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normally severely toxic concentrations in their shoots(Ernst 1968; Baker & Brooks 1989; Reeves 1992). Most ofthese species are more or less restricted to strongly metal-enriched soil types but some of them are also commonlyfound on non-metalliferous soil (Ingrouille & Smirnoff1986; Baker & Proctor 1990; Schat, Llugany & Bernhard1999).There is considerable debate concerning the ultimateevolutionary explanation of the hyperaccumulation trait.The herbivore defence hypothesis, which states that metalhyperaccumulation is a way to reduce damage by herbivoryand parasitism (Boyd & Martens 1992) is presentlyfavoured by most authors and supported by circumstantialexperimental evidence (Boyd & Martens 1994; Martens &Boyd 1994; Boyd, Shaw & Martens 1994; Pollard & Baker1997; Davis & Boyd, 2000; Ghaderian, Lyon & Baker 2000).

In comparison with normal plants, hyperaccumulatorsare characterized by strongly enhanced rates of uptake, tol-erance and root-to-shoot transport of the metals in ques-tion (Lasat, Baker & Kochian 1996; Schat et al. 1999). Theunderlying mechanisms and their precise inter-relation-ships are largely unknown yet. In F2 crosses between theZn hyperaccumulator Arabidopsis halleri and the non-hyperaccumulator Arabidopsis petraea, the Zn hyperaccu-mulation and tolerance traits segregated independently(Macnair et al. 1999). Additionally, hyperaccumulation, tol-erance and root-to-shoot transport of Zn varied indepen-dently between Thlaspi caerulescens accessions fromdifferent soil types (Schat et al. 1999). These results suggestthat hyperaccumulation might be a complex trait, withuptake, internal transport and tolerance being at leastpartly under independent genetic control. Transmembranemetal transporters may be decisively involved in uptake,xylem loading and unloading (Lasat, Baker & Kochian1998) and vacuolar sequestration of heavy metals, particu-larly in the leaf epidermal cells (Vázquez et al. 1994;Küpper, Zhao & McGrath 1999), trichomes (Krämer et al.1997), or stomatal guard cells (Heath, Southworth &Dallura 1997). The molecular basis of Zn uptake and trans-port in plants is, as yet, largely unexplored.

Grotz et al. (1998) have isolated and functionally charac-terized three Zn transporter genes from Arabidopsis, calledZIP1, ZIP2 and ZIP3 (ZIP: ZRT, IRT-like protein), by

ABSTRACT

Heavy metal hyperaccumulation in plants is an intriguingand poorly understood phenomenon. Transmembranemetal transporters are assumed to play a key role in thisprocess. We describe the cloning and isolation of three zinctransporter cDNAs from the Zn hyperaccumulator Thlaspicaerulescens. The ZTP1 gene is highly similar to the Arabidopsis ZAT gene. Of the other two, one is most prob-ably an allele of the recently cloned ZNT1 gene from T. caerulescens (Pence et al; Proceedings of the NationalAcademy of Science USA 97, 4956–4960, 2000). The second,called ZNT2, is a close homologue of ZNT1. All three zinc transporter genes show increased expression in T. caerulescens compared with the non-hyperaccumulatorcongener T. arvense, suggesting an important role in heavymetal hyperaccumulation. ZNT1 and ZNT2 are predomi-nantly expressed in roots and ZTP1 is mainly expressed inleaves but also in roots. In T. arvense, ZNT1 and ZNT2 areexclusively expressed under conditions of Zn deficiency.Their expression in T. caerulescens is barely Zn-responsive,suggesting that Zn hyperaccumulation might rely on adecreased Zn-induced transcriptional downregulation ofthese genes. ZTP1 expression was higher in plants fromcalamine soil than in plants from serpentine or normal soil.The calamine plants were also the most Zn tolerant,suggesting that high ZTP1 expression might contribute toZn tolerance.

Key-words: Thlaspi caerulescens; heavy metals; hyperaccu-mulation; tolerance; transporters; zinc/nickel.

INTRODUCTION

A limited number of plant species, called hyperaccumula-tors, accumulate certain heavy metals to extremely high,

Correspondence: Dr M.G.M. Aarts. Fax: + 31 317418094;e-mail: [email protected]

*Present address: Academisch Ziekenhuis Utrecht, Department ofHaematology, Heidelberglaan 100, 3508 GA Utrecht.

Elevated expression of metal transporter genes in threeaccessions of the metal hyperaccumulator Thlaspicaerulescens

A. G. L. ASSUNÇÃO,1 P. DA COSTA MARTINS,2* S. DE FOLTER,2 R. VOOIJS,1 H. SCHAT1 & M. G. M. AARTS2

1Department of Ecology and Ecotoxicology of Plants, Faculty of Biology, Vrije Universiteit, De Boelelaan 1087, 1081 HVAmsterdam, The Netherlands and 2BU Plant Development and Reproduction, Plant Research International, Postbus 16,6700 AA Wageningen, The Netherlands

