Citation for published version (APA): Shahbaz, M. (2011). Copper toxicity and sulfur metabolism in Chinese cabbage. Groningen: s.n.

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1 University of Groningen Copper toxiity nd sulfur metolism in Chinese ge Shhz, Muhmmd IMPORTANT NOTE: You re dvised to onsult the pulisher's version (pulisher's PDF) if you wish to ite from it. Plese hek the doument version elow. Doument Version Pulisher's PDF, lso known s Version of reord Pulition dte: 2011 Link to pulition in University of Groningen/UMCG reserh dtse Cittion for pulished version (APA): Shhz, M. (2011). Copper toxiity nd sulfur metolism in Chinese ge. Groningen: s.n. Copyright Other thn for stritly personl use, it is not permitted to downlod or to forwrd/distriute the text or prt of it without the onsent of the uthor(s) nd/or opyright holder(s), unless the work is under n open ontent liense (like Cretive Commons). Tke-down poliy If you elieve tht this doument rehes opyright plese ontt us providing detils, nd we will remove ess to the work immeditely nd investigte your lim. Downloded from the University of Groningen/UMCG reserh dtse (Pure): For tehnil resons the numer of uthors shown on this over pge is limited to 10 mximum. Downlod dte:

2 COPPER TOXICITY AND SULFUR METABOLISM IN CHINESE CABBAGE

3 The studies desried in this thesis were rried out in the Lortory of Plnt Physiology, Fulty of Mthemtis nd Nturl Sienes, University of Groningen, Nijenorgh 7, 9700 CC, Groningen, The Netherlnds. The ndidte ws finnilly supported y sholrship from the Higher Edution Commission (HEC), Pkistn with the ollortion of Netherlnds orgnistion for interntionl oorportion in higher edution (Nuffi) Cover design: Dik Visser Printed y: Grfimedi, Filitir Bedrijf RUG ISBN: (ook) ISBN: (digitl)

4 RIJKSUNIVERSITEIT GRONINGEN COPPER TOXICITY AND SULFUR METABOLISM IN CHINESE CABBAGE Proefshrift ter verkrijging vn het dotort in de Wiskunde en Ntuurwetenshppen n de Rijksuniversiteit Groningen op gezg vn de Retor Mgnifius, dr. E. Sterken, in het openr te verdedigen op vrijdg 14 oktoer 2011 om 12:45 uur door Muhmmd Shhz georen op 2 pril 1981 te Shiwl, Pkistn

5 Promotor: Copromotor: prof. dr. J.T.M. Elzeng dr. L.J. de Kok Beoordelingsommissie: prof. dr. M.J. Hwkesford prof. dr. S.H. Hneklus prof. dr. M. Tusz

6 In memory of my eloved mother nd fther nd dedited to my rother Ashfq Ahmd

7

8 Contents Chpter 1. Generl introdution 9 Chpter 2. Mteril nd methods 23 Chpter 3. Copper exposure interferes with the regultion of the uptke, distriution nd metolism of sulfte in Chinese ge 33 Chpter 4. UV rdition inreses the toxiity nd impt of opper on sulfur metolism in Chinese ge 51 Chpter 5. Sulfte deprivtion overrules the toxiity nd impt of opper on sulfur metolism in Chinese ge 71 Chpter 6. Atmospheri H 2 S nutrition hrdly ffets the toxiity nd impt of opper on sulfur metolism in Chinese ge 81 Chpter 7. Generl disussion 101 Referenes 111 Summry 129 Smenvting (Summry in Duth) 135 Khuls (Summry in Urdu) 141 Aknowledgements 147

9

10 Chpter 1. Generl introdution

11

12 Chpter 1 Copper (Cu) is n essentil redox-tive trnsition metl for norml plnt growth nd development nd oftor in mny metlloproteins (Kopsell nd Kopsell 2007; Burkhed et l. 2009; Yreul 2005, 2009). Prior to its identifition s plnt nutrient, Cu ws used in griulture for hemil weed ontrol nd s fungiide (Kopsell nd Kopsell 2007). Despite its essentility, Cu is phytotoxi t enhned onentrtions (Kopsell nd Kopsell 2007; Burkhed et l. 2009; Yruel 2005, 2009). The typil Cu ontent in plnts rnges from 0.08 to 0.24 µmol g -1 dry weight, nd Cu toxiity in ge generlly ours when the lef tissue level exeeds 0.4 µmol g -1 dry weight (Mniol nd Bekett 1985; Krämer 2010). The Cu levels in griulturl soil my strongly e enhned s onsequene of nthropogeni tivities through the pplition of orgni fertilizers, use of sewge sludge s fertilizer, pplition of sewge wter for rop prodution nd the use of Cu-ontining fungiides (Dh nd Strmns 2005; Zhou et l. 2005; Yruel 2005, 2009). The Cu ontent in pig slurries is times higher thn in soil, nd the mount of opper exreted vi fees orresponds to 72-80% of the mount ingested y the nimls (Mntovi et l. 2003). Tle 1. Input nd output of hevy metls for rle lnd in the Netherlnds in 1980 nd 2003 (dpted from Dh nd Strmns 2005). Vlues in 10 5 moles. Totl input By niml mnure By minerl fertilizers By deposition Other soures Totl output Cu Zn Cd In the Netherlnds rle soil ontins high levels of Cu nd other hevy metls s the onsequene of intensive griulture nd the use of niml mnure s orgni fertilizers. Currently, the input of Cu nd other hevy metls for rle lnd in the Netherlnds still exeeds the output, despite strit regultory legisltion, whih hs resulted in strongly deresed input s ompred to tht t the eginning of the eighties (Tle 1). Cu nd other hevy metls re not very moile in most of the soils, whih is quite lrming, sine presumly it will tke severl 11

13 hundreds or even thousnds of yers for the nturl re-equilirtion of hevy metls. For the Netherlnds it hs een estimted tht the nturl re-equilirtion of Cd nd Zn will only e rehed fter 100-1,000 yers, wheres tht of P nd Cu my even tke 3,000 nd 4,000 yers, respetively (Dh nd Strmns 2005). Tle 2. Hevy metl onentrtion (µmol kg -1 dry weight) in hrvested vegetles irregted with nl or sewge wter in the viinity of Fisld, Pkistn (Butt et l. 2005). In developing ountries Brssi nd other vegetle rops re often grown in the viinity of ig ities nd industril res, where they my e sujeted to ir nd hevy metls pollution (Yng et l. 2006). The ltter my originte from sewge, whih is diretly pplied to vegetle rops. Sewge is not only the soure of mny nutrients, ut it is often ontminted with high levels of Cu nd other hevy metls. As onsequene of untreted sewge pplition, hevy metls not only umulte in the soil ut lso in vegetles (Tle 2; Youns et l. 1998; Butt et l. 2005). Enhned Cu levels in rop plnts might not only negtively ffet plnt growth nd funtioning, ut will lso enter the food hin (Brun et l. 2001). Uptke nd distriution of Cu in plnts Copper is tken up y plnts with the lowest rte mong ll the essentil elements (Kt-Pendis nd Pendise 1992; Kopsell nd Kopsell 2007). In the soil, Cu n e inessile due to its tendeny to e present predominntly in n insolule form euse of its 12

14 Chpter 1 dsorption to ly, CCO3 or orgni mtter nd high ph (MBride et l. 1997; Suvé et l. 1997; Kopsell nd Kopsell 2007; Burkhed et l. 2009, Yruel 2005, 2009; Plmer nd Guerinot 2009). Plnts my moilize essentil metls y the seretion of heltors y the root nd/or y idifition of the rhizosphere (Clemens et l. 2002). Cu is found predominntly s Cu2+ in the soil, whih might e redued y FRO2 (ferri redutse oxidse), prior to its uptke y high ffinity COPT trnsporters (Roinson et l. 1999; Plmer nd Guerinot 2009; Fig. 1). Fig. 1. Sheme of Cu uptke nd distriution in plnt ells. Copper proteins re indited in open retngles; (CSD1, ytosoli Cu/Zn superoxide dismutse; CSD2, hloroplsti Cu/Zn superoxide dismutse; CSD3, peroxisoml Cu/Zn superoxide dismutse; ER, endoplsmi retiulum), trnsporters in light gry retngles (COPT, Cu trnsporter; HMA, hevy metl P-type ATPse; RAN1, responsive-to-ntgonist 1; PAA, P-type ATPse of Aridopsis; ZIP, Zn trnsporters), Cu-metllohperones in losed retngles (CCH, Cu hperone; ATX1, ntioxidnt 1; CCS, Cu hperone for Cu/Zn superoxide dismutse; COX, ytohrome- oxidse; SCO, synthesis of ytohrome- oxidse), FRO (Fe(III) redutse oxidse) nd possile metlinding ompounds, viz. metllothioneins (MTs) nd phytoheltins (PCs). Adpted from Pilon et l. (2006) nd Yruel (2009). 13

15 The uptke nd trnsport of Cu in plnts nd its suellulr distriution nd homeostsis re medited y high ffinity Cu trnsporter proteins (COPT trnsporters), other metllotrnsporter proteins (e.g. Cu-trnsporting P-type ATPses) nd metllohperone proteins (ATX, CCS nd COX17 like Cu hperone; Snenon et l. 2003, 2004; Yruel 2005, 2009; Burkhed et l. 2009; Plmer nd Guerinot 2009; Fig. 1). Plnts my lso tke up Cu s the more undnt Cu 2+ vi memer of the ZIP fmily, trnsporter fmily known to preferentilly trnsport divlent tions. Both ZIP2 nd ZIP4 re upregulted upon Cu defiieny (Clemens 2001; Wintz et l. 2003; Plmer nd Guerinot 2009). Cu is removed from the ytosol y the HMA5 trnsporter (Andrés-Colás et l 2006) nd exessive metl ions my omplex with viz. glutthione (GSH), phytoheltins (PCs) nd metllothioneins (MTs) nd my e sequestered in the vuole (Clemens et l. 2002; Pilon et l. 2006; Yruel 2009; Fig. 1). Cu hs limited moility in plnts nd exessive Cu tken up is often found in the root tissue (Brun et l. 2001; Qurti et l. 2003; Kopsell nd Kopsell 2007; Guerr et l. 2009; Andrde et l. 2010). Exessive Cu tken up y roots is ound to the ell wlls due to its ffinity to ronyli, roxyli, phenoli nd sulfydryl groups, ioni onds with negtively hrged sites nd N, O nd S onds in ell memrnes (Qurti et l. 2003; Kopsell nd Kopsell 2007). The trnslotion of Cu to ove-ground prts seems to e well regulted, s it is not orrelted to the Cu onentrtion in the root environment (Ginohio et l. 2002; Kopsell nd Kopsell 2007). Funtions nd toxiity of Cu in plnts Copper is essentil for plnt growth nd tivity of mny enzymes, with requirement mongst the lowest of ll elements (Kopsell nd Kopsell, 2007). Cu is n integrl omponent of photosyntheti eletron trnsport (plstoynin), mitohondril respirtion (ytohrome oxidse), ell wll metolism (mine oxidse), hormone signling (ethylene reeptors), thylkoids (polyphenol oxidse) nd retive oxygen metolism (superoxide dismutse; Clemens 2001; Hll nd Willims 2003; Yruel 2005, 2009; Burkhed et l. 2009). The reversile oxidtion-redution of Cu mkes it very vlule s oftor in enzymes suh s Cu/Zn-superoxide 14

16 Chpter 1 dismutse (Cu/ZnSOD), ytohrome oxidse, sorte oxidse, mino oxidse, lse, plstoynin (PC), nd polyphenol oxidse (Yruel 2005, 2009; Pilon et l. 2006). Plnts like ll other orgnisms possess homeostti mehnisms to mintin the orret onentrtion of essentil metl ions in different ellulr omprtments nd to minimize the dmge from exposure to enhned metl ions (Clemens 2001; Plmer nd Guerinot 2009). One inside the ell, Cu hs to e distriuted in proper mounts to ellulr omprtments nd to proteins. Enhned levels hve to e rendered innouous either y timely seretion, omprtmenttion nd/or omplextion in order to void their interferene with the norml ellulr metolism (Hll 2002; Plmer nd Guerinot 2009). The toxi effets of Cu depend on the onentrtion of Cu umulted, growth stge of plnts nd the durtion of the exposure (Clemens 2001; Medoz-Cóztl et l. 2005). In living orgnisms the redox-tive metl Cu is present in oth the Cu 2+ nd Cu + form. However, exessive free Cu ions my tlyze the formtion of the highly toxi hydroxyl rdils (OH. ) from hydrogen peroxide (H 2 O 2 ) or superoxide nions (O 2 - ) vi the Her-Weiss retion, whih my indue oxidtive stress nd use lipid peroxidtion, protein denturtion nd DNA muttion (Pinto et l. 2003; Morelli nd Srno 2004). Exessive ioni Cu is phytotoxi nd generlly results in stunted root growth nd shoot development nd in lef hlorosis (Yruel 2005; Kopsell nd Kopsell 2007). The phytotoxiity of Cu is generlly sried to its possile retion with thiol groups of proteins nd glutthione (De Vos et l. 1993; Yruel 2009). Cu toxiity my lso e due to redued nd/or ltered uptke nd distriution of other essentil nutrients (Shivon et l. 2007), e.g. derese in Fe uptke essentil in protein funtioning (Pätsikkä et l. 1998, 2002; Kopsell nd Kopsell 2007; Yruel 2009). Enhned levels of Cu my injure the plsm memrne, resulting ion lekge from roots (Hll 2002). Moreover, Cu my hinder the iosynthesis of photosyntheti pprtus, resulting in n ltered pigment nd protein omposition of photosyntheti memrnes. It hs een oserved tht espeilly photosystem II is sensitive site to enhned Cu levels (Brón et l. 1995; Yruel 2005, 2009). 15

17 Sulfte uptke nd metolism in plnts Plnts depend on their roots for the uptke of sulfur nd other nutrients. Generlly sulfte tken up y the roots is the primry sulfur soure for plnt growth. The sulfte uptke y the roots is energy dependent (driven y proton grdient generted y ATPses) through proton/sulfte (presumly 3H + /SO 4 2- ) o-trnsport nd the sulfte trnsporter-medited uptke is likely one of the primry regultory sites of sulfur metolism (De Kok et l. 2002; Sito 2004; Hwkesford nd De Kok 2006). Distint sulfte trnsporter proteins medite the uptke, trnsport nd su-ellulr distriution of sulfte nd their expression nd tivity re highly modulted y the sulfur sttus of the plnt (Glss et l. 2002; Hwkesford nd De Kok 2006; Korlewsk et l. 2007, 2008, 2010, 2011; De Kok et l. 2011; Fig. 3). Sulfte needs to e redued prior to its metolism into orgni sulfur ompounds (Fig. 2). The redution of sulfur to sulfide my tke ple in the plstids in the root s well s in the hloroplsts in the shoot, sine oth root plstids nd hloroplsts ontin ll sulfte redution enzymes (De Kok et l. 2002, 2011; Hwkesford nd De Kok 2006). However, on sis of the shoot to root prtitioning in hereous speies the min proportion of sulfte will e redued in the shoot. Sulfte needs to e tivted to denosine 5'- phosphosulfte (APS) prior to its redution to sulfite y ATP sulfurylse (Fig. 2). APS is susequently redued to sulfite y APS redutse with likely glutthione s redutnt (Leustek nd Sito 1999; Kopriv nd Koprivov 2003; Sito 2004). Sulfite is redued with high ffinity y sulfite redutse to sulfide with ferredoxin s redutnt. Sulfide is inorported into ysteine y O-etylserine(thiol)lyse, with O-etylserine s sustrte. O- etylserine is synthesized y serine etyltrnsferse nd together with O-etylserine(thiol)lyse it is ssoited s enzyme omplex lled ysteine synthse (Hell 2003; Sito 2004). The formtion of ysteine is diret oupling step etween sulfur nd nitrogen ssimiltion in plnts, nd it is the preursor of redued sulfur nd sulfur donor for most other orgni sulfur ompounds present in plnt tissue (Brunold 1993; De Kok et l. 2002). 16

18 Chpter 1 Fig. 2. Sulfte redution nd ssimiltion, nd metolism of H 2 S in plnts. APS redutse, denosine 5 -phosphosulfte redutse; Fd red, Fd ox, redued nd oxidized ferredoxin; GSH, GSSG, redued nd oxidized glutthione (dpted from De Kok et l. 2007). Different sulfte trnsporters re involved in the uptke nd distriution of sulfte in plnts. Aording to their ellulr nd suellulr expression nd possile funtioning, the sulfte trnsporters gene fmily hs een lssified in up to 5 different 17

19 groups (Buhner et l. 2004; Sito 2004; Hwkesford nd De Kok 2006; Korlewsk et l. 2007, 2008, 2009,, 2010; Fig. 3). Different isoforms re speifilly expressed in the root or in the shoot or expressed oth in root nd shoot. The Group 1 trnsporters re 'high ffinity sulfte trnsporters' (K m 10 μm), whih re involved in the uptke of sulfte y the roots. The Group 2 trnsporters re vsulr trnsporters nd re 'low ffinity sulfte trnsporters'. The role of Group 3 trnsporters is not well known yet. The Group 4 trnsporters my e involved in the trnsport of sulfte from the vuole (Ktok et l. 2004; Hwkesford 2007, 2008). The Group 5 trnsporters is distint group nd one of the isoforms (Sultr5;2) in Aridopsis is hrterized s molydenum trnsporter (Tomtsu et l. 2007; Bxter et l. 2008). It is still unresolved, whether sulfte itself or metoli produts of the sulfur ssimiltion (viz. O-etylserine, ysteine, glutthione) t s signls in the regultion of the expression nd tivity of the sulfte trnsporters (Buhner et l. 2004; Hwkesford nd De Kok 2006; Korlewsk et l. 2007, 2008, 2009,, 2010). The mjority of genes enoding the enzymes of sulfte ssimiltion, exept sulfite redutse, re present in plnts in multiple isoforms (Kopriv nd Koprivov 2003; Tkhshi et l. 2011). The in situ sulfte onentrtion in the plstid/hloroplst might e the limiting/regultory step in the sulfur redution pthwy, sine the ffinity of ATP sulfurylse for sulfte is rther low (K m pproximtely 1 mm; Stulen nd De Kok 1993). Regultion of the tivity of APS redutse is presumly the primry regultion point in the sulfte redution, sine the tivity of APS redutse is the lowest of the enzymes of the sulfte redution pthwy nd it hs fst turnover rte (Brunold 1993; Leustek nd Sito 1999; Kopriv nd Koprivov 2003; Sito 2004). The sulfte frtion, whih is not diretly redued, is present in the vuoles. The rte of remoiliztion of sulfte from the vuole my e rther slow nd sulfur-defiient plnts often ontin detetle levels of sulfte (Dvidin et l. 2000; Hwkesford nd Wry 2000; Buhner et l. 2004). 18

20 Chpter 1 Fig. 3. Phylogeneti neighour-joining tree of the oding DNAs of the Aridopsis nd Brssi olere sulfte trnsporter fmily (dpted from Buhner et l. 2004). In ddition to sulfte tken up y the roots, plnts re le to utilize folirly sored sulfurous ir pollutnts (viz. SO 2, H 2 S) s sulfur soure for growth. It hs een well estlished tht folirly sored H 2 S is diretly metolized with high ffinity into ysteine 19

21 nd susequently into other orgni sulfur ompounds (De Kok et l. 1998, 2002, 2007, 2009, 2011). There is ler evidene for intertion etween tmospheri nd pedospheri sulfur utiliztion, s H 2 S exposure resulted in downregultion of the uptke nd ssimiltion of sulfte (Westermn et l. 2000, 2001; Buhner et l. 2004; De Kok et l. 2007; Korlewsk et l. 2008). Intertion of Cu with the sulfur metolism Glutthione (GSH) is the predominnt wter-solule non-protein thiol ompound present is plnt tissue nd its onentrtion my e s high s 10 mm, though it vries with plnt development nd plnt nutrition (Leustek et l. 2000). GSH is synthesized in oth the ytosol nd hloroplst nd its level nd redox stte (GSH/GSSG rtio) my e ffeted y environmentl onditions, e.g. temperture, drought, slinity, ir pollution, irrdition (Tusz 2001). Moreover, the level of glutthione nd other non-protein thiols inresed upon exposure to hevy metls (Noito et l. 2002, 2006; Sun et l. 2007). It hs een oserved tht enhned suellulr Cu levels (nd other hevy metls) my indue the synthesis of phytoheltins (PCs), whih re derived from glutthione (nd in some plnt speies from relted ompounds, suh s homo-glutthione; Coett 2003; Verkleij et l. 2003). PCs re synthesized enzymtilly y induile - glutmylysteine dipeptidyltrnspeptidses (PC synthses) with GSH s sustrte: ( GluCys) n Gly + ( GluCys) n Gly ( GluCys) n+1 Gly + ( GluCys) n-1 Gly The numer of -glutmylysteine residues ( GluCys) n in the phytoheltins is reported to e s high s 11, ut normlly in the rnge from 2-5. The indution of PCs synthesis upon Cu exposure is rpid proess (Grill et l. 1987; Zenk 1996). The length of PCs hin shows metl-speifi differenes with Cu 2+ fvoring PC 2 (Ernst et l. 2008). Phytoheltins my omplex with Cu, nd together with ysteine-rih metllothionein proteins (t lest in some plnt speies) they my uffer the Cu onentrtion in the ytosol (De Vos et l. 1992; Ruser 2001; Coett nd Goldsrough 2002; Ernst et l. 2008; Burkhed et l. 2009). However, the signifine of 20

22 Chpter 1 phytoheltins in Cu detoxifition ppers to e restrited (De Vos et l. 1992; Hll 2002; Sht et l. 2002; Coett 2003; Coett nd Goldsrough 2002; Verkleij et l. 2003; Lee nd Kng 2005; Ernst et l. 2008; Yruel 2005, 2009). In ddition to the sulfur-rih PCs lso GSH itself (or even free ysteine), metllothionins nd low moleulr weight ysteine-rih proteins my e involved in Cu inding nd detoxifition in plnts, whih would imply diret interferene with sulfur metolism (Coett nd Goldsrough 2002; Coett 2003; Verkleij et l. 2003; Lee nd Kng 2005; Clemens nd Peršoh 2009; Sirko nd Gotor 2007). There is evidene tht sulfur nutrition nd its metolism my e involved in tolerne nd detoxifition of hevy metls (Noito et l. 2006; Sirko nd Gotor 2007; Sun et l. 2007). The uptke of sulfte y the root nd its ssimiltion re modulted y oth the plnt sulfur sttus nd the sulfur demnd for growth nd is ffeted y environmentl onditions (Hwkesford nd De Kok 2006; Hneklus et l. 2007; De Kok et l. 2011). Synthesis of PCs, metllothionins nd glutthione upon hevy metl exposure might require n inresed sulfte uptke nd ssimiltion (Anderson nd MMhon 2001; Noito et l. 2006; Sun et l. 2007; Ernst et l. 2008). For instne, Cd exposure indued n upregultion of expression nd tivity of the sulfte trnsporters nd severl enzymes of the sulfur ssimiltion pthwy nd glutthione synthesis, viz. ATP sulfurylse, APS sulfotrnsferse, -glutmylysteine synthetse nd glutthione synthetse (Anderson nd MMhon 2001). Exposure of plnts to enhned levels of hevy metls (e.g. Cd, Cu) resulted in n enhned expression nd tivity of high ffinity sulfte trnsporters (Sultr1;1, Sultr1;2; Noito et l. 2006; Sun et l. 2007). Moreover, the expression of Sultr4;1, whih is involved in the efflux of sulfte from the vuole ws upregulted upon exposure to Cu (Shivon et l. 2007). Aim nd outline of the thesis Anthropogeni tivities hve resulted in widespred pollution of soils with Cu nd other hevy metls. Cu nd other hevy metl pollution will presumly further inrese due to n inresing glol 21

