A 1 -----------------------------------MASKKMTKSYFDVLGICCTSEVPLIENILNSMDGVKEFSVIVPSRTVIVVHDTLILSQFQIVKALNQAQLEANVRVTG--ETNFK 1 -------------------------MALQNKEEEKKKVKKLQKSYFDVLGICCTSEVPIIENILKSLDGVKEYSVIVPSRTVIVVHDSLLISPFQIAKALNEARLEANVRVNG--ETSFK 1 -----------------------------MAEGEESKKMNLQTSYFDVVGICCSSEVSIVGNVLRQVDGVKEFSVIVPSRTVIVVHDTFLISPLQIVKALNQARLEASVRPYG--ETSLK 1 ---------------------------------MAAEGGRCQKSYFDVLGICCPSEVPLVEKLLQPLEGVQKVTVIVPSRTVIVVHDVDAISQSQIVKALNQARLEASVRAYGNGSEKIT 1 MAGKDEAEGLEARLLLLPPEAAAEEPTRCGGGDGGGGGRKRKKTYLDVLGVCCSAEVALVERLLAPLDGVRVVSVVVASRTVVVEHDPAAAPESAIVKALNKAGLEASVRAYG--SSGVV 84 NKWPSPFAVVSGILLLLSFFKYLYSPFRWLAVAAVVAGIYPILAKAVASLARFRIDINILVVVTVGATIGMQDYTEAAVVVFLFTIAEWLQSRASYKASAVMQSLMSLAPQKAVIAETGE 94 NKWPSPFAVVSGLLLLLSFLKFVYSPLRWLAVAAVAAGIYPILAKAFASIKRPRIDINILVIITVIATLAMQDFMEAAAVVFLFTISDWLETRASYKATSVMQSLMSLAPQKAIIAETGE 9 SQWPSPFAIVSGVLLVLSFFKYFYSPLEWLAIVAVVAGVFPILAKAVASVTRFRLDINALTLIAVIATLCMQDFTEAATIVFLFSVADWLESSAAHKASIVMSSLMSLAPRKAVIADTGL 88 NKWPSPYVLLCGLLLVVSLFEHFWHPLKWFALVAAAAGLPPIVLRSIAAIRRLTLDVNILMLIAVAGAIALKDYSEAGFIVFLFTTAEWLETRASHKATAGMSALMSMAPQKAILAETGE 119 SRWPSPYIVASGVLLTASFFEWLFPPLQCLAVAAVVAGAPPMVRRGFAAASRLSLDINVLMLIAVAGALCLGDYTEAGAIVFLFTTAEWLETLACTKASAGMSSLMGMLPVKAVIATTGE 24 EVEVDELKTNTVIAVKAGETIPIDGVVVDGNCEVDEKTLTGEAFPVPKLKDSTVWAGTINLNGYITVNTTALAEDCVVAKMAKLVEEAQNSKTETQRFIDKCSKYYTPAIILISICFVAI 214 EVEVDEVKVDTVVAVKAGETIPIDGIVVDGNCEVDEKTLTGEAFPVPKQRDSTVWAGTINLNGYICVKTTSLAGDCVVAKMAKLVEEAQSSKTKSQRLIDKCSQYYTPAIILVSACVAIV 21 EVDVDEVGINTVVSVKAGESIPIDGVVVDGSCDVDEKTLTGESFPVSKQRESTVMAATINLNGYIKVKTTALARDCVVAKMTKLVEEAQKSQTKTQRFIDKCSRYYTPAVVVSAACFAVI 28 VVAARDVKVNTVIAVKAGEVIPIDGVVVDGRSEVDESTLTGESFPVSKQPDSQVWAGTLNIDGYIAVRTTAMADNSAVAKMARLVEEAQNSRSSTQRLIDTCAKYYTPAVVVMAGSVAAI 239 VVSVRDVRVGDVVAVRAGEIVPVDGVVVDGQSEVDERSLTGESFPVPKQPHSEVWAGTMNFDGYIAVRTTALAENSTVAKMERLVEAAQNSRSKTQRLIDSCAKYYTPAVVVVAAGVALI 324 PFALKVHNLKHWVHLALVVLVSACPCGLILSTPVATFCALTKAATSGLLIKGADYLETLAKIKIVAFDKTGTITRGEFIVMDFQSLS-EDISLQSLLYWVSSTESKSSHPMAAAVVDYAR 334 PVIMKVHNLKHWFHLALVVLVSGCPCGLILSTPVATFCALTKAATSGLLIKSADYLDTLSKIKIVAFDKTGTITRGEFIVIDFKSLS-RDINLRSLLYWVSSVESKSSHPMAATIVDYAK 33 PVLLKVQDLSHWFHLALVVLVSGCPCGLILSTPVATFCALTKAATSGFLIKTGDCLETLAKIKIVAFDKTGTITKAEFMVSDFRSLS-PSINLHKLLNWVSSIECKSSHPMAAALIDYAI 328 