Altered physiology, cell structure, and gene expression of Theobroma cacao seedlings subjected to Cu toxicity

Transcription

1 University of Nersk - Lincoln DigitlCommons@University of Nersk - Lincoln Pulictions from USD-RS / UNL Fculty U.S. Deprtment of griculture: griculturl Reserch Service, Lincoln, Nersk 213 ltered physiology, cell structure, nd gene expression of Theorom cco seedlings sujected to Cu toxicity Vâni L. Souz Instituto Federl de Educção, vlsio@yhoo.com.r lex-ln F. de lmeid Universidde Estdul de Snt Cruz, lexln.uesc@gmil.com Jdiel de S. Souz jdielsntn_@hotmil.com Pedro. O. Mngeir Universidde Estdul de Snt Cruz, pmngeir@uesc.r Rildo M. de Jesus Universidde Estdul de Snt Cruz, pirovnicp@gmil.com See next pge for dditionl uthors Follow this nd dditionl works t: Souz, Vâni L.; de lmeid, lex-ln F.; de S. Souz, Jdiel; O. Mngeir, Pedro.; de Jesus, Rildo M.; Pirovni, Crlos P.; hnert, Dário; ligr, Virupx C.; nd Loguercio, Lendro L., "ltered physiology, cell structure, nd gene expression of Theorom cco seedlings sujected to Cu toxicity" (213). Pulictions from USD-RS / UNL Fculty This rticle is rought to you for free nd open ccess y the U.S. Deprtment of griculture: griculturl Reserch Service, Lincoln, Nersk t DigitlCommons@University of Nersk - Lincoln. It hs een ccepted for inclusion in Pulictions from USD-RS / UNL Fculty y n uthorized dministrtor of DigitlCommons@University of Nersk - Lincoln.

2 uthors Vâni L. Souz, lex-ln F. de lmeid, Jdiel de S. Souz, Pedro. O. Mngeir, Rildo M. de Jesus, Crlos P. Pirovni, Dário hnert, Virupx C. ligr, nd Lendro L. Loguercio This rticle is ville t DigitlCommons@University of Nersk - Lincoln:

3 DOI 1.17/s RESERCH RTICLE ltered physiology, cell structure, nd gene expression of Theorom cco seedlings sujected to Cu toxicity Vâni L. Souz & lex-ln F. de lmeid & Jdiel de S. Souz & Pedro. O. Mngeir & Rildo M. de Jesus & Crlos P. Pirovni & Dário hnert & Virupx C. ligr & Lendro L. Loguercio Received: 3 pril 213 /ccepted: 2 July 213 # Springer-Verlg erlin Heidelerg 213 strct Seedlings of Theorom cco CCN 51 genotype were grown under greenhouse conditions nd exposed to incresing concentrtions of Cu (.5, 1, 2,, 8, 16, nd 32 mg Cu L 1 ) in nutrient solution. When doses were equl or higher thn8mgcul 1, fter 2 h of tretment ppliction, lef gs exchnge ws highly ffected nd chnges in chloroplsts thylkoids of lef mesophyll cells nd plsmolysis of cells from Responsile editor: Philippe Grrigues V. L. Souz Deprtmento de Ensino, Instituto Federl de Educção, Ciênci e Tecnologi d hi, Rod. 18, km, n. 18, Vil Espernç, 9- Irecê,, rzil e-mil: vlsio@yhoo.com.r.<. F. de lmeid (*) : J. de S. Souz : P.. O. Mngeir : C. P. Pirovni : D. hnert : L. L. Loguercio Deprtmento de Ciêncis iológics, Universidde Estdul de Snt Cruz, Rod. R 15, km 16, Ilhéus,, rzil e-mil: lexln.uesc@gmil.com J. de S. Souz e-mil: jdielsntn_@hotmil.com P.. O. Mngeir e-mil: pmngeir@uesc.r C. P. Pirovni e-mil: pirovnicp@gmil.com D. hnert e-mil: drio@uesc.r L. L. Loguercio e-mil: lendro@uesc.r R. M. de Jesus Deprtmento de Ciêncis Exts e Tecnológics, Universidde Estdul de Snt Cruz, Rod. R 15, km 16, Ilhéus,, rzil e-mil: rildomot@gmil.com V. C. ligr USD-RS Sustinle Perennil Crops Lortory, 13 ltimore venue, eltsville, MD, US e-mil: v.c.ligr@rs.usd.gov the root corticl region were oserved. In ddition, cell memrnes of roots nd leves were dmged. In leves, 96 h fter tretments strted, increses in the percentge of electrolyte lekge through memrnes were oserved with increses of Cu in the nutrient solution. Moreover, there ws n increse in the concentrtion of thiorituric cid-rective sustnces in roots due to lipid peroxidtion of memrnes. Chemicl nlysis showed tht increses in Cu concentrtions in vegettive orgns of T. cco incresed with the increse of the metl in the nutrient solution, ut there ws greter ccumultion of Cu in roots thn in shoots. The excess of Cu interfered in the levels of Mn, Zn, Fe, Mg, K, nd C in different orgns of T. cco. nlysis of gene expression vi RTq-PCR showed incresed levels of MT2, SODCyt, nd PER-1 expression in roots nd of MT2, PS, PSO, SODCyt, nd SODChI in leves. Hence, it ws concluded tht Cu in nutrient solution t doses equl or ove 8 mg L 1 significntly ffected lef gs exchnge, cell ultrstructure, nd trnsport of minerl nutrients in seedlings of this T. cco genotype. Keywords Cco. Hevy metl. Lipid peroxidtion. Photosynthesis. Ultrstructure. Gene expression Introduction Copper is hevy metl nd micronutrient essentil for plnt metolism, occurring under physiologicl conditions s Cu 2+ nd Cu +. Cu ions re vitl components of vriety of enzymes nd other proteins, including trnscription fctors. Thus, for the plnt to grow nd develop in helthy mnner, Cu hs to e tken from the soil, trnsported cross memrnes, distriuted nd comprtmentlized in different tissues, with its mounts precisely regulted within the cells (Yruel 25). Excess of Cu inhiits the ctivity of severl enzymes nd interferes with vrious spects of plnt iochemistry, including photosynthesis, metolism of ftty

