Running head: EFFECTS OF Cd ON CORK OAK GROWN IN HYDROPONICS. Effects of cadmium on cork oak (Quercus suber L.) plants grown in hydroponics

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1 Running hed: EFFECTS OF Cd ON CORK OAK GROWN IN HYDROPONICS Effects of cdmium on cork ok (Quercus suer L.) plnts grown in hydroponics Yolnd Gogorcen 1,2, Ajmi Lri 3,4, Sofi Andluz 3,5, Rmón O. Crpen 6, Anuncición Adí 3, Jvier Adí 3 Deprtments of Pomology 1 nd Plnt Nutrition 3, Estción Experimentl de Aul Dei, Consejo Superior de Investigciones Científics (CSIC), P.O. Box , E Zrgoz, Spin. 2 Corresponding uthor, Yolnd Gogorcen, Tel: Fx: E-mil ddress: oiz@eed.csic.es. 4 Present ddress: Institut de l Olivier. BP. 208, Tunis. Cité Mhrjène 1082, Tunisi. 5 Present ddress: Zeu-Inmunotec, C/Bri, n 25, Duplicdo, E Zrgoz, Spin. 6 Agriculturl Chemistry Deprtment, Fculty of Sciences, Universidd Autónom de Mdrid, E Mdrid, Spin 1

2 Summry Cork ok (Quercus suer L.) is n utochthonous tree species tht is eing used for reforesttion in hevy metl-contminted res in Spin. A hydroponics experiment ws crried out to chrcterize the effects of Cd on severl morphologicl nd physiologicl prmeters in this species, including shoot length, nutrient concentrtions nd lloction in different orgns, lef pigment concentrtions, photosynthetic efficiency, root ferric chelte reductse ctivity, nd orgnic cid concentrtions in xylem sp. Four different Cd tretments were pplied, dding Cd chelted with EDTA or s chloride slt t two different concentrtions (10 nd 50 µm Cd). After one month of Cd-tretment, plnt growth ws significntly inhiited in ll tretments. Results indicte tht Cd ccumultes in ll orgns 7- to 500-fold when compred to control plnts. The highest Cd concentrtion ws found in the 50 M CdCl 2 tretment, which led to concentrtions of pproximtely 30, 123 nd 1153 µg Cd g -1 DW in leves, stems nd roots, respectively. In the strongest Cd tretments the concentrtions of P nd C decresed in some plnt prts, wheres the Mn lef concentrtions decresed with three of the four Cd tretments pplied. The concentrtions of chlorophyll nd crotenoids on n re sis decresed, wheres the (zexnthin plus ntherxnthin)/(totl violxnthin cycle crotenoids) rtio nd the non-photochemicl quenching incresed significntly in ll Cd tretments. Cdmium tretments cused significnt increses in the ctivity of the enzyme ferric chelte reductse in roots nd in the concentrtions of orgnic cids in the xylem sp. Some of the physiologicl chnges found support tht Cd induces deficiency of Fe in cork ok, lthough the plnt Fe concentrtions were not reduced significntly. At higher concentrtions the effects of Cd were more pronounced, nd were more mrked when Cd ws in the free ion form thn when present in the form of Cd-EDTA. 2

3 Key words: Cdmium, ferric chelte reductse, hevy metls, iron chlorosis, orgnic cids, photosynthetic pigments, PS(II) efficiency. Introduction Cork ok (Quercus suer L.) is sclerophyllous evergreen tree species dpted to dry summer conditions (Fri et l. 1996, Puss et l. 2009), which covers over 2.4 million h in different Mediterrnen countries (Montero nd Cñells 1999). This woody species is exploited for cork production (nnul production over 300,000 t cork) in Portugl nd Spin, in more thn million h of otherwise poor, clcreous soils (Bueno et l. 1992, Montero nd Cñells 1999, Toriio et l. 2005). The knowledge on the physiologicl responses of trees to environmentl chnges is still incomplete, despite the fct tht over two thirds of glol terrestril productivity occurs in forest ecosystems. The existing dt suggest tht long-lived trees re likely more sensitive to environmentl chnges, nd the study of the impct on forest trees of different stresses such s climte chnge nd contmintion due to humn ctivities is necessry to chieve sustinle mngement of forestry resources. One of the mjor environmentl prolems cused y humn ctivities is the contmintion y hevy metls (Nrigu 1990). Cdmium, hevy metl not essentil for plnt metolism, hs ecome mjor environmentl pollutnt, which rises not only from industril processes ut lso from the use of phosphte fertilizers tht contin significnt mounts of Cd (Kopittke et l. 2010). Cdmium is cquired from the rizhosphere y plnts minly vi divlent metl trnsporters (for reviews see DlCorso et l. 2008, Verruggen et l. 2009). Once in the plnt, Cd could e trnsferred to the food chin, constituting potentil thret for humn helth (Dudk nd Miller 1999, Moulis nd Thévenod 2010, Thévenod 2010). Therefore, there is need to understnd 3

4 the role of plnts in Cd-contminted environments (Norderg 2004). The effects of Cd toxicity on the physiology of plnts hve een studied under different viewpoints, generlly using crop plnt species (López-Millán et l. 2009, Rodríguez-Celm et l. 2010). Woody plnts hve the potentil to ct s long-term sinks of hevy metls. Although numer of studies hve een crried out with woody plnts treted with hevy metls, including Cd (Arduini et l. 1996, Kim et l. 2003, Cpun 2011, Domínguez et l. 2011), the only pulished study on the effects of hevy metls on cork ok ws crried out using Zn (Disnte et l. 2011). The complexity of the toxicity effects of Cd on plnts mkes it difficult to distinguish etween direct nd indirect mechnisms of ction (Bszynski 1986, Brceló nd Poschenrieder 1990, Krup nd Bszynski 1995, Siedleck nd Krup 1999, Ae et l. 2008). One of the most common symptoms of Cd toxicity is lef chlorosis, nd this is generlly ttriuted to the interction etween Cd nd Fe (Siedleck nd Bszynski 1993, Jlil et l. 1994, López-Millán et l. 2009). Cdmium exposure leds to serious disturnces of physiologicl processes, such s n inhiition of chlorophyll synthesis, diminution of the pools of Fe-contining electron crriers in the photosynthetic pprtus nd decresed growth of roots nd leves (Miller et l. 1984, Vázquez et l. 1992, Siedleck nd Bszynski 1993, Krup nd Bszynski 1995, Fodor et l. 2005, DlCorso et l. 2008, Gonçlves et l. 2009, Sndlio et l. 2009). Also, it hs een demonstrted tht the chnges in thylkoid structure nd composition re similr in Cdtreted nd Fe-deficient poplr plnts (Sárvári et l. 2001, 2011). Cdmium is usully immoile in soils, nd removl nd replcement of contminted soils is very expensive (c. 3 million $ h -1 ) nd, therefore, unrelistic in view of the widespred occurrence of Cd in the environment (Moulis nd Thévenod 2010). Therefore, phytoremedition mngement strtegies, including phytoextrction nd 4