functional complementation of a yeast mutant defective inZn uptake. They also identified a related genomic DNAsequence predicted to encode the ZIP4 protein. ZIP genesbelong to a growing family of putative metal transportergenes with members in the fungal, plant and animalkingdom (Grotz et al. 1998; Eng et al. 1998). The proteinsencoded by ZIP genes have a high degree of similarity withthe yeast ZRT1 and ZRT2 proteins that are involved in thehigh and low-affinity Zn uptake system (Zhao & Eide1996a,b), and with the Arabidopsis IRT1 transporter thatmediates Fe uptake (Eide et al. 1996). ZIP4 is more closelyrelated to Arabidopsis IRT1 than to ZIP1, ZIP2 or ZIP3(Eng et al. 1998). ZIP gene expression is Zn-regulated.ZIP1 and ZIP3 are induced in roots and ZIP4 in both rootsand shoots of Zn-limited plants. The ZIP1 and ZIP3 pro-teins, which are presumably plasma membrane located, aresuggested to play a role in the uptake of Zn from the rhi-zosphere, whereas ZIP4, which contains a potential chloro-plast targeting sequence, was suggested to mediatetransport of Zn into plastids (Grotz et al. 1998).

Recently, using the complementation strategy applied byGrotz et al. (1998), a Zn transporter cDNA was isolatedfrom T. caerulescens by Pence et al. (2000). This transportergene named ZNT1, is also a member of the ZIP gene familyand is highly homologous to Arabidopsis ZIP4 (Grotz et al.1998). ZNT1 is highly expressed in roots and shoots of T. caerulescens, both under conditions of Zn deficiency andat normal nutritional Zn supply. In the related non-hyper-accumulator species, Thlaspi arvense, it is expressed underZn deficient conditions, but shows strong downregulationat normal Zn supply. The high expression of Zn trans-porters in T. caerulescens, irrespective of Zn availability, hasbeen suggested to be the major reason for the enhanced Znuptake of this species. In general, alterations of the patternsof Zn-responsive transcriptional regulation of Zn trans-porters might play a pivotal role in Zn hyperaccumulation(Lasat et al. 2000; Pence et al. 2000).

The ZAT gene encodes another Zn transporter known inplants.The ZAT cDNA was isolated from Arabidopsis (Vander Zaal et al. 1999) and is homologous to the mammalianZn transporter genes ZnT2 (Palmiter, Cole & Findley 1996)and ZnT3 (Wenzel et al. 1997), which are involved in Znvesicular sequestration, and ZnT4 (Huang & Gitschier1997), which is involved in Zn transport into milk. Trans-genic Arabidopsis which overexpressed the ZAT geneexhibited enhanced Zn resistance and an increased Zncontent in roots under high Zn exposure suggesting that theZAT protein is involved in the plant-internal compartmen-tation of this metal (Van der Zaal et al. 1999).

The present study aims to identify additional Zn trans-porters in T. caerulescens and to characterize the variationin metal preference patterns with respect to uptake, root-to-shoot transport and tolerance among T. caerulescensaccessions from contrasting soil types (calamine, serpentineand non-metalliferous soil) in relation to the expression ofZn transporter genes.We identified three cDNAs putativelyencoding Zn transporter proteins and tried to establishtheir role in T. caerulescens Zn hyperaccumulation.

218 A. G. L. Assunção et al.

MATERIALS AND METHODS

Plant material and plant culture

Seeds were collected from three T. caerulescens accessions.Accession La Calamine (LC) originated from a calamineore waste, enriched in Zn, Cd and Pb, at La Calamine,Belgium. Accession Monte Prinzera (MP) originated fromNi-enriched serpentine soil at Monte Prinzera, Italy andaccession Lellingen (LE) originated from a non-metalliferous soil, at Lellingen, Luxembourg.The T. arvensenon-hyperaccumulator reference accession originated froma roadside in Amsterdam, the Netherlands. Arabidopsisthaliana, ecotype Columbia, was obtained from the Nottingham Arabidopsis Stock Centre, UK.

To grow plants, seeds of T. caerulescens and T. arvenseaccessions were sown on moist peat. Three-week-oldseedlings were transferred to 600 mL polyethylene pots(one plant per pot), filled with modified half-strengthHoagland’s nutrient solution (Schat et al. 1996), supple-mented with ZnSO4 and/or NiSO4 at the desired concen-trations. The solutions were replaced twice a week.Germination and plant culture were performed in a climatechamber (20/15 °C day/night; 250 mmol m-2 s-1 at plantlevel, 14 h d-1; 75% relative humidity).

Zn and Ni uptake, root-to-shoot transport andtolerance assays

For the measurement of Zn and Ni uptake, plants of eachof the T. caerulescens and T. arvense accessions wereexposed to nutrient solution supplemented with 1, 10 and100 mm of ZnSO4 or 0, 1, 10 and 100 mm of NiSO4, suppliedalone or together in factorial combinations. Five plantswere used per treatment. After 3 weeks of growth, theplants were harvested, after desorbing the roots systemswith ice-cold 5 mm PbNO3 (1 h). Roots and shoots weredried at 80 °C, wet-ashed in a 4 : 1 mixture of HNO3 (65%)and HCl (37%), in Teflon bombs at 140 °C for 7 h andanalysed for Zn and Ni, using flame atomic absorption spec-trometry (Perkin Elmer 1100B; Perkin Elmes, Norwalk, CT,USA). Total uptake was calculated on a total plant dryweight basis. Shoot-to-root metal concentration ratios wereused as an estimate of root-to-shoot transport.