4 cids nd proteins, respirtion, nd integrity of memrnes (lmeid et l. 27). The most importnt of these effects is relted to photosynthesis, in which Cu excess leds to chnges in the ultrstructure of chloroplsts, cusing reduction of electron trnsport in photosystems I (PS I) nd II (PS II); it lso interferes with the ctivity of Clvin cycle enzymes, such s the Ruisco (Pnou-Filotheou et l. 21; Pätsikkä et l. 22; Shul 22). Therefore, decreses in net photosynthetic rte (P N ), stomtl conductnce (gs), nd trnspirtion (E) re common in plnts exposed to excess of Cu (Moustks et l. 1997; Vssilev et l. 22; Shi-Sheng 27; Cmrollé et l. 211). In ddition, Cu excess promotes the production of free rdicls tht cuse dmge to proteins nd lipids of cellulr memrnes, therey ltering their permeility nd selectivity for intke/exit of nutrients to/from the cell, which cn cuse electrolytes lekge (Shi- Sheng 27). The mlondildehyde (MD), originted from rection etween free rdicl nd polyunsturted ftty cid, is commonly used s n indictor of lipid peroxidtion in memrnes of plnt cells under stress conditions y Cu excess (Pätsikkä et l. 22; ouzizi et l. 21; Jns et l. 21). Despite tht Cu ccumultes minly in roots, it interferes with the chemicl composition of different plnt orgns, which levels of nutritionl imlnce is speciesdependent (Shi-Sheng 27; ouzizi et l. 21; Cmrollé et l. 211). For instnce, in Glucium flvum exposed to excess of Cu, P concentrtion incresed in roots nd leves, wheres C nd Mg levels decresed in leves (Cmrollé et l. 211). ouzizi et l. (21) oserved decrese in the levels of Fe, Zn nd K in the leves of Phseolus vulgris;the sme ws oserved for Fe, K, nd Mg contents in seedlings of mrnthus tricolor (Shi-Sheng 27). Plnts hve severl potentil strtegies nd mechnisms t the cellulr level tht my e involved in detoxifiction nd tolernce to stresses cused y hevy metls. Tolernt plnts show n increse in severl homeosttic mechnisms, which contriute to prevent the metl trnsloction (Hll 22), or to keep it in stle mnner. With regrds to Cu, form of protection employed y some plnt species consists in incresing metllothionein levels, which ct minly s cheltor of the metl in excess (Coett nd Goldsrough 22). Metllothioneins re low moleculr weight proteins, rich in cysteine residues tht ind to copper nd protect the cells ginst the toxicity of this element (vn Hoof et l. 21). ntioxidnt enzymes, such s superoxide dismutses (SODs) nd clss III peroxidses, represent nother mechnism of tolernce to Cu stress. These enzymes ct y limiting the formtion nd y removing the rective oxygen species (ROS) (lscher et l. 22). The SOD enzymes re clssified into three groups: Fe SOD, Mn SOD, nd Cu Zn SOD, which re locted in different cell comprtments (lscher et l. 22). The Cu Zn SODs enzymes re divided into two clsses of isoforms, one locted in the cytoplsm nd the other in the chloroplsts (Kurep et l. 1997). The clss III peroxidses re locted in vcuoles nd cell wlls, elong to multigene fmily involved in severl physiologicl processes, nd ct in wide vriety of sustrtes, showing moderte specificity for phenols (lmgro et l. 29). Seeds of Theorom cco (Mlvcee) re commercilly explored for chocolte production, s well s for other derivtives nd y-products such s cosmetics, fine drinks, jms, icecrems nd juices (lmeid nd Vlle 21). The southestern region of hi, rzil, hs een the min cco-growing region in the world for over hundred yers. However, with the outrek of witches room disese of cco t the end of the 198s, cused y the sidiomycete Moniliophthor pernicios, n economic, socil, nd environmentl disster struck this region, s consequence of drstic reduction in cco ens production (Pereir et l. 1989). In the pst 15 yers, mjor control method of this nd other diseses (such s the lck-pod rot cused y Phytophthor spp.), hs een the development nd use of resistnt nd tolernt cco genotypes, coupled with other culturl nd chemicl control mesures under n integrted mngement pproch. couple of these mesures hve een the use of phosphte fertilizers nd cupric fungicides (Fonsec 199). Therefore, in this cco-producing region, the ccumultion of Cu in soils, plnts nd, consequently, in cco ens is mtter of concern, ecuse the control of lck-pod nd witches room with cupric fungicides hs een common prctice throughout ll these yers, s prt of the technologicl pckge recommended for this crop (Veloso nd Sntn 2). It ws found tht the content of Cu in these cco-crop soils (t the hin municiplities of Cmcn, Ilhéus, Itun, Pu rsil, nd Uruçuc) hs incresed proportionlly to the time of its pplictions. In plces where this prctice hs een systemticlly employed from 5 to 2 yers, the ville nd totl soil Cu contents were found to e ove the pproprite levels (Veloso nd Sntn 2). Tking this context into considertion, the min ojectives of this study were to evlute the tolerle physiologicl limits of exposure to Cu y T. cco seedlings, when grown in nutrient solution with different concentrtions of this element, s well s to ssess trnsloction of Cu to the shoots nd the overll chnges on plnts exposed to high levels of this metl. These ojectives were chieved y verifying the vrition cused y Cu in lef gs exchnge, ultrstructure of cellulr orgnelles, peroxidtion of cell memrnes nd minerl nutrients composition, oth in roots nd shoots. Mteril nd methods Plnt mteril nd growth conditions The experiment ws conducted under greenhouse conditions t the Universidde Estdul de Snt Cruz (UESC), Ilhéus, hi, rzil. Seeds of the T. cco genotype CCN 51 were

