Comparative transcriptome analysis of cadmium responses in Solanum nigrum and Solanum torvum

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1 Reserch Comprtive trnscriptome nlysis of cdmium responses in Solnum nigrum nd Solnum torvum Jin Xu 1,2, Jinhng Sun 1, Liguo Du 1 nd Xiojing Liu 1,2 1 Key Lortory of Agriculturl Wter Resources, Center for Agriculturl Resources Reserch, Institute of Genetics nd Developmentl Biology, Chinese Acdemy of Sciences, 28 Huizhong RD, Shijizhung 521, Chin; 2 Heei Province Engineering Lortory for Plnt Breeding nd Germplsm Enhncement of Stress-Tolernt Plnts, 28 Huizhong RD, Shijizhung 521, Chin Author for correspondence: Jin Xu Tel: Emil: xujin@sjzim.c.cn Received: 29 Ferury 2 Accepted: 3 June 2 New Phytologist (2) 19: 11 4 doi: /j x Key words: ccumultion, cdmium (Cd), iron (Fe), Solnum nigrum, Solnum torvum, tolernce, trnscriptome nlysis. Summry Solnum nigrum is cdmium (Cd) ccumultor, wheres Solnum torvum is low Cd-ccumulting plnt. The moleculr mechnisms tht re responsile for differentil cdmium (Cd) ccumultion in the two Solnum species re poorly understood. Here, grfting experiments confirmed tht incresed Cd loding into the root xylem ws responsile for the differentil Cd ccumultion in the two Solnum species. An iron (Fe) supply ssy indicted tht low Fe ccumultion in leves is relted to its Cd sensitivity. Trnscriptome nlyses reveled higher expression of the genes tht encode severl metl trnsporters s well s ntioxidnt-relted genes, nd severl orgnic nd mino cid iosynthesis metolism-relted genes in Cd-treted. Our dt lso indicted tht the different responsive mechnisms of the trnsporter genes to Fe deficiency might e responsile for differentil uptke nd redistriution of metls in the two Solnum species These results form sis upon which to further explore the moleculr mechnisms of Cd ccumultion nd tolernce, nd provide n insight into novel strtegies tht cn e used for phytoremedition nd food sfety. Introduction Cdmium (Cd) is one of the most toxic nonessentil elements, nd it hs strong inhiitory effect on plnt growth nd reproduction (Mcek et l., 22). In leves, concentrtions of Cd tht re higher thn 5 1 lg g )1 DW re toxic to most plnts (White & Brown, 21; Lux et l., 211). By contrst, some species cn hyperccumulte Cd to concentrtions in excess of 1 lg g )1 DW in their leves without showing ny negtive symptoms. Another strtegy tht llows plnts to void Cd toxicity hs emerged through nturl evolution; some plnts sequester Cd in their roots nd prevent the trnsloction of Cd into the shoots, or remoilize Cd from shoots to roots y excluding Cd from leves through the phloem (Chen et l., 2; Mendoz-Cóztl et l., 211). These plnts re referred to s low Cd-ccumulting plnts (Ymguchi et l., 21). The typicl chrcteristics of hyperccumultor plnts include: the high efficient uptke of Cd in roots; xylem loding nd trnsport from the roots to the shoots; nd the cpcity to effectively detoxify Cd. Mny studies regrding the moleculr mechnisms of Cd hyperccumultion hve een reported; however, the moleculr mechnisms tht re responsile for low Cd ccumultion in plnts re poorly understood (Ymguchi et l., 21). Cdmium is chemiclly similr to certin metl elements, including iron (Fe), zinc (Zn) nd clcium (C), nd, therefore, 11 New Phytologist (2) 19: 11 4 could displce these elements from metlloproteins (Clemens et l., 1998; Cohen et l., 1998; Verruggen et l., 29). Cd toxicity cn e ttriuted to its competition with essentil metls, especilly Fe, for metl-inding molecules (Schutzenduel & Polle, 22). Cd enters plnt cells through Fe, C nd Zn trnsporters chnnels (Wu et l., 2). Severl studies hve shown tht Cd toxicity led to Fe deficiency in plnts (Lomi et l., 22; Yoshihr et l., 2; Besson-Brd et l., 29). However, the moleculr mechnism of Fe ccumultion on Cd tolernce nd ccumultion is still not fully understood. Solnum nigrum is Cd ccumultor tht is widely grown in Asi, Europe, nd Americ. In pot-culture experiment, ccumulted 5 lg Cdg )1 of lef DW without showing ny phytotoxic symptoms or visile growth reduction (Sun et l., 2). Most Cd hyperccumultors grow slowly nd hve low iomss; however, hs fster growth rte nd higher iomss, nd therefore shows more promise for use in phytoremedition (Wei et l., 24). Solnum torvum cv. Torumuig is low Cd-ccumulting plnt. Aro et l. (28) found tht, lthough the Cd concentrtion in shoots ws lower thn tht found in S. melongen, there ws no difference in the Cd concentrtion tht ws mesured in the roots of nd S. melongen. Further studies indicted tht lthough ll of the root uptke, xylem loding, nd sequestrtion re responsile for the Cd ccumultion in plnts, the low Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust

2 New Phytologist Reserch 111 loding rte of Cd into the xylem sp in the roots is the mjor contriutor for the low Cd trnsloction to the ove-ground prts of (Mori et l., 29). The distinct types of Cd ccumultion in the two Solnum species mke Solnum n idel genus in which to compre the physiologicl nd moleculr mechnisms tht re involved in differentil Cd ccumultion. In this study, we compre Cd tolernce nd ccumultion etween the Cd ccumultor nd the low Cd ccumultor. We investigted the moleculr mechnisms tht re responsile for Cd tolernce nd ccumultion using comintion of physiologicl nd trnscriptome nlyses. These results form sis upon which to further explore the moleculr mechnisms of Cd ccumultion nd tolernce, nd provide n insight into novel strtegies tht cn e used for phytoremedition nd food sfety. Mterils nd Methods Plnt mterils nd growth conditions The seeds of L. nd Sw. were kindly provided y the Germplsm Bnk of Wild Species in Southwest Chin. To otin seedlings, the seeds were sown under sterile conditions in Petri dishes tht contined MS medium (Murshige & Skoog, 192) nd were solidified with.8% (w v) gr (Sigm). Seven-dy-old seedlings were trnsferred into Hoglnd solution (Hoglnd & Arnon, 195) nd were grown in sterilized, pthogen-free glsshouse. The cultures were mintined t C under 1 h photoperiod. The tretment with CdCl 2 nd or 5 lm Fe-EDDHA (ethylendimine-di (o-hydroxyphenylcetic) cid) ws pplied to 4-wk-old seedlings tht were grown in Hoglnd solution. The culture solution ws replced every 3 d. Mesurement of Cd 2+ flux with the scnning ion-selective electrode technique (SIET) The net Cd 2+ flux ws mesured noninvsively using SIET (BIO-1A; Younger USA Sci. & Tech. Corp., Beijing, Chin). SIET is technique tht specificlly detects ion or molecule flow nd velocity. Ion-selective microelectrodes with n externl tip dimeter of c. 3lm were mnufctured nd silnized with triutylchlorosilne, nd the tips were ckfilled with commercilly ville ionselective cocktil (Cd Ionophore I, 299, Fluk, Switzerlnd). Previous study hs proved tht the Cd 2+ electrode ws highly discrimintory ginst other ctions nd demonstrted the utility of n ion-selective Cd 2+ microelectrode s reserch tool to study hevy-metl trnsport in iologicl systems (Piñeros et l., 1998). The microelectrodes were clirted in 5 nd 5 lm Cd 2+ efore the flux mesurement. Only the electrodes with Nernstin slopes > 25 mv per decde were used (M et l., 21). After exposure to 5 lm CdCl 2 for 24 h, the root segments were smpled for the Cd 2+ flux mesurement. The mesuring solution contined 5 lm CdCl 2,1lM KCl, 2 lm CCl 2,2lM MgCl 2, 5 lm NCl, 1 lm N 2 SO 4 nd 3 lm 2(N-morpholino) ethne sulfonic cid (MES), ph 5.7. SIET mesures the concentrtion grdient of Cd 2+ y mens of Cd 2+ -specific microelectrode virted etween two different detection points on the root surfce t distnce of 1.5 lm. Ion flux dt were susequently clculted. The Cd 2+ flux dt were recorded for period of 1 15 min. The flux dt were otined with the ASET softwre, which is prt of the SIET system. The three-dimensionl ionic fluxes were clculted using MgeFlux ( The negtive vlues in the figure represent the ction influx or nion efflux nd vice vers. Phenotypic nlysis Four-week-old plnts tht were grown in Hoglnd solution were treted with CdCl 2 (2, 5, or 1 lm) for 7 d. The reltive root growth ws clculted s the root length grown in the presence of Cd divided y the men root length under control conditions s descried y Freemn et l. (21). Nine replicte plnts were mesured for ech plnt species nd tretment. Inductively coupled plsm mss spectroscopy (ICP-MS) nlysis Four-week-old plnts tht were grown in Hoglnd solution were treted with 5 lm CdCl 2 for h, or 1, 3, or 5 d. The treted roots were immersed in solution tht contined 1 mm EDTA for 2 h nd then thoroughly rinsed with distilled wter. The smples were oven-dried t 75 C for 48 h. The dried plnt tissues were ground nd digested in concentrted nitric cid for 2 3 d t room temperture. The smples were then oiled for 1 2 h until they were completely digested. After dding 4 ml of Millipore-filtered deionized wter nd rief centrifugtion, the contents of Cd, Zn, Fe, Mn nd Cu were determined using ICP-MS. Ech experiment ws repeted t lest five times. Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust Grfting experiment Grfting ws performed in 3-wk-old plnts, nd this included reciprocl nd self-grfting. In this experiment, the plnts were grfted onto (St Sn) or (St St, self-grfting), nd the plnts were grfted onto (Sn St) or (Sn Sn, self-grfting). After llowing the grft to estlish for 2 wk, the plnts were trnsferred to fresh hydroponic medium contining 5 lm CdCl 2 for 24 h, nd the susequent determintion of Cd ccumultion in the scion shoots ws performed s descried erlier. At lest six replicte plnts were mesured for ech plnt species nd tretment. Digitl trnscriptomics Four-week-old nd plnts were treted with 5 lm CdCl 2 for 24 h. The RNA ws extrcted from the roots of the control nd the two treted Solnum species using TRIzol (Gico BRL, Life Technologies, Grnd Islnd, NY, USA). The detiled experimentl procedure nd ioinformtics nlysis for the digitl gene expression (DGE) profiling re descried in the Supporting Informtion (Methods S1 nd Figs S3 S5). New Phytologist (2) 19: 11 4

3 1 Reserch New Phytologist Reverse trnscription polymerse chin rection (RT-PCR) nlysis of gene expression The plnt smple preprtion, RNA extrction nd RNA qulity nd integrity check hve lredy een descried. For the semiquntittive RT-PCR, we performed control rections using the 18S rrna nd UBQ14 primers to ensure tht n equl mount of RNA ws used in ech set of rections. We optimized the cycle numers to ensure tht the mplifiction rection ws performed in the exponentil phse. The trnscriptome results were lso verified using RT-quntittive PCR (RT-qPCR) ccording to the Minimum Informtion for Puliction of Quntittive Rel-Time PCR Experiments (MIQE) guidelines (Bustin et l., 29). The detiled experimentl procedure for the RT-qPCR nlysis is descried in Methods S1. Mesurement of photosynthesis The photosynthetic rte (Pn) ws recorded on fully expnded leves of the second youngest node t, 1, nd 5 d seprtely fter 5 lm CdCl 2 tretment using n intelligent portle photosynthesis system (LCpro+, ADC, UK). These oservtions were recorded on six to eight plnts per tretment. Detection of rective oxygen species (ROS) concentrtions To mesure the O 2 content, the treted plnt mterils (.5 g) were ground in liquid nitrogen. The otined powder ws suspended in 5 mm phosphte-uffered sline (PBS) uffer (ph 7.8). After centrifugtion (15 min, g), the superntnt ws used for O 2 content mesurements s previously descried (Verm & Mishr, 25). For H 2 O 2 content determintion, the tissue powder ws suspended in 1 mm PBS uffer (ph 7.8) tht contined 1% (w v) polyvinylpyrrolidone (PVP). After centrifugtion (2 min, g), the superntnt ws used for H 2 O 2 content mesurements s descried y Verm & Mishr (25). Mesurement of oxidtive dmge The Cd-induced oxidtive dmge (memrne liquid peroxidtion) ws estimted y mesuring the mlondildehyde (MDA) concentrtions. Fresh plnt tissues were homogenized in.1% (w v) trichlorocetic cid (TCA) solution. After centrifugtion (15 min, g), n liquot of the superntnt ws dded to.5% thiorituric cid (TBA) in 2% TCA nd heted t 9 C for 3 min. After cooling on ice, the mixture ws centrifuged t 8 g for 5 min. The sornce ws recorded t 532 nd nm. The MDA concentrtion ws clculted from