Evolution of metal hyperaccumulation required cis-regulatory changes and triplication of HMA4

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1 Vol My 28 doi:1.138/nture6877 LETTERS Evolution of metl hyperumultion required is-regultory hnges nd triplition of HMA4 Mr Hnikenne 1 {*, In N. Tlke 1 {*, Mihel J. Hydon 1 {, Christ Lnz 2, Andre Nolte 1 {, Ptrik Motte 3,4, Juergen Kroymnn 5, Detlef Weigel 2 & Ute Krämer 1 { Little is known out the types of muttions underlying the evolution of speies-speifi trits. The metl hyperumultor Aridopsis hlleri hs the rre ility to olonize hevy-metlpolluted soils, nd, s n extremophile sister speies of Aridopsis thlin, it is powerful model for reserh on dpttion 1 3. A. hlleri nturlly umultes nd tolertes lef onentrtions s high s 2.2% zin nd.28% dmium in dry iomss 4.On the sis of trnsriptomis studies, metl hyperumultion in A. hlleri hs een ssoited with more thn 3 ndidte genes tht re expressed t higher levels in A. hlleri thn in A. thlin 4 6. Some of these genes hve een genetilly mpped to rod hromosoml segments of etween 4 nd 24 M o-segregting with Zn nd Cd hypertolerne 7 9. However, the in plnt loss-of-funtion pprohes required to demonstrte the ontriution of given ndidte gene to metl hyperumultion or hypertolerne hve not een pursued to dte. Using RNA interferene to downregulte HMA4 (HEAVY METAL ATPASE 4) expression, we show here tht Zn hyperumultion nd full hypertolerne to Cd nd Zn in A. hlleri depend on the metl pump HMA4. Contrry to postulted glol trns regultory ftor governing high expression of numerous metl hyperumultion genes, we demonstrte tht enhned expression of HMA4 in A. hlleri is ttriutle to omintion of modified is-regultory sequenes nd opy numer expnsion, in omprison to A. thlin. Trnsfer of n A. hlleri HMA4 gene to A. thlin repitultes Zn prtitioning into xylem vessels nd the onstitutive trnsriptionl upregultion of Zn defiieny response genes hrteristi of Zn hyperumultors. Our results demonstrte the importne of is-regultory muttions nd gene opy numer expnsion in the evolution of omplex nturlly seleted extreme trit 1. The eluidtion of nturl strtegy for metl hyperumultion enles the rtionl design of tehnologies for the len-up of metl-ontminted soils nd for io-fortifition. HMA4 (ref. 4) is mong lrge numer of genes more highly expressed in A. hlleri thn in A. thlin 5,6 nd enodes plsm memrne protein of the 1 B fmily of trnsition metl pumps in the P-type ATPse superfmily 8,11. To investigte whether A. hlleri HMA4 (HMA4) funtions in metl hyperumultion or hypertolerne of A. hlleri, we redued the expression of HMA4 y RNA interferene (RNAi) 12 (Supplementry Fig. 1). For this, we developed geneti trnsformtion system, whih hs not so fr een ville for hyperumultor speies. HMA4 trnsript levels were deresed to etween 45% nd 1% of wild-type levels in different RNAi lines (Fig. 1, Supplementry Fig. 2), whih ppered morphologilly norml. After ultivtion of HMA4 RNAi lines in hydroponi solutions, their shoots ontined only 12 35% of the Zn onentrtions found in wild-type A. hlleri (Fig. 1, Supplementry Fig. 3). These levels were similr to shoot Zn onentrtions in A. thlin s representtive non-umultor 4. In roots of wildtype A. hlleri, metl onentrtions re low, refleting high root-toshoot metl fluxes 4,13,14. By omprison, roots of HMA4 RNAi lines umulted 49- to 134-fold higher Zn onentrtions, gin similr to the non-umultor A. thlin (Fig. 1). Prtitioning nd umultion of metls other thn Zn nd Cd (Supplementry Fig. 4) were not onsistently hnged in HMA4 RNAi lines. Together these results demonstrte tht high HMA4 trnsript levels re required for highly effiient root-to-shoot Zn flux nd for Zn hyperumultion in the shoots of A. hlleri. This is distint from the funtion of A. thlin HMA4 (AtHMA4) in root-to-shoot trnslotion of Zn for the mintenne of Zn-dependent proesses in the shoot 15,16. We next exmined how HMA4 lters the distriution of Zn within the roots of A. hlleri, y imging with the fluoresent Zn inditor Zinpyr In the wild type, fluoresene ws most intense in the xylem vessels loted inwrds from the periyle ell lyer of the root