5 grown in lck plstic tues contining Pinus rk nd turf+triturted coconut fier (1:1) s sustrte. pproximtely 3 dys fter emergence, plnts were trnsplnted into 35-L plstic trys (eight plnts per try), contining nutrient solution t 1/2 ionic strength, prepred ccording to Hoglnd nd rnon (195). For cclimtion, the plnts remined in these conditions for 2 months. Once this period ws completed, the tretments with incresing concentrtions of Cu (.5, 1, 2,, 8, 16, nd 32 mg L 1 ) in the form of CuSO 7H 2 O were pplied. The concentrtion of.5 mg Cu L 1 ws considered s the control concentrtion, ecuse Cu is n essentil micronutrient for growth nd development of plnts. During the experimentl period, the solutions were monitored dily for ph, which ws djusted to 5.5 using NOH nd/or HCl. The nutrient solution ws kept under constnt ertion nd its level mintined y ddition of deionized wter. Renewl of the nutrient solution ws mde weekly. Lef gs exchnge During the experimentl period, the net photosynthetic rte per unit of lef re (P N ), stomtl conductnce to wter vpor (gs) nd lef trnspirtion (E) were mesured t 2, 8, 72, nd 96 h fter ppliction of tretments (T). These vriles were ssessed, etween 8 nd 9 h, on mture nd completely expnded lef from the end the orthotropic pex xis of four plnts per tretment using portle photosynthesis system LI-6 (Li-Cor, Nersk, US) equipped with n rtificil light source 6-2 Redlue. The vlues of P N, gs, nd E were used to clculte the intrinsic (P N /gs) nd the instntneous (WUE=P N /E) wter use efficiencies. For the lef gs exchnge mesurements, the rtificil light source of the system ws djusted to provide photosynthetic photon flux density of 8 μmol m 2 s 1. The men vlues of tmospheric CO 2 concentrtion, ir temperture nd ir vpor pressure deficit during the lef gs exchnge mesurements were 21.7±11.9 μmol mol 1, 26.7±.7 C, nd 1.56±. kp, respectively (men±sd, n=35, corresponding to 7 tretments 5 replictions). Ultrstructurl ssessment Ultrstructurl nlyses were performed using trnsmission electron microscopy (TEM) on the root tip nd middle portion of the second mture lef from the pex of the orthotropic xis hrvested t 96 h T. The plnt mteril ws fixed in 3 % glutrldehyde in.1 M sodium ccodylte uffer (ph 7.2). Smples were sumitted to four wshes (1 min ech) in.1 M sodium ccodylte uffer, ph 7.2, nd post-fixed in 1 % osmium tetroxide, prepred in the sme uffer for 2 h t C, nd followed y dehydrtion in n ethnol grdient (3, 5, 7, 8, nd 9 % ethnol), nd y two wshes in 1 % ethnol. Then, the smples were emedded in mixture of 1 % ethnol nd LR White resin (Sigm) in the proportions 3:1 (2 h), 1:1 (2 h), 1:3 (overnight), followed y two chnges of pure LR White resin of h ech, lwys under slow gittion. The smples were plced in geltin cpsules nd covered with pure LR White resin. Polymeriztion of the resin ws completed in 2 h t 6 C. Ultr thin sections (6 7 nm) were cut on dimond knife using ultrmicrotome (model EM FC6 LEIC Microsystems), nd collected from the knife s wter th on 3-mesh Cu grids. The sections were stined for 15 min with queous solution of 5 % urnyl cette, followed y 2 min with. % led citrte (Reynolds 1963). nlyses were done using Morgni 268D TEM (FEI Compny), with ccelertion voltge of 8 kv, equipped with CCD cmer nd controlled y softwre running under Windows OS. t lest four grids with three to five sections for ech tretment were oserved nd photogrphed. The imges tht est represented the chnges in the ultrstructure of T. cco lef mesophyll nd root cells, fter ppliction of different concentrtions of Cu, were selected. Electrolyte lekge To ssess the cell memrne stility, the electrolyte lekge technique ws used (jji et l. 21). Lef discs were cut out of plnts sumitted to different [Cu] in nutrient solution t 96 h T, nd thoroughly wshed in deionized wter. fterwrds, the lef discs were plced in vils contining 1 ml of deionized wter t 25 C for 6 h under constnt gittion. The electricl conductivity ws mesured using conductivity meter nd expressed s percentge of totl conductivity, which ws otined fter plcing the vils with the lef discs t 9 C for 2 h. Lipid peroxidtion Oxidtive dmge to lipids in cell memrnes of roots nd leves were estimted s thiorituric cid-rective sustnces (TRS), minly MD, ccording to Ckmk nd Horst (1991) with some modifictions. For ech of the different Cu tretments, 2 mg of lyophilized roots or leves were homogenized in mortr with 2 ml of.1 % (w/v) trichlorocetic cid (TC). Following centrifugtion t 1, g for 5 min, n liquot of 5 μl from the superntnt ws dded to 1.5 ml of thiorituric cid solution (.5 % in 2 % TC). Smples were incuted t 9 C for 2 min. fter heting, the rection ws stopped under ice th. Centrifugtion t 1, g for min ws performed, nd then the sornce of superntnt ws red t 532 nm nd corrected for non-specific turidity y sutrcting the sornce t 6 nm. The concentrtion of TRS ws clculted from its extinction coefficient of 155 mm 1 cm 1.

6 Minerl nutrients t the end of the experiment (96 h T), three seedlings per try were collected from the different tretments nd seprted in roots, stems, nd leves. Soon fter, wshes were crried out once in tp wter, once in 3 % HCl, nd twice in deionized wter; the different vegettive orgns were plced in n oven t 75 C to otin dry iomss. Dry plnt mterils were grinded using Wiley mill to pss 2-mesh screen, nd nlyzed chemiclly (nuncição et l. 211). Concentrtions of Cu, Fe, Mn, Zn, Mg, K, nd C were nlyzed in root, stem nd lef dry mtter, using inductively coupled plsm opticl emission spectrometer, in Vrin 71-ES model. Quntittive rel-time PCR nlysis Smples of roots nd second mture lef from the pex of the orthotropic xis were hrvested t 12 nd 96 h T, frozen in liquid nitrogen nd stored t 8 C. Prior to RN extrction, the tissues were lyophilized. RN ws extrcted from roots nd leves t four different tretments (.5, 2,, nd 8mgCuL 1 ) with RNqueous kit (mion ). The RN purity nd integrity ws checked y electrophoresis in 1 % grose gels. The RN smples were used for cdn synthesis using Revertid H-Minus Reverse Trnscriptse (Ferments) nd oligo d(t) 18 primers, ccording to mnufcturer instructions. The rections were incuted t 65 C for 5 min, 37 C for 5 min, 2 C for 6 min nd 7 C for 1 min. The qpcr ws performed in Rel Time PCR (pplied iosystems, 75 model) using non-specific sequence fluorophore SYR Green I (Ferments). The undnce of trnscripts ws nlyzed using specific primers (Tle 1). To test the qulity of these primers, the specificity nd identity of the reverse trnscription products, the qpcr products were monitored fter ech PCR, using melt-curve nlysis distinguishing gene-specific from non-specific products. The rection mix consisted of cdn templte (5 ng),.5 μm of ech primer, nd 1 μl fluorophore SYR Green I in finl rection volume of 2 μl. The temperture of PCR products ws rised from 55 to 99 C t rte of 1 C/5 s, nd the resulting dt were nlyzed using the LightCycler softwre. Only single nd with chrcteristic melting point ws oserved for ech smple, indicting tht the qpcr hd produced specific product for ech primer-pir used. To confirm tht the qpcr hd produced only genes of interest, the PCR products were seprted nd visulized in grose gel t 1 %. Threshold cycle (C T ) vlues were determined using the LightCycler softwre. Numers on the reltive expression of genes were clculted s percentge of the control tretments (.5 mg Cu L 1 ), using the 2 ΔΔCt method (Livk nd Schmittgen 21) nd β-tuulin s endogenous control in order to detect chnges in trnscript numer (Tle 1). ll rections were prepred in triplicte nd performed twice. For ech tretment t different times (12 nd 96 h T), three iologicl replics were used for ech primer evlution. Sttistics The experiment ws performed on completely rndomized rrngement, with seven levels of Cu, contining five replictes of eight seedlings ech. The results were nlyzed sed on liner nd non-liner mthemticl models, with vrition of Cu concentrtion in nutrient solution s independent vrile nd lef gs exchnge, percentge of electrolyte lekge, lipid peroxidtion, nd minerl nutrient s dependent vriles. Regression nlyses nd nlyses of vrince to define the est fitting model were done utilizing the generl liner model procedure of the Sttisticl nlysis System (SS Institute 1997), using methodology outlined y Steel nd Torrie (198). The coefficients of the equtions were tested using t test (p<.5). The sttisticl nlysis of expression levels ws performed using NOV. Men comprisons were performed y Tukey s test with sttisticl significnce t p<.5. Results Lef gs exchnge Lef gs exchnge of T. cco seedlings ws highly ffected fter 2 h exposure to concentrtions of 8, 16 nd 32 mg Cu L 1 in nutrient solution. Decreses of up to 72, 75, nd 73 % for P N, gs, nd E, respectively, were oserved (Fig. 1 c). t 96 h T for the 32 mg Cu L 1 dose, these decreses reched 1, 99, nd 98 % for P N, gs, nd E, respectively. On the other hnd, for the mg Cu L 1 tretment, the decrese in P N ws of % t 96 h T. The vlues of gs nd E for the sme concentrtion t 2 h T showed lower decreses of 39 nd 38 %, respectively, without significnt differences (p<.5) for the corresponding vlues t 96 h T. The intrinsic nd instntneous wter use efficiencies (P N /gs nd WUE) did not show significnt vrition t 2 nd 8 h T. However, decline ws found t 72 h T for oth P N /gs nd WUE (Fig. 1d, e). These results re striking in demonstrting tht seedlings of T. cco were physiologiclly ffected y s short s 2-h exposure to incresing concentrtions of Cu in nutrientsolution;vluesupto2mgcul 1 were eneficil, nd ove it were detrimentl. The results showed tht the reltionship etween P N nd gs (Fig. 2) ws liner up to gs vlue of.3 mol H 2 Om 2 s 1 forthemgcul 1 concentrtion. The lowest vlues of this rtio were otined for concentrtions equl ndove8mgcul 1, wheres the highest vlues of P N nd gs werefoundfortheconcentrtionsof1nd2mgcul 1.