5 phytostiliztion cn e n lterntive. Phytoextrction with species ccumulting lrge mounts of Cd cn provide low cost remedition (Chney et l. 2004). Phytoremedition strtegies for hevy metl pollutnts hve focused on hyperccumultion plnts, lthough tree species such s poplr or willow hve een used s well for this purpose (Cpun 2011). In Spin, studies with woody species re eing developed in contminted res long the Gudimr River, ner the Doñn Ntionl Prk, which were contminted y the 1998 toxic wste spill (Crrscl et l. 2008). The trce element ccumultion hs een mesured in severl woody plnt species in the re, ut cork ok ws not mong the species studied (Domínguez et l. 2008, 2009, 2010, 2011). However, cork ok is one of the utochthonous species tht is eing used to reforest lnd in this contminted re (Grrido 2008). The im of this work ws to provide etter understnding of the physiologicl responses of cork ok (Quercus suer L.) to Cd. Cork ok plnts were grown in nutrient solution with excess Cd, nd the effects were compred with those of Fe deficiency in the sme species (Gogorcen et l. 2001). Results otined indicte tht the growth of cork ok is lredy ffected t low concentrtions of Cd, nd lso tht this species cn withstnd high Cd concentrtions in the growth medium. Also, signs typicl of Fe deficiency pper in cork ok treted with Cd. Results otined provide prcticl informtion for the possile use of cork ok in the reforesttion of contminted res in Mediterrnen forests. Mterils nd Methods Plnt Culture Cork ok (Quercus suer L.) corns were germinted in plstic continers, filled with wet snd, t 22ºC nd during dys. When roots were pproximtely 2-3 cm long 5

6 nd the first two leves were developed, plnts were trnsferred to continuously erted hydroponics nutrient solution, nd grown for pproximtely 15 dys in the conditions indicted in Gogorcen et l. (2001). The nutrient solution hd the following composition: (in mm) 2.5 C(NO 3 ) 2, 2.5 KNO 3, 1 MgSO 4, 1KH 2 PO 4, nd (in µm) 23.1 H 3 BO 3, 4.6 MnCl 2, 0.19 CuSO 4, 1.2 ZnSO 4, 0.12 N 2 MoO 4 nd 45 Fe(III)-EDTA, ph 5.5. Then, solutions were supplemented with one of the following Cd tretments: 0 Cd (control), 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 or 50 µm CdCl 2. These Cd doses were chosen sed in previous experiments crried out with nnul plnts (Lri et l. 2002). The tretments were mintined for four weeks, djusting the ph to 5.5 every two dys nd renewing nutrient solutions t dy 15. Plnts were grown t photosynthetic photon flux density (PPFD) of µmol m -2 s -1 t 22 C, 80% reltive humidity nd photoperiod of 16 h light/ 8 h drk. Shoot length (in cm) ws mesured just efore imposing the hevy metl tretments nd gin four weeks lter in ech tretment. All experiments were crried out with t lest four iologicl replictes from t lest two independent plnt tches. Chemicl specition studies The different metl species nd concentrtions present in the solutions were estimted using MINTEQA2 softwre v (US Environmentl Protection Agency, Wshington DC). This kind of softwre is le to predict the chemicl species present nd their concentrtions, using known chemicl constnts s well s ph nd ionic force vlues, lwys considering the system hs reched chemicl equilirium. Anlysis of minerl nutrients nd cdmium The minerl composition of ech plnt frction (leves, stems nd roots) ws determined s descried previously (AOAC 1990, Igrtu et l. 2000). Roots were wshed thoroughly, first with nlyticl-grde type II wter (Milli-RX, Millipore, 6

7 Bedford, MA, USA), then with slightly cidified pure wter (with few drops of HCl) nd finlly gin with type II wter (Milli-Q, Millipore). Smples were dried in n oven t 60 C for 48 hours. Potssium ws mesured y flme emission spectroscopy, nd C (fter L ddition), Mg, Fe, Mn, Cu nd Zn were mesured y flme tomic sorption spectrophotometry. Results were expressed s percentge of dry weight (DW) for mcronutrients (P, K, Mg nd C) nd s µg g -1 of DW for micronutrients (Fe, Mn, Cu nd Zn). For Cd determintion, smples (leves, stems nd roots) were dried t 60 C nd ground, nd digestion of dried mteril nd nlysis were mde s descried in Lozno-Rodríguez et l. (1995). Results were expressed s µg Cd g -1 DW. Smples for minerl nlysis were tken pooling mteril from severl plnts. Dt shown re mens ± SE of four iologicl smples, ech from n independent plnt tch. Photosynthetic pigment nlysis After four weeks of the imposition of Cd tretment, lef disks were cut from the 2 nd nd 3 rd fully-expnded leves with clirted cork orer, wrpped in luminum foil, frozen in liquid-n 2, nd stored t -20 C. Lef pigments were lter extrcted with cetone in the presence of N scorte nd stored s descried previously (Adí nd Adí 1993). Pigment extrcts were thwed on ice, filtered through 0.45 µm filter nd nlyzed y HPLC-Vis (Lri et l. 2004). Smples were tken pooling lef disks from severl plnts. Dt shown re mens ± SE of smples, otined from plnts grown in four different tches. Modulted chlorophyll fluorescence nlysis Modulted chlorophyll (Chl) fluorescence mesurements were mde in ttched leves in the growth chmer with PAM 2000 portle fluorometer (H. Wlz, Effeltrich, Germny), s descried in detil in Lri et l. (2004) nd references therein. F O nd F O (miniml Chl fluorescence yield in the drk or during energiztion, respectively), 7

8 were mesured in presence of fr-red light (7 µmol photons m -2 s -1 ) in order to fully oxidize the PSII cceptor side (Belkhodj et l. 1998). F M nd F M were the mximl Chl fluorescence yield in the drk or during energiztion, respectively, nd F V nd F V were clculted s F M -F O nd F M -F O, respectively. The drk-dpted, mximum potentil PSII efficiency ws clculted s F V /F M. The ctul ( PSII ) nd intrinsic PSII efficiency ( exc. ) were clculted s (F M -F S )/F M nd F V /F M, respectively, photochemicl quenching (qp) ws clculted s (F M -F S )/F V, non-photochemicl quenching (NPQ) ws clculted s (F M /F M )-1, nd the frction of light sored y PSII dissipted in the ntenn (D, identicl to 1- exc. ) ws estimted s in Demmig- Adms et l. (1996). Dt shown re mens ± SE of smples (leves), otined from plnts grown in five different tches. In vivo root ferric chelte reductse ctivity The root FC-R ctivity ws ssessed y mesuring the formtion of the Fe(II)- thophennthroline-disulfonic cid (BPDS 3 ) complex from Fe(III)-EDTA. Excised root tips, 2-3 cm long, were plced in the drk in Eppendorf tues contining 930 µl 10 mm MES, ph 5.5, 400 µm BPDS nd 500 µm Fe (III)-EDTA. After 10 min, roots were removed, the solution centrifuged nd the sornce of the superntnt ws mesured t 535 nm s descried in detil in Gogorcen et l. (2001). The smple used consisted of severl root tips from different plnts. Dt re mens ± SE of 3-7 smples from two independent plnt tches. Xylem sp isoltion Xylem sp ws otined from plnts grown four weeks under the different Cd tretments. Plnts, pproximtely cm in length, were devoid of roots nd rought to the lortory immersed in wter to prevent drying. The cutting ws devoid of the sl 3-4 cm of the rk next to the cut surfce, which ws then rinsed with type II 8