vsulture (Fig. 1d, Supplementry Fig. 5). By ontrst, in the HMA4 RNAi lines signl intensity ws mximl in the periyle ell lyer, qulittively similr to the hnge in Zn loliztion reported for the A. thlin hm4 mutnt ompred to the wild type 15. This indites tht silening of HMA4 impirs the relese of Zn from the root symplsm into the poplsti xylem vessels, whih provide the primry pthwy for the movement of solutes from the root to the shoot with the trnspirtion strem 17. These results re in greement with the deresed Zn prtitioning into the shoots in HMA4 RNAi lines. If inresed HMA4 tivity is primry event in hevy metl hyperumultion, we would expet tht this ffets other ndidte genes for hyperumultion known to e highly expressed in A. hlleri 4. We therefore nlysed RNA from plnts ultivted in 5 mm Zn solution y rel-time RT PCR. Expression of Zn defiieny response genes 4,18 ws speifilly deresed in roots of the HMA4 RNAi lines. For exmple, reltive trnsript levels of IRT3 (Fig. 1e) nd ZIP4 (Fig. 1f) were orrelted with HMA4 trnsript levels ross ll genotypes (r 5.99 nd.98, respetively). The memrne trnsporters enoded y IRT3 nd ZIP4 re oth memers of the sme fmily of proteins implited in the ellulr uptke of Zn (ref. 18). Thus, the high expression of these genes in roots of wild-type A. hlleri 4 ppers to e seondry onsequene of inresed HMA4 tivity, nd is likely to further enhne shoot metl umultion. 1 Mx Plnk Institute of Moleulr Plnt Physiology, D Potsdm, Germny. 2 Mx Plnk Institute for Developmentl Biology, D-7276 Tüingen, Germny. 3 Plnt Cell Biology, Deprtment of Life Sienes, 4 Center for Assistne in Tehnology of Mirosopy, Deprtment of Chemistry, University of Liège, B-4 Liège, Belgium. 5 Mx Plnk Institute for Chemil Eology, D-7745 Jen, Germny. {Present ddresses: Plnt Cell Biology, Deprtment of Life Sienes, University of Liège, B-4 Liège, Belgium (M.H.); Deprtment of Thori, Crdi nd Vsulr Surgery, University of Tüingen, D-7276 Tüingen, Germny (A.N.); BIOQUANT Center, Heidelerg Institute of Plnt Sienes, University of Heidelerg, D-6912 Heidelerg, Germny (I.N.T., M.J.H., U.K.). *These uthors ontriuted eqully to this work. 28 Nture Pulishing Group 391

2 LETTERS NATURE Vol My 28 Heterologous expression of HMA4 in ells of the udding yest Shromyes erevisie ws reported to onfer metl tolerne 4 nd metl hypersensitivity 8. Reently, three respetive mjor QTL (quntittive trit loi) for Zn nd Cd hypertolerne were mpped in progeny from ross etween A. hlleri nd its metl-sensitive non-umultor reltive Aridopsis lyrt 8,9. Notly, mjor QTL for oth Zn nd Cd hypertolerne ontined HMA4, ut omprised severl entimorgns nd thus t lest severl hundred genes. To exmine whether HMA4 funtions not only in metl hyperumultion of A. hlleri, ut lso in hypertolerne, we determined the effets of toxi metl onentrtions on root growth of plnts in hydroponi solutions 19,2. In the presene of 3 mm Cd or 1.5 mm Zn, root elongtion in wild-type A. hlleri plnts ws still out 68% nd 6% of the elongtion under ontrol onditions, respetively. In ontrst, roots of HMA4 RNAi lines exhiited only 3 15% nd 12 37% of the elongtion under ontrol onditions, respetively (Fig. 1g, h). These dt demonstrte tht HMA4 mkes sustntil ontriution to Cd nd Zn hypertolerne in A. hlleri. Our results indite tht HMA4 hs key role in severl spets of the omplex metl hyperumultor phenotype. We therefore ddressed the moleulr sis for elevted HMA4 expression in A. hlleri ompred to the non-umultor A. thlin. Erlier, Southern lot sreening provided irumstntil evidene tht there my e more thn single HMA4 gene opy in the genome of A. hlleri 4. However, informtion on the numer of gene opies, their respetive sequenes, expression nd funtionlity hs een unville. To exmine this, two overlpping A. hlleri genomi BAC (teril rtifiil hromosome) lones were sequened. Compred to the synteni segment in A. thlin, there is omplex triplition of region tht inludes HMA4 (orresponding to At2g1911 in A. thlin) nd the two downstrem genes (At2g1912 nd At2g1913) (Fig. 2). In ddition to prtil deletions nd inversions of At2g1912 nd At2g1913 orthologues, severl trnsposon nd retrotrnsposon