7 Tle 1 Pirs of gene-specific primers used in the qrt-pcr nlysis Gene cesso Function Primer MT2 CL9Contig1 iosynthesis of metllothionein F 5 -GCCCCTTGCCTTGTTG-3 R 5 -CGCCTGGCCTTTTTCT-3 PER-1 CK iosynthesis of peroxidse clss III F 5 =CGGTGTCGTGGGTCG-3 R 5 -TGGCTCGCCTTGC-3 Cu Zn SOD Cyt CL9Contig1 iosynthesis of cytosolic Cu Zn SOD F 5 -GTGTGGCTGTGTGGTTTCTCT-3 R 5 -CCCGCTCTTCCTTTG-3 Cu Zn SOD Chl CL872Contig1 iosynthesis of chloroplstic Cu Zn SOD F 5 -TGGTGCTGTCCGGGC-3 R 5 -TGTTTCCCGGTCCCCGC-3 PSO CL326Contig1 iosynthesis of PsO protein F 5 -GCCGCTGGGGTT-3 R 5 -GGCTTGGGCTGGTC-3 PS NC_ c iosynthesis of the Ps protein or D1 protein F 5 -GGTTTGCCTTTTCCCG-3 R 5 -CTCTGGCCGCCTT-3 β-tuulin GU c Endogen F 5 -TGCCCTGGTGGTGTC-3 R 5 -CGCGGGGGGTG c Ultrstructurl ssessment The effects of incresing concentrtions of Cu on cellulr ultrstructure of developing T. cco seedlings were ssessed y TEM. Chnges in root nd lef mesophyll cortex cells were oserved (Figs. 3 nd ), with occurrence of plsmolysis in root cortexcellsminlyove8mgcul 1 (Fig. 3 f), shown y contrction of the plsm memrne nd tonoplst shrinkge. Cu tretment pprently led to the rupture of plsm memrnes in some cells of the corticl root tissue (Fig. 3g l). n evidence tht Cu concentrtions in nutrient solution etweennd8mgcul 1 seems to estlish the limit from eneficil to detrimentl effects in T. cco seedlings ws lso oserved from ultrstructurl nlysis of chloroplsts (Fig. ). Orgnelles of lef mesophyll cells of seedlings sumitted to.5 (control), 2, nd mg Cu L 1 looked norml through TEM. Chloroplsts showed structured nd orgnized thylkoid memrnes, norml presence of strch grins nd plstoglouli, nd intct doule memrnes of their envelope (Fig. c). Contrriwise, from concentrtions of 8 mg Cu L 1 nd ove, the chloroplst envelopes nd thylkoids ecme disorgnized (Fig. d f), nd sence of strch grins in the strom, swollen thylkoids, nd incresed interthylkoidl spce were verified. Furthermore, the rupture of the outer nd inner doulememrne surrounding the chloroplst ws found in some smples (Fig. d e). Electrolyte lekge nd lipid peroxidtion The dmge to memrnes cused y Cu excess cn e quntified indirectly y ssessing electrolytes lekge. s shown in Fig. 5, there ws n increse in the percentge of electrolyte lekge through lef mesophyll cells memrnes of T. cco seedlings with incresing Cu in the nutrient solution. In order to check if memrne dmge lso occurred s result of lipids peroxidtion, the concentrtion of TRS ws determined. Results indicted tht there ws no significnt difference (p<.5) etween tretments in reltion to lipid peroxidtion of lef mesophyll cell memrnes with incresing Cu in nutrient solution; however, in roots, from the 8 mg Cu L 1 tretment nd ove, significnt increse of TRS concentrtion ws oserved (Fig. 5). Minerl nutrients The effects of exposure to incresing Cu concentrtions were lso studied in terms of vritions in minerl composition of vegettive orgns of T. cco seedlings. Chemicl nlysis showed tht Cu concentrtions in roots nd shoots of T. cco ws proportionl to its incresed concentrtion in the nutrient solution, ut the ccumultion of Cu in roots ws lrgely higher thn in shoots (Fig. 6). For the tretment with the highest concentrtion of the metl t 96 h T, the concentrtion of Cu in the roots ws 1,32 mg Cu kg 1 DW, wheres in stem nd leves, the ccumultion ws only 17 nd 6 mg Cu kg 1 DW, respectively (Fig. 6). These corresponded to n ccumultion of Cu in stems nd leves tht ws pproximtely 6 nd ner 175 smller thn in roots. The increse in Cu mounts in the nutrient solution interfered with the concentrtions of Mn, Zn, Fe, Mg, K, nd C, oth in roots nd shoots. There ws shrp decline in Mn nd Zn concentrtions in roots nd stems tht level off with

