (Bio-rad Hercules CA product number 162-0115).Thefollowingtwo differentantibodieswere probedtoevaluatedthedimethylationofH3:abH3(Sigma-Aldrich St.Louis,MOproductnumberH0164)forimmunodetectionoftotal histon3 fractionandanti-dimethylatedH3K4(Sigma-Aldrich St.

Louis, MO product number D5692). Immunodetection was

performedusingsecondaryantibodyofgoatanti-rabbit horserad-ish peroxidase conjugate (Sigma-Aldrich St. Louis, MO product number A0545). To strip the nitrocellulose a stripping buffer composedby62.5mMTris–HClpH6.7,100mM 2-Mercaptoetha-nol 2%SDSwasused,andafterthestripping,thenitrocellulose paperwasincubatedwithsecondaryantibodyofgoatanti-rabbit horseradishperoxidaseconjugatetoevaluatebackgroundsignal. 2.10.Statisticalanalyses

StatisticalanalyseswereconductedusingGraphPadforMac6th Edition, and analysisofvariance (ANOVA)was usedtoidentify overallsigniﬁcantdifferencesbetweentreatments.Tukeypost-hoc test was used for a posteriori comparison of individualmeans (withatleastP<0.05assigniﬁcantlevel).

3.Results

3.1.Plantgrowth(Expt1)

IncreasingconcentrationofCdinthenutrientsolutionsreduced plant growth, with substantial declines in plant dry mass incrementsandleafelongation(Table1andFig.1).Forexample, with150

m

MCdSO4therewasnonetdrymassincrementforboth shootsandroots.TheusedCdconcentrationsresultedtoxicforroot

Table1

NetdrymassincrementsinTetradeniaripariainresponsetodifferentconcentration ofCdSO4intheculturemedia(Expt1).ValuesaremeanS.E.(n=5).Different

lowercaselettersineachcolumnindicatesigniﬁcantdifferencesbetweencontrol andtreatedplants.Initialleaf,stemandrootdry masswererespectively(g): 7.10.6;20.53.1;4.80.4.

CdSO4treatment Netdrymassincrement(gplant1)

Leaves Stems Roots 0 26.83.3a 21.17.2a 13.44.2a 30 5.30.5b 3.02.2b 3.00.8b 150 2.30.5c 1.61.6b 0.00.9b

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growth(Table1),and with150

m

MCdSO4 theentireplantroot systemwascompletelynecrotic(Fig.S1).Asforplantdrymass,leaf elongationwasstronglyinhibitedintime(morethan60%declines inbothCdtreatments,TableS1)bythestressinallCdtreatments, although thereductions wereaccentuated with150

m

MCdSO4 (Fig.1a).Cadmiumimmediatelyaffectedplantwaterrelations,and onedayafterapplyingthetreatments,independentlyfromtheCd concentrations, relative water content (RWC) of the youngest expanded leaves declined to almost 50% compared to that in controlplants(Fig.S2a).However,withtime,inplantstreatedwith 30

m

MCdSO4,RWCrecoveredtovaluessimilartothoseofcontrol plants,meanwhileinplantsexposedto150

m

MCdSO4RWCwasat mosttimeslowerthanthatincontrolplants.

3.2.Gasexchangeandchlorophyllﬂuorescence(Expt1)

In all times considered, net CO2 assimilation and stomatal conductancewerereducedbyCdtreatments(Fig.2).Onedayafter thetreatmentsstomatalconductancedecreasedby60and80%in plantsexposedto30and150

m

MCdSO4,respectively,andinboth casesvaluesneverreturnedtothosemeasuredincontrolplants. Leaf photosynthetic activity had a similar trend to the one observedforstomatalconductance.

Chlorophyll fluorescenceresults showed that themaximum quantum efficiency of PSII was not affected by Cd treatments throughout the entire experimental period (Table 2, Fig. S3). However, although after one day of treatment, for all Cd concentrations consideredtherewere nosignificant changesin

Fig.1.ResponseofTetradeniaripariatoincreasingconcentrationofCdSO4intheculturemedia(Expt1):(a)leafelongation;(b)exampleofastemofaplantgrownunder

controlconditions(scalebar,5cm);(c)exampleofastemofaplantexposedto150mMCdSO4for5weeks(scalebar,5cm).In(a)valuesaremeanS.E.(n=5).

Fig.2.NetCO2assimilation(a)andstomatalconductance(b)intimeinplantsgrownwithcontrolsolutionor30and150mMCdSO4(Expt1).ValuesaremeanS.E.(n=5).

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allmeasured chlorophyllﬂuorescenceparameters,intimethere wasaprogressivedeclineintheactualquantumyieldofPSIIandin theelectrontransportrate,associatedwithaprogressiveincrease inthenon-photochemicalquenching(Fig.S3).Forexample,with 150

m

MCdSO4,attheendofthe5weeksoftreatments,Cdstress resultedina60%and40%declinesinETRand

F

PSII,respectively, and a 2-fold increase in NPQ compared with control plants (Table2).

3.3.Totalchlorophyllandcarotenoidconcentrationsinyoungest expandedleaves(Expt1)

Forthe_ﬁrst14daysaftertreatment,forallCdconcentrations considered there were no signiﬁcant declines in both total chlorophyll and carotenoid concentrations in the youngest

expandedleaves (Fig.S4). However,5weeks of treatmentwith 150

m

MCdSO4resultedin27–37%declinesintotalchlorophylland carotenoidconcentrations.Bycontrast,30

m

MCdSO4didnotaffect the concentration of these two leaf photosynthetic pigments (Table2).