insertions re evident. The flnking sequenes re lrgely synteni with A. thlin. Within oding sequenes, the three HMA4 gene opies re on verge 99% identil to eh other, ut shre only 88% sequene identity with AtHMA4, suggesting tht the triplition my hve ourred reltively reently in the A. hlleri linege (Supplementry Tle 1). After reverse trnsription we speifilly quntified either the HMA4-1 omplementry DNA lone or oth the lmost identil HMA4-2 nd HMA4-3 DNAs y rel-time PCR (Supplementry Fig. 6). The results re onsistent with similr expression levels of ll three HMA4 gene opies (Fig. 2). Thus, oth n inrese in opy numer nd elevted expression of individul gene opies ontriute to the high trnsript levels of HMA4 in A. hlleri in omprison with A. thlin. Sequene divergene from A. thlin is most pronouned in the 59-flnking regions of HMA4 genes (Supplementry Tle 1) 21, suggesting possile regultory innovtions in A. hlleri. To determine experimentlly whether the inresed expression of individul HMA4 gene opies is due to is-regultory hnges, we generted fusions of the promoters of AtHMA4 nd of the three HMA4 gene opies to the -gluuronidse (GUS) reporter for the trnsformtion of oth A. hlleri nd A. thlin. Reporter tivity direted y the A. hlleri Reltive trnsript level HMA4 1, 2, 3, 4, S P -GUS HMA4 RNAi lines d A. hlleri Shoot Zn onentrtion (mg kg 1 DW) Root Zn onentrtion (mg kg 1 DW) , 7, 6, 5, 4, 3, 2, 1, HMA4 RNAi lines HMA4 RNAi lines At At Men root elongtion (% of ontrol) Figure 1 Chrteriztion of A. hlleri HMA4 RNAi lines., HMA4 trnsript levels reltive to EF1 in roots of A. hlleri wild type () nd 35S P -GUS trnsformnt ontrols, nd four HMA4 RNAi lines.,, Zin onentrtions in shoots () nd roots () of A. hlleri () genotypes nd A. thlin Col (At), ultivted in hydroponi solution ontining 5 mm Zn for 3 weeks (DW, dry weight). d, Zn loliztion in A. hlleri roots. Confol imges show fluoresent Zn signls (green) nd ell wlls (red). 392 g e RTL IRT3 in roots HMA4 RNAi ** ** ** HMA4 RNAi lines * RTL ZIP4 in roots A. hlleri HMA4 RNAi A. hlleri 5 A. hlleri HMA4 RNAi A. hlleri A. thlin A. thlin 1,5 3, 4,5 6, 1,5 3, 4,5 6, RTL HMA4 in roots RTL HMA4 in roots h 8 3 µm CdSO mm ZnSO 4 28 Nture Pulishing Group f Men root elongtion (% of ontrol) ** ** HMA4 RNAi lines Sle rs, 5 mm;, periyle; rrowheds highlight fluoresent Zn signls in xylem vessels. e, f, Reltive trnsript levels (RTL) of IRT3 (e) nd ZIP4 (f) plotted ginst reltive HMA4 trnsript levels, ll normlized to EF1, in different genotypes. g, Cdmium tolerne, nd h, zin tolerne of A. hlleri genotypes. Vlues re men 6 s.e.m., n 5 4 to 6 nd 12 to 18 individuls for RNAi lines nd wild type, respetively (,, g, h), n 5 3to4(), n 5 6to8 (e, f). *P,.5, **P,.1, P,.1. *

3 NATURE Vol My 28 LETTERS A. hlleri BAC 17L A. hlleri BAC 7C17 HMA4-1 HMA4-2 HMA4-3 At2g191 At2g193 At2g194 Copi At2g195 At2g196 At2g197 At2g198 At2g At2g At2g Copi 1 At2g At2g At2g () Copi At2g At2g At2g At2g At2g1915 At2g1916 At2g At2g () 2 At2g () HMA4-1 HMA4-2 + HMA4-3 Trnsript level reltive to EF1 5 1, 1,5 2, 2,5 >85% identity with A. thlin ~88% identity with A. thlin 1 k Asent from synteni region of A. thlin hromosome 2 Clss I LTR retrotrnsposon elements (- nd Copi-like) (HMA ) >9% identity with A. thlin Clss I non LTR retrotrnsposon reverse trnsriptse HMA4 AtHMA4 95% identity with A. thlin Clss II trnsposon elements (En/Spm sulss) Figure 2 Genomi orgniztion nd expression of HMA4 genes in A. hlleri., Digrm showing two overlpping A. hlleri genomi BACs. Genes re represented y oxes nd nmed y A. thlin Genome Identifier odes (lue text, regions synteni with A. thlin; green, triplited genes). Tringles indite the diretion of trnsription (filled, intt genes; open, AtHMA4 promoter (AtHMA4 P ) ws muh lower thn tht direted y ny of the three HMA4 promoters (HMA4-1 P, HMA4-2 P, HMA4-3 P ), whih were s effetive s the strong onstitutive trunted or pseudogenes). Three frgments of one At2g1912 orthologue re denoted (), () nd (); LTR, long terminl repet., Reltive trnsript levels of HMA4 gene opies in A. hlleri roots, nd of AtHMA4 in A. thlin roots for omprison (men 6 s.e.m., n 5 6). uliflower