8 8.8 PN [µmol(co2) m -2 s -1 ] gs [mol(h 2O) m -2 s -1 ] E [mmol(h2o) m -2 s -1 ] WUE [µmol(co2) mmol -1 (H2O)] c e Cu (mg L -1 ) Fig. 1 Chnges in net photosynthetic rte (P N ), stomtl conductnce to wter vpor (gs), c trnspirtion (E), d rtio of the intrinsic wter use efficiency (P N /gs), nd e instntneous wter use efficiency (WUE)in leves of young plnts of T. cco exposed to different concentrtions of Cu in nutrient solution during 2 (filled upright tringle), 8 (filled circle), 72 (open upright tringle), nd96h(open circle), n=, ±SE. The regression curve equtions were: y 2 ¼ 1:5 þ 3:96 exp ð :1xÞ ðr 2 ¼ :66Þ, y 8 ¼ 1:3 þ 6:3 expð :15xÞðr 2 ¼ :75Þ, y 72 ¼ :3 þ 6:8 exð :1xÞðr 2 ¼ :87Þ, y 96 ¼ :22 þ 6:7 exp ð :1xÞ ðr 2 ¼ :76Þ for P N ; y 2 ¼ :1 þ :31 expð :18xÞ d Cu (mg L -1 ) ðr 2 ¼ :71Þ, y 8 ¼ :6 þ :62 expð :2xÞðr 2 ¼ :88Þ, y 72 ¼ :3 þ :59 expð :17xÞðr 2 ¼ :86Þ, y 96 ¼ :6 þ :62 exp ð :2xÞ ðr 2 ¼ :7Þ for gs; y 2 ¼ :17 þ :7expð :18xÞ ðr 2 ¼ :71Þ, y 8 ¼ :11 þ 1: expð :2xÞðr 2 ¼ :89Þ, y 72 ¼ :7þ 1:1 expð :17xÞðr 2 ¼ :87Þ, y 96 ¼ :1þ 1:9 expð :16xÞ ðr 2 ¼ :77Þ for E; y 2 ¼ 12:5, y 8 ¼ 126:38, y 72 ¼ 122: :75x þ :11x 2 ðr 2 ¼ pt:57þ, y 96 ¼ 128:97 1:8x ðr 2 ¼ :6Þ for P N /gs; y 2 ¼ 9:3, y 8 ¼ 6:73, y 72 ¼ 6:97 :32x þ :73x 2 ðr 2 ¼ :7Þ, y 96 ¼ 6:3 :28x þ :6x 2 ðr 2 ¼ :66Þ for WUE. The mesurements were mde t 8 μmol m 2 s 1 PPFD 8 PN/gs [µmol(co 2) mol -1 (H2O)] the increse of Cu in the nutrient solution (Fig. 7). The lowest concentrtions of Mn nd Zn in the roots were 9 nd 38 mgkg 1 DW, representing decline of 81 nd 55 %, respectively, when compred to control plnts. In the stems, the lowest concentrtions of these micronutrients were higher thn in roots, with vlues of 115 mg Mn kg 1 DW nd 79 mg Zn kg 1 DW representing decrese of 52 nd %, respectively, when compred to controls. On the other hnd, Fe incresed in roots nd leves with incresing Cu mounts in the nutrient solution (Fig. 7), with 95 nd 61 % increses, respectively. The mximum vlues oserved for roots nd leves were 77 nd 79 mg Fe kg 1 DW, while the control plnts hd vlues of 2 nd 9 mg Fe kg 1 DW, respectively. The reduction oserved in Mg oth for stems nd leves ws lso proportionl to the incresed exposure to Cu. The mximum nd minimum levels of Mg in the stems were 9.2 nd 7.16 g- Mg kg 1 DW, respectively, wheres in leves they were 5.2 nd 3.83 g Mg kg 1 DW, respectively, representing decrese of 2 % in stems nd 26 % in leves (Fig. 8). There ws decrese in K concentrtions in roots nd stems s Cu incresed in the nutrient solution. The minimum vlues of K found in roots nd stemswere15nd16gkkg 1 DW, respectively, representing

9 PN [µmol(co2) m -2 s -1 ] gs [mol(h 2 O) m -2 s -1 ] Fig. 2 Reltionship etween net photosynthetic rte per unit lef re (P N ) nd stomtl conductnce to wter vpor (gs) in young plnts of T. cco sumitted to the concentrtions.5 (open circle), 1 (open squre), 2 (open upright tringle), (filled circle), 8 (filled squre), 16 (filled dimond), nd 32 (filled upright tringle) mgcul 1 in nutrient solution during 2, 8, 72, nd 96 h (n=). The mesurements were mde t 8 μmol m 2 s 1 PPFD decrese of 33 nd 17 % compred to control (Fig. 8). The C concentrtions incresed up to 22 % in root tissues t the highest Cu concentrtion in the nutrient solution (Fig. 8). Conversely, in stems, there ws decrese of 2 % in C concentrtion in reltion to control plnts. Quntittive rel-time PCR nlysis From the morpho-physiologicl chnges in roots nd leves of seedlings when exposed to incresing concentrtions of Cu, it ws possile to select the doses tht est represented the ehvior of T. cco to toxic Cu levels to llow ssessment of gene expression t the trnscriptionl level. significnt increse (p<.5) in the trnscription levels of the MT2, SODCyt nd PER-1 genes in roots, nd PS nd PSO genes in leves ws oserved t 12 h T for the concentrtion 8 mg Cu L 1,when compred to controls (Fig. 9 e). However, t 96 h TT in the roots, only the expression of the PER-1 gene remined two times greter thn the control (Fig. 9c); in leves, compred to control, PS expression incresed for ll concentrtions of Cu (Fig. 9d) Fig. 3 TEM microgrphs of roots cross sections of young T. cco plnts exposed to different concentrtions of Cu in nutrient solution for 96 h..5 (, g), 2 (, h), (c, i), 8 (d, j), 16 (e, k) nd 32 mg Cu L 1 (f, l). rrows in d f indicte shrinkge of the plsm memrne nd tonoplst. rrows in g i showed intct memrne. rrows in j l indicte memrne rupture. cw cell wll, m mitochondri. rs.5 μm (g), 1 μm (h, i, k, nd l), 2 μm ( e nd j), 5 μm (, c, nd f)

10 Fig. TEM microgrphs of cross sections of lef mesophyll of young T. cco plnts exposed to different concentrtions of Cu in nutrient solution for 96 h..5 (), 2 (), (c), 8 (d), 16 (e) nd 32 mg Cu L 1 (f). rrows in,, nd c indicte the thylkoid memrnes. rrows in d, e, nd f show rupture of the doule memrne surrounding the chloroplst, thylkoids swollen, nd increse in intrthylkoidl spces. cw cell wll, m mitochondri, n nucleus, pg plstoglouli, sg strch grins. rs 1 μm (,, nd f), 2 μm (c, d, nd e) wheres expression of PSO incresed only for the low concentrtionsof2ndmgcul 1 (Fig. 9e). Furthermore, trnscriptions of MT2 nd SODCyt genes in the leves t 12 h T for the 2 mg Cu L 1 dose were repressed when compred to control, wherestthend8mgcul 1 concentrtions, genes trnscription incresed (Fig. 9f, g). Expression of MT2 nd SODCyt t 96 h T, t ll concentrtions of Cu, ws greter thn t 12 h T, nd incresed with incresing concentrtion of Cu in the nutrient solution (Fig. 9f, g). The SODChl gene trnscriptsincresedt12htforthe8mgcul 1 dose, nd t 96 h T for the concentrtion 2 mg Cu L 1, wheres its expressionlevelsweresuppressedt96htforthe8mgcu L 1 tretment (Fig. 9h). Discussion Concerns out the ccumultion of Cu in T. cco ens up to levels potentilly inpproprite for humn consumption hve een recently rised in southestern hi, the most importnt cco-producing region in rzil. These concerns resulted from studies showing tht direct proportion exists etween incresed Cu levels in cco-growing soils nd the numer of yers of ppliction of Cu-sed fungicides (Veloso nd Sntn 2). Hence, it ecme necessry to investigte if such increses in soil Cu levels could led to corresponding increses in the levels of Cu in cco tissues, or to n ltered physiology tht could ffect cco production. Due to much more precise control of Cu levels in nutrient solution, cco seedlings were used s experimentl models in the present work, iming to quntify potentil Cu increses in tissues, s well s to ssess their effects in physiology, cellulr ultrstructure, minerl composition nd stress-response gene expression. The inhiitory effects of excessive Cu on photosynthetic processes hve een investigted in different plnt species (Mksymiec 1997; Pätsikkä et l. 22; Shi-Sheng 27; Cmrollé et l. 211). Results from studies in Triticum estivum