To measure tolerance, plants of each of the T.caerulescens and T. arvense accessions were exposed tonutrient solution supplemented with a series of Zn or Niconcentrations. The tolerance was inferred from the pres-ence or absence of chlorosis after 3 weeks of growth undermetal exposure and represented by the first concentrationof an increasing series at which chlorosis was observed.

Library construction and screening

A cDNA library was prepared from mRNA extracted fromroots of T. caerulescens, accession LC, grown hydroponi-cally in a solution containing 10 mm ZnSO4. The cDNAlibrary with a primary titre of 1·9 ¥ 107 pfu mL-1 was con-structed in a phagemid vector (pAD-GAL4–2·1) of the

© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 217–226

HybriZap-2·1 two-hybrid cDNA cloning system (Strata-gene, La Jolla, CA, USA). About 9.5 ¥ 104 plaques of theamplified library were screened. An Arabidopsis ZAT1(Van der Zaal et al. 1999) partial cDNA clone (EST143A21, GenBank accession no. 46380; Newman et al. 1994)was used as probe, together with cloned DNA fragmentsobtained by polymerase chain reaction (PCR) using degen-erate primers ZTPfor (5¢-TTY GCI GCI GGI GTI ATHCTN GCN AC-3¢) and ZTPrev (5¢-GCI AGI ARR TCIACN AGN GCC ATR TA-3¢). Degenerate primers werebased on conserved DNA sequences in the ArabidopsisZIP genes (Grotz et al. 1998) and six additional Arabidop-sis homologues. Plaques were lifted and blotted onto anylon membrane (Amersham Pharmacia Biotech, Uppsala,Sweden), according to the recommended procedures andhybridized with the 32P-labelled DNA probes (random-primed DNA labelling system, Amersham PharmaciaBiotech, Uppsala, Sweden). Pre-hybridization andhybridization were performed in hybridization solution[10% dextran sulphate, 1 m NaCl, 1% sodium dodecyl sulphate (SDS)] supplemented with denatured salmonsperm DNA (100 mg mL-1). After an overnight incubationat 65 °C the membranes were rinsed twice in 2 ¥ SSC(300 mm NaCl, 30 mm Na Citrate, pH 7.0) for 2 min at roomtemperature and twice in 2 ¥ SSC, 1% SDS for 20 min at65 °C. Positive plaques were purified and the phagemidvector was extracted by in vivo excision according to theinstruction provided by the manufacturer.

DNA and predicted amino acid sequence analysis

DNA sequences of both strands of each cDNA were deter-mined by automated sequencing. DNA homology searchesand sequence analyses were performed using the BasicLocal Alignment Search Tool (BLAST) (Altschul et al.1997). Multiple sequence alignments were performed byusing the CLUSTAL program (DNASTAR, Madison, WI,USA). Potential protein targeting signals were predicted byPSORT (Klein, Kanehisa & DeLisi 1985), potential trans-membrane sequences were predicted using TMHMM(Sonnhammer, von Heijne & Krogh 1998).

RNA isolation and RNA blot analysis

Total RNA was extracted using the RNeasy Extraction Kit(Qiagen GmbH, Hilden, Germany) from leaves and rootsof T. caerulescens, accessions LC and MP and Arabidopsisecotype Col (all grown on normal potting soil) and fromleaves and roots of hydroponically grown T. arvense and T.caerulescens accessions LC, MP and LE, exposed to 0, 2 and10 mm Zn. Ten micrograms of total RNA was separated bygel-electrophoresis using a 1% agarose gel and capillaryblotted onto Hybond N+ nylon membrane (Amersham),according to standard procedures.

Genomic DNA fragments representing ZIP2, ZIP3 andZIP4 (obtained by PCR) and the insert of EST clone143A21 (Acc. no. T46380) representing ZAT1, as well as

Zinc transporters of Thlaspi caerulescens 219

DNA fragments representing the full cDNAs (ZTP1, ZNT1and ZNT2) were used as probes for RNA blot hybridiza-tion. A PCR fragment of 0·6 kb representing the T. arvenseZTP1 homologue (ZTP1-arvense) was also used as probefor RNA blot hybridization. The forward primer used inthis PCR reaction was 5¢-GGC AGA CTT ACG GGT TCTTCA GG-3¢ and the reverse primer was 5¢-GTG AGAACG GAA AAG CCA ATA GC-3¢. Membranes were pre-hybridized, hybridized and washed as described aboveexcept that additional washes were performed at high strin-gency (in 0·1 ¥ SSC, 1% SDS for 20 min at 65 °C). Mem-branes were stripped using a solution of 0·1% SDS, 2 mmTris-HCl (pH 8·0), 1 mm EDTA for 10 min at 65 °C. Themembranes were checked for removal of the probe beforere-probing. The hybridization signals were scanned using aFuji Phosphor Imager (BAS 2000; Fuji Photo Film Co.,Tokyo, Japan) and, if necessary, quantified using the TINAprogramme provided by the manufacturer.

DNA isolation and DNA blot analysis

DNA was extracted from leaves of T. arvense,T. caerulescens, accessions LC, MP and LE and Arabidop-sis as described by Aarts, Koncz & Pereira (2000). Onemicrogram of Thlaspi and 0·5 mg of Arabidopsis genomicDNA, separately digested with HindIII, was separated bygel-electrophoresis using a 1% agarose-TAE (Tris-Acetate40 mm, EDTA 1 mm) gel and vacuum blotted onto HybondN+ nylon membrane (Amersham) according to standardprocedures. DNA blot hybridization was performed as theRNA blot hybridization described before.