3.4.CdandKconcentrations(Expt1)

InallCdtreatments,Cdaccumulationwaslimitedinstemsand especiallyinleavesincomparisontoroots(Table3).Independently fromtheCdconcentrationintheculturemedia,Cdconcentrations inbothleavesandstemsremainedwithinorclose0.1

m

molCdg1 drymass.Attheendoftheexperiment,forbothCdtreatments, rootKconcentrationdecreasedascomparedwithcontrolplants.In both leaves and stems, K concentrations didnot varybetween

Table2

ChlorophyllfluorescenceparametersandtotalchlorophyllandcarotenoidconcentrationsinyoungleavesofTetradeniaripariaexposedtoincreasingconcentrationofCdSO4 intheculturemedia(Expt1).ValuesaremeanS.E.(n=5).Differentlowercaselettersineachrowindicatesignificant(P<0.05)differencesbetweencontrolandtreated plants.Fv/Fm,maximumpotentialphotosystemIIefficiency;ETR,electrontransportrate;FPSII,actualphotosystemIIefficiency;NPQ,non-photochemicalquenching.

Parameter CdSO4treatment(mM)

0 30 150

Fv/Fm 0.930.00a 0.910.02a 0.900.02a

ETR(mmolem2s1) 93.483.74a 75.134.33b 35.598.79c

FPSII 0.180.01a 0.140.01b 0.110.01c

NPQ 1.590.04a 2.040.20b 3.160.36c

Totalchlorophyll(mgmg1_dry_mass) _8.51_0.62a _8.68_0.57a _6.10_0.51b

Carotenoid(mgmg1drymass) 1.520.10a 1.710.11a 0.960.07b

Table3

CdandKconcentrationsinleaves,stemsandrootsofTetradeniaripariaexposedtoincreasingconcentrationofCdSO4intheculturemedia(Expt1).ValuesaremeanS.E.

(n=5).Differentlowercaselettersineachrowindicatesigniﬁcantdifferencesbetweencontrolandtreatedplants.nd,notdetectable. Parameter CdSO4treatment(mM)

Leaves Stems Roots

0 30 150 0 30 150 0 30 150

Cd(mmolg1drymass) nd 0.030.01a 0.070.01c nd 0.060.01a 0.120.01b nd 8.552.62a 23.943.47b K(mmolg1_dry_mass) ₁₁₂₀_25a ₁₁₇₇_35a ₉₉₄_71a ₁₀₉₃_132a ₁₀₀₉_99a,b ₉₀₂_38b ₈₅₄_32a ₄₉₈_53b ₅₃₃_76b

Fig.3. LossofhistoneH3de-dimethylationatlysine4inTetradeniaripariaafterexposurefor5weeksto150mMCdSO4.(a)WsternBlotanalysison20mgofacidicsoluble

extractsfromtreatedandcontrolleaves,showsthatHistoneH3lacksdimethylationinlysine4underCadmiumtreatment.(b)Gelloadingcontrol:twodifferentamountof protein(10and20mg)wereloadedinthegelandthenstainedwithCoomassiebrilliantblue.

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controlplants and plants treated with30

m

M CdSO4 (Table 3), whereas at 150

m

M CdSO4, leaf and stem K concentrations decreasedby approximately10–15%ascompared with concen-trationsincontrolplants.

3.5.HistoneposttranslationalmodiﬁcationsfollowingCdstress(Expt 1)

Thedynamicsofhistonemodificationsafter5weeksofCdSO4 treatmentswereanalyzed,focusingonthedemethylationoflysine 4,asthis modificationhaspreviouslybeenassociated inplants withdecondensed,transcriptionallyactivechromatin(Pflugerand Wagner2007; Granot etal., 2009).Compared withthecontrol plants,exposurefor5weeksto150

m

MCdSO4ledtothelossofthe demethylationinlysine4(Fig.3)meanwhilethisdrasticchange did not occur in plants treated with 30

m

M CdSO4 (Fig. S5). Moreover,we loaded on theSDS-PAGE 10and 20

m

g of acidic extractsand then performed a Comassie Brillant Blue staining (Fig.3b),inordertoconﬁrmtheestimationofproteinsthrough BradfordAssay.

3.6.GrowthrecoveryandadventitiousrootformationfollowingCd stress(Expt2)

Cdstressstronglyinducedtheinitiationofrootprimordiaon thestemoftreatedplants,resultingintheformation,after2–3 weeksoftreatment,ofseveralprimordia,mostlyintheﬁrstfew internodesofthestemsofCd-treatedplants(Table4andFig.4). Therefore,oncecuttingstakenfromthesetopinternodesfrom Cd-treatedplantsweretransferredinaCd-freemedia,thesenewroot primordiaquicklydevelopedintoadventitiousroots,despitetheir activationanddevelopmentinadventitiousrootswasstrictlylocal, andprimordiathatremaineddrynevergotactivated.Indeed,in Cd-treated cuttings, adventitious root growth occurred very quickly,withvisiblerootsalreadyafter24hofrecoverytreatment meanwhile in control plants root primordia initiation and subsequentadventitiousrootformationoccurredonaverageonly after6–10days(Table4andFigs.4and5).