11 Electrolyte lekge (%) TRS (nmol g - 1 DW) Cu (mg L -1 ) Fig. 5 Electrolyte lekge nd lipid peroxidtion in leves nd roots of young T. cco plnts exposed to different concentrtions of Cu in nutrient solution for 96 h, n=, ±SE. Percentge of electrolyte lekge in leves, the eqution of the regression curve ws y ¼ 12:98 þ 1:28x :2x 2 ðr 2 ¼ :9Þ. Concentrtion of thiorituric cid-rective sustnces (TRS) in roots (filled circle) nd leves (open upright tringle), the equtions of regression curves were: y ¼ 15:88 þ 17:8x :33x 2 ðr 2 ¼ :9Þ for roots; y ¼ 65:26 for leves Zn (mg kg -1 ) Mn (mg kg -1 ) Fe (mg kg -1 ) c (Moustks et l. 1997), Hordeum vulgre (Vssilev et l. 22), nd G. flvum (Cmrollé et l. 211) hve shown to e similr to those of T. cco, with oserved decreses in lef gs Cu, roots (mg kg -1 ) Cu in nutrient solution (mg L -1 ) Fig. 6 Vrition ofcuconcentrtion inroots(filled circle), stems (open circle), nd leves (open upright tringle) ofyoung T. cco plnts exposed to incresing concentrtions of Cu in nutrient solution for 96 h, n=3, ±SE. The equtions of regression curves were: y ¼ 133:212 ð1 :95 x Þðr 2 ¼ :95Þ for roots; y ¼ 2:78 þ :89x :1x 2 ðr 2 ¼ :99Þ for stems; y ¼ 2:77 þ :25x :x 2 ðr 2 ¼ :77Þ for leves Cu, stem nd lef (mg kg -1 ) Cu in nutrient solution (mg L -1 ) Fig. 7 Chnges in the concentrtions of Mn, Fe, nd Zn in roots (filled circle), stems (open circle), nd leves (open upright tringle) ofyoungt. cco plnts exposed to incresing concentrtions of Cu in nutrient solution for 96 h, n=3, ±SE. The equtions of regression curves for Mn were y ¼ 6:69 :9x þ 188:5 ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :8Þ for roots; y ¼ 19:92 1:1x þ 13:23 ðexp ð ðx 2 Þ= 2ÞÞðr 2 ¼ :79Þ for stems nd y ¼ 98:9 þ 67:78x 1:93x 2 ðr 2 ¼ :52Þ for leves; for Zn were y ¼ 38:36 þ 8:37ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :56Þ for roots; y ¼ 78:72þ 5:8ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :62Þ for stems nd y ¼ 227:1 þ 51:71 ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :51Þ for leves; for Fe were y ¼ 76:58 232:5 3 ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :56Þ for roots; y ¼ 55:73 for stems nd y ¼ 79:3 3:7ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :86Þ for leves exchnge prmeters fter tretment with excess of Cu. In our study, seedlings exposure to Cu concentrtions ove 8 mg Cu L 1 showed to e highly toxic to T. cco, s indicted y the shrp net photosynthetic decline fter only 2 h of tretment ppliction. This decrese ws interpreted to e directly relted to the oserved decrese in gs, which indictes tht the stomt hve closed, with consequent increse in diffusive resistnce to

12 Mg (g kg -1 ) K (g kg -1 ) C (g kg -1 ) c Cu in nutrient solution (mg L -1 ) Fig. 8 Vrition of the levels of Mg, K, nd C in roots (filled circle), stems (open circle) nd leves (open upright tringle) of young T. cco plnts exposed to incresing concentrtions of Cu in nutrient solution for 96 h, n=3, ±SE. The equtions of regression curves for Mg were y ¼ :73 for roots; y ¼ 78:72 þ 5:8 ðexpð ðx 2 Þ=2ÞÞðr 2 ¼ :62Þ for stems nd y ¼ :6 for leves; for K were y ¼ 22:6 :82xþ :18x 2 ðr 2 ¼ :65Þ for roots; y ¼ 19:23 :1xðr 2 ¼ :5Þ for stems nd y ¼ 21:15 for leves; for C were y ¼ :76 :63xþ :3x 2 ðr 2 ¼ :65Þ for roots; y ¼ 18:76 þ 5:8 ðexpð ðx 2 Þ= 2ÞÞ ðr 2 ¼ :77Þ for stems nd y ¼ 18:35 for leves CO 2 nd reduction in wter loss y trnspirtion, confirmed y the oserved reduction in E (Figs. 1 nd 2). It ws found tht T. cco plnts tolerted exposures up to mg Cu L 1, despite the oserved decrese in P N t 96 h T, which cn e considered n indirect effect of the decreses in gs nd E oserved s erly s 2 h T. Exposure to lower Cu concentrtions (1 nd 2mgL 1 ) did not ffect the cco physiology negtively, or lterntively, it might hve hd eneficil effect to the plnts, possily s micronutrient within the fvorle limits. t lest prt of the Cu effects in photosynthesis derives from the fct tht this metl is component of plstocynin, nd s such, hs direct prticiption in the electron trnsport etween PS II nd PS I in the photochemicl phse of photosynthesis. In ddition, Cu excess cn cuse chnges in proteins nd lipids of thylkoid memrnes, leding to susceptiility of PS II to photoinhiition in vivo nd to impirment of the electron trnsport etween the two photosystems (Mksymiec 1997; Pätsikkä et l. 1998; 22). The reduction in E nd gs vlues s result of stomtl closure promoted y higher Cu concentrtions hs likely fvored the decline in P N /gs nd WUE t72ht(fig.1d, e). This ws possily due to n inefficient sorption of wter y the roots nd poor trnsloction of wter nd minerl nutrients to the shoots, s suggested y the ultrstructurl chnges in cells of the root tip cortex. Concentrtions equl nd ove 8mgCuL 1 considerly ffected root cells y promoting plsmolysis, likely due to osmotic effects nd disruption of the plsm memrne (Fig. 3). Pnou-Filotheou nd oslidis (2) hve lso oserved plsmolysis, disintegrtion of cell memrnes nd non-recognition of ny orgnelles in root cells of Orignum vulgre plnts exposed to Cu stress. In leves of T. cco seedlings exposed to higher levels of Cu, the dmge imposed to the thylkoid memrnes nd chloroplst s doule memrne certinly contriuted to the oserved chnges in lef gs exchnge. Storge of strch grins in chloroplsts ws not oserved, likely due to shrp decline in cron ssimiltion nd to moiliztion of ll ssimilted compounds from the current photosynthesis to other metolic drins. Symptoms of strch grins sence nd swelling of chloroplsts doule memrne hve lso een identified in leves of O. vulgre fter Cu tretment (Pnou-Filotheou et l. 21). In Populus tremul plnts grown in soil contining mixture of hevy metls including Cu, rekges in thylkoid memrnes nd chloroplst envelope were oserved, with extrusion of wste mteril to the cytoplsm (Hermle et l. 27). Swelling of thylkoid nd incresed interthylkoidl spces were lso fetured in chloroplsts of T. estivum under Zn-deficiency stress (Peck nd McDonld 21). The loss of memrne integrity in root nd lef cells of T. cco ws evluted y the levels of lipid peroxidtion. TRS concentrtions incresed up to 15 in roots for the highest Cu dose, suggesting tht, ove mg Cu L 1, chnges in cell memrnes were due to lipids degrdtion. On the other hnd, in lef mesophyll cells, the dmge to chloroplsts (Fig. ) nd the increse in electrolyte lekge (Fig. 5)pper to e relted to possile effects of Cu on memrne proteins, ecuse there ws no significnt difference (p<.5) etween the concentrtion of TRS in control nd in Cu tretments. However, s we discuss elow, Cu ws mostly retined in the root system, therey suggesting tht the ultrstructurl effects on roots nd lef cells re of different nture. Excessive