23 popultion nd the susequent intensifition of plnt nd niml prodution (Dh nd Strmns 2005). Enhned Cu levels in plnts my intert with nd distur metolism in severl wys nd sulfur metolites might hve potentil in its detoxifition. The generl im of this projet ws to get more insight in the physiologil sis of the phytotoxiity of Cu nd the signifine of sulfur metolism in its detoxifition. In this thesis the followings topis were investigted: і) the impt of enhned Cu onentrtions on physiologil funtioning nd its intertion with the regultion of uptke, distriution nd metolism of sulfte, іі) the impt of light qulity (UV rdition) on Cu toxiity, iii) the intertion etween enhned Cu 2+ levels nd sulfte deprivtion, iv) the signifine of pedospheri (sulfte) nd tmospheri (H 2 S) sulfur nutrition in Cu detoxifition. Chinese ge [Brssi pekinensis (Lour.) Rupr.] elongs to the Brssiee (the mustrd fmily; Shrnz et l. 2006). Brssi speies re hrterized y high sulfur requirement for growth, nd the impt of sulfur nutrition on its physiology hs extensively een studied (De Kok et l. 2000; Buhner et l. 2004; Korlewsk et l. 2007, 2008, 2009,, 2011; Prmr et l. 2007; Stuiver et l. 2009). Exeptionlly, plnt speies elonging to the fmily Brssiee re not le to evolve Cu tolerne on Cu-enrihed soils (Wlker nd Bernl 2004). Chpter 2 desries the mteril nd methods used in the present study. In Chpter 3 the interferene of Cu exposure with the regultion of the uptke, distriution nd metolism of sulfte, nd the minerl nutrient omposition ws studied. In Chpter 4 the impt of UV rdition on Cu toxiity nd sulfur metolism ws investigted. In Chpter 5 the impt of enhned Cu onentrtions nd sulfte deprivtion on the uptke nd metolism of sulfte ws studied. In Chpter 6 the signifine of pedospheri (sulfte) nd tmospheri sulfur nutrition (H 2 S) on Cu toxiity ws evluted. In Chpter 7 the phytotoxiity of enhned Cu onentrtions nd possile signifine of sulfur metolism in its detoxifition in Chinese ge is disussed. 22

24 Chpter 2. Mteril nd methods

25

26 Chpter 2 Plnt mteril nd growth onditions Chinese ge (Brssi pekinensis (Lour.) Rupr. v. Ksumi F1 (Nikerson-Zwn, Mde, The Netherlnds) ws used in the present studies. Seeds were germinted in vermiulite in limte ontrolled room. Dy nd night tempertures were 22 nd 18 C, respetively, reltive humidity ws 60-70% nd the photoperiod ws 14 h t photon fluene rte of μmol m 2 s 1 (within the nm rnge) t plnt height, supplied y Philips Mster 58W/83 nd 84 fluoresent tues in rtio of 1: dy-old seedlings were trnsferred to n erted 25% Hoglnd nutrient solution, ph , onsisting of 1.25 mm C(NO 3 ) 2.4H 2 O, 1.25 mm KNO 3, 0.25 mm KH 2 PO 4, 0.5 mm MgSO 4.7H 2 O, 11.6 μm H 3 BO 3, 2.4 μm MnCl 2.4H 2 O, 0.24 μm ZnSO 4.7H 2 O, 0.08 μm CuSO 4.5H 2 O, 0.13 μm N 2 MoO 4.2H 2 O nd 22.5 μm Fe 3+ -EDTA, ontining supplementl CuCl 2 onentrtions rnging from 1-15 μm in 13 l stinless steel ontiners (10 sets of plnts per ontiner, 3 plnts per set) or in 30 l ontiners (20 sets of plnts per ontiner, 3 plnts per set) in limte-ontrolled room. For the experiments on sulfte deprivtion ll sulfte slts were repled y hloride slts. H 2 S fumigtion Two 13 l ontiners (10 sets of plnts per ontiner, 3 plnts per set) were pled in 150 l ylindril stinless steel inets (0.6 m dimeter) with poly(methyl methrylte) top. Dy nd night tempertures were 22 nd 18 C ( 1 C), respetively, nd the reltive humidity ws 60-70%. The photoperiod ws 14 h t photon fluene rte of μmol m 2 s 1 (within the nm rnge) t UV rdition level of 1.0 mw m -2 (UV-A+B; within the nm rnge) t plnt height, supplied y Philips HPL(R)N (400 W) lmps s light soure. The temperture ws ontrolled y djusting the inet wll temperture; the ir exhnge ws 40 l min -1 nd the ir inside the inets ws stirred ontinuously y two ventiltors. Pressurized H 2 S diluted with N 2 (1 ml l -1 ) ws injeted into the inoming ir strem nd djusted to the desired level y ASM eletroni mss flow ontrollers (Bilthoven, The Netherlnds). H 2 S 25

27 levels in the inets were ontrolled y n SO 2 nlyzer (model 9850) equipped with H 2 S onverter (model 8770, Monitor Ls, Mesurement Controls Corportion, Englewood, CO, USA). Seling of the lid of the ontiners nd plnt sets prevented the sorption of tmospheri H 2 S y the solution. UV-A+B rdition ws mesured with Digitl Ultrviolet Rdiometer model 5.0 (Solrteh In. Fenton, MI 48430, USA). Plnt hrvest Plnts were hrvested 3 h fter strt of the light period, nd for nion determintion, roots were rinsed in ie-old de-minerlized wter (for 3 x 20 s) in order to remove sulfte from the free spe (Kronzuker et l. 1995). Roots were seprted from the shoots, weighed, nd for pigment, phytoheltins nd RNA isoltion plnt mteril ws frozen immeditely in liquid N 2 nd stored t -80 C until further use. For nlysis of the wter-solule non-protein thiol ontent freshly hrvested plnt mteril ws used. For determintion of dry mtter, totl sulfur, nitrogen nd Cu ontent, plnt tissue ws dried t 80 C for 48 h. For dt on dry mtter, totl sulfur nd Cu ontent, presented in Chpter 6, plnt mteril ws freeze dried t -60 C for h. Pigment ontent Shoots or lef diss were homogenized in 100% etone using n Ultr Turrx (10 ml per g fresh weight) nd entrifuged t 800 g for 20 minutes. The hlorophyll, nd totl rotenoids ontent were determined ording to Lihtenthler (1987). Chlorophyll fluoresene Chlorophyll fluoresene ws mesured on the dxil side of the youngest fully developed leves using modulted fluorometer (PAM 2000, H. Wlz GmH, Effeltrih, Germny). Mesurements were performed on intt plnts in drk room under dim green light t 26

28 Chpter 2 room temperture fter drk dpttion for t lest 30 min. The mximum hlorophyll fluoresene level of drk-dpted leves (F m ) ws determined using sturting flsh of white light during 1 s t photon fluene rte of 4,000 µmol photons m -2 s -1. The initil hlorophyll fluoresene level of drk-dpted leves (F 0 ) ws sensitized with red light t photon fluene rte of out 0.1 µmol m -2 s -1 t modultion of 1.6 khz. The quntum yield of photosystem II photohemistry (F v /F m ) ws lulted from the rtio of vrile (F v = F m - F 0 ) to mximum hlorophyll fluoresene. Speifi lef re Lef re ws mesured y seling the leves in plsti sheets. The projeted lef re ws mesured y snning nd lef re ws determined y ImgeJ (see: The speifi lef re rtio (m -2 g -1 FW) ws lulted. Photosynthesis nd drk respirtion Net photosynthesis nd drk respirtion rtes were reorded in the limte room using portle, open-flow gs exhnge nlyzer (TPS-2, PP systems In., Amesury, MA, USA). Mesurements were tken of 2.5 m 2 re of the youngest fully expnded lef tthed to the plnt. The flow rte of ir through the lef hmer ws set t 300 ml min 1 nd the mesurement onditions were similr to the mient growth onditions. The photosynthesis rte ws mesured t photon fluene rte of µmol m 2 s 1 (within the nm rnge). Drk respirtion ws mesured fter drk dpttion for t lest 10 min. Cu nd minerl nutrient ontent Dried plnt mteril ws pulverized y Retsh Mixer-Mill (type MM2; Hn, Germny) nd Cu nd minerl nutrient ontent were nlyzed y indutively oupled plsm optil emission spetrosopy (ICP- OES). 5 ml of 85:15 (v/v) mixture of nitri id (Primr, Aristr s.g 27

29 1.42, 70%) nd perhlori id (Aristr/Primr, 70%) were dded to 250 mg of oven dried plnt mteril nd llowed to stnd t room temperture for t lest two hours. Smples were digested in heting lok (Crolite, London, UK) for 3 h t 60 C, 1 h t 100 C, 1 hour t 120 C nd 2.5 h t 200 C. Tempertures were rmped t either 1 or 2 C min -1 for the first nd lst or 2 nd nd 3 rd periods. Susequently 5 ml of 9.25% (v/v) HCl (AR grde) ws dded to the dried smples nd heted for 1 h t 80 C. After further ddition of 20 ml of wter, the smples were heted for 30 min t 80 C. Finlly, extrt were mde up to 25 ml with wter nd sujeted to ICP-OES (Applied Reserh Lortories, Vllire, Eulens, Switzerlnd). Totl nitrogen nd sulfur ontent The totl nitrogen ontent ws determined with modified Kjeldhl method (Brneix et l. 1988). 50 mg of dried, pulverized plnt tissue (y Retsh Mixer-Mill, type MM2; Hn, Germny) ws extrted in 2 ml extrtion solution (33.3 g N sliylte l -1 onentrted H 2 SO 4 ) in presene of 0.33 g N 2 S 2 O 3.5H 2 O nd smll mount of tlyst (K 2 SO 4 :CuSO 4 :N 2 SeO 3, 15:5:0.085 (w/w/w) folded in smll sh free filter until the smples were ompletely ler. The smples were mde up to 50 ml with de-minerlized wter nd NH 4 + ws mesured olorimetrilly t 410 nm fter retion with Nessler s regent. The totl sulfur ontent ws determined ording to modifition of the method of Jones (1995). Dried plnt tissue ws pulverized nd drop y drop, 50% Mg(NO 3 ) 2.6H 2 O (w/v) solution ws dded to mg dry mteril just to sturte the tissue nd the smples were dried overnight in n oven t 100 C. Susequently, the trys were overed with lids nd the smples were shed in n oven (Crolite BWF 11/13, Hope Vlley, Englnd) t 650 C until the residue hd eome ompletely white. The residue ws dissolved in 10 ml 20% qu regi (50 ml on. HNO 3 nd 150 ml on. HCl in 1 l distilled wter), quntittively trnsferred to mesuring flsk nd dded up to 50 or 100 ml with de-minerlized wter. To 10 or 25 ml of extrt, one SulfVer 4 Regent Powder Pillow (HACH, Permhem regents, Lovelnd, USA) ontining BCl 2 ws dded, nd the turidity ws mesured with spetrophotometer (HACH DR/400V, Lovelnd, USA) t 450 nm. 28

30 Chpter 2 Nitrte nd sulfte ontent Frozen mteril ws homogenized in de-minerlized wter (10 ml per g fresh weight) with n Ultr Turrx for 30 s (0 C), the homogente ws filtered through one lyer of Mirloth nd inuted t 100 C in oiling wter th for 10 min. The filtrte ws entrifuged t 30,000 g for 15 min (0 C). The nions were seprted y HPLC on n IonoSpher A nion exhnge olumn (250 x 4.6 mm; Vrin Benelux, Bergen op Zoom, The Netherlnds) nd determined refrtometrilly ording to Ms et l. (1986) using Knuer differentil refrtometer (model 98.00, Bd Homurg, Germny). 25 mm potssium iphthlte (ph 4.3) ontining 0.02% NN 3 (w/v) ws used s moile phse. The HPLC pprtus onsisted of Seprtions high preision pump (model 300; H.I. Amht, The Netherlnds) provided with Rheodyne smple injetor (model 7175; loop volume 20 l; Cotti, CA). The flow rte ws 1 ml min -1 nd the detetor temperture ws kept t 25 C y wter th. Pek nlysis ws performed with Shimdzu Chromtop C-R8A dt proessor (Kyoto, Jpn). Amino id ontent The mino ids were extrted s desried for nitrte nd sulfte, nd fter deproteiniztion of the extrts, its ontent ws determined with the ninhydrin olor regent ording to Rosen (1957) nd mesured olorimetrilly t 578 nm. Wter-solule non-protein thiols For nlysis of the wter-solule non-protein thiols, fresh plnt mteril ws homogenized in n extrtion medium ontining 80 mm sulfosliyli id, 1 mm EDTA, nd 0.15% (w/v) sori id with n Ultr Turrx t 0 C (10 ml per g fresh weight). Oxygen ws removed from the solution y spirtion with N 2. The homogente ws filtered through one lyer of Mirloth nd the filtrte ws entrifuged t 29

31 30,000 g for 15 min (0 C). Totl wter-solule non-protein thiol ontent ws determined olorimetrilly t 413 nm fter retion with 5,5'-dithiois[2-nitroenzoi id] ording to De Kok et l. (1988). Phytoheltins Frozen plnt mteril ws dried in freeze dryer (Heto Lyol 3000; Holten, The Netherlnds) for hours. The freeze-dried plnt mteril ws pulverized y Retsh Mixer-Mill (type MM2; Hn, Germny) nd phytoheltins were nlyzed ording to Wojs et l. (2008). Sulfte nd nitrte uptke nd sulfte uptke pity For mesurement of sulfte nd nitrte uptke, seedlings were grown on 25% Hoglnd solution t vrious CuCl 2 onentrtions, nd susequently trnsferred to pots ontining 1 l freshly prepred, erted 25% Hoglnd solution in presene of CuCl 2 in limteontrolled room, for onditions see ove. At the eginning of the uptke mesurement nd fter 24 h, liquots were tken from the nutrient solution (djusted to the originl volume with de-minerlized wter y weighing) nd nlyzed for sulfte nd nitrte ontent y HPLC. Sulfte uptke pity ws determined s desried y Korlewsk et l. (2007). Three sets of plnts (3 plnts per set) per tretment were trnsferred to 25% Hoglnd solution leled with 35 S-sulfte (2 MBq l -1 ) nd inuted for 30 min t 25 C t t photon fluene rte of μmol m 2 s 1 (within the nm rnge). Susequently, plnts were removed nd roots rinsed in ieold non-leled nutrient solution (3 x 20 s). Roots nd shoots were seprted nd digested in 1 N HCl t room temperture for 7 dys. The extrts were filtered through one lyer of Mirloth (Cliohem Corportion, L Joll, CA, USA) nd 100 μl of the filtrte ws mixed with 1 ml Emulsifier Sintilltor Plus (Perkin Elmer, Boston, MA, USA). Rdiotivity ws mesured with liquid sintilltion ounter (TRI- CARB 2,000 CA Liquid Sintilltion Anlyzer, Perkin Elmer, Wlthm, MA, USA). 30

32 Chpter 2 Totl RNA isoltion Totl RNA from root nd shoot ws isolted y using TRI REAGENT TM (Sigm), mixture of gunidine thioynte nd phenol in monophse solution, whih involved n dditionl phenol-hloroformisomyl lohol extrtion of the queous phse fter the first entrifugtion (Verwoerd et l. 1989). The finl ir-dried pellet ws dissolved in n pproprite volume of diethyl pyroronte-treted wter. The qulity of the RNA smples ws heked y eletrophoresis of 2 µg liquot on 1% (w/v) Tris-ette/grose gel. The RNA onentrtion ws mesured t 260 nm using NnoDrop ND1000 UV/VIS Spetrophotometer (Wilmington, USA). Northern nlysis Northern hyridiztion of the sulfte trnsporters nd APS redutse trnsripts ws performed ording to Churh nd Gilert (1984), with pre-hyridiztion/hyridiztion t 65 nd 60 C, respetively. 10 μg of totl RNA per slot ws seprted on n grose/formldehyde gel nd lotted onto positively hrged nylon memrne (Hyond- N+). Sequene diversity, espeilly in the 3' non-oding region, llowed the use of prtil DNA frgments for gene-speifi hyridiztion to the respetive Brssi sulfte trnsporter mrna (Buhner et l. 2004). For APS redutse, DNA frgment of APS redutse 2 (APR2) from Aridopsis thlin (L.) Heynh ws used s proe (Durenkmp et l. 2007). DNA frgments were leled with 32 P-dCTP nd used s hyridiztion proes. After hyridiztion with proes for sulfte trnsporters memrnes were wshed t 65 C twie with 2 x SSC, 0.1% SDS for 5 nd 30 min, one with 1 x SSC, 0.1% SDS nd twie with 0.1 x SSC, 0.1% SDS for t lest 30 min eh, nd exposed to Cylone MultiPurpose Phosphor Sreen (Perkin Elmer, U.K.). After hyridiztion with proe speifi for APS redutse, memrnes were wshed t 60 C twie with 2 x SSC, 0.1% SDS for 10 min nd twie with 1 x SSC, 0.1% SDS for 15 min, nd exposed to Cylone MultiPurpose Phosphor Sreen (Perkin Elmer, U.K.). The signl ws visulized nd the reltive expression estimted y using OptiQunt Aquisition nd Anlysis (Pkrd 31

33 Instrument Co.) softwre in se of exposure to Cylone MultiPurpose Phosphor Sreen (Perkin Elmer, U.K.). Sttistis For the sttistil nlysis of the dt n unpired Student s t-test (p 0.01) ws used. Further detils on the numer of experiments, mesurements per experiment nd numer of plnts re given in the legends of the tles nd figures. 32

34 Chpter 3. Copper exposure interferes with the regultion of the uptke, distriution nd metolism of sulfte in Chinese ge

35

36 Chpter 3 Astrt Exposure of Chinese ge (Brssi pekinensis) to enhned Cu 2+ onentrtions (1-10 μm) resulted in lef hlorosis, loss of photosyntheti pity nd lower iomss prodution t 5 µm. The derese in pigment ontent ws likely not the onsequene of degrdtion, ut due to hindered hloroplst development upon Cu exposure. The Cu ontent of the root inresed with the Cu 2+ onentrtion (up to 40-fold), though only minor proportion (4%) of it ws trnsferred to the shoot. Enhned Cu 2+ levels sustntilly ffeted the overll minerl nutrient omposition t 5 µm Cu 2+. The nitrte uptke y the root ws sustntilly redued t 5 µm Cu 2+. The nitrogen ontent of the root ws ffeted little t lower Cu 2+ levels, wheres tht in the shoot ws deresed t 5 µm Cu 2+. Cu ffeted the uptke, distriution nd metolism of sulfte in Chinese ge. The totl sulfur ontent of the shoot ws inresed t 2 µm Cu 2+, whih ould e ttriuted minly to n inrese in sulfte ontent. Moreover, there ws strong inrese in wter-solule nonprotein thiol ontent in the root nd to lesser extent in the shoot t 1 µm, whih ould only prtilly e sried to Cu-indued enhnement of the phytoheltin ontent. The nitrte uptke y the root ws sustntilly redued t 5 µm Cu 2+, oiniding with the derese in iomss prodution. However, the tivity of the sulfte trnsporters in the root ws slightly enhned t 2 nd 5 µm Cu 2+, ompnied y n enhned expression of the Group 1 high ffinity trnsporter Sultr1;2, nd the Group 4 trnsporters Sultr4;1 nd Sultr4;2. In the shoot there ws n indution of expression of Sultr4;2 t 5 nd 10 µm Cu 2+. The expression of APS redutse ws ffeted little in the root nd shoot up to 10 µm Cu 2+. The upregultion of the sulfte trnsporters my e due not only to greter sulfur demnd t higher Cu levels ut lso the onsequene of interferene of Cu with the signl trnsdution pthwy regulting the expression nd tivity of the sulfte trnsporters. 35

37 Introdution Copper is nutrient essentil for plnt funtioning, with requirement mongst the lowest of ll elements. However, t elevted levels, it my rpidly eome phytotoxi (Sheldon nd Menzies 2005; Xiong et l. 2006; Kopsell nd Kopsell 2007; Hn et l. 2008). Exessive ioni Cu is phytotoxi nd generlly results in stunted root growth nd shoot development nd in lef hlorosis (Yruel 2005; Kopsell nd Kopsell 2007). The phytotoxiity of Cu is generlly sried to its possile retion with thiol groups of proteins nd glutthione, nd its potentil to indue the prodution of retive oxygen speies vi the metl-tlyzed Her-Weiss retion, nd the susequent lipid peroxidtion, protein denturtion nd DNA muttion (De Vos et l. 1993; Pinto et l. 2003; Morelli nd Srno 2004; Yruel 2009). Furthermore, Cu toxiity my e used y ntgonisti effets with other tions, suh s Fe, in protein funtioning (Pätsikkä et l. 1998, 2002; Kopsell nd Kopsell 2007; Yruel 2009). Enhned suellulr Cu levels generlly indue the synthesis of phytoheltins, whih re derived from glutthione (nd in some plnt speies from relted ompounds, suh s homo-glutthione). Phytoheltins my omplex with Cu, nd together with ysteine-rih metllothionein proteins (t lest in some plnts speies) nd my uffer the Cu onentrtion in the ytosol (De Vos et l. 1992; Ruser 2001; Coett nd Goldsrough 2002; Ernst et l. 2008; Burkhed et l. 2009). Still, the signifine of phytoheltins in Cu detoxifition ppers to e very restrited (De Vos et l. 1992; Coett nd Goldsrough 2002; Sht et l. 2002; Coett 2003; Verkleij et l. 2003; Yruel 2005, 2009; Ernst et l. 2008). The uptke nd ssimiltion of sulfur in plnts is generlly demnd driven nd is regulted y the tul sulfur requirement for growth (Hwkesford nd De Kok 2006). Brssi speies re hrterized y high sulfur requirement for growth nd the impt of sulfur nutrition on expression nd tivity of the sulfte trnsporters nd its ssimiltion hs extensively een studied (Buhner et l. 2004; Korlewsk et l. 2007, 2008, 2009,; Prmr et l. 2007; Stuiver et l. 2009). Exposure of plnts to exessive toxi metls might ffet the regultion of uptke nd ssimiltion of sulfte, due to their retion with sulfur metolites nd the possile indution of synthesis of sulfur-rih phytoheltins nd metllothioneins (Ernst et 36