9 wter. The cutting ws then plced in Schölnder chmer with the distl end, including leves, inside the pressure chmer. Pressure ws then pplied in step-wise mnner, to pproximtely r. Xylem sp ws collected for 3-5 min in Eppendorf tues, fter discrding the first few drops of exudte to void contmintion (Engels nd Mrschner 1992). Totl xylem sp volume collected ws pproximtely 500 µl per plnt. Mlte dehydrogense ctivity (MDH, EC ) ws used s cytosolic contmintion mrker. The level of contmintion found ws lwys elow the limit of detection. Orgnic cid nlysis Xylem sp smples were first filtered with 0.5 µm filter (LIDA, Kenhos, WI, USA), nd orgnic cids were nlysed y HPLC-UV using 300 x 7.8 mm Aminex ionexchnge column (HPX-87H from Bio-Rd, Hercules, CA, USA) using the method descried elsewhere (López-Millán et l. 2000). Peks corresponding to citrte, mlte, mlonte, fumrte nd oxlte were identified y comprison of their retention times with those of known stndrds from Bio-Rd nd Sigm-Aldrich (St Louis, MO, USA). Dt shown re mens ± SE of 4-11 replictes from three independent plnt tches. Sttisticl nlysis Sttisticl nlyses were crried out y one fctor ANOVA, using the SPSS v softwre (Chicgo, IL, USA). For ANOVA nlysis, we tested normlity, homogeneity of vrinces nd independency. Normlity ws tested using Kolmogorov-Smirnov nd Shpiro-Wilk tests nd the homocedsticity using Levene test. Only the vrile Finl shoot length ws trnsformed nd nlyzed s ln to correct heterocedsticity. A Duncn s test ws used to seprte mens otined from different tretments. The specific numer of replictes nd sttisticl significtions re indicted in ech tle nd figure cption. 9

10 Results Chemicl specition In the CdCl 2 tretments, the mjor Cd chemicl species predicted to occur in the nutrient solution ws free Cd 2+, ccounting for pproximtely 86-87% of totl Cd (Tle 1); in these tretments, CdSO 0 4 AQ ccounted for pproximtely 7% of totl Cd. In the Cd(II)-EDTA tretments, the mjor chemicl species predicted ws Cd(II)- EDTA 2-, ccounting for pproximtely 86 nd 96 % of totl Cd in the 10 nd 50 µm Cd tretments, respectively (Tle 1). Free Cd 2+ in the 10 µm nd 50 µm Cd(II)-EDTA tretments ccounted for pproximtely 12 nd 3%, respectively. Therefore, the 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 M CdCl 2 nd 50 µm CdCl 2 tretments led to estimted free Cd 2+ concentrtions of 1.2, 1.6, 8.6 nd 43.3 µm, respectively. Plnt growth All Cd tretments hmpered plnt growth compred to control plnts, lthough root morphology ws not ffected y the hevy metl tretments used (not shown). Before imposing Cd stress (fter two weeks of growth under controlled conditions), plnts were pproximtely cm in height, nd no significnt differences were found mong plnts selected for the different tretments (Tle 2). At the end of the Cd tretment period, four weeks lter, plnts hd grown pproximtely 7.9, 2.8, 2.6, 1.6 nd <0.1 cm in the 0 Cd, 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2 tretments, respectively, indicting tht growth hd prcticlly cesed in the 50 M CdCl 2 tretment nd ws significntly decresed in the rest of the Cd tretments (Tle 2). Plnt Cd concentrtions 10

11 Cdmium tretments induced increses in the Cd concentrtions in leves, stems nd especilly roots of cork ok plnts (Figure 1). The highest Cd concentrtions in ll orgns were found with the 50 M CdCl 2 tretment, which led to concentrtions (in leves/stems/roots) of pproximtely 30/123/1153 µg Cd g -1 DW, respectively (Figure 1). In the rest of the Cd tretments Cd concentrtions were not s high: in the 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA nd 10 µm CdCl 2 tretments, concentrtions (leves/stems/roots) were 7/11/28, 20/43/65 nd 8/12/65 µg Cd g -1 DW, respectively (Figure 1). Plnt minerl composition Cdmium tretments induced chnges in the concentrtions of severl nutrients in different cork ok tissues. The concentrtions of P decresed in leves, stems nd roots in the strongest Cd tretments when compred to the controls, with differences eing sttisticlly significnt in the 50 µm CdCl 2 tretment in ll mterils, nd in leves nd roots in the 50 µm Cd(II)-EDTA tretment (Figure 2). The concentrtions of C decresed significntly in leves nd stems in the 50 µm CdCl 2 tretment nd in leves in the 50 µm Cd(II)-EDTA tretment. Potssium nd Mg concentrtions did not chnge significntly with Cd tretments: K concentrtions were in the rnges , nd % DW in leves, stems nd roots, respectively, wheres Mg concentrtions in the sme smples were in the rnges , nd % DW (dt not shown). Iron concentrtions did not chnge significntly with Cd tretments, eing in the rnges 48-58, nd µg Fe g -1 DW in leves, stems nd roots, respectively (dt not shown). Similr lef Fe concentrtions (51 nd 45 µg Fe g -1 DW) were found in control nd mild Fe-deficiency conditions in the sme species, wheres in 11

12 young, severely yellow leves Fe concentrtions were pproximtely 26 µg Fe g -1 DW (Gogorcen et l. 2001). Mngnese concentrtions decresed significntly in leves in the 50 µm Cd(II)-EDTA nd the 10 nd 50 µm CdCl 2 tretments, without reching deficiency levels. On the other hnd, Mn concentrtion incresed in roots in the 10 µm CdCl 2 tretment, wheres stem Mn concentrtions were not ffected (Figure 2). Copper concentrtions were unchnged y Cd tretments in leves nd incresed significntly in stems nd roots in the 50 M CdCl 2 tretment (Figure 2). Zinc concentrtions decresed significntly in leves in the 10 nd 50 µm Cd(II)-EDTA nd 50 M CdCl 2 tretments nd in stems in the 10 nd 50 µm Cd(II)-EDTA nd 10 µm CdCl 2 tretments, nd incresed in roots with 50 M CdCl 2 (Figure 2). Lef photosynthetic pigment composition Cdmium toxicity led to visul symptoms of chlorosis in young leves, wheres old leves remined green. The picl prt of shoots grew in rosettes, especilly in the tretments with Cd(II)-EDTA. The concentrtions per unit re of ll mjor photosynthetic pigments were similrly ffected y ll Cd tretments, with decreses of 45-52% for totl Chl nd 40-47% for crotenoids (Tle 3). However, when considering the violxnthin cycle pigments, violxnthin (V) plus ntherxnthin (A) nd zexnthin (Z), therefter referred to s (V+A+Z) pigments, no significnt chnges were found in the Cd tretments when compred to the control, lthough the differences in the (V+A+Z) concentrtions found etween the 10 nd 50 M Cd(II)-EDTA were sttisticlly significnt (Tle 3). On the other hnd, the Chl / rtio incresed significntly in the leves of plnts treted with 10 nd 50 M Cd(II)-EDTA, from 3.3 to (Tle 3). The rtios (V+A+Z) pigments/totl Chl incresed significntly in ll Cd tretments, wheres the (Z+A)/(V+A+Z) rtios showed increses with Cd, 12