PAIAKAHNLKHWFQLALVLLVSACPCALVLSTPIATFCALLRAARTGLLIKGGDVLESLASIKVAAFDKTGTITRGEFSVEEFQPVG-ERVSLQQLLYWVSSVESRSSHPMASVLVDYAQ 359 PALLGADGLEQWWKLALVMLVSACPCALVLSTPVASFCAMLRAARMGIFIKGGDVLESLGEIRAVAFDKTGTITRGEFSIDSFHLVGDHKVEMDHLLYWIASIESKSSHPMAAALVEYAQ 443 SVSVEPKPEAVEDYQNFPGEGIYGKIDGKEVYIGNKRIASRAGCLS--VPDIDVDTKGGKTIGYVYVGETLAGVFNLSDACRSGVAQAMKELKSLGIKIAMLTGDNHAAAMHAQEQLGNA 453 SVSVEPRPEEVEDYQNFPGEGIYGKIDGNDIFIGNKKIASRAGCST--VPEIEVDTKGGKTVGYVYVGERLAGFFNLSDACRSGVSQAMAELKSLGIKTAMLTGDNQAAAMHAQEQLGNV 449 SVSVEPKPDIVENFQNFPGEGVYGRIDGQDIYIGNKRIAQRAGCLTDNVPDIEATMKRGKTIGYIYMGAKLTGSFNLLDGCRYGVAQALKELKSLGIQTAMLTGDNQDAAMSTQEQLENA 447 SKSVEPKSENVSEFQIYPGEGIYGEIDGAGIYIGNKRILSRASCET----VPDMKDMKGVTIGYVACNNELIGVFTLSDACRTGSAEAIKELRSLGIKSVMLTGDSSAAATYAQNQLGNI 479 SKSIQPNPENVGDFRIYPGEGIYGEIHGKHIYIGNRRTLARASSPQ--STQEMGEMIKGVSIGYVICDGELAGVFSLSDDCRTGAAEAIRELGSLGIKSVMLTGDSSAAATHAQGQLGGV 561 MDIVRAELLPEDKSEIIKQLKREEGPTAMVGDGLNDAPALATADIGISMGVSGSALATETGNIILMSNDIRRIPQAIKLAKRAKRKVVENVVISITMKGAILALAFAGHPLIWAAVLADV 571 LDVVHGDLLPEDKSRIIQEFKKE-GPTAMVGDGVNDAPALATADIGISMGISGSALATQTGNIILMSNDIRRIPQAVKLARRARRKVVENVCLSIILKAGILALAFAGHPLIWAAVLVDV 569 LDIVHSELLPQDKARIIDDFKIQ-GPTMMVGDGLNDAPALAKADIGISMGISGSALATETGDIILMSNDIRKIPKGMRLAKRSHKKVIENVVLSVSIKGAIMVLGFVGYPLVWAAVLADA 563 LAEVHAELLPEDKVRIVGELKEKDGPTLMVGDGMNDAPALAKADVGVSMGVSGSAVAMETSHVALMSNDIRRIPKAVRLARRTHRTIIVNIIFSVITKLAIVGLAFAGHPLIWAAVLADV 597 MEELHSELLPEDKVRLVSGLKARFGPTMMVGDGMNDAAALAAADVGVSMGISGSAAAMETSHATLMSSDVLRVPEAVRLGRCARRTIAVNVAGSVAVKAAVLALAAAWRPVLWAAVLADV 681 GTCLLVILNSMLLLSDKHKTGNK----CYRESSSSSVLIAEKLEGDAAGDMEAGLLPKISDKHCKPGCCGTKTQEKAMKPAKASS----DHSHSGCCETKQKDNVTVVKKS-CCAEPVDL 69 GTCLLVIFNSMLLLREKKKIGNK---KCYRASTSKLNGRKLEGDDDYVVDLEAGLLTKSGNGQCKSSCCGDKKNQENVVMMKPSSKTSSDHSHPGCCGDKKEEKVKPLVKDGCCSEKTRK 688 GTCLLVILNSMILLRDEREAVST----CYRSSTSSPVKLEEDEVEDLEVGLLQKSEETSKKSCCSGCCSGPKDNQQK------------------------------------------- 683 GTCLLVIMYSMLLLREKDSRKAK---KCAASHHGSPKKCCSSSHHGSHAKKNHGVSHHCSDGPCKSMVSCKESSVAKNACHDHHHEHNHHEEPAHKHSSNQHGCHDHSHGHSNCKEPSNQ 717 GTCLLVVLNSMTLLREEWKGGAKEDGACRATARSLVMRSQLAADSQAPNAADAGAAGREQTNGCRCCPKPGMSPEHSVVIDIRADGERQEERPAEAAVVAKCCGGGGGEGIRCGASKKPT 792 GHGHDSGCCGDKSQQPHQHEVQVQQSCHNKPSGLDSG----------------------------------------------------------------------------------- 87 