mosi virus (CMV) 35S promoter, 35S P (Fig. 3 d). Sptil ptterns of reporter tivity for ll three HMA4 P onstruts were highly similr in oth A. hlleri nd A. thlin (Fig. 3e, g, A. hlleri A. thlin AtHMA4 P HMA4-1 P HMA4-2 P HMA4-3 P AtHMA4 P HMA4-1P HMA4-2 P HMA4-3 P Speifi reporter tivity (mmol MU mg 1 h 1 ) Shoot Root d Speifi reporter tivity (mmol MU mg 1 h 1 ) Shoot Root AtHMA4 P HMA4-1 P HMA4-2 P HMA4-3 P 35S P AtHMA4 P HMA4-1 P HMA4-2 P HMA4-3 P 35S P e f g h xp xp HMA4-2 P HMA4-2 P Figure 3 Levels nd ell speifiity of HMA4 promoter tivity in A. hlleri nd A. thlin.,, e, f, A. hlleri (left);, d, g, h, A. thlin (right).,, Histohemil detetion of GUS tivity (lue), direted y the A. thlin or A. hlleri HMA4 promoters, in leves of 5-week-old plnts., d, Speifi GUS tivity in protein extrts (men 6 s.e.m., n 5 4to7 independent lines; MU, 4-methylumelliferone). Vlues for the CMV 35S promoter re shown for omprison. e h, Reporter tivity in whole mounts 28 Nture Pulishing Group (e, g) nd trnsverse setions (f, h) of root tips from HMA4-2 P -GUS trnsformnts representtive of ll HMA4 promoters (Supplementry Figs 7, 8 nd 1). Tissues were stined for 18 h (, ) or.5 h (e to h). The inset (f) shows lose-up of the vsulr ylinder. Sle rs:,, 5 mm; e, g, 1 mm; f, 25mm; nd h, 15mm., periyle; xp, xylem prenhym. Root ntomy differs slightly etween A. thlin nd A. hlleri. 393

4 LETTERS NATURE Vol My 28 Supplementry Figs 7 1). Qulittively, tivity ptterns were lso similr for promoters originting from oth speies (Supplementry Figs 7, 8) 15. The HMA4 expression pttern ws onfirmed y in situ hyridiztion in wild-type A. hlleri (Supplementry Figs 9, 1). This reveled speifi messenger RNA umultion in the root periyle nd xylem prenhym (Fig. 3f, h, Supplementry Fig. 1), supporting funtion for HMA4 in xylem loding of Zn (see Fig. 1d). Expression in the xylem prenhym nd the mium of leves (Supplementry Fig. 9) is onsistent with possile role of HMA4 in metl distriution within the lef lde nd the exlusion of metls from speifi ell types. Together, our dt onfirm tht high HMA4 trnsript levels in A. hlleri re the result of gene opy numer triplition omined with the enhned expression of ll three A. hlleri HMA4 genes speified in is. Finlly, we determined whether inresed HMA4 tivity is not only neessry ut lso suffiient for ltered hevy metl umultion nd tolerne. To this end, we trnsformed A. thlin with n HMA4 mini-gene, onsisting of n HMA4 DNA linked to the HMA4-1 promoter. Primry trnsformnts ontined modertely elevted HMA4 trnsript levels ( nd fold in roots nd shoots, respetively, ompred to wild type, men 6 s.e.m., ZIP4 IRT3 A. thlin HMA4 P -HMA4 Trnsript level reltive to EF A. thlin HMA4 P -HMA4 n 5 6; Supplementry Fig. 9m o), t the lower end of the rnge expeted sed on previous omprisons etween A. hlleri nd A. thlin 4. Zinpyr-1 imging showed high levels of Zn in xylem tissues of roots of trnsgeni plnts (Fig. 4), mimiking the distriution of Zn in wild-type A. hlleri (see Fig. 1d). By ontrst, in non-trnsgeni A. thlin Zn levels were highest in the root periyle ell lyer (Fig. 4), resemling the loliztion in A. hlleri HMA4 RNAi lines (see Fig. 1d). Thus, trnsformtion of A. thlin with HMA4 is suffiient to repitulte Zn distriution typil of A. hlleri in the roots of the non-hyperumultor A. thlin. Roots of HMA4- trnsformed A. thlin lso ontined inresed trnsript levels of the Zn defiieny response genes ZIP4 nd IRT3 (Fig. 4), in nlogy with wild-type A. hlleri (see Fig. 1e, f). These results further support the model tht in roots of A. hlleri, the high HMA4-dependent Zn flux into the xylem depletes symplsti Zn pools, therey triggering the upregultion of Zn defiieny response genes in the roots 4. A. thlin HMA4 trnsformnts grown in medi supplemented with toxi onentrtions of 15 mmznor4mm Cd developed enhned lef hlorosis nd smller rosettes, whih re signs of Zn nd Cd hypersensitivity of the shoots (Fig. 4, Supplementry Fig. 11). When ultivted in 5 mm Zn, HMA4 trnsformnts were helthy nd umulted slightly higher Zn onentrtions thn non-trnsgeni plnts ( fold, men 6 s.e.m., n 5 4 independent experiments, P,.5; dt not