RESULTS

Physiological characterization of T.caerulescens accessions

The Zn and Ni uptake, shoot-to-root concentration ratioand tolerance characteristics were determined for hydro-ponically grown T. caerulescens and non-hyperaccumulatorT. arvense plants (Tables 1 and 2). In comparison with T. arvense, the T. caerulescens accessions La Calamine (LC),Monte Prinzera (MP) and Lellingen (LE) were all charac-terized by a strongly enhanced Zn uptake, Zn shoot-to-rootconcentration ratio and Zn tolerance. However, there werepronounced differences between these T. caerulescens


Table 1. Tolerance to Zn and Ni of Thlaspi arvense and T. caerulescens plants. Tolerance was inferred from the presenceor absence of chlorosis after 3 weeks of growth under metalexposure. The figures represent the first concentration of anincreasing series at which chlorosis was observed

Zn (mm) Ni (mm)

T. arvense < 25 < 25T. caerulescens LC 1000 100T. caerulescens MP 100 250T. caerulescens LE 50 100


Table 2. Uptake (a), shoot concentration (b), and shoot-to-root concentration ratio (c) of Zn and Ni in the non-hyperaccumulatorThlaspi arvense (Ta) and accessions (acc.) LC, MP and LE of the hyperaccumulator T. caerulescens (Tc), after 3 weeks of growth in anutrient solution with factorial combinations of Zn and Ni concentrations. Uptake is expressed on a whole plant dry weight basis (mmolmetal g-1 total plant dry weight), shoot concentration is given as mmol metal g-1 shoot dry weight. nt = not tested. Standard errors variedbetween 3 and 20% of the means

Zn supply (mm)

Species (acc.) Ni supply (mm) 1 10 100 1 10 100

(a) Uptake of Zn and NiZn uptake Ni uptake

Ta 0 1·0 3·8 nt nt nt nt1 nt nt nt 0·2 nt nt

10 nt nt nt 1·8 nt nt100 nt nt nt nt nt nt

Tc (LC) 0 4·6 8·1 35·7 nt nt nt1 5·5 9·4 37·5 0·2 0·2 0·2

10 5·0 10·0 21·3 1·5 1·7 1·9100 7·4 9·9 30·5 21·8 17·8 14·2

Tc (MP) 0 13·2 53·9 118·7 nt nt nt1 15·5 61·8 125·1 8·4 1·3 0·6

10 12·2 55·1 134·3 48·3 10·0 3·6100 11·6 42·1 108·9 104·7 34·2 20·5

Tc (LE) 0 6·9 42·1 nt nt nt nt1 4·4 30·6 nt 1·4 0·7 nt

10 5·8 28·4 nt 17·3 2·9 nt100 nt nt nt nt nt nt

(b) Shoot concentration of Zn and NiZn shoot concentration Ni shoot concentration



Tc (LC) 0 5·6 4·4 42·2 nt nt nt1 6·5 11·0 44·8 0·2 0·2 0·2

10 5·8 13·1 25·8 1·5 1·9 2·0100 9·0 11·5 34·8 25·5 20·1 15·8

Tc (MP) 0 14·1 51·5 98·6 nt nt nt1 15·2 54·3 106·6 9·9 1·4 0·6

10 9·5 48·8 116·6 49·0 10·3 4·1100 7·9 33·7 90·9 106·8 35·2 23·8



(c) Shoot-to-root concentration ratio of Zn and NiZn shoot-to-root ratio Ni shoot-to-root ratio



Tc (LC) 0 7·1 4·1 4·5 nt nt nt1 4·8 4·2 6·1 0·9 0·8 1·0

10 4·1 5·9 7·7 0·9 1·6 1·9100 5·7 4·5 6·3 3·6 2·4 2·1

Tc (MP) 0 1·5 0·8 0·5 nt nt nt1 0·9 0·6 0·5 4·2 1·7 1·8

10 0·4 0·6 0·6 1·1 1·2 2·7100 0·3 0·5 0·5 1·1 1·2 2·4



accessions. As far as tolerance is concerned, the LC plantsmaintained normal growth and leaf pigmentation at1000 mm Zn in the nutrient solution. The MP and LE plantsalready showed chlorosis at 100 and 50 mm Zn, respectively(Table 1). The analysis of Zn uptake showed that LC plantsaccumulated significantly less Zn (P < 0·01) than MP andLE plants, both at low and high supply levels (Table 2a.).Root-to-shoot transport of Zn, as estimated from the ratiobetween shoot and root Zn concentration, was significantlyhigher (P < 0·01) in all T. caerulescens accessions than in T. arvense and, among accessions, higher in LE than in LCand MP plants (Table 2c).