the difference etween the sornce vlues t 532 nd nm (Ben Amor et l., 25). The degree of memrne integrity ws lso ssessed y the percentge of electrolyte lekge. Leves nd roots were immersed in 1 ml of ddh 2 O (doule-distilled wter) nd incuted t 25 C for 2 h. The suspension medium ws mesured for the initil electricl conductivity (EC1). The smples were then oiled t New Phytologist (2) 19: C for 15 min to relese ll the electrolytes, cooled nd the finl electricl conductivity (EC2) ws mesured. The electrolyte lekge ws clculted using the formul ðec1/ec2þ1% (Wng et l., 28). Sttisticl nlysis For ech tretment, t lest eight plnts were nlyzed; ll the experiments were repeted t lest three times. The results re presented s mens ± SD. For sttisticl nlysis, we used ANOVA nd Tukey s test with the SPSS 1. softwre pckge (SPSS, Chicgo, Illinois, USA). Differences etween the tretments were tested y the lest significnt difference (LSD) test t.5 proility level. All sequence dt for this study were rchived t the Ntionl Center for Biotechnology Informtion s Short Red Archive (SRA) under ccession no SRA5399. Results Cd tolernce ssy To compre the Cd tolernce in nd, 28-d-old plnts were exposed to three Cd concentrtions, 2, 5, nd 1 lm, for 7 d. When the plnts were grown without Cd, the roots of grew 1.14-fold longer thn those of (dt not shown). Therefore, we used reltive root growth to evlute the Cd tolernce of the two species (Fig. 1). At 2 lm Cd, the reltive root growth of the two Solnum species ws nerly identicl. When the plnts were exposed to 5 nd 1 lm Cd, the root growth rte of ws nd 1.27-fold higher, respectively, thn tht of ; these results indicted tht is more tolernt to Cd stress thn. No ovious stress phenotype ws oserved in either of the plnts fter 7 d of 5 lm Cd tretment; however, 1 lm Cd led to visile lef chlorosis nd necrosis (dt not shown). Therefore, we selected Cd concentrtion of 5 lm for use in this study. Cd uptke nd ccumultion We mesured the Cd content in oth species. As shown in Fig. 1(,c), the Cd ccumultion in the plnts incresed with the durtion of the tretment. After 1 d exposure to 5 lm CdCl 2, the Cd in the leves reched concentrtion tht ws c. 5% of the Cd ccumultion level oserved fter 3 5 d. Compred with, the Cd concentrtion tht ws oserved in ws threefold higher in the leves nd 1.2-fold higher in the roots. lso showed higher Cd lef : root rtio thn (Fig. 1d). The low Cd lef : root rtio nd Cd concentrtions in the leves indicted tht hs comprtively low Cd-ccumulting cpcity. To compre the root Cd uptke in the two Solnum species, we used SIET to investigte the Cd 2+ flux in the roots of nd. The CdCl 2 tretment cused stedy net Cd 2+ influx t the region tht ws locted 29 lm from Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust

4 New Phytologist Reserch 113 () Reltive root growth (%) (c) Cd ccumultion (μg g 1 DW) Cd concentrtion (μm) Root h h 1d 3d 5d Cd lef : root rtio Lef h h 1d 3d 5d h 1d 3d 5d Fig. 1 () Reltive root growth of Solnum nigrum nd Solnum torvum plnts tht were exposed to Cd 2+ for 7 d. The Cd content in the 4-wk-old () leves, (c) roots, nd (d) the Cd lef : root rtios of the plnts tht were grown in Hoglnd solution nd treted with 5 lm CdCl 2. The sterisks indicte the vlues tht were significntly different from those of (P <.5). Error rs indicte ± SD. () Cd ccumultion (μg g 1 DW) (d) () Net Cd 2+ flux (nmol cm 2 s 1 ) Distnce from root tip (μm) () Net Cd 2+ flux (nmol cm 2 s 1 ) Time (min) (c) Fig. 2 The net Cd 2+ fluxes in the roots of Solnum nigrum nd Solnum torvum. () Effects of Cd tretment on the net Cd 2+ fluxes in nd roots. () Net Cd 2+ fluxes in nd roots. (c) The men fluxes of Cd 2+ within the mesuring periods. The sterisks indicte the vlues tht were significntly different from those of (P <.5). Ech point represents the men of five to six individul roots, nd the rs represent the stndrd error of the men the pex in oth of the root tips (Fig. 2). We lso mesured the site tht ws locted 5 lm from the root pex, in which vigorous Cd 2+ flux ws often oserved. As shown in Fig. 2(,c), there were mrked differences in the Cd 2+ fluxes etween the two species. The roots exhiited 2.78-fold higher Cd 2+ influx thn tht of, which indicted tht greter Cd uptke cpcity existed in the roots thn in the roots. Effects of Cd on micronutrient ccumultion Cdmium toxicity interferes with the ccumultion of micronutrients in plnts. Becuse of this, the concentrtions of Zn, Fe, nd Cu in nd were compred (Fig. 3). There were no significnt differences in the Cu concentrtions etween the two species with or without Cd tretment. When the plnts were grown without Cd, there were no significnt differences in the Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust New Phytologist (2) 19: 11 4

5 114 Reserch New Phytologist Zn content (μg g 1 DW) R R L L Cu content (mg g 1 DW) R R L L Time fter tretment (d) Time fter tretment (d) Lef Fe content (μg g 1 DW) Time fter tretment (d) 5 L L 4 Root Fe content (μg g 1 DW) R R Time fter tretment (d) Fig. 3 Metl contents in the cdmium (Cd)-treted Solnum nigrum nd Solnum torvum plnts. Symols: purple tringles, roots; white squres, roots; lue dimonds, leves; white circles, leves. The sterisks indicte the vlues tht were significntly different from those of (P <.5). Error rs indicte ± SD. lef Zn concentrtions etween the two species, nd the ccumultion of Zn in the root of ws higher thn in the roots, wheres the ccumultion of Fe ws lower in the roots nd higher in the leves of compred with. Both species showed reduced ccumultions of Zn, Fe, nd Cu under Cd stress. After 5 d exposure to 5 lm CdCl 2, there were no significnt differences in the Zn concentrtions etween the two plnt species. However, the Fe concentrtions in the roots were 1.54-fold lower thn those in the roots, wheres the Fe concentrtions in the leves were 2.92-fold higher thn those in the leves. Grfts confirmed the efficient root-to-shoot Cd trnsport in roots It hs recently een demonstrted tht the low loding rte of Cd into the xylem sp is responsile for the low Cd ccumultion in shoots (Mori et l., 