38 Chpter 3 l. 2008; Sirko nd Gotor 2007). In the present study, therefore, the impt of Cu 2+ (up to 10 µm) in the root environment on physiologil funtioning of Chinese ge (Brssi pekinensis) ws studied nd the onsequenes for the uptke, distriution nd metolism of sulfte were evluted. Plnt growth onditions dy-old seedlings were trnsferred to 25% Hoglnd nutrient solution, ontining supplementl onentrtions of 0, 1, 2, 5 nd 10 μm CuCl 2, in 13 l stinless steel ontiners (10 sets of plnts per ontiner, 3 plnts per set) in limte ontrolled room for 7 dys. Dy nd night tempertures were 25 nd 20 C ( 1 C), respetively, nd the reltive humidity ws 60-70%. The photoperiod ws 14 h t photon fluene rte of 340 ± 20 µmol m 2 s 1 (within the nm rnge) nd 1.5 mw m -2 UV-A+B rdition (within the nm rnge) t plnt height, supplied y Philips HPI-T (400 W) lmps s light soure. UV-A+B rdition ws mesured with Digitl Ultrviolet Rdiometer model 5.0 (Solrteh In. Fenton, MI 48430, USA). Results Impt of Cu on growth, pigments nd ontent of Cu nd other minerl nutrients Exposure of Chinese ge to enhned Cu 2+ onentrtions ( 2 µm) in the nutrient solution resulted in hlorosis of the shoot whih egn in young developing leves, nd in deresed iomss prodution of oth root nd shoot (Fig. 1, Tle 1 nd 2). A 3 dy exposure resulted in signifint derese in pigment ontent t 2 µm Cu 2+. In ddition, onentrtions 5 µm Cu 2+ ffeted plnt iomss prodution s demonstrted y the inrese in the shoot to root rtio, inditing tht t the onset of exposure, root growth ws reltively more redued thn shoot growth (Fig. 1). 37

39 4 3 dys 5 dys 7 dys Shoot iomss (g) Root iomss (g) Shoot/root rtio Chl + (mg g -1 FW) d d d d d Chl / 4 3 d d Chl/Cr Cu 2+ onentrtion ( M) Fig. 1. Impt of Cu 2+ on iomss prodution nd pigment ontent of Chinese ge. 10 to 14 dy-old seedlings were grown on 25% Hoglnd solution ontining 0, 1, 2, 5 nd 10 µm CuCl 2 for 3, 5 nd 7 dys. The initil fresh weight of shoot nd root ws nd g, respetively. The initil hlorophyll ontent ws mg g -1 fresh weight, the hlorophyll / nd hlorophyll/rotenoid rtios were nd , respetively. All dt represent the men of 3 mesurements on 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 38

40 Chpter 3 After 5 nd 7 dys the toxi effets of Cu 2+ onentrtions of 5 µm eme more pronouned nd oth shoot nd root iomss prodution were strongly redued, wheres the pigment ontent of the shoot ws sustntilly deresed (Fig. 1, Tle 1 nd 2). At 5 µm Cu 2+ there ws n inrese in dry mtter ontent in oth root nd shoot (Tle 2). It ws evident from the hlorophyll / nd hlorophyll/rotenoid rtios tht the ontent of the different pigments deresed similrly with time upon exposure to enhned Cu 2+ onentrtions (Fig. 1). Upon exposure to 10 µm Cu 2+ shoot hlorosis strted to develop rpidly nd hlorophyll ontent deresed signifintly fster thn tht of hlorophyll nd rotenoids, resulting in signifintly deresed hlorophyll / nd hlorophyll/rotenoid rtio (Fig. 1, Tle 2), wheres fter 7 dys lef injury (lef edge nerosis) strted to develop. The Cu ontent of oth root nd shoot inresed with the Cu 2+ onentrtion in the nutrient solution (Tle 2). However, the level of inrese ws quite different in root nd shoot. At 10 µm Cu 2+ the Cu ontent of roots inresed 40-fold upon 7 dys of exposure, where in the shoot it only inresed up to 4.6-fold (Tle 2). It ws evident tht the exessive Cu tken up y the root ws rther immoile. From the root nd shoot iomss nd their Cu ontent it ould e estimted tht the trnslotion of Cu from root to the shoot deresed with inresing Cu onentrtions from 28% in ontrol to 4% t 10 µm Cu 2+. The overll minerl nutrient ompositions of root nd shoot were lso sustntilly ffeted t 5 µm Cu 2+ (Tle 3). Exposure of Chinese ge to Cu 2+ resulted in derese in the ontent of C nd Mg in root nd tht of Fe in the shoot, nd derese in the ontent of Mn, P, K in root nd shoot, wheres tht of N nd Zn inresed (Tle 3). Impt of Cu on hlorophyll fluoresene, photosynthesis nd drk respirtion Despite the derese in pigment ontent upon exposure to high Cu 2+ onentrtions, the quntum yield of photosystem II photohemistry (F v /F m ) in leves ws ffeted little t 2 µm (Tle 1). The F v /F m rtio ws slightly deresed only t 10 µm Cu 2+, wheres the pigment ontent hd deresed to 26% of tht of ontrol plnts. On lef re 39

41 sis, the derese in pigment ontent ws ompnied y derese in the rte of photosynthesis. However, when expressed on hlorophyll sis, the rte of photosynthesis ws hrdly ffeted t levels up to 10 µm Cu 2+ (Tle 1). The rte of drk respirtion ws signifintly deresed t toxi Cu 2+ levels ( 5 µm; Tle 1). Tle 1. Impt of Cu 2+ on hlorophyll ontent, hlorophyll fluoresene, photosynthesis nd drk respirtion in leves of Chinese ge. 10 to 14 dy-old seedlings were grown on 25% Hoglnd nutrient solution ontining 0, 1, 2, 5 nd 10 µm CuCl 2 for 7 dys. Photosynthesis nd drk respirtion (µmol m -2 s -1 ) of the youngest fully expnded leves (3 rd or 4 th ) were mesured nd represent the men of 3 to 4 mesurements on 3 to 4 plnts ( SD). Chlorophyll ontent (mg m -2 ) of lef diss punhed from the sme leves ws mesured nd represents the men of 3 mesurements ( SD). Chlorophyll fluoresene (F v /F m rtio) represents the men of 5 to 10 mesurements ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Impt of Cu on nitrogen nd sulfur metolite ontent The totl nitrogen ontent of the root of Chinese ge ws ffeted little upon exposure to enhned Cu 2+ onentrtions, wheres tht in the shoot ws deresed t 5 µm Cu 2+ (Tle 2). Furthermore, the nitrte nd mino id ontents of the root were hrdly ffeted y Cu 2+ ; their ontents were only sustntilly enhned t 10 µm. Cu 2+ onentrtions of 2 µm hd sustntil effet on the totl sulfur ontent of the shoot, wheres the ontent of the root remined unltered (Tle 2). The enhnement in totl sulfur of the shoot ould e ttriuted to n inrese in sulfte ontent t 2 µm Cu 2+. Moreover, upon exposure to 1 µm Cu 2+, there ws strong inrese in wter-solule non-protein thiol ontent in the root nd, to lesser extent in the shoot (t 2 µm Cu 2+ ). However, this inrese in thiol ontent ould only prtilly e sried 40

42 Chpter 3 Tle 2. Impt of Cu 2+ on iomss prodution, dry mtter ontent nd ontent of pigments, opper, totl nitrogen nd sulfur, nitrte, sulfte, mino ids, wter-solule non-protein thiols nd phytoheltins. For experimentl detils see legends Tle 1. The initil fresh weight of shoot nd root ws nd g, respetively. Dt on iomss (g FW) nd dry mtter ontent (DMC; %) represent the men of 6 nd 3 independent experiments, respetively, with 3 mesurements on 6 nd 3 plnts in eh, respetively ( SD). Pigment ontent (mg g -1 FW) represents the men of 2 independent experiments with 3 mesurements on 3 shoots in eh ( SD). Copper ontent (µmol g -1 DW) represents the men of 3 mesurements with 9 plnts in eh ( SD). Totl nitrogen (mmol g -1 DW) nd sulfur ontent (µmol g -1 DW) represent the men of 1 nd 2 independent experiments, respetively, with 3 to 6 mesurements on 3 plnts in eh ( SD). Nitrte, sulfte nd wter-solule non-protein thiol ontent (µmol g -1 FW) re the men of 2 independent experiments with 3 mesurements on 3 plnts in eh ( SD). Phytoheltins (PC 2 + PC 3 ) nd mino ids (µmol g -1 FW) represent the men of 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t- test). 41

43 to Cu-indued enhnement of the phytoheltin ontent, of whih thiol residues in the root ounted for 42% of the umulted wter-solule non-protein thiols (Tle 2). In the root, PC 2 nd PC 3 phytoheltins were indued in nerly equl mounts, wheres in the shoot PC 2 ws the pre-dominnt phytoheltin present (dt not presented). Tle 3. Impt of Cu 2+ levels on minerl ontent of Chinese ge. For experimentl detils see legends Tle 1. Dt re expressed s μmol g -1 dry weight nd represent the men of 3 mesurements with 9 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Impt of Cu on nitrte nd sulfte uptke, nd expression of sulfte trnsporters nd APS redutse The uptke of sulfte nd nitrte were ffeted differently upon exposure to enhned Cu 2+ onentrtions. The uptke of sulfte ws slightly enhned t 2 nd 5 µm Cu 2+, respetively, wheres it ws deresed t 10 µm Cu 2+ (Fig. 2). The uptke of nitrte y Chinese ge ws sustntilly redued t 5 µm Cu 2+ (Fig. 2). From the sulfte to nitrte uptke rtio, it ws ovious tht the sulfte uptke inresed t 2 µm, ut ws most pronouned t 5 µm Cu 2+ (Fig. 2). 42

44 Chpter 3 The inrese in sulfte uptke upon Cu 2+ exposure ws lso evident from sulfte uptke pity mesurements, whih showed tht the tivity of the sulfte trnsporters in the root ws upregulted t 2 nd 5 µm Cu 2+ (Fig. 3). At 10 µm Cu 2+ the sulfte uptke pity did not signifintly differ from tht of the ontrol plnts (Fig. 3). The Group 1 high ffinity sulfte trnsporters re involved in the primry uptke of sulfte y the root, nd Sultr1;2 ws highly expressed in the root of Chinese ge, wheres Sultr1;1 ws hrdly expressed (Fig. 4). The impt of Cu 2+ on the expression of Sultr1;2 ws miguous, sine there ws 1.8-fold inrese in expression t 5 µm Cu 2+, wheres it deresed y pprox. 50% t 10 µm Cu 2+ (Fig. 4). The Group 4 sulfte trnsporters re involved in vuolr efflux of sulfte nd Sultr4;1 ws highly expressed in oth root nd shoot. The expression of Sultr4;1 ws hrdly ffeted t 5 µm Cu 2+, ut it ws pprox. 60% deresed in the root nd inresed 1.5-fold in the shoot t 10 µm Cu 2+ (Fig. 4). Sultr4;2 ws slightly expressed in oth the root nd shoot, though its expression inresed more thn 2-fold nd 4-fold in the shoot t 5 nd 10 µm Cu 2+, respetively (Fig. 4). The expression of APS redutse ws not ffeted in either root or shoot t < 10 µm Cu 2+, however, t 10 µm expression ws pprox. 50% deresed in the root nd inresed 1.5- fold in the shoot (Fig. 4). Disussion Cu toxiity is generlly hrterized y n inhiition of shoot nd root iomss prodution, lef hlorosis, loss of photosyntheti tivity (Moquot et l. 1996; Yruel 2005, 2009; Xiong et l. 2006; Hn et l. 2008; Burkhed et l. 2009) nd n ltered root morphology (Pnou-Filotheou nd Boslidis 2004; Sheldon nd Menzies 2005). In Chinese ge, the derese in iomss prodution upon exposure to high Cu levels ws preeded y derese in pigment ontent of the shoot. The Cu-indued derese in the pigment ontent ws ompnied with derese in photosyntheti tivity nd the rte of drk respirtion; however, expressed on hlorophyll sis, the rte of photosynthesis ws not notiely ffeted. The ltter indited tht hlorosis upon high Cu onentrtions ws most likely not the onsequene of pigment 43

45 degrdtion, ut due to hindered hloroplst development upon Cu exposure, s hs lso een oserved in leves of oregno plnts (Pnou-Filotheou et l. 2001). This ws lso supported y hlorophyll fluoresene mesurements, whih showed tht the F v /F m rtio, representing the quntum yield of photosystem II, ws only slightly redued t 10 µm Cu 2+. This result indites tht the remining hloroplsts were funtionl nd either did not or only mrginlly suffered from photoinhiition. The impt of enhned Cu levels on hloroplst development nd/or funtioning my e ttriuted to Cu-indued Fe defiieny or the exhnge of Mg ion in its hlorin ring y Cu (Pätsikkä et l. 2002; Küpper et l. 2003). High Cu levels resulted in sustntil inrese in the Cu ontent of oth root nd shoot of Chinese ge. Despite the reltively strong enhnement of the Cu ontent in the root upon exposure to high levels (up to 40-fold), the exessive Cu tken up y the root ws reltively immoile, sine only minor proportion ws trnsferred to the shoot. However, root growth ws only slightly more ffeted thn shoot growth t the higher Cu 2+ onentrtions ( 5 µm), though the root morphology ws ltered nd more undeveloped side roots were formed (dt not shown). The nture of these ltertions needs to e studied further. On the sis of the urrent results, it is diffiult to ssess the ritil Cu onentrtion, though evidently more thn 3- fold inrese in Cu ontent of the shoot of Chinese ge lredy initited toxi effets. Altertions in minerl omposition t exessive Cu levels hve lso een oserved in other plnt speies (Es nd Kohin 1997; Ouzounidou et l. 1998; Pätsikkä et l. 1998, 2002; Ali et l. 2002; Fgeri 2002; Aloui-Sossé et l. 2004; Sheldon nd Menzies 2005; Kováčik et l. 2009). The intertions of Cu with miro nd mronutrients re either synergisti, ntgonisti or hve no effets, differ with the rop speies nd plnt growing onditions (Fgeri 2002). To wht extent the hnges in minerl omposition re the onsequene of n ltered ompetition in the uptke nd trnsport of the minerls or n ltered root rhiteture t the ourrene of root injury t toxi levels of Cu 2+ levels needs to e further evluted (Sheldon nd Menzies 2005). It is not yet estlished to wht extent hnge in minerl omposition is involved in the onset of the phytotoxity of Cu. 44

46 Chpter Sulfte uptke ( mol g -1 FW root 24 h -1 ) Nitrte uptke ( mol g -1 FW root 24 h -1 ) Sulfte/Nitrte uptke rtio Cu 2+ onentrtion ( M) Fig. 2. Impt of Cu 2+ on the uptke of sulfte nd nitrte y Chinese ge nd sulfte/nitrte uptke rtio. For experimentl detils see legends Tle 1. Dt on sulfte nd nitrte uptke (μmol g -1 FW root 24 h -1 ) represent the men of 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 45

47 Exposure of Chinese ge to enhned Cu onentrtions did not mrkedly ffet nitrogen ontent. Only t 10 µm Cu 2+, where shoots hd eome severely hloroti nd visile injury symptoms (lef edge nerosis) strted to develop, there ws sustntil inrese in nitrte nd mino id ontent, inditing tht nitrogen metolism ws distured. Exposure of Chinese ge to enhned Cu onentrtions sustntilly ffeted the ontent nd distriution of sulfur ompounds in root nd shoot. There ws n umultion of wtersolule non-protein thiols in the root nd, to lesser extent, in the shoot. However, this enhnement ould only prtilly e sried to Cu-indued umultion of phytoheltins (PC 2 nd PC 3 ). The enhned thiol level ws most likely due to n umultion of redued glutthione (nd mye for lesser prt to ysteine), regulrly the pre-dominnt thiol ompound present in plnt tissue (De Kok nd Stulen 1993; De Kok et l. 2007; Rennenerg 2001). The possile impt of high Cu levels on the glutthione redox sttus, s hs een oserved upon exposure of Brssi npus to ute toxi levels of Cu (100 µm; Russo et l. 2007), needs to e studied further. Under norml onditions, the uptke, distriution nd ssimiltion of sulfur re under strit regultory ontrol nd the rte of sulfte uptke y the root will equl the overll sulfur demnd to mintin plnt growth (Hwkesford nd De Kok 2006; Korlewsk et l. 2007, 2008; Rouhed et l. 2009). Brssi speies re hrterized y high sulfur requirement for growth. However, the greter proportion of the sulfte tken up y the root my not diretly e metolized, ut stored s sulfte in the shoot (Cstro et l. 2004; Korlewsk et l. 2007, 2008, 2009, ). It hs een presumed tht the synthesis of sulfur-rih metl-helting ompounds upon exposure of plnts to potentilly toxi metls might require n enhned sulfur demnd, viz. the rte of uptke nd ssimiltion of sulfte (Sirko nd Gotor 2007; Ernst et l. 2008). Indeed, exposure of Chinese ge to high Cu 2+ onentrtions resulted in upregulted expression of Sultr1;2 nd Sultr4;1 sulfte trnsporters ompnied with slightly upregulted sulfte uptke pity t 2 nd 5 µm Cu 2+. In ontrst, the uptke of nitrte ws redued t 5 µm Cu 2+, whih oinided with the derese in iomss prodution. In generl, the net nitrte uptke is losely linked to the reltive growth rte (Ter Steege et l. 1999; Westermn et l. 2000). 46

48 Chpter Cu 2+ onentrtion ( M) Fig. 3. Impt of Cu 2+ on the sulfte uptke pity of Chinese ge. For experimentl detils see legends Tle 1. Dt on sulfte uptke pity (μmol g -1 FW root h -1 ) represents the men of two independent experiments with 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). However, it ws doutful s to whether the oserved upregultion of the sulfte trnsporters in Chinese ge ws solely due to higher sulfur demnd t higher Cu tissue ontents. It is unlikely tht the oserved enhnement of the thiol ompounds in Chinese ge t high Cu 2+ onentrtions would require sustntil upregultion of the sulfte uptke, sine this sulfur proportion never exeeded more thn 5% of the totl sulfur ontent. The upregultion of the sulfte trnsporters in the root ws ompnied with n enhned totl sulfur ontent of the shoot, whih ould e ttriuted to sulfte umultion. It my e tht this umultion of sulfte ws the onsequene of Cu-indued disturne in the signl trnsdution pthwy regulting the expression nd tivity of the sulfte trnsporters. Evidently, Cu exposure ffeted the regultion of sulfte trnsporters in the root in suh wy tht more sulfte ws tken up thn ould e metolized nd utilized for growth, resulting in enhned sulfte ontent of the shoot. 47

49 Fig. 4. Impt of Cu 2+ on mrna undne of sulfte trnsporters (Sultr) nd APS redutse (APR) (Northern-lot nlysis) in root nd shoot of Chinese ge. Equl RNA loding ws determined y ethidium romide stining of gels (shown in the ottom pnels). For experimentl detils see legends Tle 1. A representtive set of dt from two independent experiments is presented. Regultion of sulfte uptke nd its distriution my e ontrolled t trnsriptionl, trnsltionl nd/or post-trnsltionl level. Both externl nd internl sulfte onentrtion nd tht of thiol ompounds viz. glutthione, hve een presumed to e involved in the ontrol of the expression nd tivity of sulfte trnsporters (Hwkesford nd De Kok 2006; Rouhed et l. 2009). The high ffinity sulfte trnsporters Sultr1;1 nd Sultr1;2 re responsile for the primry uptke of sulfte y the root, though their expression nd tivity re differently regulted (Hwkesford nd De Kok 2006; Korlewsk et l. 2008; Rouhed et l. 2008, 2009). The expression nd tivity of the onstitutive sulfte trnsporter Sultr1;2 ppered to e relted to metoli demnd (Korlewsk et l. 2008; Rouhed et l. 2008, 2009). This trnsporter ws only upregulted t sulfte 48

50 Chpter 3 onentrtions in the root environment lose to the K m vlue (5-10 µm) or upon sulfte deprivtion, presumly triggered y low in situ thiol onentrtion. Sultr1;1 is n induile sulfte trnsporter, whih is generlly lmost not expressed in sulfte-suffiient plnts, s ws lso ovious from the urrent study. However, the expression of Sultr1;1 is highly upregulted in the root upon sulfte deprivtion nd is triggered y the in situ (internl or externl) sulfte onentrtion rther thn the thiol onentrtion (Buhner et l. 2004; Korlewsk et l. 2007, 2008, 2009, ; Prmr et l. 2007; Stuiver et l. 2009; Rouhed et l. 2008, 2009). The onstitutive sulfte trnsporter Sultr4;1 nd the induile sulfte trnsporter Sultr4;2 re generlly only upregulted upon sulfur defiieny, when plnts try to remoilize nd redistriute ll possile ville sulfte from, for instne, the vuoles (Buhner et l. 2004; Prmr et l. 2007; Korlewsk et l. 2009,; Stuiver et l. 2009). The expression nd tivity of APS redutse is generlly downregulted t high thiol levels (Westermn et l. 2001; Durenkmp et l. 2007; Korlewsk et l. 2008). From the present study, however, it ws evident tht upon exposure of plnts to enhned Cu 2+ levels, the presumed signl trnsdution pthwy of the regultion of the expression nd the tivity of the sulfte trnsporters nd tht of APS redutse were ypssed or overruled. Here the upregulted expression of Sultr1;2 (root), Sultr4;1 (shoot) nd the Sultr4;2 (shoot) sulfte trnsporter genes resulted in modest inrese in sulfte uptke pity, nd net uptke ourred oth t norml or even enhned thiol levels (root, shoot) nd norml (root) or even enhned sulfte levels (shoot). Moreover, the strongly enhned thiol levels in the root nd to lesser extent in the shoot t high Cu levels did not result in downregultion of the expression of APS redutse. The mjority of ells in oth shoot nd root hve the pity to redue nd ssimilte sulfte in their plstids, enling lol signling of sulfte uptke, distriution nd redution/ssimiltion t ellulr level. The possile signifine of the diret retion of Cu with sulfide nd/or other metolites involved in the sulfur metolism signl trnsdution pthwy needs further to e evluted. 49

51 Conlusions High Cu 2+ onentrtions in the root environment were toxi for Chinese ge, whih my in prt e sried to Cu-indued distured development of the hloroplsts, resulting in hlorosis nd hindered iomss prodution. Furthermore, enhned Cu ontents in the root ffeted the regultion of the uptke nd distriution of sulfte, whih ould not e sried to n impt on the presumed signl trnsdution pthwy therein involved. It remins unler to wht extent the oserved upregultion of the sulfte trnsporters ws the onsequene of higher sulfur demnd t higher Cu onentrtion or the result of n interferene/retion of Cu with the signl ompounds regulting the expression nd tivity of the sulfte trnsporters. 50