13 lthough in the 50 µm Cd(II)-EDTA tretment chnges were not sttisticlly significnt t P<0.05 (Tle 3). Chlorophyll fluorescence prmeters Cdmium tretments induced moderte decrese (9-18%) in the drk-dpted efficiency of PSII, ssessed y the F V /F M rtio, when compred to control vlues (Figure 3A). The PSII efficiency t stedy-stte photosynthesis ( PSII ) ws lso decresed y pproximtely 30-50% y ll Cd tretments (Figure 3B). This decrese ws ssocited with 15-30% decrese in intrinsic PSII efficiency ( exc ) (Figure 3C) nd 14-27% decrese in the proportion of open, oxidized PSII rection centers (estimted y qp, Figure 3D). The decreses were most mrked in the 50 M CdCl 2 tretment (18, 50, 30 nd 27% for F V /F M, PSII, exc nd qp, respectively). On the other hnd, non-photochemicl quenching (NPQ) incresed with ll Cd tretments (y %) when compred to control vlues, with the lrgest increse eing found in the 50 M CdCl 2 tretment (Figure 3E). Root Fe(III)-chelte reductse ctivity All Cd tretments induced sttisticlly significnt increses in the FCR ctivity of cork ok roots when compred to control plnt vlues (Figure 4). Increses were in the rnge 2.0- to 2.4-fold in the 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA nd 10 µm CdCl 2 tretments, nd pproximtely 4-fold in the 50 µm CdCl 2 tretment (Figure 4). Orgnic cid concentrtions in xylem sp The concentrtions of the orgnic cids citrte, mlte, mlonte, oxlte nd fumrte were determined in xylem sp of cork ok. Significnt xylem sp increses were found for mlonte in the 10 µm Cd(II)-EDTA tretment, for citrte in the 10 µm CdCl 2 13

14 tretment nd for citrte, mlte nd mlonte in the 50 µm CdCl 2 tretment (Figure 5). No significnt chnges in xylem sp croxyltes were found in the 50 µm Cd(II)- EDTA tretment (Figure 5). Fumrte nd oxlte xylem sp concentrtions did not chnge significntly with Cd (oxlte concentrtions were in the rnge µm, dt not shown). Discussion The results indicte tht the growth nd physiology of cork ok is lredy ffected t low concentrtions of Cd in the nutrient solution. The lightest Cd tretments used, 10 µm Cd(II)-EDTA (corresponding to pproximtely 1 µm free Cd 2+ in the nutrient solution), led to very modest increse in lef Cd concentrtion (7 µg Cd g -1 DW) ut lredy resulted in mjor decreses in shoot growth (64% when compred to control vlues), significnt decreses in the lef concentrtions of photosynthetic pigments (lef chlorosis), s well s increses in the reltive mounts nd de-epoxidtion of protective xnthophyll cycle pigments, chnges in chlorophyll fluorescence nd increses in root ferric chelte reductse. The reltively low lef Cd concentrtions found support tht t low Cd concentrtions cork ok plnts cn void mjor metl uptke, wheres developing significnt physiologicl responses to overcome stress. However, cork ok ppers to e plnt species tht cn withstnd high Cd concentrtions in the growth medium. Further increses in Cd in the nutrient solution (50 µm Cd(II)-EDTA nd 10 µm CdCl 2, corresponding to pproximtely 2 nd 9 µm free Cd 2+ in the nutrient solution, respectively) led to still moderte lef Cd concentrtions (20 nd 8 µg Cd g -1 DW in the 50 µm Cd(II)-EDTA nd 10 µm CdCl 2 tretments, respectively), nd resulted in further decreses in plnt growth (67-80% when compred to control vlues), wheres chnges in photosynthetic pigments nd chlorophyll fluorescence were very similr to those oserved with the lightest Cd 14

15 tretment. Cdmium concentrtions found in roots nd eril prts of cork ok were much lower thn those found in poplr grown with 10 µm Cd(NO 3 ) 2 (Fodor et l. 2005). The hrshest Cd tretment, 50 µm CdCl 2 (corresponding to 43 µm free Cd 2+ ) led to complete cesstion of growth, nd to the highest Cd concentrtions in cork ok leves (30 µg Cd g -1 DW), stems nd roots. This strong Cd tretment led to increses in root FCR ctivity, increses in xylem sp croxyltes, nd decreses in P nd C concentrtions. A deleterious effect of Cd on growth hs een found in other plnts, including nnul nd woody species. In sugr eet plnts grown with similr Cd tretments in nutrient solution, growth ws lso more ffected with 50 M CdCl 2 (pproximtely 43 µm free Cd 2+ ) thn with µm Cd(II)-EDTA nd 10 µm CdCl 2 (1-8 µm free Cd 2+ ) (Lri et l. 2002). In other plnt species such s tomto, poplr, Holm ok or Pinus sylvestris, plnt growth ws lso mrkedly reduced with Cd stress (Morl et l. 1994, Kim et l. 2003, Fodor et l. 2005, López-Millán et l. 2009, Domínguez et l. 2011, Sárvári et l. 2011). In Holm ok, iomss ws lso ltered y exposure to Cd in controlled conditions, lthough seedlings showed reltively high tolernce to the extremely high Cd ccumultion in the roots nd in the leves (Domínguez et l. 2011). Cork ok plnts seem to e very efficient in preventing lrge mounts of Cd to enter the plnt. The Cd concentrtions found in the eril prt of cork ok (7-20 nd µg Cd g -1 DW in leves nd stems, respectively) were similr to those found in Holm ok (Domínguez et l. 2011) ut mrkedly lower thn those found with the sme tretments in sugr eet (>200 µg Cd g -1 DW in ll tretments; Lri et l. 2002), nd lso much lower thn the concentrtions found in tomto with µm CdCl 2 ( µg Cd g -1 DW; López-Millán et l. 2009) nd in poplr with 10 µm Cd(NO 3 ) 2 (>400 µg Cd g -1 DW; Fodor et l. 2005). Concerning root Cd concentrtions, they were 15