Intherecoveryexperiment,mostplantspreviouslyexposedto Cd(80%ofallplantsintherecoveryexperimentand100%ofall plantsforthecuttingexperiment,seeTable4)wereabletoform

Table4

Numberofstemswithpreformedrootprimordia,recoveryrate,timerequiredfortheemergenceofrootprimordiaandnewadventitiousrootsformation(scoredwithavisual evaluation)inCd-treatedplantstransferredintoaCd-freemediaandincuttingstakenfromcontrolandCd-treatedplantsandplacedintoaCd-freemediafor3weeks.Priorto therecoverytreatment,plantswereeithergrowninacontrolsolutionorinasolutionwith150mMCdSO4for5weeks.ValuesaremeanS.E.(n=5).Differentlowercase

lettersineachcolumnindicatesigniﬁcantdifferencesbetweentreatments.nd,notdetectedasrootprimordiawerevisibleonlyonthesoft-woodstems.*rootprimordiawere alreadyvisiblepriortotherecoverytreatment.

Stemswithvisiblerootprimordia (%)

Recoveryrate (%)

Rootprimordiavisible(daysfrom recovery)

Emergencenewroots(daysfrom recovery)

Controlplants Cuttings 96a 100 6.50.2a 9.70.4a Cd-treated plants Entire plants nd 80 5.01.7a 10.23.6a Cuttings 8810b 100 0b* 1.20.2b

Fig.4.RootprimordiaandadventitiousrootsinstemsofTetradeniaripariaplants(Expt2).(a)Constitutivelypreformedrootprimordiapresentonallinternodesinallplants, whichhoweverdidnotalwayshastenintoadventitiousrootformation;(b)typicalstemofacontrolplant;(c)Cdinducedinitiationofnewrootprimordiaonthestemofa plantgrownunderCdstress;(d)sectionofastemofT.ripariawithoutrootprimordia(scalebar,200mm);(e)sectionofarootprimordiaonastemofaCd-treatedplant(scale bar,200mm);(f)theCd-inducedrootprimordiadevelopedin24–48hintoadventitiousrootswheninaCd-freemedia.

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newrootsandleavesonceplacedinaCd-freemedia,asshownby theprogressive increase in leaf elongation, leaf photosynthetic activityand stomatal conductance (Figs. 5 and 6, Table S2). In particular, when cuttings were taken from plants previously treatedwith150

m

MCdSO4,leafphotosyntheticactivityincreased fromanaveragevalueof0.3 toanaveragevalueof5.6

m

MCO2 m2s1duringthe3-weekrecoveryperiod(Fig.6a).Despitethe observedincreasesinleafelongationandphotosyntheticactivity incuttingspreviouslytreatedwithCd,3weeksaftertherecovery

treatment, leafandrootlengths ofcuttings fromcontrol plants wereapproximatelydouble thanthose ofcuttings fromtreated plants(Fig.5).Whenconsideringtherecoveryoftheentireplants (i.e.,withonlytherootsystemandthebasalportionofthestem bathingintheCd-freemedia),recoverywassigniﬁcantlyslower,as the initiation of root primordial in Cd-treated plants occurred mainlyintheﬁrstfewinternodesofthestems,andadventitious rootformationwasseenonaverageonly10daysaftertherecovery treatment(Table4,Fig.5).

Fig.5.Recoveryofplantspreviouslyexposedto150mMCdSO4andofcuttings(ﬁrst3–4internodesofthestems)ofTetradeniaripariaplantspreviouslyexposedtoacontrol

solutionor150mMCdSO4andthentransferredtoaCd-freemedia:(a)totalrootlength;(b)numberofmainadventitiousroots;(c)leafelongationrate.Cuttingsfromcontrol

plantsweretakentoevaluatetheirrecoveryspeedandrate.ValuesaremeanS.E.(n=5).Asterisksindicatesigniﬁcantdifferencesbetweencuttingstakenfromcontrol plantsandcuttingstakenfromCdtreatedplants.*P<0.05.

Fig.6.NetCO2assimilation(a)andstomatalconductance(b)intimemeasuredintheleavesofplantspreviouslyexposedto150mMCdSO4andinleavesofcuttings(ﬁrst3–4

internodesofthestems)ofplantspreviouslyexposedtocontrolor150mMCdSO4andsubsequentlytransferredintoaCd-freemedia.Cuttingsfromcontrolplantsweretaken

toevaluatetheirrecoveryspeed.ValuesaremeanS.E.(n=5).Asterisksindicatesigniﬁcantdifferencesbetweencuttingstakenfromcontrolplantsandcuttingstakenfrom Cdtreatedplants.*P<0.05.NetCO2assimilationandstomatalconductanceincontrolandCd-treatedplantsintheconsideredperiodwere,respectively:9.970.21and

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4.Discussion

The quiescence strategy has been considered pivotal in prolonging plant survival under stressful conditions and to improve survival rateand generation of newtissues after the stresshasreceded(GreenwayandGibbs2003;Fukaoand Bailey-Serres,2004;Bailey-Serres andVoesenek,2008; Colmeret al., 2009;Luoetal.,2011).Weobservedthat,followingCdstress,T. riparia adopted a quiescence-like response and there was an enhancedformationofadventitiousrootprimordiainthestems. Wetherefore subsequently tested and conﬁrmed the following hypotheses:(i)thequiescentresponseandtheprolongedsurvival ofT.ripariatoCdstressisassociatedwithlowCdconcentrationsin shoottissues; (ii) theCd-induced quiescence enablesplants to recover rapidly, with minimal damage, once the stress has receded; and (iii) root primordia will quickly develop into adventitious roots in more favourable conditions, therefore enabling the plants to rapidly exploit new environmental opportunities.