52 Chpter 4. UV rdition inreses the toxiity nd impt of opper on sulfur metolism in Chinese ge

53

54 Chpter 4 Astrt Biomss prodution, dry mtter ontent, speifi lef re nd pigment ontent of Chinese ge were ll quite similr whether plnts were grown with HPI-T (2.2 mw m -2, UV-A+B) or SON-T (0 mw m -2, UV-A+B) lmps s light soure. Exposure of plnts to enhned Cu 2+ onentrtions (2 10 μm) resulted in more rpid nd stronger derese in plnt iomss prodution nd pigment ontent in presene of UV rdition (HPI-T) ompred to the sene of UV rdition (SON-T). The F v /F m rtio ws only deresed t 5 μm Cu 2+ in the presene of UV rdition, when lef tissue strted to eome neroti. The ontent of Cu in oth root nd shoot of Chinese ge ws strongly ffeted y the level of UV rdition, oth in sene nd presene of enhned Cu 2+ onentrtions in the root environment. The oserved enhned Cu toxiity in presene of UV is proly due lrgely to higher umultion of Cu in oth root nd shoot. The totl sulfur ontent of the shoot ws inresed t 2 µm Cu 2+ in presene of UV nd t 5 µm Cu 2+ in sene of UV, whih ws minly ttriuted to n inrese in sulfte ontent. Moreover, there ws strong inrese in wter-solule non-protein thiol ontent upon Cu 2+ exposure in the root nd, to lesser extent in the shoot, oth in presene nd sene of UV. The expression nd tivity of the high ffinity sulfte trnsporter, Sultr1;2, ws enhned t 2 µm in presene of UV, nd t 5 µm Cu 2+ in sene of UV. Furthermore, in the shoot, the expression of vuolr sulfte trnsporter, Sultr4;1, ws upregulted t 5 µm Cu 2+ in presene nd sene of UV, whilst the expression of seond vuolr sulfte trnsporter, Sultr4;2, ws upregulted t 10 µm Cu 2+ in presene of UV. The expression of APS redutse in the root ws hrdly ffeted nd it ws slightly downregulted t 2 µm in presene of UV nd t 10 µm, in sene of UV. The expression nd tivity of sulfte trnsporters were enhned upon exposure t enhned Cu 2+ onentrtions; this my e due not only to greter sulfur demnd t higher Cu levels, ut more likely ws the onsequene of Cu toxiity, sine it ourred more rpidly in presene of ompred to the sene of UV. 53

55 Introdution Exposure of Chinese ge (Brssi pekinensis) to 5 µm Cu 2+ resulted in lef hlorosis nd derese in plnt iomss prodution nd pigment ontent (Chpter 3). The derese in pigment ontent ws ompnied y loss of photosyntheti pity, however, expressed on hlorophyll sis, the rte of photosynthesis ws hrdly ffeted t levels up to 10 µm Cu 2+ (Chpter 3). Lef hlorosis, whih ws first visile in young developing leves, ws proly not the onsequene of pigment degrdtion, ut due to hindered hloroplst development t high Cu 2+ onentrtions (Chpter 3). Enhned Cu onentrtions ffeted the uptke, distriution nd ssimiltion of sulfte in Chinese ge. Both the expression nd tivity of the sulfte trnsporters were enhned t pprox. 2-5 µm Cu 2+. At 1 µm Cu 2+ there ws n inrese in wter-solule nonprotein thiol ontent of the root nd to lesser extent of the shoot of Chinese ge. This inrese ould only e prtilly ttriuted to Cu-indued umultion of the phytoheltins (Chpter 3). Cu 2+ exposure hrdly ffeted the totl sulfur ontent of the root of Chinese ge, wheres tht of the shoot ws inresed t 1 µm, the ltter minly due to n inrese in sulfte ontent (Chpter 3). It remins to e resolved to wht extent hnges in sulfte uptke nd metolism upon Cu 2+ exposure were the onsequene of higher sulfur demnd in order to detoxify the exess Cu, or the result of diret interferene of Cu with the signl trnsdution pthwy involved in sulfur metolism. It hs een predited tht there will e n inrese in UV rdition on the erth surfe due to ozone depletion in the strtosphere nd redution of erosols nd louds (MKenzie et l. 2007). UV-B ( nm) is the most hrmful prt of the UV spetrum for plnts rehing the surfe of the erth. UV-B sorption y the folige my result in the formtion of retive oxygen speies, espeilly in the hloroplsts (Tusz 2001). Consequently, high UV-B levels my lter thylkoid integrity, indue pigment degrdtion nd derese hlorophyll / rtio, Ruiso tivity nd stomtl ondutivity (Jnsen et l. 1998; Lrsson et l. 1998) nd sustntilly ffet plnt performne. The level of pigment degrdtion ws dependent on the length nd intensity of the UV-B rdition (Kkni et l. 2003). Plnt speies differ in their ility to tolerte UV-B rdition nd 54

56 Chpter 4 pigment degrdtion ourred more rpidly in diotyledons thn in monootyledons (Kkni et l. 2003). Cu hs potentil lso to elerte the formtion of retive oxygen speies in plnt tissue (Pinto et l. 2003) nd the high light intensities whih my enhne the toxiity of Cu to hloroplsts funtioning, ws presumly due to n enhned prodution of hydroxyl rdils (Yruel et l. 1996; Yruel 2009). The impt of Cu exposure nd UV rdition on plnts my e synergisti. For instne, oth growth nd hlorophyll ontent were redued to greter extent when the quti plnt Lemn gi ws simultneously exposed to Cu nd UV rdition (Bu et l. 2003). Moreover, Cu toxiity ws inresed upon exposure to high light intensities nd/or UV rdition in hloroplsts (Pätsikkä et l. 1998), green lg (West et l. 2003, Lupi et l. 1998), ynoteri (Ri et l. 1998; Gouvê et l. 2008) nd duk weed (Bu et l. 2003), with symptoms inditive of synergisti oxidtive dmge. The intertive effets of Cu nd UV rdition hve only een studied in ynoteri nd quti plnt speies. There is little known out the omined effets of enhned Cu levels nd light qulity (viz. UV rdition) in plnts. The present study ws undertken to investigte (і) the impt of light qulity (UV rdition) on Cu toxiity, nd (іі) the intertion of Cu toxiity with the regultion of the uptke nd metolism of sulfte in Chinese ge (Brssi pekinensis). Plnt growth onditions 10-dy-old seedlings were trnsferred to 25% Hoglnd nutrient solution, ontining supplementl onentrtions of 0, 2, 5 nd 10 μm CuCl 2 in 13 l stinless steel ontiners (10 sets of plnts per ontiner, 3 plnts per set) in limte-ontrolled room for 7 dys. Dy nd night tempertures were 25 nd 20 C ( 1 C), respetively, nd the reltive humidity ws 60-70%. The photoperiod ws 14 h t photon fluene rte of µmol m 2 s 1 (within the nm rnge) t plnt height. For the different light qulity nd UV-A+B rdition studies, the limte-ontrolled room ws split into two prts y non-trnsprent thermopore sheet. In one prt light ws supplied y Philips HPI-T lmps (400 W) nd in the other prt y 55

57 Philips SON-T lmps (600 W). HPI-T lmps hd 2.2 mw m -2 UV-A+B (within the nm rnge) nd 0.06 mw m -2 UV-B rdition (within the nm rnge), wheres SON-T lmps hd lmost no UV rdition, mesured with Digitl Ultrviolet Rdiometer model 5.0 nd model SM 6.0, respetively (Solrteh In. Fenton, MI 48430, USA). The spetrum of light from oth the lmps is shown in figure 1. In experiments with UV filtered out, light ws provided y HPI-T lmps nd the UV (A+B) rdition ws filtered out with Perspex sheet (ISPA Plstis Groningen, The Netherlnds) UV (A+B) Philips HPI-T UV (A+B) Philips SON-T 800 mw m -2 s nm PAR nm PAR 2500 mw m -2 s nm nm Fig. 1. Spetrum of light t plnt height from Philips HPI-T nd Philips SON-T lmps. UV (A+B), ultrviolet irrdition; PAR, photosynthetilly tive rdition. 56

58 Chpter 4 Results Impt of Cu nd light qulity on growth, dry mtter ontent, pigment ontent nd Cu ontent Irrespetive of whether Chinese ge ws grown with HPI-T nd SON-T lmps s the light soure, plnt iomss prodution, dry mtter ontent, shoot pigment ontent nd the speifi lef re were ll quite similr, however, shoot to root rtio ws slightly lower with HPI-T (Fig. 2 nd 3). However, if plnts were exposed to enhned Cu 2+ onentrtions in the nutrition solution, iomss prodution ws more rpidly ffeted with the HPI-T ompred to SON-T light soure. With HPI-T, Cu 2+ exposure resulted in signifint derese in plnt iomss prodution t 2 µm (up to 70% t 10 µm) ompnied y derese in speifi lef re, wheres the shoot to root rtio ws only slightly inresed (Fig. 2). However, with SON-T, plnt iomss prodution ws only slightly deresed t 5 µm Cu 2+ (up to 34% t 10 µm), wheres shoot to root rtio only signifintly inresed t 10 µm (Fig. 2). At 5 µm Cu 2+ there ws n inrese in dry mtter ontent in oth root nd shoot with HPI-T, wheres it ws hrdly ffeted t ll with SON-T (Fig. 2). The pigment ontent strted to derese with oth HPI-T nd SON- T t 2 µm Cu 2+, ut similrly to the oservtions on iomss prodution, the derese ourred more rpidly with HPI-T ompred to SON-T (Fig. 3). For exmple, t 2 µm Cu 2+, pigment ontent hd deresed to 55 nd 21% with HPI-T nd SON-T, respetively (Fig. 3). If plnts were exposed to 5 µm Cu 2+ nd HPI-T, not only the pigment ontent ws deresed, ut there ws lso hnge in pigment omposition. Here, hlorophyll ontent deresed signifintly fster thn tht of hlorophyll nd rotenoids, resulting in signifintly deresed hlorophyll / nd hlorophyll/rotenoid rtio (Fig. 3). With SON-T, the hlorophyll / nd hlorophyll/rotenoid rtios were only slightly deresed t 5 µm Cu 2+ (Fig. 3). The quntum yield of photosystem ІІ photohemistry (F v /F m ) only deresed in Chinese ge leves upon exposure to 5 µm Cu 2+ with HPI-T wheres, with SON-T it ws not ffeted (Fig. 3). 57

59 Biomss prodution (g FW) Root Light soure HPI-T SON-T e e e d Biomss prodution (g FW) Shoot f f f e d DMC (%) DMC (%) d Shoot/root rtio SLA (m -2 g -1 FW) Cu 2+ onentrtion ( M) Fig. 2. Impt of Cu 2+ nd light qulity on iomss prodution, dry mtter ontent nd speifi lef re of Chinese ge. 10-dy-old seedlings of Chinese ge were grown to 25% Hoglnd solution ontining 0, 2, 5 nd 10 µm CuCl 2 for 7 dys. Plnts were grown with HPI-T (lk rs) nd SON-T lmps s light soure (grey rs). The initil shoot nd root weight ws nd g fresh weight, respetively. The dt on iomss prodution (g FW), dry mtter ontent (DMC; %) nd shoot/root rtio represent the men of 6 experiments with 9 mesurements with 3 plnts in eh ( SD), nd speifi lef re (SLA; m -1 g -1 FW) represents the men of 6 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t- test). 58

60 Chpter 4 Chl + (mg g -1 FW) Light soure HPI-T SON-T d d Chl / d de e Chl/Cr Fv/Fm Cu 2+ onentrtion ( M) Fig. 3. Impt of Cu 2+ nd light qulity on pigment ontent, pigment omposition nd hlorophyll fluoresene. For experimentl detils see legends Fig. 2. Plnts were grown with HPI-T (lk rs) nd SON-T lmps s light soure (grey rs). The pigment ontent (mg g -1 FW) represents the men of 2 experiments with 3 mesurements in eh ( SD) nd hlorophyll fluoresene (F v /F m rtio) the men of 9 mesurements ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Chinese ge grown with HPI-T hd 40% higher Cu ontent in oth root nd shoot ompred to plnts grown with SON-T (Fig. 4). With oth HPI-T nd SON-T, the Cu ontent of oth root nd shoot inresed with the Cu 2+ onentrtion in the nutrient solution, however, plnts grown with HPI-T umulted more Cu thn with SON-T upon exposure to Cu 2+ (Fig. 4) For exmple, t 2 µm Cu 2+, the Cu ontent in root of HPI-T nd SON-T ws 4.18 nd 1.54 μmol g -1 DW, respetively. The umultion of Cu upon exposure of plnts to high Cu onentrtions in the nutrient solution differed strongly etween root nd shoot, sine the exessive Cu tken up y the root ppered to e rther immoile. For instne, t 10 µm Cu 2+ the Cu ontent of root inresed 11-fold fter 7 dys of exposure, wheres in 59

61 the shoot it ws only inresed up to 1.3-fold with HPI-T. With SON-T the ontent of Cu inresed in root up to 14-fold nd in the shoot, up to 2.8-fold with 10 µm Cu 2+ (Fig. 4). In order to determine the signifine of UV rdition on the impt of enhned Cu onentrtion on Chinese ge, plnts were grown under onditions where the UV-A+B rdition of the HPI- T lmps ws filtered out y UV-soring Perspex sheet (HPI-T*). Biomss prodution nd pigment ontent of Chinese ge grown with HPI-T nd HPI-T* were quite similr (Fig. 5). However, the oserved derese in plnt iomss prodution, pigment ontent nd hlorophyll fluoresene t 2 µm Cu 2+ with HPI-T ws lrgely diminished with HPI-T*, demonstrting tht UV-A+B rdition strongly enhned the phytotoxiity of enhned Cu 2+ onentrtions (Fig. 5). Cu ( mol g _1 DW) Root 20 Light soure g 15 HPI-T SON-T f 10 e 5 d Cu ( mol g _1 DW) Cu 2+ onentrtion ( M) Shoot d d Fig. 4. Impt of Cu 2+ nd light qulity on Cu ontent. For experimentl detils see legends Fig. 2. Plnts were grown with HPI-T (lk rs) nd SON-T lmps s light soure (grey rs). The Cu ontent (µmol g -1 DW) represents the men of 3 mesurements with 9 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Impt of Cu nd light qulity on nitrogen nd sulfur metolite ontent The nitrte, mino id nd sulfur metolite ontents of Chinese ge were quite similr for oth HPI-T nd SON-T grown plnts. Upon Cu 2+ exposure, the nitrte ontent ws slightly deresed in the 60

62 Chpter 4 root t 2 µm nd t 10 µm Cu 2+ in the shoot with oth HPI-T nd SON-T (Fig. 6 nd 7). There ws strong inrese in mino id ontent of the shoot upon Cu 2+ exposure t 2 µm nd to lesser extent in the root with HPI-T, wheres it ws hrdly ffeted with SON-T (Fig. 6). At 2 µm Cu 2+ there ws sustntil inrese in totl sulfur ontent of the shoot nd t 5 µm in the root, with HPI-T. However, with SON-T, Cu 2+ onentrtions of 5 µm hd sustntil effet on totl sulfur ontent of the shoot wheres the ontent of the root ws only inresed t 10 µm Cu 2+. The inrese in totl sulfur ontent upon Cu 2+ exposure ould for the greter prt e ttriuted to enhne sulfte ontent (Fig. 7). Moreover, upon Cu 2+ exposure t 2 µm, there ws strong inrese in wter-solule non-protein thiol ontent in the root nd to lesser extent in the shoot (t 5 µm Cu 2+ ) for oth HPI-T nd SON-T (Fig. 7). Impt of Cu nd light qulity on sulfte uptke pity nd expression of sulfte trnsporters nd APS redutse Both the expression of the high ffinity sulfte trnsporter Sultr1;2 nd the sulfte uptke pity of the root of Chinese ge were slightly higher with SON-T ompred to HPI-T illumintion (Fig. 8 nd 9). The expression of the Group 2 sulfte trnsporter Sultr2;2, whih is involved in vsulr trnsport, ws lso slightly more expressed with SON-T ompred to HPI-T grown plnts, in root (Fig. 9). The expression of the Group 4 sulfte trnsporter Sultr4;1, whih is involved in vuolr efflux of sulfte, ws quite similr for oth HPI-T nd SON-T illumintion, ut in the shoot expression ws 40% lower with SON-T (Fig. 9). Upon Cu 2+ exposure of HPI-T grown plnts, there ws strong inrese in sulfte uptke pity t 2 µm (out 2-fold), wheres it ws slightly deresed t 10 µm Cu 2+ (Fig. 8). With SON-T illumintion, sulfte uptke pity ws inresed t 5 µm Cu 2+ (Fig. 8). Both for HPI-T nd SON-T illumintion, the inrese in sulfte uptke pity upon Cu 2+ exposure ws ompnied with n enhned expression of Sultr1;2 in the root, wheres Sultr1;1 ws hrdly expressed (Fig. 8 nd 9). With HPI-T illumintion, the expression of Sultr1;2 in roots ws inresed y 159 nd 77% t 2 nd 5 µm Cu 2+, respetively, wheres with SON-T illumintion, expression ws inresed y 40% t 5 µm Cu 2+ (Fig. 9). Upon Cu 2+ 61

63 exposure with HPI-T illumintion, the expression of Sultr2;2 ws slightly upregulted in root t 2 µm Cu 2+, however with SON-T there ws no respone (Fig. 9). Upon Cu 2+ exposure, the expression of Sultr4;1 ws hrdly ffeted in the root under oth HPI-T nd SON-T illumintion. However, for HPI-T illumintion, the expression of Sultr4;1 ws inresed y 53 nd 166% t 5 nd 10 µm Cu 2+ respetively, nd for SON-T illumintion, its expression ws inresed y 50% t 10 µm Cu 2+ in shoot (Fig. 9). Sultr4;2 ws slightly expressed in root with SON-T illumintion, ut upon Cu 2+ exposure expression ws strongly deresed. However, in shoot, the expression of Sultr4;2 ws inresed 1.4-fold nd 3.6-fold t 5 nd 10 µm Cu 2+, respetively, under HPI-T illumintion, ut ws hrdly expressed under SON-T illumintion (Fig. 9). The expression of APS redutse in oth root nd shoot ws quite similr for oth HPI-T nd SON-T grown plnts, nd expression ws slightly enhned in root upon Cu 2+ exposure. However, in shoot the expression of APS redutse ws slightly deresed t 2 µm Cu 2+ under HPI-T, nd t 10 µm Cu 2+ under SON-T illumintion (Fig. 9). Disussion A simultneous exposure of plnts to enhned Cu levels nd UV rdition my result in synergisti negtive effets on plnt growth nd funtioning (Lupi et l. 1998; Bu et l. 2003). From the urrent study it ws evident tht growth nd metolite ontent of Chinese ge ws hrdly ffeted upon illumintion with HPI-T nd SON-T lmps s light soures, despite the lrge differenes in light spetrum (viz. UV level). However, if Chinese ge ws exposed to enhned Cu 2+ onentrtions in the root environment, toxi effets ourred more rpidly with HPI-T ompred to SON-T illumintion. The differenes in the development of toxi effets of Cu 2+ t different light onditions, ould e solely sried to differenes in the level of UV rdition upon illumintion with HPI-T (UV-A+B) nd SON-T (no UV-A+B) lmps. The negtive effets of enhned Cu 2+ onentrtions on iomss prodution, pigment ontent nd the F v /F m rtio in Chinese ge ws strongly deresed in sene of UV-A+B (SON- T lmps) nd when the UV-A+B of the HPI-T lmps ws filtered out. Moreover, with HPI-T lmps, enhned Cu 2+ onentrtions lso 62

64 Chpter 4 resulted in hnge in lef morphology of Chinese ge, illustrted y derese in speifi lef re (SLA). Plnt iomss (g) Light soure HPI-T HPI-T* d d d S/R rtio Chl + (mg g -1 FW) Chl / d d Chl/Cr 3 2 Fv/Fm Cu 2+ onentrtion ( M) Fig. 5. Impt of Cu 2+ nd UV rdition on iomss prodution, pigment ontent nd hlorophyll fluoresene. 10-dy-old seedlings of Chinese ge were grown on 25% Hoglnd solution ontining 0, 2, nd 5 µm CuCl 2 for 7 dys. Plnts were grown with HPI-T (lk rs) nd UV rdition filtered out from HPI-T* lmps y Perspex sheet (white rs). The initil shoot nd root weight ws nd g fresh weight, respetively. The dt on iomss prodution (g FW) nd shoot/root rtio represent the men of 9 mesurements with 3 plnts in eh ( SD). The pigment ontent (mg g -1 FW) represents the men of 2 experiments with 3 mesurements in eh ( SD) nd hlorophyll fluoresene (F v /F m rtio) the men of 9 mesurements ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 63

65 Nitrte ( mol g -1 FW) Root 100 Light soure HPI-T 80 SON-T Shoot 0 Amino ids ( mol g -1 FW) d Cu 2+ onentrtion ( M) Fig. 6. Impt of Cu 2+ nd light qulity on the ontent of nitrte nd mino ids. For experimentl detils see legends Fig. 2. Plnts were grown with HPI-T (lk rs) nd SON-T lmps s light soure (grey rs). The nitrte nd mino id ontent (µmol g -1 FW) represent the men of 2 experiments with 3 mesurements in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). The ontent of Cu in oth root nd shoot of Chinese ge ws strongly ffeted y the level of UV, oth in sene nd presene of enhned Cu 2+ onentrtions in the root environment. In sene of enhned Cu 2+ onentrtions, Chinese ge grown with HPI-T illumintion hd 40% higher Cu ontent in root nd shoot ompred to SON-T grown plnts. Moreover, the umultion of Cu t enhned Cu 2+ onentrtions ws lwys sustntilly higher in oth root nd shoot of Chinese ge grown with HPI-T rther thn SON-T illumintion (in root even up 2.5-fold). The oservtion tht Cu ws more toxi in presene thn in sene of UV is proly lrgely due to the oserved differenes in umultion of Cu in root nd shoot of Chinese ge. Similr to previous oservtions (Chpter 64

66 Chpter 4 3), the exessive Cu tken up y the root ws rther immoile nd on whole plnt sis not more thn 20% ws trnsferred to the shoot. Totl sulfur ( mol g _1 DW) Root Light soure HPI-T SON-T Shoot 0 25 Sulfte ( mol g -1 FW) d d d d Thiols ( mol g -1 FW) d d Cu 2+ onentrtion ( M) Fig. 7. Impt of Cu 2+ nd light qulity on the ontent of totl sulfur, sulfte nd wter-solule non-protein thiols. For experimentl detils see legends Fig. 2. Plnts were grown with HPI-T (lk rs) nd SON-T lmps s light soure (grey rs). The sulfte nd wter-solule nonprotein thiol ontent (µmol g -1 FW) represent the men of 2 experiments with 3 mesurements in eh ( SD). Totl sulfur ontent (µmol g -1 DW) represents the men of 3 mesurements ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 65