13 Fig. 9 mount of trnscripts in the roots ( c) nd leves (d h) of young T. cco plnts exposed to incresing concentrtions of Cu in nutrient solution for 12 nd 96 h (n=3, ±SE). The mrn levels were quntified y quntittive rel-time PCR. The mrn levels were normlized with respect to tuulin, nd re expressed reltive to those of control plnts tht were given vlue of 1. Uppercse letters compre verges etween tretments 12 h T nd lowercse letters compre verges etween tretments 96 h T. Mens followed y the sme letter re not significntly different t p<.5, ccording to Tukey s test Fold increse in MT2 expression Fold increse in PER expression Fold increse in PsO expression c e 12 h 96 h Root Root Lef d f Root Lef Lef c C Fold increse in cytoplsmic SOD expression Fold increse in MT2 expression Fold increse in Ps expression Fold increse in cytoplsmic SOD expression g Lef C D h Lef Fold increse in chloroplst SOD expression Cu mg L -1 Cu mg L -1 mounts of Cu showed to induce oxidtive stress responses, with the formtion of ROS tht cuse lipid peroxidtion of cell memrnes nd increses in TRS levels (minly MD) in plnt cells (Pätsikkä et l. 22; Jns et l. 21). Moreover, Cu cn lso dmge the memrnes y recting with sulfhydryl groups, which led to proteins denturtion (Pnou-Filotheou nd oslidis 2). The oserved low moility of Cu within the T. cco plnt, s suggested y its mjor ccumultion in the root system (Fig. 6), confirms similr results in other studies in

14 vrious plnt species (Pätsikkä et l. 22; Pnou-Filotheou nd oslidis 2; Shin-Sheng 27; ouzizi et l. 21; Cmrollé et l. 211). It hs een suggested tht the preferentil ccumultion of Cu in roots is the result of tolernce mechnism in plnts to cope with the stress cused y this metllic element t high concentrtions (ouzizi et l. 21). When such mechnisms of Cu tolernce in the root zone ecome overloded, Cu +2 is then trnslocted to shoots through xylem nd phloem. When it reches the leves, smll proportion of Cu ccumultes in the chloroplsts, which is likely responsile for the ultrstructurl dmges oserved (Figs. 3 nd ). Tken together, these results suggest tht the trnsport of this metl is highly regulted process, even in plnts exposed to Cu excess (Pätsikkä et l. 22). Ptterns of stress-response gene expression otined (nd discussed further elow) come into support of this view. crop-cultivting soil is physicochemiclly nd iologiclly dynmic entity, such tht distriution nd vilility of ny element to plnts tend not to occur in uniform nd homogenous mnner; soil ptches of different sizes, composition nd loctions compose mosic tht will disply distinct forms nd concentrtions of the vrious soil elements ville to plnts. Hence, in cco production field, we cn resonly speculte tht the protection ginst excess of Cu in dult plnts opertes t the level of morphologicl nd gene expression chnges in the root system, likely to void sorption nd/or prevent this element to trnslocte to more sensitive tissues/cells of the plnt. Despite its low moility, nd ccumultion minly in the root system, Cu ffects the trnsport of other essentil minerl nutrients for plnt metolism (Shul 22; Xiong et l. 22), s shown in this study. The uptke of Mn nd Zn y roots of T. cco seedlings ws significntly ffected with incresing Cu in the nutrient solution. However, the levels of these elements in leves were prcticlly unchnged, possily due to the fct tht they re criticl for photosynthetic processes. Mn is n essentil cofctor in the wter oxidtion pthwy of PS II (Dučić nd Polle 25). Zn occurs in high concentrtions in iologicl systems when compred with other micronutrients. Zn helps mintining the structurl integrity nd controlling the permeility of cell memrnes, it is criticlly importnt for cells protection from ROS-induced dmges (Ckmk 2). Thus, the decrese of Zn in T. cco roots my hve contriuted to the disruption nd lipid peroxidtion of cell memrnes, s oserved y ultrstructurl nlysis (Figs. 3 nd ) nd TRS quntifiction (Fig. 5). Moreover, in mny monocots nd eudicots, Zn deficiency cuses ccumultion of Fe in roots nd shoots, which is ssocited with incresed lipid peroxidtion nd dmge to the chlorophyll molecules in plnts under stressing conditions (Ckmk 2). The Cu-ssocited increse in Fe content tht occurred in roots nd leves of the studied seedlings ws likely coupled to the decline in Zn nd my hve lso influenced the dmge oserved in cell memrnes nd in photosynthetic rtes. The Mg element in plnts hs the ility to interct with nucleophilic lignds (Shul 22). It is the centrl tom of the chlorophyll molecule nd is n importnt element for riosoml suunits ggregtion during protein iosynthesis. In chloroplsts, Mg 2+ is required to ctivte Ruisco, therey resulting in CO 2 fixtion (Shul 22). When plnt species re sumitted to excessive mounts of Cu, Mg cn e chemiclly replced y this element in the chlorophyll molecule, leding to decline in photosynthesis (Kupper et l. 1998). Hence, the decrese in Mg concentrtion in shoots of T. cco nd its replcement y Cu in chlorophyll my hve contriuted to the decline in ssimiltion rtes of CO 2 in this species. With regrds to K, decline in its levels often indictes efflux of this element cross plsm memrne, which ws likely dmged y the lipid peroxidtion promoted y Cu excess, resulting in loss of memrne selectivity nd increse in its permeility (Murphy et l. 1999; Jns et l. 21). Toxic levels of Cu cn lso inhiit memrne TPses nd promote incresed cytoplsmic concentrtions of C, due to lockge in TP-dependent pumping of C to the outside of the cell. The Cu-induced increse of cytoplsmic C levels triggers vrious ctolic processes y ctivtion of phospholipses, which result in the formtion of free rdicls (Mksymiec 1997). Trnscriptionl nlysis of gene expression y qpcr suggests tht the ntioxidnt defense systems nd iotic stress tolernce in seedlings were shrply stimulted y Cu excess in nutrient solution. Up-regultion of metllothioneins in plnts hs een constntly relted to Cu homeostsis y detoxifiction nd protection ginst oxidtive stress (Coett nd Goldsrough 22; Cozz et l. 212). The incresed expression of MT2 only 12 h T implies tht the protection strtegy dopted y T. cco is to tolerte nd ccumulte high levels of Cu in the root system, in order to ensure the mintennce of metolism in leves. Studies with Silene vulgris tolernt to Cu hve shown higher levels of mrn trnscripts for MT2 (vn Hoof et l. 21). On their turn, the incresed expression of SODCyt nd PER-1 in roots, nd SODChl nd SODCyt in leves of T. cco suggest the existence of defense mechnism ginst oxidtive stress promoted y Cu, y the expression of genes encoding ntioxidnt enzymes (Lee et l. 27). The SOD enzymes constitute the first line of cellulr defense ginst ROS (lscher et l. 22), wheres the clss III peroxidses re involved in the formtion of lignin nd suerin, s well s in mintining dequte levels of ROS (lmgro et l. 29). The gene pso encodes protein tht is extrinsic to the oxygen evolution center locted in PS II, eing considered s the min protein responsile for wter photolysis (De Ls Rivs et l. 2). Mutnts of ridopsis thlin tht lck the pso gene hve shown decresed ctivity of the PS II (Murkmi et l. 22). In turn, ps is locted in the chloroplst genome nd encodes protein, Ps or D1 tht is intrinsic to the PS II (Nelson nd Yocum 26). Since