Cadmium affected T. riparia growth in a dose dependent manner,and150

m

MCdSO4quicklyarrestedleafelongationrates andresultedinwidespreadnecrosisandeventuallydeathofthe entirerootsystemafter5weeksoftreatment(Fig.S1).Thisstrong reductionin the rootgrowth, and subsequentrootdeath, with 150

m

MCdSO4couldbedirectlylinkedwiththelarge accumula-tionofCdinroottissues,asalreadyobservedforotherspecies(e.g.,

Perfus-Barbeoch et al., 2002). Cadmium concentrations were foundto be several folds higher in roots than in shoots, with concentrations up to 241

m

molg1 dry mass. By contrast, Cd concentrationsinshootsremainedwithinorcloseto0.1

m

molg1 drymass,a clearindicationofrestrictedaccumulationof Cdin shoots.However, withboth 30 and 150

m

MCdSO4, Cd concen-trationsinleavesandstemsexceededbyfarthelimitformaximum Cdlevelsformedicinalplants(0.3

m

gg1drymass,i.e.0.003

m

mol g1 dry mass), indicating that this species is not suitable as a medicinalplantwhencultivatedinsubstrateswithevenmoderate Cd concentrations. Nevertheless, this exclusion of Cd fromthe above-ground tissues was sufﬁcient to avoid the irreversible damageof these tissues, and shoots of treated plantremained alive,asclearlyshownbythefastresumptionofphotosynthesis andleafelongationoncethestressreceded.

Cadmium stress quickly affected shoot water relations, as shownbythesubstantialdeclinesinleafrelativewatercontent andstomatalconductanceintheﬁrst24hoftreatments.Indeed, asdeclinesinleafphotosyntheticratewereparalleledbydeclines instomatalconductanceandCi(Fig.S2b),theseresultssupport the view that photosynthesis was initially limited by CO2 diffusionasa consequenceof lowleaf stomatal conductance(

Medrano et al., 2002). This result is in accordance with the increasing evidence suggesting that exposure to toxic metal concentrationsinitiallyaffectsplantwaterrelations(Barceloand Poschenrieder,1990;Sagardoyetal.,2010;Bazihizinaetal.,2014). Forinstance,in Arabidopsisthaliana,exposureto50–100

m

MCd resultedin a progressivedecline in leaf stomatal conductance, witha completestomatalclosure 4–6daysafterthetreatments (Perfus-Barbeochetal.,2002).Severalfactorscouldexplainthese initialdeclinesinstomatalopeningfollowingCdstress.Cadmium hasbeenfoundtocausestrongdepolarizationofrootcorticalcells, downtovalueswithintherangeofthediffusionpotential(Sanz et al., 2009). This altered root membrane functionality could explaintherapiddeclinesin stomatalconductanceasit isnow wellestablishedthatchangesattherootlevelcanquickly,through rapidsystemiclong-distancesignals,regulatestomatalbehaviour (Comstock 2002;Christmannet al., 2007;Choi et al.,2014).In alternative,ithasbeensuggestedthatCdcanentertheguardcells throughcalciumchannelsthusaffectingguardcellregulationand

stomatalopening(Perfus-Barbeochetal.,2002).However,given theobservedresponsesinT.riparia,thissecondhypothesisseems lesslikelyasCdconcentrationsmeasuredinshootswaslow,even after5weeksoftreatments.Nevertheless,fromtheseventhday on, prolonged Cd stress led substantial declines in the actual efﬁciency of PSII and ETR and increases in non-photochemical quenching, which coincided with great reductions in CO2 assimilation rates. This would indicate that, although initially decreasedphotosynthesiswasassociatedwithdeclinesin stoma-tal conductance, subsequently Cd impaired leaf photosynthetic machinery.Indeed,althoughthetypeofdamagevariesbasedon thespeciesusedandtheenvironmentalcontingencies(e.g.,Krupa etal.,1993;Küpperetal.,2007),photosyntheticreactionsbelong to the most important sites affected by Cd, with PSII often identiﬁedasoneofthemaintarget(Küpperetal.,2007).

ThehypothesisthatCdinducedaquiescent-likeresponsein T.ripariawas furtherconfirmedby thehistoneH3 posttransla-tionalmodifications,whichresultedinthelossofthemethylation of Lys4 of histone H3 (H3-K4) after Cd stress. Indeed, histone modifications, such as methylation and acetylation, in the chromatinsurroundinggenesarethoughttoregulate transcrip-tionalactivityandgeneexpression,withthemethylationofH3-K4 generally associated with transcriptionally active chromatin (Pflugerand Wagner 2007; Granot et al., 2009). As plants are sessile organisms it has been shown that gene expression is dynamically controlled in response to the appearance or disappearance of an environmental stress through reversible anddynamicchangesinhistonemodification(Tsujietal.,2006), anddeclinesindimethylationofH3-K4havebeenlinkedwiththe entryoftheplantsintoaquiescentstateduringstressfulperiods (Granotetal.,2009).