shown). These results suggest more effiient trnsfer of metls from roots to leves in HMA4 trnsformnts ompred to non-trnsgeni A. thlin. The metl sensitivity of shoots of A. thlin expressing HMA4 indites tht dditionl genes re required for metl detoxifition in order to ommodte the high HMA4-dependent metl flux into the shoots of A. hlleri 7,9,22. In summry, using series of funtionl riteri 23, we hve demonstrted mjor role for HMA4 in nturlly seleted Zn hyperumultion nd ssoited Cd nd Zn hypertolerne in A. hlleri. High HMA4 expression in A. hlleri is speified in is nd mplified y gene opy numer expnsion. Inresed expression of HMA4 in A. hlleri supports the enhned Zn flux from the root symplsm into the xylem vessels neessry for shoot Zn hyperumultion, nd ts s physiologil mster swith to upregulte Zn defiieny response gene expression in roots (Supplementry Fig. 1). These findings re of entrl importne for the development of phytoremedition nd io-fortifition strtegies. Tken together, our results provide evidene for previously proposed roles of is regultory diversifition 24 nd opy numer expnsion 25,26 in eukryoti dpttion µm ZnSO 4 15 µm ZnSO 4 µm CdSO 4 4 µm CdSO 4 A. thlin HMA4 P -HMA4 Figure 4 Chrteriztion of A. thlin expressing HMA4.,Zn loliztion in roots of 1-dy-old seedlings. Confol imges show fluoresent Zn signls (green) nd ell wlls (red). Sle rs, 25 mm;, periyle; rrowheds highlight fluoresent Zn signls in xylem vessels., Reltive trnsript levels of ZIP4 nd IRT3 in roots of hydroponilly ultivted 5.5-week-old plnts (men 6 s.e.m., n 5 6)., Zn nd Cd toxiity in shoots of 17-dy-old HMA4 trnsformnts seven dys fter trnsfer to ontrol medium (5 mm Zn, no Cd) or medium supplemented with 15 mm Zn or 4 mm Cd Nture Pulishing Group METHODS SUMMARY Plnt mteril. A. hlleri (L.) O Kne nd Al-Shehz ssp. hlleri (ession Lngelsheim) or A. thlin L. Heynhold (ession Columi) were used in ll experiments. Plnts were ultivted hydroponilly 4,5 or on solid medium ontining the hydroponi solution (HD pltes) in plsti Petri dishes 28 (Methods). Plnt trnsformtions. Construts were generted y loning of PCR produts into GATEWAY-omptile inry vetors (Methods; Supplementry Methods for primer sequenes). A. thlin ws trnsformed y florl dip 29. Agroterium tumefiens-medited stle trnsformtion of A. hlleri ws performed using tissue-ulture sed proedure 3 (Supplementry Methods). Genomi DNA nd RNA gel lot nlyses were performed for the primry hrteriztion of A. hlleri HMA4 RNAi trnsformnts (Supplementry Methods). Physiologil hrteriztion of plnts. For the determintion of zin umultion nd trnsript levels, plnts were ultivted hydroponilly t 5 mm ZnSO 4 in ontrolled growth hmer for 3 weeks (Supplementry Methods). The Zn inditor Zinpyr-1 (Sigm) ws used for onfol fluoresene imging of Zn in roots of A. hlleri nd A. thlin plnts 16. For the determintion of metl tolerne, sequentil root elongtion ssys nd growth ssys were performed in metl-supplemented hydroponi solutions nd on HD pltes, respetively (Methods). BAC nlysis nd sequening. Two A. hlleri BACs (7C17, 17L7) overing the region orthologous to the HMA4 region of the A. thlin genome nd ontining totl of three genomi HMA4 opies of A. hlleri, were isolted nd ompletely sequened (Methods). Gene nd trnsposle element nnottions were

5 NATURE Vol My 28 LETTERS mde on the sis of similrity serhes performed using the BLASTN nd BLASTX progrms ginst the TAIR dtse ( nd the non-redundnt dtse (nr, Expression nlyses. Reltive trnsript levels were determined y rel-time RT PCR 4. Histohemil GUS stining, fluorimetri quntittive GUS tivity ssys nd in situ hyridiztions were rried out ording to stndrd proedures (Supplementry Methods). Full Methods nd ny ssoited referenes re ville in the online version of the pper t Reeived 2 Deemer 27; epted 28 Ferury 28. Pulished online 2 April Mithell-Olds, T. Aridopsis thlin nd its wild reltives. Trends Eol. Evol. 16, (21). 2. Koornneef, M., Alonso-Blno, C. & Vreugdenhil, D. Nturlly ourring geneti vrition in Aridopsis thlin. Annu. Rev. Plnt Biol. 55, (24). 3. Bker, A. J. M., MGrth, S. P., Reeves, R. D. & Smith, J. A. C. in Phytoremedition of Contminted Soil nd Wter (eds Terry, N. & Bñuelos, G. S.) (CRC Press LLC, Bo Rton, Florid, 1999). 