The Ni tolerance was higher in T. caerulescens than in T. arvense, particularly in the MP plants originating fromserpentine soils (Table 1). The Ni accumulation variedstrongly between the accessions (P < 0·01). The MP plantsand, to a lower degree also the LE plants, hyperaccumu-lated Ni, although only at low external Zn concentrations(1 mm). Higher Zn concentrations strongly inhibited Niuptake whereas Ni had no effect on Zn uptake (Table 2a).The LC plants did not show any Ni hyperaccumulation atall, irrespective of the concentration of Zn in the nutrientsolution (Table 2a). Remarkably, there was no inhibitoryeffect of Zn on Ni uptake in these plants. Transport of Niwas consistently higher in T. caerulescens than in T. arvensewith the highest shoot-to-root concentration ratios for LEand the lowest for LC. The MP and LE plants exhibited apronounced inhibitory effect of Ni on Zn transport and viceversa. This was not apparent in LC plants (Table 2c).

Identification of Zn transporter genes in T. caerulescens

To assess whether any of the known Arabidopsis Zn trans-porter genes might be differentially expressed in T. caerulescens, Northern blot experiments were performedusing genomic DNA fragments representing the ZIP2,ZIP3 and ZIP4 genes and a partial cDNA clone represent-ing ZAT1 as probes. The blots contained RNA from leavesand roots of T. caerulescens accessions LC and MP and Ara-bidopsis ecotype Col, all grown on normal potting soil.


ZAT1 was clearly overexpressed in T. caerulescens leavescompared to roots, or to Arabidopsis leaves and roots(Fig. 1). Transcription of ZIP2 and ZIP3 was not detectedin any of the samples, but weak ZIP4 transcription wasfound for all samples (data not shown).The ZIP2 and ZIP3probes hybridized strongly to a T. caerulescens DNA-blot(data not shown), proving that the inability to detect ZIP2and ZIP3 transcripts was not due to lack of homology.

In order to obtain Thlaspi-specific probes for ZIP homo-logues, a degenerate PCR approach was chosen. From data-base searches it became apparent that in Arabidopsis at least10 ZIP-like gene sequences are present, including the knownZIP and IRT genes. Degenerate primers were designed ontwo conserved DNA sequences found within this genefamily (see ‘Materials and Methods’). In total, PCR frag-ments for five different ZIP-like genes were isolated from T.caerulescens accession LC, which were homologous to Ara-bidopsis genes ZIP1, ZIP3 and ZIP4 (data not shown).

Isolation of T. caerulescens Zn transporter cDNAs

The Arabidopsis partial ZAT1 cDNA insert and DNAfragments L13 and L3, homologous to, respectively, ZIP1and ZIP4, were used as probes to screen a T. caerulescenscDNA library. The library was made with root RNA fromLC plants grown hydroponically on medium containing10 mm Zn. With probe L13 no positive clones were found.With each of the other two probes about 40 positive cloneswere obtained. After sequencing the 5¢ ends of the longestinserts, three different full-length cDNA sequences wereidentified, one from a ZAT1-specific clone and two from L3-specific clones. The complete DNA sequence for each full-length clone was determined and compared to sequences inthe GenBank database. The cDNA fragment obtained withthe Arabidopsis ZAT1 partial cDNA probe, is 1340 basepairs (bp) long and encodes a predicted protein of 393amino acids. The cDNA has 90% DNA identity and 75%amino acid identity with Arabidopsis ZAT1 cDNA (Van derZaal et al. 1999). We called this gene ZTP1 for Zn Trans-Porter 1 (Fig. 2).The ZTP1 protein is predicted to be an inte-gral membrane protein with six potential transmembranedomains (Fig. 2), just like the Arabidopsis ZAT protein ofwhich it is most likely the T. caerulescens orthologue. Thesubcellular location of ZTP1 is unclear. Although previousstudies with the Arabidopsis ZAT gene suggested targetingof the protein to the vacuole (Van der Zaal et al. 1999), anobvious vacuolar targeting signal was not detected.

The cDNA clones obtained with the L3 probe are 1375and 1520 bp long and encode predicted proteins of 409 and423 amino acids, respectively. They share 90 and 83% DNAidentity and 76 and 65% amino acid identity with the pre-dicted Arabidopsis ZIP4 DNA and protein sequences(Grotz et al. 1998). By database search we found the firstclone to be nearly identical (99% DNA identity) to theZNT1 Zn transporter gene recently cloned from T. caerulescens accession Prayon (ZNT1-PR; Pence et al.2000) and we believe it to be the LC allele of ZNT1 (ZNT1-


Figure 1. Northern blot analysis of Arabidopsis ZAT geneexpression in leaves and roots of T. caerulescens, accessions LCand MP (TcLC and TcMP) and Arabidopsis thaliana ecotypeColumbia (AtCol), grown in normal potting soil. The second rowrepresents the hybridization of the blot with 16S rRNA used as aloading control. The blot was washed under low-stringencyconditions.