Adventitious roots develop ectopically from aboveground organsandformnaturallyfromstemtissueand/orunderstressful environmental conditions (e.g. ﬂooding, drought or salt stress,

Wangetal.,2009;Liaoetal.,2012;SteffensandRasmussen,2016) toreplacetheexistingrootsystemthathaseitherbeenkilledor whose function is impaired by the stress (Pezeshki 2001; Dawoodetal.,2014).Inparticular,despitebeingphytotoxic,heavy metal stress has been found to induce speciﬁc ‘stress-induced morphogenic responses’ (cf. Potters, 2007; Xu et al., 2011), including the enhanced formation of adventitious roots (Gad andAtta-Aly,2006).In agreementwiththeseﬁndings,ourdata show that highCd stress leadsto characteristic stress-induced morphogenicresponses:areductioninprimaryrootgrowthand, at 150

m

M CdSO4, a concomitant induction of root primordia formation in most stems (Fig. 4). As reported for Solanum dulcamara underﬂooded conditions(Dawoodet al.,2014), root primordiaactivationanddevelopmentintoadventitiousrootswas local,asprimordiathatremaineddryneverdevelopedintoaroot (data not shown). These Cd-induced root primordia enabled a muchfasterresumptionofplantgrowth(rootandleafelongation andleafphotosyntheticactivity)comparedwithcontrolcuttings andtreatedplantsthathadonlytheprimordia-freeportionofthe stem exposed to the Cd-free, where de novoinitiation of root primordiawasrequired(Table4).Thisresultcanbeexplainedby consideringthatincuttingspreviouslytreatedwithCd,duringthe ﬁrstweekofrecovery,leafphotosyntheticactivitywascorrelated withrootelongationratherthanleafgrowth(TableS3).Itwould thereforebereasonabletoexpectthatcarbohydrateretainedor newlysynthesizedinleavesafterrecoverywereinitiallyusedfora quick adventitious rootgrowth. Subsequently, fromthe second week of recovery, there was a change in the trend, and leaf photosynthetic activitywas correlated mainlywiththe relative leafelongation,which would suggesta shiftinphotoassimilate partitioning.

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5.Conclusion

BasedontheremarkablesurvivalcapacityofT.ripariaunderCd stressandthegrowthresumptionunderoptimalconditions,we bring forward the hypothesis that this species adopted the quiescencestrategyinresponsetoCdstress.Indeedtheresponse ofT. ripariatoCd stresswas associatedwith: (a)a ‘quiescence response’,i.e.,nobiomassaccumulation,associatedwithhistone H3modification;(b)alimitedCdaccumulationinshoottissues; and (c) the induction of root primordia. What might be the physiologicalandecologicalsignificanceoftheobservedresponses inT.ripariainaCd-richenvironment?Ithasbeensuggestedthat both quiescence and stress induced morphogenic responses in generalareamechanismforstressevasion(Potters,2007).Given thatT.riparia is nota metallophyte,and bydefinitionadaptive responsestoCdcouldnothaveevolvedundernaturalselection,the responsedescribedinthepresentstudyarelikelytorepresentan inherentgeneralpurposeresponseofT.ripariatotransientadverse growthconditions,whichenablesplantstoquicklyresumenormal growthinmorefavourableconditions,increasingplants’abilityto rapidlyexploitnewenvironmentalopportunities.

Funding

This research was supported by funding from the Italian Defence Ministry (research project: “VESPA-VEgetal System for PollutionAvoidance”).

Acknowledgement

We thank the Shangrila association for providing the plant material.

AppendixA.Supplementarydata

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. envexpbot.2016.05.006.

References

Annan,K.,Dickson,R.A.,Amponsah,I.K.,Nooni,I.K.,2013.Theheavymetalcontents ofsomeselectedmedicinalplantssampledfromdifferentgeographical locations.Pharmacogn.Res.5,103–108.

Bailey-Serres,J.,Voesenek,L.A.C.J.,2008.Floodingstress:acclimationsandgenetic diversity.Annu.Rev.PlantBiol.59,313–339.

Bailey-Serres,J.,Lee,S.C.,Brinton,E.,2012.Waterprooﬁngcrops:effectiveﬂooding survivalstrategies.PlantPhysiol.160,1698–1709.

Barcelo,J.,Poschenrieder,C.,1990.Plantwaterrelationsasaffectedbyheavy-metal stress—areview.J.PlantNutr.13,1–37.

Barthwal,J.,Nair,S.,Kakkar,P.,2008.Heavymetalaccumulationinmedicinalplants collectedfromenvironmentallydifferentsites.Biomed.Environ.Sci.21,319– 324.

Baryla,A.,Carrier,P.,Franck,F.,Coulomb,C.,Sahut,C.,Havaux,M.,2001.Leaf chlorosisinoilseedrapeplants(Brassicanapus)grownoncadmium-polluted soil:causesandconsequencesforphotosynthesisandgrowth.Planta212,696– 709.

Bazihizina,N.,Taiti,C.,Marti,L.,Rodrigo-Moreno,A.,Spinelli,F.,Giordano,C.,etal., 2014.Zn2+

-inducedchangesattherootlevelaccountfortheincreasedtolerance ofacclimatedtobaccoplants.J.Exp.Bot.65,4931–4942.

Bazihizina,N.,Colzi,I.,Giorni,E.,Mancuso,S.,Gonnelli,C.,2015.Photosynthesizing onmetalexcess:copperdifferentlyinducedchangesinvariousphotosynthetic parametersincoppertolerantandsensitiveSileneparadoxaL.populations. PlantSci.232,67–76.