4. Tlke, I. N., Hnikenne, M. & Krämer, U. Zin-dependent glol trnsriptionl ontrol, trnsriptionl deregultion, nd higher gene opy numer for genes in metl homeostsis of the hyperumultor Aridopsis hlleri. Plnt Physiol. 142, (26). 5. Beher, M., Tlke, I. N., Krll, L. & Krämer, U. Cross-speies mirorry trnsript profiling revels high onstitutive expression of metl homeostsis genes in shoots of the zin hyperumultor Aridopsis hlleri. Plnt J. 37, (24). 6. Weer, M. et l. Comprtive mirorry nlysis of Aridopsis thlin nd Aridopsis hlleri roots identifies niotinmine synthse, ZIP trnsporter nd other genes s potentil metl hyperumultion ftors. Plnt J. 37, (24). 7. Dräger, D. B. et l. Two genes enoding Aridopsis hlleri MTP1 metl trnsport proteins o-segregte with zin tolerne nd ount for high MTP1 trnsript levels. Plnt J. 39, (24). 8. Courot, M. et l. A mjor QTL for Cd tolerne in Aridopsis hlleri o-lolizes with HMA4, gene enoding hevy metl ATPse. Plnt Physiol. 144, (27). 9. Willems, G. et l. The geneti sis of zin tolerne in the metllophyte Aridopsis hlleri ssp. hlleri (Brssiee): An nlysis of quntittive trit loi. Genetis 176, (27). 1. Hoekstr, H. E. & Coyne, J. A. The lous of evolution: evo devo nd the genetis of dpttion. Evolution Int. J. Org. Evolution 61, (27). 11. Axelsen, K. B. & Plmgren, M. G. Inventory of the superfmily of P-type ion pumps in Aridopsis. Plnt Physiol. 126, (21). 12. Smith, N. A. et l. Totl silening y intron-splied hirpin RNAs. Nture 47, (2). 13. Krämer, U. et l. Free histidine s metl heltor in plnts tht hyperumulte nikel. Nture 379, (1996). 14. Lst, M. M. et l. Moleulr physiology of zin trnsport in the Zn hyperumultor Thlspi erulesens. J. Exp. Bot. 51, (2). 15. Hussin, D. et l. P-type ATPse hevy metl trnsporters with roles in essentil zin homeostsis in Aridopsis. Plnt Cell 16, (24). 16. Sinlir, S. A. et l. The use of the zin-fluorophore, Zinpyr-1, in the study of zin homeostsis in Aridopsis roots. New Phytol. 174, (27). 17. Clemens, S., Plmgren, M. G. & Krämer, U. A long wy hed: understnding nd engineering plnt metl umultion. Trends Plnt Si. 7, (22). 18. Grotz, N. et l. Identifition of fmily of zin trnsporter genes from Aridopsis tht respond to zin defiieny. Pro. Ntl Ad. Si. USA 95, (1998). 19. Bert, V. et l. Geneti sis of Cd tolerne nd hyperumultion in Aridopsis hlleri. Plnt Soil 249, 9 18 (23). 2. MNir, M. R. et l. Zin tolerne nd hyperumultion re genetilly independent hrters. Pro. R. So. Lond. B 266, (1999). 21. Windsor, A. J. et l. Prtil shotgun sequening of the Boeher strit genome revels extensive mirosynteny nd promoter onservtion with Aridopsis. Plnt Physiol. 14, (26). 22. Küpper, H., Lomi, E., Zho, F. J. & MGrth, S. P. Cellulr omprtmenttion of dmium nd zin in reltion to other elements in the hyperumultor Aridopsis hlleri. Plnt 212, (2). 23. Weigel, D. & Nordorg, M. Nturl vrition in Aridopsis. How do we find the usl genes? Plnt Physiol. 138, (25). 24. Clrk, R. M., Wgler, T. N., Quijd, P. & Doeley, J. A distnt upstrem enhner t the mize domestition gene t1 hs pleiotropi effets on plnt nd infloresent rhiteture. Nture Genet. 38, (26). 25. Bekmnn, J. S., Estivill, X. & Antonrkis, S. E. Copy numer vrints nd geneti trits: loser to the resolution of phenotypi to genotypi vriility. Nture Rev. Genet. 8, (27). 26. Sugino, R. P. & Innn, H. Seletion for more of the sme produt s fore to enhne onerted evolution of duplited genes. Trends Genet. 22, (26). 27. Zhong, S., Khodursky, A., Dykhuizen, D. E. & Den, A. M. Evolutionry genomis of eologil speiliztion. Pro. Ntl Ad. Si. USA 11, (24). 28. Arrivult, S., Senger, T. & Krämer, U. The Aridopsis metl tolerne protein AtMTP3 mintins metl homeostsis y mediting Zn exlusion from the shoot under Fe defiieny nd Zn oversupply. Plnt J. 46, (26). 29. Clough, S. J. & Bent, A. F. Florl dip: simplified method for Agroteriummedited trnsformtion of Aridopsis thlin. Plnt J. 16, (1998). 