LC). The second clone, which is a close homologue ofZNT1-LC with 80% DNA identity, was labelled ZNT2(Fig. 3). One recently deposited EST sequence represent-ing the ZIP4 gene was found in the GenBank database(Acc. No. AV441840), which starts at nearly the same posi-tion as the ZNT1-LC cDNA clone and downstream of the5¢ end of the ZNT2 cDNA. After comparison of this ESTsequence to the genomic DNA sequence of ZIP4 in Ara-bidopsis, we detected an in-frame stop codon only 3 basesupstream of the EST 5¢ end. As no putative intron–exonboundary sequences were found between the stop codonand the EST sequence start, we concluded that the pre-dicted start codon of ZIP4 corresponds to the first ATGcodon we identified in both the ZNT1-LC and the ZNT2cDNA clones, which is thus most likely the protein transla-tion start codon. With this new ZIP4 cDNA sequence, thepredicted ZIP4 protein sequence is extended at the N-terminus by 34 amino acids compared with earlier publica-tions (Grotz et al. 1998; Pence et al. 2000). This start codonis not present in the ZNT1-PR cDNA sequence reportedby Pence et al. (2000) and consequently the predicted aminoacid sequence of ZNT1-LC is 30 amino acids longer at itsN-terminus compared to ZNT1-PR. The ZNT1-LC andZNT2 proteins are predicted to have a N-terminal signalsequence (Fig. 3), as well as the eight potential transmem-brane domains previously reported for ZNT1-PR by Penceet al. (2000). ZNT1-LC and ZNT2 are likely to be targetedto the plasma membrane.

Expression of Zn transporter genes in differentT. caerulescens accessions and in T. arvense

To determine the expression of the identified Zn transportergenes, low stringency hybridizations were performed usingthe ZTP1, ZNT1-LC and ZNT2 cDNA inserts as probes on blots containing RNA from the three different


T. caerulescens accessions and the non-hyperaccumulator T. arvense. Compared to T. arvense, ZTP1 is higherexpressed in both roots and leaves of T. caerulescens acces-sions LC,MP and LE,grown hydroponically at 0,2 and 10 mmZn (Fig. 4).The expression in T. caerulescens LC was slightlyhigher than in the other two T. caerulescens accessions. InMP and LE the difference in expression between roots andleaves was more pronounced, with enhanced mRNA levelsin leaves.To confirm that the overexpression of ZTP1 in LCwas not due to preferential hybridization of the LC probe tothe LC RNA, an additional hybridization was performedusing a 0·6 kb PCR-fragment of the ZTP1 homologue fromT. arvense as a probe (ZTP1-arvense). The T. arvense probepreferentially hybridized to T. arvense RNA compared to T.caerulescens RNA. However, as with the ZTP1 probe, theZTP1-arvense probe hybridized stronger to T. caerulescensLC RNA than to T. arvense RNA. After image analysis, weestimated that the ZTP1 mRNA hybridization signal wasabout five times higher in LC than in T. arvense.

The ZNT1 expression levels in root and leaf of all acces-sions are very similar to the ZNT2 expression levels. Incomparison with T. arvense, both genes are highly expressedin all three T. caerulescens accessions, at all tested Zn con-centrations (Fig. 5). The expression is higher in roots thanin leaves, which is most pronounced for ZNT1. The


Figure 2. Amino acid sequence alignment of the T. caerulescensZTP1 zinc transporter with the A. thaliana ZAT zinc transporter(GenBank accession no. AF072858). The sequences were alignedusing the CLUSTAL method (DNAstar). The putative transmem-brane domains, predicted by TMHMM (Sonnhammer et al. 1998),are overlined and numbered. Identical residues are shaded.

Figure 3. Amino acid sequence alignment of ZNT1 (LC) andZNT2 with ZNT1 (Prayon) (GenBank accession no. AF133267)and ZIP4, predicted from genomic and partial cDNA sequences(GenBank accessions no. ATU95973 and AV441840). Thesequences were aligned using the CLUSTAL method included inthe DNAstar programme (DNAstar). The putativetransmembrane domains, predicted by TMHMM (Sonnhammeret al. 1998), are numbered and overlined. Identical residues areshaded.

T. arvense ZNT1 transcript was only detected in roots andleaves of plants grown at 0 mm Zn. Also ZNT2 is expressedonly at 0 mm Zn, although it is barely detectable afterhybridization (Fig. 5). Under these conditions, and espe-cially in roots, the expression is much lower in T. arvensethan in T. caerulescens.To establish whether ZNT1 is down-regulated by high zinc concentrations, we hybridized aRNA-blot, containing root and leaf RNA of LC plantssampled in a 48 h time period after transfer from 0 mm Znto 1 mm Zn, with a ZNT1-LC-specific probe (Fig. 6). Therewas no apparent reduction in hybridization signal in this48 h period, either in roots or in shoots. To determine thelevel of cross hybridization of the T. caerulescens LC probesto MP, LE and T. arvense DNA and RNA, low and highstringency DNA blot hybridizations were performed.Under low stringency conditions, the ZTP1 probe detectedthe corresponding ZTP1 genomic fragment as well as onecross-hybridizing ZTP1-homologous sequence, present inboth Thlaspi species and in Arabidopsis (Fig. 7). Upon highstringency washing only the ZTP1-containing fragmentswere observed. The Southern analysis with ZNT1 andZNT2 probes showed the respective homologues in T. arvense, Arabidopsis and all T. caerulescens accessions.Under reduced stringency conditions each of the twoprobes cross-hybridized to their respective DNA frag-ments, but not to any additional DNA fragments (Fig. 7),suggesting that these two genes are the only closely relatedZIP4 homologues present in T. caerulescens and T. arvense.

DISCUSSION

The physiological analysis of three Zn hyperaccumulator T. caerulescens accessions and a non-hyperaccumulator


T. arvense accession confirms that the Zn hyperaccumula-tor species is characterized by a much higher Zn uptake,shoot-to-root concentration ratio and tolerance than therelated non-hyperaccumulator species. Additionally, wehave observed a high and independent inter-accession vari-ability for these physiological properties in T. caerulescens.