29). We therefore ssumed tht the higher Cd ccumultion tht ws oserved in the leves ws not only the result of the greter Cd uptke cpcity of the roots, ut lso possily the result of more efficient loding cpcity of Cd into the root xylem. Furthermore, we wondered whether Cd loding in the roots ws dependent on the shoot genotype. To test these hypotheses, grfting experiment nd susequently determintion of the scions Cd content were performed in nd to nlyze the longdistnce trnsport of Cd in whole plnts (Fig. 4). After 24 h of tretment, the nongrfted nd self-grfted plnts showed virtully identicl Cd ccumultion. Compred with the self-grfted plnts ( plnts), the scions from the plnts ccumulted 2.89-fold higher New Phytologist (2) 19: 11 4 Cd ccumultion (μg g 1 DW) S.torum/ S.nigrum/ / / Fig. 4 The effect of grfting on the scions cdmium (Cd) ccumultion in Solnum nigrum nd Solnum torvum. The left side of the division sign ( ) represents the scions, nd the right side represents the rootstocks. The columns tht re leled with different letters were significntly different t P <.5. Error rs indicte ± SD. concentrtion of Cd, wheres the scions from the plnts ccumulted only 33.4% of the Cd concentrtion. It ws cler tht the Cd content of the scions lrgely depended on the rootstocks, which suggests tht oth the Cd uptke cpcity in roots nd the differentil Cd loding cpcity into the root xylem re responsile for the differentil Cd ccumultion etween nd. Gene expression nlysis The results in the previous sections indicte tht oth the Cd uptke cpcity nd its loding into the root xylem re primrily responsile for Cd ccumultion. To further elucidte the mechnisms underlying the differentil Cd ccumultion in Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust

6 New Phytologist Reserch 115 nd, we investigted the trnscriptionl regultion profiles in the two Solnum species roots using tg-sed DGE system. DGE nlysis is n extremely sensitive method for the detection of differences in gene expression nd fcilittes the utiliztion of nonmodel species. We used 24 h time course of Cd exposure to evlute the Cd-responsive genes, ecuse this time course ccounted for 5% of the mximum Cd ccumultion in the ove-ground portions of oth species, nd the exponentil increse stge implied n intense metolism modultion in plnts; nd therey induced lrge mount of Cd-responsive gene expression. Twelve DGE lirries were creted from three independent iologicl smples of four tretments nd sequenced using Solex (Illumin) Technology (Sn Diego, Cliforni, USA). After the 3 dptor frgments, few low-qulity sequences nd severl types of impurities were filtered. The sequencing qulity evlution is presented in Fig. S1. The sturtion nlysis showed tht when the sequencing mount reched 2 M or higher, the numer of detected genes lmost cesed to increse (dt not shown). The expressed tgs tht were otined from the control nd Cd-treted nd roots nd tht ligned to the reference genes generted expression dt for 9387, 9211, 1 159, nd 1 84 genes, respectively. The genes in ll four groups of differentil expression re listed in Tle S2. Genes involved in hevy metl trnsport nd detoxifiction The trnscriptionl regultion of metl trnsport nd detoxifiction-relted genes tht might e involved in the Cd uptke, trnsport nd sequestrtion processes during Cd tretment were exmined (Tles 1, 2). Two metl trnsporters, n Mg trnsporter MGT nd n HMA gene, showed constitutively nd inducily higher expression levels in roots. A PDR-type ABC trnsporter PDR2 showed constitutively higher expression levels in roots, wheres, it ws more highly expressed in roots with Cd tretment. Memers of the ZIP fmily re involved in the trnsport of Zn Fe (Koe et l., 24). A ZIP trnsporter, IRT1, ws more highly expressed in Cd-treted roots of ; wheres IRT2 nd Zn trnsporter, ZIP11, were more highly expressed in the roots of. The other differentilly expressed metl ion trnsport gene tht displyed higher expression levels in ws Cu trnsport protein, COPT5. Antioxidnt-relted genes Cdmium stress induced mrked ccumultion of ROS nd ffected the ntioxidnt content in the plnts (Xu et l., 29). Fifteen ntioxidtive stress-relted genes were differentilly regulted etween nd during tretment with Cd. Among these genes, six memers of the peroxidse superfmily were up-regulted in the roots when compred with under either constitutive ()Cd) or induced (+Cd) conditions. By contrst, five peroxidse fmily genes, n scorte peroxidse gene (APX1), n NADPH oxidse gene (gp91-phox), peptidyl-cysteine S-nitrosyltionrelted gene (peroxiredoxin-2e), nd nonsymiotic hemogloin gene (ns-h1) showed higher expression levels in roots thn in. Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust When gene expression levels were compred etween the nd roots, n ntioxidtive gene, ns-h1, ws more up-regulted in thn ll of the other genes tht were tested (17.4-fold higher expression in ). The ns-h1 protein hs superior ffinity for oxygen nd its overexpression in plnts ws shown to increse the ROS scvenging cpcity, therey improving stress tolernce (Borisjuk et l., 27; Thiel et l., 211). Genes involved in metolic processes The expression levels of eight genes tht re involved in mino cid iosynthesis nd metolism were higher in the roots thn in the roots with or without Cd tretment. These genes re involved in the iosynthesis nd metolism of severl mino cids, such s romtic mino cids, sprtte, serine, nd threonine. Three mino cid trnsporters lso showed higher expression levels in roots with or without Cd tretment. Cdmium toxicity mrkedly ffected crohydrte metolism in these plnts. Two citrte synthse CSY nd CLA genes, nd two mlte dehydrogense MDH genes were more highly expressed in the roots thn in the roots. Cd tretment lso mrkedly modulted cell wll metolism processes. Three genes (RGP9, XTH9, ndexpa) tht re involved in cell wll metolism nd iosynthesis were more highly expressed in the roots with or without Cd tretment. Verifiction of the DGE results To verify the gene expression ptterns tht were oserved in the DGE studies y using n independent experimentl pproch, semiquntittive