Besson-Bard,A.,Gravot,A.,Richaud,P.,Auroy,P.,Duc,C.,Gaymard,F.,etal.,2009. NitricoxidecontributestocadmiumtoxicityinArabidopsisbypromoting cadmiumaccumulationinrootsandbyup-regulatinggenesrelatedtoiron uptake.PlantPhysiol.149,1302–1315.

Caldas,E.D.,Machado,L.L.,2004.Cadmium:mercuryandleadinmedicinalherbsin Brazil.FoodChem.Toxicol.42,599–603.

Chinnusamy,V.,Zhu,J.K.,2009.Epigeneticregulationofstressresponsesinplants. Curr.Opin.PlantBiol.12,133–139.

Choi,W.G.,Toyota,M.,Kim,S.H.,Hilleary,R.,Gilroy,S.,2014.Saltstress-inducedCa2+

wavesareassociatedwithrapid:long-distanceroot-to-shootsignalingin plants.Proc.NatlAcad.Sci.U.S.A.111,6497–6502.

Christmann,A.,Weiler,E.W.,Steudle,E.,Grill,E.,2007.Ahydraulicsignalin root-to-shootsignallingofwatershortage.PlantJ.52,167–174.

Clemens,S.,Aarts,M.G.M.,Thomine,S.,Verbruggen,N.,2013.Plantscience:thekey topreventingslowcadmiumpoisoning.TrendsPlantSci.18,92–99.

Colmer,T.D.,Vos,H.,Pedersen,O.,2009.Toleranceofcombinedsubmergenceand salinityinthehalophyticstem-succulentTecticorniapergranulata.Ann.Bot. 103, 303–312.

Comstock,J.P.,2002.Hydraulicandchemicalsignallinginthecontrolofstomatal conductanceandtranspiration.J.Exp.Bot.53,195–200.

Cosio,C.,Vollenweider,P.,Keller,C.,2006.Localizationandeffectsofcadmiumin leavesofacadmium-tolerantwillow(SalixviminalisL:)I.Macrolocalizationand phytotoxiceffectsofcadmium.Environ.Exp.Bot.58,64–74.

Dawood,T.,Rieu,I.,Wolters-Arts,M.,Derksen,E.B.,Mariani,C.,Visser,E.J.W.,2014. Rapidﬂooding-inducedadventitiousrootdevelopmentfrompreformed primordiainSolanumdulcamara.AoBPlants doi:http://dx.doi.org/10.1093/ aobpla/plt058.

Dwivedi,S.K.,Dey,S.,2002.Medicinalherbs:apotentialsourceoftoxicmetal exposureformanandanimalsinIndia.Arch.Environ.Health57,229–231.

Fukao,T.,Bailey-Serres,J.,2004.Plantresponsestohypoxia—issurvivalabalancing act?TrendsPlantSci.9,449–456.

Gad,N.,Atta-Aly,M.A.,2006.Effectofcobaltontheformation:growthand developmentofadventitiousrootsintomatoandcucumbercuttings.JAppSci Res.2,423–429.

Gairola,S.,Naidoo,Y.,Bhatt,A.,Nicholas,A.,2009.Aninvestigationofthefoliar trichomesofTetradeniariparia(Hochst.)CoddLamiaceae:animportant medicinalplantofSouthernAfrica.Flora204,325–330.

Gallego,S.M.,Pena,L.B.,Barcia,R.A.,Azpilicueta,C.E.,Lannone,M.F.,Rosales,E.P.,et al.,2012.Unravellingcadmiumtoxicityandtoleranceinplants:insightinto regulatorymechanisms.Environ.Exp.Bot.83,33–46.

Gazim,Z.C.,Demarchi,I.G.,Lonardoni,M.V.C.,Amorim,A.C.L.,Hovell,A.M.C., Rezende,C.M.,Ferreira,G.A.,Lima,E.L.,Cosmo,F.A.,Cortez,D.A.G.,2011. AcaricidalactivityoftheessentialoilfromTetradeniariparia(Lamiaceae)onthe cattletickRhipicephalus(Boophilus)microplus(Acari,Ixodidae).Exp.Parasitol. 129,175–178.

Gazim,Z.C.,Rodrigues,F.,LourencoAmorin,A.C.,deRezende,C.M.,Sokovic,M., Tesevic,V.,etal.,2014.Newnaturalditerpene-typeabietanefromtetradenia ripariaessentialoilwithcytotoxicandantioxidantactivities.Molecules19,514– 524.

Granot,G.,Sikron-Persi,N.,Gaspan,O.,Florentin,A.,Talwara,S.,Paul,L.K.,etal., 2009.Histonemodiﬁcationsassociatedwithdroughttoleranceinthedesert plantZygophyllumdumosumBoiss.Planta231,27–34.

Grant,C.A.,Buckley,W.T.,Bailey,L.D.,Selles,F.,1998.Cadmiumaccumulationin crops.Can.J.PlantSci.78,1–17.

Greenway,H.,Gibbs,J.,2003.Mechanismsofanoxiatoleranceinplants.II.Energy requirementsformaintenanceandenergydistributiontoessentialprocesses. Funct.PlantBiol.30,999–1036.

Gurib-Fakim,A.,2006.Medicinalplants:traditionsofyesterdayanddrugsof tomorrow.Mol.AspectsMed.27,1–93.

Haag-Kerwer,A.,Schafer,H.J.,Heiss,S.,Walter,C.,Rausch,T.,1999.Cadmium exposureinBrassicajunceacausesadeclineintranspirationrateandleaf expansionwithouteffectonphotosynthesis.J.Exp.Bot.50,1827–1835.