16 only high in the 50 µm CdCl 2 tretment, wheres in the other tretments they were quite low, in the rnge µg Cd g -1 DW, vlues much lower thn those found with the sme externl Cd concentrtions in roots of sugr eet ( µg Cd g -1 DW; Lri et l. 2002), tomto (1600 µg Cd g -1 DW; López-Millán et l. 2009) nd poplr (>1300 µg Cd g -1 DW; Fodor et l. 2005). Cork ok hs lso een reported to exclude Zn, since plnts grown with 50 µm Zn in the nutrient solution led to only modertely high lef Zn concentrtions (75 µg Zn g -1 DW; Disnte et l. 2011), vlues much lower thn the Zn concentrtions found in similr conditions in sugr eet leves (240 µg Zn g -1 DW; Sgrdoy et l. 2010). In cork ok, plnt Fe concentrtions were unffected y Cd, in spite of the ctivtion of the mechnisms for Fe root cquisition nd xylem trnsport (see elow). Similr lef Fe concentrtions (51 nd 45 µg Fe g -1 DW) were found in control nd mild Fedeficiency conditions in the sme species, wheres in young, severely yellow leves Fe concentrtions were pproximtely 26 µg Fe g -1 DW (Gogorcen et l. 2001). Decreses in lef Mn concentrtions were found, ut concentrtions did not rech deficiency levels. Disturnces in the uptke nd distriution of micronutrients with Cd toxicity hve een found in other species. A decrese in lef Mn with Cd ws lso oserved in other plnt species grown with Cd (Hernández et l. 1998, Lri et l. 2002, Clemens 2006, Rodríguez-Serrno et l. 2009, Kopittke et l. 2010). An ccumultion of Cu nd Zn in roots hs een oserved in sugr eet nd tomto plnts grown with CdCl 2 (Lri et l. 2002, López-Millán et l. 2009). The decreses in Mn (in leves) nd Zn (leves nd stems), nd the increses in Cu (roots nd stems) nd Zn (roots) re likely e ssocited to competition with Cd in some step(s) of the divlent metl cquisition or trnsport processes, since mny metl trnsporters cn work with different divlent metl ions (Küpper nd Kochin 2010, Solti et l. 2011). 16

17 Although Cd tretments led to some decreses in the P nd C concentrtions in cork ok, the miniml vlues otined were pproximtely 0.2% nd 0.5% DW, respectively, within the rnges common in other Quercus species (Benton Jones et l. 1991), nd therefore do not suggest the presence of deficiency. A deficiency of P hs lso een found in leves of Quercus ilex in soils contminted with Cd (Domínguez et l. 2010). The reltively low C concentrtion in leves nd stems of cork ok in the presence of Cd is likely to e due to the known competition etween C nd Cd in the C plsm memrne trnsporters (Clemens 2006, Rodríguez-Serrno et l. 2009, Solti et l. 2011). Chnges in the concentrtions of mcronutrients with Cd hve een reported in other species. For instnce, the lef concentrtions of C nd Mg decresed in pe plnts grown with 50 µm CdCl 2 (Rodríguez-Serrno et l. 2009), nd C nd K concentrtions decresed with Cd toxicity in needles of Pinus sylvestris (Kim et l. 2003) nd leves of ens nd tomto plnts (Ouriti et l. 1997). The photosynthetic pigment nd chlorophyll fluorescence responses of cork ok to Cd stress indicte moderte photosynthetic pprtus stress, nd er some similrities with those occurring in the sme species with Fe deficiency. In Fe-deficient cork ok plnts, mrked decrese (87%) of photosynthetic pigments on n re sis ws found, long with significnt Chl / rtio increses (from 3.1 to 4.8; Gogorcen et l. 2001). The reltive increse in the mount of protective pigments s well s the functioning of the (V+A+Z) cycle, demonstrted y the increses in the (V+A+Z) pigments/totl Chl nd (Z+A)/(V+A+Z) rtios, often occurs in response to Fe deficiency (Lri et l. 2006) nd hs lso een found in Cd stress in sugr eet (Lri et l. 2002, Chng et l. 2003), potto (Gonçlves et l. 2009) nd tomto (López-Millán et l. 2009). In the cse of cork ok, the increses in the Chl /, (V+A+Z) pigments/totl Chl nd (Z+A)/(V+A+Z) rtios with Cd were less mrked thn those found with Fe deficiency 17

18 (Gogorcen et l. 2001). Also, the decreses in photosynthetic pigments, including Chl, were similr in ll Cd tretments nd much lower thn those present in the sme species upon Fe deficiency (Gogorcen et l. 2001). On the other hnd, chnges found in chlorophyll fluorescence were similr to those found in Fe-deficient cork ok plnts (8, 43, 21 nd 24% decreses for F V /F M, PSII, exc nd qp, respectively, nd 38% increses in NPQ; Gogorcen et l. 2001), Cd-treted sugr eet (Lri et l. 2002) nd Holm ok (Domínguez et l. 2011). These chnges in Chl fluorescence prmeters indicte tht high Cd in the nutrient solution leds to n impired PSII ctivity, which occurs concomitntly to reltive increse in the mount of (V+A+Z) pigments per Chl nd to n increse in the de-epoxidtion stte of the violxnthin cycle pigments [s indicted y the (Z+A)/(V+A+Z) rtio], similrly to wht occurs with Fe deficiency (Lri et l nd references therein). Similr decreses were found in F V /F M in cork ok when plnts were treted with Zn concentrtions over 150 µm (Disnte et l. 2011). The increses in root FCR ctivity nd xylem sp croxyltes point out to the presence of Cd-induced Fe shortge, even though demnd for Fe ws low due to stopped growth nd plnt Fe concentrtions were still within the dequte rnge. Chnges in root FCR ctivities found were similr to those found in cork ok grown in Fe deficiency conditions (4-fold increses in FCR, Gogorcen et l. 2001). Also, the root FCR ctivity showed similr increses in sugr eet plnts treted during 7-10 dys with Cd (Lri et l. 2002, Chng et l. 2003). However, in tomto plnts grown with 10 nd 100 M CdCl 2 the root FCR ctivity decresed when compred to the controls (López-Millán et l. 2009). On the other hnd, increses in croxyltes, especilly citrte nd mlte, re known to occur in the xylem sp of mny plnts under Fe deficiency (Nikolic nd Römheld 1999, Adí et l. 2002, Romolà et l. 2002, López- Millán et l. 2009, Lri et l. 2010, Rellán et l. 2010, Adí et l. 2011) nd Zn 18

19 toxicity (Sgrdoy et l. 2011). Increses in the mount nd ctivity of enzymes involved in croxylte synthesis hve een found in roots with Fe deficiency (Adí et l. 2011), Cd toxicity (López-Millán et l. 2009, Rodríguez-Celm et l. 2010) nd Zn toxicity (Sgrdoy et l. 2011). Regrding the possile use of cork ok to reforest metl contminted res, results indicte tht t low Cd concentrtions cork ok my e used to cover the lnd (i.e., in phytostiliztion prctices), lthough significnt decreses in tree growth cn e expected. The low concentrtions of Cd in ll plnt prts nd the cesstion of growth found t high Cd concentrtions strongly suggest tht cork ok is not suitle for extrcting lrge mounts of metls. In ny cse, if cork ok is grown in contminted res trees re likely to hve reltively low Cd concentrtions in the oveground prts, therefore limiting dverse effects on the environment. Further experiments with dult trees should e envisged to identify the mechnism y which Cd is excluded. Acknowledgments We grtefully cknowledge A. Poc for technicl support nd R. Giménez for her help with figures. In memorim of Isidoro Gogorcen who pssed wy while this work ws crried out. Funding Supported y the Spnish Ministry of Science nd Innovtion (MICINN, projects AGL nd AGL , co-finnced with FEDER), nd the Argón Government (groups A03 nd A44). 19