67 Enhned Cu levels s well s UV rdition hve the potentil to indue oxidtive stress y the formtion of retive oxygen speies (Lupi et l. 1998; Tusz 2001; Bu et l. 2003), whih might lso hve ontriuted the development of lef nerosis t 5 µm Cu 2+ with HPI-T illumintion. Exposure of Lemn gi to UV rdition nd enhned Cu onentrtions resulted in n enhned tivity of Cu/ZnSOD (Bu et l. 2003). Cu/ZnSOD performs signifint role in the detoxifition of retive oxygen speies nd expression in the ytosol nd hloroplsts ws upregulted t high light nd enhned Cu levels (Tusz 2001; Pilon et l. 2011). This enzyme is the most undnt Cu protein long with plstoynin in green plnts (Yruel 2005, 2009, Cohu nd Pilon 2007). To wht extent the oserved enhnement of Cu ontent in Chinese ge with HPI-T illumintion ws the onsequene of UV rdition-indued enhned Cu uptke for synthesis of Cu/ZnSOD is not known. Similr to previous oservtions (Chpter 3), the derese in pigment ontent t enhned Cu 2+ onentrtions in the root environment with HPI-T (nd UV-filtered out HPI-T) nd SON-T illumintion ws not ompnied with hnge quntum yield of photosystem II photohemistry (F v /F m rtio) inditing tht the remining hloroplsts were funtionl. Only t 5 μm Cu 2+ upon HPI-T illumintion, where lef tissue strted to eome neroti, there ws derese in F v /F m rtio, onfirming higher toxiity of Cu in presene of UV. Exposure of Chinese ge to enhned Cu onentrtions sustntilly ffeted the ontent nd distriution of sulfur ompounds in the root nd shoot of Chinese ge (Chpter 3). There ws strong umultion of wter-solule non-protein thiols in the root nd, to lesser extent in the shoot. However, inrese in thiol ontent in Chinese ge ould only prtilly e sried to Cu-indued synthesis of phytoheltins (Chpter 3). There ws enhned totl sulfur ontent in oth root nd shoot whih ould e ttriuted to sulfte umultion. The enhned sulfur ontent upon Cu 2+ exposure ws most likely the onsequene of upregultion of the expression nd tivity of sulfte trnsporters in the root of Chinese ge. Expression nd tivity of sulfte trnsporters in Chinese ge were slightly ffeted y HPI-T nd SON-T illumintion, however, expression ws strongly inresed t enhned Cu 2+ onentrtions. Expression nd tivity of Sultr1;2 in the root of 66

68 Chpter 4 Chinese ge were slightly enhned with SON-T ompred to HPI-T illumintion, whih ws most likely due to slightly higher shoot iomss prodution. It ws evident tht the rte of sulfte uptke pity y the root ws determined y the sink pity of the shoot (Korlewsk et l. 2009) Light soure HPI-T SON-T e d e Cu 2+ onentrtion ( M) Fig. 8. Impt of Cu 2+ nd light qulity on sulfte uptke pity. For experimentl detils see legends Fig. 2. Plnts were grown with HPI-T (lk rs) nd SON-T lmps s light soure (grey rs). Dt on sulfte uptke pity (µmol g -1 FW root h -1 ) represent the men of 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). The uptke, distriution nd ssimiltion of sulfur t whole plnt level re ontrolled y the plnt sulfur sttus nd the sulfur demnd to mintin the plnt growth (Hwkesford nd De Kok 2006; Rouhed et l. 2009). It hs een proposed tht oth low nd highffinity sulfte trnsporters my ply key roles in sulfte uptke, trnslotion nd distriution in higher plnts (Buhner et l. 2004; Hwkesford nd De Kok 2006). However, the mehnism y whih Cu influenes the sulfur sttus of the plnts nd the ltered gene expression of oth sulfte trnsporters nd APS redutse is not ler. It my e tht Cu itself interferes/rets with the regultory 67

69 signl ompounds involved trnsporters (Chpter 3). in the regultion of the sulfte Fig. 9. Impt of Cu2+ nd light qulity on mrna undne of sulfte trnsporters (Sultr) nd APS redutse (APR; Northern-lot nlysis) in the root nd shoot of Chinese ge. Equl RNA loding ws determined y ethidium romide stining of gels (shown in the ottom pnels). For experimentl detils see legends Fig. 2. A representtive set of dt from two independent experiments is given. The expression of the onstitutively expressed sulfte trnsporter, Sultr4;1, nd the wholly induile sulfte trnsporter, Sultr4;2, ws upregulted in the shoot upon Cu2+ exposure t enhned levels ( 5 μm); generlly these sulfte trnsporters only upregulted upon sulfte defiieny (Buhner et l. 2004; Prmr et l. 2007; Korlewsk et l. 2009,; Stuiver et l. 2009). The expression nd tivity of APS redutse is generlly downregulted t enhned thiol levels (Westermn et l. 2001; Durenkmp et l. 2007; Korlewsk et l. 2008). However, similrly to previous oservtions, the strongly enhned thiol levels in the root nd to lesser extent in the shoot did not result in downregultion of the expression of APS redutse (Chpter 3). Wheres, the expression of APS redutse ws slightly 68

70 Chpter 4 downregulted t 2 nd 10 µm Cu 2+ with HPI-T nd with SON-T illumintion respetively, whih my e due to n enhned sulfte ontent of the shoot. It my e tht the sulfte ontent rther thn the thiol onentrtion ws of greter signifine in the regultion of the expression of APS redutse in the shoot. From the present dt it ws evident tht the upregulted expression nd tivity of sulfte trnsporters upon Cu 2+ exposure t enhned levels ws likely not due to higher sulfur need t higher tissue Cu ontents, sine plnts took up more sulfte thn ws metolized, resulting in n enhned sulfte ontent of the shoot (Chpter 3). Furthermore, the wter-solule non-protein thiols whih re presumed to e involved in inding/helting exessive Cu in plnt tissue, umulted t quite similr levels with HPI-T nd SON-T illumintion irrespetive of the development of Cu toxiity. However, the oserved enhnement in expression nd tivity of sulfte trnsporters, totl sulfur ontent nd sulfte ontent upon Cu 2+ exposure, ourred more rpidly in presene of UV rdition (HPI-T) thn in sene of UV rdition (SON-T). Therefore, inreses in sulfte uptke, thiol ontent nd totl sulfur ontent my not only e due to higher sulfur need, ut my e speifilly response to the ourrene of Cu toxiity, s the inreses pper more rpidly in presene of UV rdition. Conlusions Enhned Cu 2+ onentrtions in the root environment nd UV rdition hd negtive synergisti effets in Chinese ge. The negtive effets of enhned Cu 2+ onentrtions on iomss prodution, pigment ontent nd the F v /F m rtio in Chinese ge were strongly inresed in presene of UV illumintion. Enhned Cu toxiity in presene of UV ws lrgely due to UV-indued enhned umultion of Cu ontent in oth root nd shoot of Chinese ge. Enhned Cu ontent in the root ffeted the regultion of the uptke nd distriution of sulfte, whih ws due not only to higher sulfur need for its detoxifition. It is suggested tht the regultion lso responded to the ourrene of Cu toxiity diretly, sine this ourred more rpidly in presene of UV rdition ompred to the sene of UV rdition. Furthermore, it is suggested tht the 69

71 enhned Cu ontent in the roots interferes/rets with the signl ompounds involved in the regultion of expression nd tivity of sulfte trnsporters. 70

72 Chpter 5. Sulfte deprivtion overrules the toxiity nd impt of opper on sulfur metolism in Chinese ge

73

74 Chpter 5 Astrt Exposure of Chinese ge (Brssi pekinensis) to n enhned Cu 2+ level (4 µm) resulted in redued plnt iomss prodution nd n inresed shoot to root rtio t oth sulfte-suffiient nd sulftedeprived onditions. However, sulfte deprivtion hd more rpid negtive effet on plnt iomss prodution thn enhned Cu 2+ level. The expression nd tivity of the sulfte trnsporters nd the expression of APS redutse in Chinese ge were rpidly upregulted (lredy fter 1 or 2 dys) upon exposure to 4 μm Cu 2+, sulfte deprivtion nd their omintion. Though, the impt of sulfte deprivtion on the expression nd tivity of the sulfte trnsporters ws hrdly further ffeted y Cu

75 Introdution The uptke, distriution nd ssimiltion of sulfur re modulted y the plnt sulfur sttus nd the sulfur demnd for growth (Hwkesford nd De Kok 2006). Sulfte deprivtion of Chinese ge resulted in rpidly indued expression of Sultr1;1 nd n enhned expression of the onstitutively expressed Sultr1;2 in the root, ompnied with n inresed sulfte uptke pity (Korlewsk et l. 2008; Stuiver et l. 2009). It hs lso een reognized tht enhned Cu levels intert with sulfte metolism. For exmple, one week exposure of Chinese ge to enhned Cu levels ( 5 μm), resulted in deresed plnt iomss prodution, enhned expression of the Group 1 high ffinity sulfte trnsporters nd enhned sulfte uptke pity (Chpter 3 nd 4). The upregultion of the sulfte trnsporters in Chinese ge upon Cu 2+ exposure ws likely not only due to higher sulfur demnd neessry for the synthesis of metl-inding ompounds (viz. phytoheltins), ut might lso e the onsequene of diret interferene/retion of Cu with the signl trnsdution pthwy involved in the regultion of the sulfte trnsporters (Chpter 3). In the present hpter, the intertion etween n enhned Cu 2+ level nd sulfur nutrition ws studied. Plnt growth onditions 10-dy-old seedlings were trnsferred to 30 l ontiners nd grown on 25% Hoglnd nutrient solution ontining 0.5 mm sulfte for 7 dys nd susequently trnsferred to fresh nutrient solution ontining 0.5 mm sulfte (+S) or 0 mm sulfte (-S) t 0 nd 4 µm CuCl 2 for 1, 2, 3 nd 4 dys. Dy nd night tempertures were 25 nd 20 C ( 1 C), respetively, nd the reltive humidity ws 60-70%. The photoperiod ws 14 h t photon fluene rte of µmol m 2 s 1 (within the nm rnge) nd 2.2 mw m -2 UV-A+B rdition (within the nm rnge) t plnt height, supplied y Philips HPI-T (400 W) lmps s light soure. UV-A+B rdition ws mesured with Digitl Ultrviolet Rdiometer model 5.0 (Solrteh In. Fenton, MI 48430, USA). 74

76 Chpter 5 Results nd disussion Exposure of Chinese ge to 4 µm Cu 2+ resulted in deresed plnt iomss prodution, n inresed dry mtter ontent nd n inresed shoot to root rtio, s it hs een oserved previously (Fig. 1; Chpter 3 nd 4). Root growth ws more rpidly ffeted thn shoot growth, resulting in n inresed shoot to root rtio lredy fter 2 dys (Fig. 1). Similrly to previous oservtions (Korlewsk et l. 2008; Stuiver et l. 2009), sulfte deprivtion of Chinese ge resulted in deresed plnt iomss prodution nd in hnge in shoot to root iomss prtitioning in fvor of tht of the root, s illustrted y derese in shoot to root rtio (Fig. 1). Plnt iomss (g) S +S,Cu -S -S,Cu Plnt d S/R rtio Shoot/root rtio 1 2 DMC (%) Root DMC (%) Shoot Dys of exposure Fig. 1. Impt of Cu 2+ nd sulfte deprivtion on plnt iomss prodution (g FW), dry mtter ontent (%) nd shoot to root rtio of Chinese ge. Seedlings were grown on 25% Hoglnd nutrient solution ontining 0.5 mm sulfte in limte-ontrolled room for 7 dys nd susequently trnsferred to fresh nutrient solution t 0.5 mm sulfte (+S) or 0 mm sulfte (-S) ontining supplementl 0 or 4 µm CuCl 2 for 1, 2, 3 nd 4 dys. Dt represent the men of 15 mesurements with 3 plnts in eh (± SD). Different letters indite signifint differenes etween tretments within the dy of exposure (p < 0.01, Student s t-test). 75

77 Root Shoot Nitrte ( mol g -1 FW) S +S,Cu -S -S,Cu 0 20 Sulfte ( mol g -1 FW) Thiols ( mol g -1 FW) d d d d Dys of exposure Fig. 2. Impt of Cu 2+ nd sulfte deprivtion on nitrte, sulfte nd thiol ontent (µmol g -1 FW) of Chinese ge. For experimentl detils see legends Fig. 1. Dt represent the men of 3 mesurements with 3 plnts in eh (± SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). A simultneous exposure of plnts to sulfte deprivtion nd 4 μm Cu 2+ resulted in n lmost similr derese of plnt iomss prodution s oserved upon Cu 2+ exposure of sulfte-suffiient plnts. The shoot to root iomss prtitioning, however, ws quite 76

78 Chpter 5 similr to tht oserved for Cu 2+ -exposed sulfte-suffiient plnts (Fig. 1). There ws n inrese in dry mtter ontent of the root of the Cu 2+ -exposed sulfte-deprived plnts fter 3 dys, wheres tht of the shoot remined unffeted (Fig. 1). Upon sulfte deprivtion the iomss prodution t n enhned Cu 2+ level ws only redued fter 4 dys of exposure. At sulfte-suffiient onditions, Cu 2+ exposure resulted in rpid inrese in wter-solule non-protein thiol ontent in oth root nd shoot. However, the thiol umultion ws muh more pronouned in the root thn in the shoot; fter 4 dys its ontent hd inresed 4- fold nd 1.5-fold in root nd shoot, respetively (Fig. 2). It ws evident tht in Chinese ge only smll proportion of the inrese in thiol ontent ould e sried to Cu 2+ -indued synthesis of phytoheltins (Chpter 3). The sulfte ontent of the root ws hrdly ffeted upon Cu 2+ exposure, ut its ontent in the shoot strted to inrese fter 2 dys, up to 1.5-fold fter 4 dys of exposure (Fig. 2). In ontrst, the nitrte ontent deresed in oth root nd shoot upon Cu 2+ exposure (Fig. 2). Upon sulfte deprivtion the thiol nd sulfte ontent were strongly deresed in oth root nd shoot. However, the nitrte ontent of the shoot ws inresed, wheres tht of the root ws not ffeted upon sulfte deprivtion (Fig. 2; Korlewsk et l. 2008; Stuiver et l. 2009). The thiol ontent in the root of plnts simultneously exposed to sulfte deprivtion nd 4 μm Cu 2+ ws inresed fter 1 dy, therefter it remined unltered (Fig. 2). In the shoot, however, the thiol ontent ws inresed fter 1 dy nd remined higher thn tht of the sulfte-deprived plnts up to 4 dys of exposure (Fig. 2). The strong derese in thiol ontent in oth root nd shoot upon sulfte deprivtion ws most likely due to growth dilution nd/or metolism of thiol ompounds to support the synthesis of other essentil orgni sulfur-ontining ompounds (e.g. proteins). The thiol ontent of plnts simultneously exposed to sulfte deprivtion nd 4 μm Cu 2+ remined unltered/higher in the root nd shoot thn in sulftedeprived plnts. This might indite tht prt of the umulted thiols upon Cu 2+ exposure, presumly the phytoheltins frtion ould not e re-metolized. The nitrte ontent of Chinese ge simultneously exposed to sulfte deprivtion nd Cu 2+ ws not sustntilly ffeted in the oth root nd shoot (Fig. 2). 77

79 S +S,Cu -S -S,Cu d d d Dys of exposure Fig. 3. Impt of Cu 2+ nd sulfte deprivtion on sulfte uptke pity (µmol g -1 FW root h -1 ) of Chinese ge. For experimentl detils see legends Fig. 1. Dt represent the men of 3 mesurements with 3 plnts in eh (± SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Exposure of Chinese ge to 4 µm Cu 2+ resulted in n enhned expression nd tivity of the sulfte trnsporters in the root (Fig. 3 nd 4). The expression of the onstitutively undnt sulfte trnsporter Sultr1;2 nd the sulfte uptke pity were lredy upregulted fter 1 dy of exposure (Fig. 3 nd 4). In oth root nd shoot, lso the Group 4 sulfte trnsporter Sultr4;1 ws lredy upregulted upon 1 dy of Cu 2+ exposure, wheres in the root lso Sultr4;2 ws slightly upregulted upon 4 dys of exposure (Fig. 4). The expression of APS redutse, the key regulting enzyme in sulfte redution pthwy, ws first upregulted upon 1 dy of Cu 2+ exposure in the root, ut fter 2 dys it ws downregulted gin to level similr to tht of non-exposed ontrol plnts (Fig. 4). The expression of APS redutse in the shoot, however, ws not ffeted upon Cu 2+ exposure (Fig. 4). 78

80 Chpter 5 Fig. 4. Impt of Cu2+ nd sulfte deprivtion on mrna undne of sulfte trnsporters (Sultr) nd APS redutse (APR; Northern-lot nlysis) in the root nd shoot of Chinese ge. Equl RNA loding ws determined y ethidium romide stining of gels (shown in the ottom pnels). For experimentl detils see legends Fig. 1. The trnsient upregultion of APS redutse in the root my illustrte temporry upregultion of the sulfte redution pthwy possily needed for Cu-indued synthesis of thiols (presumly glutthione nd for lesser prt phytoheltins; Chpter 3), in response to exessive Cu tken up y the root (Fig. 3 nd 4). Sulfte deprivtion lso resulted in strongly enhned expression of Sultr1;1 nd Sultr1;2 in the root, n enhned expression of Sultr2;2, Sultr4;1, Sultr4;2 nd APS redutse in oth root nd shoot, nd in 79

81 n inresed sulfte uptke pity of the root (Fig. 3 nd 4; Korlewsk et l. 2008; Stuiver et l. 2009). A simultneous exposure of plnts to sulfte deprivtion nd 4 μm Cu 2+ hrdly ffeted the oserved upregulted expression of the sulfte trnsporters nd APS redutse, ut fter 2 dys it resulted in slightly less upregultion of Sultr1;1 in the root long with the derese in the sulfte deprivtionindued upregultion of the sulfte uptke pity of the root (Fig. 3 nd 4). Conlusions From the urrent results it ws evident tht sulfte deprivtion hd more rpid negtive effet on plnt iomss prodution thn n enhned Cu 2+ level. Cu 2+ exposure distured the sulfte metolism very rpidly nd lredy fter one dy the expression nd the tivity of the sulfte trnsporters were upregulted. It is unlikely tht the upregultion of the sulfte uptke ould solely e ttriuted to higher sulfur need upon Cu 2+ exposure, sine Cu 2+ exposure lso resulted in enhned sulfte levels in the shoot. The onsequenes of sulfte deprivtion on plnt growth nd the expression nd tivity of the sulfte trnsporters, suh s the indution nd upregultion of sulfte trnsporters nd APS redutse were hrdly further ffeted y n enhned Cu 2+ level. This indited tht sulfte deprivtion more or less surpssed the development of Cu toxiity. 80

82 Chpter 6. Atmospheri H 2 S nutrition hrdly ffets the toxiity nd impt of opper on sulfur metolism in Chinese ge

83

84 Chpter 6 Astrt H 2 S exposure did not ffet plnt iomss prodution, dry mtter, pigment nd totl sulfur ontent, if plnts were grown t n mple sulfte supply to the root. There ws diret intertion etween tmospheri H 2 S nd pedospheri sulfte utiliztion nd H 2 S exposure resulted in downregultion of the sulfte uptke pity nd the expression of Sultr1;2 in the root. H 2 S exposure did not ffet the toxiity of Cu 2+, inditing tht higher sulfur sttus of the plnt did not hve ny signifine in Cu toxiity. There ws sustntil inrese in thiol ontent of the shoot (2-fold) nd to lesser extent of tht in the root in H 2 S exposed plnts. If plnts were simultneously exposed to H 2 S nd enhned Cu 2+ onentrtions, the thiol umultion in the root even further inresed. Both in sene nd presene of H 2 S, high Cu 2+ onentrtions (> 10 µm) resulted in n upregultion of the sulfte uptke pity, however, H 2 S-indued prtil downregultion of the sulfte uptke pity lso ourred t ll Cu 2+ onentrtions. The H 2 S-indued downregultion of the expression of Sultr1;2 ws hrdly ffeted t inresing Cu 2+ onentrtions. Sulfte deprivtion of Chinese ge resulted in deresed iomss prodution, deresed shoot to root rtio, n inresed dry mtter ontent (espeilly in the shoot), nd in deresed pigment nd sulfur metolite ontent, nd in strongly inresed sulfte uptke pity of the root. The Group 1, 2 nd 4 sulfte trnsporters were ll upregulted in oth root nd shoot. The impt of sulfte deprivtion on growth nd the expression nd tivity of the sulfte trnsporters were hrdly further ffeted t enhned Cu 2+ levels. If sulfte-deprived plnts were simultneously exposed to H 2 S, iomss prodution ws quite similr to tht of sulfte-suffiient plnts, though the shoot to root rtio remined lower. The upregultion of the expression of the sulfte trnsporters upon sulfte deprivtion nd tht of APS redutse in sene nd presene of enhned Cu 2+ in the shoot ws lrgely or ompletely llevited upon H 2 S exposure. At ll onditions, the regulr signl trnsdution pthwy of sulfte trnsporters nd APS redutse ws overruled t high Cu tissue levels. 83

85 Introdution Enhned Cu 2+ onentrtions ( 5 μm) in the root environment were toxi for Chinese ge, whih ould in prt e sried to Cuindued distured development of the hloroplsts, resulting in hlorosis nd redued iomss prodution (Sheldon nd Menzies 2005; Yruel 2005; Chpter 3 nd 4). Upon UV rdition, enhned Cu 2+ onentrtions hd negtive synergisti effets in Chinese ge, whih ws proly lrgely due to higher umultion of Cu in oth root nd shoot (Chpter 4). Sulfte deprivtion hd more rpid negtive effet on plnt iomss prodution thn n enhned Cu 2+ level in Chinese ge (Chpter 5). There ws strong umultion of wter-solule non-protein thiols in the root nd, to lesser extent in the shoot upon Cu 2+ exposure. The inrese in thiol ontent in Chinese ge ould only prtilly e sried to Cuindued synthesis of phytoheltins (Chpter 3). Phytoheltins nd other thiols my e involved in the inding of exessive free Cu nd its detoxifition, nd the Cu-indued synthesis of these thiols might require n enhned sulfte uptke nd ssimiltion (Inouhe 2005; Lee nd Kng 2005; Sirko nd Gotor 2007). Indeed, enhned Cu ontents in the root ffeted the regultion of the uptke nd distriution of sulfte; there ws n inrese in expression nd tivity of the high ffinity sulfte trnsporter Sultr 1;2. This inrese ws ompnied with n inrese in totl sulfur ontent in the shoot, whih ws minly due to n umultion of sulfte (Chpter 3 nd 4). However, it needs still to e resolved to wht extent the upregultion of the sulfte trnsporters ws the onsequene of higher sulfur demnd t higher Cu onentrtions or the result of n interferene/retion of Cu with the signl ompounds regulting the expression nd tivity of the sulfte trnsporters (Chpter 3). In ddition to sulfte tken up y the root, plnts re le to utilize the folirly-sored sulfurous ir pollutnts (SO 2, H 2 S) s sulfur soure for growth (De Kok et l. 2002, 2007, 2009; Yng et l. 2006; Korlewsk et l. 2008). Atmospheri H 2 S is diretly metolized with high ffinity into ysteine nd susequently into other orgni sulfur ompounds (De Kok et l. 1998, 2002, 2007, 2009). There ws diret intertion etween tmospheri nd pedospheri sulfur utiliztion in Brssi, nd H 2 S exposure resulted in downregultion of sulfte uptke y the root nd its nd ssimiltion in the shoot 84