3. Chteu, S., Sngwn, R. S. & Sngwn-Norreel, B. S. Competene of Aridopsis thlin genotypes nd mutnts for Agroterium tumefiens-medited gene trnsfer: role of phytohormones. J. Exp. Bot. 51, (2). Supplementry Informtion is linked to the online version of the pper t Aknowledgements We thnk D. Burin, D. Wlther, C. Glnte, T. Werner nd the grdeners of the Mx Plnk Institute of Moleulr Plnt Physiology for ssistne, R. Shmidt for A. thlin 35S P -GUS lines, I. Somssih for pjawohl8, nd S. Thomine for omments on the mnusript. This work ws funded y: Germn Reserh Foundtion Kr1967/3-1, Heisenerg Fellowship Kr1967/4-1; Germn Federl Ministry of Edution nd Reserh Biofuture nd GABI-ADVANCIS 31537A; Europen Union RTN METALHOME HPRN CT , InP PHIME FOOD-CT (U.K.). Further funding ws from Fonds spéiux pour l Reherhe, University of Liège, Belgium (M.H., P.M.), Fonds de l Reherhe Sientifique FNRS, Belgium (M.H.), nd the Mx Plnk Soiety (D.W.). Author Contriutions I.N.T., M.H., M.J.H., A.N., U.K., P.M. nd J.K. performed experiments, C.L. the BAC sequening nd ssemly, M.H. ssemly nd BAC nnottion; D.W. nd J.K. provided the BAC lirry nd filters; U.K., M.H. nd I.N.T. jointly designed experiments; D.W. gve experimentl dvie nd edited the mnusript; U.K. oneived of the study nd direted the reserh; U.K., M.H. nd I.N.T. wrote nd edited the mnusript; ll uthors ommented on the mnusript. Author Informtion Reprints nd permissions informtion is ville t BAC sequenes re ville online (Gennk ession numers EU38272, EU38273). Correspondene nd requests for mterils should e ddressed to U.K. (ute.kremer@ioqunt.uni-heidelerg.de). 28 Nture Pulishing Group 395

6 doi:1.138/nture6877 METHODS Plnt ultivtion. Plnts were ultivted in limte-ontrolled glsshouse t 21 uc dy/17 uc night, onstnt humidity of 5%, with supplementry lighting from equl mounts of HPIT nd Son-T Agro lmps (Philips) providing photoperiod of 16 h, or in growth hmer t 2 uc dy/18 uc night, humidity of 6% dy/75% night, nd photoperiod of 11 h t photon flux density 145 mmol m 22 s 21. Genertion of onstruts. All HMA4 sequenes were PCR-mplified using proofreding polymerse (Pfu Turo, Strtgene), nd verified y sequening fter diretionl loning into pentr/d TOPO (Invitrogen) nd gin fter sitedireted reomintion into the respetive GATEWAY-omptile inry vetor efore plnt trnsformtion, unless indited otherwise. To generte the HMA4 RNAi onstrut for trnsformtion of A. hlleri, frgment orresponding to p 2,541 2,997 of the open reding frme of HMA4 ws mplified from A. hlleri DNA nd suloned into the inry vetor pjawohl8 (GenBnk ession AF48413), generting n intronsplied hirpin onstrut with ntisense HMA4 frgment intron sense HMA4 frgment onfigurtion downstrem of the CMV 35S promoter 12. To generte promoter-reporter onstruts for the trnsformtion of A. thlin nd A. hlleri, n AtHMA4 promoter frgment of 2,625 p ws mplified from genomi DNA of A. thlin. The HMA4-1, HMA-2 nd HMA4-3 promoter frgments of 2,326, 1,311 nd 1,82 p, respetively, were mplified from A. hlleri genomi DNA. All promoter frgments, whih inlude promoter nd 59-UTR regions nd end fter the first 3 p of the respetive HMA4 oding sequene, were suloned into the pmdc163 inry vetor 31. The HMA4 P -HMA4 onstrut for trnsformtion of A. thlin ws generted y inserting oth the HMA4-1 promoter frgment nd the full-length HMA4 oding sequene into promoter-less vrint of the pmdc32 vetor 31 (Supplementry Methods). Physiologil hrteriztion of plnts. A. hlleri plnts were mintined in hydroponi ulture 4,5 in limte-ontrolled glsshouse. A. hlleri HMA4 RNAi lines nd wild type ontrol individuls were propgted vegettively vi rooting of shoot uttings on snd for 5 weeks nd susequently ultivted in hydroponis for 4 5 weeks efore inititing experimentl tretments. Results for wild type ontrols were verged from the following A. hlleri individuls: (1) untrnsformed, (2) regenerted from tissue ulture following mok trnsformtion nd (3) trnsformed with uliflower mosi virus (CMV) 35S promoter-gus- Intron onstrut 32. A. thlin plnts were grown from seeds in hydroponi ulture 4,5 for 6 weeks efore experimentl tretments were initited. Metl tolerne of A. hlleri genotypes ws determined in limte-ontrolled glsshouse y mesuring root elongtion in slightly modified hydroponi ontrol solution (5 mm Zn, no