reverse trnscription (RT)-PCR nd RT-qPCR nlyses were performed for selected genes. We selected UBQ14, 18S RNA, ACT, TUBST1, S23, RPL8, nd UBI2 s the cndidte reference genes for RT-qPCR normliztion. The genorm softwre nlysis indictes tht the expression of 18S RNA ws the most stle in the two Solnum species, nd the pirwise vrition V vlue of 18S RNA, ACT, UBQ14 nd UBI2 ws.15 (Fig. S2), which suggested tht these genes were suitle reference genes for RT-qPCR normliztion. The gene expression levels were normlized using the geometric men of the four most stle expressed reference gene quntities, s descried in the genorm mnul ( genorm_mnul.pdf). The results presented in Fig. 5 showed good greement with the DGE dt. The expression levels of MGT, IRT1, PDR2, HMA, ns-h1, MEE, nd CYS genes were higher in, wheres IRT2, ZIP11, nd COPT5 were more highly expressed in under Cd stress conditions. The high confirmtion rte indictes the reliility of our dt. Effect of Fe on lef Cd tolernce Cdmium toxicity strongly impired the seedling growth nd led to Fe deficiency. ICP-MS nlysis indicted tht the ccumultion of Fe in leves is lower thn in. To determine whether the lower Fe ccumultion is relted to the low Cd tolernce in leves, we nlyzed the impct of New Phytologist (2) 19: 11 4

7 11 Reserch New Phytologist Tle 1 Constitutive differences ()Cd; Solnum nigrum Solnum torvum) of 4-wk-old plnts tht were grown in Hoglnd solution for 1 d Unigene TPM-Snck TPM-Stck Stck Snck P-vlue FDR Annottion Most relted Aridopsis gene Metl trnsport nd detoxicity gnl UG Les#S ± ± E Iron-regulted trnsporter 2 (IRT2) IRT2 (AT4G198.1) YFR2I11A 2.9 ± ± E E 8 Zinc trnsporter ZIP11 ZIP11 (AT1G5591.1) MLF3E7A 1. ± ± Copper trnsporter 5 (COPT5) COPT5 (AT5G25.1) TVR5N17C 58.5 ± ± PDR-type ABC trnsporter 2 (PDR2) PDR (AT1G1552.1) gnl UG Les#S ±.52.7 ±.3 ) E 3 1.5E 29 Mgnesium trnsporter (MGT) MGT2 (AT1G11.1) PLA8N18C ± ±.52 ) E E 28 CPx-type hevy metl ATPses (Cu-HMA) HMA mrna (AT5G2458.1) Antioxidnt-relted genes gnl UG Les#S ± ± Pericrp peroxidse 3 PCA (AT3G4911.1) TVL29A5A 5.51 ± ± E E 13 Secretory peroxidse PRXR1 (AT4G219.1) gnl UG Les#S ± ± Cell wll peroxidse POD mrna (AT1G524.1) SmFL2N14A ± ± E E 13 Anionic peroxidse swp7 POD mrna (AT4G3342.1) TVR9D9C 7.7 ± ± E E 13 Peroxidse 1 precursor POD 1 (AT2G1898.1) TVR2O5A ± ± E E 13 Peroxidse POD mrna (AT4G21.1) TVR2B24C.57 ± ± Peroxidse RCI3 (AT1G52.1) OVS1L14C ± ±.3 )1.91.3E 1 2.5E 15 L-scorte peroxidse 1 (APX1) APX3 (AT4G35) gnl UG Les#S ± ± 1.72 ) E 72 7.E 72 gp91-phox RBOH F (AT1G4) gnl UG Les#S ± ±.82 ) E E 5 Peroxiredoxin-2E Peroxiredoxin-2E (AT3G529) gnl UG Les#S ± ± 1.42 ) E E 54 Ctionic peroxidse 1 POD 52 (AT5G534) gnl UG Les#S ± ± 4.42 )4.89 Peroxidse 27 (POD27) POD 27 (AT3G119) gnl UG Les#S ± ±.7 ) E E 2 Peroxidse isoform 1 PRXR1 (AT4G219) gnl UG Les#S ± ± 2.3 ) E E 24 Peroxidse 31-like POD 3 (AT5G415) gnl UG Les#S ± ±.1 )1.98 Nonsymiotic hemogloin clss 1 HB1 (At2g1) Amino cid iosynthesis nd metolism processes gnl UG Les#S ± ± 1.45 ) E E 18 Crmoyl phosphte synthse (CARA) CARA (AT3G2774) gnl UG Les#S ± ±.92 ) E E 51 Dehydroquinte dehydrtse (MEE) MEE32 (AT3G35) gnl UG Les#S ± ±.3 ) E E 11 Cysteine desulfurse NFS1 (AT5G572) SmFL2A1A 7.7 ± ±.19 ) E 1 3.4E 1 Dihydroxy-cid dehydrtse (DHAD) DHAD (AT3G2394) gnl UG Les#S ± ±.41 ) E E 25 Asprtte minotrnsferse-like ASP5 (AT4G3199) gnl UG Les#S ± ±.1 ) E E 22 SAT1 SERAT2;1 (AT1G5592) MLF1C11A 2.14 ± ± 1.51 )4 3.71E E 87 DAHP synthse 1 (DHS1) DHS1 (AT4G3998) gnl UG Les#S ± ± 2.98 ) E.11E Chloroplst threonine deminse 1 (OMR1) OMR1 (AT3G15) SmFL4D23A 15.3 ± ±.52 ) E E 1 Serine rcemse SR (AT4G114) Amino cid trnsporter SmFL25G5A 25.3 ± ±.48 ) E E 29 Amino cid trnsporter CAT (AT5G477) gnl UG Les#S ± ±.32 ) E E 1 Amino cid permese 2 AAP2 (AT5G922) SmFLE21A ± ±.1 ) E 7 4.1E 9 Amino cid trnsporter AAP (AT5G418) Crohydrte nd Cell wll metolism processes PLA7L14C ± ±.24 ) E E 28 ATP-citrte synthse (CSY) CSY4 (AT2G4435.1) gnl UG Les#S ± ± 1.82 ) E E 22 ATP citrte synthse ctivity (CLA) ACLA-3 (AT1G943.1) SmFL11P19A ± ±.4 ) E E 35 Mlte dehydrogense (PMDH) PMDH1 (AT2G2278) SmFL29H15A 51.7 ± ± E E RGP2 RGP2 (AT5G155.1) TVR4LC ± ± XTH9 XTH9 (AT4G321.2) TVR31P1A 27.7 ± ± E E 13 O-methyltrnsferse (OMT) OMT1 (AT5G541) TVR34MC 1.87 ± ± Expnsin (EXPA) EXLB1 (AT4G173) Snck, control for roots; Stck, control for roots; TPM, normlized expression level of genes, men ± SE, n = 3; Stck Snck, log2 (multiples of differentilly expressed); P-vlue, P-vlue from difference test; FDR, flse discovery rte. Ornge, up-regulted genes; green, down-regulted genes. New Phytologist (2) 19: 11 4 Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust

8 New Phytologist Reserch 117 Tle 2 Induced differences (+ Cd; Solnum nigrum ) of 4-wk-old plnts tht were grown in Hoglnd solution nd treted with 5 lm CdCl 2 for 1 d Unigene TPM-SntrR TPM-SttrR Sttr Sntr P-vlue FDR Annottion Most relted Aridopsis gene Metl trnsport nd detoxicity gnl UG Les#S ± ± Iron-regulted trnsporter 2 (IRT2) IRT2 (AT4G198.1) YFR2I11A 4.1 ± ± E 8.33E Zinc trnsporter ZIP11 ZIP11 (AT1G5591.1) MLF3E7A ± ± Copper trnsporter 5 (COPT5) COPT5 (AT5G25.1) gnl UG Les#S ± ±.85 ) E 1 1.2E 15 Mgnesium trnsporter (MGT) MGT2 (AT1G11.1) gnl UG Les#S ± ± 1.4 ) E E 81 Iron-regulted trnsporter 1 (IRT1) IRT1 (AT4G199.1) TVR5N17C ± ± ) E 3.71E PDR-type ABC trnsporter 2 (PDR2) PDR (AT1G1552.1) PLA8N18C 13.9 ± ±.4 ) E E 2 CPx-type hevy metl ATPses (Cu-HMA) HMA mrna (AT5G2458.1) Antioxidnt-relted genes gnl UG Les#S ± ± E E Pericrp peroxidse 3 PCA (AT3G4911.1) TVL29A5A ± ± E E Secretory peroxidse PRXR1 (AT4G219.1) gnl UG Les#S ± ± E 8.34E Cell wll peroxidse POD mrna (AT1G524.1) TVR9D9C.2 ± ± Peroxidse 1 precursor POD 1 (AT2G1898.1) TVR2O5A 8.4 ± ± Peroxidse POD mrna (AT4G21.1) TVR2B24C.4 ± ± Peroxidse RCI3 (AT1G52.1) gnl UG Les#S ± ± 3.23 ) E E 85 Peroxidse POD 59 (AT5G1989) gnl UG Les#S ± ± 1.13 ) E E 13 Peroxidse 21 POD mrna (AT2G3713) OVS1L14C 17. ± ±.1 ) E E 17 L-scorte peroxidse 1 (APX1) APX3 (AT4G35) gnl UG Les#S ± ± 2.4 ) E E 8 gp91-phox RBOH F (AT1G4) gnl UG Les#S ± ± 1.7 ) E E 1 Peroxiredoxin-2E Peroxiredoxin-2E (AT3G529) gnl UG Les#S ± ± 1.4 )5.5 Ctionic peroxidse 1 POD 52 (AT5G534) gnl UG Les#S ± ±.2 )1.5 Peroxidse 27 (POD27) POD 27 (AT3G119) gnl UG Les#S ± ±.33 ) E E 33 Peroxidse isoform 1 prxr1 (AT4G219) gnl UG Les#S ± ±.24 )17.44 Nonsymiotic hemogloin clss 1 HB1 (At2g1) Amino cid iosynthesis nd metolism processes gnl UG Les#S ± ± 3.5 ).1 4.3E E 133 Dehydroquinte dehydrtse (MEE) MEE32 (AT3G35) gnl UG Les#S ± ±.15 ) E E 17 Cysteine desulfurse NFS1 (AT5G572) SmFL2A1A.89 ± ±.15 ) E E 21 Dihydroxy-cid dehydrtse (DHAD) DHAD (AT3G2394) gnl UG Les#S ± ±.4 ) E E 3 Asprtte minotrnsferse-like ASP5 (AT4G3199) gnl UG Les#S ± ± 2.32 ) E E 4 SAT1 SERAT2;1 (AT1G5592) MLF1C11A ± ± 1.17 ) E E 38 DAHP synthse 1 (DHS1) DHS1 (AT4G3998) gnl UG Les#S ± ± 4.7 ) E 8 1.E 7 Chloroplst threonine deminse 1 (OMR1) OMR1 (AT3G15) SmFL4D23A ± ±.17 ) E E 17 Serine rcemse SR (AT4G114) Amino cid trnsporter SmFL25G5A ± ±.17 ) E E 2 Amino cid trnsporter CAT (AT5G477) gnl UG Les#S ± ±.21 ) E 17.34E 17 Amino cid permese 2 AAP2 (AT5G922) SmFLE21A ± ± 1.1 ) E E 33 Amino cid trnsporter AAP (AT5G418) Crohydrte nd cell wll metolism processes PLA7L14C 73. ± ± 1. ) E E 11 ATP-citrte synthse (CSY) CSY4 (AT2G4435.1) gnl UG Les#S ± ± 17.1 )3.25 ATP citrte synthse ctivity (CLA) ACLA-3 (AT1G943.1) TVR3E9W 4.2 ± ±.22 ) E 5 7.5E 5 Mlte dehydrogense (MDH) MDH (AT5G5833) gnl UG Les#S ± ±.7 ) E E 1 Mlte dehydrogense (mmdh) mmdh1 (AT1G5324) SmFL29H15A ± ± E E RGP2 RGP2 (AT5G155.1) TVR4LC 9.57 ± ± XTH9 XTH9 (AT4G321.2) TVR34MC ± ± E E 13 Expnsin (EXPA) EXLB1 (AT4G173) Sntr, Cd-treted roots; Sttr, Cd-treted roots; TPM, normlized expression level of genes, men ± SE, n = 3; Sttr Sntr, log2 (multiples of differentilly expressed); P-vlue, P-vlue from difference test; FDR, flse discovery rte. Ornge, up-regulted genes; green, down-regulted genes. Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust New Phytologist (2) 19: 11 4

9 118 Reserch New Phytologist () gnl UG Les#S gnl UG Les#S gnl UG Les#S5872 PLA8N18C TVR5N17C gnl UG Les#S PLA7L14C gnl UG Les#S58833 YFR2I11A MLF3E7A TVR18P14C X7238 ck Cd ck Cd Annottion MGT IRT1 ns-h1 HMA PDR 2 MEE CSY IRT2 ZIP11 COPT5 UBQ14 18S RNA () Trnscript level gnl UG Les#S (MGT) Snck Sntr Stck Sttr gnl UG Les#S (IRT1) PLA7L14C (CSY) gnl UG Les#S5872 (ns-h1) 9 3 PLA8N18C (HMA) 8 4 gnl UG Les#S58833 (IRT2) 9 3 gnl UG Les#S (MEE) TVR5N17C (PDR2) Fig. 5 Semiquntittive reverse trnscription polymerse chin rection (RT-PCR) () nd quntittive RT-PCR () confirmtion of the digitl gene expression (DGE) dt from Solnum nigrum nd Solnum torvum. ck, 4-wk-old untreted control; Cd, treted with 5 lm CdCl 2 for 24 h. Brs represent the 95% CI of the normlized expression. Fe supply on lef physiology in Cd-treted plnts. Supplementtion with Fe did not ffect Cd ccumultion in the leves of the two species fter 5 d of tretment. Fe deficiency incresed Cd ccumultion in leves fter 5 d of tretment; however, it did not ffect Cd ccumultion in leves (Fig. ). The Pn of oth species continued to decrese during the exposure to 5 lm Cd stress. Fe supplementtion did not ffect the Pn in the Cd-treted plnt; however, the rte of reduction ws lower in Fe-supplemented plnts (Fig. 7). By contrst, Fe deficiency reduced the Pn to greter extent in oth nd thn did Cd tretment lone. Similrly, the O 2 nd H 2O 2 ccumultion in leves of supplemented with 5 lm Fe ws lower thn with Cd tretment lone, New Phytologist (2) 19: 11 4 nd Fe deficiency induced higher O 2 nd H 2O 2 ccumultion in oth nd leves (Fig. 7,c). Consistent with the phenomenon of ROS ccumultion, Fe deficiency incresed the MDA concentrtion nd electrolyte lekge, wheres Fe supplementtion effectively reduced the MDA concentrtion nd electrolyte lekge in leves of Cd-treted (Fig. 8,), indicting tht there is positive effect of Fe supplementtion on lleviting Cd-induced oxidtive dmge in plnts. These results indicte tht Fe deficiency incresed Cd ccumultion only in leves fter 5 d of tretment. IRT1 nd IRT2 re two importnt Fe trnsporter genes involved in the Cd ccumultion nd tolernce in plnts (Vert et l., 22, 29). To explore the possile involvement of these genes in modulting Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust

10 New Phytologist Reserch 119 () 75 Lef Fe content (μg g 1 DW) () Lef Cd content (μg g 1 DW) the differentil Fe Cd ccumultion in the two species, we exmined the gene expression of IRT1 nd IRT2 in the roots of plnts treted with Cd or without Fe supply. As shown in Fig. 9, oth Cd tretment nd Fe deficiency up-regulted the expression of IRT1 in. By contrst, Cd tretment nd Fe deficiency down-regulted the expression of IRT1 nd IRT2 in the roots of. The results of these experiments re discussed in the following section. Discussion + Cd + Cd + Fe + Cd Fe + Cd + Cd + Fe + Cd Fe 1 5 Time fter tretment (d) + Cd + Cd + Fe + Cd Fe + Cd + Cd + Fe + Cd Fe 1 5 Time fter tretment (d) Fig. Effects of Fe supplementtion nd deficiency on Cd ccumultion in Solnum nigrum nd Solnum torvum leves. The lef Fe ccumultion () nd the lef Cd ccumultion () were recorded seprtely t 1 nd 5 d fter Cd tretment. Asterisks indicte vlues significntly different from those of the plnts treted with Cd lone (P <.5). Error rs indicte ± SD. The trnscriptionl regultion of genes plys n importnt role in metl homeostsis (Wintz et l., 23). The mjority of trnscriptomics nlyses of differentil Cd ccumultion re sed on the comprtive nlysis etween hyperccumultor nd relted nonhyperccumultor species. The reported Cd hyperccumultors re lso Zn hyperccumultors, which suggests tht Cd nd Zn ccumultion t lest prtilly rely on common genetic determinnts (Xing et l., 28; Verruggen et l., 29). However, in this study, we found tht lthough ccumulted lower Cd, the Zn concentrtion ws unffected, implying tht specific mechnism exists in for low Cd ccumultion. Therefore, in this study, we chose the Cd ccumultor nd its low Cd-ccumulting reltive for comprtive trnscriptome nlysis. Also, to the est of our knowledge, this Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust () () O 2. production (nmol g 1 FW) (c) H 2 O 2 production (μmol g 1 FW) Pn (μmol m 2 s 1 ) Cd + Cd + Fe + Cd Fe + Cd + Cd + Fe + Cd Fe 1 5 Time fter tretment (d) + Cd + Cd + Fe + Cd Fe + Cd + Cd + Fe + Cd Fe 1 5 Time fter tretment (d) + Cd + Cd + Fe + Cd Fe + Cd + Cd + Fe + Cd Fe study represents the first trnscriptome nlysis of using tg-sed DGE system, which llowed us to identify new genes tht my contriute to the differentil Cd ccumultion nd tolernce phenotype of the two Solnum species. The enhnced expression of hevy metl trnsporter genes in hyperccumultors is universl nd is regrded s centrl chrcteristic of hevy metl hyperccumultion (Weer et l., 2; Verruggen et l., 29). However, in this study, we found tht severl metl trnsporters showed higher expression in, which suggests tht these genes re involved in low Cd ccumultion. These results re discussed in the following. Our study supports the ide tht the modultion of Cd distriution in plnts nd intrcellulr redistriution (vi sequestrte metl in vcuoles or orgnelles in roots or long-distnce trnsport from root to 1 5 Time fter tretment (d) Fig. 7 Effects of Fe supplementtion nd deficiency on Cd tolernce in Solnum nigrum nd Solnum torvum leves. Photosynthetic rte (Pn) (), production of superoxide (O 2 ) () nd hydrogen peroxide (H 2O 2 ) (c) were recorded seprtely t, 1, nd 5 d fter Cd tretment. Asterisks indicte vlues significntly different from those of the plnts treted with Cd lone (P <.5). Error rs indicte ± SD. New Phytologist (2) 19: 11 4

11 Reserch New Phytologist MDA level (μmol g 1 FW) Electrolyte lekge (%) () () ck Fe Cd Cd+Fe Cd Fe ck Fe Cd Cd+Fe Cd Fe Fig. 8 Effects of Fe supply on Cd tolernce in Solnum nigrum (open rs) nd Solnum torvum (closed rs). Mlondildehyde (MDA) content () nd electrolyte lekge () in the leves of 4-wk-old nd plnts tht were grown in Hoglnd solution treted with 5 lm CdCl 2 (Cd), 5 lm Fe (Fe), 5 lm CdCl 2 plus 5 lm Fe (Cd+Fe) or Fe deficiency plus 5 lm CdCl 2 (Cd Fe) for 5 d. The different letters indicte the vlues tht were significntly different from their pproprite controls (without Cd) t P <.5. Error rs indicte ± SD. Reltive expression level IRT1 ck Cd Fe ck Cd Fe shoot nd storge in lef vcuoles) ply vitl role in differentil Cd ccumultion. The Cd ccumultor ws more tolernt to Cd stress thn (Figs 1, 7, 8). The lef Cd ccumultion ws threefold higher in thn in, which demonstrtes the high Cd detoxifiction cpility of. A previous study indicted tht, lthough ccumulted similr Cd concentrtions in its roots, it ccumulted lower Cd concentrtions in its shoots compred with eggplnt (S. melongen; Aro et l., 28), which suggested tht the roots contined IRT2 c c ck Cd Fe ck Cd Fe Fig. 9 The expression profile of IRT1 nd IRT2 were monitored y RT-qPCR in the 4-wk-old Solnum nigrum nd Solnum torvum plnts grown under conditions without (ck) nd with 5 lm CdCl 2 (Cd) s well s without Fe supply (Fe)) t 1 d. The sterisks indicte the vlues tht were significntly different from their pproprite controls (ck) (P <.5) Error rs indicte ± SD. New Phytologist (2) 19: 11 4 c c d d vitl fctor tht controlled the Cd loding cpcity into the xylem. In support of this result, y using horticulturl nd physiologicl pproches, we demonstrted tht the root-to-shoot trnsloction plys mjor role in the differentil Cd ccumultion etween nd. Susequently, our trnscriptomic studies offered some insight into the moleculr mechnisms tht potentilly medite these differences in Cd ccumultion. Severl studies hve indicted tht high ntioxidtive cpcity is responsile for hevy metl hyperccumultion in plnts (Schutzenduel & Polle, 22; Cho & Seo, 25; Wng et l., 28). ccumulted lower ROS concentrtions thn in the presence of Cd (Fig. 7,c), which suggests tht hs high free rdicl scvenging cpcity. However, in this study, we found tht most differentilly expressed ntioxidnt-relted genes re peroxidse fmily genes. Other severl importnt ntioxidtive enzyme genes, such s SOD nd CAT, did not show differentil expression etween the two species. The trnscriptome nlysis lso identified n ns-h1 gene, which my function s n ntioxidnt. Of ll of the genes tht were tested, ns-h1 ws the most up-regulted gene in the roots compred with tht in the roots. Plnt ns-hs function in vriety of cellulr processes nd its overexpression in plnts were shown to enle the cell to mintin high quntities of ATP when under stress (Borisjuk et l., 27; Thiel et l., 211). Previous studies indicted tht purified recominnt Aridopsis ns-hs displyed intrinsic peroxidse-like ctivity (Skmoto et l., 24). Overexpression of GhH1 gene decresed the concentrtion of cellulr NO in Aridopsis seedlings (Qu et l., 2), indicting tht ns-hs plys role in modulting the NO concentrtions nd the rtio of H 2 O 2 NO in the defense process (Igmerdiev et l., 24; Qu et l., 2). In this study, we found tht roots ccumulted higher level of ns-h1 trnscript thn tht in. Moreover, we found tht Cd tretment further reduced ns-h1 expression in roots ut did not hve significnt effect on ns-h1 expression in roots (Tle S1). These findings imply tht this H1 gene my ply n importnt role in the Cd stress-response process of seedlings. Interestingly, we lso found tht peptidyl-cysteine S-nitrosyltion-relted gene, peroxiredoxin-2e, ws more highly expressed in roots. The post-trnsltionl modifiction (S-nitrosyltion) of cysteine thiol to form nitrosothiol (SNO) is key feture of NO nd is coupled to the stimultion of ll isoforms of NO synthse (Stmler, 1994; Stmler et l., 21; Liu et l., 24). The mjority of ll NO-ffected proteins seem to e regulted y the S-nitrosyltion of single criticl Cys residue (Lindermyr et l., 25). Our previous studies hve indicted tht NO is ssocited with long-term Zn nd Cd tolernce nd ccumultion in seedlings (Xu et l., 21, 211). The enhnced expression of the peroxiredoxin-2e gene in suggests tht the gene my e involved in modulting Cd tolernce nd ccumultion y the NO signling pthwy nd requires further investigtion. Trnscriptome nlysis lso reveled tht n Asp iosynthesisrelted gene (ASP), cysteine desulfurse gene (NFS) tht cn Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust

12 New Phytologist Reserch 1 ctlyze Cys to Al, n romtic mino cid iosynthesis-relted gene (MEE), nd other five mino cid metolism-relted genes showed higher expression in thn in, implying tht the Cd response in mino cid iosynthesis nd metolism is greter in the roots of thn in the roots of. Amino cid ccumultion is centrl plnt response to hevy metl exposure. Further metolomic nlysis of mino cids will help to elucidte the roles of mino cids in controlling Cd tolernce nd ccumultion in hyperccumulting nd low Cd-ccumulting plnts. The ZIP fmily trnsporters re the importnt Zn Fe trnsporters (Tlke et l., 2; Plz et l., 27) nd the COPT fmily trnsporters re the importnt Cu trnsport proteins (Sncenon et l., 23; Penrrui et l., 21). Two ZIP trnsporters (IRT2 nd ZIP11) nd COPT trnsporter (COPT5) displyed lower level of gene expression in thn in. IRT2 my comprtmentlize Fe Cd into vesicles, to prevent toxicity y excess free Fe Cd in the cytosol (Vert et l., 29). The results were consistent with the oserved phenotype of high Fe ccumultion in roots nd implied tht my rely on other trnsporters to cquire Cd. ZIP11 is n endomemrne system-locted Zn Cd trnsporter (Plz et l., 27; Antosiewicz et l., 28). COPT5 is locted in the tonoplst nd functions s vcuolr Cu trnsporter (Klumnn et l., 211). An Aridopsis copt5 mutnt showed mrkedly reduced root growth under Cd toxicity (Klumnn et l., 211), which implies tht COPT5 is lso involved in Cd tolernce in plnts. High expression of IRT2, ZIP11, nd COPT5 in roots my promote Cd into the vcuolr or endomemrne systems, therey incresing root Cd sequestrtion nd reducing Cd trnsport from the roots to the shoots. In this study, three hevy metl trnsporters, PDR2, IRT1, nd n HMA trnsporter, showed higher expression in Cd-treted roots thn in roots. IRT1 is the min route of Fe entry into the plnt nd medites the ccumultion of dditionl metl ions (Vert et l., 22). Becuse irt1 muttion is lethl, it is cler tht no other ZIP gene cn sustitute for its loss. Even overexpression of IRT2, which is the closest homolog of IRT1, cnnot compenste for the loss of IRT1 (Vrotto et l., 22). IRT1 my lso e involved in the high-ffinity Cd uptke in Thlspi cerulescens Gnges roots (Lomi et l., 22). PDR2 is memer of the pleiotropic drug resistnce (PDR) sufmily of the ATP-inding cssette (ABC) fmily of trnsporters. In Aridopsis, two PDR genes, AtPDR8 nd AtPDR, hve een suggested to trnsport hevy metl ions nd confer Cd nd P resistnce in Aridopsis (Lee et l., 25; Kim et l., 27). The most closely relted Aridopsis gene to the Solnum PDR2 is AtPDR, which suggests tht the Solnum PDR2 gene my e involved in hevy metl resistnce nd trnsmemrne trnsport in plnts. Memers of the HMA fmily re thought to e involved in the trnsport of hevy metls (Axelsen & Plmgren, 21), nd severl HMA genes hve een shown to e involved in hevy metl long-distnce trnsport nd detoxifiction (Hussin et l., 24; Wong & Coett, 29). HMA2 hs een suggested to trnsport Zn nd Cd in Aridopsis (Wong et l., 29). TcHMA3 plys key role in the extreme Cd tolernce in T. cerulescens (Ueno Ó 2 The Authors New Phytologist Ó 2 New Phytologist Trust et l., 211). HMA4 is responsile for the efficient xylem loding of Cd (Bernrd et l., 24; Verret et l., 24) nd hs een implicted s key gene in Cd hyperccumultor species. However, severl similr ppers filed to phenocopy Cd hyperccumultion y overexpressing HMA4 (Hnikenne et l., 28; Brsz et l., 21, 2; Sieminowski et l., 211), which suggests tht Cd hyperccumultion nd tolernce re the results of mny gene networks working simultneously. In this study, n HMA gene showed higher expression in roots thn in roots under Cd toxicity, which suggests tht the gene is linked to Cd ccumultion nd tolernce in. The most closely relted Aridopsis gene of the Solnum HMA is hevy metl trnsport detoxifiction domin-contining protein (AT5G2458). However, the chrcteriztion of the Aridopsis HMA gene remins uncler nd requires further investigtion. Compred with, roots ccumulted only 2% more Cd; however, the leves ccumulted 179% more Cd, which indictes tht low Cd long-distnce trnsport rte is n importnt mechnism for low Cd ccumultion in. Ymguchi et l. (211) reported tht the rrier function of the root endoderml Csprin nd is one mechnism tht is responsile for low Cd loding into the stele in. In this study, severl Csprin nd iosynthesis-relted genes, including RGP2 (UDP-glucose: protein trnsglucosylse), XTH9 (xyloglucn endotrnsglucosylse-hydrolse), nd OMT1 (O-methyltrnsferse) tht is involved in Csprin nd suerin iosynthesis (Held et l., 1993), displyed higher constitutive expression levels in roots. A metolite nlysis lso identified n mino cid tht is n importnt component of the Csprin nd, hydroxy-l-proline (Hyp), nd shows greter undnce in the Cd-treted roots (dt not shown). Although our present results, which were otined from the trnscriptome nd metolite nlyses, supported this hypothesis, we did not oserve ny morphologicl differences etween the roots endoderml Csprin nds of the two Solnum species (dt not shown). In ddition, it is difficult to explin why only Cd nd Fe trnsport ws restrined, while Zn nd Cu trnsport ws unffected. Our study showed tht during Cd tretment, no significnt differences in Zn ccumultion existed in nd. However, ccumulted higher Fe concentrtion in the roots nd lower Fe in the leves. These results suggest tht hs lower cpcity of Fe loding into the xylem, which therey limited its long-distnce root-to-shoot trnsport. A similr phenotype of low Cd nd Fe ccumultion in leves implies tht the modulting or trnsport pthwys tht were responsile for Cd nd Fe loding into the xylem were similr. Cdmium toxicity led to Fe deficiency in plnts. Decresed Fe ccumultion in leves mgnified the mlfunction of the photosynthetic system nd susequent oxidtive dmge (Siedleck et l., 1997; Solti et l., 28). In this study, we found tht the ccumultion of Fe in leves is lower thn tht in. Therefore, we hypothesize tht low Fe ccumultion my e relted to the high Cd sensitivity in leves compred with. Further study on the supplementtion with Fe in Cd-treted plnts supported the ssumption. New Phytologist (2) 19: 11 4