20 References Adí, J. nd A. Adí Iron nd plnts pigments. In Iron Cheltion in Plnts nd Soil Microorgnisms. Eds. L. Brton nd B. Hemming. Acdemic Press, Sn Diego, pp Adí, J., A.F. López-Millán, A. Romolà nd A. Adí Orgnic cids nd Fe deficiency: review. Plnt Soil 241: Adí, J., S. Vázquez, R. Rellán-Álvrez, H. El-Jendoui, A. Adí, A. Álvrez- Fernández nd A.F. López-Millán Towrds knowledge-sed correction of iron chlorosis. Plnt Physiol. Biochem. 49: Ae, T., M. Fukmind nd M. Ogswr Cdmium ccumultion in the shoot nd roots of 93 weed species. Soil Sci. Plnt Nutr. 54: A.O.A.C Plnts. In Officil Methods of Anlysis of the Assocition of Officil Anlyticl Chemists. Ed. K Hedrich. Acdemic Press, Wshington DC, pp Arduini I., D.L. Godold nd A. Onnis Cdmium nd copper uptke nd distriution in Mediterrnen tree seedlings. Physiol. Plnt. 97: Brceló, J. nd Ch. Poschenrieder Plnt wter reltions s ffected y hevy metl stress: review. J. Plnt Nutr. 13:1-37. Bszynski, T Interference of Cd in functioning of the photosynthetic pprtus of higher plnts. Act Soc. Bot. Polon., 55: Belkhodj, R., F. Morles, R. Quílez, A.F. López-Millán, A. Adí nd J. Adí Iron deficiency cuses chnges in chlorophyll fluorescence due to the reduction in the drk of the photosystem II cceptor side. Photosynth. Res. 25:

21 Benton Jones, J., H.A. Mills nd B. Wolf Plnt Anlysis Hndook: A Prcticl Smpling, Preprtion, Anlysis nd Interprettion Guide. Micro-Mcro. Pulishing, Athens, GA. USA, 213 p. Bueno, M.A., R. Astorg nd J.A. Mnzner Plnt regenertion thought somtic emryogenesis in Quercus suer. Physiol. Plnt. 85: Cpun, M Hevy metls nd woody plnts-iotechnologies for phytoremedition. iforest 4:7-15. Crrscl, F., L. Cordero nd I. Redondo Corredor verde del Gudimr. Blnce de 10 ños de resturción. Ed. Consejerí de Medio Amiente. Junt de Andlucí, Spin, 12 p. Chney, R.L., P.G. Reeves, J.A. Ryn, R.W. Simmons, R.M. Welch nd J.S. Angle An improved understnding of soil Cd risk to humns nd low cost methods to phytoextrct Cd from contminted soils to prevent soil Cd risks. Biometls 17: Chng, Y.C., M. Zouri, Y. Gogorcen, J.J. Lucen nd J. Adí Effect of cdmium nd led on ferric chelte reductse ctivities in sugr eet roots. Plnt Physiol. Biochem. 41: Clemens, S Toxic metl ccumultion, responses to exposure nd mechnisms of tolernce in plnts. Biochimie 88: DlCorso, G., S. Frinti, S. Mistri nd A. Furini How plnts cope with cdmium: stking ll on metolism nd gene expression. J. Integrt. Plnt Biol. 50: Disnte, K.B., D. Fuentes nd J. Cortin Response to drought of Zn-stressed Quercus suer L. seedlings. Environ. Exp. Bot. 70:

22 Demmig-Adms, B, W.W. Adms III, D.H. Brker, B.A, Logn, D.R. Bowling nd A.S. Verhoeven Using chlorophyll fluorescence to ssess the frction of sored light llocted to therml energy dissiption of excess excittion. Physiol. Plnt. 98: Domínguez, M.T., T. Mrñón, J.M. Murillo, R. Schulin nd B.H. Roinson Trce element ccumultion in woody plnts of the Gudimr Vlley, SW Spin: A lrge-scle phytomngement cse study. Environ. Pollut.152: Domínguez, M.T., F. Mdrid, T. Mrñón nd J.M. Murillo Cdmium iovilility in soils nd ccumultion in Holm ok roots: potentil for phytostiliztion. Chemosphere 76: Domínguez, M.T., T. Mrñón, J.M. Murillo, R. Schulin nd B.H. Roinson Nutritionl sttus of Mediterrnen trees Growing in contminted nd remedited re. Wter Air Soil Pollut. 205: Domínguez, M.T., T. Mrñón, J.M. Murillo nd S. Redondo-Gómez Response of Holm ok (Quercus ilex susp. llot) nd mstic shru (Pistci lentiscus L.) seedlings to high concentrtions of Cd nd Tl in the rizhosphere. Chemosphere 83: Dudk, S. nd W.P. Miller Accumultion of potentilly toxic elements in plnts nd their trnsfer to humn food chin. J. Environ. Sci. Helth B 34: Engels, C. nd H. Mrschner Adpttion of potssium trnsloction into the shoot mize (Ze mys L.) to shoot demnd: Evidence for xylem loding s regulting step. Physiol. Plnt. 86: Fri, T., J.I. Grcí-Plzol, A. Adí, S. Cersoli, J.S. Pereir nd M.M. Chves Diurnl chnges in photoprotective mechnisms in leves of cork ok (Quercus suer) during summer. Tree Physiol. 16:

23 Fodor, F., L. Gspr, F. Morles, Y. Gogorcen, J.J. Lucen, E. Cseh, K. Kröpfl, J. Adí nd E. Sárvári Effects of two iron sources on iron nd cdmium lloction in poplr (Populus l L.) plnts exposed to cdmium. Tree Physiol. 25: Grrido, H Gudimr, cienci, técnic y resturción. El ccidente minero 10 ños después. Ed. Consejo Superior de Investigciones Científics. Mdrid, Spin, 207 p. Gogorcen, Y., N. Molís, A. Lri, A. Adí nd J. Adí Chrcteristion of the responses of cork ok (Quercus suer) to iron deficiency. Tree Physiol. 21: Gonçlves, J.F., L.A. Tldi, D. Crgnelutti, L.B. Pereir, J. Mldner, A.G. Becker, L.V. Rossto, R. Ruer, M.D. Bgtini, D.A. Bisognin, M.R.C. Schetinger nd F.T. Nicoloso Cdmium-induced oxidtive stress in two potto cultivrs. Biometls 22: Hernández, L.E., E. Lozno-Rodríguez, A. Gárte nd R. Crpen-Ruiz Influence of cdmium on the uptke, tissue ccumultion nd sucellulr distriution of mngnese in pe seedlings. Plnt Sci. 132: Igrtu, E., R. Grs, M. Snz, A. Adí nd J. Adí Prognosis of iron chlorosis from the minerl composition of flowers in pech. J. Hort. Sci. Biotechnol. 75: Jlil, A, F. Selles nd J.M. Clrke Effect of cdmium on growth nd the uptke of cdmium nd other elements y durum whet. J. Plnt Nutr. 17: Kim, C.G., J.N.B. Bell nd N.W. Menzies Effects of soil cdmium on Pinus sylvestris L. seedlings. Plnt Soil 257:

24 Kopittke, P.M., F.P.C. Blmey nd N.W. Menzies Toxicity of Cd to signl grss (Brchiri decumens Stpf.) nd Rhodes grss (Chloris gyn Kunth.). Plnt Soil 330: Krup, Z. nd T. Bszynski Some spects of hevy metls toxicity towrds photosynthetic pprtus-direct nd indirect effects on light nd drk rections. Act Physiol. Plnt. 17: Küpper, H. nd L.V. Kochin Trnscriptionl regultion of metl trnsport minerl nutrition during cclimtiztion zinc in the Cd Zn hyperccumultor, Thlspi cerulescens (Gnges popultion). New Phytol. 185: Lri, A., F. Morles, A. Adí, Y. Gogorcen, J.J. Lucen nd J. Adí Effects of Cd nd P in sugr eet plnts in nutrient solution: induced Fe deficiency nd growth inhiition. Funct. Plnt Biol. 29:1-12. Lri, A., A. Adí, F. Morles nd J. Adí Fe resupply to Fe-deficient sugr eet plnts leds to rpid chnges in the violxnthin cycle nd other photosynthetic chrcteristics without significnt de novo chlorophyll synthesis. Photosynth. Res. 79: Lri, A., A. Adí, J. Adí nd F. Morles Down co-regultion of light sorption, photochemistry, nd croxyltion in Fe-deficient plnts growing in different environments. Photosynth. Res. 89: Lri, A., F. Morles, A. Adí nd J.Adí Chnges in iron nd orgnic cid concentrtions in xylem sp nd poplstic fluid of iron-deficient Bet vulgris plnts in response to iron resupply. J. Plnt Physiol. 167: López-Millán, A.F., F. Morles, S. Andluz, Y. Gogorcen, A. Adí, J. De Ls Rivs nd J. Adí Responses of sugr eet roots to iron deficiency. Chnges in cron ssimiltion nd oxygen use. Plnt Physiol. 124:

25 López-Millán, A.F., F. Morles, Y. Gogorcen, A. Adí nd J. Adí Metolic responses in iron deficient tomto plnts. J. Plnt Physiol. 166: López-Millán, A.F., R. Sgrdoy, M. Solns, A. Adí nd J. Adí Cdmium toxicity in tomto (Lycopersicon esculentum) plnts grown in hydroponics. Environ. Exp. Bot. 65: Lozno-Rodríguez, E., M. Luguer M, J.J. Lucen nd R.O. Crpen-Ruiz Evlution of two different cid digestion methods in closed systems for trce element determintions in plnts. Químic Anlític 14: Miller G.W., S.C. Pushnik nd G.W. Welkie Iron chlorosis, world wide prolem. The reltion of chlorophyll iosynthesis to iron. J. Plnt Nutr. 7:1-22. Montero, G. nd I. Cñells Mnul de reforestción del lcornoque (Quercus suer L.). MAPA-INIA. Mdrid, Espñ, 103p. Morl, R., I. Gómez, J. Nvrro Pedreno nd J. Mtix Effects of cdmium on nutrient distriution, yield, nd growth of tomto grown in soilless culture. J. Plnt Nutr. 17: Moulis, J.M. nd F. Thévenod New perspectives in cdmium toxicity: n introduction. Biometls 23: Nikolic, M. nd V. Römheld Mechnism of Fe uptke y the lef symplst: is Fe inctivtion in lef cuse of Fe deficiency chlorosis? Plnt Soil 215: Norderg, M Helth impcts of cdmium exposure nd its prevention. Biometls 17: Nrigu, J.O Glol metl pollution poisoning the iosphere? Environment 32:

26 Ouriti, O., H. Goui nd M.H. Ghorl Responses of en nd tomto plnts to cdmium: Growth, minerl nutrition, nd nitrte reduction. Plnt Physiol. Biochem. 35: Puss, J., J. Pereir nd J. Aronson The tree. In Cork Ok Woodlnds on the Edge. Ecology. Adptive Mngement nd Restortion. Eds. J. Aronson, J.S. Pereir nd J.G. Puss. Islnd Press, Wshington DC, pp Rellán-Álvrez, R., J. Giner-Mrtínez-Sierr, J. Ordun, I. Orer, J.A. Rodríguez- Cstrillón, J.I. Grcí-Alonso, J. Adí nd A. Álvrez-Fernández Identifiction of tri-iron(iii), tri-citrte complex in the xylem sp of iron-deficient tomto resupplied with iron: new insights into plnt iron long-distnce trnsport. Plnt Cell Physiol. 51: Rodríguez-Celm, J., R. Rellán-Álvrez, A. Adí, J. Adí nd A.F. López-Millán Chnges induced y two levels of cdmium toxicity in the 2-DE protein profile of tomto roots. J. Proteom. 73: Rodríguez-Serrno, M., M.C. Romero-Puerts, D.M. Pzmiño, P.S. Testillno, M.C. Risueño, L.A. del Río nd L.M. Sndlio Cellulr response of pe plnts to cdmium toxicity: cross tlk etween rective oxygen species, nitric oxide, nd clcium. Plnt Physiol. 150: Romolà, A.D., W. Bruggemnn, A.F. López-Millán, M. Tglivini, J. Adí, B. Mrngoni nd P.R. Moog Biochemicl responses of tolernce to Fe deficiency in kiwifruit (Actinidi delicios). Tree Physiol. 22: Sgrdoy, R., S. Vázquez, I.D. Florez-Srs, A. Alcete, M. Ris-Cró, J. Flexs, J. Adí nd F. Morles Stomtl nd mesophyll conductnces to CO 2 re the min limittions to photosynthesis in sugr eet (Bet vulgris) plnts grown with excess Zinc. New Phytol. 187:

27 Sgrdoy, R., F. Morles, R. Rellán-Álvrez, A. Adí, J. Adí nd A.F. López- Millán Croxylte metolism in sugr eet plnts grown with excess Zn. J. Plnt Physiol. 168: Sndlio, L.M., M. Rodríguez-Serrno nd M.C. Romero-Puerts Rective oxygen species in cdmium toxicity. In Rective Oxygen Species in Plnt Signlling. Eds. L.A. del Río nd A. Puppo. Springer-Verlg, Berlin Heidelerg, pp Sárvári, É., Z. Szigeti, F. Fodor, E. Cseh, K. Tussor, Gy. Záry, Sz. Veres nd I. Mészáros Reltionship of iron deficiency nd the ltered thylkoid development in Cd treted poplr plnts. In Proc. 12th Congress on Photosynthesis, S3-25. Sárvári, E., A. Solti, B. Bs, I. Mészáros, L. Lévi nd F. Fodor Impct of moderte Fe excess under Cd stress on the photosynthetic performnce of poplr (Populus jcquemontin vr. gluc cv. Kopeczkii). Plnt Physiol. Biochem. 49: Siedleck, A. nd T. Bszynski Inhiition of electron flow round photosystem I in chloroplsts of Cd-treted mize is due to Cd-induced iron deficiency. Physiol. Plnt. 87: Siedleck, A. nd Z. Krup Cd/Fe interction in higher plnts-its consequences for the photosynthetic pprtus. Photosynthetic 36: Solti, A., E. Sárvári, B. Tóth, B. Bs, L. Levi nd F. Fodor Cd ffects the trnsloction of some metls either Fe-like or C-like wy in poplr, Plnt Physiol. Biochem. 49: Thévenod, F Ctch me if you cn!: Novel spects of cdmium trnsport. Biometls 23:

28 Toriio, M., C. Celestino nd M. Molins Cork ok Quercus suer L. In Protocol for Somtic Emryogenesis in Woody Plnts. Eds. S.M. Jin nd P.K. Gupt. Springer, Dordrecht, Netherlnds, pp Vázquez, M.D., Ch. Poschenrieder nd J. Brceló Ultrstructurl effects nd locliztion of low cdmium concentrtions in en roots. New Phytol. 120: Verruggen, N., Ch. Hermns nd H. Scht Moleculr mechnisms of metl hyperccumultion in plnts. Curr. Op. Plnt Biol. 12:

29 Tle 1. Mjor Cd chemicl species (in % of totl Cd) in the nutrient solution, s estimted with chemicl specition softwre. Tretment Cd 2+ Cd(II)EDTA 2- CdNO 3+ CdSO 4 0 AQ CdHPO 4 0 AQ 10 µm Cd(II)-EDTA µm Cd(II)-EDTA µm CdCl µm CdCl

30 Tle 2. Shoot length (in cm) of cork ok plnts efore nd fter the Cd tretments. Plnts were grown for two weeks in control conditions, nd then Cd tretments (0 Cd, 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2 ) were pplied for four weeks. Dt re mens ± SE of plnts from t lest two different tches. Vlues in the sme column followed y different letter re significntly different t P 0.05 (Duncn s multiple rnge test). Shoot length (cm) Tretment Before Cd tretment After Cd tretment Control (0 µm Cd) 14.1 ± ± 0.6 d 10 M Cd(II)-EDTA 14.8 ± ± 0.5 c 50 M Cd(II)-EDTA 13.2 ± ± M CdCl ± ± M CdCl ± ±

31 Tle 3. Lef photosynthetic pigment concentrtions in cork ok control plnts treted during four weeks with 0 Cd, 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2. Chl+, Crotenoids nd V+A+Z re in µmol m -2, (V+ A + Z)/Chl rtios re in mmol VAZ mol -1 Chl, nd Chl / nd (Z+A)/(V+A+Z) re in molr rtios. Smples were tken pooling lef disks from severl plnts. Dt shown re mens ± SE of smples, otined from plnts grown in 4 different tches. Vlues in the sme column followed y different letter re significntly different t P 0.05 (Duncn s multiple rnge test). Tretment Chl +/re Crot./re (V+A+Z)*/re Chl / (V+A+Z)/Chl (Z+A)/(V+A+Z) µmol m -2 µmol m -2 µmol m -2 rtio rtio rtio Control 608 ± ± 6 18 ± ± ± ± M Cd(II)-EDTA 331 ± ± ± ± ± ± M Cd(II)-EDTA 309 ± ± ± ± ± ± µm CdCl ± ± 6 15 ± ± ± ± µm CdCl ± ± ± ± ± ± 0.05 *(V+A+Z): violxnthin (V) plus ntherxnthin (A) plus zexnthin (Z); (Z+A): zexnthin (Z) plus ntherxnthin (A). 31

32 Figure cptions Figure 1. Cdmium concentrtions (in µg g -1 DW) in leves, stems nd roots of cork ok plnts treted during four weeks with 0 Cd (control), 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2. Smples were tken pooling mteril from severl plnts. Dt shown re mens ± SE of four iologicl smples, ech from n independent plnt tch. Brs mrked with the sme letter were not significntly different (Duncn s multiple rnge test) t the P 0.05 proility level. Plese note the Y-xes vlues re different for ech mteril. Figure 2. Mcro- (P nd C, in % of DW) nd micro-nutrients (Mn, Cu, nd Zn, in µg g - 1 DW) concentrtions in leves, stems nd roots of cork ok plnts treted during four weeks with 0 Cd (control), 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2. Smples were tken pooling mteril from severl plnts. Dt shown re mens ± SE of four iologicl smples, ech from n independent plnt tch. Brs mrked with the sme letter were not significntly different (Duncn s multiple rnge test) t the P 0.05 proility level. Figure 3. Modulted Chl fluorescence prmeters in cork ok plnts treted during four weeks with 0 Cd (control), 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2. Dt shown re mens ± SE of smples (leves), otined from plnts grown in 5 different tches. Brs mrked with the sme letter were not significntly different (Duncn s multiple rnge test) t the P 0.05 proility level. Figure 4. Root ferric chelte reductse ctivity of cork ok plnts treted during four weeks with 0 Cd (control), 10 µm Cd(II)-EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2. The smple used consisted of severl root tips from different plnts. Dt re mens ± SE of 3-7 smples from 2 independent plnt tches. Brs mrked 32

33 with the sme letter were not significntly different (Duncn s multiple rnge test) t the P 0.05 proility level. Figure 5. Orgnic cid concentrtions (in mm, excepting for fumrte in µm) in xylem sp of cork ok plnts treted during four weeks with 0 Cd (control), 10 µm Cd(II)- EDTA, 50 µm Cd(II)-EDTA, 10 µm CdCl 2 nd 50 µm CdCl 2. Dt shown re mens ± SE of 4-11 replictes from three independent plnt tches. Brs mrked with the sme letter were not significntly different (Duncn s multiple rnge test) t the P 0.05 proility level. 33

34 50 40 Leves d 30 c Stems Cd (µg g -1 DW) Roots Control 10 µm Cd-EDTA 50 µm Cd-EDTA 10 µm CdCl 2 50 µm CdCl 2 Fig.1 34

35 0,8 Leves Stems Roots P (% of DW) 0,6 0,4 c c c 0,2 2,0 0,00 C (% of DW) 1,5 1,0 c c c 0, , c Mn (µg g -1 DW) c Cu (µg g -1 DW) Zn (µg g -1 DW) c c c c 0 0 Fig. 2 Control 10 µm 50 µm Cd-EDTA Cd-EDTA 10 µm 50 µm CdCl 2 CdCl 2 Control 10 µm 50 µm Cd-EDTA Cd-EDTA 10 µm 50 µm CdCl 2 CdCl 2 Control 10 µm 50 µm Cd-EDTA Cd-EDTA 10 µm 50 µm CdCl 2 CdCl 2 35

36 1,0 0,8 c A F V / F M 0,6 0,4 0,2 1,0 0,00 B 0,8 PSII 0,6 0,4 c 0,2 0,0 1,0 0 C 0,8 c exc 0,6 0,4 0,2 0,0 1,0 0 0,8 c D 0,6 qp 0,4 0,2 1,5 0,00 c c E 1,0 NPQ 0,5 0,0 0 Fig. 3 Control 10 µm Cd-EDTA 50 µm Cd-EDTA 10 µm CdCl 2 50 µm CdCl 2 36