The metallicolous T. caerulescens accessions LC and MPare specifically adapted to their native soils, since theobserved metal tolerance characteristics correspond wellwith the soil metal composition at the sites of seed collec-tion. The LC and MP plants exhibited elevated toleranceto, respectively, Zn and Ni. The high level of Zn tolerancein LC plants is associated with decreased uptake and trans-port of this metal, compared to the non-metallicolousaccession LE. Reduced Zn accumulation and transport inT. caerulescens and Arabidopsis halleri accessions fromcalamine soil, as compared to accessions from non-metal-liferous soil, has been reported previously (Meerts & VanIsacker 1997; Bert et al. 2000; Escarré et al. 2000), indicat-ing that Zn tolerance and accumulation in these species areindependent traits. Schat et al. (1999) however, reported avery low Zn and Cd shoot-to-root concentration ratio in anon-metallicolous accession originating from another sitenear Lellingen, about 4 km distant from the site of originof the accession LE used in the present study. More exten-sive comparisons of non-metallicolous and metallicolousaccessions have shown that the low transport in the formeraccession from Lellingen is probably highly exceptional(data not shown).

In contrast to the observations for Zn uptake by the Zn-tolerant LC accession, the highly Ni-tolerant MP plantsshowed an increased rather than decreased uptake of Niand Zn, compared to the non-metallicolous accession LE.


Figure 4. Northern blot analysis of ZTP1 (A)and ZTP1-arvense (B) expression in leaves androots of T. arvense (Ta) and T. caerulescens,accessions LC, MP and LE (TcLC, TcMP andTcLE), grown at 0, 2 and 10 mm Zn.Approximately equal loading of the RNAs inblots A and B is shown in C and D,respectively, after hybridization with a 16SrRNA-specific probe. The blots were washedunder low-stringency conditions.

Figure 5. Northern blot analysis of ZNT1 (A)and ZNT2 (B) expression in leaves and roots ofT. arvense (Ta) and T. caerulescens accessionsLC, MP and LE (TcLC, TcMP and TcLE),grown at 0, 2 and 10 mm Zn. Approximatelyequal loading of the RNAs in blots A and B isshown in C and D, respectively, afterhybridization with a 16S rRNA-specific probe.The blots were washed under low-stringencyconditions.

This indicates once more that metal tolerance and metaluptake are independent traits.

The observed metal uptake, transport and tolerancecharacteristics suggest an important role for transmem-brane metal transporters in the metal hyperaccumulationmechanism. Thorough genetic and physiological analysis ofthe inter-accession variability in respect to the expressionof T. caerulescens Zn transporter genes could give moreinsight into the function of this genes and the mechanismof metal hyperaccumulation. In this work it was shown thatT. caerulescens contains at least three different expressedgenes with strong homology to Zn transporters. ZTP1 is theclosest homologue of the Arabidopsis ZAT Zn transportergene and most likely the Thlaspi orthologue. Based onSouthern analysis there appears to be one other ZAT/ZTP1homologous DNA sequence present in both Thlaspi andArabidopsis. A corresponding cDNA has not been foundand expression has thus not been tested. Overexpressingthe ZAT gene in Arabidopsis caused an increased Zncontent in roots as well as enhanced Zn tolerance. This sug-gests that the ZAT protein is involved in the internal com-partmentation of this metal (Van der Zaal et al. 1999).Based on the Northern analysis, we conclude that ZTP1 isclearly overexpressed in T. caerulescens compared to T. arvense, with the highest expression in LC. LC is also theaccession with the highest tolerance to Zn. Together withits predominant expression in the leaves this emphasizesthe proposed role for ZTP1/ZAT-like transporters in Zncompartmentation and also suggests an important contri-bution of ZTP1 expression to Zn tolerance.

ZNT1 and ZNT2 resemble the Arabidopsis ZIP4 gene,suggested to encode a Zn transporter (Grotz et al. 1998).Experimental evidence supporting this view was recently


provided by Pence et al. (2000), also mentioned by Lasatet al. (2000), who described the cloning of a ZNT1 partialcDNA from T. caerulescens accession Prayon, by functionalcomplementation of a Zn uptake deficient yeast mutant.Wehave been unable to show any functional complementationusing the same zinc uptake-deficient yeast mutant zhy3(Zhao & Eide 1996b) transformed with overexpression con-structs containing either the ZTP1, ZNT1 or ZNT2 cDNAsequences. It may be that the presence of the plant N-terminal signal sequence interfered with proper intracellu-lar localization of the heterologous protein.The presence ofan N-terminal signal sequence may be also the reason thatthe ZNT2 cDNA was not identified in the functional com-plementation experiment that yielded the ZNT1 cDNA(Pence et al. 2000; Lasat et al. 2000), although the ZNT2mRNA is not considerably less abundant than the ZNT1mRNA. On the other hand, the presence of a signalsequence did not disturb the functional complementation ofthe yeast mutant by the Arabidopsis ZIP1, ZIP2 and ZIP3genes (Grotz et al. 1998), which belong to the same ZIP-likegene family, although not as closely related to ZNT1 andZNT2 as ZIP4. As for ZNT2 in T. caerulescens, neither afull-length nor a partial ZIP4 cDNA clone was picked upfrom the Arabidopsis seedling cDNA-expression librarythat yielded the ZIP1, ZIP2 and ZIP3 cDNAs (Grotz et al.1998), although its transcript should not be less abundant.