15 stresses cused y hevy metls promote reduction of ps gene trnscripts, nd so, interfere with the opertion of PS II (Geiken et l. 1998; llkhverdiev et l. 22), the decreses in T. cco lef gs exchnge is likely relted to dmge to PS II s descried for other plnt species sujected to stress y Cu (Mksymiec 1997; Pätsikkä et l. 22). The exposure of T. cco seedlings to 8 mg Cu L 1 stimulted n increse in PS nd PSO trnscripts t 12 h T, likely to prevent dmge to PS II. However, t 96 h T, their expression ws lower thn in the 2 nd mg Cu L 1 doses (Fig. 9), suggesting tht inhiition in their trnscription hs likely strted from this level on. This would e in greement with the fct tht there ws no recovery of the lef gs exchnge fter tretments with higher Cu doses, s previously discussed (Fig. 1). The levels of trnscripts of PS nd PSO t 96 h T incresed for the 2 nd mg Cu L 1,suggestingthtT. cco tolertes well up to these Cu concentrtions. Despite tht exposure to Cu ws only during very short period of time, not ddressing effects of long-term stress, or cclimtion/dpttion types of response, ll the dmges identified t the cellulr level cn e regrded s irreversile to those cells nd le to ffect downstrem processes nd metolisms of the plnt, s long s the Cu stress is mintined (Lichtentler 1996). From the present studies with seedlings, we conclude tht the T. cco plnts re likely cple of tolerting Cu concentrtions from to 8 mg Cu L 1 t their root system. Cu doses ove these levels (t lest in conditions tht surround the entire root system in homogeneous nd constnt mnner, s provided y nutrient solutions) pper to e very toxic, remrkly interfering with the overll metolism of this species. s tolernce strtegy, Cu ws minly ccumulted in roots nd remined mostly restricted to this orgn, indicting low moility within the plnt. In this sense, this plnt species ppers to e rhizofilterer of this metl. In spite of the increse expression of genes involved in tolernce to stresses, such s tht cused y Cu, the excess of this metllic element in the roots cused the loss of integrity of cell memrnes through lipid peroxidtion, leding to n imlnced uptke nd trnsloction of wter nd minerl nutrients to shoots. These events irreversily compromised the ultrstructure of chloroplsts nd the lef gs exchnge. Tken together, ll these ltertions oserved in T. cco seedlings sujected to incresing levels of Cu suggest tht plnts grown in soils with high levels of copper cn e negtively ffected in their growth nd metolism. Our results lso indicte tht the mjor deleterious effects of high levels of Cu t the root system is more likely relted to potentil losses in cco productivity due to the physiologicl nd the ultrstructurl chnges oserved, rther thn to the ccumultion of Cu in shoot tissues t toxic levels (lthough this effect should not e neglected). Therefore, ccumultion of Cu in the cco ens t toxic levels for consumption ppers not to ecome prolem, s environmentl pollution y copper will likely strike directly on cco production resulting in the lck of ens ville for consumption. cknowledgments We thnk Mr. lerto José dos Sntos Júnior nd Ms. Vléri Ferreir Fernndes of the Electron Microscopy Center-UESC for the technicl ssistnce on EM. V. L. Souz ws supported y FPES (Fundção de mpro Pesquis do Estdo d hi). This work ws funded y grnt from USD, within n interntionl coopertive greement with CEPLC, UESC, FUNP, nd MRS Ccu Inc. References llkhverdiev SI, Nishiym Y, Miyiri S, Ymmoto H, Ingki N, Kneski Y, Murt N (22) Slt stress inhiits the repir of photodmged photosystem II y suppressing the trnscription nd trnsltion of ps genes in Synechocystis. Plnt Physiol 13: lmgro L, Gómez Ros LV, elchi-nvrro S, ru R, Ros rceló, Pedreño M (29) Clss III peroxidses in plnt defence rections. J Exp ot 6: lmeid -F, Vlle RR, Mielke MS, Gomes FP (27) Tolernce nd prospection of phytoremeditor woody species of Cd, P, Cu nd Cr. rz J Plnt Physiol 19:83 98 lmeid -F, Vlle RR (21) Cco: Ecophysiology of Growth nd Production. In: DMtt FM (ed) Ecophysiology of tropicl tree crops. Nov Science, Huppuge, pp 37 7 lscher RG, Erturk N, Heth LS (22) Role of superoxide dismutses (SODs) in controlling oxidtive stress in plnts. J Exp ot 53: nuncição DS, Leo DJ, Jesus RM, Ferreir SLC (211) Use of multivrite nlysis techniques for evlution of nlyticl dt determintion of the minerl composition of cge (rssic olerce). Food nl Methods : jji M, Kinet J-M, Lutts S (21) The use of the electrolyte lekge method for ssessing cell memrne stility s wter stress tolernce test in durum whet. Plnt Growth Regultion :1 1 ouzizi H, Jouili H, Geitmnn, El Ferjni E (21) Copper toxicity in expnding leves of Phseolus vulgris L.: ntioxidnt enzyme response nd nutrient element uptke. Ecotoxicol Environ Sfety 73: Ckmk I, Horst WJ (1991) Effect of luminum on lipid peroxidtion, superoxide dismutse, ctlse nd peroxidse ctivities in root tips of soyen (Glycine mx). Physiol Plnt 83:63 68 Ckmk I (2) Possile roles of zinc in protecting plnt cells from dmge y rective oxygen species. New Phytol 16: Cmrollé J, Mteos-Nrnjo E, Redondo-Gómez S, Luque T, Figuero ME (211) Growth, reproductive nd photosynthetic responses to copper in the yellow-horned poppy, Glucium flvum Crntz. Environ Exp ot 71:57 6 Coett C, Goldsrough P (22) Phytocheltins nd metllothioneins: roles in hevy metl detoxifiction nd homeostsis. nnu Rev Plnt iol 53: Cozz R, runo L, itonti M (212) Expression pttern of type-2 metllothionein gene in wild popultion of the psmmophyte Silene niceensis. Protoplsm. doi:1.17/s De Ls RJ, lser M, rer J (2) Evolution of oxygenic photosynthesis: genome-wide nlysis of the OEC extrinsic proteins. Trends Plnt Sci 9:18 25 Dučić T, Polle (25) Trnsport nd detoxifiction of mngnese nd copper in plnts. rz J Plnt Physiol 17: Fonsec SE (199) vlição de resistênci Crinipellis pernicios em novos clones de Theorom cco. Rev grotrópic 2:137 13