Haider,S.,Naithani,V.,Barthwal,J.,Kakkar,P.,2004.Heavymetalcontentinsome therapeuticallyimportantmedicinalplants.Bull.Environ.Contam.Toxicol.72, 119–127.

He,J.L.,Qin,J.J.,Long,L.Y.,Ma,Y.L.,Li,H.,Li,K.,Jiang,X.N.,Liu,T.X.,Polle,A.,Liang,Z. S.,Luo,Z.B.,2011.Netcadmiumfluxandaccumulationrevealtissue-specific oxidativestressanddetoxificationinPopulusxcanescens.Physiol.Plant143, 50–63.

He,J.L.,Li,H.,Luo,J.,Ma,C.F.,Li,S.J.,Qu,L.,Gai,Y.,Jiang,X.N.,Janz,D.,Polle,A.,Tyree, M.,Luo,Z.B.,2013a.Atranscriptomicnetworkunderliesmicrostructuraland physiologicalresponsestocadmiuminPopulusxcanescens.PlantPhysiol.162, 424–439.

He,J.L.,Ma,C.F.,Ma,Y.L.,Li,H.,Kang,J.Q.,Liu,T.X.,Polle,A.,Peng,C.H.,Luo,Z.B., 2013b.Cadmiumtoleranceinsixpoplarspecies.Environ.Sci.Pollut.Res.20, 163–174.

He,J.,Li,H.,Ma,C.,Zhang,Y.,Polle,A.,Rennenberg,H.,Cheng,X.,Luo,Z.-B.,2015. Overexpressionofbacterial<gamma>-glutamylcysteinesynthetasemediates changesincadmiuminﬂuxallocation,anddetoxiﬁcationinpoplar.NewPhytol. 205,240–254.

Julkowska,M.M.,Testerink,C.,2015.Tuningplantsignalingandgrowthtosurvive salt.TrendsPlantSci.20,586–594.

Küpper,H.,Parameswaran,A.,Leitenmaier,B.,Trtilek,M.,Setlik,I.,2007. Cadmium-inducedinhibitionofphotosynthesisandlong-termacclimationtocadmium stressinthehyperaccumulatorThlaspicaerulescens.NewPhytol.175,655–674.

Kieffer,P.,Planchon,S.,Ouﬁr,M.,Ziebel,J.,Dommes,J.,Hoffmann,L.,etal.,2009. Combiningproteomicsandmetaboliteanalysestounravelcadmium stress-responseinpoplarleaves.J.ProteomeRes.8,400–417.

Kim,J.M.,To,T.K.,Ishida,J.,Morosawa,T.,Kawashima,M.,Matsui,A.,etal.,2008. AlterationsoflysinemodiﬁcationsonthehistoneH3N-tailunderdrought stressconditionsinArabidopsisthaliana.PlantCellPhysiol.49,1580–1588.

Krupa,Z.,Öquist,G.,Huner,N.P.A.,1993.Theeffectsofcadmiumonphotosynthesis ofPhaseolusvulgaris—aﬂuorescenceanalysis.Physiol.Plant88,626–630.

(10)

Lawlor,D.W.,2013.Geneticengineeringtoimproveplantperformanceunder drought:physiologicalevaluationofachievements,limitations,and possibilities.J.Exp.Bot.64,83–108.

Lefevre,I.,Vogel-Mikus,K.,Jeromel,L.,Vavpetic,P.,Planchon,S.,Arcon,I.,etal., 2014.Differentialcadmiumandzincdistributioninrelationtotheir physiologicalimpactintheleavesoftheaccumulatingZygophyllumfabagoL. PlantCellEnviron.37,1299–1320.

Liao,W.B.,Huang,G.B.,Yu,J.H.,Zhang,M.L.,2012.Nitricoxideandhydrogen peroxidealleviatedroughtstressinmarigoldexplantsandpromoteits adventitiousrootdevelopment.PlantPhysiol.Biochem.58,6–15.

Lopez-Millan,A.F.,Sagardoy,R.,Solanas,M.,Abadia,A.,Abadia,J.,2009.Cadmium toxicityintomato(Lycopersiconesculentum)plantsgrowninhydroponics. Environ.Exp.Bot.65,376–385.

Luo,F.L.,Nagel,K.A.,Scharr,H.,Zeng,B.,Schurr,U.,Matsubara,S.,2011.Recovery dynamicsofgrowth:photosynthesisandcarbohydrateaccumulationafter de-submergence:acomparisonbetweentwowetlandplantsshowingescapeand quiescencestrategies.Ann.Bot.107,49–63.

Luo,M.,Liu,X.,Singh,P.,Cui,Y.,Zimmerli,L.,Wu,K.,2012.Chromatinmodiﬁcations andremodelinginplantabioticstressresponses.Biochim.Biophys.Acta1819, 129–136.

Lux,A.,Martinka,M.,Vaculik,M.,White,P.J.,2011a.Rootresponsestocadmiumin therhizosphere:areview.J.Exp.Bot.62,21–37.

Lux,A.,Vaculik,M.,Martinka,M.,Liskova,D.,Kulkarni,M.G.,Stirk,W.A.,VanStaden, J.,2011b.Cadmiuminduceshypodermalperidermformationintherootsofthe monocotyledonousmedicinalplantMerwillaplumbea.Ann.Bot.107,285–292.