86 Chpter 6 (Westermn et l. 2000, 2001; Buhner et l. 2004; De Kok et l. 2007; Korlewsk et l. 2008). In sene of sulfte in the root environment, n tmospheri level of 0.2 µl l -1 H 2 S ppered to e suffiient to over the sulfur demnd for growth of Brssi (Korlewsk et l. 2008). In the present hpter, the intertion etween pedospheri sulfte nd tmospheri H 2 S nutrition, nd enhned Cu 2+ levels ws studied, in order to get insight into the signifine of the plnt sulfur sttus in Cu toxiity, nd the interferene of Cu with the signl trnsdution pthwy regulting the expression nd tivity of the sulfte trnsporters (nd APS redutse). Plnt growth onditions 10-dy-old seedlings of Chinese ge were trnsferred to 25% Hoglnd solution t 0.5 mm sulfte (+S, sulfte-suffiient) or 0 mm sulfte (-S, sulfte-deprived), ontining supplementl onentrtions of 0, 5, 10 nd 15 μm CuCl 2 in 13 l ontiners (10 sets of plnts per ontiner, 3 plnts per set) in fumigtion inets nd fumigted with 0 or 0.2 µl l -1 H 2 S for 11 dys. Results Impt of Cu, H 2 S nd sulfte deprivtion on growth, pigment ontent nd Cu ontent Exposure of Chinese ge to 0.2 µl l -1 H 2 S for 11 dys did not ffet plnt iomss prodution, shoot to root rtio, dry mtter ontent nd pigment ontent t sulfte-suffiient ondition (Fig. 1 nd 2). However, the Cu ontent ws slightly higher in root nd slightly lower in shoot t sulfte-suffiient onditions upon H 2 S exposure (Fig. 4). If plnts were exposed to enhned Cu 2+ onentrtions ( 5 µm) t sulfte-suffiient onditions, the iomss prodution of oth root nd shoot ws deresed in oth sene (+S) nd presene of H 2 S (+S, H 2 S), wheres the shoot to root rtio ws hrdly ffeted (Fig. 1). At 10 µm Cu 2+ there ws n inrese in dry mtter ontent in oth root nd shoot (Fig. 2). 85

87 5 4 +S +S,H 2 S -S -S,H 2 S Shoot iomss (g) d d e d d Root iomss (g) e e d d f e d Shoot/root rtio d Cu 2+ onentrtion ( M) Fig. 1. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on iomss prodution of Chinese ge. 10-dy-old seedlings of Chinese ge were grown on 25% Hoglnd solution ontining 500 µm sulfte (+S, light grey rs), fumigted with 0.2 µl l -1 H 2 S (+S,H 2 S, lk rs) or 0 µm sulfte (-S, white rs), fumigted with 0.2 µl l -1 H 2 S (-S,H 2 S, drk grey rs) for 11 dys. Plnts were exposed to 0 to 15 µm Cu 2+ in the root environment. The initil shoot nd root weight ws nd g fresh weight, respetively. Dt on iomss prodution (g FW) nd shoot/root rtio represent the men of 2 experiments with 9 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 86

88 Chpter 6 At sulfte-suffiient onditions pigment ontent ws only deresed t 15 µm Cu 2+, wheres the hlorophyll / nd hlorophyll/rotenoid rtio were hrdly ffeted in oth sene nd presene of H 2 S (Fig. 3). The Cu ontent in oth root nd shoot of Chinese ge inresed with the Cu 2+ onentrtion (Fig. 4). At sulfte-suffiient onditions, the Cu ontent ws inresed up to 40-fold in root nd up to 5-fold in shoot t 15 µm Cu 2+. H 2 S exposure of Cu 2+ -exposed plnts resulted in lower Cu ontent t 15 µm nd in root, nd in shoot t ll Cu 2+ onentrtions (Fig. 4). The plnt iomss prodution ws strongly deresed upon 11- dy sulfte deprivtion, wheres the shoot to root rtio ws deresed nd the dry mtter ontent sustntilly inresed (Fig. 1 nd 2). Exposure of sulfte-deprived plnts to enhned Cu 2+ onentrtions resulted in deresed iomss prodution of oth root nd shoot t 15 µm, wheres the shoot to root rtio ws inresed t 5 µm, ut dry mtter ontent of oth root nd shoot ws hrdly ffeted (Fig. 1 nd 2). Sulfte deprivtion resulted in deresed pigment ontent, nd upon exposure to Cu 2+ the hlorophyll ontent nd the hlorophyll / nd hlorophyll/rotenoid rtio were hrdly further ffeted (Fig. 3). The Cu ontent of sulftedeprived plnts ws rther high in oth root nd shoot; it inresed up to 56-fold in root nd up to 5.5-fold in shoot t 15 µm Cu 2+ (Fig. 4). H 2 S exposure of sulfte-deprived (-S, H 2 S) plnts llevited the development of sulfur defiieny symptoms. The plnt iomss prodution, dry mtter nd pigment ontent were quite similr to tht of sulfte-suffiient (+S) plnts, however, the hlorophyll / ws signifintly higher in the presene of H 2 S (Fig. 1, 2 nd 3). A simultneous exposure of sulfte-deprived plnts to Cu 2+ nd H 2 S resulted in deresed plnt iomss prodution t 5 µm, n inresed shoot to root rtio t 15 µm, nd n inresed dry mtter ontent of root t 10 µm (Fig. 1 nd 2). There ws derese in pigment ontent t 10 µm Cu 2+, however, the hlorophyll / nd hlorophyll/rotenoid rtio were hrdly ffeted (Fig. 3). The Cu ontent of H 2 S-fumigted, sulfte-deprived plnts ws higher in root, nd t enhned Cu 2+ onentrtions, its ontent inresed in oth root nd shoot. However, the Cu ontent of H 2 S-fumigted, sulftedeprived plnts remined signifintly lower thn tht of sulftesuffiient plnts t ll Cu 2+ onentrtions (Fig. 4). The exessive Cu tken up y the root ws reltively immoile nd only minor 87

89 proportion ws trnsferred to the shoot (Fig. 4). The ltter further deresed with inresing Cu 2+ onentrtions in the root environment t oth sulfte-suffiient nd sulfte-deprived onditions (Fig. 4). Shoot S +S,H 2 S -S -S,H 2 S d DMC (%) 10 d d 5 0 Root 12 DMC (%) e d d Cu 2+ onentrtion ( M) Fig. 2. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on dry mtter ontent of Chinese ge. For experimentl detils see legends Fig. 1. Dt on dry mtter ontent (DMC; %) represent the men of 2 experiments with 9 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Impt of Cu, H 2 S nd sulfte deprivtion on nitrogen nd sulfur metolite ontent The ontent of nitrte, mino ids nd totl sulfur ws not ffeted in sulfte-suffiient, H 2 S-exposed plnts (Fig. 5 nd 6). However, H 2 S exposure resulted in slightly lower sulfte ontent of oth root nd shoot, strong inrese in the thiol ontent of the shoot, nd to 88

90 Chpter 6 lesser extent in the root (Fig. 6 nd 7). Exposure to Cu 2+ t enhned onentrtions ( 10 µm) resulted in deresed nitrte ontent of oth root nd shoot in sene (+S) nd presene of H 2 S (+S, H 2 S). The mino id ontent ws hrdly ffeted upon Cu 2+ exposure in oth root nd shoot in sene nd presene of H 2 S (Fig. 5). The totl sulfur ontent of the shoot, oth in sene nd presene of H 2 S, ws sustntilly inresed t 10 µm Cu 2+, wheres tht of the root remined unltered (Fig. 6). The inrese in totl sulfur ontent upon exposure to Cu 2+ ould for the greter prt e ttriuted to n enhne sulfte ontent (Fig. 6). There ws n inrese in wtersolule non-protein thiol ontent in the root nd to lesser extent in the shoot t 5 µm Cu 2+ t sulfte-suffiient onditions. The inrese in thiol ontent of the root ws further inresed upon simultneous exposure to Cu 2+ nd H 2 S, the thiol ontent of shoot ws hrdly further ffeted (Fig. 7). Sulfte-deprived plnts (-S) ontined very low levels of totl sulfur, sulfte nd thiols in oth root nd shoot. Moreover, there ws strong inrese in mino id ontent, wheres the nitrte ontent ws hrdly ffeted (Fig. 5 nd 6). If sulfte-deprived plnts were exposed to enhned Cu 2+ onentrtions, the totl sulfur nd thiol ontent in shoot nd sulfte ontent in oth root nd shoot were hrdly ffeted (Fig. 5, 6 nd 7). However, the totl sulfur nd thiol ontent in the root ws inresed t 5 µm nd the nitrte ontent deresed t 15 µm, nd the nitrte ontent in the shoot inresed t 5 µm Cu 2+ (Fig. 5, 6 nd 7). H 2 S exposure of sulfte-deprived plnts (-S, H 2 S) resulted in sustntil inrese in totl sulfur nd thiol ontent of oth root nd shoot, wheres the sulfte ontent remined low (Fig. 6). The mino id ontent ws quite similr to tht of sulfte-suffiient plnts (Fig. 5). A simultneous exposure of sulfte-deprived plnts to Cu 2+ nd H 2 S resulted in slight derese in nitrte ontent in oth root nd shoot t 15 µm, wheres the mino id, totl sulfur nd sulfte ontents were hrdly ffeted. Moreover, there ws strong inrese in thiol ontent in the root t 5 µm, nd slight inrese in the shoot t 10 µm upon simultneous Cu 2+ nd H 2 S exposure of sulfte-deprived plnts (Fig. 5, 6 nd 7). 89

91 Chl + (mg g -1 FW) S +S,H 2 S -S -S,H 2 S d d Chl / d d Chl/Cr Cu 2+ onentrtion ( M) Fig. 3. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on pigment ontent of Chinese ge. For experimentl detils see legends Fig. 1. Dt on hlorophyll ontent (hl +; mg g -1 FW), hlorophyll to hlorophyll rtio (Chl /), nd hlorophyll to rotenoid rtio (Chl/Cr) represent the men of 2 experiments with 3 mesurements in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t- test). 90

92 Chpter 6 Cu ( mol g _1 DW) Shoot 2.5 +S +S,H 2 S Root 25 d -S -S,H 2 S d e d f f e Cu ( mol g _1 DW) f e e e d d d Cu 2+ onentrtion ( M) Fig. 4. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on opper ontent of Chinese ge. For experimentl detils see legends Fig. 1. Dt represent the men of 3 mesurements with 9 plnts eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). Impt of Cu, H 2 S nd sulfte deprivtion on sulfte uptke pity nd expression of sulfte trnsporters nd APS redutse Exposure of Chinese ge to H 2 S resulted in downregultion of the sulfte uptke pity t sulfte-suffiient onditions (+S; Fig. 8). Both in sene nd presene of H 2 S, onentrtions > 10 µm Cu 2+ resulted in n upregultion of the sulfte uptke pity, however, the H 2 S-indued prtil downregultion of the sulfte uptke pity lso ourred t ll Cu 2+ onentrtions. There ws n inrese in sulfte uptke pity t enhned Cu 2+ onentrtions in oth sene (+S) nd in presene of H 2 S (+S, H 2 S) t 10 µm 91

93 Cu 2+ (Fig. 8). This inrese in sulfte uptke pity ws ompnied with n enhned expression of Sultr1;2 in root, wheres Sultr1;1 ws hrdly expressed t sulfte-suffiient onditions. The expression of Sultr1;2 ws downregulted y 70% upon H 2 S exposure nd its expression ws inresed y 190% upon exposure to 15 µm Cu 2+ in root (Fig. 9). H 2 S exposure to sulftesuffiient Chinese ge resulted in downregultion of expression of the sulfte trnsporters nd tht of APS redutse in oth root nd shoot (Fig. 9). The expression of Sultr2;2 ws enhned y 120% t 5 µm Cu 2+ in root, wheres Sultr2;2 ws hrdly expressed in the shoot of Chinese ge. The Cu-indued inresed expression of Sultr2;2 ws sent upon H 2 S exposure (Fig. 9). The expression of Sultr4;1 inresed with 50% in the root t 15 µm Cu 2+ in sene nd presene of H 2 S, wheres, its expression in the shoot ws slightly deresed (30%). The expression of Sultr4;2 ws inresed y 70% in root t 15 µm Cu 2+ in sene nd presene of H 2 S, wheres, its expression ws hrdly expressed in the shoot (Fig. 9). The expression of APS redutse ws inresed y 60% in root t 15 µm Cu 2+ in sene nd presene of H 2 S, wheres its expression ws hrdly ffeted y Cu 2+ exposure nd upon H 2 S exposure down regulted y 70% in shoot (Fig. 9). Sulfte deprivtion (-S) resulted in n inresed sulfte uptke pity of the root of Chinese ge, whih ws even higher upon H 2 S exposure (Fig. 8). Exposure of sulfte-deprived plnts to enhned Cu 2+ onentrtions did not ffet the sulfte uptke pity. A simultneous Cu 2+ nd H 2 S exposure of sulfte-deprived plnts resulted in n inresed sulfte uptke pity only t 15 µm (Fig. 8). The expression of the sulfte trnsporters nd APS redutse ws strongly enhned upon sulfte deprivtion in oth root nd shoot (Fig. 9). The expression of Sultr1;1 ws deresed y 80% t 15 µm Cu 2+ in root nd ws hrdly ffeted upon H 2 S exposure t sulfte-deprived onditions. Sultr2;2 ws slightly expressed t sulfte-deprived onditions ut it ws hrdly ffeted y Cu 2+ exposure in oth root nd shoot. Upon H 2 S exposure Sultr1;2 nd Sultr2;2 were hrdly expressed in shoot in oth sene nd presene of enhned Cu 2+ onentrtions in the root environment (Fig. 9). 92

94 Chpter 6 Shoot Nitrte ( mol g -1 FW) S +S,H 2 S -S -S,H 2 S d d e 0 Amino ids ( mol g -1 FW) d Nitrte ( mol g -1 FW) Root de d e e Amino ids ( mol g -1 FW) d Cu 2+ onentrtion ( M) Fig. 5. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on nitrte nd mino ids ontent of Chinese ge. For experimentl detils see legends Fig. 1. Dt on nitrte nd mino ids ontent (µmol g -1 FW) represent the men of 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 93

95 Totl sulfur ( mol g _1 DW) Sulfte ( mol g -1 FW) Shoot Root 600 +S +S,H 2 S d -S -S,H 2 S d d d d Totl sulfur ( mol g _1 DW) d d d d e 0 20 Sulfte ( mol g -1 FW) d Cu 2+ onentrtion ( M) Fig. 6. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on totl sulfur nd sulfte ontent of Chinese ge. For experimentl detils see legends Fig. 1. Dt on totl sulfur (µmol g -1 DW), nd sulfte ontent (µmol g -1 FW) represent the men of 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 94

96 Chpter 6 Upon sulfte deprivtion, the expression of Sultr4;1 nd Sultr4;2 in root ws hrdly ffeted y Cu 2+ nd y simultneous Cu 2+ nd H 2 S exposure. The expression of these trnsporters ws deresed y 80% upon H 2 S exposure in shoot in sene nd presene of enhned Cu 2+ onentrtions. The expression of APS redutse ws hrdly ffeted in root upon Cu 2+ nd upon simultneous Cu 2+ nd H 2 S exposure t sulfte-deprived onditions. However, its expression ws downregulted y 75-90% in shoot upon H 2 S exposure in sene nd presene of enhned Cu 2+ onentrtions in the root environment (Fig. 9). 2.0 Shoot +S +S,H 2 S -S -S,H 2 S Thiols ( mol g -1 FW) d d d d d d 0.0 Root 4 Thiols ( mol g -1 FW) f f e g d d f e d d Cu 2+ onentrtion ( M) Fig. 7. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on thiol ontent of Chinese ge. For experimentl detils see legends Fig. 1. Dt on thiol ontent (µmol g -1 FW) represent the men of 3 mesurements with 3 plnts in eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 95

97 Disussion Similr to previous oservtions, Chinese ge ws le to utilize folirly sored H 2 S nd reple sulfte, tken up y the root, s sulfur soure for growth (Korlewsk et l. 2008). At sulfte-suffiient onditions, H 2 S exposure did not ffet plnt iomss prodution, dry mtter, pigment nd totl sulfur ontent, ut it resulted in sustntil inrese in thiol ontent of the shoot (2-fold) nd to lesser extent of tht in the root. If sulfte-suffiient plnts were simultneously exposed to H 2 S nd enhned Cu 2+ onentrtions in the root environment, the thiol umultion in the root ws further inresed. Exposure of sulfte-suffiient Chinese ge to H 2 S did not ffet the toxiity of Cu 2+ nd the Cu-indued derese in plnt iomss prodution ws quite omprle, even though the Cu ontent of the shoot ws signifintly lower upon H 2 S exposure. This indited tht higher sulfur sttus of the plnt did not hve ny signifine in Cu toxiity. Sulfte uptke pity ( mol g -1 FW root h -1 ) S +S,H 2 S -S -S,H 2 S Cu 2+ onentrtion ( M) Fig. 8. Impt of Cu 2+ nd H 2 S exposure nd sulfte deprivtion on sulfte uptke pity of Chinese ge. For experimentl detils see legends Fig. 1. Dt on sulfte uptke pity (µmol g -1 FW root h -1 ) represent the men of two independent experiments with 3 mesurements with 3 plnts eh ( SD). Different letters indite signifint differenes etween tretments (p < 0.01, Student s t-test). 96

98 Chpter 6 Fig. 9. Impt of Cu2+ nd H2S exposure nd sulfte deprivtion on mrna undne of sulfte trnsporters (Sultr) nd APS redutse (APR) (Northernlot nlysis) in the root nd shoot of Chinese ge. Equl RNA loding ws determined y ethidium romide stining of gels (shown in the ottom pnels). A representtive set of dt from two independent experiments is given. For experimentl detils see legends Fig

99 Similr to previous oservtions, there ws diret intertion etween tmospheri H 2 S nd pedospheri sulfte utiliztion in Chinese ge (Westermn et l. 2000, 2001; Buhner et l. 2004; De Kok et l. 2007; Korlewsk et l. 2008). At sulfte-suffiient onditions, H 2 S exposure resulted in downregultion of the sulfte uptke pity nd the expression of Sultr1;2 in the root. Both in sene nd presene of H 2 S, inresing Cu 2+ onentrtions resulted in n upregultion of the sulfte uptke pity. However, H 2 S- indued prtil downregultion of the sulfte uptke pity ourred t ll Cu 2+ onentrtions. The H 2 S-indued downregultion of the expression of Sultr1;2 ws hrdly ffeted t inresing Cu 2+ onentrtions nd it even depressed the Cu-indued upregultion of this trnsporter. Moreover, the Cu-indued upregultion of Sultr1;1 nd Sultr2;2 were llevited upon H 2 S exposure, the ltter trnsporter is involved in the vsulr trnsport of sulfte (Buhner et l. 2004; Prmr et l. 2007; Korlewsk et l. 2010). The expression of Sultr4;1 nd Sultr4;2 ws upregulted t 15 μm Cu 2+ in root t sulfte-suffiient onditions. Generlly, Group 4 sulfte trnsporters re only upregulted upon sulfur limittion, when plnts remoilize nd/or redistriute ville sulfte, for instne from vuoles (Buhner et l. 2004; Prmr et l. 2007; Korlewsk et l. 2009,, 2010; Stuiver et l. 2009; Chpter 5). Sulfte deprivtion of Chinese ge resulted in deresed iomss prodution, derese in shoot to root rtio, n inresed dry mtter ontent (espeilly in the shoot) nd in derese in pigment nd sulfur metolite ontent. As oserved efore, sulfte deprivtion resulted in strongly inresed sulfte uptke pity of the root, nd the Group 1, 2 nd 4 sulfte trnsporters were ll upregulted in oth root nd shoot (De Kok et l. 1997; Westermn et l. 2000; Buhner et l. 2004; Yng et l. 2006; Korlewsk et l. 2007, 2008; Chpter 5). Similr to previous oservtions (Chpter 5), the onsequenes of sulfte deprivtion on growth nd the expression nd tivity of the sulfte trnsporters were hrdly further ffeted y enhned Cu 2+ levels. If sulfte-deprived plnts were simultneously exposed to H 2 S, plnt iomss prodution ws quite similr to tht of sulfte-suffiient plnts. However, the shoot to root rtio remined lower, demonstrting tht upon H 2 S exposure in the sene of sulfte in the root environment, plnts still invested reltively more iomss in 98

100 Chpter 6 root, even though the sulfur supply ws suffiient to over the sulfur demnd for growth (Buhner et l. 2004; Korlewsk et l. 2007, 2008). If plnts were grown with H 2 S s the sole sulfur soure, the impt of enhned Cu 2+ onentrtions on iomss prodution ws omprle to tht oserved for sulfte-suffiient plnts nd the degree of Cu toxiity ws similr. However, the upregultion of the expression of the sulfte trnsporters upon sulfte-deprivtion nd APS redutse in sene nd presene of enhned Cu 2+ in the shoot ws lrgely or ompletely llevited upon H 2 S exposure. It is generlly presumed tht metoli produts of sulfte ssimiltion, viz. thiols s sulfide, ysteine, nd glutthione t s signls in the regultion of the expression nd tivity of the sulfte trnsporters (Hwkesford nd De Kok 2006). However, t enhned Cu 2+ onentrtions, there ws hrdly ny reltion etween the undnt thiol levels nd the expression nd tivity of the sulfte trnsporters t sulfte-suffiient, sulfte-deprived onditions, oth in presene nd sene of H 2 S. Apprently, the regulr signl trnsdution pthwy of the sulfte trnsporters ws overruled t high Cu tissue levels. The expression nd tivity of APS redutse is generlly downregulted t enhned thiol levels (Westermn et l. 2001; Durenkmp et l. 2007; Korlewsk et l. 2008). However, in sulfte-suffiient plnts its expression ws hrdly ffeted in the root in presene of H 2 S nd enhned Cu 2+ onentrtions, irrespetive of the high thiol levels present under these onditions. Only in the shoot the inrese in thiol ontent upon H 2 S exposure ws ompnied with slight derese in expression of APS redutse. Despite the oservtions tht enhned Cu 2+ onentrtions interfered with the regultion of the uptke nd ssimiltion of sulfur in plnts, there ws no diret reltion etween their sulfur sttus nd Cu toxiity. Evidently, totl sulfur, sulfte nd thiol ontent, nd the level of tivity nd expression of the sulfte trnsporters nd of APS redutse, whih ll strongly differed in presene nd sene of sulfte nd/or H 2 S s sulfur soure for growth, hd hrdly impt on the development of Cu toxiity in Chinese ge. 99