SEGDMVSLSSCKKSSHVKHDLKMKGGSGCCASKNEKGKEVVAKSCCEKPKQQVESVGDCKSGHCEKKKQAEDIVVPVQIIGHALTHVEIELQTKETCKTSCCDSKEKVKETGLLLSSENT ------------------------------------------------------------------------------------------------------------------------ 8 LITNKHACHDGHNHCADTSNLHDTKKHDCHGHEHSTCKEELN------------------------------------------------------------------------------ 837 ATVVVAKCCGGGGGGEGTRCGASKNPATAAVVAKCCSGGGGE------------------------------------------------------------------------------ 829 -----------CCGGKSQQPHQHELQQSCHDKPSGLDIGTGPKHEGSS--------------------------------------------------TLVNLEGDAKEELKVLVNG--- 927 PYLEKGVLIKDEGNCKSGSENMGTVKQSCHEKGCSDEKQTGEITLASEEETDDQDCSSGCCVNEGTVKQSFDEKKHSVLVEKEGLDMETGFCCDAKLVCCGNTEGEVKEQCRLEIKKEEH ------------------------------------------------------------------------------------------------------------------------ 842 ----------------ALPPTNDHACHGHEHSHCEEPVALHSTGEHACHEHEHEHIHCDEPIGSHCADKHACHDHEQVHEHHCCDEQQTPHTADLHPCHDHDHDNLEVEEVKDCHAEPPH 879 ----------GIGCGASKKPTATAVVAKCCGGGGEGTRCAASKKPATAAVVAKCCGG--------------------------------------------------------------- 885 ------------------------------------------FCSSPADLAITSLKVKSDSHCKS---------------------NCSSRERCHHGSNCCRSYAKESCSHDHHHTRAHG 147 CKSGCCGEEIQTGEITLVSEEETESTNCSTGCCVDKEEVTQTCHEKPASLVVSGLEVKKDEHCESSHRAVKVETCCKVKIPEACASKCRDRAKRHSGKSCCRSYAKELCSHRHHHHHHHH ------------------------------------------------------------------------------------------------------------------------ 946 HHNHCCHEPHDQVKNDTHPVQEHSISIEESSDHHEHHHNEEHKAEDCGHHPKPKDCAPPPTDCISRNCCSNTSKGKDICSSLHRDHHTSQASRCCRSYVKCSRPSRSCCSHSIVKLPEIV 926 ------------------------------------------DGGEGTGCGASKRSPPAEGSCSGGEGGTNGVGRCCTSVKRPTCCDMGAAEVSDSSPETAKDCRNGRCCAKTMNSGEVK B 942 VGTLKEIVIE 1167 HHHVSA---- ---------- 166 VE-------- 4 G--------- C WT Ubiquitin Supplementl Figure S1. Sequene, struture nd knokout mutnts of. (A) Alignment of HMA2-like proteins in Arbidopsis thlin (, 3 nd 4) nd rie ( nd ). Core regions without N- nd C-terminus vrible regions for similrity omprison re indited by vertil broken lines. (B) Struture of gene. The gene onsists of 1 exons nd 9 introns. The exons re indited s white boxes. Positions of the Tos17 insertion muttions re indited. (C) Expression of mrna in wild type rie (v. ) nd two Tos17 insertion mutnts heked by RT-PCR.