Maxwell,K.,Johnson,G.N.,2000.Chlorophyllﬂuorescence—apracticalguide.J.Exp. Bot.51,659–668.

Medrano,H.,Escalona,J.M.,Bota,J.,Gulìas,J.,Flexas,J.,2002.Regulationof photoshyntesisofC3plantsinresponsetoprogressivedrought:stomatal conductanceasareferenceparameter.Ann.Bot.89(7),895–905.

Pan,J.,Plant,J.A.,Voulvoulis,N.,Oates,C.J.,Ihlenfeld,C.,2010.Cadmiumlevelsin Europe:implicationsforhumanhealth.Environ.Geochem.Health32, 1–12.

Parmar,P.,Kumari,N.,Sharma,V.,2013.Structuralandfunctionalalterationsin photosyntheticapparatusofplantsundercadmiumstress.Bot.Stud.54,45– 51.

Perfus-Barbeoch,L.,Leonhardt,N.,Vavasseur,A.,Forestier,C.,2002.Heavymetal toxicity:cadmiumpermeatesthroughcalciumchannelsanddisturbstheplant waterstatus.PlantJ.32,539–548.

Pezeshki,S.R.,2001.Wetlandplantresponsestosoilﬂooding.Environ.Exp.Bot.46, 299–312.

Pﬂuger,J.,Wagner,D.,2007.Histonemodiﬁcationsanddynamicregulationof genomeaccessibilityinplants.Curr.Opin.PlantBiol.10,645–652.

Potters,G.,2007.Stress-inducedmorphogenicresponses:growingoutoftrouble? TrendsPlantSci.12,98–105.

Redondo-Gomez,S.,Wharmby,C.,Castillo,J.M.,Mateos-Naranjo,E.,Luque,C.J.,de Cires,A.,etal.,2006.Growthandphotosyntheticresponsestosalinityinan extremehalophyteSarcocorniafruticosa.Physiol.Plant.128,116–124.

Sagardoy,R.,Vazquez,S.,Florez-Sarasa,I.,Albacete,A.,Ribas-Carbo,M.,Flexas,J.,et al.,2010.StomatalandmesophyllconductancestoCO2arethemainlimitations

tophotosynthesisinsugarbeet(Betavulgaris)plantsgrownwithexcesszinc. NewPhytol.187,145–158.

Sandalio,L.M.,Dalurzo,H.C.,Gomez,M.,Romero-Puertas,M.C.,delRio,L.A.,2001. Cadmium-inducedchangesinthegrowthandoxidativemetabolismofpea plants.J.Exp.Bot.52,2115–2126.

Sanz,A.,Llamas,A.,Ullrich,C.I.,2009.DistinctivephytotoxiceffectsofCdandNion membranefunctionality.PlantSignal.Behav.4,980–982.

Sasidharan,R.,Voesenek,L.A.C.J.,2013.Lowlandrice:high-endsubmergence tolerance.NewPhytol.197,1029–1031.

Steffens,B.,Rasmussen,A.,2016.Thephysiologyofadvantageousadventitious roots.PlantPhysiol.170,603–617.

Tsuji,H.,Saika,H.,Tsutsumi,N.,Hirai,A.,Nakazono,M.,2006.Dynamicand reversiblechangesinhistoneH3-Lys4methylationandH3acetylation occurringatsubmergence-induciblegenesinrice.PlantCellPhysiol.47,995– 1003.

Ueno,D.,Kono,I.,Yokosho,K.,Ando,T.,Yano,M.,Ma,J.F.,2009.Amajorquantitative traitlocuscontrollingcadmiumtranslocationinrice(Oryzasativa).NewPhytol. 182,644–653.

Uraguchi,S.,Fujiwara,T.,2012.Cadmiumtransportandtoleranceinrice: perspectivesforreducinggraincadmiumaccumulation.Rice5.doi:http://dx. doi.org/10.1186/1939-8433-5-5.

Uraguchi,S.,Fujiwara,T.,2013.Ricebreaksgroundforcadmium-freecereals.Curr. Opin.PlantBiol.16,328–334.

Waalkes,M.P.,2003.Cadmiumcarcinogenesis.Mutat.Res.Fund.Mol.M533,107– 120.

Wang,Y.,Li,K.,Li,X.,2009.Auxinredistributionmodulatesplasticdevelopmentof rootsystemarchitectureundersaltstressinArabidopsisthaliana.J.PlantPhysiol. 166,1637–1645.

Wellburn,A.R.,1994.Thespectraldeterminationofchlorophyllaandchlorophyllb, aswellastotalcarotenoids,usingvarioussolventswithspectrophotometersof differentresolution.J.PlantPhysiol.144,307–313.

WorldHealthOrganization.(1998).Qualitycontrolmethodsformedicinalplant materials.Geneva,Switzerland.

Wu,H.L.,Chen,C.L.,Du,J.,Liu,H.,Cui,Y.,Zhang,Y.,etal.,2012.Co-overexpressionFIT withAtbHLH38orAtbHLH39inArabidopsis-enhancedcadmiumtolerancevia increasedcadmiumsequestrationinrootsandimprovedironhomeostasisof shoots.PlantPhysiol.158,790–800.

Xu,S.,Zhang,B.,Cao,Z.Y.,Ling,T.F.,Shen,W.B.,2011.Hemeoxygenaseisinvolvedin cobaltchloride-inducedlateralrootdevelopmentintomato.Biometals24, 181– 191.