101 Conlusions In Chinese ge there ws diret intertion etween tmospheri H 2 S nd pedospheri sulfte nutrition nd H 2 S exposure resulted in downregultion of sulfte uptke y the root. There ws n upregultion of the sulfte uptke pity t high Cu 2+ onentrtions (> 10 µm) in oth sene nd presene of H 2 S, though the H 2 S-indued prtil downregultion of the sulfte uptke pity ourred t ll Cu 2+ onentrtions. Upon sulfte deprivtion, folirly sored H 2 S ould reple sulfte s sulfur soure for growth, however, the toxiity of Cu 2+ ws similr to tht oserved in sulftesuffiient plnts oth in sene nd presene of H 2 S, inditing tht the sulfur sttus of the plnt did not hve ny signifine in Cu tolerne. Moreover, the presumed signl trnsdution pthwy involved in the regultion of the expression nd tivity of the sulfte trnsporters (APS redutse) ppered to e overruled t high Cu tissue levels. 100

102 Chpter 7. Generl disussion

103

104 Chpter 7 The physiologil sis for the toxiity of Cu in Chinese ge Cu is n essentil nutrient for plnt growth nd development, ut t enhned levels in the root environment it my rpidly eome phytotoxi (Chpter 3, 4, 5 nd 6). The toxiity of Cu depends on the onentrtion of Cu umulted, growth stge of plnts nd the durtion of the exposure (Clemens 2001; Medoz-Cóztl et l. 2005; Chpter 3, 4, 5 nd 6) nd on the light qulity, viz. UV undne (Chpter 4). Generlly, Cu toxiity resulted in n inhiition of root nd shoot iomss prodution, lef hlorosis, loss of photosyntheti tivity (Moquot et l. 1996; Yruel 2005, 2009; Xiong et l. 2006; Hn et l. 2008; Burkhed et l. 2009; Chpter 3, 4, 5 nd 6) nd n ltered root morphology (Pnou-Filotheou nd Boslidis 2004; Sheldon nd Menzies 2005). Root growth of Chinese ge ws slightly more ffeted thn shoot growth t high Cu 2+ onentrtions ( 5 µm; Chpter 3, 4 nd 5). Exposure of Chinese ge to enhned Cu 2+ onentrtions in the root environment resulted in sustntil inrese in the Cu ontent of oth root nd shoot. The exessive Cu umulted in the root ws reltively immoile nd only minor proportion ws trnsferred to the shoot (Chpter 3, 4 nd 6). Cu hs high ffinity for ronyli, roxyli, phenoli nd sulfydryl groups nd therefore it is strongly ound to the ell wll, whih presumly limits oth influx of Cu into the symplst nd its trnslotion to the shoot (Qurti et l. 2003). A 3- to 5-fold inrese in Cu ontent of the shoot of Chinese ge lredy resulted in 50% derese in plnt iomss prodution (Tle 1). Under norml onditions the Cu ontent in plnts rnges from 0.08 to 0.24 µmol g -1 dry weight, wheres in ge Cu lredy eme toxi when the lef tissue level exeeded 0.4 µmol g -1 dry weight (Mniol nd Bekett 1985; Mrshner 1995; Krämer 2010). A simultneous exposure of plnts to enhned Cu levels nd UV rdition my result in synergisti negtive effets on plnt growth nd funtioning (Lupi et l. 1998; Bu et l. 2003). If Chinese ge ws exposed to enhned Cu 2+ onentrtions in the root environment, its toxi effets ourred more rpidly in presene thn in sene of UV rdition (Chpter 4). The ontent of Cu in oth root nd shoot of Chinese ge ws strongly enhned upon UV 103

105 rdition (up 40%). The higher Cu toxiity in presene of UV my lrgely e ttriuted to the oserved higher Cu ontent in root nd shoot (Chpter 4). Tle 1. Cu ontent in plnt tissue t ontrol nd EC 50 (hlf mximl effetive onentrtion t whih 50% plnt iomss prodution ws deresed). Cu ontent (μmol g -1 FW) Control EC 50 Root Shoot Plnt The physiologil sis for the phytotoxiity of Cu is still mivlent. Cu hs the potentil to elerte the formtion of retive oxygen speies in plnt tissue (Pinto et l. 2003). For instne, the toxiity of Cu for hloroplsts funtioning ws enhned t high light intensities, whih ws presumed to e due to n enhned prodution of hydroxyl rdils (Yruel et l. 1996; Yruel 2009). The toxiity of Cu my lso e sried to its possile retion with thiol groups of proteins, therey inhiiting enzyme tivity nd protein funtioning; moreover it my reple other essentil metls in proteins (De Vos et l. 1993; Yruel 2009). Enhned Cu onentrtions my interfere with the iosynthesis of photosynthesis mhinery nd modify the pigments nd proteins of the thylkoid memrne network (Pätsikkä et l. 2002; Yruel 2005, 2009). From the urrent study it eme evident tht hlorosis of the shoot of Chinese ge t high Cu onentrtions ws likely not due to pigment degrdtion, ut the onsequene of hindered hloroplst development (Chpter 3 nd 4). The Cu-indued hlorosis in Chinese ge ws ompnied y derese in photosyntheti tivity, however, oth the rte of photosynthesis, expressed on hlorophyll sis, nd the quntum yield of photosystem II (F v /F m rtio) were hrdly ffeted up to 10 µm Cu 2+. The F v /F m rtio ws only ffeted when leves of Chinese ge hd strted to eome neroti (Chpter 3 nd 4). The impt of enhned Cu levels on hloroplst development nd/or funtioning my e ttriuted to Cu-indued Fe defiieny or y the sustitution of the entrl Mg ion of hlorophyll y Cu (Pätsikkä et l. 2002; Küpper et l. 2003). 104

106 Chpter 7 The overll minerl nutrient omposition of Chinese ge ws ffeted upon Cu 2+ exposure t enhned onentrtions in the root environment. Cu umultion t enhned levels in the roots my result in n unlned minerl nutrient uptke nd distriution in Chinese ge. It is not yet well hrterized to wht extent hnge in minerl omposition is involved in the onset of the phytotoxiity of Cu (Chpter 3). Intertion etween Cu nd sulfur metolism Similr to ll other orgnisms, plnts possess homeostti mehnisms to ontrol the onentrtions of essentil metl ions in different ellulr omprtments, in order to prevent the ourrene of toxi effets of exessive levels of free metl ions in the ytoplsm (Clemens 2001; Plmer nd Guerinot 2009). Cu is strong tivtor of phytoheltins iosynthesis, nd n form omplex with phytoheltins. However, it is still doutful to wht extent phytoheltin-cu omplexes re sequestered in the vuole (Coett nd Goldrough 2002; Yruel 2009). In ddition to the sulfur-rih phytoheltins, glutthione itself (or even free ysteine) nd low moleulr weight ysteine-rih proteins my e involved in Cu inding nd its detoxifition in plnts. This would imply diret interferene of enhned tissue Cu levels with the regultion of sulfte uptke nd its ssimiltion in the plnt (Coett nd Goldsrough 2002; Coett 2003; Verkleij et l. 2003; Lee nd Kng 2005; Clemens 2001; Sirko nd Gotor 2007). Exposure of Chinese ge to enhned Cu 2+ onentrtions in the root environment resulted in n umultion of wter-solule non-protein thiols in the root, nd to lesser extent in the shoot (Chpter 3, 4, 5 nd 6). However, this enhnement ould only prtilly e sried to Cu-indued synthesis of phytoheltins (PC 2 nd PC 3 ; Chpter 3). The enhned thiol levels were most likely due the umultion of redued glutthione nd my e for lesser prt to ysteine (De Kok et l. 2007; Chpter 3). The high ffinity sulfte trnsporters Sultr1;1 nd Sultr1;2 re involved in the primry uptke of sulfte y the root (Hwkesford 2003, 2007; Korlewsk et l. 2008; Tkhshi nd Sito 2008). From the present oservtions it ws evident, tht t mple sulfte 105

107 supply Sultr1;2 ws the prinipl sulfte trnsporter responsile for sulfte uptke y Chinese ge (Chpter 3, 4, 5 nd 6). Exposure of Chinese ge to enhned Cu 2+ onentrtions resulted in upregultion of expression of Sultr1;2, ompnied with n inresed sulfte uptke pity of the root (Fig. 1; Tle 2; Chpter 3, 4, 5 nd 6) Cu ontent in root ( mol g -1 FW) Fig. 1. Reltionship etween Cu ontent in the root tissue nd sulfte uptke pity of Chinese ge (derived from Chpter 3, 4 nd 6). It ws doutful whether the oserved upregultion of the expression nd tivity of sulfte trnsporters in Chinese ge ws solely due to higher sulfur demnd t higher Cu tissue ontents. Sine, it is unlikely tht the oserved enhnement of the thiol ompounds in Chinese ge t high Cu 2+ onentrtions would require sustntil upregultion of the sulfte uptke, sine this sulfur proportion never exeeded more thn 5 % of the totl sulfur ontent (Chpter 3). Moreover, the toxiity of Cu ws hrdly ffeted y the plnt sulfur nutritionl sttus. Biomss prodution of Chinese ge upon sulfte deprivtion to Chinese ge ws severely ffeted nd the simultneous exposure of sulfte-deprived plnts to enhned 106

108 Chpter 7 Cu 2+ onentrtions hd hrdly dditionl effets. It ws ovious, tht the development of sulfur defiieny ws more rigorously nd more or less overruled the ourrene of Cu toxiity (Chpter 5 nd 6). Moreover, if Chinese ge ws grown t sulfte, t H 2 S or t oth simultneously s sulfur soure, the Cu-indued derese in plnt iomss prodution ws quite omprle t ll onditions (Chpter 6). From the reltionship etween Cu ontent of root tissue nd sulfte uptke pity it ws ovious, tht the tivity of the sulfte trnsporter Sultr1;2 inresed with the Cu ontent up to μmol g -1 FW root, t whih visile injury symptoms strted to develop (hlorosis nd nerosis; Fig. 1). At higher onentrtions is deresed gin. Tle 2. Sulfur metolite ontent nd regultion of sulfur metolism s ffeted y enhned Cu 2+ onentrtions in the root environment., upregultion;, downregultion;, not ffeted. Upon Cu 2+ exposure Root Shoot Sulfur metolite ontent Totl sulfur Sulfte Thiols Regultion of sulfur metolism Sulfte uptke Sultr1;1 Sultr1;2 APS redutse It is generlly presumed tht sulfte itself or metoli produts of the sulfte ssimiltion, viz. sulfide, ysteine, glutthione, would t s signls in the regultion of the expression nd tivity of the sulfte trnsporters (Hwkesford nd De Kok 2006). At n mple sulfur supply the levels of these ompounds would t s repressors of the sulfte trnsporters. Likewise, high levels of redued sulfur ompounds (sulfide, ysteine nd glutthione) would downregulte the expression nd tivity of APS redutse (Westermn et l. 2001; Durenkmp et l. 2007; Korlewsk et l. 2008). Wheres, upon sulfte deprivtion, low tissue levels of these sulfur metolites re generlly ompnied y n upregulted (de-repressed) expression nd tivity of the sulfur trnsporters nd APS redutse (Chpter 5 107

109 nd 6; Buhner et l. 2004; Prmr et l. 2007; Korlewsk et l. 2009,; Stuiver et l. 2009). However, if Chinese ge ws exposed to enhned Cu 2+ onentrtions, the expression nd tivity of Sultr1;2 in roots nd the expression of Sultr4;1 nd Sultr4;2, nd of APS redutse in oth root nd shoot were upregulted, even t n enhned sulfte nd thiol ontent in root nd shoot (Chpter 3, 4, 5 nd 6, Tle 2). Fig. 2. The possile signifine of H 2 S in the ross-tlk etween the sulfte redution pthwy in the hloroplsts/plstids nd the trnsription of sulfte trnsporters in the nuleus. The mjority of plnt ells in oth root nd shoot re le to redue nd ssimilte sulfte, enling lol ellulr signling of the uptke, suellulr distriution nd redution of sulfte (Chpter 3). However, key unresolved issue is the signl trnsdution in the ross-tlk etween the sulfte redution pthwy in the hloroplsts/plstids nd the trnsription of sulfte trnsporters/sulfte reduing enzymes in 108

110 Chpter 7 the nuleus (Fig. 2). From humn nd niml physiology it hs eome evident tht in ddition to nitri oxide (NO) nd ron monoxide (CO), lso H 2 S might funtion s n endogenous gseous trnsmitter, where it my trget K ATP hnnel proteins nd the AMPdependent protein kinse pthwy (Wng 2002; Mnrdi et l. 2009). In prokryotes is hs een shown tht sulfide is involved in trnsriptionl regultion of the ys-operon, for genes involved in sulfur uptke nd ssimiltion (Kredih 1993). H 2 S might hve similr funtion in plnts - it is the first produt of the sulfte redution pthwy - s n endogenous gseous trnsmitter in the ross-tlk etween the sulfte redution pthwy in hloroplst/plstid nd the trnsription of sulfte trnsporters/sulfte reduing enzymes in the nuleus (De Kok et l. 2011; Fig. 2). At the ellulr ph, H 2 S is lrgely undissoited nd in this form it my esily pss memrnes (De Kok et l. 2007). Evidently, plnts grown under norml onditions emit minute levels of H 2 S, whih emission hs een presumed to e regultory step in the homeostsis of the sulfur pools in plnts (Rennenerg 1984; Shröder 1993; Bloem et l. 2007). At whole plnt level, is hs een shown tht H 2 S exposure my diminish the tivity of sulfte redution in the shoot y downregultion of the expression nd tivity of APS redutse (Durenkmp et l. 2007; Chpter 6), the key-regulting enzyme in the sulfte redution pthwy, nd it my result in downregultion of the expression of sulfte trnsporters in the shoot (Westermn et l. 2000, 2001; Buhner et l. 2004; Durenkmp et l. 2007; Korlewsk et l. 2008; Chpter 6). From the urrent study it ws evident tht the presumed signl trnsdution pthwy in the regultion of expression nd the tivity of the sulfte trnsporters nd APS redutse (sulfte nd thiols) ws y-pssed or overruled, if Chinese ge ws exposed to enhned Cu 2+ in the root environment. H 2 S is rpidly reting with free Cu 2+ ions yielding in the formtion of CuS s preipitte (Dvidson et l. 2001; Trn et l. 2003; Yşşyerli et l. 2003). If H 2 S would funtion s n endogenous gseous trnsmitter in the ross-tlk etween the sulfte redution pthwy in hloroplst/plstid nd the trnsription of sulfte trnsporters/sulfte reduing enzymes in the nuleus, Cu-indued derese in H 2 S onentrtion might e responsile for the oserved upregultion of the expression nd tivity of the sulfte trnsporters nd the expression of APS redutse. 109

111 110

112 Referenes

113

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125 Russo M., Sgherri C., Izzo R. nd Nvri-Izzo F. (2007) Brssi npus sujet to opper exess: Phospholipses C nd D nd gluththione system in signlling. Environmentl nd Experimentl Botny 62: Sito K. (2004) Sulfur ssimiltory metolism. The long smelling rod. Plnt Physiology 136: Snenon V., Puig S., Mir H., Thiele D.J. nd Penrrui L. (2003) Identifition of opper trnsporter fmily in Aridopsis thlin. Plnt Moleulr Biology 51: Snenon V., Puig S., Mteu-Andres I., Dorey E., Thiele D.J. nd Penrrui L. (2004) The Aridopsis opper trnsporter COPT1 funtions in root elongtion nd pollen development. Journl of Biologil Chemistry 279: Suvé S., MBride M.B., Norvell W.A. nd Hendershot W.H. (1997) Copper soluility nd speition of in situ ontminted soils: Effets of opper level, ph nd orgni mtter. Wter Air nd Soil Pollution 100: Sht H., Lluggny M., Vooijs R., Hrtley-Withker J. nd Bleeker P.M. (2002) The role of phytoheltins in onstitutive nd dptive metl tolernes in hyperumultor nd non-hyperumultor metlophytes. Journl of Experimentl Botny 53: Shivon M., Lihong Z., Adel-Ghny S.E., Pilon M., Mlgoli M., nd Pilon-Smits E.A.H. (2007) Vrition in opper tolerne in Aridopsis thlin essions Columi, Lndserg eret nd Wssilewskij. Physiologi Plntrum 129: Shrnz M.E., Lysk M.A. nd Mithell-Olds T. (2006) The ABC s of omprtive genomis in the Brssiee: uilding loks of ruifer genomes. Trends in Plnt Sienes 11: Shröder P. (1993) Plnts s soure of tmospheri sulfur. In: Sulfur Nutrition nd Assimiltion in Higher Plnts: Regultory, Agriulturl nd Environmentl Aspets (eds. De Kok L.J., Stulen I., Rennenerg H. nd Ruser W.E.). SPS Ademi Pulishing, The Hgue. pp Sheldon A. nd Menzies N.W. (2005) The effet of opper toxiity on growth nd morphology of Rhodes grss (Chloris gyn) in solution ulture. Plnt nd Soil 278: Sirko A. nd Gotor C. (2007) Moleulr links etween metls in the environment nd plnt sulfur metolism. In: Sulfur in Plnts - An 124

126 Referenes Eologil Perspetive (eds. Hwkesford M.J. nd De Kok, L.J.). Springer, pp Stuiver C.E.E., Korlewsk A., Posthumus F.S. nd De Kok L.J. (2009) Impt of sulfur deprivtion on root formtion, nd tivity nd expression of sulfte trnsporters in Chinese ge. In: Sulfur Metolism in Plnts: Regultory Aspets, Signifine of Sulfur in the Food Chin, Agriulture nd the Environment (eds. Sirko A., De Kok L.J., Hneklus S., Hwkesford M.J., Rennenerg H., Sito K., Shnug E. nd Stulen I.). Weikersheim, Mrgrf Pulishers, pp Stulen I. nd De Kok L.J Whole plnt regultion of sulfte uptke nd metolism - theoretil pproh nd omprison with urrent ides on regultion of nitrogen metolism. In: Sulfur Nutrition nd Assimiltion in Higher Plnts: Regultory, Agriulturl nd Environmentl Aspets (eds. De Kok L.J., Stulen I., Rennenerg H. nd Ruser W.E.). SPS Ademi Pulishing, The Hgue. pp Sun X.M., Lu B., Hung S.Q., Meht S.K., Xu L.L. nd Yng Z.M. (2007) Coordinted expression of sulfte trnsporters nd its reltion with sulfur metolites in Brssi npus exposed to dmium. Botnil Studies 48: Tkhshi H. nd Sito K. (2008) Uptke, distriution nd suellulr trnsport of sulfte. In: Sulfur Metolism in Phototrophi Orgnisms (eds. Hell R., Dhl C., Knff D.B. nd Leustek T.). Springer Dordreht, pp Tkhshi H., Kopriv S., Giordno M., Sito K. nd Hell R. (2011) Sulfur ssimiltion in photosyntheti orgnisms: Moleulr funtions nd regultions of trnsporters nd ssimiltory enzymes. Annul Review of Plnt Biology 62: Tusz M. (2001) The role of glutthione in plnt response nd dpttion to nturl stress In: Signifine of Glutthione to Plnt Adpttion to the Environment (eds. Grill D., Tusz M. nd De Kok L.J.). Kluwer Ademi Pulishers, Dordreht, pp Ter Steege M.W., Stulen I., Wiersem P.K., Posthumus F.S. nd Vlurg W. (1999) Effiieny of nitrte uptke in spinh: Impt of externl nitrte onentrtion nd reltive growth rte on nitrte influx nd efflux. Plnt nd Soil 208: Tomtsu H., Tkno J., Tkhshi H., Wtne-Tkhshi A., Shigki N. nd Fujiwr T. (2007) An Aridopsis thlin high- 125

127 ffinity molydte trnsporter required for effiient uptke of molydte from soil. PNAS 104: Trn T.T.M., Fiud C., Sutter E.M.M. nd Villnov A. (2003) The tmospheri orrosion of opper y hydrogen sulphide in underground onditions. Corrossion Siene 45: Verwoerd T.T., Dekker B.M.M. nd Hoekem A. (1989) A smll-sle proedure for the rpid isoltion of plnt RNAs. Nulei Aids Reserh 17: Verkleij J.A.C., Sneller F.E.C. nd Sht H. (2003) Metllothioneins nd phytoheltins: eophysiologil spets. In: Sulphur in Plnts (eds. Arol Y.P. nd Ahmd A.). Kluwer Ademi Pulishers, pp Wlker D.J. nd Bernl M.P. (2004) The effets of opper nd led on growth nd zin umultion of Thlspi erulesens J. nd C. Presl: Implitions for phytoremedition of ontminted soils. Wter, Air nd Soil Pollution 151: Wng R. (2002) Two s ompny, three s rowd: n H 2 S e the third endogenous gseous trnsmitter. FASEB Journl 16: West L.J.A., Li K., Greenerg B.M., Mierle G. nd Smith R.E.H. (2003) Comined effet of opper nd ultrviolet rdition on mirosopi green lg in nturl soft lke wters of vrying dissolved orgni ron ontent. Aquti Toxiology 64: Westermn S., De Kok L.J. nd Stulen I. (2000) Intertion etween metolism of tmospheri H 2 S in the shoot nd sulfte uptke y the roots of urly kle (Brssi olere L.). Physiologi Plntrum 109: Westermn S., Stulen I., Suter M., Brunold C. nd De Kok L.J. (2001) Atmospheri H 2 S s sulfur soure for Brssi olere: onsequenes for the tivity of the enzymes of the ssimiltory sulfte redution pthwy. Plnt Physiology nd Biohemistry 39: Wintz H., Fox T., Wu Y-Y., Feng V., Chen W., Chng H-S., Zhu T. nd Vulpe C. (2003) Expression profiles of Aridopsis thlin in minerl defiienies revel novel trnsporters involved in metl homeostsis. Journl of Biologil Chemistry 278: Wojs S., Clemens S., Hennig J., Sklodowsk A., Koper E., Sht H., Bl W. nd Antosiewiz D.M. (2008) Overexpression of phytoheltin synthse in too: distintive effets of AtPCS1 126

128 Referenes nd CePCS genes on plnt response to dmium. Journl of Experimentl Botny 59: Xiong Z-T., Liu C. nd Geng B. (2006) Phytotoxi effets of opper on nitrogen metolism nd plnt growth in Brssi pekinesis Rupr. Eotoxiology nd Environment Sfety 64: Yng L., Stulen I. nd De Kok L.J. (2006) Impt of sulfte nutrition on utiliztion of tmospheri SO 2 s sulfur soure for Chinese ge. Journl of Plnt Nutrition nd Soil Siene 169: Yşşyerli S., Dogu G., Irfn A.R. nd Dogu T. (2003) Brekthrough nlysis of H 2 S removl on CU-V-MO, CU-V, nd CU-MO mixed oxides. Chemil Engineering Communitions 190: Youns M., Shhzd F., Afzl S., Khn M.I. nd Ali K. (1998) Assessment of Cd, Ni, Cu nd P pollution in Lhore, Pkistn. Environment Interntionl 24: Yruel I. (2005) Copper in plnts. Brzilin Journl of Plnt Physiology 17: Yruel I. (2009) Copper in plnts: quisition, trnsport nd intertions. Funtionl Plnt Biology 36: Yruel I., Pueyo J.J., Alonso P.J. nd Piorel R. (1996) Photoinhiition of photosystem ΙΙ from higher plnts: effet of opper inhiition. Journl Biologil Chemistry 271: Zenk M.H. (1996) Hevy metl detoxifition in higher plnts - review. Gene 179: Zhou D.-M., Ho X-Z., Wng Y-J., Dong Y-H. nd Cng L. (2005) Copper nd Zn uptke y rdish nd pkhoi s ffeted y pplition of livestok nd poultry mnures. Chemosphere 59:

129 128

130 Summry

131

132 Summry Copper is n essentil nutrient for plnts, however, t enhned onentrtions in the root environment it my rpidly eome phytotoxi (Chpter 1). The toxi effets of Cu depend on the growth stge of the plnt, durtion of the exposure nd the level of Cu umulted. The typil Cu ontent in plnts rnges from 0.08 to 0.24 µmol g -1 dry weight, nd Cu toxiity generlly ours when the lef tissue ontent exeeds 0.4 µmol g -1 dry weight. Arle soils my ontin high levels of Cu nd/or other toxi metls s the onsequene of intensive griulturl prtie, the use of niml mnure s orgni fertilizers, nd of Cu-ontining fungiides. Enhned Cu levels in rop plnts might not only negtively ffet plnt growth nd funtioning, ut might lso enter the food hin. Sulfur is mronutrient, essentil for plnt growth nd its dpttion to the environment. Generlly, sulfte tken up y the root is the primry sulfur soure for plnts. Moreover, plnts re lso le to utilize folirly sored sulfurous ir pollutnts (viz. SO 2, H 2 S) s sulfur soure. Distint sulfte trnsporter proteins medite the uptke, trnsport nd su-ellulr distriution of sulfte. Their expression nd tivity re modulted y the sulfur sttus of the plnt. The synthesis of sulfur-rih metolites (viz. PCs, GSH) is generlly elerted upon Cu exposure nd my e involved in Cu inding, implying diret signifine of sulfur metolism in Cu detoxifition. In this thesis the physiologil sis of the phytotoxiity of Cu nd signifine of sulfur metolism in its detoxifition hve een investigted in Chinese ge. Chpter 2 gives n overview of mterils nd methods used in this thesis. In ll experiments Chinese ge (Brssi pekinensis (Lour.) Rupr. v. Ksumi F1 (Nikerson-Zwn, Mde, The Netherlnds) ws used. In ll experiments plnts were ultivted on nutrient solution. In Chpter 3 the toxiity of enhned Cu onentrtions nd its interferene with the regultion of the uptke, distriution nd metolism of sulfte ws studied. Exposure of Chinese ge to enhned Cu 2+ onentrtions resulted in lef hlorosis, loss of photosyntheti pity nd lower iomss prodution t 5 µm. The derese in pigment ontent ws likely not the onsequene of degrdtion, ut due to hindered hloroplst development upon Cu exposure. The Cu ontent of the root inresed with the Cu 2+ onentrtion, though only minor proportion of it ws trnsferred to 131

133 the shoot. Enhned Cu 2+ onentrtions sustntilly ffeted the overll minerl nutrient omposition t 5 µm Cu 2+. A high Cu ontent in the root ffeted the uptke, distriution nd metolism of sulfte in Chinese ge. At 1 µm Cu 2+ there ws strong inrese in wter-solule non-protein thiol ontent in the root nd to lesser extent in the shoot, whih ould only prtilly e sried to Cu-indued enhnement of the phytoheltins ontent. The tivity of the sulfte trnsporters in the root ws slightly enhned t 2 nd 5 µm Cu 2+, ompnied y n enhned expression of the Group 1 high ffinity trnsporter Sultr1;2, nd the Group 4 trnsporters Sultr4;1 nd Sultr4;2. The expression of APS redutse ws hrdly ffeted in root nd shoot t enhned Cu 2+ onentrtions. The upregultion of the sulfte trnsporters ws likely not only due to higher sulfur demnd t higher Cu onentrtions, ut lso the onsequene of interferene of Cu itself with the regultory signl trnsdution pthwy involved in the expression nd tivity of the sulfte trnsporters. In Chpter 4 the impt of UV rdition on Cu toxiity nd its intertion with the regultion of the uptke nd metolism of sulfte ws investigted. The negtive effets of enhned Cu 2+ onentrtions on iomss prodution nd pigment ontent of Chinese ge were strongly inresed in presene of UV. Chlorophyll fluoresene (F v /F m ) ws only deresed when lef tissue strted to eome neroti. The expression nd tivity of the high ffinity sulfte trnsporter Sultr1;2 were enhned t 2 µm in presene of UV, nd t 5 µm Cu 2+ in sene of UV. Furthermore, in the shoot, the expression of Sultr4;1 ws upregulted t 5 µm Cu 2+ in presene nd sene of UV nd the expression of Sultr4;2 ws upregulted t 10 µm Cu 2+ in presene of UV. The enhned Cu toxiity in presene of UV ws lrgely due to UV-indued enhned umultion of Cu in oth root nd shoot. In Chpter 5 the impt of enhned Cu onentrtions nd sulfte deprivtion on the uptke nd metolism of sulfte in Chinese ge ws studied. Exposure of plnts to 4 µm Cu 2+ resulted in redued plnt iomss prodution nd n inresed shoot to root rtio t oth sulfte-suffiient nd sulfte-deprived onditions. However, sulfte deprivtion hd more rpid negtive effet on plnt iomss prodution thn enhned Cu 2+ onentrtions. The expression nd tivity of the sulfte trnsporters nd the expression of APS 132

134 Summry redutse in Chinese ge were rpidly up-regulted (lredy fter 1 or 2 dys) upon exposure to 4 μm Cu 2+, sulfte deprivtion nd their omintion. The impt of sulfte deprivtion on the expression nd tivity of the sulfte trnsporters ws hrdly further ffeted y enhned Cu 2+ onentrtions, whih indited tht ourrene of sulfur defiieny symptoms upon sulfte deprivtion more or less overruled the development of Cu toxiity. In order to get more insight into the signifine of the plnt sulfur sttus in Cu toxiity, the intertion etween pedospheri sulfte nd tmospheri H 2 S nutrition, nd enhned Cu 2+ onentrtions ws studied in Chpter 6. There ws diret intertion etween tmospheri nd pedospheri sulfur nutrition in Chinese ge nd H 2 S exposure resulted in downregultion of sulfte uptke y the root. Exposure to H 2 S resulted in n inresed thiol ontent in the shoot nd to lesser extent in the root. The thiol ontent in the root ws further inresed upon simultneous exposure to H 2 S nd enhned Cu 2+ onentrtions. There ws n upregultion of the sulfte uptke pity t high Cu 2+ onentrtions (> 10 µm) in oth sene nd presene of H 2 S, though the H 2 S-indued prtil downregultion of the sulfte uptke pity ourred t ll Cu 2+ onentrtions. The presumed signl trnsdution pthwy involved in the regultion of the expression nd tivity of the sulfte trnsporters (APS redutse) ppered to e overruled t high Cu tissue levels. Upon sulfte deprivtion, folirly sored H 2 S ould reple sulfte sulfur soure for growth, however, the toxiity of Cu 2+ ws quite similr to tht oserved in sulfte-suffiient plnts, lso in presene of H 2 S. Evidently, the plnt sulfur sttus did not hve ny signifine in Cu tolerne. From the reserh presented in this thesis it n e onluded (Chpter 7) tht enhned Cu 2+ onentrtions in the root environment re toxi for Chinese ge. The presene of UV strongly enhned Cu toxiity. Enhned Cu 2+ onentrtions my interfere with the regultion of the uptke nd ssimiltion of sulfur in Chinese ge, ut there ws no diret reltion etween the plnt sulfur sttus nd Cu toxiity. It is generlly presumed tht sulfte itself or metoli produts of the sulfte ssimiltion, viz. thiols (glutthione), would t s signls in the regultion of the expression nd tivity of the sulfte trnsporters. From the present study it ws evident tht the presumed signl trnsdution pthwy in regultion 133

135 of expression nd tivity of the sulfte trnsporters nd APS redutse (sulfte nd thiols) were y-pssed or overruled, if Chinese ge ws exposed to enhned Cu 2+ in the root environment. It is suggested tht undissoited H 2 S my funtion s n endogenous gseous trnsmitter in the ross-tlk etween the sulfte redution pthwy in hloroplsts/plstids nd the trnsription of sulfte trnsporters/sulfte reduing enzymes in the nuleus. A Cu-indued derese in suellulr H 2 S onentrtion might e responsile for the oserved upregultion of the expression nd tivity of the sulfte trnsporters nd the expression of APS redutse. 134

136 Smenvtting (Summry in Duth)

137

138 Smenvtting (Summry in Duth) Koper (Cu) is een essentiële voedingsstof voor plnten, mr ij hoge onentrties in het wortelmilieu kn het ook toxish worden (Hoofdstuk 1). De toxishe effeten vn Cu zijn fhnkelijk vn het groeistdium vn de plnt, de duur vn de lootstelling en de onentrtie vn Cu in de plnt. Het Cu gehlte in plnten vrieert vn 0,08 tot 0,24 µmol g -1 drooggewiht, en Cu toxiiteit treedt in het lgemeen op wnneer het ldweefsel meer dn 0,4 µmol g -1 drooggewiht evt. Lndouwgronden kunnen verontreinigd zijn met Cu en ndere giftige metlen, ls gevolg vn intensieve lndouw, het geruik vn dierlijke meststoffen en vn Cu-houdende fungiiden. Te hoge Cu onentrties heen niet lleen een negtieve invloed op de groei en fysiologie vn plnten, mr vormen ook een risio voor de voedselketen. Zwvel is een mronutriënt die essentieel is voor de groei vn plnten en hun npssing n het milieu. Onder normle omstndigheden wordt sulft, dt wordt opgenomen door de wortels, geruikt ls zwvelron voor de plnt. Plnten kunnen ook zwvelgssen (m.n. SO 2, H 2 S), welke door het ld (vi de huidmondjes) worden opgenomen, geruiken ls zwvelron voor de groei. Vershillende sulfttrnsporter eiwitten zijn etrokken ij de opnme, het trnsport en de su-ellulire verdeling vn sulft in de plnt. Hun expressie en tiviteit zijn fgestemd op de zwvelehoefte vn de plnt. Indien plnten worden lootgesteld n hoge Cu onentrties in het wortelmilieu dt leidt dit in het lgemeen tot een verhoogde synthese vn zwvelrijke verindingen (m.n. fytoheltinen en glutthion), die toxishe metlen zols Cu kunnen inden, en zodnig mogelijk een rol spelen in de Cu ontgifting. In dit proefshrift is enerzijds de fysiologishe sis vn de toxiiteit vn Cu en nderzijds het elng vn het zwvelmetolisme hierij onderzoht in Chinese kool. In Hoofdstuk 2 wordt een overziht gegeven vn mteril en methoden die in dit proefshrift zijn geruikt. In lle experimenten werd Chinese kool (Brssi pekinensis (Lour.) Rupr. v. Ksumi F1 (Nikerson-Zwn, Mde, Nederlnd) geruikt ls proefplnt en de proeven werden uitgevoerd met plnten gekweekt op voedingsoplossing. 137

139 In Hoofdstuk 3 werd de toxiiteit vn verhoogde Cu onentrties en de invloed vn dit metl op de regultie vn de opnme, de verdeling en het metolisme vn sulft estudeerd. Het lootstellen vn Chinese kool n verhoogde Cu 2+ onentrties leidde tot ldvergeling (hlorose), een verlies vn fotosynthetishe piteit en een verminderde groei (iomssprodutie) ij onentrties 5 μm. De dling vn het pigmentgehlte ws wrshijnlijk niet het gevolg vn frk mr vn een verstoorde hloroplstontwikkeling onder invloed vn Cu. Het Cu gehlte in de plnt nm toe met de Cu onentrtie in het wortelmilieu. Slehts een klein deel vn het opgenomen Cu door de wortels werd nr de spruit getrnsporteerd. Verhoogde Cu 2+ onentrties ( 5 μm) eïnvloedden sterk het gehlte vn de ndere minerlen in de plnt. Er ws een sterke stijging in het gehlte vn wter-oplosre niet-eiwit geonden thiolverindingen in de wortel, en in mindere mte in de spruit ij 1 μm Cu 2+. Deze stijging kon slehts gedeeltelijk worden toegeshreven n een verhoging vn het fytoheltinen gehlte. De tiviteit vn de sulfttrnsporters in de wortel ws iets verhoogd ij 2 en 5 μm Cu 2+. Er ws tevens een verhoging vn de (gen)expressie vn de Groep 1 hoge ffiniteit sulfttrnsporter Sultr1;2 (verntwoordelijk voor de opnme vn sulft), en de Groep 4 sulft trnsporters Sultr4;1 en Sultr4;2 (spelen een rol in het sulfttrnsport vnuit de vuole). Verhoogde Cu 2+ onentrties hdden nuwelijks invloed op de expressie vn APS redutse (een elngrijk enzym in de reduktie vn sulft), zowel in de wortel ls in de spruit. De verhoogde expressie en tiviteit vn de sulfttrnsporters werden niet lleen veroorzkt door een hogere zwvelehoefte vn de plnt ij verhoogde Cu onentrties, mr ws mogelijk ook het gevolg vn intertie/retie vn Cu met verindingen die een signlfuntie vervullen in de regultie vn de expressie en tiviteit vn sulfttrnsporters. In Hoofdstuk 4 werd de invloed vn UV op de Cu toxiiteit en de regultie vn de opnme, de verdeling en het metolisme vn sulft in Chinese kool estudeerd. De negtieve effeten vn verhoogde Cu 2+ onentrties op de iomssprodutie en het

140 Smenvtting (Summry in Duth) pigmentgehlte werden versterkt in nwezigheid vn UV. De hlorofyl fluoresentie (F v /F m ) werd lleen negtief eïnvloed ndt het ldweefsel zihtr eshdigd (nerotish) ws. De expressie en tiviteit vn sulfttrnsporter Sultr1;2 werden verhoogd ij 2 μm in nwezigheid vn UV, en ij 5 μm Cu 2+ in fwezigheid vn UV. Bovendien werd in de spruit de expressie vn Sultr4;1 sterk verhoogd ij 5 μm Cu 2+ in nwezigheid en fwezigheid vn UV, en de expressie vn Sultr4;2 werd verhoogd ij 10 μm Cu 2+ in nwezigheid vn UV. De verhoogde Cu toxiiteit in nwezigheid vn UV kon grotendeels worden toegeshreven n een door UV geïndueerde verhoging vn de Cu umultie in zowel de wortel ls de spruit. In Hoofdstuk 5 werd de invloed vn de omintie vn verhoogde Cu 2+ onentrties in fwezigheid vn sulft in het wortelmilieu op de opnmepiteit en het metolisme vn sulft in Chinese kool onderzoht. Blootstelling vn plnten n 4 μm Cu 2+ leidde tot een lgere iomssprodutie vn de plnt en een verhoogde spruit/wortel verhouding zowel in n- ls fwezigheid vn sulft. De fwezigheid vn sulft hd sneller een negtief effet op de iomssprodutie vn de plnt dn de verhoogde Cu 2+ onentrties. De expressie en tiviteit vn de sulftrnsporters en de expressie vn APS redutse in de Chinese kool werden l n 1 of 2 dgen verhoogd n lootstelling n 4 μm Cu 2+, ij n- en fwezigheid vn sulft. De invloed vn sulftonttrekking op de expressie en tiviteit vn de sulfttrnsporters werd nuwelijks verder eïnvloed door verhoogde Cu 2+ onentrties. Hieruit leek dt het onstn vn de negtieve effeten vn zwveldefiiëntie sneller verliep dn dt vn Cu toxiiteit. Om meer inziht te krijgen in de rol die de zwvelvoorziening vn de plnt speelt in de ontwikkeling vn Cu toxiiteit, werden de interties tussen sulftvoeding en H 2 S egssing enerzijds, en verhoogde Cu 2+ onentrties nderzijds, estudeerd in Chinese kool (Hoofdstuk 6). Het lootstellen vn plnten n H 2 S leidde tot een verminderde sulftopnme door de wortel. De opnmepiteit vn sulft werd verhoogd door Cu 2+ (>10 μm), zowel in fwezigheid ls nwezigheid vn H 2 S. Ehter, H 2 S egssing leidde ij lle Cu

141 onentrties tot een verminderde opnmepiteit vn sulft. H 2 S egssing resulteerde in een verhoogd gehlte vn wter-oplosre niet-eiwit geonden thiol verindingen, m.n. in de spruit, en in mindere mte in de wortels. Het thiolgehlte in de wortels steeg verder ij een gelijktijdige lootstelling n H 2 S en verhoogde Cu 2+ onentrties. Er estond geen enkel vernd tussen het thiolgehlte en de expressie en tiviteit vn de sulfttrnsporters (en APS redutse). Plnten wren in stt om in fwezigheid vn sulft, H 2 S te geruiken ls zwvelron voor de groei, mr de toxiiteit vn Cu 2+ ws vergelijkr met die in nwezigheid vn sulft (met of zonder H 2 S). Blijkr speelde de zwvelvoorziening geen rol vn etekenis in de Cu tolerntie vn de plnt. Uit het onderzoek gepresenteerd in dit proefshrift kn geonludeerd worden (Hoofdstuk 7), dt verhoogde Cu 2+ onentrties (> 2 μm) in het wortelmilieu toxish zijn voor Chinese kool. De nwezigheid vn UV leidde tot een sterk verhoogde Cu toxiiteit in de plnt. Verhoogde Cu 2+ onentrties verstoorden de regultie vn de opnme en ssimiltie vn zwvel, mr er ws geen direte reltie tussen de zwvelvoorziening vn de plnt en de Cu toxiiteit. Het wordt lgemeen ngenomen dt sulft zelf, of produten vn de zwvelssimiltie, o.. glutthion, fungeren ls signlverindigen in de regultie vn de expressie en tiviteit vn de sulfttrnsporters (en APS redutse). Ehter ij verhoogde Cu onentrties leek er geen enkel vernd te estn tussen de gehltes vn deze verindingen en expressie en tiviteit vn de sulfttrnsporters (en APS redutse). Mogelijk vervult H 2 S ls gs een signlfuntie in de intertie tussen de sulftredutie in de hloroplst/plstide en de trnsriptie vn de sulfttrnsporters in de elkern. Een Cu-geïndueerde fnme vn de suellulire H 2 S onentrtie, ls gevolg vn een direkte retie vn Cu met H 2 S, zou verntwoordelijk kunnen zijn voor de wrgenomen vernderingen in de expressie en tiviteit vn de sulfttrnsporters (en APS redutse) in Chinese kool. 140

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148 Aknowledgements With gret plesure I would like to express my grtitude to ll who hve ontriuted to this thesis. First nd foremost I would like to thnk Allh Almighty for His immense lessings nd help provided to me throughout my life. I would like to thnk the Higher Edution Commission (HEC), Pkistn for providing me finnil support during my whole Ph.D. period, in ollortion with the Netherlnds orgnistion for interntionl ooportion in higher edution (Nuffi). I m hertily thnkful to my supervisor, Luit J. De Kok, whose enourgement, guidne nd support from the eginning to the end, enled me to develop n understnding of the sujet. I would like to thnk him for tehing me sientifi writing nd for the opportunity of prtiiption in interntionl workshops nd symposi. One simply ould not wish etter or friendlier supervisor. Bep nd Freek, thnk you very muh for your exellent tehnil help. You oth ontriuted lot to this thesis, nd without your help it would hve not een possile to do some very ig nd extensive experiments. I would lso like to thnk Bep for the ritil reding nd lnguge orretions of my mnusripts nd thesis, nd for epting to e my prnymph. I would like to thnk Ineke Stulen for her interest in my projet nd her vlule ontriution t our weekly work disussion. I would lso like to thnk Ger for helping me to rrnge nd hnge lmps nd the tops of the fumigtion inets, nd for the finnil mtters. I would like to thnk Prof. Dr. Theo Elzeng, my promotor, for epting me s Ph.D. student. Thnk you for your support nd for the rrngement of pper work. I m very grteful to Prof. Dr. Mlolm Hwkesford for his strong interest in my reserh nd his visits to our l during my Ph.D. period. Thnk you for the vlule disussions nd your enourgement. Peter Buhner, thnk you for the sulfte trnsporter DNA proes nd for your vlule suggestions. I would lso like to thnk Mrs. Sroj Prmr for the minerl nutrient nlysis. I m grteful to Dr. Ather Ndeem, for his fvor nd help to finish my mster in time. Aleksndr, thnk you for tehing me moleulr tehniques nd for the wonderful time we hve hd. I enjoyed ll the friendly onverstion on siene nd soil spets with you. I would like to thnk Mei for eing nie ollegue nd for pulishing together. I would lso like to thnk Jn Henk for his help with the photosynthesis mesurements. Jnnie, thnk you for your help for rrnging ll douments nd sending letters. Dik Visser, I m very grteful for designing my thesis over pge. 147

149 I would like to show my grtitude to ll the other memers of Plnt Physiology for reting nie tmosphere t work nd during offee/te reks. Thnks to Ftm, Wouter, Ik, Cordul, Jqueline, Mrtin Stl, He, Thereh, Sumithr, Jn, Eelo, Sio, Mri, Eri, Leen nd Mrtin Reih for eing suh nie ollegues nd for your help during this period. I would like to thnk Ijz, Kmrn nd Sujeeth for their kind help nd dvie on moleulr tehniques nd for shring lunh. I would lso like to thnk Arjo Bunskoeke (Isotope Lortory) for his kind help nd support during Northern hyridiztion nd sulfte uptke mesurements. I owe my deepest grtitude to Tyy Akhtr for eing suh ring nd wonderful friend sine we met 10 yers go nd for epting to eome one of my prnymphs. I would like to thnk Neem Mushtq for trnslting my thesis summry in Urdu. I would lso like to thnk my housemtes Ehsn nd Rshid for their gret ooking ilities nd for eing so nie. Ehsn, thnk you for your hospitlity during my homeless period here in Groningen. Munir, thnk you for your help in finding house nd for your guidne. I should not forget to knowledge Youns nd Bhhi for their kindness nd ll their ffetion. During the lst 4 yers whenever I met Youns or ontted him, he lwys offered me to hve dinner with him nd most of the time I hd no hoie thn epting it. I would lso like to thnk Zi for his support nd entertinment he provided. It is plesure to thnk my Pkistni ollegues Bill Nizi, Ali, Arsln, Sulmn, Smin nd Imrn Fzl here in Groningen nd Muhmmd Khurshid, Frz, Mzhr, Imrn Gujr, Shkeel, Sjid, Shuj, Junid nd Amir outside of Groningen for ll their support, help nd for the nie time we spent together. Finlly, I wnt to thnk my fmily. Unfortuntely, my prents re not here to shre the Ph.D. elertion with me, ut I m sure tht they keep n eye on me from heven. I m relly grteful to ll my rothers nd sisters for their ontinuous love, support, wishes nd pryers. Adul Ghfoor, thnk you for your support nd guidne in the eginning of my university edution. I would lso like to thnk to my elders nd my friends k home in Pkistn, Smd, Zeeshn, Sfdr, Asif for their ontinuous love, support nd for ll the good wishes. If I did not mention someone s nme here, it does not men tht I do not knowledge your support nd help. Agin, I would like to thnk everyone who supported nd helped me during my Ph.D. study. 148