91 69 OsHMA1 AtHMA1 99 AtHMA6 OsHMA7.1 OsHMA4 OsHMA5 AtHMA5 AtHMA7 /RAN1 OsHMA9 OsHMA6 AtHMA8 OsHMA8 Supplementl Figure S2. Phylogeneti reltionship of HMA proteins in rie (blk) nd Arbidopsis thlin (blue). Bootstrp vlues from trils re indited. The.1 sle shows substitution distne.
A B C D E F (e) Supplementl Figure S3. Immunohistohemil stining of nd GFP. (A-D) Stining with nti- polylonl ntibody in 2 mm from the pex of seminl root of (A) nd (B) mutnts, unelongted bsl node (C) nd node III (D) of wild type rie. Stining with nti-gfp ntibody in root (E) nd node I (F) of promoter-gfp trnsgeni rie. Red olor indites the signl from ntibody. Sle br = µm.
Mirosome 2/3 Surose (%) 3/4 4/5 5/6 Pellet H + -ATPse Plsm membrne V type-atpse Tonoplst Bip ER Arf1 Golgi Supplementl Figure S4. Subellulr loliztion of by western blot nlysis. Mirosome extrted from whole roots of wild type rie (v. ) grown hydroponilly were used for western blot nlysis. The mirosome frtion ws frtionted by surose-density grdient. Polylonl ntibodies of nti-, nti-h + -ATPse (plsm membrne mrker), nti-v-type ATPse (tonoplst mrker), nti-bip (ER mrker) nd Arf1 (Golgi mrker) were used.
K onentrtion (mg / kg DW) 5 4 3 2 Mg onentrtion (mg / kg DW) 6 5 4 3 2 C onentrtion (mg / kg DW) 4 3 2 Mn onentrtion (mg / kg DW) 3 2 Fe onentrtion (mg / kg DW) 14 12 8 6 4 2 Cu onentrtion (mg / kg DW) 25 2 15 1 5 Zn onentrtion (mg / kg DW) 8 6 4 2 Cd onentrtion (mg / kg DW) 3 2 1 Supplementl Figure S5. Minerl onentrtion of the shoots t the vegettive stge. Shoots were tken from five week-old seedlings grown hydroponilly treted with nm Cd for lst one week. The onentrtion of K, Mg, C, Mn, Fe, Cu, Zn nd Cd in the shoots re shown. Error brs represent ±SD of three independent biologil replites. Sttistil omprison ws performed by one-wy ANOVA followed by the Tukey s multiple omprison test. All dt were ompred with. P <.5, P <.1.