Initially the ZIP4 protein was predicted to contain apotential chloroplast targeting sequence. Based on therecently deposited ZIP4 partial cDNA sequence (acc. no.AV441840) the predicted protein contains an N-terminalsignal sequence and is most likely targeted to the plasmamembrane (PSORT, Klein et al. 1985).This is more in accor-dance with expression in both roots and shoots.


Figure 6. Northern blot analysis of ZNT1expression in roots (R) and leaves (L) of T. caerulescens accessions LC grown at1 mm Zn for a 48 h period. The blot waswashed under low-stringency conditions.

Figure 7. Southern blot analysis of ZTP1(A), ZNT1 (B) and ZNT2 (C).GenomicDNA from T. arvense (Ta), T. caerulescensaccessions LC (TcLC), MP (TcMP), LE(TcLE) and A. thaliana ecotype Columbia(At) was digested with HindIII. The figurerepresents low-stringency (A and C) andhigh-stringency (B) washings. Under lowstringency washing, the ZTP1 (A) andZNT2 (C) probes cross-hybridize to onlyone other homologous gene copy (*). Forthe ZNT2 probe, this is the ZNT1 gene, asthe weaker hybridization signals in (C) arethe very strong hybridizing signals in (B).

The ZNT1 and ZNT2 genes are clearly part of the ZIP-like gene family. First of all they encode proteins with eightpredicted transmembrane domains, as found in the Ara-bidopsis ZIP4 protein and other related ZIP proteins. Thepredicted proteins contain a histidine-rich region betweentransmembrane domains III and IV (Fig. 3), which is pro-posed to be the heavy metal binding sequence (Eng et al.1998). Finally, the proteins contain the conserved ZIP sig-nature sequence in transmembrane domain IV. Thissequence is fully conserved among all members of the ZIPfamily (Grotz et al. 1998; Eng et al. 1998) and suggested toplay a role in substrate transport over a membrane.

In comparison with T. arvense, ZNT1 and ZNT2 arehighly expressed in at least four, respectively, three,T. caerulescens accessions. The high expression concernspredominantly root tissue, which is a strong indication of arole for these genes in enhanced Zn uptake from the soil.This is in line with the strongly enhanced Zn uptake in T. caerulescens compared with T. arvense (Table 2). The dif-ferences in ZNT1/ZNT2 expression among T. caerulescensaccessions are less pronounced and also much less associ-ated with differences in Zn uptake between these acces-sions. It is clear that expression of ZNT1 and ZNT2 doesnot account for all of the observed inter-accession differ-ences in Zn uptake, transport to the shoots, or in Zn toler-ance. Of course it would be very unlikely that zinc uptakeis solely controlled by the expression of two zinc trans-porter genes. Additional zinc transporters, like the non-metal-specific Nramp transporters (Thomine et al. 2000) orother ZIP-like proteins are also likely to be involved in zincuptake. Alternatively, zinc transporter regulation may acton the post-transcriptional level as was reported for theyeast ZRT1 zinc transporter (Gitan et al. 1998; Gitan &Eide, 2000).

The Ni hyperaccumulation in MP and, to a lower degree,in LE and the complete absence of this phenomenon in LC, suggests the presence of at least two different uptake systems involved in Zn/Ni hyperaccumulation in T.caerulescens:a Zn-specific high-affinity uptake system anda low-affinity Zn/Ni uptake system, which prefers Zn overNi. The latter seems to be suppressed in the LC accession,but overexpressed in the MP accession.The relatively smalldifference in ZNT1 and ZNT2 expression between the dif-ferent accessions suggests that other genes are responsiblefor the enhanced Ni accumulation in MP and LE.

All three Zn transporters are highly expressed in T. caerulescens at all Zn concentrations tested. The highestconcentration of 10 mm approaches the Zn concentrationwhich is available as water-soluble Zn in heavily Zn-contaminated soils (Ernst & Nelissen, 2000). Pence et al.(2000) observed that ZNT1 expression is downregulatedonly after prolonged exposure to 50 mm Zn, whereas in T.arvense it is downregulated at 1 mm. In addition, weobserved that the transcriptional downregulation of ZNT1was not yet obvious after 48 h exposure to 1 mm Zn. Theapparent decrease in Zn-imposed downregulation of zinctransporter genes in T. caerulescens has been conservedamong at least four accessions and may well be the first evo-


lutionary step that led to the ability to accumulate and tol-erate high Zn levels in this hyperaccumulator species.

ACKNOWLEDGMENTS

We thank Martijn Fiers for making the cDNA library,Richard Immink and Marco Busscher for their support inperforming the automatic sequence reactions, Dr DavidEide for providing the zinc uptake-deficient yeast strain, DrArle Kruckeberg for providing plasmids and technicalsupport and Paul Koevoets and Professor Dr Wilfried Ernstfor critical reading of the manuscript.

Part of this work was supported by the Portuguese Foun-dation for Science and Technology, programme PRAXISXXI (grant no. BD/16152/98).

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Received 14 June 2000; received in revised form 9 October 2000;accepted for publication 9 October 2000


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