dded Cd,.14 mm KH 2 PO 4 to void preipittion t high metl onentrtions), nd susequently in the presene of sequentilly inresed metl onentrtions, in 5-dy intervls (Zn: 8 mm, 1.5 mm; Cd: 1 mm, 3 mm) 2. Two experiments were performed for eh metl. For metl tolerne ssys in A. thlin HMA4 trnsformnts (T1 genertion), 1-dy-old hygromyin-resistnt seedlings were trnsferred onto HD pltes ontining 5 mm ZnSO 4 (ontrol), 15 mm ZnSO 4,or4mM CdSO 4 (nd 5 mm ZnSO 4 ) nd mintined in limte-ontrolled growth hmer. Photogrphs of representtive individuls were tken one week fter trnsfer. The experiment ws repeted three times, with three pltes per genotype nd 1 replite seedlings per plte in eh experiment. For Zn imging, segments of newly formed distl roots were used of A. hlleri plnts grown in hydroponi medium ontining 1 mm ZnSO 4. For A. thlin, seedlings (T3 genertion) were grown for 1 dys on HD pltes in limte-ontrolled growth hmer nd inuted in hydroponi solution with 25 mm ZnSO 4 for 2.5 h efore stining. Fluoresene of the Zn-Zinpyr-1 helte ws oserved using onfol lser snning mirosopy (Lei SP2) with 488 nm exittion using FITC nd Texs Red filters. For A. thlin, two trnsgeni HMA4 lines showing inresed HMA4 trnsript levels were nlysed. For A. hlleri, HMA4 RNAi lines 4.2.1, nd were nlysed (4.2.1 is shown in Fig. 1d). Imges shown re representtive of the results from t lest 6 wild type plnts nd 6 trnsgeni plnts. Rel-time RT PCR nlysis. Preprtion of totl DNse-treted RNA, synthesis of DNA, primer design nd sequenes, qulity ontrol nd dt nlysis were s desried 4. Vlues shown in Fig. 1 re verges from three to four independent iologil experiments, vlues shown in Figs 1e, f, 2 nd 4 re verges from six to eight tehnil replites from one experiment representtive of totl of three independent iologil experiments. BAC nlysis nd sequening. A BAC lirry ws onstruted in the pindigobac-536 vetor using HindIII-digested totl genomi DNA of 8 individuls from A. hlleri ssp. hlleri, essions Roderherrunn nd Stutenkmm (Germny) (Keygene), nd sreened with n HMA4 proe 4 s desried 33. A totl of 18 positive BAC lones were otined. A omintion of Southern lots nd restrition pttern nlyses, together with BAC end sequening, demonstrted tht two BACs (7C17, 17L7) overed ll of totl of three genomi HMA4 opies of A. hlleri, nd the region orthologous to the HMA4 region of the A. thlin genome. For omplete sequening, BAC 7C17 nd 17L7 sulone lirries were prepred with n verge insert size of 5 1 k, nd totl of 1,152 positive lones for eh BAC were end-sequened to 13-fold overge s desried 34. Sequene reds were se-lled using the LifeTre softwre 35 nd ssemled into ontigs with the PHRAP nd CONSED softwre pkges ( 36. Remining gps nd miguously ssemled regions were losed nd polished y primer wlking nd PCR with oligonuleotides designed sed on the flnking sequenes. Comprtive genomi sequene nlyses were done with the GenomeVISTA softwre ( 37,38 to identify synteni regions. Sttistis. All dt evlution nd sttistis were done using Mirosoft Exel. Sttistil nlysis of dt from A. hlleri HMA4 RNAi lines ws performed y multiple omprisons sed on ANOVA. 31. Curtis, M. D. & Grossniklus, U. A gtewy loning vetor set for high-throughput funtionl nlysis of genes in plnt. Plnt Physiol. 133, (23). 32. Vnnneyt, G. et l. Constrution of n intron-ontining mrker gene: spliing of the intron in trnsgeni plnts nd its use in monitoring erly events in Agroterium-medited plnt trnsformtion. Mol. Gen. Genet. 22, (199). 33. Benderoth, M. et l. Positive seletion driving diversifition in plnt seondry metolism. Pro. Ntl Ad. Si. USA 13, (26). 34. Eppinger, M. et l. Who te whom? Adptive Helioter genomi hnges tht ompnied host jump from erly humns to lrge felines. PLoS Genet. 2, e12 (26). 35. Wlther, D., Brth, G. & Morris, M. Bselling with LifeTre. Genome Res. 11, (21). 36. Gordon, D., Ajin, C. & Green, P. Consed: grphil tool for sequene finishing. Genome Res. 8, (1998). 37. Bry, N., Duhk, I. & Phter, L. AVID: A glol lignment progrm. Genome Res. 13, (23). 38. Couronne, O. et l. Strtegies nd tools for whole-genome lignments. Genome Res. 13, 73 8 (23). 28 Nture Pulishing Group