K onentrtion (mg / kg DW) 3 25 2 15 5 Mg onentrtion (mg / kg DW) 8 6 4 2 C onentrtion (mg / kg DW) 14 12 8 6 4 2 Mn onentrtion (mg / kg DW) 3 25 2 15 1 5 Fe onentrtion (mg / kg DW) 2 15 5 Cu onentrtion (mg / kg DW) 25 2 15 5 Zn onentrtion (mg / kg DW) 6 4 2 Cd onentrtion (mg / kg DW) 12 8 4 Supplementl Figure S6. Minerl onentrtion of the root t the vegettive stge. Root were tken from five week-old seedlings grown hydroponilly treted with nm Cd for lst one week. The onentrtion of K, Mg, C, Mn, Fe, Cu, Zn nd Cd in the roots re shown. Error brs represent ±SD of three independent biologil replites. Sttistil omprison ws performed by one-wy ANOVA followed by the Tukey s multiple omprison test. All dt were ompred with. P <.5, P <.1.
A B.5 Shoot dry weight (g).4.3.2.1. C.1 Root dry weight (g).8.6.4.2. Supplementl Figure S7. Growth of wild-type rie nd mutnts t the vegettive stge. Wild type rie (v. ) nd two oshm2 mutnts ( nd ) were grown hydroponilly for 5 weeks (A) nd hrvested. Dry weight of the shoot (B) nd root (C) were reorded. Error brs represent ±SD of three independent biologil replites. Sttistil omprison ws performed by one-wy ANOVA followed by the Tukey s multiple omprison test. All dt were ompred with. P <.5.
K onentrtion (mg / kg DW) 6 5 4 3 2 Mg onentrtion (mg / kg DW) 6 5 4 3 2 C onentrtion (mg / kg DW) 12 8 6 4 2 Mn onentrtion (mg / kg DW) 24 18 12 6 Fe onentrtion (mg / kg DW) 8 6 4 2 Cu onentrtion (mg / kg DW) 8 6 4 2 Cd onentrtion (mg / kg DW) 2. 1.5 1..5. Supplementl Figure S8. Minerl onentrtion of the shoot bsl meristem t the vegettive stge. Shoots were tken from five week-old seedlings grown hydroponilly treted with nm Cd for lst one week. The onentrtion of K, Mg, C, Mn, Fe, Cu nd Cd in the shoots bsl meristem (2 m-lowest prt of lef sheth of youngest expnded lef nd inner orgns) re shown. Error brs represent ±SD of three independent biologil replites. Sttistil omprison ws performed by one-wy ANOVA followed by the Tukey s multiple omprison test. All dt were ompred with. P <.5, P <.1.
3 6 6 K onentrtion (mg / kg) 2 b b b I II Mg onentrtion (mg / kg) 4 2 b b d d dd I II C onentrtion (mg / kg) 4 2 b b b I II Mn onentrtion (mg / kg) 6 4 2 b b b b I II Fe onentrtion (mg / kg) 3 2 b b b b b b b I II Cu onentrtion (mg / kg) 8 6 4 2 b b b b b b b b I II Supplementl Figure S9. Minerl onentrtion in the nodes. Different nodes (node I to III from the top of the ulm) were smpled from wild type rie nd two oshm2 mutnts ( nd ) grown in field t the hrvest nd the onentrtion of K, Mg, C, Mn, Fe nd Cu were determined with ICP-MS. Error brs represent ±SD of three independent biologil replites. Sttistil omprison ws performed by one-wy ANOVA followed by the Tukey s multiple omprison test. Different letters indite signifine. P <.5.
A B -Cd -Zn 1 mm Zn 2 µm Cd C -Zn 3 µm Cd Supplementl Figure S1. Yest trnsport ssy for Cd nd Zn. (A) Growth of wild type yest strin INVS1 trnsformed with empty vetor, nd in the presene of, 2 or 3 µm CdSO4 with gltose for 3 dys. (B) Growth of wild type yest strin BY4741 trnsformed with empty vetor nd in the presene of or 1 mm ZnSO4 with gltose for 3 dys. (C) Growth of Zn hypersensitive yest strin zr1ot1 trnsformed with empty vetor nd in the presene of, 2 or 4 µm ZnSO4 with gltose for 3 dys. 2 µm Zn 4 µm Zn